CHEN Yi-guang , LI Hui-ming , LI Qin-yuan , CHEN Wei , CUI Xiao-Long
2007, 47(4):571-577.
Abstract:The microbial diversity of cultivable bacteria,isolated from the ancient salt deposits from the Yipinglang Salt Mine(YPL)in the Yunnan Province,P. R. China,was investigated by using conventional culture-dependent method and phylogenetic analyses based on 16S rRNA gene sequence comparisons. 38 bacteria strains were isolated from the brine,halite and saline soil samples on MBA (marine broth agar 2216,Difco) and ISP 2 (International Streotomyces Project medium 2) media supplemented with 0.5~3.5mol/L NaCl. The genomic DNAs of the isolates were extracted and their 16S rRNA genes were amplified by PCR using bacterial universal primers. The resulting 16S rRNA gene sequences were compared with sequences obtained from public databases to find the most closely related species. Phylogenetic analyses were performed using the software packages MEGA after multiple alignment of sequence data by CLUSTAL X. The evolutional instances (corrected by Kimura's 2-parameter model) were calculated and clustering was performed with the neighbor-joining method. The results showed that the isolates are members of twenty-four genera (Acinetobacter,Agromyces,Arthrobacter,Bacillus,Brevundimonas,Chromohalobacter,Dietzia,Erythrobacter,Exiguobacterium,Halomonas,Idiomarina,Kocuria,Marinobacter,Micrococcus,Paracoccus,Planomicrbium,Porphyrobacter,Pseudomonas,Psychrobacter,Roseivivax,Saccharospirillum,Salegentibactor,Salinicoccus,Streptomyces) of seventeen families (Alteromonadaceae,Bacillaceae,Caulobacteraceae,Flavobacteriaceae,Halomonadaceae,Idiomarinaceae,Microbacteriaceae,Micrococcaceae,Moraxellaceae,Planococcaceae,Pseudomonadaceae,Rhodobacteraceae,Dietziaceae,Saccharospirillaceae,Sphingomonadaceae,Staphylococcaceae,Streptomycetaceae) in four major phylogenetic groups (Actinobacteria,Bacteroidetes,Firmicutes,Proteobacteria). The most abundant and diverse isolates were within the phyla of Proteobacteria(47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)and Firmicutes(34.2%). The phylogenetic distance matrix results suggested that out of 38 isolates 32 are different strains of 27 known species,and that at least 3 stains represent new species within 3 characterized genera. Y3 (Accession No. EF177665) and Y25 (EF177670) represent new species of the genera Idiomarina and Saccharospirillum,respectively. Y15 (DQ837380),Y16 (EF177680) and Y22 (EF177689) represent a new species of the genus Salinicoccus. And strain Y21 (EF177692) may represent a novel species of a possible new genus of the family Staphylococcaceae. The results presented above shown that there are abundant bacterial species diversity and phylogenetic diversity in the ancient salt deposits from the Yipinglang Salt Mine.
CHEN Yi-guang , LI Hui-ming , LI Qin-yuan , CHEN Wei , CUI Xiao-Long
2007, 47(4):571-577.
Abstract:The microbial diversity of cultivable bacteria,isolated from the ancient salt deposits from the Yipinglang Salt Mine(YPL)in the Yunnan Province,P. R. China,was investigated by using conventional culture-dependent method and phylogenetic analyses based on 16S rRNA gene sequence comparisons. 38 bacteria strains were isolated from the brine,halite and saline soil samples on MBA (marine broth agar 2216,Difco) and ISP 2 (International Streotomyces Project medium 2) media supplemented with 0.5~3.5mol/L NaCl. The genomic DNAs of the isolates were extracted and their 16S rRNA genes were amplified by PCR using bacterial universal primers. The resulting 16S rRNA gene sequences were compared with sequences obtained from public databases to find the most closely related species. Phylogenetic analyses were performed using the software packages MEGA after multiple alignment of sequence data by CLUSTAL X. The evolutional instances (corrected by Kimura's 2-parameter model) were calculated and clustering was performed with the neighbor-joining method. The results showed that the isolates are members of twenty-four genera (Acinetobacter,Agromyces,Arthrobacter,Bacillus,Brevundimonas,Chromohalobacter,Dietzia,Erythrobacter,Exiguobacterium,Halomonas,Idiomarina,Kocuria,Marinobacter,Micrococcus,Paracoccus,Planomicrbium,Porphyrobacter,Pseudomonas,Psychrobacter,Roseivivax,Saccharospirillum,Salegentibactor,Salinicoccus,Streptomyces) of seventeen families (Alteromonadaceae,Bacillaceae,Caulobacteraceae,Flavobacteriaceae,Halomonadaceae,Idiomarinaceae,Microbacteriaceae,Micrococcaceae,Moraxellaceae,Planococcaceae,Pseudomonadaceae,Rhodobacteraceae,Dietziaceae,Saccharospirillaceae,Sphingomonadaceae,Staphylococcaceae,Streptomycetaceae) in four major phylogenetic groups (Actinobacteria,Bacteroidetes,Firmicutes,Proteobacteria). The most abundant and diverse isolates were within the phyla of Proteobacteria(47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)and Firmicutes(34.2%). The phylogenetic distance matrix results suggested that out of 38 isolates 32 are different strains of 27 known species,and that at least 3 stains represent new species within 3 characterized genera. Y3 (Accession No. EF177665) and Y25 (EF177670) represent new species of the genera Idiomarina and Saccharospirillum,respectively. Y15 (DQ837380),Y16 (EF177680) and Y22 (EF177689) represent a new species of the genus Salinicoccus. And strain Y21 (EF177692) may represent a novel species of a possible new genus of the family Staphylococcaceae. The results presented above shown that there are abundant bacterial species diversity and phylogenetic diversity in the ancient salt deposits from the Yipinglang Salt Mine.
NI Hui-juan , BAO Qiu-hua , SUN Tian-song , CHEN Xia , ZHANG He-ping
2007, 47(4):578-582.
Abstract:A total of 87 yeast strains were isolated from 28 home-made koumiss samples,a traditional fermented mare milk product in Xinjiang of China. The isolates were identified by standard physiological and biochemical tests and analysis of the large-subunit (26S) rDNA gene D1/D2 domain sequences. They are proved to be Saccharomyces unisporus (48.3% of the isolates),Kluyveromyces marxianus (27.6%) and Pichia membranaefaciens (15.0%),Saccharomyces cerevisiae (9.2%). Among them,six isolates and a standard yeast strain were selected for analysis of D1/D2 domain sequences. They are indicated as S. unisporus,K. marxianus,S. cerevisiae,P. membranifaciens,P. fermentans,P. galeiformis and the standard yeast strain is indicated as K. lactis (100%). The results obtained demonstrate the value of using analysis of D1/D2 domain sequences methods,in conjunction with the traditional taxonomic methods based on phenotypic characteristics. This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the wealth of yeast biodiversity of the koumiss in Xinjiang Province. The result obtained shown that S. unisporus and K. marxianus were the predominant strains of koumiss in Xingjiang of China.
NI Hui-juan , BAO Qiu-hua , SUN Tian-song , CHEN Xia , ZHANG He-ping
2007, 47(4):578-582.
Abstract:A total of 87 yeast strains were isolated from 28 home-made koumiss samples,a traditional fermented mare milk product in Xinjiang of China. The isolates were identified by standard physiological and biochemical tests and analysis of the large-subunit (26S) rDNA gene D1/D2 domain sequences. They are proved to be Saccharomyces unisporus (48.3% of the isolates),Kluyveromyces marxianus (27.6%) and Pichia membranaefaciens (15.0%),Saccharomyces cerevisiae (9.2%). Among them,six isolates and a standard yeast strain were selected for analysis of D1/D2 domain sequences. They are indicated as S. unisporus,K. marxianus,S. cerevisiae,P. membranifaciens,P. fermentans,P. galeiformis and the standard yeast strain is indicated as K. lactis (100%). The results obtained demonstrate the value of using analysis of D1/D2 domain sequences methods,in conjunction with the traditional taxonomic methods based on phenotypic characteristics. This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the wealth of yeast biodiversity of the koumiss in Xinjiang Province. The result obtained shown that S. unisporus and K. marxianus were the predominant strains of koumiss in Xingjiang of China.
SUN Chang-po , SONG Fu-ping , ZHANG Jie , HUANG Da-fang
2007, 47(4):583-587.
Abstract:The spoIVF operon exists in Bacillus universally. Two proteins encoded by the spoIVF operon are essential for the sporulation of Bacillus subtilis. In this study,a spoIVF operon disruption mutant G03(spoIVF-),in which the spoIVF operon was deleted,was constructed by homologous recombination. The result showed that the mutant strain lost the ability of sporulation. At the same time,the expression of Insecticidal Crystal Protein(ICP) was severely reduced in G03(spoIVF-)mutant strain and resulted in no crystals. The lacZ gene was fused with the promoter of the cry1Aa gene and expressed in mutant strain G03(spoIVF-)and G03 wild strain. The activity of β-galactosidase much lower in mutant G03(spoIVF-) strain than in the wild-type strain. This further suggested that the activity of σE and σK factors was affected in mutant G03(spoIVF-) strain. The ability of sporulation and production of Insecticide Crystal Protein was complemented by the expression of spoIVF operon through the vector of pSTK in the mutant strain. In all,The spoIVF operon is essential for the sporulation and the expression of cry gene controlled by σE and σK factors.
SUN Chang-po , SONG Fu-ping , ZHANG Jie , HUANG Da-fang
2007, 47(4):583-587.
Abstract:The spoIVF operon exists in Bacillus universally. Two proteins encoded by the spoIVF operon are essential for the sporulation of Bacillus subtilis. In this study,a spoIVF operon disruption mutant G03(spoIVF-),in which the spoIVF operon was deleted,was constructed by homologous recombination. The result showed that the mutant strain lost the ability of sporulation. At the same time,the expression of Insecticidal Crystal Protein(ICP) was severely reduced in G03(spoIVF-)mutant strain and resulted in no crystals. The lacZ gene was fused with the promoter of the cry1Aa gene and expressed in mutant strain G03(spoIVF-)and G03 wild strain. The activity of β-galactosidase much lower in mutant G03(spoIVF-) strain than in the wild-type strain. This further suggested that the activity of σE and σK factors was affected in mutant G03(spoIVF-) strain. The ability of sporulation and production of Insecticide Crystal Protein was complemented by the expression of spoIVF operon through the vector of pSTK in the mutant strain. In all,The spoIVF operon is essential for the sporulation and the expression of cry gene controlled by σE and σK factors.
LIN Chun-hua , HE Chun-ping , WANG Kui-di , LIAO Qi-heng , ZHENG Fu-cong
2007, 47(4):588-592.
Abstract:Using thermal asymmetric interlaced PCR(TAIL-PCR),39 specifical fragments of Magnaporthe grisea genomic DNA flanked on the T-DNA were successfully amplified from 37 M. grisea transformants randomly selected from the mutants induced by T-DNA insertion in our laboratory. These fragments were sequenced and then compared by BLAST with the sequences of M. grisea genomic DNA published in netwwork. T-DNA insertion positions for 17 transformants were spotted on the genome of M. grisea. Of all the 39 amplified specifical fragments,19 were M. grisea genomic DNA and 20 contained the vector backbone sequences. Of the 19 M. grisea genomic DNA fragments,10 flanked on the right and 9 on the left border of T-DNA inserted. Of the 10 M. grisea genomic DNA fragments flanked on the right border of T-DNA,9 had a 102bp identical sequence. However,the 7 fragments flanked on the left border of T-DNA did not have this regularity. The above results demonstrated that T-DNA right nick positions were relatively fixed on the M. grisea genomic DNA,similar to the ones on plant genomic DNA. The spotted positions of T-DNA on the genome for 17 M. grisea transformants established a solid foundation for further functional genomic research of M. grisea.
LIN Chun-hua , HE Chun-ping , WANG Kui-di , LIAO Qi-heng , ZHENG Fu-cong
2007, 47(4):588-592.
Abstract:Using thermal asymmetric interlaced PCR(TAIL-PCR),39 specifical fragments of Magnaporthe grisea genomic DNA flanked on the T-DNA were successfully amplified from 37 M. grisea transformants randomly selected from the mutants induced by T-DNA insertion in our laboratory. These fragments were sequenced and then compared by BLAST with the sequences of M. grisea genomic DNA published in netwwork. T-DNA insertion positions for 17 transformants were spotted on the genome of M. grisea. Of all the 39 amplified specifical fragments,19 were M. grisea genomic DNA and 20 contained the vector backbone sequences. Of the 19 M. grisea genomic DNA fragments,10 flanked on the right and 9 on the left border of T-DNA inserted. Of the 10 M. grisea genomic DNA fragments flanked on the right border of T-DNA,9 had a 102bp identical sequence. However,the 7 fragments flanked on the left border of T-DNA did not have this regularity. The above results demonstrated that T-DNA right nick positions were relatively fixed on the M. grisea genomic DNA,similar to the ones on plant genomic DNA. The spotted positions of T-DNA on the genome for 17 M. grisea transformants established a solid foundation for further functional genomic research of M. grisea.
LIU Wen-ping , ZENG Hong-mei , LIU Yan-feng , YUAN Jing-jing , QIU De-wen
2007, 47(4):593-597.
Abstract:A positive clone was screened from Alternaria tenuissima expression library . The result of sequencing and gene analysis indicated that the cloned DNA fragment has a complete ORF,which was named peaT2 (Protein Elicitor from Alternaria tenuissima 2) with the GenBank accession number EF212880. The gene was amplified by PCR and subcloned into the pPIC9K of Pichia pastoris expression system. The resulting recombinant plasmid pPIC9K/peaT2 was verified by sequencing and digested by SacⅠ. The linearized DNA was transformed into P. pastoris GS115 by electroporation. By means of MD and G418-YPD plates and PCR, the recombinant P. pastoris strains (his- mut+) were obtained. A recombinant clone cultivated on YPD plate with high concentration of G418 was randomly selected as expression strain. The protein expression was induced by methanol and analyzed by 12% SDS-PAGE. The results of SDS-PAGE and Western blot indicated that this gene was expressed successfully in P. pastoris with the induction of methanol. In BMMY culture medium, the expressed protein reached its maximum amount at 72h, whereas no corresponding protein was detected in the negative control. Bioassay was performed with the expressed protein. After soaking with the expressed protein for 8h, wheat seeds were cultured, the height of seedlings was measured after 24h, 36h, 48h, 7d, respectively, and the root length was measured after 7 days. The results showed that the expressed protein can promote seeding growth and root length obviously in appropriate concentration. It is revealed that PeaT2 can act as protein elicitor.
LIU Wen-ping , ZENG Hong-mei , LIU Yan-feng , YUAN Jing-jing , QIU De-wen
2007, 47(4):593-597.
Abstract:A positive clone was screened from Alternaria tenuissima expression library . The result of sequencing and gene analysis indicated that the cloned DNA fragment has a complete ORF,which was named peaT2 (Protein Elicitor from Alternaria tenuissima 2) with the GenBank accession number EF212880. The gene was amplified by PCR and subcloned into the pPIC9K of Pichia pastoris expression system. The resulting recombinant plasmid pPIC9K/peaT2 was verified by sequencing and digested by SacⅠ. The linearized DNA was transformed into P. pastoris GS115 by electroporation. By means of MD and G418-YPD plates and PCR, the recombinant P. pastoris strains (his- mut+) were obtained. A recombinant clone cultivated on YPD plate with high concentration of G418 was randomly selected as expression strain. The protein expression was induced by methanol and analyzed by 12% SDS-PAGE. The results of SDS-PAGE and Western blot indicated that this gene was expressed successfully in P. pastoris with the induction of methanol. In BMMY culture medium, the expressed protein reached its maximum amount at 72h, whereas no corresponding protein was detected in the negative control. Bioassay was performed with the expressed protein. After soaking with the expressed protein for 8h, wheat seeds were cultured, the height of seedlings was measured after 24h, 36h, 48h, 7d, respectively, and the root length was measured after 7 days. The results showed that the expressed protein can promote seeding growth and root length obviously in appropriate concentration. It is revealed that PeaT2 can act as protein elicitor.
MA Zheng , RAO Zhi-ming , SHEN Wei , FANG Hui-ying , ZHUGE Jian
2007, 47(4):598-603.
Abstract:1,3-Propanediol is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1,3-propanediol has been a novel and competitive way. In our previous job,the gene dahB encoding for glycerol dehydratase from Klebsiella and the gene yqhD encoding for 1,3-propanediol oxidoreductase isoenzyme from E. coli were cloned respectively. The two genes were then tandemly ligated and expressed successfully in E. coli. The recombinant E. coli strain could produce 1,3-propanediol from D-glycerol. In the current research,the expression vectors including pGAPZB-yqhD,pGAPZB-dhaB and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB were furtherly constructed on the basis of our previous job. Then the vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB was introduced into Saccharomyces cerevisiae W303-1A using LiAc transformation method successfully. D-glucose is used as substrate to produce 1,3-propanediol with the recombinant strain after fermentation for 72h. SDS-PAGE analysis showed recombinant products at about 61kD、43kD、21kD、15kD,consistent with the molecular weight from the report. The specific enzymatic activity of the glycerol dehydratase and 1,3-propanediol oxidoreductase isoenzyme of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD -pGAP-dhaB were 24U/mg protein and 15U/mg protein,respectively,while those of the control were undetectable. In contrast to the wild strain without 1,3-propanediol output,1,3-propanediol concentration of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB reaches about 1.5g/L. The above results showed that the engineered S. cerevisiae strain which can convert the D-glucose as substrate to produce 1,3-propanediol by one-step fermentation was constructed successfully. This accomplishment bodes well for future construction of recombinant yeast strain which could overproduce 1,3-propanediol with the lower cost feedstock D-glucose by introducing the two genes yqhD and dhaB into the yeast strain overproducing glycerol with D-glucose (e.g. Candida glycerinogenes WL2002-5,which is capable of producing glycerol more than 120g/L with D-glucose as substrate and has been used for the commercial production of glycerol).
MA Zheng , RAO Zhi-ming , SHEN Wei , FANG Hui-ying , ZHUGE Jian
2007, 47(4):598-603.
Abstract:1,3-Propanediol is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1,3-propanediol has been a novel and competitive way. In our previous job,the gene dahB encoding for glycerol dehydratase from Klebsiella and the gene yqhD encoding for 1,3-propanediol oxidoreductase isoenzyme from E. coli were cloned respectively. The two genes were then tandemly ligated and expressed successfully in E. coli. The recombinant E. coli strain could produce 1,3-propanediol from D-glycerol. In the current research,the expression vectors including pGAPZB-yqhD,pGAPZB-dhaB and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB were furtherly constructed on the basis of our previous job. Then the vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB was introduced into Saccharomyces cerevisiae W303-1A using LiAc transformation method successfully. D-glucose is used as substrate to produce 1,3-propanediol with the recombinant strain after fermentation for 72h. SDS-PAGE analysis showed recombinant products at about 61kD、43kD、21kD、15kD,consistent with the molecular weight from the report. The specific enzymatic activity of the glycerol dehydratase and 1,3-propanediol oxidoreductase isoenzyme of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD -pGAP-dhaB were 24U/mg protein and 15U/mg protein,respectively,while those of the control were undetectable. In contrast to the wild strain without 1,3-propanediol output,1,3-propanediol concentration of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB reaches about 1.5g/L. The above results showed that the engineered S. cerevisiae strain which can convert the D-glucose as substrate to produce 1,3-propanediol by one-step fermentation was constructed successfully. This accomplishment bodes well for future construction of recombinant yeast strain which could overproduce 1,3-propanediol with the lower cost feedstock D-glucose by introducing the two genes yqhD and dhaB into the yeast strain overproducing glycerol with D-glucose (e.g. Candida glycerinogenes WL2002-5,which is capable of producing glycerol more than 120g/L with D-glucose as substrate and has been used for the commercial production of glycerol).
XU Bo , CAO Yu-sheng , CHEN Yan , GUO Xing-hua
2007, 47(4):604-609.
Abstract:The food-grade inducible gene expression system in L. lactis was constructed for expression of cytoplasmic and anchored heterologous proteins. Gene α-aga encoding α-galactosidase was used as food-grade selectable marker instead of antibiotic resistance gene. Firstly,a food-grade cytoplasmic inducible expression vector pRNA48 containing α-aga,theta replicon from pRAF800,and PnisA-MCS-TpepN from pNZ8048 was constructed. Then the cell wall anchored expression vector pRNV48 containing α-aga,theta replicon,and PnisA-SPUsp45-nucA-CWAM6-t1t2 was constructed based on the plasmids pRNA48 and pVE5524,which was suitable for the heterologous proteins anchored to the cell wall of L.lactis NZ9000. The fusion OprF/H derived from Pseudomonas aeruginosa was cloned into plasmids pRNA48 and pRNV48 to construct the pRNA48-OprF/H and 3RNV48-OprF/H for the expression of OprF/H. OprF/H was produced by the recombinant strains when induced with nisin. The highest yield of active OprF/H was 9.6% of intracellular soluble protein and 9.8% of cell wall anchored protein in L.lactis NZ9000,respectively. The immunogenicity and specificity of the expressed protein from recombinant were tested by animal immunization and Western blot.
XU Bo , CAO Yu-sheng , CHEN Yan , GUO Xing-hua
2007, 47(4):604-609.
Abstract:The food-grade inducible gene expression system in L. lactis was constructed for expression of cytoplasmic and anchored heterologous proteins. Gene α-aga encoding α-galactosidase was used as food-grade selectable marker instead of antibiotic resistance gene. Firstly,a food-grade cytoplasmic inducible expression vector pRNA48 containing α-aga,theta replicon from pRAF800,and PnisA-MCS-TpepN from pNZ8048 was constructed. Then the cell wall anchored expression vector pRNV48 containing α-aga,theta replicon,and PnisA-SPUsp45-nucA-CWAM6-t1t2 was constructed based on the plasmids pRNA48 and pVE5524,which was suitable for the heterologous proteins anchored to the cell wall of L.lactis NZ9000. The fusion OprF/H derived from Pseudomonas aeruginosa was cloned into plasmids pRNA48 and pRNV48 to construct the pRNA48-OprF/H and 3RNV48-OprF/H for the expression of OprF/H. OprF/H was produced by the recombinant strains when induced with nisin. The highest yield of active OprF/H was 9.6% of intracellular soluble protein and 9.8% of cell wall anchored protein in L.lactis NZ9000,respectively. The immunogenicity and specificity of the expressed protein from recombinant were tested by animal immunization and Western blot.
YAN Zhuo-yan , XU Zhen-jian , XU Guang-zhi , TIAN Bing , HUA Yue-jin
2007, 47(4):610-615.
Abstract:Dps (DNA protection during starvation) is a member of the iron-binding protein family in prokaryotes. It has been shown previously that Dps possesses ferroxidase activity and the ability to sequester iron that seems to protect DNA from oxidative damage. Based on the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the gene (DRB0092) encoding a Dps protein homology in the extremely radioresistant bacterium Deinococcus radiodurans was deleted from the wild type strain R1 genome. The obtained mutant was designated as Kdps and further verified by PCR and sequencing. Survival rates of the mutant and wild type strain were investigated after challenged with different doses of hydrogen peroxide (H2O2). Results showed that the survival rate of dps mutant reduced rapidly under the low concentration of H2O2 (≤10mmol/L), while the wild type strain showed no sudden decrease. When the H2O2 concentration was higher than 30mmol/L, the difference of the survival rates between the mutant and wild type was more than 50-folds. The result demonstrated that the loss of dps gene in D. radiodurans made cells become more sensitive to oxidative damage. An iron staining method was used to determinate catalase activity in native polyacrylamide electrophoresis gels. The result displayed that two catalases in dps mutant were enhanced about 2-folds than that of wild type. The soluble Dps protein was obtained after construction of expression plasmid and inducement in E. coli transformant. The Dps protein showed the capacity of DNA binding and protected DNA from hydroxyl free radical cleavage in vitro. This study demonstrates that Dps protein of D. radiodurans plays an important role in its antioxidant system, which may contribute to its extreme resistance of this bacterium.
YAN Zhuo-yan , XU Zhen-jian , XU Guang-zhi , TIAN Bing , HUA Yue-jin
2007, 47(4):610-615.
Abstract:Dps (DNA protection during starvation) is a member of the iron-binding protein family in prokaryotes. It has been shown previously that Dps possesses ferroxidase activity and the ability to sequester iron that seems to protect DNA from oxidative damage. Based on the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the gene (DRB0092) encoding a Dps protein homology in the extremely radioresistant bacterium Deinococcus radiodurans was deleted from the wild type strain R1 genome. The obtained mutant was designated as Kdps and further verified by PCR and sequencing. Survival rates of the mutant and wild type strain were investigated after challenged with different doses of hydrogen peroxide (H2O2). Results showed that the survival rate of dps mutant reduced rapidly under the low concentration of H2O2 (≤10mmol/L), while the wild type strain showed no sudden decrease. When the H2O2 concentration was higher than 30mmol/L, the difference of the survival rates between the mutant and wild type was more than 50-folds. The result demonstrated that the loss of dps gene in D. radiodurans made cells become more sensitive to oxidative damage. An iron staining method was used to determinate catalase activity in native polyacrylamide electrophoresis gels. The result displayed that two catalases in dps mutant were enhanced about 2-folds than that of wild type. The soluble Dps protein was obtained after construction of expression plasmid and inducement in E. coli transformant. The Dps protein showed the capacity of DNA binding and protected DNA from hydroxyl free radical cleavage in vitro. This study demonstrates that Dps protein of D. radiodurans plays an important role in its antioxidant system, which may contribute to its extreme resistance of this bacterium.
YING Jiao-yan , YANG Su-sheng , LIU Shuang-jiang , JIANG Cheng-ying
2007, 47(4):616-621.
Abstract:Comamonas sp. strain CNB-1 degrades chloronitrbenzene and nitrobenzene for carbon and nitrogen sources. In this study,accumulation of polyhydroxyalkanoic acids (PHAs) within strain CNB-1 cells was investigated under various conditions. Results indicated that strain CNB-1 was able to synthesize PHA from various short-chain fatty acid and alcohols,and 57 w% of the dry cell weight (DCW) PHA was obtained when valerate and 1,4-butanediol were co-fed. Supplements of short-chain alcohols stimulated the accumulation of PHAs,and this stimulatory effect was attributed to the more amount of reductant generated from alcohol dehydrogenation. The genes encoding for PHA polymerase (phaC),for acetoacetyl-CoA thiolase (phaA),and acetoacetyl-CoA reductase (phaB) were cloned in Escherichia coli,and the recombinant E. coli synthesized PHA and showed enzymatic activities of PHA polymerase,acetoacetyl-CoA thiolase,and acetoacetyl-CoA reductase. The three genes occurred as a cluster of pha(C-A-B). To optimize their expression,the three genes were cloned to the pET vector and expressed respectively. Mass of expressed protein was detected and the enzyme activities increased greatly in contrast to wild CNB-1 strain,which is about 4.1,71,and 2882 folds of activities of CNB-1.
YING Jiao-yan , YANG Su-sheng , LIU Shuang-jiang , JIANG Cheng-ying
2007, 47(4):616-621.
Abstract:Comamonas sp. strain CNB-1 degrades chloronitrbenzene and nitrobenzene for carbon and nitrogen sources. In this study,accumulation of polyhydroxyalkanoic acids (PHAs) within strain CNB-1 cells was investigated under various conditions. Results indicated that strain CNB-1 was able to synthesize PHA from various short-chain fatty acid and alcohols,and 57 w% of the dry cell weight (DCW) PHA was obtained when valerate and 1,4-butanediol were co-fed. Supplements of short-chain alcohols stimulated the accumulation of PHAs,and this stimulatory effect was attributed to the more amount of reductant generated from alcohol dehydrogenation. The genes encoding for PHA polymerase (phaC),for acetoacetyl-CoA thiolase (phaA),and acetoacetyl-CoA reductase (phaB) were cloned in Escherichia coli,and the recombinant E. coli synthesized PHA and showed enzymatic activities of PHA polymerase,acetoacetyl-CoA thiolase,and acetoacetyl-CoA reductase. The three genes occurred as a cluster of pha(C-A-B). To optimize their expression,the three genes were cloned to the pET vector and expressed respectively. Mass of expressed protein was detected and the enzyme activities increased greatly in contrast to wild CNB-1 strain,which is about 4.1,71,and 2882 folds of activities of CNB-1.
JIANG Yun , HUANG Li-li , CHEN Chang-qing , QIAO Hong-ping , KANG Zhen-sheng
2007, 47(4):622-627.
Abstract:An actinomycete strain xjy was isolated from the soil of cotton field in Xinjiang against pathogenic fungus Fulvia fulva. The growth of 23 plant pathogens and 6 bacteria were strongly inhibitded by strain xjy on PDA plate. Antimicrobial spectrum of fermentation filtrate of strain xjy is extensive and selective to different pathogens. The morphology,cultural characteristics,physiological and biochemical properties,chemotaxonomy and 16S rDNA sequences of this strain were studied. The strain showed faint yellow vegetative mycelium,spiral spore-bearing filaments and column spores with smooth surface. No pigment was produced in culture. The cell wall typeⅠ and sugar type C showed the strain with streptomyces character. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related species and showed 99.6% identity of nucleotide sequence of 16S rDNA with Streptomyces lavendulae. From the polyphasic taxonomical view,the strain xjy falls into Streptomyces lavendulae. The optimum fermentation condition of strain xjy for producing the most effective ferment filtrate were cultured in 2% soybean flour,2% glucose,0.8% NaCl,0.2% CaCO3,0.32% (NH4)2SO4,the initial pH of 7.0,at 28℃ and shaked at 180r/mim for 6d. These results are valuable to strain application,antibiotics purification and its industrialization.
JIANG Yun , HUANG Li-li , CHEN Chang-qing , QIAO Hong-ping , KANG Zhen-sheng
2007, 47(4):622-627.
Abstract:An actinomycete strain xjy was isolated from the soil of cotton field in Xinjiang against pathogenic fungus Fulvia fulva. The growth of 23 plant pathogens and 6 bacteria were strongly inhibitded by strain xjy on PDA plate. Antimicrobial spectrum of fermentation filtrate of strain xjy is extensive and selective to different pathogens. The morphology,cultural characteristics,physiological and biochemical properties,chemotaxonomy and 16S rDNA sequences of this strain were studied. The strain showed faint yellow vegetative mycelium,spiral spore-bearing filaments and column spores with smooth surface. No pigment was produced in culture. The cell wall typeⅠ and sugar type C showed the strain with streptomyces character. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related species and showed 99.6% identity of nucleotide sequence of 16S rDNA with Streptomyces lavendulae. From the polyphasic taxonomical view,the strain xjy falls into Streptomyces lavendulae. The optimum fermentation condition of strain xjy for producing the most effective ferment filtrate were cultured in 2% soybean flour,2% glucose,0.8% NaCl,0.2% CaCO3,0.32% (NH4)2SO4,the initial pH of 7.0,at 28℃ and shaked at 180r/mim for 6d. These results are valuable to strain application,antibiotics purification and its industrialization.
CHEN Chun-feng , YANG Xiao-tong , LI Xu-quan , MI Ke , YANG Qing-yao
2007, 47(4):628-633.
Abstract:Five Ganoderma species,including G. lucidum,G. tsugae,G. oerstedii,G. resinaceum and G. subamboinens,were parallel studied under an identical condition. These species were cultivated using liquid fermentation and their mycelia polysaccharides were extracted and compared on the physical/chemical properties and in vitro immunomodulatory activities. These results showed that the polysaccharide yields varied markedly among different species,and G. oerstedii was the highest among the five. However,HPLC analysis showed all the polysaccharide extracts had similar molecular weight distributions and monosaccharide compositions. They all contained glucose,galactose,mannose,glucosamine hydrochloride and fucose. In vitro assays,these polysaccharide extracts significantly stimulated phagocytosis and nitric oxide production by RAW 264.7 macrophage cell line,and G. subamboinens exerted the strongest potency. When Con A was not or presented,they all showed an up-or-down immunomodulatory effect on mouse splenocyte proliferation. The results illustrate that in addition to G. lucidum and G. tsugae,which are the two mostly studied and applied species,other Ganoderma species can also produce polysaccharides with similar physical/chemical properties and with similar immunomodulatory activities.
CHEN Chun-feng , YANG Xiao-tong , LI Xu-quan , MI Ke , YANG Qing-yao
2007, 47(4):628-633.
Abstract:Five Ganoderma species,including G. lucidum,G. tsugae,G. oerstedii,G. resinaceum and G. subamboinens,were parallel studied under an identical condition. These species were cultivated using liquid fermentation and their mycelia polysaccharides were extracted and compared on the physical/chemical properties and in vitro immunomodulatory activities. These results showed that the polysaccharide yields varied markedly among different species,and G. oerstedii was the highest among the five. However,HPLC analysis showed all the polysaccharide extracts had similar molecular weight distributions and monosaccharide compositions. They all contained glucose,galactose,mannose,glucosamine hydrochloride and fucose. In vitro assays,these polysaccharide extracts significantly stimulated phagocytosis and nitric oxide production by RAW 264.7 macrophage cell line,and G. subamboinens exerted the strongest potency. When Con A was not or presented,they all showed an up-or-down immunomodulatory effect on mouse splenocyte proliferation. The results illustrate that in addition to G. lucidum and G. tsugae,which are the two mostly studied and applied species,other Ganoderma species can also produce polysaccharides with similar physical/chemical properties and with similar immunomodulatory activities.
XU Shu-jing , JU Jian-song , MA Yan-he
2007, 47(4):634-638.
Abstract:In the previous study we have isolated DNA fragment containing an alanine racemase gene (dadX) from Pseudomonas fluorescens TM5-2. Adjacent to dadX one ORF similar to a putative glycine/D-amino acid oxidase gene have been found. The same gene organization is found in several Pseudomonas species. Here, author would characterize this ORF to determine what kind of enzyme this gene encodes. DNA fragment containing gene encoding putative glycine/D-amino acid oxidase was cloned into the expression vector. Firstly oxidase activity in cell lysates prepared from the recombinant cells was measured, however, neither glycine nor D-alanine were oxidized judging from hydrogen peroxide formation. Secondly when the amino acid sequence deduced from the oxidase gene was compared to dye-linked D-amino acid dehydrogenases, all the important residues including FAD-binding motif were conserved. This gene was transformed and checked on TTC plate, it showed some activities of D-amino acid dehydrogenase. D-amino acid dehydrogenase activity was also detected when D-alanine and DCIP were used. The best substrate of this enzyme is D-histdine, which is different from some reports. Author will be in progress to purify the dehydrogenase and determine enzyme characteristics.
XU Shu-jing , JU Jian-song , MA Yan-he
2007, 47(4):634-638.
Abstract:In the previous study we have isolated DNA fragment containing an alanine racemase gene (dadX) from Pseudomonas fluorescens TM5-2. Adjacent to dadX one ORF similar to a putative glycine/D-amino acid oxidase gene have been found. The same gene organization is found in several Pseudomonas species. Here, author would characterize this ORF to determine what kind of enzyme this gene encodes. DNA fragment containing gene encoding putative glycine/D-amino acid oxidase was cloned into the expression vector. Firstly oxidase activity in cell lysates prepared from the recombinant cells was measured, however, neither glycine nor D-alanine were oxidized judging from hydrogen peroxide formation. Secondly when the amino acid sequence deduced from the oxidase gene was compared to dye-linked D-amino acid dehydrogenases, all the important residues including FAD-binding motif were conserved. This gene was transformed and checked on TTC plate, it showed some activities of D-amino acid dehydrogenase. D-amino acid dehydrogenase activity was also detected when D-alanine and DCIP were used. The best substrate of this enzyme is D-histdine, which is different from some reports. Author will be in progress to purify the dehydrogenase and determine enzyme characteristics.
ZHANG De-yong , CHENG Fei-xue , CHENG Ju-e , ZHANG Zhan-hong , LIU Yong
2007, 47(4):639-644.
Abstract:5-aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (ALAS). However, the fidelity of ALAS gene among species is low. The ALAS gene of photosynthetic bacteria Rhodoblastus acidophilus was cloned from its genomic DNA by conventional PCR and Veterette PCR and further sequenced. The identity of ALAS gene among photosynthetic bacteria species is from 64.0% to 95.1% according to phylogenic analysis. Furthermore, the ALAS gene was subcloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAS was overexpressed in Escherichia coli strains JM109, M15 and BL21(DE3), respectively. The expected 44kD protein was detected by SDS-PAGE in three E. coli strains after IPTG induction and further purified by affinity purification on Ni-NTA. The conditions including strain, medium, substrate of ALA synthesize (glycine and succinic acid), and ALA dehydratase inhibitor (levulinic acid) were optimized for attainning the maximum yield of ALA in E. coli. The ALA production was established on E.coli M15, medium 1 supplied with 100mmol/L glycine and 50mmol/L succinic acid, and 40mmol/L levulinic acid. The activity of ALAS was up to 333U/min·mg of protein. Meanwhile, the output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. ALA has a variety of agricultural applications not only as an herbicide, insecticide, and growth promoting factor, but also based on its ability to confer salt and cold temperature tolerance in plants. Our recombinant bacteria are of great potential in the production of ALA. Our results offer an easy and simple ALA mass production method and may stimulate the application of ALA in agriculture.
ZHANG De-yong , CHENG Fei-xue , CHENG Ju-e , ZHANG Zhan-hong , LIU Yong
2007, 47(4):639-644.
Abstract:5-aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (ALAS). However, the fidelity of ALAS gene among species is low. The ALAS gene of photosynthetic bacteria Rhodoblastus acidophilus was cloned from its genomic DNA by conventional PCR and Veterette PCR and further sequenced. The identity of ALAS gene among photosynthetic bacteria species is from 64.0% to 95.1% according to phylogenic analysis. Furthermore, the ALAS gene was subcloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAS was overexpressed in Escherichia coli strains JM109, M15 and BL21(DE3), respectively. The expected 44kD protein was detected by SDS-PAGE in three E. coli strains after IPTG induction and further purified by affinity purification on Ni-NTA. The conditions including strain, medium, substrate of ALA synthesize (glycine and succinic acid), and ALA dehydratase inhibitor (levulinic acid) were optimized for attainning the maximum yield of ALA in E. coli. The ALA production was established on E.coli M15, medium 1 supplied with 100mmol/L glycine and 50mmol/L succinic acid, and 40mmol/L levulinic acid. The activity of ALAS was up to 333U/min·mg of protein. Meanwhile, the output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. ALA has a variety of agricultural applications not only as an herbicide, insecticide, and growth promoting factor, but also based on its ability to confer salt and cold temperature tolerance in plants. Our recombinant bacteria are of great potential in the production of ALA. Our results offer an easy and simple ALA mass production method and may stimulate the application of ALA in agriculture.
JIA Tian-jun , LIU Dian-wu , LUO Jian-hua , ZHONG Guang-ming
2007, 47(4):645-648.
Abstract:To characterize the hypothetical protein CT249 using antibodies raised with CT249 fusion protein. The open reading frame (ORF) coding for CT249 in the Chlamydia trachomatis serovar L2 genome was cloned into the pGEX-6p2 vector using the restriction enzymes BamHⅠ and NotⅠ. The recombinant plasmid pGEX-6p2-CT249 was transformed into XL1-blue bacteria and the gene CT249 was expressed as fusion proteins with the glutathione -s-transferase (GST) tagged to the N-terminus. The GST-CT249 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CT249 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). The CT249 gene with 351bps in length was successfully cloned and expressed as GST fusion protein with molecular weight of 38.2kDa. The anti-fusion protein antibodies produced from mice detected the hypothetical protein CT249 in the inclusion membrane of Chlamydia trachomatis-infected cells. Using antibodies raised with GST-CT249 fusion protein, the hypothetical protein CT249 have been identified as a Chlamydia trachomatis inclusion membrane protein. Given the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells, this finding has provided a useful tool for further understanding the mechanisms of chlamydial intracellular parasitism.
JIA Tian-jun , LIU Dian-wu , LUO Jian-hua , ZHONG Guang-ming
2007, 47(4):645-648.
Abstract:To characterize the hypothetical protein CT249 using antibodies raised with CT249 fusion protein. The open reading frame (ORF) coding for CT249 in the Chlamydia trachomatis serovar L2 genome was cloned into the pGEX-6p2 vector using the restriction enzymes BamHⅠ and NotⅠ. The recombinant plasmid pGEX-6p2-CT249 was transformed into XL1-blue bacteria and the gene CT249 was expressed as fusion proteins with the glutathione -s-transferase (GST) tagged to the N-terminus. The GST-CT249 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CT249 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). The CT249 gene with 351bps in length was successfully cloned and expressed as GST fusion protein with molecular weight of 38.2kDa. The anti-fusion protein antibodies produced from mice detected the hypothetical protein CT249 in the inclusion membrane of Chlamydia trachomatis-infected cells. Using antibodies raised with GST-CT249 fusion protein, the hypothetical protein CT249 have been identified as a Chlamydia trachomatis inclusion membrane protein. Given the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells, this finding has provided a useful tool for further understanding the mechanisms of chlamydial intracellular parasitism.
LI Ke , ZHENG Tian-ling , TIAN Yun , YUAN Jian-jun
2007, 47(4):649-653.
Abstract:The composition of bacterial community in the intestine of the white shrimp, Litopenaeus vannamei under laboratory culture condition was determined using the 16S rDNA clone library. 16s rRNA gene was amplified and a library was constructed by using the genomic DNA extracted from the bacteria in the shrimp intestine as template. 12 different RFLP patterns of the clones were obtained by restriction fragment length polymorphism analysis using AfaⅠ and MspⅠ. Compared with the published sequences in GenBank database, sequencing results of cloned 16S rDNA amplicons revealed a diverse community including γ-proteobacteria and Firmicutes in the intestine of artificial diet-fed shrimp. Results showed that the Firmicutes group can be a dominant component (75.4%) in the shrimp intestinal microflora and other clones belong to γ-proteobacteria (24.6%) which were identified as Shewanella sp., Pantoea sp., Aranicola sp., Pseudomonas sp. and Vibrio sp., respectively. These results provide the first comprehensive description of microbial diversity of the white shrimp intestine and suggest that most of the bacteria associated with shrimp intestine are uncultured and novel species.
LI Ke , ZHENG Tian-ling , TIAN Yun , YUAN Jian-jun
2007, 47(4):649-653.
Abstract:The composition of bacterial community in the intestine of the white shrimp, Litopenaeus vannamei under laboratory culture condition was determined using the 16S rDNA clone library. 16s rRNA gene was amplified and a library was constructed by using the genomic DNA extracted from the bacteria in the shrimp intestine as template. 12 different RFLP patterns of the clones were obtained by restriction fragment length polymorphism analysis using AfaⅠ and MspⅠ. Compared with the published sequences in GenBank database, sequencing results of cloned 16S rDNA amplicons revealed a diverse community including γ-proteobacteria and Firmicutes in the intestine of artificial diet-fed shrimp. Results showed that the Firmicutes group can be a dominant component (75.4%) in the shrimp intestinal microflora and other clones belong to γ-proteobacteria (24.6%) which were identified as Shewanella sp., Pantoea sp., Aranicola sp., Pseudomonas sp. and Vibrio sp., respectively. These results provide the first comprehensive description of microbial diversity of the white shrimp intestine and suggest that most of the bacteria associated with shrimp intestine are uncultured and novel species.
ZHU Liang , XU Xiang-yang , CAO Dan-feng , LUO Wei-guo , YANG Yan-ni
2007, 47(4):654-661.
Abstract:The granulation of aerobic sludges for high-rate biodegradation of organic wastewater containing aniline and chloroanilines was investigated in a laboratory-scale sequencing airlift bioreactor (SABR). Aerobic granules were observed in 15days after start-up in SABR. After subsequent 83days, SABR was operated sequentially in superficial air velocity of 2.4cm/s, COD loadings of 1.0~3.6kg/(m3·d) and (chloro-)anilines loadings increased stepwise to 1kg/(m3·d), a steady-state performance of aerobic granular SABR was achieved at last, as evidenced by high and stable COD and (chloro-)anilines removal efficiencies of above 90% and 99.9%, respectively. Mature granules with median size of 0.45~2.5mm, minimal settling velocity of 62.1m/h, and SVI of 56mL/g were developed. Aerobic granular sludge displayed noteworthy SOUR, specific (chloro-)anilines degradation rates, PN content and PN/PS ratio in EPS extracts as 154mgDO/(gVSS·h), 0.18g/(gVSS·d), 28.0±1.9mg/gVSS and 6.5mg/mg respectively, indicating that they had high activity and ability to withstand high (chloro-)anilines loadings. Phylogenetic analysis of (chloro-)anilines-degrading aerobic granules indicated that β-、γ-Proteobacteria and Flavobacteria were dominant classes and the predominance bacteria were closely related to Pseudomonas sp. and Flavo-bacterium sp. Compared to chloroanilines-degrading aerobic granules, the population diversity was higher in the aniline and chloroaniline-degrading aerobic granules.
ZHU Liang , XU Xiang-yang , CAO Dan-feng , LUO Wei-guo , YANG Yan-ni
2007, 47(4):654-661.
Abstract:The granulation of aerobic sludges for high-rate biodegradation of organic wastewater containing aniline and chloroanilines was investigated in a laboratory-scale sequencing airlift bioreactor (SABR). Aerobic granules were observed in 15days after start-up in SABR. After subsequent 83days, SABR was operated sequentially in superficial air velocity of 2.4cm/s, COD loadings of 1.0~3.6kg/(m3·d) and (chloro-)anilines loadings increased stepwise to 1kg/(m3·d), a steady-state performance of aerobic granular SABR was achieved at last, as evidenced by high and stable COD and (chloro-)anilines removal efficiencies of above 90% and 99.9%, respectively. Mature granules with median size of 0.45~2.5mm, minimal settling velocity of 62.1m/h, and SVI of 56mL/g were developed. Aerobic granular sludge displayed noteworthy SOUR, specific (chloro-)anilines degradation rates, PN content and PN/PS ratio in EPS extracts as 154mgDO/(gVSS·h), 0.18g/(gVSS·d), 28.0±1.9mg/gVSS and 6.5mg/mg respectively, indicating that they had high activity and ability to withstand high (chloro-)anilines loadings. Phylogenetic analysis of (chloro-)anilines-degrading aerobic granules indicated that β-、γ-Proteobacteria and Flavobacteria were dominant classes and the predominance bacteria were closely related to Pseudomonas sp. and Flavo-bacterium sp. Compared to chloroanilines-degrading aerobic granules, the population diversity was higher in the aniline and chloroaniline-degrading aerobic granules.
TANG Li-hua , PAN Zhi-ming , CHENG Ning-ning , JIAO Xin-an , ZHANG Xiao-ming
2007, 47(4):662-666.
Abstract:The hemagglutinin protein (HA) gene of avian influenza virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-HA, and subcloned into eukaryotic expression vector pmcDNA3.1+. The HA gene was identified by sequencing. The recombinant plasmids were transformed into attenuated Salmonella typhimurium SL7207*, and the recombinants were designated as SL7207(pmcDNA3.1-HA). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3.1-HA is apparently higher than pcDNA3.1-HA in SL7207*. In order to compare the immune response induced by those two recombinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2×109CFU respectively. Both SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) initiated HA-specific mucosal antibodies in immunized mice. Furthermore, commercial ISA brown chickens were immunized with SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) at the dosage of 5×109CFU and boosted two weeks later with the same dose. Intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive control. The result of protective immunity showed that the chicken immunized with SL7207*(pmcDNA3.1-HA) had the protective rate of 79.3%, higher than that of SL7207*(pcDNA3.1-HA) with 56.7%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of avian influenza in the poultry.
TANG Li-hua , PAN Zhi-ming , CHENG Ning-ning , JIAO Xin-an , ZHANG Xiao-ming
2007, 47(4):662-666.
Abstract:The hemagglutinin protein (HA) gene of avian influenza virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-HA, and subcloned into eukaryotic expression vector pmcDNA3.1+. The HA gene was identified by sequencing. The recombinant plasmids were transformed into attenuated Salmonella typhimurium SL7207*, and the recombinants were designated as SL7207(pmcDNA3.1-HA). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3.1-HA is apparently higher than pcDNA3.1-HA in SL7207*. In order to compare the immune response induced by those two recombinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2×109CFU respectively. Both SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) initiated HA-specific mucosal antibodies in immunized mice. Furthermore, commercial ISA brown chickens were immunized with SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) at the dosage of 5×109CFU and boosted two weeks later with the same dose. Intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive control. The result of protective immunity showed that the chicken immunized with SL7207*(pmcDNA3.1-HA) had the protective rate of 79.3%, higher than that of SL7207*(pcDNA3.1-HA) with 56.7%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of avian influenza in the poultry.
XU Yi-gang , CUI Li-chun , GE Jun-wei , ZHAO Li-li , LI Yi-jing
2007, 47(4):667-672.
Abstract:Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L.casei 393 giving rise to recombinant strain pPG-VP2-E290/L.casei 393. The recombinant L.casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L.casei393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.
XU Yi-gang , CUI Li-chun , GE Jun-wei , ZHAO Li-li , LI Yi-jing
2007, 47(4):667-672.
Abstract:Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L.casei 393 giving rise to recombinant strain pPG-VP2-E290/L.casei 393. The recombinant L.casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L.casei393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.
CAI Kun , WANG Hui , BAO Shi-zhong , SHI Jing , HOU Xiao-jun
2007, 47(4):673-676.
Abstract:To constructed the recombinant human anti-rabies virus ScdsFv, cys sites were introduced into framework region (FR) of VH and VL genes which were amplified from human anti-rabies virus ScFv respectively using genetic point mutation technology. Cloned the ScdsFv gene into expression vector pET22b(+) and transformed into E.coli BL21 (DE3). The target protein was expressed by inducing with IPTG. Followed by renaturation in vitro and purified by Ni-NTA. The binding activity of ScdsFv was identified by Fluorescent antibody test (FAT) and ELISA. Results showed that recombinant ScdsFv were expressed at high level. Purity of the protein >90% after purified by Ni-NTA and renaturaton in vitro. FAT and ELISA results demonstrated that ScdsFv could binding antigen specificity and was more stable than ScFv. Recombinant ScdsFv provided experiment materials for further functional study.
CAI Kun , WANG Hui , BAO Shi-zhong , SHI Jing , HOU Xiao-jun
2007, 47(4):673-676.
Abstract:To constructed the recombinant human anti-rabies virus ScdsFv, cys sites were introduced into framework region (FR) of VH and VL genes which were amplified from human anti-rabies virus ScFv respectively using genetic point mutation technology. Cloned the ScdsFv gene into expression vector pET22b(+) and transformed into E.coli BL21 (DE3). The target protein was expressed by inducing with IPTG. Followed by renaturation in vitro and purified by Ni-NTA. The binding activity of ScdsFv was identified by Fluorescent antibody test (FAT) and ELISA. Results showed that recombinant ScdsFv were expressed at high level. Purity of the protein >90% after purified by Ni-NTA and renaturaton in vitro. FAT and ELISA results demonstrated that ScdsFv could binding antigen specificity and was more stable than ScFv. Recombinant ScdsFv provided experiment materials for further functional study.
WANG Qing-hua , WANG Xi-jun , HU Sen , GE Jin-ying , BU Zhi-gao
2007, 47(4):677-681.
Abstract:DNA vaccines have successfully induced effective antibody and cellular immune response to many viral pathogens. The antibody response of DNA immunization induction in mouse model with envelope glycoproteins of Rift Valley Fever Virus(RVFV),G(N+C),GN and GC was investigated. For this purpose, three codon G(N+C),GN and GC gene were insert into mammalian expression vector pCAGGS under chicken β-actin promoter to construct pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G(N+C). The expression of recommbinant GN or / and GC protein in BHK cells transfected with pCAGG-RVFV-GC or pCAGG-RVFV-G(N+C) DNA were confirmed by immunoprecipitation. Six-week-old female BALB/c mice were intramuscularly primed with 100(g pCAGG-RVFV-GN+pCAGG-RVFV-GC+pCAGG-RVFV-G(N+C), and boosted with same dose after 4 weeks. The serums were collected at 3 weeks post final boost. The serum IgG against Rift Valley Fever Virus G(N+C) protein were detect by indirect ELISA using recombinant Baculovirus expressed Rift Valley Fever Virus GN and GC glycoprotein. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GCand pCAGG-RVFV-G(N+C) elicited much strong IgG response. For serum neutralization antibody assay, a recombinant Vesicular Stomatitis Virus pseudotype, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVΔG*G, Whitt M A) and complemented with Rift Valley Fever Virus G(N+C) glycoprotein expressed in transient (VSVΔG* RVFV-G), was use to replace the authentic Rift Valley Fever Virus. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GCand pCAGG-RVFV-G(N+C) also induced high titer of neutralization antibody response. These result indicates that DNA immunization is an efficient vaccine strategy against Rift Valley Fever Virus.
WANG Qing-hua , WANG Xi-jun , HU Sen , GE Jin-ying , BU Zhi-gao
2007, 47(4):677-681.
Abstract:DNA vaccines have successfully induced effective antibody and cellular immune response to many viral pathogens. The antibody response of DNA immunization induction in mouse model with envelope glycoproteins of Rift Valley Fever Virus(RVFV),G(N+C),GN and GC was investigated. For this purpose, three codon G(N+C),GN and GC gene were insert into mammalian expression vector pCAGGS under chicken β-actin promoter to construct pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G(N+C). The expression of recommbinant GN or / and GC protein in BHK cells transfected with pCAGG-RVFV-GC or pCAGG-RVFV-G(N+C) DNA were confirmed by immunoprecipitation. Six-week-old female BALB/c mice were intramuscularly primed with 100(g pCAGG-RVFV-GN+pCAGG-RVFV-GC+pCAGG-RVFV-G(N+C), and boosted with same dose after 4 weeks. The serums were collected at 3 weeks post final boost. The serum IgG against Rift Valley Fever Virus G(N+C) protein were detect by indirect ELISA using recombinant Baculovirus expressed Rift Valley Fever Virus GN and GC glycoprotein. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GCand pCAGG-RVFV-G(N+C) elicited much strong IgG response. For serum neutralization antibody assay, a recombinant Vesicular Stomatitis Virus pseudotype, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVΔG*G, Whitt M A) and complemented with Rift Valley Fever Virus G(N+C) glycoprotein expressed in transient (VSVΔG* RVFV-G), was use to replace the authentic Rift Valley Fever Virus. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GCand pCAGG-RVFV-G(N+C) also induced high titer of neutralization antibody response. These result indicates that DNA immunization is an efficient vaccine strategy against Rift Valley Fever Virus.
YANG Bing , HE Jin-sheng , SHI Chang-xin , ZHANG Mei , YU Jie-mei
2007, 47(4):682-685.
Abstract:To construct a helper-dependent adenoviral vector expressing human respiratory syncytial virus (RSV) subgroup A F gene, and finish large scale preparation, purification and identification of the vector. F gene under the control of CMV promoter was subcloned into a shutle vector pSC11, and then cloned into HADd plasmid pSC15B. The HDAd/F genome was liberated by removing the bacterial sequences from the resulting plasmid pSC15B/F digested with restriction enzyme PmeⅠ, and then the linear HDAd/F DNA was transfected into 293Cre4 cells with calcium phosphate transfection method. The cells were infected by helper virus 16 hours after transfection. HDAd/F was amplified by serial coinfection of 293Cre4 cells by helper virus and the crude lysates from previous passage until it reached plateau of amplification by BFU staining of parallel amplified control vector pSC9A. HDAd/F was purified by CsCl gradient ultracentrifugation and expression of F protein was identified by RT-PCR and Western blot. HDAd/F was constructed, purified successfully. The expression of F protein was detected. The successful construction and preparation of HDAd/F is the foundation for the further investigation of potential immune protection in vivo and opens a new window for the RSV vaccine research.
YANG Bing , HE Jin-sheng , SHI Chang-xin , ZHANG Mei , YU Jie-mei
2007, 47(4):682-685.
Abstract:To construct a helper-dependent adenoviral vector expressing human respiratory syncytial virus (RSV) subgroup A F gene, and finish large scale preparation, purification and identification of the vector. F gene under the control of CMV promoter was subcloned into a shutle vector pSC11, and then cloned into HADd plasmid pSC15B. The HDAd/F genome was liberated by removing the bacterial sequences from the resulting plasmid pSC15B/F digested with restriction enzyme PmeⅠ, and then the linear HDAd/F DNA was transfected into 293Cre4 cells with calcium phosphate transfection method. The cells were infected by helper virus 16 hours after transfection. HDAd/F was amplified by serial coinfection of 293Cre4 cells by helper virus and the crude lysates from previous passage until it reached plateau of amplification by BFU staining of parallel amplified control vector pSC9A. HDAd/F was purified by CsCl gradient ultracentrifugation and expression of F protein was identified by RT-PCR and Western blot. HDAd/F was constructed, purified successfully. The expression of F protein was detected. The successful construction and preparation of HDAd/F is the foundation for the further investigation of potential immune protection in vivo and opens a new window for the RSV vaccine research.
LIU Guo-ping , WU Bin , LIN Yi-yuan , JIN Mei-lin , CHEN Huan-chun
2007, 47(4):686-691.
Abstract:Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3B. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund's incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5×OD50 Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease.
LIU Guo-ping , WU Bin , LIN Yi-yuan , JIN Mei-lin , CHEN Huan-chun
2007, 47(4):686-691.
Abstract:Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3B. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund's incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5×OD50 Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease.
ZHAI Rong-ling , XU Huai-ying , WANG You-ling , QIN Zhuo-ming , JIANG Shi-jin
2007, 47(4):692-696.
Abstract:Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore,it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.
ZHAI Rong-ling , XU Huai-ying , WANG You-ling , QIN Zhuo-ming , JIANG Shi-jin
2007, 47(4):692-696.
Abstract:Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore,it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.
CHEN Wei , HANG Feng , ZHAO Jian-xin , TIAN Feng-wei , ZHANG Hao
2007, 47(4):697-701.
Abstract:Alteration of cell membrane permeability is speculated to be one of the mechanisms by which microwave kills microorganisms. It has been reported that permeability alteration may be reflected by cell shape changes observed under electron microscopy, or detected by measuring the leakage of intracellular protein and DNA using spectrophotometry. These methods, however, suffer from accuracy and sensitivity. Calcium is an important cell signaling molecule. Its level is tightly regulated with an intracellular to extracellular differential of approximately 1 to 10,000. Damage of cells will lead to alterations in membrane permeability and consequently influx of extracellular Ca2+. In the present study two probes, fluorescein diacetate (FDA) and fluo-3/AM, were used to quantify membrane permeability of E.coli and S.aureus after microwave treatment. These chemical probes, after metabolized by intracellular esterases and binding to Ca2+, emit strong fluorescence. Our data showed 20.7%, 28.1%, 74.8% and 89.8% increases in cel membrane permeability of E.coli after 50, 55, 60 and 65℃ microwave treatment, respectively, compared to untreated controls. Modest membrane permeability increases of 4.1%, 6.0%, 21.9% and 19.7% were seen for S.aureus. The permeability levels correlate with the fatality rates of microbes. These results suggest that alteration in cell membrane permeability contributes, in part, to the nonthermal-effect of cell killing by microwave.
CHEN Wei , HANG Feng , ZHAO Jian-xin , TIAN Feng-wei , ZHANG Hao
2007, 47(4):697-701.
Abstract:Alteration of cell membrane permeability is speculated to be one of the mechanisms by which microwave kills microorganisms. It has been reported that permeability alteration may be reflected by cell shape changes observed under electron microscopy, or detected by measuring the leakage of intracellular protein and DNA using spectrophotometry. These methods, however, suffer from accuracy and sensitivity. Calcium is an important cell signaling molecule. Its level is tightly regulated with an intracellular to extracellular differential of approximately 1 to 10,000. Damage of cells will lead to alterations in membrane permeability and consequently influx of extracellular Ca2+. In the present study two probes, fluorescein diacetate (FDA) and fluo-3/AM, were used to quantify membrane permeability of E.coli and S.aureus after microwave treatment. These chemical probes, after metabolized by intracellular esterases and binding to Ca2+, emit strong fluorescence. Our data showed 20.7%, 28.1%, 74.8% and 89.8% increases in cel membrane permeability of E.coli after 50, 55, 60 and 65℃ microwave treatment, respectively, compared to untreated controls. Modest membrane permeability increases of 4.1%, 6.0%, 21.9% and 19.7% were seen for S.aureus. The permeability levels correlate with the fatality rates of microbes. These results suggest that alteration in cell membrane permeability contributes, in part, to the nonthermal-effect of cell killing by microwave.
ZHENG Gui-zhen , ZHAO Ying , DAI Meng , LIU Jing , WANG Fu-qiang
2007, 47(4):702-705.
Abstract:Peroxisomes are important subcellular organelles that are present in almost all eukaryotic cells. They are involved in a variety of metabolic functions include fatty acid β-oxidation, H2O2-based respiration and so on. The last step of penicillin biosynthetic is also located in peroxisome in Penicillium chrysogenum. Peroxisome biogenesis has been well elucidated in Saccharomyces cerevisiae and a lot of yeast peroxisome-deficient strains were available to validate the functions of peroxisome genes from other organisms. On the base of vector pYES2, the yeast expression vector pYES2G was constructed, which containing GFP-SKL reporter gene that fused the peroxisomal targeting signal 1 (PTS1) and used TEF1 as a promotor. Cells of INVScl transformed with vector pYES2G displayed a punctate fluorescence pattern; while transformants of ATCC4003603 (a pex5-deficient yeast strain) with pYES2G showed a diffuse fluorescence pattern, which indicated that GFP-SKL can be localized in peroxisome effectively by PEX5p. Furthemore, the plasmids of pYES2G/ScPEX5 and pYES2G/PcPEX5 were created by cloning PEX5p encoding genes of S. cerevisiae and P. chrysogenum into the multiple cloning site of pYES2G, and then transformed into the yeast strain ATCC4003603, respectively. Both transformants showed punctate fluorescence patterns, which suggested ATCC4003603 was complemented by the foreign ScPEX5p and PcPEX5p. The plasmid pYES2G provides a visible and effective method for studying the functions of fungal peroxisome related genes.
ZHENG Gui-zhen , ZHAO Ying , DAI Meng , LIU Jing , WANG Fu-qiang
2007, 47(4):702-705.
Abstract:Peroxisomes are important subcellular organelles that are present in almost all eukaryotic cells. They are involved in a variety of metabolic functions include fatty acid β-oxidation, H2O2-based respiration and so on. The last step of penicillin biosynthetic is also located in peroxisome in Penicillium chrysogenum. Peroxisome biogenesis has been well elucidated in Saccharomyces cerevisiae and a lot of yeast peroxisome-deficient strains were available to validate the functions of peroxisome genes from other organisms. On the base of vector pYES2, the yeast expression vector pYES2G was constructed, which containing GFP-SKL reporter gene that fused the peroxisomal targeting signal 1 (PTS1) and used TEF1 as a promotor. Cells of INVScl transformed with vector pYES2G displayed a punctate fluorescence pattern; while transformants of ATCC4003603 (a pex5-deficient yeast strain) with pYES2G showed a diffuse fluorescence pattern, which indicated that GFP-SKL can be localized in peroxisome effectively by PEX5p. Furthemore, the plasmids of pYES2G/ScPEX5 and pYES2G/PcPEX5 were created by cloning PEX5p encoding genes of S. cerevisiae and P. chrysogenum into the multiple cloning site of pYES2G, and then transformed into the yeast strain ATCC4003603, respectively. Both transformants showed punctate fluorescence patterns, which suggested ATCC4003603 was complemented by the foreign ScPEX5p and PcPEX5p. The plasmid pYES2G provides a visible and effective method for studying the functions of fungal peroxisome related genes.
ZHU Lei , CHANG Hui-ping , TANG Xin-yun , LI Kun-Yang , CAO Yuan-yuan
2007, 47(4):706-709.
Abstract:Monascus pigment is a natural, safe pigment and preservative. Six inhibitors of key enzymes from three metabolic pathways were chosen according to databases, and were used in basic medium to study their effects on the pigment synthesis in Monascus anka strain M5034. Trimethylamine, inhibitor of shikimic acid pathways, and anthranilic acid and 3,4-dihydroxybenzoic acid, inhibitors of mevalonic acid pathways, had no effects on the pigment biosynthesis. Pigment biosynthesis was severely inhibited by three inhibitors of the key enzymes in the polyketide pathways at the concentrations with no effects for growth of the strain. Iodiacetamide (lower than 0.5mmol/L), specific inhibitor of β-ketoacyl-acylcarrier protein (ACP) synthase, reduced remarkably the pigment synthesis by 64.7%; 1.0 mmol/L imidazole, being nonspecific inhibitor of ACP synthase, could strongly suppress the synthesis of pigment by 60%, and 0.5mmol/L 2,4-dinitrofluorobenzene, inhibiting the activity of thioesterase, strongly limited the pigment production with inhibitory extent up to 91.5%. All data implied that Monascus pigments might be synthesized through polyketide pathway.
ZHU Lei , CHANG Hui-ping , TANG Xin-yun , LI Kun-Yang , CAO Yuan-yuan
2007, 47(4):706-709.
Abstract:Monascus pigment is a natural, safe pigment and preservative. Six inhibitors of key enzymes from three metabolic pathways were chosen according to databases, and were used in basic medium to study their effects on the pigment synthesis in Monascus anka strain M5034. Trimethylamine, inhibitor of shikimic acid pathways, and anthranilic acid and 3,4-dihydroxybenzoic acid, inhibitors of mevalonic acid pathways, had no effects on the pigment biosynthesis. Pigment biosynthesis was severely inhibited by three inhibitors of the key enzymes in the polyketide pathways at the concentrations with no effects for growth of the strain. Iodiacetamide (lower than 0.5mmol/L), specific inhibitor of β-ketoacyl-acylcarrier protein (ACP) synthase, reduced remarkably the pigment synthesis by 64.7%; 1.0 mmol/L imidazole, being nonspecific inhibitor of ACP synthase, could strongly suppress the synthesis of pigment by 60%, and 0.5mmol/L 2,4-dinitrofluorobenzene, inhibiting the activity of thioesterase, strongly limited the pigment production with inhibitory extent up to 91.5%. All data implied that Monascus pigments might be synthesized through polyketide pathway.
FAN Hong-jie , HE Shang , LU Cheng-ping
2007, 47(4):710-713.
Abstract:Streptococcus equi subsp. zooepidemicus belongs to lancefield group C streptococcus, which can cause disease both in animals and humans. It has been associated with a wide variety of serious infections, including meningitis, pneumonia, septic arthritis and mastitis. The M like proteins on the surface of S. equi subsp. zooepidemicus have an antiphagocytic role analogous to that of group A streptococcal M proteins that are essential in establishing infection. In the present study, the M-like gene without partial signal peptide sequence was amplified from genomic DNA of S. equi subsp. zooepidemicus ATCC35246 strain isolated from pig by polymerase chain reaction (PCR). Then the amplified fragment was cloned in the proper orientation into the site between EcoRⅠ and XhoⅠof pET32-a(+) via restriction endonuclease EcoRⅠ and XhoⅠ. The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing, then transformed into E. coli BL21. An fusion protein was expressed in BL21 after induced by IPTG, SDS-PAGE analysis showed that the recombinant protein had a molecular weight of 60 kD, Western blotting showed a positive reaction with the antiserum against ATCC35246. To prepare the monoclonal antibodies (McAbs) against the M-like protein, 6~8 weeks old BABL/c mice were immunized endermicly with purified recombinant M-like protein by Ni-nitrilotriacetic acid affinity chromatography. Splenocytes from the immuniszed mice were fused with SP2/0 and indirect ELISA was used to screen hybridoma cells. 12 hybridoma cell lines secreting McAbs against M-like protein of Streptococcus equi subsp. zooepidemicus were generated, and indirect ELISA confirmed that these McAbs only reacted with M-like protein, but not reacted with other bacteria such as group A Streptococci, Streptococcus suis type 2, Streptococcus equi. The indirect ELISA titers of these 12 ascites McAbs were about 2.56×104 to 1.01×105,and the subtype of these McAbs belong to IgG2b、IgG1、IgM. The results of adhersion inhibition showed McAbs 2C8 could inhibit the adhersion of M-like protein to HEp-2 cell.
FAN Hong-jie , HE Shang , LU Cheng-ping
2007, 47(4):710-713.
Abstract:Streptococcus equi subsp. zooepidemicus belongs to lancefield group C streptococcus, which can cause disease both in animals and humans. It has been associated with a wide variety of serious infections, including meningitis, pneumonia, septic arthritis and mastitis. The M like proteins on the surface of S. equi subsp. zooepidemicus have an antiphagocytic role analogous to that of group A streptococcal M proteins that are essential in establishing infection. In the present study, the M-like gene without partial signal peptide sequence was amplified from genomic DNA of S. equi subsp. zooepidemicus ATCC35246 strain isolated from pig by polymerase chain reaction (PCR). Then the amplified fragment was cloned in the proper orientation into the site between EcoRⅠ and XhoⅠof pET32-a(+) via restriction endonuclease EcoRⅠ and XhoⅠ. The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing, then transformed into E. coli BL21. An fusion protein was expressed in BL21 after induced by IPTG, SDS-PAGE analysis showed that the recombinant protein had a molecular weight of 60 kD, Western blotting showed a positive reaction with the antiserum against ATCC35246. To prepare the monoclonal antibodies (McAbs) against the M-like protein, 6~8 weeks old BABL/c mice were immunized endermicly with purified recombinant M-like protein by Ni-nitrilotriacetic acid affinity chromatography. Splenocytes from the immuniszed mice were fused with SP2/0 and indirect ELISA was used to screen hybridoma cells. 12 hybridoma cell lines secreting McAbs against M-like protein of Streptococcus equi subsp. zooepidemicus were generated, and indirect ELISA confirmed that these McAbs only reacted with M-like protein, but not reacted with other bacteria such as group A Streptococci, Streptococcus suis type 2, Streptococcus equi. The indirect ELISA titers of these 12 ascites McAbs were about 2.56×104 to 1.01×105,and the subtype of these McAbs belong to IgG2b、IgG1、IgM. The results of adhersion inhibition showed McAbs 2C8 could inhibit the adhersion of M-like protein to HEp-2 cell.
HU Xiao-na , ZHU Rui-liang , LIU Hong-zhen , BI Jian-min
2007, 47(4):714-717.
Abstract:To study the immunogenicity of Bordetella avium OMP, ultrasonic dispersion、TritonX-100 technique were used to extract Bordetella avium OMP. Its content was determined by Bradford method and it was detected by SDS-PAGE. Then OMP immunizing antigen was prepared and 1-day chicken was vaccinated by hypodermic inoculation, with the immunizing does of 0.3mL(OMP90μg), 0.5mL(OMP150μg), 0.8mL(OMP240μg), respectively. Results showed that content of Bordetella avium OMP is 300 g/mL, the best immunizing does is 0.5mL each one and Chickens can be protected against Bordetella avium at fetal dose if antibody titer is over 1∶28. We can see from the antibody level detected by indirect ELISA that antibody can last long enough to help chickens pass susceptible period, so OMP has good immunogenicity. Results of this study lay good foundation for the development of monoclonal antibody to OMP, rapid diagnosis kit and subunit vaccine.
HU Xiao-na , ZHU Rui-liang , LIU Hong-zhen , BI Jian-min
2007, 47(4):714-717.
Abstract:To study the immunogenicity of Bordetella avium OMP, ultrasonic dispersion、TritonX-100 technique were used to extract Bordetella avium OMP. Its content was determined by Bradford method and it was detected by SDS-PAGE. Then OMP immunizing antigen was prepared and 1-day chicken was vaccinated by hypodermic inoculation, with the immunizing does of 0.3mL(OMP90μg), 0.5mL(OMP150μg), 0.8mL(OMP240μg), respectively. Results showed that content of Bordetella avium OMP is 300 g/mL, the best immunizing does is 0.5mL each one and Chickens can be protected against Bordetella avium at fetal dose if antibody titer is over 1∶28. We can see from the antibody level detected by indirect ELISA that antibody can last long enough to help chickens pass susceptible period, so OMP has good immunogenicity. Results of this study lay good foundation for the development of monoclonal antibody to OMP, rapid diagnosis kit and subunit vaccine.
LI Xiao-hua , LONG Ci-fanq , ZHOU Xiu-fen , DENG Zi-xin
2007, 47(4):718-720.
Abstract:Electroporation of Micromonospora sp.40027 isolated from soil was studied with Streptomyces plasmid pSET152, an integrative vector commonly used in Streptomyces genetic manipulation. Transformant was not obtained by electroporation with germinated spores of Micromonospora sp.40027 as recipient, but plasmid pSET152 can be electroporated into the fresh mycelium of Micromonospora sp.40027, and the highest electroporation efficiency was yielded under the electric field strength of 13kV/cm. Plasmid stability experiment and southern blot showed that pSET152 could stably exist in the Micromonospora sp. 40027, and was integrated into its chromosome via the attP site, originated from Streptomyces phage C31. These data suggested that plasmid pSET152 was successfully electroporated into Micromonospora, and that the integrase gene and attP site of Streptomyces phage ΦC31 could play the same role in Micromonospora.
LI Xiao-hua , LONG Ci-fanq , ZHOU Xiu-fen , DENG Zi-xin
2007, 47(4):718-720.
Abstract:Electroporation of Micromonospora sp.40027 isolated from soil was studied with Streptomyces plasmid pSET152, an integrative vector commonly used in Streptomyces genetic manipulation. Transformant was not obtained by electroporation with germinated spores of Micromonospora sp.40027 as recipient, but plasmid pSET152 can be electroporated into the fresh mycelium of Micromonospora sp.40027, and the highest electroporation efficiency was yielded under the electric field strength of 13kV/cm. Plasmid stability experiment and southern blot showed that pSET152 could stably exist in the Micromonospora sp. 40027, and was integrated into its chromosome via the attP site, originated from Streptomyces phage C31. These data suggested that plasmid pSET152 was successfully electroporated into Micromonospora, and that the integrase gene and attP site of Streptomyces phage ΦC31 could play the same role in Micromonospora.
ZHENG Yan , LIU Xi-peng , LIU Jian-hua
2007, 47(4):721-724.
Abstract:Attempts to transform Klebsiella pneumoniae resulted in very low efficiencies because of capsule polysaccharide (CPS). It was reported that some chelating agents could reduce CPS production and improve transformation efficiency. These methods mentioned above could not improve transformation efficiency apparently by incorporating such agents to liquid medium. However, this method introduces a simple way for efficient transformation of K. pneumoniae. In this method, K. pneumoniae strains NTUH-K2044 and magA- mutant are envolved as recipients. The plasmids used in this way are composed of pIP843T,pIP843TdhaB,pIP843TdhaT with different sizes. The sole critical step is to harvest bacteria on LB plates to prepare competent cells. 150±10, 1.3×103±100, 2×105±300, and 3.4×107±500 transformants were obtained per microgram plasmid DNA with NTUH-K2044 liquid cells, magA- liquid cells, NTUH-K2044 solid cells, and magA- solid cells, respectively. The number of transformants per μg DNA obtained by electroplating solid cells is at least 103 fold higher than that of transformants with liquid-cultured bacteria. This method will benefit gene manipulation and genetic study in K. pneumoniae.
ZHENG Yan , LIU Xi-peng , LIU Jian-hua
2007, 47(4):721-724.
Abstract:Attempts to transform Klebsiella pneumoniae resulted in very low efficiencies because of capsule polysaccharide (CPS). It was reported that some chelating agents could reduce CPS production and improve transformation efficiency. These methods mentioned above could not improve transformation efficiency apparently by incorporating such agents to liquid medium. However, this method introduces a simple way for efficient transformation of K. pneumoniae. In this method, K. pneumoniae strains NTUH-K2044 and magA- mutant are envolved as recipients. The plasmids used in this way are composed of pIP843T,pIP843TdhaB,pIP843TdhaT with different sizes. The sole critical step is to harvest bacteria on LB plates to prepare competent cells. 150±10, 1.3×103±100, 2×105±300, and 3.4×107±500 transformants were obtained per microgram plasmid DNA with NTUH-K2044 liquid cells, magA- liquid cells, NTUH-K2044 solid cells, and magA- solid cells, respectively. The number of transformants per μg DNA obtained by electroplating solid cells is at least 103 fold higher than that of transformants with liquid-cultured bacteria. This method will benefit gene manipulation and genetic study in K. pneumoniae.
NIAN Si-ji , YIN You-ping , YUAN Qing , XIA Yu-xian , WANG Zhong-kang
2007, 47(4):725-728.
Abstract:A reliable and simple polymerase chain reaction method for TCK pathogen was established firstly. A 1322bp unique fragment of TCK was amplified and identified by the technique of semi-specific random amplified polymorphism (RM-PCR). Two pairs of species-specific primers CQUK2/ CQUK3 and CQUK4/ CQUK5 were designed according to the unique fragment of TCK. The first pair primers were capable to stably amplify target DNA band of 747bp from chromosomal DNA of 18 strains of TCK isolates without any DNA bands obtained from 29 strains of TCT. The second pair primers could produce a 200bp target DNA band stably, while no band was amplified from teliospore or mycelium DNA of TCT of strains. Tilletia genus primers were used as internal control of molecular detection system, which can detect whether the PCR inhibitors exist in testing sample or avoid pseudo-negative and pseudo-positive of PCR reaction. The molecular detection approach could rapidly, accurately detect and identify the DNA of teliospore or mycelium of TCK from wheat tissues.
NIAN Si-ji , YIN You-ping , YUAN Qing , XIA Yu-xian , WANG Zhong-kang
2007, 47(4):725-728.
Abstract:A reliable and simple polymerase chain reaction method for TCK pathogen was established firstly. A 1322bp unique fragment of TCK was amplified and identified by the technique of semi-specific random amplified polymorphism (RM-PCR). Two pairs of species-specific primers CQUK2/ CQUK3 and CQUK4/ CQUK5 were designed according to the unique fragment of TCK. The first pair primers were capable to stably amplify target DNA band of 747bp from chromosomal DNA of 18 strains of TCK isolates without any DNA bands obtained from 29 strains of TCT. The second pair primers could produce a 200bp target DNA band stably, while no band was amplified from teliospore or mycelium DNA of TCT of strains. Tilletia genus primers were used as internal control of molecular detection system, which can detect whether the PCR inhibitors exist in testing sample or avoid pseudo-negative and pseudo-positive of PCR reaction. The molecular detection approach could rapidly, accurately detect and identify the DNA of teliospore or mycelium of TCK from wheat tissues.
LI Jian-li , ZHANG Wan-po , BI Ding-ren
2007, 47(4):729-733.
Abstract:NS1 protein is the only non-structural protein of influenza A virus,which is encoded by the collinear mRNA transcribed from the viral RNA segment 8. It is a RNA-banding protein which has important posttranscriptional regulatory activity. It accumulates in the nucleus of the infected cell at early times but is also present in the cytoplasm later in the infection, Two functional domains have been identified in the NS1protein: a RNA-binding domain at the N-terminus, and an effector domain at the C-terminus, which are associated with the inhibiting host celluar protein synthesis, inducing apoptosis and resisting the production of interferon-α/β. In addition, NS1protein could provide a means for the distinction between vaccinated and virus-infected poultry and act as a vaccine vector because it can tolerate large inserts of foreign sequences. Moreover, it might be an attractive target for antiviral drug design. All of these demonstrate that NS1 protein have the potential application benefits.
LI Jian-li , ZHANG Wan-po , BI Ding-ren
2007, 47(4):729-733.
Abstract:NS1 protein is the only non-structural protein of influenza A virus,which is encoded by the collinear mRNA transcribed from the viral RNA segment 8. It is a RNA-banding protein which has important posttranscriptional regulatory activity. It accumulates in the nucleus of the infected cell at early times but is also present in the cytoplasm later in the infection, Two functional domains have been identified in the NS1protein: a RNA-binding domain at the N-terminus, and an effector domain at the C-terminus, which are associated with the inhibiting host celluar protein synthesis, inducing apoptosis and resisting the production of interferon-α/β. In addition, NS1protein could provide a means for the distinction between vaccinated and virus-infected poultry and act as a vaccine vector because it can tolerate large inserts of foreign sequences. Moreover, it might be an attractive target for antiviral drug design. All of these demonstrate that NS1 protein have the potential application benefits.
2007, 47(4):734-737.
Abstract:Many microorganisms could use nonribosomal peptide synthetases (NRPSs) to synthesize various bioactive peptides with complicated structures. Because of their special physical-chemical and pharmacological properties, the nonribosomal peptides (NRPs) had been extensively studied, and would have great potential for commercial application. NRPSs are composed of many iterative modules whose different assembling orders determine the specificity of amino acid sequences of their peptide products. The NRPs are assembled by NRPSs via multiple-carrier thiotemplate mechanism, and the substrate specificity of NRPSs is determined by both adenylation domain and condensation domain. Recently, many expected peptides were synthesized using natural whole NRPSs, some specific domains of NRPSs, or novel NRPSs which were constructed by the combination or hybrid combination of specific modules or domains of some known NRPSs.
2007, 47(4):734-737.
Abstract:Many microorganisms could use nonribosomal peptide synthetases (NRPSs) to synthesize various bioactive peptides with complicated structures. Because of their special physical-chemical and pharmacological properties, the nonribosomal peptides (NRPs) had been extensively studied, and would have great potential for commercial application. NRPSs are composed of many iterative modules whose different assembling orders determine the specificity of amino acid sequences of their peptide products. The NRPs are assembled by NRPSs via multiple-carrier thiotemplate mechanism, and the substrate specificity of NRPSs is determined by both adenylation domain and condensation domain. Recently, many expected peptides were synthesized using natural whole NRPSs, some specific domains of NRPSs, or novel NRPSs which were constructed by the combination or hybrid combination of specific modules or domains of some known NRPSs.
CHEN Jian-shun , JIANG Ling-li , FANG Wei-huan
2007, 47(4):738-742.
Abstract:The genus Listeria consists of six species: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri and L. grayi. Two of the species, L. monocytogenes and L. ivanovii are pathogenic. The heterogeneity of remaining species, previously assumed to be nonpathogenic, regarding their capability of acquiring virulence-associated genes may reflect their potential ability to be causative agents of diseases, especially in immunocompromised mannals. Virulence determinants involved in environmental tolerance, adhesion and invasion of eukaryotic cells and intracellular life function interactively. The virulence genes are mostly organized into discrete genetic units known as pathogenicity islands (PAIs), among which Listeria pathogenicity island 1 (LIPI-1) and island 2 (LIPI-2) are the most important. During the evolution of pathogenicity, a common ancestor bearing PAIs gave rise to the currently prevailing typical strains of six species through horizontal transfer of virulence determinants or by events such as recombination and natural selection. Bacteriophages, transposons and plasmids might play critical roles in these processes as the executants. Compred to pathogenic species, the nonpathogenic species lost LIPI-1 (L. innocua, L. welshimeri and L. grayi) or harbored corrupted LIPI-1 (L. innocua, L. welshimeri). Some types of natural atypical Listeria strains such as nonhemolytic L. seeligeri and hemolytic L. innocua, although complicating taxonomic identification, should contribute fruitful insights into the evolution events of pathogenicity underlying the phylogeny of the genus Listeria.
CHEN Jian-shun , JIANG Ling-li , FANG Wei-huan
2007, 47(4):738-742.
Abstract:The genus Listeria consists of six species: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri and L. grayi. Two of the species, L. monocytogenes and L. ivanovii are pathogenic. The heterogeneity of remaining species, previously assumed to be nonpathogenic, regarding their capability of acquiring virulence-associated genes may reflect their potential ability to be causative agents of diseases, especially in immunocompromised mannals. Virulence determinants involved in environmental tolerance, adhesion and invasion of eukaryotic cells and intracellular life function interactively. The virulence genes are mostly organized into discrete genetic units known as pathogenicity islands (PAIs), among which Listeria pathogenicity island 1 (LIPI-1) and island 2 (LIPI-2) are the most important. During the evolution of pathogenicity, a common ancestor bearing PAIs gave rise to the currently prevailing typical strains of six species through horizontal transfer of virulence determinants or by events such as recombination and natural selection. Bacteriophages, transposons and plasmids might play critical roles in these processes as the executants. Compred to pathogenic species, the nonpathogenic species lost LIPI-1 (L. innocua, L. welshimeri and L. grayi) or harbored corrupted LIPI-1 (L. innocua, L. welshimeri). Some types of natural atypical Listeria strains such as nonhemolytic L. seeligeri and hemolytic L. innocua, although complicating taxonomic identification, should contribute fruitful insights into the evolution events of pathogenicity underlying the phylogeny of the genus Listeria.
2007, 47(4):743-745.
Abstract:Helicobacter pylori is a human-specific gastric pathogen that colonizes over half the worlds population. Infection with this bacterium is associated with a spectrum of gastric pathologies ranging from mild gastritis to peptic ulcers and gastric cancer. A strong predictor of severe disease outcome is infection with a bacterial strain harbouring the cag (cytotoxin associated gene) pathogenicity island (PAI), a 40 kb stretch of DNA that encodes homologues of several components of a type Ⅳ secretion system (TFSS). One gene within the cag PAI,cagA, has been shown to encode a substrate for the TFSS which is translocated into host cells,inducing the dephosphrylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. It appeears to play a key role in H. pylori pathogenesis. Very little is known about the H. pylori cag PAI-encoded TFSS, the expression of Cag proteins in H. pylori, and the functions of individual proteins encoded by the cag PAI. Only by exploring the mechanistic details of the interplay between H. pylori and eukaryotic cells can we endeavour to understand how these cellular interactions play out at the tissue and organismal level during the lifelong coexistence of bacterium and host.
2007, 47(4):743-745.
Abstract:Helicobacter pylori is a human-specific gastric pathogen that colonizes over half the worlds population. Infection with this bacterium is associated with a spectrum of gastric pathologies ranging from mild gastritis to peptic ulcers and gastric cancer. A strong predictor of severe disease outcome is infection with a bacterial strain harbouring the cag (cytotoxin associated gene) pathogenicity island (PAI), a 40 kb stretch of DNA that encodes homologues of several components of a type Ⅳ secretion system (TFSS). One gene within the cag PAI,cagA, has been shown to encode a substrate for the TFSS which is translocated into host cells,inducing the dephosphrylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. It appeears to play a key role in H. pylori pathogenesis. Very little is known about the H. pylori cag PAI-encoded TFSS, the expression of Cag proteins in H. pylori, and the functions of individual proteins encoded by the cag PAI. Only by exploring the mechanistic details of the interplay between H. pylori and eukaryotic cells can we endeavour to understand how these cellular interactions play out at the tissue and organismal level during the lifelong coexistence of bacterium and host.
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