Abstract:In order to understand the phylogenetic relationships among Nocardiopsis species more rationally, the gyrB, sod and rpoB gene partial sequences of 24 validly published Nocardiopsis species were determined. The phylogenetic trees were reconstructed including 16S rRNA gene and above-mentioned three housekeeping genes. The average similarities of gyrB，sod and rpoB of Nocardiopsis species were 87.7%、87.3 % and 94.1%, respectively. Meanwhile, the average similarity of 16S rRNA gene of Nocardiopsis species was 96.65%. The variabilities of gyrB，sod and rpoB gene in Nocardiospis are greater than that of 16S rRNA gene. By comparing the phylogenetic trees, the topology of gyrB gene tree is almost consistent with that of 16S rRNA gene tree. Consequently, the gyrB gene possesses the superiority in phylogenetic taxonomy of the genus Nocardiopsis.

Abstract:In order to understand the phylogenetic relationships among Nocardiopsis species more rationally, the gyrB, sod and rpoB gene partial sequences of 24 validly published Nocardiopsis species were determined. The phylogenetic trees were reconstructed including 16S rRNA gene and above-mentioned three housekeeping genes. The average similarities of gyrB，sod and rpoB of Nocardiopsis species were 87.7%、87.3 % and 94.1%, respectively. Meanwhile, the average similarity of 16S rRNA gene of Nocardiopsis species was 96.65%. The variabilities of gyrB，sod and rpoB gene in Nocardiospis are greater than that of 16S rRNA gene. By comparing the phylogenetic trees, the topology of gyrB gene tree is almost consistent with that of 16S rRNA gene tree. Consequently, the gyrB gene possesses the superiority in phylogenetic taxonomy of the genus Nocardiopsis.

Abstract:TPR repeat was originally defined as a 34 amino acid structural repeat (TPR-34). Equal length tandem repeats (ELTR) was proposed to represent the ancestral repeat pattern. Length polymorphism of TPR repeats was analyzed using PATTINPROT, two new versions of TPR repeat of 40 and 42 amino acids were identified. These ‘long' TPRs endow new functional capacities to the resulting proteins. A strong correlation between varied lengths and new functions supports the hypothesis that length variation is an underlying mechanism for the function evolution of repeat containing proteins.

Abstract:TPR repeat was originally defined as a 34 amino acid structural repeat (TPR-34). Equal length tandem repeats (ELTR) was proposed to represent the ancestral repeat pattern. Length polymorphism of TPR repeats was analyzed using PATTINPROT, two new versions of TPR repeat of 40 and 42 amino acids were identified. These ‘long' TPRs endow new functional capacities to the resulting proteins. A strong correlation between varied lengths and new functions supports the hypothesis that length variation is an underlying mechanism for the function evolution of repeat containing proteins.

Abstract:The culture fluids of Escheriachia coli with shuttle plasmid and Bacillus subtilis strains were mixed and coincubated for 40 minutes after culturing respectively in LB and minimal media. The steadily plasmid transfer by natural genetic transformation between these two gram-negative and gram-positive bacteria has been demonstrated by the methods of selective medium screening, DNaseⅠsensitivity test and plasmid detection. In contrast to MM culture B. subtilis LB culture can be competent and has equivalent transformation frequency. Furthermore, the maximal transformation frequency was obtained when cells in exponential phase served as donors or recipients. It is suggested that B. subtilis solid transformation is different from liquid plasmid transformation including the whole process of DNA plasmid competence producing. Understanding the mechanisms of gene transfer between bacteria may aid in assessing the potential risk associated with the release of recombinant organisms into the environment.

Abstract:The culture fluids of Escheriachia coli with shuttle plasmid and Bacillus subtilis strains were mixed and coincubated for 40 minutes after culturing respectively in LB and minimal media. The steadily plasmid transfer by natural genetic transformation between these two gram-negative and gram-positive bacteria has been demonstrated by the methods of selective medium screening, DNaseⅠsensitivity test and plasmid detection. In contrast to MM culture B. subtilis LB culture can be competent and has equivalent transformation frequency. Furthermore, the maximal transformation frequency was obtained when cells in exponential phase served as donors or recipients. It is suggested that B. subtilis solid transformation is different from liquid plasmid transformation including the whole process of DNA plasmid competence producing. Understanding the mechanisms of gene transfer between bacteria may aid in assessing the potential risk associated with the release of recombinant organisms into the environment.

Abstract:To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxⅦ (subunit Ⅶ of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxVⅡ-PTEN-EYFPN1 as a template, CoxⅦ-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites XhoⅠand XbaⅠ. The shuttle plasmid was linearize with PmeⅠand co-transformed with adenoviral backbone vector pAdeasy-1 into E.coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E.coli DH5α for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using PacⅠ and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID₅₀ as 10⁷pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.

Abstract:To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxⅦ (subunit Ⅶ of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxVⅡ-PTEN-EYFPN1 as a template, CoxⅦ-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites XhoⅠand XbaⅠ. The shuttle plasmid was linearize with PmeⅠand co-transformed with adenoviral backbone vector pAdeasy-1 into E.coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E.coli DH5α for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using PacⅠ and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID₅₀ as 10⁷pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.

Abstract:An Actinobacillus pleuropneumoniae apxⅡC mutant was constructed by transconjugation and counterselection method. Briefly，a transconjugation plasmid pEHA1 was constructed，and transformed into donor strain Escherichia coli β2155. After mixed the donor cells with A. pleuropneumoniae acceptor cells，the mixture was cultivated for about 5 hours and plated on solid medium containing chloromycetin. Then the Cm^R positive clones were picked and inoculated into liquid medium in the absence of any antibiotic. Cultures were pelleted，plated on sucrose plates and incubated overnight. Finally，Sucrose-resistant colonies (Suc^R) were selected and considered as mutant. The mutant was verified by PCR，heredity stability，exotoxin secretion and sequence analysis，suggested that the construction of the mutant was sucessful. The biological characteristics of this mutant strain was further investigated. Compared with parental strain，the results indicated that the mutant hold the same growth rate in vitro and reduced virulence on mice. Altogether，this mutation system will facilitate development of live attenuated vaccines and research on functions of novel genes of A. pleuropneumoniae.

Abstract:An Actinobacillus pleuropneumoniae apxⅡC mutant was constructed by transconjugation and counterselection method. Briefly，a transconjugation plasmid pEHA1 was constructed，and transformed into donor strain Escherichia coli β2155. After mixed the donor cells with A. pleuropneumoniae acceptor cells，the mixture was cultivated for about 5 hours and plated on solid medium containing chloromycetin. Then the Cm^R positive clones were picked and inoculated into liquid medium in the absence of any antibiotic. Cultures were pelleted，plated on sucrose plates and incubated overnight. Finally，Sucrose-resistant colonies (Suc^R) were selected and considered as mutant. The mutant was verified by PCR，heredity stability，exotoxin secretion and sequence analysis，suggested that the construction of the mutant was sucessful. The biological characteristics of this mutant strain was further investigated. Compared with parental strain，the results indicated that the mutant hold the same growth rate in vitro and reduced virulence on mice. Altogether，this mutation system will facilitate development of live attenuated vaccines and research on functions of novel genes of A. pleuropneumoniae.

Abstract:Vaccination has not been used widely because of the interference in the discrimination between infected and vaccinated animals in immune-screening procedures. In the present study，chloramphenicol resistance gene（Cm^r）was cloned into the genomic DNA of brucella suis S2 strain by homologous recombination with knocking out the WbkC gene，and obtained the recombinant rS2-WbkC. Further study confirmed that rS2-WbkC was conversed into rough-phenotype form smooth-phenotype. The recombinant keeps the ability to chloramphenicol resistance after 25 passages in tryptic soy agar (TSA). Mice tests showed rS2-WbkC offered similar protection to S2 strain，but more safe than S2. Serum collected form rS2-WbkC immunized mice could be easily distinguished from antiserum produced by smooth-phenotype brucella abortus. In view of these result，rS2-WbkC is a promising candidate for vaccine strain.

Abstract:Vaccination has not been used widely because of the interference in the discrimination between infected and vaccinated animals in immune-screening procedures. In the present study，chloramphenicol resistance gene（Cm^r）was cloned into the genomic DNA of brucella suis S2 strain by homologous recombination with knocking out the WbkC gene，and obtained the recombinant rS2-WbkC. Further study confirmed that rS2-WbkC was conversed into rough-phenotype form smooth-phenotype. The recombinant keeps the ability to chloramphenicol resistance after 25 passages in tryptic soy agar (TSA). Mice tests showed rS2-WbkC offered similar protection to S2 strain，but more safe than S2. Serum collected form rS2-WbkC immunized mice could be easily distinguished from antiserum produced by smooth-phenotype brucella abortus. In view of these result，rS2-WbkC is a promising candidate for vaccine strain.

Abstract:Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli，being a candidate for enzymic detection of PQQ，is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21(DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method，over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain，named MP606，was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC，absorption spectra assay，and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences，overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95%. The highest value is with two strains of Methylovorus，which reached at 99%.

Abstract:Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli，being a candidate for enzymic detection of PQQ，is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21(DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method，over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain，named MP606，was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC，absorption spectra assay，and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences，overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95%. The highest value is with two strains of Methylovorus，which reached at 99%.

Abstract:Type 1 diabetes mellitus（T1DM） is an auto-immune disease while oral administrating its autoantigens could be a treatment of T1DM. To express human insulin gene (ins) in lactic acid bacteria(LAB) for oral vaccine，ins gene was replaced by LAB bias codon and an 8-amino-acid-residue linker peptide forming a β-turn was designed to link insulin chain A and B. After synthesized by primer annealing method，the whole ins gene was fused with signal peptide sequence sp_Usp45，subcloned into a LAB secretory expressive vector pSW501 and then introduced to Lactococcus lactis(L. lactis) MG1363 and Lactobacillus casei(Lb. casei) ATCC27092 respectively. Western blot showed that the expression product (SP_Usp45-INS protein) targeted mainly at the cell wall while little was found in cytoplasm or supernatant. The highest expression level emerged in exponential phase when the optical density at 600nm of the culture was 0.4. The culture of the recombinant strain Lb. casei/pSW501 was administered to non-obese diabetic (NOD) mice orally. ELISA and Western blot results showed that the recombinant strain could induce SP_Usp45-INS -specific antibodies and raise IL-4 level (38.583±2.083pg/mL，P<0.05) in the mice's sera. Expression of insulin in the food-grade vehicle LAB could induce oral immune tolerance in NOD mice and protect it from pancreas injury，suggesting it might be a new way to the treatment of T1DM.

Abstract:Type 1 diabetes mellitus（T1DM） is an auto-immune disease while oral administrating its autoantigens could be a treatment of T1DM. To express human insulin gene (ins) in lactic acid bacteria(LAB) for oral vaccine，ins gene was replaced by LAB bias codon and an 8-amino-acid-residue linker peptide forming a β-turn was designed to link insulin chain A and B. After synthesized by primer annealing method，the whole ins gene was fused with signal peptide sequence sp_Usp45，subcloned into a LAB secretory expressive vector pSW501 and then introduced to Lactococcus lactis(L. lactis) MG1363 and Lactobacillus casei(Lb. casei) ATCC27092 respectively. Western blot showed that the expression product (SP_Usp45-INS protein) targeted mainly at the cell wall while little was found in cytoplasm or supernatant. The highest expression level emerged in exponential phase when the optical density at 600nm of the culture was 0.4. The culture of the recombinant strain Lb. casei/pSW501 was administered to non-obese diabetic (NOD) mice orally. ELISA and Western blot results showed that the recombinant strain could induce SP_Usp45-INS -specific antibodies and raise IL-4 level (38.583±2.083pg/mL，P<0.05) in the mice's sera. Expression of insulin in the food-grade vehicle LAB could induce oral immune tolerance in NOD mice and protect it from pancreas injury，suggesting it might be a new way to the treatment of T1DM.

Abstract:In this paper，a recombinant baculovirus containing the ORF of bovine interferon-β (BoIFN-β) gene，rBac-BoIFN-β，was generated to express recombinant BoIFN-β (rBoIFN-β) in sf9 insect cells. The expression of rBoIFN-β in rBac-BoIFN-β infecting sf9 cells and its supernatants was confirmed by indirect immunofluorescence assay and Western blot. The antiviral activity of rBoIFN-β in the supernatant can reach 10^6.0AU/mL evaluated by the antiviral assay with VSV*GFP that expressed green fluorescence protein，and rBoIFN-β could stimulate the expression of luciferase reporter gene controlled by chicken Mx promoter. All the results showed that rBac-BoIFN-β constructed here could express high level recombinant BoIFN-β in secreted form that had the bioactivity of natural type Ⅰ IFN.

Abstract:In this paper，a recombinant baculovirus containing the ORF of bovine interferon-β (BoIFN-β) gene，rBac-BoIFN-β，was generated to express recombinant BoIFN-β (rBoIFN-β) in sf9 insect cells. The expression of rBoIFN-β in rBac-BoIFN-β infecting sf9 cells and its supernatants was confirmed by indirect immunofluorescence assay and Western blot. The antiviral activity of rBoIFN-β in the supernatant can reach 10^6.0AU/mL evaluated by the antiviral assay with VSV*GFP that expressed green fluorescence protein，and rBoIFN-β could stimulate the expression of luciferase reporter gene controlled by chicken Mx promoter. All the results showed that rBac-BoIFN-β constructed here could express high level recombinant BoIFN-β in secreted form that had the bioactivity of natural type Ⅰ IFN.

Abstract:An antimicrobial substance produced by Brevibacillus laterosporus isolated from the sea sediment was purified and characterized. The antimicrobial substance was purified by ultrafiltration，DEAE-Sepharose Fast flow chromatography，CM-Sepharose Fast flow chromatography and HPLC reversed phase column chromatography，and after the final purification step，one active fraction was obtained，designated R-1. The molecular weight (MW) was accurately determined by MALDI-TOF-MS as 1608.023 Da. And its pI was determined with Rotofor Cell BIO-RAD to be 8.55. Amino acid analysis of the purified R-1 showed that it was composed of Leu，Tyr，Val，Ile，Lys，Gly，Met，Ser and Ala. Most of them were hydrophobic and neutral amino acid except Lys which was a basic amino acid. And this accorded with pI of R-1. R-1 remained active over a wide temperature range and it also was active over a broad pH rang. R-1 was insusceptible to pancreatin，pepsin and alkaline proteinase. Agar radial diffusion assay showed that R-1 had low minimun bactericidal concentration against Gram-Positive Bacteria such as Streptococcus mutans，Staphylococcus aureaus，Clostridium and Gram- Negative Bacteria such as Escherichia coli，Pseudomonas putrefaciens. And R-1 had antibacterial activities against Candida albicans.

Abstract:An antimicrobial substance produced by Brevibacillus laterosporus isolated from the sea sediment was purified and characterized. The antimicrobial substance was purified by ultrafiltration，DEAE-Sepharose Fast flow chromatography，CM-Sepharose Fast flow chromatography and HPLC reversed phase column chromatography，and after the final purification step，one active fraction was obtained，designated R-1. The molecular weight (MW) was accurately determined by MALDI-TOF-MS as 1608.023 Da. And its pI was determined with Rotofor Cell BIO-RAD to be 8.55. Amino acid analysis of the purified R-1 showed that it was composed of Leu，Tyr，Val，Ile，Lys，Gly，Met，Ser and Ala. Most of them were hydrophobic and neutral amino acid except Lys which was a basic amino acid. And this accorded with pI of R-1. R-1 remained active over a wide temperature range and it also was active over a broad pH rang. R-1 was insusceptible to pancreatin，pepsin and alkaline proteinase. Agar radial diffusion assay showed that R-1 had low minimun bactericidal concentration against Gram-Positive Bacteria such as Streptococcus mutans，Staphylococcus aureaus，Clostridium and Gram- Negative Bacteria such as Escherichia coli，Pseudomonas putrefaciens. And R-1 had antibacterial activities against Candida albicans.

Abstract:A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins，the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process，the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein，there were some bipy ramidal crystals with a size of 1.5×3.0μm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10μg/mL and 11.34μg/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with hightoxicity.

Abstract:A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins，the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process，the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein，there were some bipy ramidal crystals with a size of 1.5×3.0μm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10μg/mL and 11.34μg/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with hightoxicity.

Abstract:Rice bacterial blight and blast are the most crucial rice disease. Xa21 confers resistance to bacterial blight，while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor kinase-like proteins. Biochemical properties of XA21 kinase expressed in bacterial were characterized in our previous report. In this study，both XA21 and PI-D2 kinase domain were PCR amplified and cloned into yeast expression vector pEGH via recombinational cloning strategy，kinase proteins expressed in eukaryotic yeast system was purified and autophosphorylation assay was carried out. The results indicated that XA21 and PI-D2 protein can be detected by SDS-PAGE and showed expected molecular weight. Autophosphorylation assay indicated that yeast expressed XA21 and PI-D2 were active when incubated with P32 labelled ATP. The experiment provided basic materials for biochemical prosperity analysis，protein-protein interaction and substrate screening research.

Abstract:Rice bacterial blight and blast are the most crucial rice disease. Xa21 confers resistance to bacterial blight，while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor kinase-like proteins. Biochemical properties of XA21 kinase expressed in bacterial were characterized in our previous report. In this study，both XA21 and PI-D2 kinase domain were PCR amplified and cloned into yeast expression vector pEGH via recombinational cloning strategy，kinase proteins expressed in eukaryotic yeast system was purified and autophosphorylation assay was carried out. The results indicated that XA21 and PI-D2 protein can be detected by SDS-PAGE and showed expected molecular weight. Autophosphorylation assay indicated that yeast expressed XA21 and PI-D2 were active when incubated with P32 labelled ATP. The experiment provided basic materials for biochemical prosperity analysis，protein-protein interaction and substrate screening research.

Abstract:Genemonic DNA and cDNA homologous fragments of the scd(scytalone dehydratase) gene were obtained by polymerase chain reaction（PCR）amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of scytalone dehydratase and polyketide synthase domains from others fungis. The completed cDNA sequence of scd in E. turcica was obtained by the method of SMART-RACE and 3′RACE. There is one open reading frame composed of 181 codons and two deduced introns of 50 and 78 nucleotides in the scd gene. The deduced amino acid sequence of the scd showed high similarity to the amino acid sequence of scytalone dehydratase from Bipolaris oryzae. Carpropamid，a specific inhibitor，could inhibit the conidial germination and appressorium production of E. turcica within 24h treatment but no evident inhibitory effect after 24h . The experimental results also suggested that E. turcica could not penetrate the surface of corn tissue or increase in the corn tissue. It was conclusion that scd gene might play an important role in melanin biosynthetic pathway and pathogenicity of E. turcica.

Abstract:Genemonic DNA and cDNA homologous fragments of the scd(scytalone dehydratase) gene were obtained by polymerase chain reaction（PCR）amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of scytalone dehydratase and polyketide synthase domains from others fungis. The completed cDNA sequence of scd in E. turcica was obtained by the method of SMART-RACE and 3′RACE. There is one open reading frame composed of 181 codons and two deduced introns of 50 and 78 nucleotides in the scd gene. The deduced amino acid sequence of the scd showed high similarity to the amino acid sequence of scytalone dehydratase from Bipolaris oryzae. Carpropamid，a specific inhibitor，could inhibit the conidial germination and appressorium production of E. turcica within 24h treatment but no evident inhibitory effect after 24h . The experimental results also suggested that E. turcica could not penetrate the surface of corn tissue or increase in the corn tissue. It was conclusion that scd gene might play an important role in melanin biosynthetic pathway and pathogenicity of E. turcica.

Abstract:The different depth snow samples were collected from the Miaoergou glacier in East Tianshan Mountains regions，China. Total bacteria counts were established by 4′，6- diamino-2-phenylindole (DAPI). Both culture-dependent and culture-independent methods，denaturing gradient gel electrophoresis (DGGE)，were used to examine the bacterial diversity and community structure. The microbial abundance and diversity index have a close relationship with mineral particle concentration. These mineral particles，such as Ca²⁺，Mg²⁺ and Cl^- etc，are good indicators of climate and environment. The 16S rDNA gene both of cultured bacteria and DGGE band sequenced belong to following groups：proteobacteria（α-，β-，γ-），Cytophaga-Flavobacterium-Bacteroides(CFB)，High GC and Low GC. Among these，Proteobacteria and CFB are dominant groups. Compare with bacteria revealed from ice and snow in Qinghai-Tibet Plateau，South Pole and North Pole which have been reported，Paracoccu sp. and Aquasalina sp. are especially exist in this study area as far as we know. The microbial abundance and community structure are all changed obviously in different depth snow samples. It indicates that the snow bacteria community is influenced by many factors. The results show that because the special geographical position of the East Tianshan Mountains，microorganism in ice and snow of this area has its particularity.

Abstract:The different depth snow samples were collected from the Miaoergou glacier in East Tianshan Mountains regions，China. Total bacteria counts were established by 4′，6- diamino-2-phenylindole (DAPI). Both culture-dependent and culture-independent methods，denaturing gradient gel electrophoresis (DGGE)，were used to examine the bacterial diversity and community structure. The microbial abundance and diversity index have a close relationship with mineral particle concentration. These mineral particles，such as Ca²⁺，Mg²⁺ and Cl^- etc，are good indicators of climate and environment. The 16S rDNA gene both of cultured bacteria and DGGE band sequenced belong to following groups：proteobacteria（α-，β-，γ-），Cytophaga-Flavobacterium-Bacteroides(CFB)，High GC and Low GC. Among these，Proteobacteria and CFB are dominant groups. Compare with bacteria revealed from ice and snow in Qinghai-Tibet Plateau，South Pole and North Pole which have been reported，Paracoccu sp. and Aquasalina sp. are especially exist in this study area as far as we know. The microbial abundance and community structure are all changed obviously in different depth snow samples. It indicates that the snow bacteria community is influenced by many factors. The results show that because the special geographical position of the East Tianshan Mountains，microorganism in ice and snow of this area has its particularity.

Abstract:Bacterial strains CB and CS were isolated from the Anoxic Sulfide Oxidizing (ASO) reactor working under steady-state with sulfide and nitrate as electron donor and acceptor，respectively. Based on electron microscopy，physiological test and 16S rDNA phylogenetic sequence analysis，the isolate CB was very close to Bacillus pseudofirmus and the isolate CS was very close to Bacillus hemicellulosilytus and Bacillus halodurans. The 16S rDNA sequences of both isolates were submitted to GenBank，NCBI and accession numbers EF542806 and EF542807 were allotted for isolates CB and CS，respectively. According to Biolog carbon source utilization test，the isolate CB is weak to use the carbon sources，while the isolate CB can use many carbon sources. Both the isolate CB and the isolate CS are able to use nitrate for sulfide oxidation. The isolate CS has greater capability to oxidize sulfide with nitrate as electron acceptor.

Abstract:Bacterial strains CB and CS were isolated from the Anoxic Sulfide Oxidizing (ASO) reactor working under steady-state with sulfide and nitrate as electron donor and acceptor，respectively. Based on electron microscopy，physiological test and 16S rDNA phylogenetic sequence analysis，the isolate CB was very close to Bacillus pseudofirmus and the isolate CS was very close to Bacillus hemicellulosilytus and Bacillus halodurans. The 16S rDNA sequences of both isolates were submitted to GenBank，NCBI and accession numbers EF542806 and EF542807 were allotted for isolates CB and CS，respectively. According to Biolog carbon source utilization test，the isolate CB is weak to use the carbon sources，while the isolate CB can use many carbon sources. Both the isolate CB and the isolate CS are able to use nitrate for sulfide oxidation. The isolate CS has greater capability to oxidize sulfide with nitrate as electron acceptor.

Abstract:An organic-solvent-tolerant bacterium strain YP1 producing organic-solvent-stable protease was isolated from crude oil contaminant soil. Strain YP1 was strictly aerobic，motile，gram positive，spore-forming，and rod shaped. The YP1strain was identified as Bacillus licheniformis using culture system BIOLOG analysis (SIM=0.62，16-24h). The 16S rDNA sequence analysis (GenBank accession number EF105377) suggested that strain YP1 was clustered together with B. licheniformis in phylogenetic tree. Based on all the taxonomy，strain YP1 was identified as B. licheniformis. YP1 strain could tolerant organic solvents at different levels，especially it can grow well in the presence of water-miscible solvents dimethylformamide (DMF，logP=-1.0) and dimethylsulphoxide (DMSO，logP=-1.35) at a concentration of 10% ［V/V］. Strain YP1 can also tolerant middle concentrations of NaCl and extra alkaline conditions（pH12）. More than 80% of the biomass remained at pH range 10.5-12. However strain YP1 was sensitive to antibiotics such as ampicillin，tetracycline，kanamycin and chloromycetin. The protease production could be enhanced by acetone and repressed by alkanols such as dodecylalcohol and octanol during the fermentation. Compared to trypsin，the YP1 protease had a wider tolerance for organic solvents. YP1 protease tolerated up to at least 11 organic solvents with logP ranging from -1.35 to 5.6 including benzene，toluene，DMSO and DMF etc at 50% (V/V) concentration. Moreover，when solvents such as decane and dodecyl alcohol with log P values above 4.0 were added to the crude protease，the enzyme activity levels were 1.08 and 1.21 times higher than the control respectively. Its high tolerance for water-miscible solvents DMF and DMSO makes it an ideal catalyst for kinetic- and equilibrium-controlled synthesis. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.

Abstract:An organic-solvent-tolerant bacterium strain YP1 producing organic-solvent-stable protease was isolated from crude oil contaminant soil. Strain YP1 was strictly aerobic，motile，gram positive，spore-forming，and rod shaped. The YP1strain was identified as Bacillus licheniformis using culture system BIOLOG analysis (SIM=0.62，16-24h). The 16S rDNA sequence analysis (GenBank accession number EF105377) suggested that strain YP1 was clustered together with B. licheniformis in phylogenetic tree. Based on all the taxonomy，strain YP1 was identified as B. licheniformis. YP1 strain could tolerant organic solvents at different levels，especially it can grow well in the presence of water-miscible solvents dimethylformamide (DMF，logP=-1.0) and dimethylsulphoxide (DMSO，logP=-1.35) at a concentration of 10% ［V/V］. Strain YP1 can also tolerant middle concentrations of NaCl and extra alkaline conditions（pH12）. More than 80% of the biomass remained at pH range 10.5-12. However strain YP1 was sensitive to antibiotics such as ampicillin，tetracycline，kanamycin and chloromycetin. The protease production could be enhanced by acetone and repressed by alkanols such as dodecylalcohol and octanol during the fermentation. Compared to trypsin，the YP1 protease had a wider tolerance for organic solvents. YP1 protease tolerated up to at least 11 organic solvents with logP ranging from -1.35 to 5.6 including benzene，toluene，DMSO and DMF etc at 50% (V/V) concentration. Moreover，when solvents such as decane and dodecyl alcohol with log P values above 4.0 were added to the crude protease，the enzyme activity levels were 1.08 and 1.21 times higher than the control respectively. Its high tolerance for water-miscible solvents DMF and DMSO makes it an ideal catalyst for kinetic- and equilibrium-controlled synthesis. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.

Abstract:Syntrophic acetogenic bacteria, an important functional one in anaerobic habitats, were detected and counted by fluorescence in situ hybridization (FISH) technology by using 16S rRNA-based oligonucleotide probes. For enumeration and quantification of the targeted bacteria, an attempt was made to optimize the hybridization conditions. The optimum conditions are as follows: a fixation time of 19h, a dehydrated time of 5min, and a formamide concentration of 55% in hybridized solution. The abundance of syntrophic acetogenic bacteria of different environmental samples were quantified by FISH and the results showed that Upflow Anaerobic Sludge Reactor (UASB) treating STHZ〗high-concentration organic wastewater and the digestive tract of some animals were the main habitats of syntrophic acetogenic bacteria. The numbers of syntrophic acetogenic bacteria in UASB and cattle manure were 1.70×10⁹ cells/mL sample and 6.50×10⁸ cells/mL sample, respectively. Meanwhile, the sediments of rivers and lakes existed less of the bacteria and the contents of them were just about 1.20×10⁸ cells/mL sample in Taihu lake.

Abstract:Syntrophic acetogenic bacteria, an important functional one in anaerobic habitats, were detected and counted by fluorescence in situ hybridization (FISH) technology by using 16S rRNA-based oligonucleotide probes. For enumeration and quantification of the targeted bacteria, an attempt was made to optimize the hybridization conditions. The optimum conditions are as follows: a fixation time of 19h, a dehydrated time of 5min, and a formamide concentration of 55% in hybridized solution. The abundance of syntrophic acetogenic bacteria of different environmental samples were quantified by FISH and the results showed that Upflow Anaerobic Sludge Reactor (UASB) treating STHZ〗high-concentration organic wastewater and the digestive tract of some animals were the main habitats of syntrophic acetogenic bacteria. The numbers of syntrophic acetogenic bacteria in UASB and cattle manure were 1.70×10⁹ cells/mL sample and 6.50×10⁸ cells/mL sample, respectively. Meanwhile, the sediments of rivers and lakes existed less of the bacteria and the contents of them were just about 1.20×10⁸ cells/mL sample in Taihu lake.

Abstract:The DNA fragment encoding the truncated SLT-IIeB and FedF of Ee strain were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKSF. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-SF fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with mouse anti-SLT-IIeB antiserum, mouse anti-FedF antiserum and moue anti-GST monoclonal antibody respectively. The fusion protein was further purified and used as an antigen for preparation of immune serum. The anti-sera of GST-SF were able to restrain the toxicity of SLT-IIe to Vero-E6 cells and inhibit the adhesin of F18 fimbriae to brush borders of swine in vitro. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-SF, GST-B, GST-F and challenged intraperitoneally with volume of 5 LD50 Ee strain. The results show the fusion protein GST-SF had more shrong immunogenicity and better protection against Ee strain.

Abstract:The DNA fragment encoding the truncated SLT-IIeB and FedF of Ee strain were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKSF. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-SF fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with mouse anti-SLT-IIeB antiserum, mouse anti-FedF antiserum and moue anti-GST monoclonal antibody respectively. The fusion protein was further purified and used as an antigen for preparation of immune serum. The anti-sera of GST-SF were able to restrain the toxicity of SLT-IIe to Vero-E6 cells and inhibit the adhesin of F18 fimbriae to brush borders of swine in vitro. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-SF, GST-B, GST-F and challenged intraperitoneally with volume of 5 LD50 Ee strain. The results show the fusion protein GST-SF had more shrong immunogenicity and better protection against Ee strain.

Abstract:An immunogenic protein, named HM3, of Streptococcus suis type 2 HA9801 was identified by using immunoproteomic assay. Some characters of this protein were analyzed by several online bioinformatical tools, including BLAST, SinglP, HMMTOP and PSORTb. The most homologous sequence of HM3 was extracellular solute-binding protein (gi|69246104) of Enterococcus faecium. The predictions results showed that HM3 contained Signal peptide and one transmembrane region, and Non-Cytoplasmic Localization were also predicted. Partial gene of this protein were amplified from the genome of HA9801 by PCR and inserted into expression plasmid pET-32a (+) after double digested by BamH I and Sal I, then transformed into BL21 (DE3) where they were induced to express by IPTG. After induced, there was specific proteins band of approximately 45kDa on the SDS-PAGE gel. Western-blotting showed that recombinant protein could react with immune serum of HA9801 of SPF (Specefic pathogen Free)mini-swine. This protein could be taken as a vaccine candidate of SS2.

Abstract:An immunogenic protein, named HM3, of Streptococcus suis type 2 HA9801 was identified by using immunoproteomic assay. Some characters of this protein were analyzed by several online bioinformatical tools, including BLAST, SinglP, HMMTOP and PSORTb. The most homologous sequence of HM3 was extracellular solute-binding protein (gi|69246104) of Enterococcus faecium. The predictions results showed that HM3 contained Signal peptide and one transmembrane region, and Non-Cytoplasmic Localization were also predicted. Partial gene of this protein were amplified from the genome of HA9801 by PCR and inserted into expression plasmid pET-32a (+) after double digested by BamH I and Sal I, then transformed into BL21 (DE3) where they were induced to express by IPTG. After induced, there was specific proteins band of approximately 45kDa on the SDS-PAGE gel. Western-blotting showed that recombinant protein could react with immune serum of HA9801 of SPF (Specefic pathogen Free)mini-swine. This protein could be taken as a vaccine candidate of SS2.

Abstract:Candidate IBV vaccines should elicit cellular responses as well as humoral responses. S1 gene of avian infectious bronchitis virus and interleukin-2 (IL-2) gene from chicken were inserted into the bicistronic pIRES-EGFP/DsRed plasmid. The pIRES-S1, pIRES-IL2 and pIRES-S1/IL-2 plasmid expressing or co-expressing S1 and IL-2 gene were constructed. Plasmids were transfected into the Vero cells by lipofectamine, and the expressed products were detected by RT-PCR and indirect immunofluorescence assay. The 7-day-old chickens were immunized intramuscularly with plasmids encapsulated by liposome and boosted three weeks later. Two weeks after boosting, chickens were challenged by virulent IBV strain. The results showed that coadministration of a plasmid expressing IL-2 with the S1 DNA vaccine led to only a marginal increase in humoral and T cell responses. However, immunization with the bicistronic plasmid pIRES-S1/IL-2 that co-expressing S1 and IL-2 under control of a single promoter led to a dramatic augmentation of humoral and T cell responses. The protective efficacy could be significantly enhanced after injection with plasmids pIRES-S1/IL-2 or pIRES-S1+pIRES-IL2. These results demonstrate that bicistronic DNA vaccine containing IL-2 elicit remarkably immune responses and suggest that optimal humoral and cellular responses priming requires the precise temporal and spatial codelivery of Ag and IL-2.

Abstract:Candidate IBV vaccines should elicit cellular responses as well as humoral responses. S1 gene of avian infectious bronchitis virus and interleukin-2 (IL-2) gene from chicken were inserted into the bicistronic pIRES-EGFP/DsRed plasmid. The pIRES-S1, pIRES-IL2 and pIRES-S1/IL-2 plasmid expressing or co-expressing S1 and IL-2 gene were constructed. Plasmids were transfected into the Vero cells by lipofectamine, and the expressed products were detected by RT-PCR and indirect immunofluorescence assay. The 7-day-old chickens were immunized intramuscularly with plasmids encapsulated by liposome and boosted three weeks later. Two weeks after boosting, chickens were challenged by virulent IBV strain. The results showed that coadministration of a plasmid expressing IL-2 with the S1 DNA vaccine led to only a marginal increase in humoral and T cell responses. However, immunization with the bicistronic plasmid pIRES-S1/IL-2 that co-expressing S1 and IL-2 under control of a single promoter led to a dramatic augmentation of humoral and T cell responses. The protective efficacy could be significantly enhanced after injection with plasmids pIRES-S1/IL-2 or pIRES-S1+pIRES-IL2. These results demonstrate that bicistronic DNA vaccine containing IL-2 elicit remarkably immune responses and suggest that optimal humoral and cellular responses priming requires the precise temporal and spatial codelivery of Ag and IL-2.

Abstract:Lentivectors have drawn considerable attention recently and become important delivery vehicles for gene transfer manipulation. By Transiently co-transfecting 293T packaging cells with three DNA plasmids system encoding lentivector constituents, a protocol for bulky preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentivector with high titer has been established. Transient co-transfection of 293T packaging cells resulted in production of high-titer vector (1.1×107IU/ml), which can be further concentrated over 100-fold through a single step centrifugation. The vector was capable of efficiently transducing a variety of cells from both primate and non-primate sources, including of human T-lymphoblastoid cell line. Long-term culture of vector transduced cells showed a stable expression of foreign gene over 18 months detected by RT-PCR. Assessment of potential generation of replication-competent virus revealed no detection of p24 antigen protein or infectious particles in vector-transduced cells.

Abstract:Lentivectors have drawn considerable attention recently and become important delivery vehicles for gene transfer manipulation. By Transiently co-transfecting 293T packaging cells with three DNA plasmids system encoding lentivector constituents, a protocol for bulky preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentivector with high titer has been established. Transient co-transfection of 293T packaging cells resulted in production of high-titer vector (1.1×107IU/ml), which can be further concentrated over 100-fold through a single step centrifugation. The vector was capable of efficiently transducing a variety of cells from both primate and non-primate sources, including of human T-lymphoblastoid cell line. Long-term culture of vector transduced cells showed a stable expression of foreign gene over 18 months detected by RT-PCR. Assessment of potential generation of replication-competent virus revealed no detection of p24 antigen protein or infectious particles in vector-transduced cells.

Abstract:Citrus bacterial canker disease caused by the gram negative bacterium Xanthomonas axonopodis pv. citri (XAC) is a severe bacterial disease of most commercial citrus species and cultivars around the world. Single chain variable fragment (ScFv) is artificial construction small molecular antibody produced by genetic engineering which may be used to identify target pathogens and prevent plant diseases including XAC. To express ScFv against XAC and obtain functional ScFv, single-chain antibody fragments (ScFv 95) selected by ribosome display was amplified using an assembly of polymerase chain reaction (PCR), and a recombined plasmid pET30a(+)-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a(+). PET30a(+)-XAC-ScFv was transformed into Escherichia coli BL21 (DE3) and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process was done. The results showed that ScFv recombined antibody of XAC with a molecular of 32kDa was expressed successfully as inclusion bodies and the functional ScFv was obtained through purification and renaturation. Meanwhile, the Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac, which paves a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease.

Abstract:Citrus bacterial canker disease caused by the gram negative bacterium Xanthomonas axonopodis pv. citri (XAC) is a severe bacterial disease of most commercial citrus species and cultivars around the world. Single chain variable fragment (ScFv) is artificial construction small molecular antibody produced by genetic engineering which may be used to identify target pathogens and prevent plant diseases including XAC. To express ScFv against XAC and obtain functional ScFv, single-chain antibody fragments (ScFv 95) selected by ribosome display was amplified using an assembly of polymerase chain reaction (PCR), and a recombined plasmid pET30a(+)-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a(+). PET30a(+)-XAC-ScFv was transformed into Escherichia coli BL21 (DE3) and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process was done. The results showed that ScFv recombined antibody of XAC with a molecular of 32kDa was expressed successfully as inclusion bodies and the functional ScFv was obtained through purification and renaturation. Meanwhile, the Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac, which paves a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease.

Abstract:To accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different equine infectious anemia virus (EIAV) strains, the cDNA of EIAV receptor, ELR1, was cloned and inserted in an eukaryotic expression vector pcDNA3.1(+). This recombinant plasmid was designated as pELR1. The 293 cell line was transiently transfected with pELR1 and the expression of ELR1 on transfected cells was verified by Western blot and indirect immunofluorescence assay (IFA). Furthermore, the transcription regulatory region, long terminal repeat (LTR), of an EIAV vaccine strain and a reporter of firefly luciferase gene were tandemly cloned into pELR1. The resultant expression vector, which was designated as pELR1-LTR-Luc, was used to transfect 293 cells. A transfected cell line, ELR1-LTR-Luc (293E) which consistently expressed ELR1 and produced luciferase under the regulation of LTR, was isolated and further characterized. The entrance and replication of EIAV in ELR1-LTR-Luc (293E) cells were verified by IFA. The luciferase activity in the cell line treated with 1000 TCID50 of an EIAV vaccine strain for 24h was increased by 2.15 folds when compared with the activity in untreated cells. Furthermore, the luciferase activities in the cell line were linearly correlated with the doses of inoculated EIAV virulent stain L21 diluted at 10^-2～10^-7. The transfected genes in the ELR1-LTR-Luc (293E) cell line were consistently expressed during 35 passages of the host cells. This ELR1-LTR-Luc report system can be used for the study of interaction between EIAV strains and the receptor, as well as for the evaluation of neutralizing antibodies raised by EIAV.

Abstract:To accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different equine infectious anemia virus (EIAV) strains, the cDNA of EIAV receptor, ELR1, was cloned and inserted in an eukaryotic expression vector pcDNA3.1(+). This recombinant plasmid was designated as pELR1. The 293 cell line was transiently transfected with pELR1 and the expression of ELR1 on transfected cells was verified by Western blot and indirect immunofluorescence assay (IFA). Furthermore, the transcription regulatory region, long terminal repeat (LTR), of an EIAV vaccine strain and a reporter of firefly luciferase gene were tandemly cloned into pELR1. The resultant expression vector, which was designated as pELR1-LTR-Luc, was used to transfect 293 cells. A transfected cell line, ELR1-LTR-Luc (293E) which consistently expressed ELR1 and produced luciferase under the regulation of LTR, was isolated and further characterized. The entrance and replication of EIAV in ELR1-LTR-Luc (293E) cells were verified by IFA. The luciferase activity in the cell line treated with 1000 TCID50 of an EIAV vaccine strain for 24h was increased by 2.15 folds when compared with the activity in untreated cells. Furthermore, the luciferase activities in the cell line were linearly correlated with the doses of inoculated EIAV virulent stain L21 diluted at 10^-2～10^-7. The transfected genes in the ELR1-LTR-Luc (293E) cell line were consistently expressed during 35 passages of the host cells. This ELR1-LTR-Luc report system can be used for the study of interaction between EIAV strains and the receptor, as well as for the evaluation of neutralizing antibodies raised by EIAV.

Abstract:An endophytic antagonistic bacterium was isolated from Amorphophallus konjac calli. In order to identify this bacterium, 16S rDNA was amplified and partially sequenced. Sequence comparison showed that this sequence has the highest similarity to that in Bacillus subtilis，with 99.0％ identities. That demonstrated this bacterium belongs to Bacillus subtili, named BSn5. The extracted extracellular protein from strain BSn5 had antibacterial activity against Erwinia carotovora subp. carotovora, which was unstable after heated, sensitive to proteinase K and resistant to trypsin. There was only a 31.6kDa protein component as by SDS-PAGE detection. Nondenaturing polyacrylaminde gel was used to purify this protein. The purified 31.6kDa protein exhibited inhibitory activity against Erwinia carotovora subp. carotovora. This protein is different from all known metabolites from Bacillus subtilis, suggesting that it may be a novel antibacterial protein.

Abstract:An endophytic antagonistic bacterium was isolated from Amorphophallus konjac calli. In order to identify this bacterium, 16S rDNA was amplified and partially sequenced. Sequence comparison showed that this sequence has the highest similarity to that in Bacillus subtilis，with 99.0％ identities. That demonstrated this bacterium belongs to Bacillus subtili, named BSn5. The extracted extracellular protein from strain BSn5 had antibacterial activity against Erwinia carotovora subp. carotovora, which was unstable after heated, sensitive to proteinase K and resistant to trypsin. There was only a 31.6kDa protein component as by SDS-PAGE detection. Nondenaturing polyacrylaminde gel was used to purify this protein. The purified 31.6kDa protein exhibited inhibitory activity against Erwinia carotovora subp. carotovora. This protein is different from all known metabolites from Bacillus subtilis, suggesting that it may be a novel antibacterial protein.

Abstract:Multigene-based phylogenetic analyses are becoming more widely used in molecular taxonomy because of its lab-to-lab portability and reproducibility. The first step that needs to be settled for this approach is to amplify and sequence several housekeeping genes. Four housekeeping genes atpD, recA, rpoB and trpB were chosen for streptomycetes, which are representatives of high G+C mol% Gram-positive bacteria. Primer pairs for amplification and sequencing of the gene fragments were designed according to the known genome sequences of two streptomycetes and three mycobacteria, as well as the available recA gene sequences of another two streptomycetes in NCBI database, by using software packages Primer premier 5.0, Oligo 6.0 and SPCR 3.0, and NCBI BLAST program. Performance of the primer sets were validated by specific amplification of all gene fragments from the 55 streptomycete tested strains under optimized PCR reaction conditions, and by successful sequencing of the amplification products. It is concluded that the primer sets designed here are effective, and the primer design procedure and guidelines are valuable for other housekeeping genes of high G+C mol% bacteria.

Abstract:Multigene-based phylogenetic analyses are becoming more widely used in molecular taxonomy because of its lab-to-lab portability and reproducibility. The first step that needs to be settled for this approach is to amplify and sequence several housekeeping genes. Four housekeeping genes atpD, recA, rpoB and trpB were chosen for streptomycetes, which are representatives of high G+C mol% Gram-positive bacteria. Primer pairs for amplification and sequencing of the gene fragments were designed according to the known genome sequences of two streptomycetes and three mycobacteria, as well as the available recA gene sequences of another two streptomycetes in NCBI database, by using software packages Primer premier 5.0, Oligo 6.0 and SPCR 3.0, and NCBI BLAST program. Performance of the primer sets were validated by specific amplification of all gene fragments from the 55 streptomycete tested strains under optimized PCR reaction conditions, and by successful sequencing of the amplification products. It is concluded that the primer sets designed here are effective, and the primer design procedure and guidelines are valuable for other housekeeping genes of high G+C mol% bacteria.

Abstract:In order to probe into the antagonistic mechanism of bacteria against Verticillium dahliae kleb., twelve endophytic bacterial strains were isolated from the Urtica cannabina L. in XinJiang.Through antagonistic experiments, the antagonistic charts of the twelve endophytic bacterial strains against eleven crop diseases have been obtained,respectively,and the XJUL-6 which has higher antagonistic activity against Verticillium dahliae kleb. was screened from the endophytes with the Joan-board diffusion method. The optimum growing temperature range of XJUL-6 is 36℃～38℃, and the optimum growing pH is pH6-8. Phylogenetic analysis of the XJUL-6 based on comparison of 16S rRNA sequence revealed that it is closely related to Bacillus cereus p221 with 98% identity.Moreover, the (G+C)mo1% content of the genomic DNA was 38.84 mol% for XJUL-6.According to the observation of the morphology,the study of the physiological and biochemical,16S rDNA and (G+C)mo1% content, the XJUL-6 was identified as a new members of the species Bacillus cereus. The XJUL-6 will offer potential material for studying the mechanism of bacteria against verticillium dahliae kleb. in cotton.

Abstract:In order to probe into the antagonistic mechanism of bacteria against Verticillium dahliae kleb., twelve endophytic bacterial strains were isolated from the Urtica cannabina L. in XinJiang.Through antagonistic experiments, the antagonistic charts of the twelve endophytic bacterial strains against eleven crop diseases have been obtained,respectively,and the XJUL-6 which has higher antagonistic activity against Verticillium dahliae kleb. was screened from the endophytes with the Joan-board diffusion method. The optimum growing temperature range of XJUL-6 is 36℃～38℃, and the optimum growing pH is pH6-8. Phylogenetic analysis of the XJUL-6 based on comparison of 16S rRNA sequence revealed that it is closely related to Bacillus cereus p221 with 98% identity.Moreover, the (G+C)mo1% content of the genomic DNA was 38.84 mol% for XJUL-6.According to the observation of the morphology,the study of the physiological and biochemical,16S rDNA and (G+C)mo1% content, the XJUL-6 was identified as a new members of the species Bacillus cereus. The XJUL-6 will offer potential material for studying the mechanism of bacteria against verticillium dahliae kleb. in cotton.

Abstract:To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD.

Abstract:To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD.

Abstract:To enhance the effects of ectomycorrhizal fungi to promote the growth of Korea spruce seedlings, based on previous research, the combinations of different ectomycorrhizal fungi strains were screened by dual culture method. 3 years transplanting seedlings of Korea spruce were inoculated by different combinations of ectomycorrhizal fungi strains using lister inoculating method in the field, and those were inoculated by different single strains were designed as control respectively. Thus the effects of different combinations and different single fungus strains to promote the growth of Korea spruces were studied. The results showed that all single strains and combinations in this experiment can promote the growth of Korea spruce seedlings. The growth characteristics of seedlings were observed 100 days after inoculation. The growth promoting effect of strain L15 was the best in all combinations and single strains. Comparing with control, the average height of seedlings inoculated by strain L15 was increased 30.88%, the average collar diameter of these was increased 15.29%. The growth promoting effect of combinations L15/025 or L15/009 were better than strain 009 or 025. Comparing with control, the leaf chlorophyll contents of seedling inoculated by strain 010 and combination L15/025 were increased significantly, the contents of chlorophyll a were increased 59.15% and 54.61% respectively, and the contents of chlorophyll b were increased 76.34% and 67.78% respectively. Except seedling inoculated by strain 010, the activities of hydrogen peroxidase of other treated seedlings were lower than one of control. The root activities of all treated seedlings were lower than one of control. In conclusion, inoculation by the mixture of high-effect strain and other single strain weakens the effect of high-effect strain to promote the growth of Korea spruce seedlings, the activity of hydrogen peroxidase and the root activity of seedling are not correlated with its biomass.

Abstract:To enhance the effects of ectomycorrhizal fungi to promote the growth of Korea spruce seedlings, based on previous research, the combinations of different ectomycorrhizal fungi strains were screened by dual culture method. 3 years transplanting seedlings of Korea spruce were inoculated by different combinations of ectomycorrhizal fungi strains using lister inoculating method in the field, and those were inoculated by different single strains were designed as control respectively. Thus the effects of different combinations and different single fungus strains to promote the growth of Korea spruces were studied. The results showed that all single strains and combinations in this experiment can promote the growth of Korea spruce seedlings. The growth characteristics of seedlings were observed 100 days after inoculation. The growth promoting effect of strain L15 was the best in all combinations and single strains. Comparing with control, the average height of seedlings inoculated by strain L15 was increased 30.88%, the average collar diameter of these was increased 15.29%. The growth promoting effect of combinations L15/025 or L15/009 were better than strain 009 or 025. Comparing with control, the leaf chlorophyll contents of seedling inoculated by strain 010 and combination L15/025 were increased significantly, the contents of chlorophyll a were increased 59.15% and 54.61% respectively, and the contents of chlorophyll b were increased 76.34% and 67.78% respectively. Except seedling inoculated by strain 010, the activities of hydrogen peroxidase of other treated seedlings were lower than one of control. The root activities of all treated seedlings were lower than one of control. In conclusion, inoculation by the mixture of high-effect strain and other single strain weakens the effect of high-effect strain to promote the growth of Korea spruce seedlings, the activity of hydrogen peroxidase and the root activity of seedling are not correlated with its biomass.

Abstract:To study the fast bio-degradation of nicotine and chlorogenic acid in tobacco residues, the effective streptomycetes on degradation of nicotine and chlorogenic acid were screened. The cultural characteristics of Z6 and Z8 strains on different media were inspected. The bio-degradation characters of two strains were discussed. The results showed that the nicotine degradation rate of streptomycete Z8 reached to 83.9% after cultured for 48h in tobacco solid media and reached to 93.7% after cultured for 72h, when the nicotine content of tobacco reduce to 0.38mg/g, which is lower than the innocuity standard (European Union Regulations and Italian law classify them as “toxic and hazardous wastes" when the nicotine content exceeds 500 mg/kg d. w.). Z6 is a good strain for chlorgenic acid bio-degradation. The chlorogenic acid degradation rate of Z6 is 57.1% for 48h and 67.5% for 72h respectively after cultured.

Abstract:To study the fast bio-degradation of nicotine and chlorogenic acid in tobacco residues, the effective streptomycetes on degradation of nicotine and chlorogenic acid were screened. The cultural characteristics of Z6 and Z8 strains on different media were inspected. The bio-degradation characters of two strains were discussed. The results showed that the nicotine degradation rate of streptomycete Z8 reached to 83.9% after cultured for 48h in tobacco solid media and reached to 93.7% after cultured for 72h, when the nicotine content of tobacco reduce to 0.38mg/g, which is lower than the innocuity standard (European Union Regulations and Italian law classify them as “toxic and hazardous wastes" when the nicotine content exceeds 500 mg/kg d. w.). Z6 is a good strain for chlorgenic acid bio-degradation. The chlorogenic acid degradation rate of Z6 is 57.1% for 48h and 67.5% for 72h respectively after cultured.

Abstract:Internalin B of Listeria monocytogenes TA wild strain was amplified by PCR method and sequenced, then subcloned into prokaryotic vector pET28a for expression. The complete length of InlB gene was 1893 bp, encoding 630 amino acids. The front 35 amino acids consisted of signal peptide. There were one cap domain with α-helix region, 6 leucine-rich repeat motifs, one immunoglobulin (Ig)-like domain, one B repeat sequence, 3 direct repeat GW domains and 5 N-glycosylation sites. The amino acid of leucine amounted to 10.2 percent in all amino acids of deduced sequence. Compared the InlB genes with that of other 18 isolates reported in GenBank, the identities of nucleotide sequence and deduced amino acid sequence were 91.1%～99.6% and 92.3%～99.8%, respectively. Expressed InlB protein was detected by SDS-PAGE and confirmed by western blot. Recombinant protein was successfully purified by Ni2+ affinity chromatograph column.

Abstract:Internalin B of Listeria monocytogenes TA wild strain was amplified by PCR method and sequenced, then subcloned into prokaryotic vector pET28a for expression. The complete length of InlB gene was 1893 bp, encoding 630 amino acids. The front 35 amino acids consisted of signal peptide. There were one cap domain with α-helix region, 6 leucine-rich repeat motifs, one immunoglobulin (Ig)-like domain, one B repeat sequence, 3 direct repeat GW domains and 5 N-glycosylation sites. The amino acid of leucine amounted to 10.2 percent in all amino acids of deduced sequence. Compared the InlB genes with that of other 18 isolates reported in GenBank, the identities of nucleotide sequence and deduced amino acid sequence were 91.1%～99.6% and 92.3%～99.8%, respectively. Expressed InlB protein was detected by SDS-PAGE and confirmed by western blot. Recombinant protein was successfully purified by Ni2+ affinity chromatograph column.

Abstract:A plate assay based on the formation of haloes on Petri dishes, containing the trypan blue dye and polysaccharides as substrates, provides a specific, reliable and rapid detection of corresponding polysaccharide degrading enzymes and their producing microorganisms. A blue complex was formed by mixing trypan blue and polysaccharides as substrates. It has been proved by testing three strains that the trypan blue was neither harmful to microorganisms nor enzymes and could stand the normal sterilization. It's optimum concentration was from 0. 005 % to 0. 01 % (W/V). It do not need to prepare dye-labelled polysaccharides, so is a money and time-consuming method. The sensitivity of trypan blue method was the same as traditional method and it has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, especially for large-scale searching programs, such as screening of large numbers of natural samples and engineering bacteria. Using this method, polysaccharide-degrading enzyme genes also has potential of as a new kind of marker gene in gene engineering techniques. According to the result, this method is suitable for detecting cellulase, amylase, pullulanase and mannase, but not suitable for detecting xylanase and inulinase.

Abstract:A plate assay based on the formation of haloes on Petri dishes, containing the trypan blue dye and polysaccharides as substrates, provides a specific, reliable and rapid detection of corresponding polysaccharide degrading enzymes and their producing microorganisms. A blue complex was formed by mixing trypan blue and polysaccharides as substrates. It has been proved by testing three strains that the trypan blue was neither harmful to microorganisms nor enzymes and could stand the normal sterilization. It's optimum concentration was from 0. 005 % to 0. 01 % (W/V). It do not need to prepare dye-labelled polysaccharides, so is a money and time-consuming method. The sensitivity of trypan blue method was the same as traditional method and it has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, especially for large-scale searching programs, such as screening of large numbers of natural samples and engineering bacteria. Using this method, polysaccharide-degrading enzyme genes also has potential of as a new kind of marker gene in gene engineering techniques. According to the result, this method is suitable for detecting cellulase, amylase, pullulanase and mannase, but not suitable for detecting xylanase and inulinase.

Abstract:The major flavor substances Lactic acid bacteria produced include the buttermilk aroma butanedione, the yoghurt flavor acetaldehyde and the amino acid. Metabolic engineering in LAB had focused primarily on rerouting of pyruvate metabolism towards butanedione or acetaldehyde to improve the flavor of fermented milk, which has considerable economic value. The typical yogurt flavor is caused by acetaldehyde produced through many different pathways. The attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase, which encoded by the glyA gene. In addition, the efficient conversion of glucose into acetaldehyde was achieved by over-expression of pyruvate decarboxylase and NADH oxidase in LAB. The concentration of acetaldehyde derived from this regulation is higher than the other strategy. As for the regulation of butanedione, it was focused on combinating inactivation of α-acetolactate decarboxylase with low lactate dehydrogenase activity, or with overproduction of NADH-oxidase, or with overproduction of α-acetolactate synthase. Then the possibility of co-regulation with certain three kinds of enzyme above was recommended.

Abstract:The major flavor substances Lactic acid bacteria produced include the buttermilk aroma butanedione, the yoghurt flavor acetaldehyde and the amino acid. Metabolic engineering in LAB had focused primarily on rerouting of pyruvate metabolism towards butanedione or acetaldehyde to improve the flavor of fermented milk, which has considerable economic value. The typical yogurt flavor is caused by acetaldehyde produced through many different pathways. The attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase, which encoded by the glyA gene. In addition, the efficient conversion of glucose into acetaldehyde was achieved by over-expression of pyruvate decarboxylase and NADH oxidase in LAB. The concentration of acetaldehyde derived from this regulation is higher than the other strategy. As for the regulation of butanedione, it was focused on combinating inactivation of α-acetolactate decarboxylase with low lactate dehydrogenase activity, or with overproduction of NADH-oxidase, or with overproduction of α-acetolactate synthase. Then the possibility of co-regulation with certain three kinds of enzyme above was recommended.

Abstract:Sodium ion with high concentration is toxic to living cells, and microorganisms adapt to the environment containing high concentration of salt by the strategies of salt-in-cytoplasm and compatible solutes. The Na＋ extrusion system plays important roles in maintaining cytoplasmic Na^＋ homeostasis and pH level in microbial cells. Two possible mechanisms of Na⁺ circulation across the cytoplasmic membrane have been proposed, namely primary Na⁺ pump and secondary Na⁺/H⁺ antiporter. Primary sodium pumps coupled the extrusion of Na^＋ to respiration, and the activity of which was insensitive to uncoupler CCCP (carbonyl-cyanide m-chlorophenylhydrazone). There were two types of secondary Na⁺/H⁺ antiporters-encoding genes designated single gene and multiple subunits, respectively. The types of transportation systems for Na^＋, possible mechanisms of Na^＋　extrusion, and projects for further study in bacteria are reviewed.

Abstract:Sodium ion with high concentration is toxic to living cells, and microorganisms adapt to the environment containing high concentration of salt by the strategies of salt-in-cytoplasm and compatible solutes. The Na＋ extrusion system plays important roles in maintaining cytoplasmic Na^＋ homeostasis and pH level in microbial cells. Two possible mechanisms of Na⁺ circulation across the cytoplasmic membrane have been proposed, namely primary Na⁺ pump and secondary Na⁺/H⁺ antiporter. Primary sodium pumps coupled the extrusion of Na^＋ to respiration, and the activity of which was insensitive to uncoupler CCCP (carbonyl-cyanide m-chlorophenylhydrazone). There were two types of secondary Na⁺/H⁺ antiporters-encoding genes designated single gene and multiple subunits, respectively. The types of transportation systems for Na^＋, possible mechanisms of Na^＋　extrusion, and projects for further study in bacteria are reviewed.

Abstract:The growing population of overweight humans threatens both industrialized and developing countries and has been accompanied by obesity-related disorders, including typeⅡdiabetes, hypertension, cardiovascular pathology and nonalcoholic fatty liver disease. Recent researches have demonstrated that intestinal microbiota may be associated with the host's obesity. There were researches on the interaction between Bacteroides thetaiotaomicron and the energy metabolism of the host. Methanobrevibacer smithii had been improved to impact the host's energy metabolism through modulating the gene transcription of B. thetaiotaomicron. The microbiota can direct the host to increase hepatic production of triglycerides, promote storage of triglycerides in adipocytes through suppression of intestinal expression of a circulating LPL inhibitor, and have an effect on the host's energy deposition through the interaction with host's hormones (eg. Leptin). Some metabolic products of the microbiota like SCFAs, other organic acids, alcohols and gases can be used by the host directly. Researches mentioned above are just started. According to the results above, some key points remain unknown. For example, the underlying mechanism of the interaction between microbiota or some unique microbes and the host, the procedure of dietary polysaccharides degradation of the microbes, and the relationship between the microbiota and the host's hormones. In this paper, the corresponding research results of author's lab has also been reviewed and the future research prospect s have been summarized.

Abstract:The growing population of overweight humans threatens both industrialized and developing countries and has been accompanied by obesity-related disorders, including typeⅡdiabetes, hypertension, cardiovascular pathology and nonalcoholic fatty liver disease. Recent researches have demonstrated that intestinal microbiota may be associated with the host's obesity. There were researches on the interaction between Bacteroides thetaiotaomicron and the energy metabolism of the host. Methanobrevibacer smithii had been improved to impact the host's energy metabolism through modulating the gene transcription of B. thetaiotaomicron. The microbiota can direct the host to increase hepatic production of triglycerides, promote storage of triglycerides in adipocytes through suppression of intestinal expression of a circulating LPL inhibitor, and have an effect on the host's energy deposition through the interaction with host's hormones (eg. Leptin). Some metabolic products of the microbiota like SCFAs, other organic acids, alcohols and gases can be used by the host directly. Researches mentioned above are just started. According to the results above, some key points remain unknown. For example, the underlying mechanism of the interaction between microbiota or some unique microbes and the host, the procedure of dietary polysaccharides degradation of the microbes, and the relationship between the microbiota and the host's hormones. In this paper, the corresponding research results of author's lab has also been reviewed and the future research prospect s have been summarized.