Abstract:Abstract: Actinobacillus pleuropneumoniae (App) is the etiological agent of porcine pleuropneumonia which causes huge economic losses to the pig industry. RTX (repeat in the structural toxin，RTX ) is the main virulence-associated factor of the pathogen and, as a double blade sword, plays a dual role on the pathogen’s infection and immunity. The recently research progress that has been made within around ten years is updated in this paper and the necessity and techniques to initiate the pathogen-host interaction study is also raised and discussed. It is believed that the research on the interaction between host and App RTX may further elucidate the pathogen’s molecular pathogenesis.

Abstract:Abstract：Helper-dependent adenoviral vector (HDAd) lacking all viral coding sequences with the advantages of minimal immunogenicity, negligible chronic-toxicity, and durable transgene expression over first-generation adenovirus vector (FGAd). HDAd vehicles have demonstrated tremendous potential for gene therapy in animal models for inherited diseases, neurodegenerative diseases and cancer etc. Additionally, the large cloning capacity of HdAd, up to 37 kb, permits the delivery of whole genemic loci, multiple transgenes. In this review we characterize the basic features of HdAd and summarize some of their experimental and potential clinical applications both at present and in future.

Abstract:Abstract: [Objective]: Based on the antibacterial analysis, we screened Polyketide synthase (PKS I) gene from the epiphytic bacteria of Porphyra spp., in order to obtain the PKS I positive strains and detect the potential connection between the PKS pathway and the antibacterial mechanisms. [Methods]: A total of 31 bacteria with broad-spectrum antibacterial activity were screened by agar-screening methods. The 16S rDNA and the Ketosynthase gene were amplified from the genome DNA of these bacteria, which were cloned into pMD19-T vector for sequencing analysis. [Results]: Porphyra spp. epiphytic bacteria showed broad-spectrum antibacterial activity. Three PKS I positive epiphytic bacteria were obtained from Wenzhou rotten Porphyra spp. samples which had high antibacterial activity. The BLAST results indicated that the Ketosynthase fragments of PKS I from the strains of WPhG3, WPySw1 and WPySw2 shared highest similarity (98%, 99%, 98%) to the strains of Bacillus subtilis subsp. Subtilis str. 168 (NP_389602), Bacillus subtilis (ABR19776) and Aspergillus carbonarius (AAZ99721), respectively. Furthermore, the phylogenetic analysis based on 16S rDNA sequences indicated that they belonged to the genus of Bacillus. [Conclusion]: The flora of Porphyra spp. epiphytic bacteria was complex, which regulated the phycosphere in many ways. The PKS I pathway might be a performance of antibacterial function of Bacillus from Wenzhou rotten samples.

Abstract:Objective: To find catalytic center of MDV-1 UL13 and express it in vitro to investigate the function of UL13 kinase. Methods: UL13 gene was amplified by polymerase chain reaction (PCR) from MDV-1 CVI988/Rispens strain. The codon bias of UL13 in Escherichia coli was analyzed by online service GENEART (www.gcua.de）and DNAstar software. Then the UL13 truncated fragments were expressed in Escherichia coli, and mice were immunized with the expressed Glutathione S Transferase fusion protein. The conserved domain was analyzed with protein blast and Cn3D 4.1 online software of National Center for Biotechnology Information. Results: UL13 gene was successfully amplified. The sequence analysis suggests that 259-400 and 431-513 amino acid residues are low abundance for rare codon and strong antigenicity in UL13. Result of conserved domain analysis demonstrated that 152-297 residue iskinase catalytic center of UL13. However, conserved glycin in kinase subdomain Ⅶ for most protein kinase was replaced by serine in UL13 and proline in kinase subdomain Ⅷ replaced by cysteine. The serum from mice immunized with truncated fragment, 259-400 amino acids, could react with recombinant UL13 protein expressed in insect cells in immunofluorenscence assay. Conclusion: The 152-297 residue is kinase catalytic center of MDV-1 UL13; UL13 protein expressed in vitro induced specific antibodies against UL13.

Abstract:Abstract: [Objectives] Characteristic of energy metabolism in ruminant is negative energy balance in perinatal period. Propionic acid from ruminal microbes fermentation is a vital glyconeogenesis substrate for prevention of negative energy balance. We intend to isolate and screen a strain of Propionibacterium acnes from health cow rumen fluid, and study its rumen fermentation characteristics. [Methods] A strain of Propionibacterium acnes from rumen fluid of health cow with permanent rumen fistula under sterile condition was isolated by segregation procedure of anaerobic bacterium and specific medium SLB, and identified by extraction of the genome DNA of isolation bacterium, clone of the 16S rRNA gene, and sequencing of the strain. We study effect of the stain on pH, volatile fatty acid and lactic acid in rumen fluid by vitro and vivo test. [Results] The results showed that a strain of bacterium isolated from health cow rumen fluid was identified as the Propionibacterium acnes by morphology, biochenmical characteristics, sequence homology. In vitro test of the strain, pH in rumen fluid first dropped, to the lowest at 12 h, then increased gradually; concentration of volatile fatty acid, including acetic acid, propionic acid and butyric acid, first increased, to the highest at 12 h, then decreased gradually; the lactic acid concentration and ratio of acetate to propionate ratio decreased overall. In vivo test of the strain, pH in rumen fluid decrease overall; concentration of the volatile fatty acid increased overall. [Conclusions] A strain of Propionibacterium acnes was first isolated successfully from health cow rumen fluid at home, it is an important basis for us to develop microecological preparation for prevention of cows’ negative energy balance in perinatal period in future.

Abstract:Abstract: [Objective] Studying the kinetics of conjugated linoleic acid (CLA) bioconversion by Lactobacillus plantarum ZS2058. [Methods] We investigated the effects of the substrate concentration, cell mass, reaction pH and temperature on the reaction rate of producing CLA by L. plantarum ZS2058, and established the equations of reaction rate at the initial response stage under different substrate concentrations by double-reciprocal plot and Hanes-Woolf plot method. [Results] The results showed that there existed in a marked substrate inhibition effect, and the early reaction rate of producing c9, t11-CLA reached the maximum of 15.99 μg/(mL?h) when linoleic acid concentration was 0.4 mg/mL. The early reaction rate ascended with the cell mass increasing, and when the cell mass was at 5×1010 cfu/mL, the response rate close to the maximum. The optimum pH and temperature of bioconversion of CLA were 6.5 and 40℃ respectively. Michaelis constant was obtained by double-reciprocal plot and Hanes-Woolf plot method, the reaction rate equation followed the classic Michaelis-Mentent equation at the low substrate concentration, while there existed in a marked substrate inhibition effect at the high substrate concentration. [Conclusion] Through exploring the various factors of CLA bioconversion by L. plantarum ZS2058, we established the reaction rate equations of the initial response stage under different substrate concentrations, and got the optimal reaction conditions. These results will contribute to the CLA production and its physiological functions research.

Abstract:Abstract: [Objective] In this study we investigated characteristics of ChiB (chitinase B) from Bt 15A3(Bacillus thuringiensis subsp. colmeri 15A3) and evaluated its potential in antagonism and insecticidal enhancing effects. [Methods] The ChiB was expressed in E.coli(Escherichia coli). Expression product was purified by ammonium sulfate precipitation, dialysis and Sephadex G-200 gel filtration chromatography. The molecular mass of ChiB was directly estimated by zymogram after SDS-PAGE(sodium dodecyl sulfate-polyacryamide gel electroresis). The proteins with different molecular weights were identified by MS(mass spectrum) and analyzed through bio-informatics. We studied some kinds of metal ions’ impacts on the chitinase activity; the optimal temperature, pH and its stability in different temperature and pH. We also studied the inhibition effects on sporangia germination of fungi and the synergistic effects of ChiB on larvicidal activity. [Results] The molecular mass of ChiB was estimated 70 kDa by zymogram. The protein with 64 kDa was the product of C-terminal degradation of ChiB in E.coli. The chitinase activity was improved by Fe3+ and inhibited by Zn2+, Ag2+. The optimal temperature for ChiB was 60 ℃, whereas, the optimal pH was 5.0. The enzyme was quite stable at temperature less than 60 ℃ and pH between 4.0 and 8.0. ChiB inhibited sporangia germination and the IC50 (50 % inhibited concentration) was about 4 μg/mL. Moreover, the bioassays showed that ChiB could reduce the LC50 (50 % lethal concentration) of the crystal protein of Bt. against S.exigua and H.armigera larvae by approximately 26 % and 30 % respectively.[Conclution] ChiB not only had stable characteristics, but also had good antagonism activity and insecticidal enhancing effects.

Abstract:Abstract: [Objective] Sortase is a novel anti-infection target enzyme for its critical action of anchoring surface proteins to the cell wall. [Methods] We amplified the srtA gene from Staphylococcus aureus chromosomal DNA by PCR technique, and then constructed two prokaryotic expression vectors pet22-srtA and pTRX-srtA with regular molecular cloning operation. The pet22-srtA and pTRX-srtA were transformed into E. coli BL21 (DE3) competent cells and overexpressed under 1 mmol/L IPTG (isopropy-β-D-thiogalactoside) induction. [Results] SDS-PAGE and western blot results show that approximately 45 kDa and 39 kDa proteins were expressed by pet22-srtA and pTRX-srtA respectively. [Conclusion] The molecular chaperone thioredoxin was beneficial to the prokaryotic expression of srtA gene. Moreover, the experiment laid solid foundation to study sortase’s enzymatic property and inhibitors screening especially.

Abstract:Abstract: [Objective] To characterize the GDSL (glycine, asparticacid, serine and leucine motif in protein sequence) esterase Xcc_est from Xanthomonas campestris pv. campestris (Xcc) 8004. [Methods] Xcc_est gene and different domains of Xcc_est gene were PCR amplified and expressed in Escherichia coli, the HIS-Tagged fusion proteins were purified by Ni-NTA chromatography. [Results] The optimum pH and temperature of partly purified Xcc_est were pH 8.0 and 52 °C when pNPB (4- nitrophenylbutyrate) was used as substrate. The Km and Vmax value of Xcc_est and the passenger domain (Xcc_estN1-334) for pNPB were 47.6 ± 4.6 μmol/L, 67.6 ± 7.8 U/mg and 469.4 ± 9.8 μmol/L, 2.5 ±0.9 U/mg respectively. Inclusion bodies of mature domain Xcc_est (Xcc_estN26-606) could be refolded but inclusion bodies of the passenger domain (Xcc_estN26-334) could not be refolded. Refolded mature domain had broad substrate spectrum and showed higher stability than Xcc_est when stored at 25 °C. [Conclusions] To some extend, refolded Xcc_estN26-606 is a candidate for biotransformation application.

Abstract:[Objective] To compare the amino acid and dipeptide composition of proteins from piezophilic and non-piezophilic microbes is of great significance for understanding the stability of piezophilic protein and directed mutation of them. [Methods] The amino acids of different secondary structure and the dipeptides of 639 orthologs proteins were counted and the deviation of them were calculated. [Results] The amino acid composition based on secondary structure and the dipeptide composition reveals some common trends.The amino acids vary widely in β sheet and coil. In β sheet, piezophilic proteins contain more amino acids such as Val, Ile and Leu, whereas less Arg, Lys and Asp; in coil, piezophilic proteins contained more amino acids such as Val and Ile, whereas less Gly and Pro. On the other hand, piezophilic proteins contain more dipeptides such as YM, MN, KD, QC, CI, MW, MM, CY, WQ, HC, RC and QH, whereas less dipeptides such as TW, MS, VD, DH, YE, CT, MW, CF, CK, CM, MY, QI, TH, MQ, QQ and MC。[Conclusion] Dipeptide contains more structure and sequence information than amino acid, and it will be more helpful for understanding the mechanism of piezophilic adaptation and guiding the engineering of proteins.

Abstract:[Objective] To prepare (S)-1-phenyl-1,2-ethanediol by a one-step method, we constructed an enzyme coupled system consisting of (R)- and (S)- specific carbonyl reductases in Escherichia coli. [Methods] The genes coding (R)- and (S)- specific carbonyl reductases from Candida parapsilosis were inserted into a co-expression vector pETDuetTM-1. The positive plasmid was transformed into codon optimized E. coli Rosetta and an enzyme-coupled recombinant strain E. coli Rosetta /pETDuet-rcr-fdh was constructed. When the OD600 value of the culture reached 0.6-0.8, IPTG with a final concentration of 1 mmol/L was added to induce both target proteins at 30 oC for 10 h. [Results] SDS-PAGE analysis showed that two target enzymes were expressed simultaneously with the relative molecular weights of 37 kDa and 30 kDa. When 5 mol/L Zn2+ was added into a phosphate buffer (pH7.0), the biotransformation results showed that the product (S)-1-phenyl-1,2-ethanediol was produced with the optical purity of 91.3% enantiomeric excess and a yield of 75.8% by E. coli Rosetta/pETDuet-rcr-scr. [Conclusion] The functions of two redox enzymes were integrated by molecular recombinant technique and a one-step method for preparation of (S)-1-phenyl-1,2-ethanediol was developed through an enzyme-coupled system. The method may simplify procedure in chiral alcohols preparation.

Abstract:[Objective] Bifidobacterium has important physiological activity in gastrointestinal tract of human and animals, and the tolerability of acid, intestinal juice and oxgall bile salt are the key factors that influence the function of living bacteria preparation. In this study, feces of 12 healthy Mongolian children were selected to isolate Bifidobacterium with probiotic properties. [Methods] Bifidobacterium was isolated from feces by selective medium and identified by physiological biochemical test. The tolerability of acid, intestinal juice and oxgall bile salt of 11 Bifidobacteria were studied and B. animalis V9 was identified further by molecular biological approach 16S rDNA. [Results] Eleven Bifidobacteria isolated from feces of 12 healthy Mongolian children were identified as B. adolescentis (A1, H3, G4, A8 and V10), B. longum (C6, C7 and D11), B. pseudocatenlatum (B2), B. bifidum (G5), B. animalis (V9). B. animalis V9 had the best acid tolerance with the survival rate 92.4% in artificial gastric juice at pH2.0 for 3h, whereas others had lower than 31.25%. B. animalis V9 also had good survival rate (99.7%) in artificial intestinal juice at pH8.0 for 8h after anaerobic cultured 3h in artificial gastric juice at pH2.0, and tolerated oxgall bile salt at concentration of 0.3%. B. animalis V9 was identified further by molecular biological approach 16S rDNA and result showed the homologies of B. animalis V9 was 99% with Bifidobacterium animalis subsp. lactic BB12. [Conclusion] B. animalis V9 had good probiotic properties to be potentiallyused in dairy products and health products.

Abstract:[Objective] To study the bacterial diversity in sediment and water from two disused thermal vents in Yangbajing, Tibet, China. [Methods] We constructed 16S rRNA gene libraries of the total DNA from three samples. Sediment sample A and water sample A were from thermal vent A. Sediment sample B was from thermal vent B. Positive clones from the libraries were analyzed randomly by amplified ribosomal DNA restriction analysis. Positive clones’ sequences of every Operational Taxonomic Units from libraries were determined, and sequence data were submitted to GenBank and contrasted to those known sequences. Phylogenetic trees were built up by using MEGA4.0 program. [Results] Most of bacteria communities of the two thermal vents were typical thermophilc inhabitants, the predominant proteobacteria communities were found in thermal vent A and thermal vent B (ratios in sediment A, water sample A and sediment B were 41.08%, 38.00% and 42.57%) One of the sub-predominant bacteria communities, Deinococcus-Thermus, was found in thermal vent A and thermal vent B (ratios in sediment A, water sample A and sediment B were 10.71%, 20.00% and 21.30%). Moreover, one of the sub-predominant bacteria communities, Acidobacteria that was rarely found in hot springs or thermal vents, was present both in sediments of thermal vent A and B (16.07%, 19.15%). The third sub-predominant community in sediment of thermal vent A was Eubacterium sp. (14.28%), belonging to phylum Firmicutes whereas no Acidobacteria was found in water sample A. Instead, Hydrogenobacter, belonging to phylum Aquificae, was another sub-predominant community (16.00%) in water sample A. In addition, Chloroflexi, Cyanobacteria and CFB group (Cytophaga-Flexibacter-Bacteroides) were detected in both thermal vents. [Conclusion] Compared with and contrasted to the references, we found that bacteria communities in Yangbajing thermal field were similar to that in some hot springs and thermal vents around the world but slightly abnormal. Some bacteria communities in thermal vent A and thermal vent B were not very popular, such as Vibrio sp., Bacteriovorax sp., Holophaga/Acidobacterium, Verrucomicrobium.

Abstract:[Objective] We studied the bacterial community during Agaricus bisporus composting phase II, to develop a quick and accurate method by modern molecular ecology technique for dynamic inspection on bacterial community during A. bisporus composting. [Methods] We selected seven A. bisporus compost samples from different stages of phase II. We used PCR to amplify the V3 regions of the 16S ribosomal DNA from those samples. We analyzed PCR products by denaturing gradient gel electrophoresis. [Results] The denaturing gradient gel electrophoresis profile showed a dramatic change of bacterial communities which related to the process of phase II of A. bisporus compost. DNA sequencing of the dominant bands of denaturing gradient gel electrophoresis profile indicated that the bacterial diversity in the phase II of A. bisporus compost was much higher than that studied by traditional method cultured-based approach. We found not only the genus of Bacillus, commonly believed to dominate high temperature compost, but also some new groups belonged to the class of Bacilli such as Trichococcus, Planococcus, Caryophanon and even the γ-subgroup of Proteobacteria. At the same time, some sequences were related to the genus of Thermus thermophilus and α-subdivision of Proteobacteria which were only recently reported in the hot compost. Several sequences display extremely low similarity with the cultivated species in the GenBank, indicating high diversity of uncultivated bacteria in the process of A. bisporus composting. [Conclusion] We found distinctly changes on bacterial communities during A. bisporus composting phase II by denaturing gradient gel electrophoresis profile. Also we found a lot of presently uncultured bacteria species during A. bisporus composting. And what then? Meaningful of your study? I don’t see any indications/conclusion from your study, as you stated in the beginning of the abstract.

Abstract:[Objective] To study the effect of clpE gene deletion on the virulence of Streptococcus pneumoniae. [Methods] The clpE-deficient strain was constructed by LFH-PCR and identified by PCR and sequencing. The impact of clpE mutant on the virulence of S. pneumoniae was evaluated in a mouse model. In addition, we also studied the effect of clpE mutant on adherence and invasion of host cells. Real time RT-PCR was used to measure the mRNA expression levels of autolysin A, pneumococcal surface adhesion A, pneumolysin, pneumococcal surface protein A and neuraminidase [Results] The clpE gene was replaced completely by erm cassette. Mice virulence experiments showed that the median lethal time of the wide-type was 54 h, whereas that of clpE mutant was 21d (P<0.01). Cell culture infection experiments indicated that adherence and invasion of clpE mutant were strongly reduced (P<0.05). The expression of virulent factors in clpE mutant was lower than that of the wild-type (P<0.05). [Conclusion] ClpE is involved in virulence by modulating the expressions of virulence factors.

Abstract:[Objective] TLR7 and TLR3 (Toll-like receptor, TLR) are the two of important pattern recognition receptors (PRRs) that recognize the single-stranded (ss) RNA and double-stranded (ds) RNA of virus-origin leading to activation of cells, respectively. RSV (respiratory syncytial virus, RSV) could be recognized by both TLR7 and TLR3. The aim of this study was to investigate the kinetics and profile of lung TLR7 and TLR3 expression during the early phase of the pathogenesis of RSV infection, and to explore its correlation with pulmonary inflammatory response. [Methods] We intranasally inoculated BALB/c mice with live RSV to induce acute lung inflammation, and detected the lung expression of TLR7 and TLR3 mRNA by semiquantitative RT-PCR analysis at the indicated times on day 1 after RSV infection. We also measured the transcription factor nuclear factor-kappaB (NF-κB) protein expression with western blot assay in nuclear extracts. Lung tissue sections were stained with hematoxylin and eosin and examined under a light microscope to perform the histopathological examination. [Results] We found that RSV infection could rapidly upregulate the lung expression levels of both TLR7 and TLR3 gene in a time-dependent manner, furthermore, the response of TLR7 to RSV (1 h) was prior to that of TLR3 (4 h). The mice inoculated intranasally with RSV resulted in activation of NF-κB as soon as 4 h later in lung tissue. Moreover, RSV mediated early transcriptional responses of TLR7 and TLR3 were parallelling with the severe extent of RSV-induced pulmonary inflammation. [Conclusion] TLR7 and TLR3 were really involved in RSV-induced lung inflammation by detecting the viral RNA. This may allowed the infected organism to detect viral infections and initiate a proinflammatory response via multiple TLRs. This study may be useful in the development of tools to modulate the activities of therapeutic TLR ligands.

Abstract:[Objective] To understand the diversity of cultivable bacteria isolated from a sea anemone collected from the coastal water of the Naozhou island in the Leizhou Bay on South China Sea. [Methods] Bacteria were isolated from a sea anemone by using conventional culture-dependent method and investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. [Results] We isolated 126 bacteria strains from the sample on marine agar 2216 (Difco), International Streotomyces Project medium 2 agar, nutrient agar, sea water agar and humic acid agar supplemented with 0–2 mol/L NaCl. Based on partial morphological, physiological and biochemical characteristics, we selected 42 strains for molecular systematic study based on 16S rRNA gene sequences. Our results showed that 42 isolates were members of eighteen genera (Alteromonas, Bacillus, Brachybacterium, Brevibacterium, Halobacillus, Halomonas, Nocardiopsis, Oceanobacillus, Piscibacillus, Planococcus, Pontibacillus, Pseudoalteromonas, Pseudonocardia, Salinicoccus, Salinivibrio, Staphylococcus, Vibrio, Virgibacillus) of eleven families (Alteromonadaceae, Bacillaceae, Brevibacteriaceae, Dermabacteraceae, Halomonadaceae, Planococcaceae, Pseudoalteromonadaceae, Pseudonocardiaceae, Nocardiopsaceae, Staphylococcaceae, Vibrionaceae) in three major phylogenetic groups (Actinobacteria, Firmicutes, Gamma-Proteobacteria). The most abundant and diverse isolates were within the phylum Firmicutes (40.5 %) and the subphylum Gamma-Proteobacteria (33.3 %). The phylogenetic distance matrix results suggested that, out of 42 isolates, 37 were different strains of 19 known species, and that at least 6 strains represented 6 new species within 6 characterized genera. [Conclusion] The results presented above showed that there were abundant species diversity and phylogenetic diversity of bacteria isolated from the sea anemone.

Abstract:[Objective] To analyze the diversity of bacterial community in Guangxi buffalo rumen and to identify the possible cellulolytic bacterial group. [Methods] Metagenomic DNAs were isolated directly from a buffalo rumen and its enriched culture, and were used as PCR templates to amplify 16S rRNA genes. Two libraries carrying 16S rRNA genes of bacteria in the two samples were constructed. The bacterial community composition was revealed by the constructed phylogenetic tree of known sequences and the sequences randomly selected from the libraries. [Results] We found that both samples contained low G+C Gram-positive bacteria (LGCGPB) and Cytophaga-Flexibacter-Bacteroides (CFB) phyla as the majorities, and Spirochaetes as the minorities. LGCGPB accounts for 56.66% and 73.33% of the bacterial communities in buffalo rumen and its enriched culture. We detected Fibrobacteres in the rumen sample (3.33%) but not in the enriched sample. Furthermore, we found Proteobacteria as a major component in the enrichment (13.33%) but not in the rumen sample. Clone R46 was not clustered into any known phyla and might belong to a novel taxonomic group. [Conclusion] The LGCGPB and Proteobacteria may play important roles in the hydrolysis of cellulose in buffalo rumen. The bacterial composition in the rumen of buffalo is quite similar to those in the rumen of yak, cattle and sheep.

Abstract:[Objective] We evaluated antioxidative effect of two antioxidative strains, isolated from the traditional fermented dairy products.[Methods] Both intact cells and cell-free extract of Lactobacillus SY13 and LJJ were used to study the inhibited effect of linoleic acid peroxidation,the ability of scavenging1,1-diphenyl-2-picrylhydrazyl r- adical,hydroxyl radical,superoxide anion radical,the ability of tolerancing hydrogen peroxide and the chelating capacity of ferrous ion and reducting activity. [Results] L-actobacillus SY13 and LJJ domonstrated highest inhibition ratio on linoleic acid pero-xidation by 62.95% and 66.16%,respectively.The cell-free extract show excellent sca-venging superoxide anion and hydroxyl radicals activity.However,the intact cells of L JJ scavenging superoxide and hydroxyl radicals capacity weren’t detected.The intact cells of SY13 and LJJ on 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability and chelating ferrous ion capacity were superior to cell-free extract.The highest reduced activity was equivalent to 305 μmol/L and 294 μmol/L L-cysteine. [Conclusion] The results showed two lactobacilli strains have good antioxidant capacity.As potential pr-obiotics,it can be used in future.

Abstract:[Objective] α-glucosidase from Aspergillus niger SG136 was expressed in Pichia pastoris. [Methods] cDNA of mature α-glucosidase(aglu) was amplified from the total DNA of A. niger SG136 by PCR and overlap-PCR with primers designed based on the sequence of A. niger CBS 513.88 in NCBI database. The gene was cloned into pMD18-T simple vector. The sequencing result showed that the gene encoded for a protein of 960 amino acids residues, which was 1 amino acid different from that of A. niger CBS 513.88. The expression vector pPIC9K-aglu was constructed by subcloning the gene into plasmid pPIC9K, and then transformed into P. pastoris through electroporation after linearized by BglⅡ digestion. The recombinant P. pastoris KM71/pPIC9K-aglu were screened in MD and YPD/G418 plates, and identified by PCR. In shaking culture condition, methanol was added to a final concentration of 1 % to induce the secretion of α-glucosidase. [Results] Electrophoresis analysis of KM71/pPIC9K-aglu culture supernatant showed that there were two major protein bands corresponding to 98 kDa and 33 kDa respectively in SDS-PAGE, and there was only one band in Native PAGE; while in the control experiment of KM71/pPIC9K, there were no visible bands. Transglucosidation reaction from crude enzyme revealed that contents of isomaltooligosaccharides were up to 26.0 % under the optimal conditions of pH 5 and 60 ℃ at 24 h. [Conclusion] A. niger α-glucosidase was expressed in P. pastoris with transglucosidation activity.

Abstract:[Objective] Listeria monocytogenes （LM） is a gram-positive facultative intracellular pathogen which can cause animal and human listeriosis. In order to use killed LM in vaccination, we compared, in the mouse model, the immunogenicity of LM, which were lethally inactivated by γ –irradiation, traditional heat or formalin treatment. [Methods] BALB/c mice were inoculated intraperitoneally with these killed vaccine candidates. We detected the serum antibody titers with indirect enzyme-linked immunosorbent assay (ELISA) and evaluated the protective efficacy of each vaccine candidates by the resistance to lethal dose challenge of homologous live LM and the effect of bacterial elimination in the spleen and liver of immunized mice. Adoptive transfer of Flow Cytometry sorted T splenocytes from immunized mice to na?ve recipients, subsequently challenged with high dose of LM, we performed to determine the possible role of T cell . [Results] The serum antibody level of mice inoculated with γ-irradiated LM was the highest, up to 1:1280 as determined by ELISA, while the level of mice immunized with heat-killed or formalin-killed LM was 1:640 or 1:160, respectively. The protective efficacy of γ-irradiated, heat-killed or formalin-killed vaccines candidates were 100%, 35% or 30%. As jadged by the bacterial elimination in the organs, mice inoculated with γ-irradiated LM were the most efficiently protected group. Adoptive T cell transfer assay showed that γ-irradiated LM can trigger T cell protective immune response. [Conclusion] All the results indicated the superiority of γ-irradiation over traditional heat or formalin treatment in generating LM killed vaccine candidate. γ-irradiation may be applied to numerous bacterial vaccine candidates, and could have important potential in development of killed vaccines.