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Abstract:To tract the intracellular localization of the hypothetical protein CT058 in the Chlamydia trachomatis-infected cells. The gene ct058 was expressed as GST-CT058 fusion protein in E. coli and its antibody was prepared by immunizing mouse with the fusion protein. Then, the anti-GST-CT058 an-tibody was used to localize the endogenous protein in cells infected by C. trachomatis serovar L2 using indirect immunofluorescence assay (IFA). Western blot was carried out to detect whether CT058 pre-sents in the purified elementary body (EB) or reticulate body (RB). The anti-GST-CT058 antibody de-tected an inclusion-inside signal in an IFA. The intra-inclusion signal could be removed by pre-absorption of the antibody with the GST-CT058 fusion protein but not control GST-CT232. Fur-thermore, the mouse anti-CT058 antibody recognized the endogenous CT058 protein from purified EBs and RBs in a Western blot assay. The results of the current study support the general consensus that the hypothetical protein CT058 was localized within the inclusion body of the C. trachomatis-infected cells.

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Abstract:The pullulanase gene of thermophilic Bacillus subtilis WY-34 was cloned in E. coli to characterize the recombinant pullulanase. The pluA gene encoding pullulanase from the thermophilic B. subtilis WY-34 was overexpressed in E. coli. The properties of recombinant pullulanase were studied. Also substrate specificity of the recombinant pullulanase was evaluated in this paper. The pluA gene was 2.2 kb in length and encoded 718 amino acid protein. The recombinant pullulanase was purified to homogeneity using Ni-IDA agarose chromatography, resulting in a specific activity of 93.2 U/mg. SDS-PAGE and gel permeation chromatography analysis showed that the molecular weight of the protein is approximately 76.2 kD and 74.3 kD respectively. The purified enzyme was optimally active at 40 °C and pH 6.0, stable at 45 °C and retained more than 80% relative activity with in pH 6.0?9.0. It exhibited high substrate specificity toward pullulan. The properties of recombinant pullulanase may be useful for application of pullulanase in industry.

Abstract:Strain producing cold-active amylase want to be isolated. Enzymatic properties of the strain will be determined. Screening strains from The Black mountant. Determine its species by its morphological, the physiological and biochemical properties and 16S rDNA sequence analysis, to study its enzymatic properties. A strain C2 producing cold-active amylase was identified as Exiguobacterium sp.. Enzymatic properties showed that the optimum pH value is 7.5 and the optimum reaction temperature is 25 °C, but the thermal stability of the enzyme is relatively poor. The amylase activity was activated by Ca2+ and Fe2+, and was inhibited by Cu2+, Ni2+ and Go2+. By the identification of TLC, the product of enzymolysis is glucose. It is indicates that the strain can product cold-active starch glucoamylase. Amylase produced by strain C2 in line with the nature of cold-active amylase, it deserves to be further researched.

Abstract:Bacterial community associated with Suaeda salsa (L.) rhizosphere in petroleum-contaminated saline-alkali soil was investigated by 16S rRNA gene clone library analysis. Halotolerant petroleum-degrading bacteria were further isolated by enrichment using petroleum as the sole source of carbon and energy. Marinobacter, Alcanivorax and Pseudomonas dominated Suaeda salsa (L.) rhizosphere in petroleum-contaminated saline-alkali soil as revealed by 16S rRNA gene clone library analysis, which were likely to have a role in the phytoremediation of such soil by Suaeda salsa (L.). Eight halotolerant petroleum-degrading bacterial strains were isolated, which could grow up to 6%?10% NaCl, and could degrade 32.3% ?57.0% petroleum hydrocarbon in 3% NaCl over a period of 14 days. They were identified to belong to the genus of Gordonia, Achromobacter, Dietzia, Bacillus and Pseudomonas by 16S rRNA gene sequence analysis. They may involve in the degradation of petroleum in the phytoremediation of such soil by Suaeda salsa (L.).

Abstract:To simplify the construction procedure of Yarrowia lipolytica secretion/expression system, eliminate the antibiotic contamination, and use gene mel (encoding tyrosinase) as a new type reporter for constructing Y. lipolytica secretion/expression vectors. Gene mel was synthesized by assembly PCR, and fused with homologous promoter pTEF, signal sequence XPR2pre and terminator LIP2t by overlap PCR. The resulted fragment was used to construct new type of secretion/expression plasmids of Yarrowia lipolytica expression system, with which human gene rho was expressed. The synthesized mel and pTEF, XPR2pre, LIP2t were successfully fused into gene fragment TXML, which was substituted for the reporter gene ura3d4 of Y. lipolytica plasmids pINA1297 and pINA1297-a, resulting in new type plasmids pINA1297-M and pINA1297-a-M. This new system could screen the yeast recombinant transformants by phenotype, with which the soluble protein Rho was successfully expressed. In this re-search, mel gene was used as a new reporter for the first time in non-conventional yeast expression system, and it also lends a basis for applying mel in other ?eukaryotic expression systems. Additionally, the resulted soluble Rho can be used for further study of its character, structure, functions and the cor-relation between other members of Rho oncogene family.

Abstract:This study aims to clone ftsb gene from Streptococcus pyogenes 5005, to express and purify FtsB, and to characterize its ferrichrome binding properties in vitro. DNA fragments encoding ftsb was obtained by PCR and inserted into plasmid pGEX4T-1, followed by transformation into Escherichia coli DH5α. The constructed recombinant plasmid was transformed into Escherichia coli BL21 to express FtsB. The conditions used to induce expression were optimized. The protein was purified using affinity chromatography. The ferrichrome binding property was initially characterized, and the binding site was investigated by blocking histidine residues. with DEPC. The 33 kD FtsB was successfully expressed and purified, and the yield was 30 mg/L. The UV-visible spectra showed the FtsB had a visible absorption peak at 425 nm with ferrichrome and that histidine didn’t participate in ferrichrome binding. This approach reports the ferrichrome binding properties and binding site of FtsB, providing theoretical foundations for the further study of ferrichrome transport mechanism in bacteria and the development of drug target and vaccine candidate.

Abstract:Zearalenone (ZEN) is a high toxic mycotoxin. How to detect ZEN from food and feed is important and challenging. Monoclonal antibody was prepared by means of immunization in mice with conjugates of ZEN and bovine serum albumin (BSA), and enzyme-linked immunosorbent assay for ZEN based on monoclonal antibodies was developed. Four hybridoma cell strains secreting anti-zearalenone monoclonal antibodies were cloned. The subclasses of the monoclonal antibody were characterized as Ig G1 for three and Ig G2b for one. One of the cell strains 2C9 was used to prepare ascites for its high bioactivity. The titer of purified ascites was 1/40 000. The indirect-competitive ELISA was developed with good specificity for ZEN, the half maximal inhibitory concentration (IC50) is 1.90 ng/mL, the detection limit is 0.051 ng/mL, and the detection range is 0.115?13.9 ng/mL. The recovery rate could get to 96.5%?113% while the sample containing ZEN 1.46?93.8 μg/kg. The method for detection was de-veloped, which could be applied to detect ZEN from a variety of grain and feed samples.

Abstract:The aim of this study was to investigate Lactobacillus plantarum IMAU10014’s growth promotion effects on tomato, biocontrol efficacy on Botrytis cinerea caused disease and its induction effects on several defense-related enzymes. Seed-soaking with IMAU10014 was used for determining its growth promotion effects on tomato, and leaf spraying and root soaking inoculation were employed to determine its biocontrol effectiveness. Seed-soaking with IMAU10014 at concentrations of 107 CFU/mL for 6 to 8 h significantly increased seed germination rates and buds size. Seed germination rate was 100%. Treatment of tomato seedlings with L. plantarum IMAU10014 reduced B. cinerea disease index from 47.22 to 32.41 and 26.39 (Biocontrol effect 31.36% and 44.11%), respectively. Significant induction of plant defense-related enzymes, including phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) was observed in tomato plants treated with L. plantarum IMAU10014. L. plantarum IMAU10014 has good potential as a biological pesticide to control Botrytis blight.

Abstract:This study was to investigate the dominant spoilage bacteria (DSB) of shrimp and detect the quorum sensing mediated by N?acyl?homoserine lactones (AHLs). The strains of DSB were identified by 16S rRNA gene sequence. AHLs of DSB were detected with strain Chromobacterium violaceum CV026. The results indicated that two strains Aci-1 and Aci-2 belong to Acinetobacter spp.. Both bacterial strains can secret AHLs, and the biofilm formation of Aci-1 were significantly affected by exogenous AHLs. There is a positive relationship between the AHLs activity of DSB and the total plate count and TVB-N content of shrimp, and the correlation coefficient is 0.846 6 (P<0.05) and 0.986 7 (P<0.01), respectively. The DSB of shrimp have the quorum sensing system mediated by AHLs, which plays an important role in the spoilage of shrimp.

Abstract:The aim of the study was explore a set of new detective targets in pathogenic Aeromonas veronii which was more specific than others at present, to develop a new duplex PCR method which could effectively detection the pathogenic A. veronii. Comparison analysis of the 16S rRNA gene sequences of Aeromonas spp. was used to explore A. veronii specific targets, which were then evaluated and used to design specific primers, and select the conservative primers of aerolysin genes in Aeromonas spp. to explore pathogenic targets. PCR parameters were optimized, its reaction system was developed, and its sensitivity and specificity were checked. 12 Aeromonas stains and 10 non-Aeromonas stains were tested for pathogenic A. veronii by duplex PCR method. The duplex PCR can clearly identify A. veronii from Aeromonas species, and can identify aerolysin-producing stains of A. veronii. The detection limit of genomic DNA by the duplex PCR was 1.35×10?3 mg/L. A novel duplex PCR method was successfully developed in this study, which could effectively detect pathogenic A. veronii with high accuracy and sensitivity.

Abstract:A plate coloration method by auxotroph strain for screening high L-arginine production mutants quickly and efficiently was established. Coynebacterum crenatum SYPA5-5 which is an L-arginine producer was mutated by N+ ion implantation with an energy level of 10 keV. Combined with the directed screening method and fermentation in flasks, a mutant C. crenatum SYPA5-5-36 with a higher L-arginine production and stability was selected. The yield of L-arginine produced by this mutant could reach 35.85 g/L which was 19.5% higher than that of the original strain. Therefore, the plate coloration method by auxotroph strain could be applied to screening high L-arginine production mutants quickly and efficiently.

Abstract:The type Ⅲ secretion system (T3SS) effector is considered as one of the key virulence factors in Xanthomonas oryzae. X. oryzae pv. oryzae and X. oryzae pv. oryzicola cause bacterial leaf blight and bacterial leaf streak in rice, which are important bacterial diseases of rice. Based on bioinformatic analysis of the bacterial genome and other recent reports, X. oryzae contains at least 28 classes of T3SS effectors, divided into two groups: TAL (transcription activator-like) effectors and non-TAL (non tran-scription activator-like) effectors. This paper reviews the number, classes, structure and host targets of T3SS effectors in X. oryzae, which may provide a new insight into the mechanism of rice-X. oryzae in-teraction, regulatory network and molecular breeding of rice.

Abstract:Constructivism learning theory is increasingly recognized in higher education reform. Using this theory, we designed a new microbiological experiment teaching course including core and optional experi-ments, established a student-teacher interactive platform integrating the multimedia and network technologies, and propounded an evaluation system which is more comprehensive, objective and reli-able. The pilot demonstrated that this new paradigm can significantly arouse the participant interest and initiative, enhance the teaching efficiency and performance as well.

Abstract:Enterohemorrhagic Escherichia coli O157:H7 is a kind of important pathogenic bacterium, and deeper studies of molecular pathogenesis will contribute better to related vaccine research and disease control. Tandem affinity purification (TAP) technique is a kind of new method which was recently developed and used to separate and purify native protein complexes and so on to study interactions between proteins. In this study, a prokaryotic TAP expression vector constructed by our laboratory was successfully used to express fusion protein GroEL-TAP in Enterohemorrhagic Escherichia coli O157:H7. An efficient method for preparation of native protein complexes was developed and all experimental parameters of tandem affinity purification were optimized. The highly pure protein complexes com-posed of both tagged GroEL-TAP and natural GroEL were obtained with our expression and purification system. These results showed that our TAP system worked well to specifically purify protein complex participated with target protein in Enterohemorrhagic Escherichia coli O157:H7. This study provided satisfactory experimental base for follow-on works on identifying virulence protein participated com-plexes.

Abstract:To develop a rapid sensitive and specific method of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H6 subtype avian influenza virus (AIV), six primers specific to the eight site of HA gene were designed according to the sequences of H6 subtype AIV hemagglutinin gene available in GenBank. The viral RNA was amplified with one-step RT-LAMP method, and the reaction conditions were optimized. The results showed that the specificity of the assay was fine without cross amplification of other respiratory tract pathogens, and the detection limit of the RT-LAMP assay was 0.01 pg. The assay is a sensitive, simple, rapid and practical method for detection of H6 subtype AIV in the field.