Objective:To get norovirus (NoV) GII.6 P protein through prokaryotic expression and prepare the polyclonal antibody.Methods:NoV GII.6 P region gene was amplified and cloned into prokaryotic expression vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein GII.6 P was expressed by induced Isopropyl β-D-Thiogalactoside (IPTG) and then purified with Ni-NTA Affinity Column. The binding ability of recombinant GII.6 P was determined by oligosaccharide binding assay and the polyclonal antibody serum was prepared by immunizing BALB/c mice. The titer of GII.6 P polyclonal antibody was determined by ELISA, and the specificity of the antibody was detected by western blot (WB). The effectiveness of GII.6 P polyclonal antibody was assessed. Results:The recombinant GII.6P-pET28a plasmid was constructed successfully and the recombinant GII.6 P protein was expressed with relative molecular mass of 40 ×10 3. The purity of GII.6P protein was more than 90% after purification. The oligosaccharide binding showed that the GII.6 P protein binds to B, le b and H2, but does not bind to A, H1, and le a type; the titer of GII.6 P polyclonal antibody was 1∶160 000. WB indicated that the antibody had high specificity and the cross experiments did not show affinity to GII.4. Conclusions:The GII.6P protein has been expressed successfully and the GII.6 P polyclonal antibody with high titer was prepared, which provides an effective tool for detection and vaccine development for NoV GII.6.

Chloramphenicol-resistant gene was cloned and analyzed by constructing genomic DNA library of Serratia marcescens KMR-3. It showed that cloned chloramphenicol-resistant gene encoded a protein product of 397 amino acids. The protein belonged to PRK10473 protein, and it showed 92% similarity to drug resistance transporter, Bcr/CflA subfamily of Serratia proteamaculans 568. Regulation elements including promoter, terminator, Shine-Dalgarno (SD) sequence and transcription start site also were identified.
Amino Acid Sequence ; Base Sequence ; Chloramphenicol Resistance ; genetics ; Cloning, Molecular ; Molecular Sequence Data ; Serratia marcescens ; classification ; genetics

Based on the constructed promoter probe vectors that could replicate both in E. coli and in a cold-adapted bacterium, several candidate promoters were isolated and their activities were evaluated by RT-PCR. The transcription initiation sites and core sequence of promoters were determined by primer extension analysis. A low-temperature expression vector was constructed by using the strongest promoter and a thermolabile alpha-amylase gene was successfully overproduced under control of this promoter at low temperature (7 degrees C), while the secreted alpha-amylase amounted up to 35% of the total extracellular proteins. The expression system is expected to be useful for the production of thermolabile exogenous proteins at low temperatures.
Acinetobacter ; genetics ; metabolism ; Adaptation, Physiological ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Base Sequence ; Cold Temperature ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Transformation, Genetic ; alpha-Amylases ; biosynthesis ; genetics

To assess the variability of adhesin gene hpaA in differene H pyloristrains with PCR restriction fragment length polymorphism (RFLP) A 710 bp gene hpaA , obtained from 9 different H pylori strains, were digested by Hha Ⅰand Hae Ⅲ individually and analyzed by agarose gel electrophoresis Four different polymorphic types were found in hpaA digested with Hae III and five types with Hha I Clinical isolates of H pylori from Chongqing showed difference among them and remarkably distinguished from foreign standard strains Mongolia gerbil adapted H pylori strain,which were obtained from Mongolia gerbil infected with clinical isolate, also showed inconsistence in hpaA RFLP The hpaA gene from different H pylori strains revealed 1 variability, and this might provide an effective method for developing molecular epidemiology of H pylori