[Cloning, expression and identification of flaB gene from a clinical isolate of Helicobacter pylori].
To clone Helicobacter pylori flagellin B gene (flaB) to construct prokaryotic expression system of the gene and to identify immunity of the fusion protein.



The flaB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted flaB gene was constructed. FlaB fusion protein was expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages. Western blot was applied to determine immunoreactivity and immunogenicity of the fusion protein and antibody against whole cell of H.pylori and rabbit antiserum immunized with the fusion protein, respectively.



In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned flaB gene was from 96.31% approximate, equals 97.73%, while the homology of its putative amino acid sequence was as high as 99.41% approximate, equals 100%. The expression output of FlaB fusion protein in pET32a-flaB-BL21DE3 system was approximately 40% of the total bacterial proteins. FlaB fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce specific antibody with high titer after the animal was immunized with the protein.



A prokaryotic expression system of H. pylori flaB gene with high efficiency has been established successfully. The expressed FlaB fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine and detect kit.
Liang
Shao-Hui
SH
Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China.
Mao
Ya-Fei
YF
Yan
Jie
J
D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005408:Flagellin / Q000235:genetics* / Q000276:immunology; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D005408:Flagellin; C416532:flaB flagellin