摘要
AIM: To clone flagellin genes A (flaA) and B (flaB) from a clinical strain of Helicobacter pylori (H pylori) and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins.METHODS: The flaA and flaBgenes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR. The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning. The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed, respectively. The expressions of FlaA and FlaB fusion proteins in E. Coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG)at different concentrations were examined by SDS-PAGE.Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA (rFlaA) or FlaB (rFlaB)recombinant proteins were applied to the determination of the fusion proteins immunity. ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori, respectively.RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28-97.13% and 96.31-97.73%, and their putative amino acid sequence homologies were 99.61-99.80% and 99.41-100% for the two genes, respectively. The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaBBL21DE3 systems was as high as 40-50% of the total bacterial proteins. Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins. Ninety-eight and zero point 4 and 92.80% of the serum samples from 125 patients infected with H pylori were positive for rFlaA and rFlaB antibodies, respectively.One hundred percent and 98.98% of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes,respectively.CONCLUSION: Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established. The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity. High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine.
AIM:To clone flagellin genes A(flaA)and B(flaB)from a clinical strain of Helicobacter pylori(H pylori)and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins. METHODS:The flaA and flaB genes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR.The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning.The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed,respectively.The expressions of FlaA and FlaB fusion proteins in E.coli BL21DE3 induced by isopropylthio-β-D-galactoside(IPTG) at different concentrations were examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA(rFlaA)or FlaB(rFlaB) recombinant proteins were applied to the determination of the fusion proteins immunity.ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori,respectively. RESULTS:In comparison with the reported corresponding sequences,the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28-97.13 % and 96.31-97.73 %,and their putative amino acid sequence homologies were 99.61-99.80 % and 99.41-100 % for the two genes,respectively.The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaB- BL21DE3 systems was as high as 40-50 % of the total bacterial proteins.Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins.Ninety-eight and zero point 4 and 92.80 % of the serum samples from 125 patients infected with H pylori were positive for rFlaA and rFlaB antibodies,respectively. One hundred percent and 98.98 % of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes, respectively. CONCLUSION:Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established.The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity.High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine.
基金
the Excellent Young Teacher Fund of Chinese Education Ministry and the General Research Plan of the Science and Technology Department of Zhejiang Province,No.001110438
