Construction of hpaA gene from a clinical isolate of Helicobacter pyloriand identification of fusion protein  

作　　者：Ya-Fei Mao Jie Yan Li-Wei Li Shu-Ping Li Department of Medical Microbiology and Parasitology,College of Medical Sciences,Zhejiang University,Hangzhou 310031,Zhejiang Province,China 

出　　处：《World Journal of Gastroenterology》2003年第7期1529-1536,共8页世界胃肠病学杂志（英文版）

基　　金：the Excellent Young Teacher Fund of Chinese Education Ministry;the General Research Plan of Science and Technology Department of Zhejiang Province,No.001110438

摘　　要：AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.METHODS: The hpaA 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 recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E. coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE.Western blot with commercial antibody against whole cell of H. pylorias well as immunodiffusion assay with selfprepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H. pylori and to examine HpaA expression of 109 clinical isolates of H. pylori.RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32ahpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H. pyloriand to induce rabbit producing specific antiserum with 1:4immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125patients infected with H.pylori(102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori(109/109) were detectable for HpaA.CONCLUSION: A prokaryotic expression system with high efficiency of H.pylorihpaA gene was successfully established.The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H. pyloriclinicalstrains and specific antibody production in H. pyloriinfecAIM:To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein. METHODS:The hpaA gene from a clinical isolate Y06 of H. pyloriwas amplified by high fidelity PCR.The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning.The recombinant expression vector inserted with hpaA gene was constructed.The expression of HpaA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H.pylori as well as immunodiffusion assay with self- prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein.ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H.pyloriand to examine HpaA expression of 109 clinical isolates of H.pylori. RESULTS:In comparison with the reported corresponding sequences,the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25- 97.32% and 95.38-98.46%,respectively.The output of HpaA fusion protein in its expression system of pET32a- hpaA-BL21DE3 was approximately 40% of the total bacterial proteins.HpaA fusion protein was able to combine with the commercial antibody against whole cell of H.pyloriand to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein.81.6% of the serum samples from 125 patients infected with H.pylori(102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori(109/ 109) were detectable for HpaA. CONCLUSION:A prokaryotic expression system with high efficiency of H.pylorihpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity.High frequencies of HpaA expression in different H.pylori clinical strains and specific antibody production in H.pylori infected patie

关 键 词：HPAA基因 幽门螺旋杆菌 融合蛋白 聚合酶链反应 

分 类 号：R573[医药卫生—消化系统]