幽门螺杆菌临床菌株hpaA基因的克隆和表达及鉴定

目的：克隆幽门螺杆菌（Hp）hpaA基因，构建hpaA基因表达载体，鉴定其表达的融合蛋白免疫性。方法：采用高保真PCR从幽门螺杆菌临床分离菌株Y06中扩增hpaA基因，T-A克隆后测定核苷酸序列，构建pET32a的hpaA表达载体，在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导表达，采用Hp全菌抗体的Western blot和兔抗融合蛋白血清的免疫扩散试验鉴定其免疫性。结果：所克隆的hpaA基因与报道的相应核苷酸序列同源性为94.25％-97.32％，氨基酸序列同源性高达95.38％-98.46％。pET32a-hpaA-BL21DE3系统的HpaA融合蛋白表达量高达细菌总蛋白的40％左右。HpaA融合蛋白能与Hp全菌抗体发生结合反应，免疫家兔能获得高效价抗体。结论：本研究成功地构建了Hp hpaA高效表达系统，所表达的HpaA融合蛋白有良好的免疫原性和免疫反应性，可作为Hp疫苗的抗原。

Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori

Objective: To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity 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 expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25%~97.32%, while the homology of its putative amino acid sequence was as high as 95.38%~98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein. Conclusion: An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.