人幽门螺杆菌热休克蛋白A编码基因的克隆、表达及抗原性研究

Cloning, expression and antigenic analysis of heat shock protein A gene of human Helicobacter pylori

目的:构建含人幽门螺杆菌(Hpylori)热休克蛋白A编码基因的重组载体、进行核甘酸序列分析,并在E.coli BL21中表达,研究其抗原性,为疫苗的开发奠定基础方法:利用分子克隆技术从Hpylori DNA染色体中,扩增热休克蛋白A编码基因片段;将目的基因与载体pET32a(+)同时经kpn Ⅰ、BamH Ⅰ双酶切、纯化、连接后,转化含有目的基出的重组载体;以含目的基因片段的重组载体转化大肠杆菌BL21(DE30)并表达;表达产物经纯化后,用Western blot法检测其抗原性结果:经酶切、测序分析表明,插入的基出片段为Hpylori热休克蛋白A编码基因,与GenBank报道的相比较,有1.6％的碱基(bp)发生变异,1.6％的氨基酸残基改变.经SDS-PAGE分析发现,融合基因表达的蛋白M_r为33×10~3。其中pET32a(+)表达的蛋白Mr约为20×10~3,可溶性表达产物占全菌总蛋白的18.96％,重组蛋白经Ni^+-NTA琼脂糖树脂纯化后,其纯度达95％以上.用Westernblot方法检测显示,该重组蛋白可被Hpylori阳性患者的血清所识别,具有良好的抗原性。结论:成功地克隆并表达了Hpylori热休克蛋白A码基因,为Hpylori蛋白质疫苗的研制和快速诊断试剂盒的研究奠定了良好的基础。

AIM: To construct a recombinant vector containing gene encoding heat shock protein A with a Mr of 13 000 from human Helicobacter pylori (H pylori)and express it in E. coli BL21, and to explore the antigenicity. METHODS: The targot gene was amplified from H pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) digested by restrictive en- donuclease enzymes of kpn I, BamH I simultaneously. The recombinant vector was transformed and expressed in E. coli BL21. The antigenicity of recombinant fusion protein was analysed by Western blot. RESULTS: Enzyme digestion and sequencing analysis showed that the target gene has been inserted into the recombinant vector, but as compared with the gene reported by GenBenk, 1.6% of gene mutation and 1.6% of amino acid residues change in H pylori occurred, respectively. SDS-PAGE analysis showed that the recombinant vector could be expressed in E. Coli BL21, the relative molecular mass (Mr) of expressed product was 33×10~3, while M_r of protein expressed by pET32a (+) was about 20×10~3, and soluble fusion expression product accounted for 18.96% of total bacterial protein. After purification with Ni^+-NTA agarose resin, the purity of recombinant fusion protein was about 95%. Western blot result showed that recombinant fusion protein could be recognized by anti-H pylori positive serum, suggesting that the protein had good antigenicity. CONCLUSION: the gene encoding H pylori heat for protein A has been cloned and expressed successfully. The results lay the foundation for development of H pylori protein vaccine and a quick diagnostic kit for detection of H pylori infection,