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目的构建幽门螺杆菌(Helicobacter pylori)IV型分泌系统cagV(hp0530)基因的原核表达系统,表达并纯化CagV蛋白,初步分析其结构和抗原性,为进一步研究幽门螺杆菌的致病机制和治疗药物的筛选奠定基础。方法以H.pylori ATCC700392株基因组为模板,采用聚合酶链反应(PCR)扩增获得目的片段,将其插入表达载体pET28a后转化大肠杆菌BL21(DE3),表达纯化后利用蛋白质印迹(Western blot)分析CagV蛋白的抗原性。结果双酶切鉴定结果证实cagV基因重组表达载体构建成功;重组表达蛋白经飞行质谱鉴定为幽门螺杆菌CagV蛋白;Western blot检测结果显示CagV蛋白具有特异抗原性。结论所克隆表达的CagV蛋白具有较好的抗原性,对后续的的H.pylori致病性和检测、分析研究有比较重要的意义。
Objective To construct prokaryotic expression system of cagV (hp0530) gene of Helicobacter pylori type IV secretion system, express and purify CagV protein, and preliminary analyze its structure and antigenicity. To further study the pathogenesis of H. pylori and its therapeutic drug The basis of the screening. Methods The target fragment was amplified by polymerase chain reaction (PCR) using the genome of H. pylori ATCC700392 as a template and inserted into the expression vector pET28a. The recombinant plasmid was transformed into E. coli BL21 (DE3), expressed and purified by Western blotting, Analysis of CagV protein antigenicity. Results The results of double enzyme digestion confirmed that cagV gene was successfully constructed. The recombinant protein was identified as CagV protein of Helicobacter pylori by fly mass spectrometry. Western blot results showed that CagV protein has specific antigenicity. Conclusion The cloned CagV protein has good antigenicity, and is of great significance to the follow-up pathogenicity, detection and analysis of H.pylori.