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目的构建幽门螺杆菌(Hp)vacA基因片段原核表达载体并进行表达。方法采用PCR技术,扩增HpvacA基因的1个片段B,并克隆入pGEM-3Zf(-)质粒。测序正确后,再克隆入质粒pRSETA中,构建重组表达载体pRSE-TA-B。结果经过转化E.coliBL21DE3(plysS)后,诱导表达出相对分子质量(Mr)为33000的蛋白。SDS-PAGE分析显示,表达量占全菌总蛋白质的47.8%,上清中目的蛋白占全菌总蛋白的10.9%。ELISA和Westernblot分析表明,表达蛋白能与兔抗VacA多抗结合。结论成功地构建原核表达载体pRSETA-B,为进一步探讨该片段的生物学活性,以及临床上通过检测患者血清预测Hp的感染提供了实验依据。
Objective To construct the prokaryotic expression vector of vacA gene of Helicobacter pylori (Hp) and express it. Methods A fragment B of HpvacA gene was amplified by PCR and cloned into pGEM-3Zf (-) plasmid. After sequencing, it was cloned into plasmid pRSETA to construct recombinant expression vector pRSE-TA-B. Results After transformation of E.coli BL21DE3 (plysS), a protein with relative molecular mass (Mr) of 33000 was induced. SDS-PAGE analysis showed that the expression level accounted for 47.8% of the total bacterial total protein, and the target protein in the supernatant accounted for 10.9% of the total bacterial total protein. ELISA and Western blot analysis showed that the expressed protein could bind to rabbit anti-VacA polyclonal antibody. Conclusion The prokaryotic expression vector pRSETA-B was successfully constructed, which provided an experimental basis for further exploring the biological activity of this fragment and clinically detecting the infection of Hp by detecting the serum of patients.