【摘要】 目的克隆表达问号钩端螺旋体毒力基因mviN并观察表达产物对血管内皮细胞ECV304及肺上皮细胞A549的毒性作用。方法将mviN基因插入原核表达载体pET32a(+),构建重组质粒pET-mviN,转化大肠杆菌BL21(DH3)以高效表达携带组氨酸标签的Trx-MviN融合蛋白,并作亲和层析纯化。以XTT法检测毒力蛋白Trx-MviN对ECV304及A549细胞增殖的抑制作用,同时以流式细胞术检测其作用于ECV304及A549后的细胞凋亡率。结果成功构建了重组质粒pET-mviN并高效表达出Trx-MviN融合蛋白;毒力蛋白Trx-MviN作用于ECV304及A549后,与对照组相比较,其细胞增殖被显著抑制(P<0.05),而细胞凋亡率显著增加(P<0.01)。结论重组质粒pET-mviN高效表达出Trx-MviN融合蛋白,纯化后的融合蛋白Trx-MviN对ECV304及A549具有细胞毒性作用。更多还原

【Abstract】 To clone and express the virulence gene mviN of Leptospira interrogans serovar Lai strain 017 in E.coli.BL21(DH3) and to investigate the cytotoxicity of the expressed product on ECV304 and A549 cells.The recombinant plasmid pET-mviN was constructed by inserting gene mviN into prokaryotic expression sector pET32a(+),then it was transformed into E.coli.BL21(DH3) to express fusion protein Trx-MviN which had a His-tag after induction by 1 mmol/L IPTG.The fusion protein was purified by using HisTrap affinity columns.The XTT method was applied to assay the inhibitory effects of the virulent protein Trx-MviN on ECV304 and A549 cell proliferation and flow cytometry(FCM) was applied to measure the apoptotic rate of ECV304 and A549 cells after incubation with the virulent protein Trx-MviN.The recombinant plasmid pET-mviN was successfully constructed and it highly expressed the fussion protein Trx-MviN.The virulent protein Trx-MviN could significantly inhibit cell proliferation(P<0.05) and promote apoptosis of ECV304 and A549 cells(P<0.01),compared with the control group.It is demonstrated that the recombinant plasmid pET-mviN highly expressed the fussion protein TrxMviN and the purified protein Trx-MviN has cytotoxicity on ECV304 and A549 cells.更多还原