问号钩端螺旋体毒力基因mviN的表达及细胞毒性作用研究  

Expression of the virulence gene mviN of Leptospira interrogans serovar Lai and its cytotoxic effect

作　　者：王中平[1] 鲍朗[1] 张会东[1] 商正玲[1] 钟琪[1] 郝牧[1] 朱海龙[1] 

机构地区：[1]四川大学华西基础医学与法医学院感染免疫研究室,成都610041

出　　处：《中国人兽共患病学报》2007年第1期23-26,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(30070670)

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。To clone and express the virulence gene mviN of Leptospira interrogans serovar Lai strain 017 in E. coll. BL21（DH3） and to investigate the cytotoxicity of the expressed product on ECV304 and A549 cells. The recombinant plasmid pET-raviN was constructed by inserting gene mv/N into prokaryotic expression sector pET32a（＋ ）, then it was transformed into E. coll. BL21（DH3） to express fusion protein Trx-MviN which had a His-tag after inductionoby 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-raviN 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 apoptnsis of ECV304 and A549 cells（P〈0. 01） ,compared with the control group. It is demonstrated that the recombinant plasmid pET-raviN highly expressed the fussion protein Trx-MviN and the purified protein Trx-MviN has cytotoxicity on ECV304 and A549 cells.

关 键 词：mviN 钩端螺旋体 毒力 XTT法 细胞凋亡率 

分 类 号：R377.5[医药卫生—病原生物学]