幽门螺杆菌分泌与重组表达的VacA蛋白的分离纯化及其致细胞空泡效应与凋亡的对比研究  

Isolation and purification of recombinant VacA and Helicobacter pylori-secreted VacA and VacA-induced cell vacuolar change and apoptosis

作　　者：常辉[1] 左钱飞[2] 敬海明[2] 邹全明[2] 兰春慧[1] 陈东风[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所消化内科,重庆400042 [2]第三军医大学药学院微生物与生化药学教研室,重庆400038

出　　处：《军事医学》2014年第9期714-718,744,共6页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81171526);重庆市自然科学基金资助项目(CSTC 2011 jjA10061)

摘　　要：目的分离纯化幽门螺杆菌分泌和重组表达的细胞空泡毒素抗原(VacA)蛋白,并评价其致细胞空泡效应及致细胞凋亡效应。方法分别从幽门螺杆菌ATCC26695菌株培养上清和重组表达VacA蛋白的pQE30-VacAE.coliM15基因工程菌中分离纯化VacA蛋白,经酸化后,以不同终浓度(5,10 ng/ml)分别与人胃腺癌AGS细胞共孵24 h,观察致空泡效应,并通过流式细胞术检测细胞凋亡。结果成功分离纯化出幽门螺杆菌分泌和重组表达的VacA蛋白;幽门螺杆菌分泌的VacA蛋白能显著引起AGS细胞的空泡样改变及凋亡(P<0.01),而重组表达的VacA蛋白致细胞空泡样改变及凋亡不显著(P>0.05)。结论幽门螺杆菌分泌的VacA蛋白有良好的空泡毒性及致凋亡效应,而重组表达的VacA蛋白无致空泡及凋亡效应,幽门螺杆菌分泌的VacA蛋白可用于VacA作用机制的研究。Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA,and to investigate the effect of VacA-induced cell vacuolar change and apoptosis. Methods VacA proteins were separated and purified from the culture supernatant of H. pylori（ ATCC26695） or from the split products of genetically engineered bacteria（ pQE30-VacA-E. coli M15） expressing recombinant VacA. The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng /ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay,respectively. Results H. pylori-secreted VacA and recombinant VacA were successfully separated and purified. The H. pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells（ P 〈0. 01） while the recombinant VacA did not. Conclusion H. pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis,but recombinant VacA can not,suggesting that the purified VacA protein secreted by H. pylori can be used to explore VacA-induced pathogenesis.

关 键 词：螺杆菌 幽门 细胞空泡毒素 蛋白纯化 细胞凋亡 胃肿瘤 

分 类 号：R735.2[医药卫生—肿瘤]