炭疽芽胞杆菌致死因子在大肠埃希菌中的表达与纯化  

Expression and purification of lethal factor of Bacillus anthracis in Escherichia coli

作　　者：魏建春[1] 张恩民[1] 张慧娟[1] 张建华[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206

出　　处：《中国媒介生物学及控制杂志》2012年第4期295-297,共3页Chinese Journal of Vector Biology and Control

摘　　要：目的在大肠埃希菌中表达炭疽致死因子,获得纯化蛋白,用于炭疽快速诊断方法以及炭疽致病机制研究。方法 PCR方法扩增炭疽致死因子,构建表达质粒pETLF,转化大肠埃希菌BL21(DE3),IPTG诱导重组蛋白表达,通过Ni离子金属螯合树脂进行纯化,Western blot(WB)试验检查免疫原性,MTT方法检查生物学活性。结果获得重组炭疽致死因子,WB结果显示重组蛋白具有良好的免疫反应性,MTT方法进行的生物学活性测定表明致死因子与保护性抗原联合,细胞毒性与文献报道相比有明显降低。结论所获得的重组炭疽致死因子,对于发展炭疽快速诊断方法,以及研究炭疽毒素的作用机制具有一定的促进作用。Objective To acquire the purified recombinant lethal factor （rLF） of Bacillus anthracis and investigate its use in the rapid diagnosis of anthrax and in the study of the mechanism of the disease. Methods The lethal factor gene was cloned into the Escherichia coli expression vector pET-30a（＋） and expressed as a fusion protein in E. coli BL21（DE3）. rLF was purification of rLF by Ni-NTA affinity chromatography. The purified rLF was subjected to immunogenicity and biological activity analysis by Western blot assay and MTT assay. Results rLF was expressed and purified. Western blot results suggested that recombinant proteins reacted specifically with anti-B. anthracis serum. The cytotoxicity analysis showed the rLF was fully functional. And rLF combined with PA could significantly decrease cytotoxicity compared with previous study. Conclusion rLF protein has tremendous potential both for the diagnosis of B. anthracis infection and for the study of the mechanism of anthrax toxin.

关 键 词：炭疽芽胞杆菌 致死因子 克隆表达 

分 类 号：R378.72[医药卫生—病原生物学]