结核分枝杆菌Rv2461c基因的克隆、表达、纯化及其抗原性的初步检测  

The Cloning,Expression,Purification and Antigenicity of Rv2461c of Mycobacterium tuberculosis

作　　者：王庆忠[1] 张鹭[2] 马国举[3] 廖海印[3] 王洪海[2] 

机构地区：[1]上海市临床检验中心,上海200126 [2]复旦大学生命科学学院遗传学研究所,遗传工程国家重点实验室,上海200433 [3]齐齐哈尔大学生命科学与工程学院,齐齐哈尔161000

出　　处：《现代生物医学进展》2008年第8期1445-1448,共4页Progress in Modern Biomedicine

基　　金：国家973国家基础研究计划(2005CB523102);中国博士后科学基金(20070410688)

摘　　要：目的:探索结核分枝杆菌Rv2461c基因编码的clpP蛋白作为结核检测抗原的可行性。方法:克隆结核分枝杆菌clpP蛋白的编码基因Rv2461c,构建pET30a-Rv2461c重组质粒,并将其成功的转化到大肠杆菌JF1125克隆载体中,测序鉴定结果正确。将pET30a-Rv2461 c重组质粒转化到大肠杆菌BL21中,表达纯化目的蛋白进行质谱分析.制备clpP蛋白的多克隆抗体,通过亚细胞分离及Western检测分析蛋白的亚细胞定位,纯化的clpP蛋白通过间接ELISA实验进行抗原性的初步检测。结果:结核分枝杆菌clpP蛋白大量存在于细胞质中,少量存在于细胞壁和细胞膜中。重组抗原在检测肺结核病人中的检出率为38.3%(23/60),检测敏感性为38.3%,特异性为90%。结论:为后续深入研究clpP蛋白的生物功能、clpP作为诊断靶标、药靶候选蛋白的可行性提供了基础。Objective：To investigate the antigenicity of clpP coded by Rv2461c.Methods：Rv2461c gene which encodes clpP protein of M.tuberculosis was cloned and transformed into E.coli JF 1125 to construct a recombinant plasmid pET30a-Rv2461 c successfully. The gene was induced to express in E.coli BL21 and the purified recombinant target protein was tested by mass spectrometry.The polyclonal antibodies of clpP were prepared to detect the subcellular localization by Western-blotting.The antigenicity of the antigen was detected by indirect enzyme-linked immunosorbent assay（ELISA）.Results：The antigens were expressed successfully in E.coli BL21. clpP was mainly found in cytoplasm,with the trace quantity in the membrane and cell wall.ELISA results showed that clinical serum positive rate of recombinant protein antigen was 38.3%（23/60）in TB patients.The specificity of this protein was 38.3%,and the sensitivity was 90%.Conclusions：These results will provide basements for further study on the biological functions of Rv2461 c and its candidate potentiality as serological diagnosis and drug-target.

关 键 词：结核分枝杆菌 clpP蛋白 Rv2461c 蛋白表达 亚细胞定位 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]