结核分枝杆菌ClpC2蛋白的纯化、抗血清的制备及抗原性分析  

Purification,antiserum preparation and antigenic characters of ClpC2 in Mycobacterium tuberculosis

作　　者：穆柳青[1] 李岱容[2] 张春燕[1] 樊宇[1] 何永林[1] 杨春[1] 

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学附属第一医院呼吸科,重庆400016

出　　处：《中国免疫学杂志》2014年第6期784-788,796,共6页Chinese Journal of Immunology

基　　金：国家青年科学基金资助项目(No.81101216)

摘　　要：目的:分别探索结核分枝杆菌(Mycobacterium tuberculosis,MTB)ClpC2蛋白和兔抗ClpC2多克隆抗体作为肺结核检测抗原或抗体的可行性。方法:诱导表达重组蛋白ClpC2(rClpC2);SDS-PAGE与Western blot鉴定rClpC2后利用亲和层析法进行纯化;纯化后的重组蛋白免疫新西兰大白兔,Western blot检测兔抗血清的特异性;双向免疫扩散法与酶联免疫吸附法(ELISA)测定兔抗血清效价;rClpC2通过间接ELISA进行抗原性的初步检测,兔抗ClpC2多克隆抗体通过间接ELISA检测肺结核病人血清中ClpC2抗原。结果:rClpC2经SDS-PAGE分析,在相对分子质量46 kD处可见特异性条带;Western blot分析rClpC2可与MTB H37Rv株感染的小鼠血清发生抗原抗体反应;经Bandscan分析rClpC2约占菌体总蛋白的58.7%,纯化后rClpC2的纯度为88.5%;Western blot验证兔抗血清可与卡介苗(Bacillus calmette-guerin,BCG)的ClpC2蛋白发生抗原抗体反应;双向免疫扩散法测定兔抗血清效价为1∶32,ELISA法测定兔抗血清效价为1∶320 000;rClpC2在检测肺结核病人中的检出率为46%,检测敏感性为46%,特异性为90%;兔抗ClpC2多克隆抗体在检测肺结核病人中的检出率为40%,检测的敏感性为40%,特异性为90%。结论:成功纯化结核分枝杆菌ClpC2蛋白,制备特异性兔抗ClpC2多克隆抗体,为进一步研究ClpC2蛋白的生物功能、ClpC2作为诊断靶标、药靶候选蛋白的可行性及兔抗ClpC2多克隆抗体生物学功能提供实验基础。Objective：To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods：rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay （ELISA）.The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results：SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion： Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.

关 键 词：结核分枝杆菌 rClpC2 兔抗血清 

分 类 号：R378.91[医药卫生—病原生物学]