结核分枝杆菌ClpP1重组蛋白的特性探讨及初步应用  

The characteristics and preliminary application of ClpP1 recombinant protein of Mycobacterium tuberculosis

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

机构地区：[1]重庆医科大学病原生物学教研室分子医学与肿瘤研究中心,400016 [2]重庆医科大学附属第一医院呼吸内科,400016

出　　处：《免疫学杂志》2014年第4期306-309,313,共5页Immunological Journal

基　　金：国家自然科学基金青年基金项目(81101216)

摘　　要：目的用纯化结核分枝杆菌(Mycobacterium tuberculosis,M.tb)ClpP1重组蛋白免疫新西兰白兔制备ClpP1多克隆抗体并检测其效价,间接ELISA法分析重组蛋白的抗原性,以初步评价其应用价值。方法通过SDS-PAGE确定ClpP1重组蛋白的主要表达形式,利用纯化包涵体方式对重组蛋白进行纯化;将纯化蛋白免疫新西兰白兔制备ClpP1多克隆抗体并检测其效价;再分别以纯化蛋白为抗原,制备抗体为一抗,间接ELISA法检测临床诊断结核病患者血清中抗M.tb抗体和M.tb抗原。结果 SDSPAGE显示ClpP1重组蛋白主要以包涵体表达形式存在,将纯化蛋白免疫新西兰白兔4次,取血测抗体效价为1∶64 000;Western blot结果显示制备抗体可较好的与ClpP1蛋白特异性结合;间接ELISA法结果表明,以重组蛋白为抗原测患者血清中抗M.tb抗体和以制备抗体为一抗测血清中的M.tb抗原的2实验组分别与对照组相比均具有显著性差异。结论制备了具有免疫原性的M.tb ClpP1重组蛋白及效价较高的多克隆抗体,并得出ClpP1多克隆抗体具有较好的免疫反应性,为进一步研究ClpP1重组蛋白的特性及其应用奠定了基础。The genome of Mycobacterium tuberculosis （M. tb）, the main causative agent for tuberculosis, encodes proteases including proteasome and Clp proteases. In this study, we aimed to prepare Clp proteases 1 （ClpP1） polyclonal antibody, analyze its antigenicity by indirect ELISA, and explore its preliminary application. Firstly, ClpP1 protein was purified and used to immunize rabbits for 4 times for preparing ClpP1 polyclonal antibody, and titer of the antibody was detected as 1：64 000. Western blot test showed that the polyclonal antibody could specifically bind with ClpP1 recombinant protein and ClpP1 protein of acilli Calmette-Guerin （BCG）. Indirect ELISA revealed the ClpP1 recombinant protein high immunogenicity and could strongly react with the prepared ClpP1 polyclonal antibody in sera of tuberculosis. All the result indicated that immunogenic ClpP1 recombinant protein and polyclonal antibody with high titer are successfully prepared, which would lay a foundation for further study on the characteristics and application of ClpP1 recombinant protein.

关 键 词：结核分枝杆菌 ClpP1重组蛋白 多克隆抗体 

分 类 号：R378.911[医药卫生—病原生物学]