艰难梭菌毒素C蛋白的克隆表达和抗体制备  

Cloning and expression of toxin C gene of Clostridium difficile and its polyclonal antibody preparation

作　　者：王建霞[1,2] 王宏伟[1] 杨锡琴[2] 黄忱[3] 罗芸[3] 金大智[3] 冯晓燕[2] 张贺秋[2] 

机构地区：[1]河北大学生命科学学院,河北保定071000 [2]军事医学科学院基础医学研究所,北京100850 [3]浙江省疾病预防控制中心,浙江杭州310051

出　　处：《中国药理学与毒理学杂志》2017年第7期760-765,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81471998);国家卫生和计划生育委员会科学研究基金-浙江省医药卫生重大科技计划(WKJ-ZJ-1507);河北大学研究生创新项目(X2016072)~~

摘　　要：目的克隆艰难梭菌毒素C(tcdC)基因并构建TcdC蛋白的原核表达载体,制备多克隆抗体。方法从艰难梭菌标准株(ATCC43255)基因组DNA中扩增获得tcdC基因的部分片段,连接到原核表达载体并转化到大肠杆菌中,诱导表达GST-TcdC/L和IL1-TcdC/L融合蛋白,前者用作免疫抗原,后者用作检测抗原。采用SDS-PAGE法鉴定融合蛋白的表达,并通过Ni柱纯化IL1-TcdC/L融合蛋白,Q柱纯化GST-TcdC/L融合蛋白。将纯化的GST-TcdC/L融合蛋白于兔背部皮内多点注射,每只1 mg,4周1次,共3次。最后1次免疫后2周,采血分离血清。用IL1-TcdC/L蛋白包被酶联板,采用ELISA法检测多抗血清效价;Western蛋白印迹法检测多抗特异性,结果 SDS-PAGE结果表明,所表达的2个融合蛋白与预期分子质量一致。ELISA结果显示,制备的TcdC多克隆抗体效价>1:6.4×10~4。Western蛋白印迹实验显示,制备的TcdC/L多克隆抗体能特异性识别艰难梭菌中的TcdC蛋白。结论成功克隆了艰难梭菌tcdC/L基因并进行了原核表达,成功制备了其多克隆抗体,为后续研究tcdC基因在艰难梭菌致病过程中的作用及机制奠定了基础。OBJECTIVE To clone and express toxin C（tcdC） gene pathogenicity locus of Clostridium difficile（CD）, and prepare the polyclonal antibody against TcdC protein. METHODS The tcdC gene was amplified from CD（ATCC43255） strain genome DNA and inserted into prokaryotic expression vectors.The correct recombinant plasmids were transformed into E.coli which was induced to express the GSTTcdC/L and IL1-TcdC/L fusion protein. Meanwhile, the fusion proteins were respectively purified through Ni-NTA agarose affinity chromatography and Q-Sepharose Fast Flow. The purified GST-TcdC/L fusion protein was used as an antigen to inoculate rabbits to produce antiserum. Two weeks after the final immunization, the rabbits were sacrificed and serum was collected. The titer of the serum was determined by ELISA and the reactivity of the polyclonal antibody was identified by Western blotting. RESULTS The SDS-PAGE result showed that GST-TcdC/L and IL1-TcdC/L fusion proteins were expressed at the size of 45 ku and 39 ku. The titer of TcdC/L polyclonal antibody was over 1：6.4×10^4. Western blotting detection demonstrated that the TcdC/L polyclonal antibody recognized the TcdC protein in CD（ATCC43255）.CONCLUSION The tcdClL gene is cloned and the polyclonal antibody against TcdC/L is prepared,which will contribute to studing the function and mechanism of tcdC gene in pathogenicity locus.

关 键 词：艰难梭菌 毒素C蛋白 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]