热休克蛋白16.3多克隆抗体的制备  

Preparation of polyclonal antibody for heat shock protein 16. 3

作　　者：高绍莹 秦欢[1] 徐林[1] 秦娜琳[1] 于伟娜[1] 丁陈波 袁建波[2] 宋丹 罗军敏[1] 

机构地区：[1]遵义医学院免疫学教研室、贵州省免疫学研究生教育创新基地 [2]遵义医学院医学检验系2011级 [3]遵义医学院护理系2011级,贵州遵义563003

出　　处：《细胞与分子免疫学杂志》2014年第8期848-851,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：贵州省科学技术基金(黔科合J字[2008]2188);贵州省优秀科技教育人才省长基金(黔科教办[2010]04号)

摘　　要：目的大肠杆菌表达Hsp16.3,制备其多克隆抗体。方法设计引物,从H37Rv基因组中扩增出目的片段Hsp16.3,pET28a-Hsp16.3质粒的构建、克隆和表达。对His-Hsp16.3进行诱导、表达和纯化。用纯化好的蛋白免疫新西兰大白兔获得多克隆抗体,用ELISA检测抗血清中多抗的效价,用Western blot法检测多克隆抗体的生物活性。结果重组质粒pET28aHsp16.3在E.coli BL21(DE3)中成功表达,SDS-PAGE结果显示在His-Hsp16.3的相对分子质量(Mr)约为20 000。制备的多克隆抗体效价为1∶800,且特异性较好。结论成功的克隆、表达、纯化出His-Hsp16.3,并且成功制备了抗Hsp16.3多克隆抗体。Objective To induce the prokaryotic expression of His-heat shock protein 16.3 （Hsp16.3） and prepare its polyclonal antibody. Methods DNA primers were designed and synthesized, and Hsp16. 3 cDNA was amplified from Mycobacterium tuberculosis H37Rv by PCR. Thereafter, the recombinant vector pET28a-Hsp16.3 was constructed, cloned and induced to express. The HisHsp16.3 protein was purified using a Ni-NTA column. After identification by SDS-PAGE, the purified protein was used to immunize the New Zealand white rabbits to prepare the polyclonal antibody. The titer and biological activity of the polyclonal antibody were analyzed by ELISA and Western blotting, respectively. Results The prokaryotic expression vector pET28a-Hsp16.3 was constructed and expressed well in E. coil BL21 （ DE3 ）. SDS-PAGE showed that the relative molecular mass was about 20 000. The titer of the polyclonal antibody reached 1：800. The purified antibody was proved to have a good specificity. Conclusion The His-Hsp16.3 has been cloned, expressed and purified successfully. Polyclonal antibody of His-Hsp16.3 protein has been obtained as well.

关 键 词：抗体 基因表达 结核分枝杆菌 

分 类 号：R392.11[医药卫生—免疫学] R392.33[医药卫生—基础医学]