肺炎链球菌热休克蛋白ClpE多克隆抗体的制备及其亚细胞定位  

Preparation of polyclonal antibody and subcellular location of heat shock protein ClpE of Streptococcus pneumoniae

作　　者：蔡莺莺[1] 闫文娟[1] 张群[2] 王虹[1] 胥文春[1] 何於娟[1] 尹一兵[1] 张雪梅[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学附属儿童医院检验科,重庆400014

出　　处：《中国生物制品学杂志》2014年第1期28-32,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81000766)

摘　　要：目的制备肺炎链球菌(Streptococcus pneumoniae,S.pn)热休克蛋白(heat shock protein,HSP)ClpE多克隆抗体,并确定ClpE在S.pn表面的亚细胞定位。方法应用PCR法从S.pn D39中扩增clpE基因全长片段,克隆入原核表达载体pET28a,转化E.coli BL21(DE3),IPTG诱导表达,经亲和层析纯化重组蛋白。用纯化的重组蛋白免疫BALB/c小鼠,制备多克隆抗体,间接ELISA法测定抗体效价,Western blot法鉴定抗体的特异性。ELISA法分析ClpE在S.pn表面的亚细胞定位。结果重组表达质粒经PCR、双酶切和测序证实构建正确;表达的重组ClpE蛋白相对分子质量约83 000,表达量约占全菌总蛋白的60%,主要以可溶性上清形式存在;纯化的重组蛋白纯度达90%;制备的ClpE多克隆抗体效价达1∶4 096 000,可与S.pn D39全菌发生特异性反应,S.pn D39在正常生长时可表达ClpE蛋白;ClpE在S.pn表面有表达。结论成功制备了高效价和特异性的ClpE多克隆抗体,ClpE在S.pn表面可正常表达,为表面蛋白。Objective To prepare the polyclonaI antibody against heat shock protein ClpE of Streptococcus pneumoniae （S. pn）, and determine its subcellular location in S. pn. Methods Full length ClpE gene of S. pn D39 was amplified by PCR and cloned into prokaryotic expression vector pET28a. The constructed recombinant plasmid pET28a： ：ClpE was transformed to E. coli BL21 （DE3） and induced by IPTG. The expressed product was purified by affinity chromatography, with which BALB/c mice were immunized. The prepared polyclonal antibody was determined for titer by indirect ELISA, and Ibr specificity by Western blot, and for subcellular location in ＆ pn by ELISA. Results PCR, restriction analysis and sequencing proved that the recombinant plasmid pET28a： ：clpE was constnlcted correctly. The expressed ClpE protein, with a relative molecular mass of about 83 000, contained about 60% of total somatic protein, mainly existed in a soluble form and reached a purity of 90%. The prepared polycional antibody against ClpE reached a titer of 1 ： 4 096 000, and showed specific reaction with S. pn D39. ClpE was expressed on the surface of S. pn D39 growing normally. Conclusion High titer specific polyclonal antibody against ClpE was prepared successfully, and ClpE was expressed normally on sur- face of S. pn.

关 键 词：肺炎链球菌 热休克蛋白 ClpE 亚细胞定位 

分 类 号：R378.14[医药卫生—病原生物学] R392.33[医药卫生—基础医学]