肺炎链球菌热休克蛋白ClpL的原核表达、纯化及抗原特性分析  

Prokaryotic expression,purification and antigenic characters of heat shock protein ClpL of Streptococcus pneumoniae

作　　者：钟文[1] 张群[2] 龚艺[1] 张彦青[1] 陈倩[1] 陈特[1] 董杰[1] 王虹[1] 张雪梅[1] 

机构地区：[1]重庆医科大学医学检验系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》2012年第4期393-397,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31070819);重庆市卫生局科技研究项目(2010-2-218)

摘　　要：目的原核表达并纯化肺炎链球菌(Streptococcus pneumonia,S.pn)热休克蛋白ClpL,并评价其作为S.pn疫苗候选蛋白的可行性。方法PCR扩增S.pn D39全长ClpL基因,克隆至原核表达载体pET-28a(+)中,转化E.coli BL21(DE3),IPTG诱导表达,表达的重组ClpL蛋白经Ni-NTA亲和层析柱纯化后,免疫BALB/c小鼠,ELISA检测抗ClpL多克隆抗体的效价及亚型;Western blot分析ClpL蛋白在5种常见S.pn中的保守性;流式细胞术及Western blot分析ClpL蛋白在S.pn中的亚细胞定位。结果重组表达质粒pET-28a(+)-ClpL经双酶切及测序证实构建正确;ClpL蛋白在E.coli BL21(DE3)中获得了可溶性表达,纯化后纯度达90%,浓度为4.97 mg/ml;免疫小鼠可产生高滴度、高特异性的IgG抗体,以IgG1和IgG2b亚型为主;ClpL蛋白在5株常见S.pn中均有表达;ClpL蛋白为分泌型蛋白,不表达于S.pn表面。结论原核表达并纯化了S.pn ClpL蛋白,其作为一种在S.pn中保守存在的分泌型蛋白,可能是一种较好的疫苗候选蛋白。Objective To express heat shock protein ClpL of Streptococcus pneumoniae （S. pn） in prokaryotic cells, and evaluate the feasibility of expressed product as a candidate protein of S. pn vaccine. Methods-, Fullqength CIpL gene of S. pn D39 was amplified by PCR and cloned into prokaryotic expression vector pET-28a（＋ ）. The constructed recombinant plasmid pET-28a （＋）- CIpL was transformed to E. coli BL21 （DE3） and induced with IPTG. The expressed product was purified by Ni-NTA affinity chromatography, with which BALB / c mice were immunized and determined for titer and subtype of induced polyclonaI antibody against ClpL. The ClpL protein was analyzed for conservativeness in five common S. pn strains by Western blot, and for subcellular location in S. pn by flow cytometry and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a （＋）-ClpL was constructed correctly. CIpL protein was expressed in a soluble form in E. coli BL21 （DE3）, reached a purity of 90% and a concentration of 4.97 mg/ ml respectively after purification, and induced highly specific IgG with high titer in mice, most of which were of subtypes IgG1 and IgG2b. ClpL was expressed in all the five common S. pn strains. However, ClpL was a secretory protein not expressed on surface of S. pn. Conclusion The ClpL protein of S. pn was expressed in prokaryotic cells and purified, which was conserved in S. pn and might be used as a satisfactory candidate protein of S. pn vaccine.

关 键 词：链球菌 肺炎 ClpL蛋白 原核细胞 基因表达 纯化 疫苗 

分 类 号：R378.1[医药卫生—病原生物学] R392.33[医药卫生—基础医学]