结核分枝杆菌Rv2460 c基因的克隆,表达及编码产物的免疫特性分析

摘要

Objective:To clone and express Mycobacterium tuberculosis(Mtb) Rv2460c gene(encoding ClpP2 protein),and evaluate the immunogenicity of its coding product. Methods: The recombinant plasmid of pET32a (+) vector-ClpP2 that H37Rv Rv2460c gene was cloned into the plasmid pET32a(+)vector,was transformed into E. coli BL21(DE3)and induced expression by IPTG,then purified by affinity chromatography. The recombinant protein was confirmed by SDS-PAGE and Western blot. The analysis of immunogenicity of Mtb ClpP2 and its epitope prediction were performed by bioinformatic methods. The antibody levels of polyclonal antibody titer against ClpP2 protein in rabbits and TB patients′ serum were detected by ELISA. Results: The recombinant ClpP2 protease was expressed as inclusion bodies in E. coli. The purity of purified protein was 93% by bandscan software analysis. The bioin-formatics analysis shows Mtb ClpP2 protein has multiple preponderant B cell and T cell epitopes. Rabbit antiserum titer was 1∶64 000;Serum anti-ClpP2 antibody levels in TB patients was higher than that in healthy control subjects. Conclusion:The recombinant ClpP2 protein was purified, and specific Rabbit anti-ClpP2 polyclonal antibody was prepared successfully. Experiment and bioinformatic information studies showed that Mtb ClpP2 protease has strong immunogenicity.%目的：：对结核分枝杆菌Rv2460c基因(编码ClpP2蛋白)进行克隆,表达,并评价其编码产物的免疫原性。方法：扩增结核分枝杆菌(Mycobacterium tuberculosis,Mtb)H37Rv 的Rv2460c基因,构建重组质粒pET32a(+)-ClpP2,用IPTG诱导表达,利用亲和层析法进行纯化。用SDS-PAGE和Western blot分析重组蛋白ClpP2的表达和鉴定。用生物信息学对Mtb ClpP2的免疫原性分析和表位预测。酶联免疫吸附法( ELISA)测定兔和TB病人血清anti-ClpP2滴度。结果：重组ClpP2蛋白酶在大肠杆菌中以包涵体形式表达,经 bandscan 软件分析纯化蛋白的纯度为93%。生物信息学分析Mtb ClpP2蛋白具有多个优势B细胞表位和T细胞表位。 ELISA法测定兔抗血清效价为1∶64000；检测肺结核病人血清中anti-ClpP2抗体水平高于健康对照人群。结论：成功纯化了重组蛋白ClpP2和制备了特异性兔抗ClpP2多克隆抗体,实验和信息学均表明Mtb ClpP2蛋白酶有较强的免疫原性。