沙眼衣原体MIP蛋白的原核表达、纯化及免疫学特性鉴定

Prokaryotic Expression,Purification and Immunological Characters of Microphage Infectivity Potentiator of Chlamydia Trachomatis

目的采用p GEX-6p载体原核表达沙眼衣原体(Ct)巨噬细胞感染增强因子(MIP),进一步纯化后免疫小鼠,评价该蛋白作为Ct疫苗候选抗原的可行性。方法聚合酶链反应技术从Ct D型基因组扩增MIP编码基因,构建p GEX-6p-1/MIP重组质粒并转化大肠杆菌,IPTG诱导重组菌表达GST-MIP融合蛋白,Pre Scission蛋白酶切除GST标签后免疫Balb/c小鼠,ELISA法检测MIP的免疫学特性。结果 MIP编码基因正确插入p GEX-6p-1载体,核苷酸序列与Gen Bank登陆的Ct D型MIP基因完全一致;重组融合蛋白在E.coli中稳定高效表达,酶切后纯度达90%以上;MIP蛋白具有良好的免疫原性,诱导小鼠产生高滴度特异性Ig G型抗体,亚型以Ig G2a为主;MIP免疫组小鼠脾淋巴细胞受MIP蛋白刺激,分泌高水平IFN-γ。结论重组表达的Ct MIP蛋白具有良好的免疫原性,能诱导小鼠产生高特异性的体液及细胞免疫应答,可进一步探讨其作为Ct亚单位疫苗的可行性。

Objective Using pGEX-6p prokaryotic vector to express macrophage infection enhancement factor （MIP） of Chlamydia trachomatis, then purify this protein to immunize mice and evaluate the feasibility of using MIP as a vaccine candidate antigen. Methods PCR amplify MIP encoding gene from C.trachomatis genome to construct pGEX-6p- 1/MIP recombinant plasmid.After transformed into E.coli XL1-Blue and induced by IPTG, PreScission protease was used to remove GST tag and GST free MIP proteins were used to immunize Balb/c mice.The immunological characters of MIP were detected by ELISA. Results Amplified MIP gene was correctly inserted into the pGEX-6p-1 vector and the nucleotide sequence was identical to C.trachomatis serovar D MIP gene in GenBank.Recombinant fusion protein was stably and highly expressed in E.coli and reached a purity of more than 90% after enzyme digestion.MIP protein has good immunogenicity as it induces high titer of the specific IgG antibody production in mice sera and high IFN-γ production upon stimulating mice spleenocytes in vitro. Conclusion Recombinant C.trachomatis MIP protein has good immunogenicity,which can induce specific humoral and cellular immune responses.h is worth to further explore the feasibility of using MIP as chlamydia subunit vaccine.