原核表达并纯化沙眼衣原体(Ct)巨噬细胞感染增强因子MIP的免疫活性分析

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

目的原核表达并纯化沙眼衣原体(Chlamydia trachomatis,Ct)巨噬细胞感染增强因子MIP,评价其作为Ct疫苗候选蛋白的可行性。方法采用PCR技术从Ct D型基因组中扩增MIP基因,构建pGEX-6p-1/MIP原核表达质粒,转化E.coli XL1-Blue,IPTG诱导重组GST-MIP融合蛋白表达,经酶切后得到无GST标签的纯蛋白免疫Balb/c鼠,ELISA法检测免疫小鼠的血清抗体和脾细胞因子的产生情况。结果成功构建了pGEX-6p-1/MIP原核表达质粒,序列测定证实重组质粒插入片段与Gen Bank登陆的Ct D型一致,长度为729 bp;GST-MIP融合蛋白在E.coli中获得稳定高效表达,纯化后纯度达90%以上;MIP蛋白免疫小鼠产生高滴度、高特异性的IgG抗体,以IgG2a亚型为主;MIP蛋白免疫小鼠的脾细胞受衣原体菌体或MIP蛋白刺激,产高水平IFN-γ。结论原核表达并纯化了Ct MIP蛋白,该蛋白具有良好的免疫原性,可诱导小鼠产生高滴度抗体和Thl型免疫应答,可能为潜在有效的Ct亚单位疫苗。

Objective In order to express microphage infectivity potentiator （MIP）of Chlamydia trachomatis （C.trachomatis）in prokaryotic cells, and evaluate the feasibility of expressed product as a candidate protein of C.trachomatis vaccine.Methods Full-length MIP gene of C.trachomatis serovar D was amplified by polymerase chain reaction （PCR） and cloned into prokaryotic expression vector pGEX-6p-l. Then the constructed recombinant plasmid pGEX-6p-l/MIP was transformed to E.coli XLl-Blue and induced with IPTG. The expressed products were purified by Glutathione Sepharose 4B Beads and the GST tag were removed by prescission protease, with which Balb/c mice were immunized and determined for titer and subtype of induced polyclonal antibody against MIP. ELISA was carried out to analyze the cytokines production by splenocytes upon Chlamydia EB stimulation. Results The pGEX-6p-l/MIP expression plasmid was successfully constructed. DNA sequencing showed that the inserted MIP was about 729 bp. The target protein was effectively expressed in E.coli, reached a purity of 90% after purification, and induced highly specific IgG with high titer in mice most of which were of subtypes IgG2a. ELISA shows that the MIP protein induce high IFN-γ production in mice splenocytes. Conclusion The MIP protein of C.trachomatis was successfully expressed and purified from prokaryotic cells, which was highly immunogenic and induce Th1 immune responds in Chlamydia infected mice. Thus, MIP might be used as a satisfactory vaccine candidate protein to provide protection against C.trachomatis infection.