沙眼衣原体多形外膜蛋白PmpG的克隆、表达、纯化及鉴定

Cloning, expression, purification and identification of Cldamydia trachomatis polymorphic membrane protein G

目的 克隆、表达、纯化E型沙眼衣原体多形外膜蛋白（PmpG）,并鉴定其免疫原性.方法 用PCR技术扩增从E型沙眼衣原体中提取的PmpG基因片段,再将其定向插入到原核表达载体PET30a（＋）中,构建重组表达质粒,将其转化入E.coli DH5α中,并用酶切分析、PCR扩增及基因序列测定等对重组质粒进行鉴定,诱导表达后用SDS-PAGE及Western印迹进行鉴定,然后进行蛋白纯化,并免疫BALB/c小鼠鉴定其免疫原性.结果 PCR扩增出约1092 bp DNA片段,序列测定证实与基因库的E型沙眼衣原体一致;表达产物的相对分子质量为55 000,与预期分子量相符,Western印迹证实表达产物为特异性蛋白,纯化后的蛋白免疫小鼠获得抗原特异性抗体.结论 构建的PmpG原核表达载体在大肠杆菌中得到了表达,且具有免疫原性.

Objective To clone, express and purify Chlamydia trachomatis polymorphic membrane protein （Pmp G）, and to identify its immunogenicity. Methods The Pmp G gene of C. trachomatis serotype E was amplified by PCR, cloned into prokaryotic expression vector PET30a （＋）. The positive recombinant was transformed into the bacterium E coli （BL-21）, identified by enzyme digestion, PCR amplification and gene sequencing. Then, it was induced to express followed by the identification of expression product with SDS-PAGE and Western blotting. The purified protein was used to immunize BALB/C mice to test its immunogenicity. Results PCR produced a 1092 bp-sized DNA fragment, which had a sequence consistent with that of PmpG gene of C. trachomatis E type in the GenBank database. The molecular weight of expression product was 55 kD, which was proved to be the expected size, and Western Blotting confirmed it to be the specific protein. Moreover, special antibodies to PmpG were induced to be generated by mice immunized with the purified protein. Conclusions The constructed prokaryotic expression vector for PmpG is expressed successfully in E. coli, and the expression product shows immunogenicity.