沙眼衣原体主要外膜蛋白在大肠杆菌中的表达与鉴定

目的 克隆沙眼衣原体主要外膜蛋白(MOMP)基因,构建原核表达重组载体,并通过E.coli BL21实现MOMP的融合表达.方法 用PCR法扩增MOMP基因,克隆插入到pUCm-T载体中,序列测定正确后,将其亚克隆到原核表达载体pET-22b(+)中,采用Ni-NTA亲和层析法纯化表达产物,对表达产物进行SDS-PAGE和蛋白质印迹鉴定.结果 PCR扩增出约1200bpDNA片段,序列测定证实与基因库登陆的D型沙眼衣原体一致;表达产物的相对分子质量为47000,与预期分子质量相符,蛋白印迹证实表达产物为特异性蛋白.结论 沙眼衣原体MOMP基因可以在大肠杆菌中得到表达,其表达产物能与相应的抗体结合,为沙眼衣原体核酸疫苗的研究奠定了基础.

Expression and Identification of Major Outer Membrane Protein of Chlamydia trachomatis in Escherichia coli

Objective To construct the recombinant plasmid containing the major outer membrane protein(MOMP) gene of Chlamydia trachomatis and expres s MOMP protein in E.coli BL21. Methods The MOMP gene was amplified by polymera se chain reaction from the genome of Chlamydia trachomatis serovar D. The amplif ied fragment was directly inserted into pUCm-T vector and verified by DNA sequen cing. MOMP gene was then subcloned into the prokaryotic expression vector pET-22 b( ). The recombinant protein of MOMP was purified by Ni-NTA affinity chromatogr aphy and identified by SDS-PAGE and Western blot. Results The MOMP gene, which is about 1 200 bp, was successfully amplified and cloned. The DNA sequence of t he cloned MOMP gene was the same as that published by the GenBank. SDS-PAGE anal ysis showed that the relative molecular weight of this fusion protein was about 47 kDa which was consistent with the theoretically predicted value, and the spec ificity of this recombinant protein was confirmed by Western blot. Conclusions The MOMP gene of Chlamydia trachomatis was successfully cloned and expressed in the prokaryotic expression system, which may lay the foundation for the developm ent of Chlamydia trachomatis vaccine.