刚地弓形虫Rop18基因的原核表达及鉴定

利用PCR技术从弓形虫RH株基因组中扩增出Rop18基因片段,克隆入pET-30a载体,将重组质粒转化入大肠杆菌Top10中,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列(登录号:JX045329.1)比对发现,核苷酸同源性为99.6%。将阳性重组质粒转化入大肠杆菌BL21中,表达产物经SDS-PAGE检测结果显示,Rop18基因在大肠杆菌中成功表达;融合蛋白的分子质量在66.2 ku左右,Western blotting显示其能被兔抗弓形虫免疫血清识别。结果表明,弓形虫RH株Rop18基因可以在原核表达系统中高效表达,该重组蛋白具有免疫原性,有望作为弓形虫疫苗候选抗原。

Prokaryotic Expression of Toxoplasma gondii Rop18 Gene and Immunogenicity Analysis of the Recombinant Protein

In this study,the Rop18 gene of T.gondii was cloned and expressed in E.coli Top10, and the immunogenicity of the recombinant protein was analyzed. The coding region of Rop18 was amplified with PCR and cloned into the prokaryotic expression vector pET-30a. The recombinant plasmid were confirmed by PCR, enzyme digestion and DNA sequencing, which was then transformed into E.coli BL21 induced by IPTG.The expressed proteins were analyzed by SDS-PAGE and Western blotting.Results indicated that the recombinant pET30a-Rop18 was correctly constructed.The results of SDS-PAGE and Western blotting revealed that the molecular weight of the recombinant protein was approximately 66.2 ku and could be recognized by rabbit antiserum against T.gondii.The results suggested that the Rop18 gene of T.gondii could be expressed in E.coli,and immunogenicity of the recombinant protein had been identifieded by Western blotting indicating that it might be used as vaccine candidate antigen in the future.