弓形虫棒状体蛋白2和膜表面蛋白1重组复性后体外免疫反应性研究

目的分析基因工程生产的弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)的体外免疫反应性,并对表达产物进行复性处理以获得表达产物的天然构象,为体内免疫学活性的研究作准备。方法重组质粒pET28b/ROP2-P30转化大肠杆菌BL21-Codon Plus(DE3)-RIL菌株,经IPTG诱导的表达产物超声破壁后,SDS-PAGE分析表达产物的表达形式,对产生的重组蛋白ROP2-P30过Sephadex G-25交联葡聚糖柱,经尿素梯度洗脱进行目的蛋白的复性,通过免疫共沉淀反应及免疫印迹实验检测其复性后的重组蛋白的免疫反应性。结果重组质粒pET28b/ROP2-P30在大肠杆菌中以融合形式表达,融合表达的产物主要以包涵体形式存在,该重组蛋白复性后具有明显的免疫反应性。结论利用SephadexG-25交联葡聚糖柱尿素梯度可以复性重组的弓形虫复合蛋白ROP2-P30,该蛋白复性后体外具有免疫反应性,可用于进一步开展弓形虫病复合型疫苗的研制工作。

Immunoreactivity of the recombinant protein after refolding of rhoptry protein 2 and major surface protein 1 from Toxoplasma gondii

The recombinant rhoptry protein 2 (ROP2) and major surface protein 1(P30) from Toxoplasma gondii produced by gene-engineering technique were analyzed for their immunoactivities in vitro, and would be refolded to obtain the native conformation as a preparation to detect its immunoactivity in vivo. In this way, the recombinant plasmid pET28b/ROP2-P30 was transformed to E.coli BL21-coden plus (DE3)-RIL strain, and was expressed under induction with IPTG. Cells were lysed by multiple rounds of sonication, and the expressed products were analyzed by using SDS-PAGE. The recombinant protein ROP2-P30 was then refolded by Sephadex G-25 in a urea gradient, and the immunoreactivity of the recombinant ROP2-P30 renatured was tested with immuno-precipitation and immuno-blotting assay. It was demonstrated that the recombinant ROP2-P30 was expressed in E.coli as a fusion protein and was formed as an inclusion body. After refolding, it could be recognized by Toxoplasma-specific IgG antibody in human sera ,as demonstrated in immuno-precipitation and immuno-blotting assay. It is evident that the recombinant protein ROP2-P30 refolded by Sephadex G25 in a urea gradient has displayed its immunoactivity in vivo.