刚地弓形虫活化蛋白激酶C受体1基因的克隆和原核表达及纯化和抗原性分析

发布时间：2018-05-16 13:18

本文选题：刚地弓形虫 + 活化蛋白激酶C受体　；参考：《环境与健康杂志》2013年07期

【摘要】：目的克隆刚地弓形虫活化蛋白激酶C受体1(TgRACK1)基因,原核表达、纯化TgRACK1,并分析其抗原性。方法制备弓形虫RH株速殖子总RNA,根据TgRACK1基因全长编码序列(GenBank登录号:AY547291)的开放阅读框设计引物并进行逆转录-聚合酶链式反应(RT-PCR)扩增,扩增产物经双酶切后连接入pGEX-6p-1载体,重组质粒转化大肠埃希菌DH5α,阳性菌落经菌液PCR、双酶切及DNA测序进行鉴定。将重组质粒pGEX-6p-1-TgRACK1转化至大肠埃希菌BL21(DE3)并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)结合考马斯亮蓝染色检测表达产物;分别以抗GST标签抗体和兔抗弓形虫血清为一抗,免疫印迹(Western blotting)分析鉴定重组蛋白及其抗原性。重组融合蛋白经GST琼脂糖凝胶亲和纯化,纯化蛋白经SDS-PAGE分离,考马斯亮蓝染色结合凝胶成像系统分析其纯度。结果 RT-PCR扩增产物约为970 bp,重组质粒pGEX-6p-1-TgRACK1构建成功。经IPTG诱导获得相对分子质量约63 kDa的可溶性重组蛋白。诱导表达的蛋白质为带GST标签的重组融合蛋白,且能被兔抗弓形虫血清识别。亲和纯化后获得纯度大于95%的重组TgRACK1蛋白质。结论克隆得到弓形虫活化蛋白激酶C受体1基因,原核表达并纯化出该基因编码的蛋白质,且该重组蛋白具有抗原性。
[Abstract]:Objective to clone Toxoplasma gondii activated protein kinase C receptor 1 (TgRACK1) gene, express and purify TgRACK1 in prokaryotic, and analyze its antigenicity. Methods Toxoplasma gondii RH strain Toxoplasma gondii Toxoplasma gondii R@@ The recombinant plasmid was transformed into Escherichia coli DH5 伪. The positive colonies were identified by PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pGEX-6p-1-TgRACK1 was transformed into Escherichia coli BL21 (DE3) and was induced by isopropyl- 尾 -D- thiogalactoside (IPTG). The expression product was detected by SDSPAGE and Coomassie brilliant blue staining. The recombinant protein and its antigenicity were identified by Western blotting analysis with anti-Toxoplasma gondii antibody and anti-Toxoplasma gondii antibody as the first antibody. The recombinant fusion protein was purified by GST agarose gel affinity. The purified protein was separated by SDS-PAGE. Coomassie brilliant blue staining and gel imaging system were used to analyze its purity. Results the RT-PCR amplification product was about 970 BP, and the recombinant plasmid pGEX-6p-1-TgRACK1 was successfully constructed. The soluble recombinant protein with relative molecular weight of 63 kDa was obtained by IPTG induction. The expressed protein is a recombinant fusion protein with GST tag and can be recognized by rabbit antitoxoplasma serum. After affinity purification, recombinant TgRACK1 protein with purity greater than 95% was obtained. Conclusion Toxoplasma gondii activated protein kinase C receptor 1 gene was cloned, and the protein encoded by this gene was expressed and purified in prokaryotic expression, and the recombinant protein was antigenicity.
【作者单位】：山西医科大学寄生虫学教研室;山西医科大学生理学系;
【基金】：国家自然科学基金(81071374) 山西省高校科技研究开发项目(20111010) 山西医科大学科技创新基金(01201103) 山西医科大学基础学院331科技启动基金(201202)
【分类号】：R382

【共引文献】