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目的克隆并原核表达刚地弓形虫(Toxoplasma gondii)Rhomboid-1(TgROM1)蛋白。方法收集、纯化弓形虫速殖子,用Trizol法提取总RNA,应用RT-PCR技术扩增TgROM1基因,回收的PCR产物与pMD18-T载体连接,构建重组克隆质粒pMD18-T-TgROM1。将重组克隆质粒亚克隆至原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-TgROM1并转化至Rosetta感受态,用IPTG诱导表达,表达产物进行SDS-PAGE和Western blot分析。结果成功克隆了643bp的TgROM1基因,双酶切鉴定重组表达质粒pGEX-4T-1-TgROM1构建正确。SDS-PAGE检测重组表达质粒表达的TgROM1蛋白分子质量约为48ku,Western blot检测表明该蛋白能被鼠抗弓形虫血清识别。结论成功克隆了弓形虫ROM1基因并原核表达了具有反应原性的重组TgROM1蛋白,为该蛋白的功能研究奠定了基础。
Objective To clone and prokaryote the Rhomboid-1 (TgROM1) protein of Toxoplasma gondii. Methods Toxoplasma gondii tachyzoites were collected and purified by Trizol method. The TgROM1 gene was amplified by RT-PCR. The recovered PCR product was ligated with pMD18-T vector to construct the recombinant plasmid pMD18-T-TgROM1. The recombinant plasmid was subcloned into the prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1-TgROM1 was constructed and transformed into Rosetta competent cells. The recombinant plasmid was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blot . Results The 643bp TgROM1 gene was cloned successfully. The recombinant plasmid pGEX-4T-1-TgROM1 was identified by double enzyme digestion. The molecular weight of TgROM1 protein expressed by recombinant expression plasmid was about 48ku by SDS-PAGE. Western blot showed that the protein could be recognized by anti-Toxoplasma gondii serum. Conclusion The Toxoplasma gondii ROM1 gene was successfully cloned and the prokaryotic expression of the recombinant prokaryotic expression vector TgROM1 protein laid the foundation for the functional study of this protein.