弓形虫Rhomboid-1蛋白基因的克隆及原核表达

目的克隆并原核表达刚地弓形虫（Toxoplasmagondii）Rhomboid-1（TgROMl）蛋白。方法收集、纯化弓形虫速殖子，用Trizol法提取总RNA，应用RTPCR技术扩增TgROMl基因，回收的PCR产物与pMDl8-T载体连接，构建重组克隆质粒pMDl8-T-TgROMl。将重组克隆质粒亚克隆至原核表达载体pGEX一4T—l中，构建重组表达质粒pGEX-4Tl-TgR（）M1并转化至Rosetta感受态，用IPTG诱导表达，表达产物进行SDS-PAGE和Westernblot分析。结果成功克隆了643bp的TgROMl基因，双酶切鉴定重组表达质粒pGEX-4T-1-TgROMl构建正确。SDS-PAGE检测重组表达质粒表达的TgROMl蛋白分子质量约为48ku，Westernblot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ROMl基因并原核表达了具有反应原性的重组TgROMl蛋白，为该蛋白的功能研究奠定了基础。

Cloning and prokaryotic expression of Rhomboid-1 gene of Toxoplasma gondii

Objective To clone and express the Rhomboid-1 gene （TgROM1） of Toxoplasma gondii. Methods T. gondii tachyzoites were harvested and purified, and their total RNA was extracted in order to amplify the TgROMI gene using RT-PCR. The amplified TgROM/ gene was then subcloned into the prokaryotic expression vector pGEX-4Tq. The constructed recombinant plasmid pGEX 4T-1-TgROM1 was transformed into Rosetta-competent cells for expression. Ex- pression was induced with IPTG, and the expressed product was identified using SDS-PAGE and Western blotting. Re- sults Restriction analysis showed that recombinant plasmid pGEX-4T-1 TgROM1 was correctly constructed. SDS~PAGE showed that the recombinant protein had a molecular mass of about 48 ku, and Western blotting showed that the recombi nant protein was recognized. Conclusion The recombinant Rhomboid protein of T. gondii was successfully expressed in Rosetta. This provides a basis for analysis of the function of this protein.