刚地弓形虫致密颗粒蛋白GRA2全基因的原核重组表达  

Cloning and prokaryotic expression of the full-length GRA2 gene of Toxoplasma gondii

作　　者：黄敏君[1,2] 薛峰[1,2] 洪彩玲[1] 孙岚[1,2] 甘绍伯[1,2] 谷俊朝[1,2] 

机构地区：[1]首都医科大学附属北京友谊医院,北京热带医学研究所,北京100050 [2]热带病防治研究北京市重点实验室,北京100050

出　　处：《中国病原生物学杂志》2013年第12期1093-1095,1112,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81071375,81301404);北京市自然科学基金项目(No.7132042);首都医科大学基础-临床科研合作基金项目(No.13JL16);首都医科大学附属北京友谊医院科研启动基金项目(No.2011_30yykyqd)

摘　　要：目的克隆弓形虫RH株致密颗粒蛋白2(GRA2)的全长基因,在大肠埃希菌工程菌中重组表达GST-HisGRA2融合蛋白。方法提取弓形虫总RNA,反转录获得cDNA,PCR扩增GRA2基因全长,与pET-41Ek/LIC载体通过基因重组直接连接,在大肠埃希菌RosettaTM(DE3)pLysS工程菌中诱导表达重组融合蛋白,然后采用SDS-PAGE和Western blot鉴定重组蛋白产物。结果 PCR鉴定弓形虫GRA2全基因重组表达质粒构建正确;诱导表达的GSTHis-GRA2融合重组蛋白分子质量单位为52ku,与预测值相符。结论本研究成功构建了弓形虫GRA2全基因重组蛋白质粒,该质粒能表达GRA2蛋白,为研究GRA2与宿主细胞的相互作用奠定了基础。Objective The total length of the GRA2 gene of Toxoplasma gondii strain RH was cloned and expressed in E. coli to produce a GST-His-GRA2 recombinant fusion protein. Methods cDNA was synthesized from the total RNA of T. gondii. The total length of GRA2 was amplified using PCR and ligated into the pET-41 Ek/LIC vector by direct nucleotide sequence recombination. The recombinant fusion protein was induced in E. coli strain RosettaTM （DE3） pLysS and analyzed with SDS-PAGE and Western blotting. Results The recombinant expression plasmid of the GRA2 gene was verified to be correct according to PCR. The molecular weight （MW） of the induced GST-His-GRA2 fusion protein was 52 ku, which agreed with the predicted MW. Conclusion A recombinant expression plasmid of the GRA2 gene was successfully constructed. The full length of the GRA2 recombinant protein obtained in this study has laid the foundation for further study of the interaction between GRA2 and host ceils.

关 键 词：刚地弓形虫 致密颗粒蛋白GRA2 反转录基因克隆 原核表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]