摘要
目的克隆弓形虫RH株致密颗粒蛋白7(GRA7)的全长基因,在大肠杆菌工程菌中重组表达GST-His-GRA7融合蛋白。方法提取弓形虫总RNA,反转录获得cDNA,PCR扩增GRA7基因全长,与pET-41 Ek/LIC载体通过基因重组直接连接,在大肠杆菌RosettaTM(DE3)pLysS工程菌中诱导表达重组融合蛋白,最后采用SDS-PAGE和West ern blotting鉴定重组蛋白产物。结果构建了弓形虫GRA7全基因的重组表达质粒,诱导表达了GST-His-GRA7融合重组蛋白,分子量为62kD,与预测结果相符。结论研究获得了弓形虫GRA7全基因的重组蛋白,为进一步研究弓形虫GRA7抗原性及其与宿主细胞的相互作用奠定了基础。
Objective To clone the total length of GRA7 gene of Toxoplasma gomh＇i strain RH expressed in E.eoli to pro- duce GST-His-GRA7 recombinant fusion protein. Methods The cDNA were synthetized from the total RNA of Toxoplasma gondii. The total length of GRA7 was amplified using PCR, and ligated into the pET-41 Ek/LIC vector by direct nucleotide se- quence recombination. The recombinant fusion protein was induced in E.eoli strain RosettaTM （DE3） pLysS, and analyzed by SDS-PAGE and Westem blotting. Results The recombinant expression plasmid of the GRA7 gene was constructed, and the 62kD GST-His-GRA7 fusion protein was induced successfully. Conclusion The full length of GRA7 recombinant protein obtained in this study set foundation for further study on the immunogenicity of GRA7 protein and the interaction between GRA7 and host cells.
出处
《中国热带医学》
CAS
2013年第9期1045-1047,1050,共4页
China Tropical Medicine
基金
国家自然科学基金面上项目(No.81071375)
北京市自然科学基金面上项目(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)
