摘要
目的在大肠杆菌工程菌中重组表达刚地弓形虫RH株棒状体蛋白5(ROP5)的融合蛋白GST-6×His-ROP5,并初步评价重组蛋白的抗原性。方法体外细胞培养弓形虫速殖子,提取总RNA,反转录获得cDNA,PCR扩增ROP5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,最后通过Western blotting鉴定重组蛋白rROP5的抗原性。结果构建了弓形虫ROP5全基因重组表达质粒,诱导表达了GST-6×His-ROP5融合蛋白,分子量为96 kDa,与预测结果相符,重组蛋白与人源抗血清有显著免疫印迹反应。结论本研究获得了弓形虫ROP5全长重组蛋白,为进一步改进弓形虫诊断抗原,研究蛋白结构以及分泌蛋白与宿主细胞的相互作用奠定了基础。
Objectives To clone and express the rhoptry protein ROP5 gene of Toxoplasma gondii strain RH and to detect the expressed the recombinant fusion protein GST-6xHis-ROP5 in E. coli for analysis of antigenicity of the recombinant protein. Methods The Toxoplasma gondii tachyzoite were cultivated in ceil line, the eDNA were synthetized from the total RNA. The total length of ROP5 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 the anfigenicity of rROP5 was analyzed by Western blotting of the recombinant protein and human anti-Z gondii serum. Results The recombinant expression plasmid of the ROP5 gene was constructed, and the 96kD GST-6xHis-ROP5 fusion protein was induced successfully. The recombinant protein can react with the human anti-T, gondii serum significantly in immunoblotting analysis. Conclosion The full length of ROP5 recombinant protein obtained in this study set foundation for further improving diagnostical antigens, study on protein structure, and protein interaction research between T. gondii secretory protein and host cells.
出处
《中国热带医学》
CAS
2014年第3期259-263,共5页
China Tropical Medicine
基金
国家自然科学基金项目(No.81071375
No.81301404)
北京市自然科学基金项目(No.7132042
No.7144196)
北京市优秀人才培养资助D类项目(No.2013D003034000021)
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)
关键词
刚地弓形虫
棒状体蛋白ROP5
原核重组表达
抗原性
Toxoplasma gondii, Rhoptry protein ROPS, Recombinant prokaryotic expression, Antigenicity
