吉林大学学报（医学版）2017，Vol.43Issue(6)：1109-1114,6.DOI:10.13481/j.1671-587x.20170608
刚地弓形虫肌动蛋白profilin的原核表达和纯化
Prokaryotic expression and purification of Toxoplasma gondii profilin protein
摘要
Abstract
Objective:To discuss the prokaryotic expression system and purification conditions of Toxoplasma gondii profilin-like protein (TgPRF), and to provide basis for the study on anti-tumor immuno-adjuvant. Methods:The coding region of TgPRF gene was amplified with a pair of specific primers which were designed according to the cDNA of tachyzoites of Toxoplasma gondii RH strain.The PCR products were cloned into the pET-28a (+ ) vector after double enzyme digestion. The recombinant pET28a (+ )-TgPRF plasmid was transformed into E.coli DH5αcells.The positive clones were selected by the double restrictive enzyme digestion and sequencing.The correct pET28a (+)-TgPRF plasmid was transformed into E.coli BL21 (DE3)and induced for 4 h by IPTG.The expression of recombinant TgPRF protein was analyzed by SDS-PAGE method;the expression of recombinant protein His-profilin was detected by Western blotting method.Results:The length of product of PCR was 492 bp.The recombinant plasmid pET28a-TgPRF was confirmed by double restriction enzyme digestion and sequencing.The SDS-PAGE results showed that the target protein was expressed in E.coli BL21 (DE3)in bacteria supernatant.The purified TgPRF protein was obtained by Ni-NTA agarose gel column chromatography with the purity>90%.The Western blotting results revealed that the recombinant TgPRF protein could be recognized by Anti-His antibody.Conclusion: The recombinant plasmid pET28a-TgPRF is successfully constructed,and the TgPRF protein is obtained with the soluble prokaryotic expression.关键词
刚地弓形虫/profilin/原核表达/蛋白纯化Key words
Toxoplasma gondii/profilin/prokaryotic expression/protein purification分类
医药卫生引用本文复制引用
李会敏,伊焕发..刚地弓形虫肌动蛋白profilin的原核表达和纯化[J].吉林大学学报（医学版）,2017,43(6):1109-1114,6.基金项目
吉林省科技厅自然科学基金资助课题(20150101127JC) （20150101127JC）
