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当前位置: 首页 > 文章 > 红笛鲷重组激活基因rag1原核表达条件的优化及纯化 广东海洋大学学报 2012,32 (1) 11-16
Position: Home > Articles > Purification and Optimization of Prokaryotic Expression of rag1 Gene in Red Snapper (Lutjanus sanguineus) Journal of Guangdong Ocean University 2012,32 (1) 11-16

红笛鲷重组激活基因rag1原核表达条件的优化及纯化

作  者：
张雪利;鲁义善;谢吉国;简纪常;吴灶和
单  位：
广东海洋大学水产学院广东省水产经济动物病原生物学及流行病学重点实验室暨广东省高等学校水产经济动物病害控制重点实验室
关键词：
红笛鲷;rag1基因;原核表达;表达条件;优化;纯化;Western blot分析
摘  要：
克隆编码红笛鲷(Lutjanus sanguineus)RAG1蛋白(recombination activating protein1)活性核心区的基因序列,并与pET-28a(+)载体连接,构建原核表达载体pET-28a-RAG1,将其转入大肠杆菌BL21(DE3)菌株,利用IPTG进行诱导表达。为提高融合蛋白的表达效率,运用传统的实验方法对诱导条件进行优化。SDS-PAGE分析表明,在37℃条件下,利用0.1 mmol/L IPTG诱导8 h后,RAG1重组融合蛋白的表达量最大,相对分子质量与预测值相符,该蛋白主要以包涵体形式高效表达,利用His Trap HP亲和柱使其得到进一步纯化;Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明表达蛋白为目的蛋白。
译  名：
Purification and Optimization of Prokaryotic Expression of rag1 Gene in Red Snapper (Lutjanus sanguineus)
作  者：
ZHANG Xue-li,LU Yi-shan,XIE Ji-guo,JIAN Ji-chang,WU Zao-he(Fisheries College of Guangdong Ocean University,Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals & Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions,Zhanjiang 524025,China)
关键词：
Lutjanus sanguineus;gene rag1;prokaryotic expression;expression conditions;optimization;purification;western blot analysis
摘  要：
In the core domain,the gene sequence of coding the Lutjanus sanguineus RAG1 protein was cloned and then inserted into the pET-28a(+) vector to construct prokaryotic expression plasmid pET-28a-RAG1.The recombinant RAG1 fusion protein was overexpressed in Escherichia coli BL21(DE3) cells in the presence of isopropyl-β-thiogalactopyranoside(IPTG).To achieve a high level expression,the optimized induction condition was identified by using classical experimental method.SDS-PAGE analysis showed that inducing the cells at 37 ℃ in 0.1 mmol/L of IPTG for 8 hours was the optimal conditions for expression of the recombinant RAG1 fusion protein.The relative molecular mass of the expressed product was identical to the expected protein which was mainly detected in the insoluble fraction of E.coli cell lysates.The recombinant RAG1 fusion protein was purified by using His Trap HP affinity column.Western blot analysis showed that the recombinant RAG1 fusion protein could be combined with mouse anti-His-Tag Mab,so the expressed protein was definitely confirmed to the aim protein.

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