溶藻弧菌dtd基因的克隆及原核表达条件优化  

Cloning and Optimization of Prokaryotic Expression of D-Tyr-tRNA^(Tyr) Deacylase(DTD) Gene from Vibrio alginolyticus

作　　者：陈立明 庞欢瑛[1,2,3] 简纪常[1,2,3] 吴灶和[2,3,4] 

机构地区：[1]广东海洋大学水产学院 [2]广东海洋大学广东省水产经济动物病原生物学及流行病学重点实验室 [3]广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088 [4]仲恺农业工程学院,广东广州510225

出　　处：《广东海洋大学学报》2014年第3期52-57,共6页Journal of Guangdong Ocean University

基　　金：国家科技支撑计划(2012BAD17B02);广东省自然科学基金(B12121)

摘　　要：根据NCBI数据库中已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列合成一对特异性引物,应用聚合酶链式反应(PCR技术)扩增溶藻弧菌HY9001菌株dtd基因,将其定向克隆到原核表达载体pET-32a构建重组表达质粒pET-DTD,并对其进行诱导温度、诱导时间、诱导IPTG浓度等条件的优化,最后探究其纯化时最佳咪唑洗脱浓度。结果表明:DTD蛋白成功表达,且其以包涵体的形式存在,在诱导温度37℃、IPTG浓度0.1 mmol/L条件下诱导5 h表达量最高,纯化最佳咪唑洗脱浓度为150 mmol/L。A pair of specific primers was designed and synthesized based on D-Tyr-tRNA^TyT Deacylase （DTD） of Vibrio alginolyticus published on GenBank. The dtd gene was amplified by PCR and then inserted into the pET-32a （＋） vector to construct the prokaryotic expression plasmid. Then the temperature, IPTG concentration, induction time, elution concentration of the pET-DTD was optimized to obtain a relatively large amount of the target protein. The result showed that the DTD protein was successfully expressed, and the expressed protein existed in the form of inclusion body. The optimization condition of inducible expression for DTD protein in Escherichia coli was at 37℃ for 5 hours, with 0.1 mmol/L of IPTG. The best elution concentration of imidazole was 150 mmol/L.

关 键 词：溶藻弧菌 DTD 基因克隆 原核表达 条件优化 

分 类 号：S941[农业科学—水产养殖]