草鱼NEDD4结合蛋白基因1原核表达条件的优化及纯化  

Purification and Optimization of Prokaryotic Expression of NEDD4 Binding Protein 1 in Grass Carp(Ctenopharyngodon idellus)

作　　者：杨林[1] 蔡佳[1] 简纪常[1] 颜鹏[1] 鲁义善[1] 吴灶和[1,2] 

机构地区：[1]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,广东湛江524025 [2]仲恺农业工程学院,广东广州510225

出　　处：《广东海洋大学学报》2013年第4期38-42,共5页Journal of Guangdong Ocean University

基　　金：国家973计划(2009CB118704)

摘　　要：通过PCR方法克隆草鱼(Ctenopharyngodon idellus)NEDD4结合蛋白基因1(简称CiN4BP1)的开放阅读框(ORF)序列,将扩增产物与pET-32a(+)表达载体相连接,构建原核表达载体pET-N4BP1,对重组质粒进行酶切和测序鉴定,然后将其导入大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,检测该蛋白表达情况。SDS-PAGE分析表明,在温度为37℃,IPTG浓度为0.06 mmol/L,诱导时间为4 h时,N4BP1重组融合蛋白的表达量最高,蛋白分子质量为40.2 ku,与软件预测值大小相符,该蛋白主要以包涵体形式表达。利用His Trap HP亲和柱纯化目的蛋白;Western blot分析表明,N4BP1融合蛋白能与鼠抗His-tag单克隆抗体发生特异性反应,说明该表达的蛋白为目的蛋白。The open reading frame （ORF） sequence of NEDD4 binding protein 1 of Grass Carp （Ctenopharyngodon idellus） was cloned by PCR and then inserted into the pET-32a（＋） vector to construct prokaryotic expression plasmid pET-32a-N4BP1. The recombinant plasmid was by enzyme digestion and sequencing identification, and then was overexpressed in Escherichia coli BL21（DE3） cells and through isopropyl-β-thiogalactopyranoside （IPTG） induction to detect the protein expression. SDS-PAGE analysis showed that including the cells at 37℃ in 0.06 mmol/L of IPTG for 4 hours were the optimal conditions for expression of the recombinant N4BP1 fusion protein. The molecular mass of the expressed protein product was 40.2 ku, which was identical to the expected protein and was mainly detected in the insoluble fraction of E. coli cell lysates. Western blot analysis showed that the recombinant N4BP1 fusion protein could be combined with mouse anti-His-Tag Mab, so the expressed protein was definitely confirmed to the aim protein.

关 键 词：草鱼 N4BP1 原核表达 表达条件 优化 纯化 WESTERN BLOT分析 

分 类 号：S942.1[农业科学—水产养殖] Q344.13[农业科学—水产科学]