作　　者： ; ; ; ; ;

机构地区： 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

出　　处： 《广东海洋大学学报》 2010年第6期25-30,共6页

摘　　要： 通过克隆编码红笛鲷(Lutjanus sanguineus)非特异性毒性细胞受体蛋白-1(nonspecific cytotoxic cell receptor protein-1,NCCRP-1)成熟肽基因序列,然后与载体连接构建pET21a-NCCRP融合蛋白表达载体,将其转入大肠杆菌BL21进行诱导表达并优化条件。SDS-PAGE分析表明,在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.8 mmol/L、37℃条件下培养3 h后表达量最大,分子大小与预期值相符,融合蛋白主要以包涵体形式高效表达,通过HisTrap HP柱子使其得到进一步纯化;Western blot分析表明,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明获得该表达产物。 The cloned mature peptide of Lutjanus sanguineus NCCRP-1 was connected with the expression vector pET-21a to construct fusion protein pET21a-NCCRP.This recombinant was transformed into Escherichia coli BL21 and mainly expressed in the form of inclusion body.The optimal condition for the expression of this protein was that the cells were induced at 37 ℃,in 0.8 mmol/L of IPTG for 3 hours.The molecular weight of the expressed product was identical to that of expected protein by SDS-PAGE analysis,then purified by HisTrap HP column.The result of Western blot show that the expressed product could be combined with mouse anti-His-tag Mab.The fusion protein which lays the basic could be obtained for the future research on function and applied in fish innate immunity.

关 键 词： 红笛鲷 基因 原核表达 条件优化 纯化

领　　域： [农业科学] [农业科学] [生物学]