黄瑜,张雪利,鲁义善,简纪常,吴灶和,黄浦江,周泽军

• 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
• 2:仲恺农业工程学院

摘要(Abstract)：

克隆编码红笛鲷(Lutjanus sanguineus)末端脱氧核糖核酸转移酶TdT蛋白(Terminal deoxynucleotidyl transferases)成熟肽基因序列,并与pET-32a(+)载体连接,构建原核表达载体pET-32a-TdT,再将其转入大肠杆菌BL21(DE3)菌株,利用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。运用传统方法优化诱导条件,以提高重组融合蛋白的表达效率。SDS-PAGE分析表明,37℃、0.7 mmol/L IPTG条件下诱导4 h后,TdT融合蛋白的表达量最大,分子质量大小与预测值相符,该蛋白主要以包涵体形式存在。利用HisTrap HP亲和层析柱使TdT蛋白得到进一步纯化,最佳咪唑洗脱浓度为300 mmol/L,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的结合,表明表达蛋白为目的蛋白。

关键词(KeyWords)： 红笛鲷;tdt基因;原核表达;优化;纯化;Western blot分析

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(41240041);; 广东省科技厅国际合作项目(2012B050600029)

作者(Author): 黄瑜,张雪利,鲁义善,简纪常,吴灶和,黄浦江,周泽军

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