文献类型：期刊文献

中文题名：红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化

英文题名：Purification and Optimization of Prokaryotic Expression of tdt Gene of Red Snapper (Lutjanus sanguineus)

作者：黄瑜[1];张雪利[1];鲁义善[1];简纪常[1];吴灶和[2];黄浦江[1];周泽军[1]

机构：[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

年份：2013

卷号：33

期号：1

起止页码：44

中文期刊名：广东海洋大学学报

外文期刊名：Journal of Guangdong Ocean University

收录：CSTPCD

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

语种：中文

中文关键词：红笛鲷;tdt基因;原核表达;优化;纯化;Western;blot分析

外文关键词：Lutjanus sanguineus; tdt gene; prokaryotic expression; optimization; purification;Western blot analysis

中文摘要：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。

外文摘要：The gene sequence of coding the mature peptide Lutjanus sanguineus Terminal deoxynueleotidyl transferases （TdT） protein was cloned and then inserted into the pET-32a（＋） vector to construct prokaryotic expression plasmid pET-32a-TdT. And then it was transferred into E. coli BL21 （DE3） strain. The recombinant TdT fusion protein was over expressed in Escherichia coli BL21（DE3） cells in the presence of isopropyl-^-thiogalactopyranoside （IPTG）. To achieve a high level expression, the optimized induction conditions were identified by using classical experimental method. SDS-PAGE analysis revealed that inducing the cells, the optimal conditions were at 37 ~C in 0.7 mmol/L IPTG for 4 hours for expression of the recombinant TdT fusion protein. The molecular mass of the expressed product was identical to the predicted protein which was mainly detected in the insoluble fraction of E. coli cell lysates. The recombinant TdT fusion protein was purified by using His Trap HP affinity column and the best elution concentration of imidazole was 300 mmol/L. Western blot analysis showed that the recombinant TdT fusion protein could be combined with mouse anti-His-Tag Mab, So the expressed protein was definitely confirmed to the aim protein.