融合蛋白IL6-OmpW的质粒构建及表达纯化  

Construction and Purification of Prokaryotic Expression of Fusion Protein IL6-OmpW

作　　者：黄浦江[1,2,3] 黄郁葱[1,2,3] 简纪常[1,2,3] 吴灶和[2,3,4] 鲁义善[1,2,3] 黄瑜[1,2,3] 樊云霞[1,2,3] 

机构地区：[1]广东海洋大学水产学院,广东湛江524025 [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524025 [3]广东省水产经济动物病害控制重点实验室,广东湛江524025 [4]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43-48,共6页Journal of Guangdong Ocean University

基　　金：国家科技支撑项目(2012BAD17B02);国家自然科学基金项目(41240041);广东省科技厅国际合作项目(2012B050600029)

摘　　要：以重组质粒pMD18-T/IL6和pMD18-T/OmpW为模板,分别扩增红笛鲷IL-6基因和哈维氏弧菌外膜蛋白OmpW基因,运用PCR重叠延伸剪切技术,将IL-6和OmpW基因融合,将融合基因定向克隆到原核表达载体pET-32a(+),转化大肠杆菌BL21(DE3)感受态,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导融合蛋白高效表达,融合蛋白分子质量约为66.6 ku。优化后表达条件为温度37℃,IPTG浓度0.2 mmol·L-1,诱导时间5 h。用HisTrap HP亲和柱纯化重组蛋白,最佳咪唑洗脱浓度为400 mmol·L-1,纯化蛋白的质量浓度为480μg·mL-1。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反应,表明目的蛋白得以正确表达。The fusion gene IL6-（Gly4Ser）3-OmpW was constructed with the DNA fragments of the red snapper IL-6 gene and Vibrio harveyi outer membrane protein OmpW gene which from recombinant plasmid pMD18-T/IL6 and pMD18-T/OmpW by Gene-SOEing method. The full length product was then cloned into the prokaryotic expression vector pET-32a（＋） for protein expression in Escherichia coli strain BL21（DE3）. The molecular weight of expression fusion protein IL6-（Gly4Ser）3-OmpW was about 66.6 ku. The recombinant protein was high expressed under induction conditions of exposure at 37℃, in 0.2 mmol·L^-1 of IPTG for 5 h. The fusion protein was purified using HisTrap^TM HP affinity column and the best elution concentration of imidazole was 400 mmol·L^-1. The concentration of purified fusion protein was 480 μg·mL^-1. Western-blot analysis showed that the recombinant fusion protein could be combined with mouse anti-His-Tag Mab, indicating that the aim protein was expressed successfully. These results could provide a foundation for further study of its biological activity.

关 键 词：哈维氏弧菌OmpW 红笛鲷IL-6 基因融合 原核表达 

分 类 号：Q78[生物学—分子生物学] S94[农业科学—水产养殖]