哲罗鱼胰岛素样生长因子-II的原核表达与活性分析  

Prokaryotic expression and bioactivity analysis of Hucho taimen insulin-like growth factor-II

作　　者：吴秀梅[1,2] 徐黎明[1] 赵景壮[1] 刘淼[1] 曹永生[1] 卢彤岩[1] 尹家胜[1] 

机构地区：[1]中国水产科学研究院黑龙江水产研究所,黑龙江哈尔滨150070 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2015年第2期243-249,共7页Journal of Fishery Sciences of China

基　　金：国家科技支撑计划项目(2012BAD25B10);公益性行业(农业)科研专项(201003055-14)

摘　　要：在鱼类胚胎发育过程中,胰岛素样生长因子-II(insulin-like growth factors-II,IGF-II)起着关键的作用。本研究根据Gen Bank收录的鲑鳟鱼IGF-II基因序列设计引物,以哲罗鱼(Hucho taimen)肝RNA提取物为模板,利用RT-PCR方法扩增出哲罗鱼IGF-II基因开放阅读框。将目的基因IGF-II与原核表达载体p SUMO连接构建出重组表达载体p SUMO-IGF。将该重组质粒转化到大肠杆菌Rosetta中进行目的蛋白的诱导表达。SDS-PAGE电泳分析显示,约在40 k D处含有清晰的条带,与预期结果相符;目的蛋白以包涵体形式存在。对包涵体进行变性/复性后获得较纯的目的蛋白,利用ELISA和MTT方法对目的蛋白进行免疫学活性及生物学活性分析。ELISA结果显示该目的蛋白能够与商品化的抗鲑鳟鱼IGF-II的抗体发生特异性反应,并且呈现抗原浓度依赖性,该结果说明本研究获得了具有良好免疫原性的IGF-II蛋白;MTT方法测定IGF-II蛋白对鲤(Cyprinus carpio)上皮细胞(epitheliaoma papulosum cyprini,EPC)和虹鳟(Oncorhynchus mykiss)性腺细胞(rainbow trout gonad,RTG-2)的增殖效果来鉴定IGF-II蛋白的生物学活性,结果显示所表达的哲罗鱼IGF-II蛋白能够有效的刺激EPC细胞和RTG-2细胞增殖。该结果表明利用原核表达系统获得的哲罗鱼IGF-II蛋白具有良好的生物学活性。本研究为哲罗鱼的生长模式和生长繁殖的研究奠定了基础。Fish insulin-like growth factor-II（IGF-II） plays a key role in the development of embryos. Our objective was to isolate and characterize Hucho taimen IGF-II. Primers were designed based on the salmon IGF-II gene sequence. An RNA extract of H. taimen liver was used as a template, and the IGF-II gene open reading frame was amplified by one step RT-PCR. The IGF-II gene was inserted into a p SUMO vector to construct an expression vector. The recombinant vector was transformed into E. coli Rosetta to induce expression of the target protein. SDS-PAGE analysis revealed a clear target band with expected size of ~40 k D, and the recombinant protein was mainly in the form of an inclusion body. Pure target fusion protein was obtained by denaturation and renaturation of the inclusion. Purified IGF-II was obtained from the fusion protein following digestion by SUMO protease and separation on a Ni2＋-NTA column. ELISA and MTT assays were used to quantify the immunological and biological activity of the target protein. The target protein can specifically react with commercial rabbit anti-salmon trout IGF-II antibodies in an antigen concentration-dependent manner. The isolated IGF-II protein has excellent immunogenicity. The MTT assay was used to measure the effect of IGF-II protein on the proliferation of Epitheliaoma papulosum cyprini cells（EPC） and rainbow trout gonad（RTG-2） cells. The expressed H. taimen IGF-II protein can effectively stimulate EPC and RTG-2 cell proliferation. The H. taimen IGF-II protein expressed by the prokaryotic system had good bioactivity. In summary, we successfully cloned the c DNA of H. taimen IGF-II from liver tissue and constructed the plasmid p SUMO-IGF used for prokaryotic expression. The plasmid was suitable for IPTG induction and expression of IGF-II in E. coli bacteria. Additionally, the plasmid p SUMO contains a SUMO tag, which can improve the level of expression and the stability of the target protein. A 6×His tag, located in the SUMO tag, can be recognized by Ni2＋-NTA, so

关 键 词：胰岛素样生长因子-II 哲罗鱼 生物活性 MTT 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]