哲罗鱼胰岛素样生长因子-II的原核表达与活性分析

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。

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

Fish insulin-like growth factor-II (IGF-II) plays a key role in the development of IGF-II. Primers were designed based on the salmon IGF-II gene sequence. An RNA extract of 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 pSUMO vector to construct an expression vector. The recombinant vector was transformed into SDS-PAGE analysis revealed a 40 kD, 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 cells (EPC) and rainbow trout gonad (RTG-2) cells. IGF-II protein can effectively stimulate EPC and RTG-2 cell proliferation. The IGF-II protein expressed by the prokaryotic system had good bioactivity. In summary, we successfully cloned the cDNA of IGF-II from liver tissue and constructed the plasmid pSUMO-IGF used for prokaryotic expression. The plasmid was suitable for IPTG induction and expression of IGF-II in bacteria. Additionally, the plasmid contains a SUMO tag, which can improve the level of expression and the stability of the target protein. A 6-NTA, so the purified IGF-II can be obtained from the fusion protein following digestion using SUMO protease and separation on a Ni²⁺-NTA column. Using this method, we obtained a high concentration of IGF-II that had high purity and exhibited the desired bioactivity. growth and reproduction.