哲罗鱼胰岛素样生长因子-II的原核表达与活性分析

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哲罗鱼胰岛素样生长因子-II的原核表达与活性分析
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作者单位: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
作者简介:吴秀梅(1988–), 女, 硕士研究生, 从事水产动物繁殖生理学研究. E-mail: wuxiumei_1988@163.com
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中图分类号:Q785; S 917
基金项目:国家科技支撑计划项目(2012BAD25B10); 公益性行业(农业)科研专项(201003055-14).

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

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1. Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

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摘要:

-II(insulin-like growth factors-II, IGF-II)利用pSUMO-IGF中进行目的蛋白的诱导表达。, 目的蛋白以包涵体形式存在。对包涵体进行变性利用方法对目的蛋白进行免疫学活性及生物学活性分析。IGF-IIIGF-II方法测定Cyprinus carpio)(epitheliaoma papulosum cyprini, EPC)Oncorhynchus mykiss的增殖效果来鉴定细胞增殖。该结果表明利用原核表达系统获得的哲罗鱼

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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.

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吴秀梅,徐黎明,赵景壮,刘淼,曹永生,卢彤岩,尹家胜.哲罗鱼胰岛素样生长因子-II的原核表达与活性分析[J].中国水产科学,2015,22(2):243-249
WU Xiumei, XU Liming, ZHAO Jingzhuang, LIU Miao, CAO Yongsheng, LU Tongyan, YIN Jiasheng. Prokaryotic expression and bioactivity analysis of Hucho taimen insulin-like growth factor-II[J]. Journal of Fishery Sciences of China,2015,22(2):243-249

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在线发布日期: 2015-06-29
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