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

Home > Archive>Volume 22, Issue 2, 2015 >243-249

Prokaryotic expression and bioactivity analysis of Hucho taimen insulin-like growth factor-II
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Affiliation: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
Clc Number:Q785; S 917
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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]. Jounal of Fishery Sciences of China, 2015,[volume_no](2):243-249

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Online: June 29,2015
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