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《Journal of Fishery Sciences of China》 2015-02
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Prokaryotic expression and bioactivity analysis of Hucho taimen insulin-like growth factor-II

WU Xiumei;XU Liming;ZHAO Jingzhuang;LIU Miao;CAO Yongsheng;LU Tongyan;YIN Jiasheng;Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences;College of Fisheries and Life Science, Shanghai Ocean University;  
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 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. Our results provide a foundation for the study of H. taimen growth and reproduction.
【Fund】: 国家科技支撑计划项目(2012BAD25B10);; 公益性行业(农业)科研专项(201003055-14)
【CateGory Index】: S917.4
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Chinese Journal Full-text Database 10 Hits
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【Co-citations】
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