Prokaryotic Expression Of Hucho Taimen Insulin-like Growth Factor-Ⅱ 、Preparation Of Its Polyclonal Antibody And Tissue Distribution In Hucho Taime

Fish insulin-like growth factor-II(IGF-II) plays a key role in the development of embryo. Hucho taimen is the biggest one in salmonidae fish and its flesh is very delicious.So Hucho taimen have high economic value, But now, there haven’t about zhe Hucho taimen IGF- II gene research in china. In this study, salmon IGF-II gene indexed by Gen Bank was used to design primers P1 and P2. RNA extract of Hucho taimen liver was used as a template, and IGF-II gene open reading frame was amplified by one step RT-PCR. Then use some biology software analysis Hucho taimen IGF- II open reading frame gene sequence, Which included using Clustal W analysis nucleotide sequence and amino acid sequence homology.Using DNAMAN analysis amino acid sequence, Protean software analysis protein molecular weight and isoelectric point prediction;Clustalx software and MEGA 1.83 1.83 software was used to construct the nucleic acid sequence system evolutionary tree. Gene expression was deduced by q RT-PCR. IGF-II gene was inserted into p SUMO vector to construct expression vector.The recombinant vector was transformed into E. Coli Rosetta to induce expression of the target protein. the recombinant protein was mainly in the form of inclusion body.After dissolution of inclusion bodies in buffer containing 8 M Urea and 2 M Urea, high purity protein was achieved. ELISA and MTT assays were utilized for immunological activity and biological activity analysis of target protein. The purified protein was used for mouse polyclonal antibody preparation. The mouse polyclonal antibody was analyzed in ELISA assay. The research lays a foundation for the study of Hucho taimen growth and reproduction. The corresponding results as follows.1. The Cloning of insulin-like growth factors-II c DNA and Tissue Distribution in Hucho taimen.IGF-II gene open reading frame was amplified by one step RT-PCR. The IGF-II contained an ORF of 645 bp encoding 214 amino acid protein with a signal peptides and five regions named B、C、A、D、E. The structure and characteristics of the IGF-II gene were analyzed using bioinformatics software. The results showed that the molecular weight was 2.4554 k Da and the isoelectric point was 9.92. Hucho taimen shared high identity with salmon and the homology at nucleotide and amino acid level of the IGF-II gene were 98%. The IGF-II m RNA expression was detected in different tissues using real-time quantitative PCR technique. The highest expression was observed in the liver, followed by in the heart, gill, spleen, hindgut, kidney, stomach, and muscle, and the lower expression in the foregut and brain.2. Prokaryotic expression and bioactivity analysis of Hucho taimen Insulin-like Growth Factor-II.IGF-II gene was inserted into p SUMO vector to construct expression vector. The recombinant vector was transformed into E. Coli Rosetta to induce expression of the target protein. SDS-PAGE analysis showed a clear target band with expected size about40 k Da, and the recombinant protein was mainly in the form of inclusion body. Pure target fusion protein was obtained by denaturation and renaturation of the inclusion.ELISA and MTT assays were utilized for immunological activity and biological activity analysis of target protein. ELISA analysis showed that the target protein can specifically react with commercial rabbit anti-salmon trout IGF-II antibodies in an antigen concentration-dependent manner. The results indicated that the obtained IGF-II protein has excellent immunogenicity. The MTT aasay 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 results showed that the expressed Hucho taimen IGF-II protein can effectively stimulate EPC cells and RTG-2 cells proliferation.The results indicated that the Hucho taimen IGF-II protein expressed by prokaryotic system had good bioactivity. In summary, we have successfully cloned the c DNA of Hucho taimen IGF-II from Hucho taimen liver and construct the plasimd p SUMO-IGF used for prokaryotic expression, which is suitable for IPTG induction and high expression of IGF-II in E. coli bacteria. The purified protein was used for mousepolyclonal antibody preparation. The mouse polyclonal antibody was analyzed in ELISA assay. The titers of the mouse polyclonal antisera with recombinant IGF-II were1:25000. Our results provide a basis for future genetic breeding 、 growth and reproduction of Hucho taimen.