In vitro Recombinant Expression of Insulin-Like Factor Ⅱfrom Cynoglossus semilaevis Günther

Abstract:

To explore the role of insulin-like factor Ⅱ(IGF-Ⅱ) in growth regulation of Cynoglossus semilaevis Günther, the IGF-Ⅱ gene was expressed in vitro and the bioactivity was determined by methyl thiazolyl tetrazolium (MTT method). The mature peptide domain of IGF-Ⅱ gene of C. semilaevis Günther was cloned by PCR amplification and sequenced for verification. The obtained mature peptide fragment was then subcloned into the prokaryotic expression vector pET-28a (IGF-Ⅱ/pET28a). The recombinant plasmid was expressed in E.coli BL21 (DE3) cells and the recombinant IGF-Ⅱ protein containing 6×His tag at N-terminus was induced by IPTG. SDS-PAGE analysis indicated that the obtained IGF-Ⅱ protein was found in the form of inclusion bodies with molecular weight of 11.4 kDa, which accounted for 43.7% of the whole bacterial protein post 2-hour induction with IPTG. Western blotting analysis indicated that the recombinant IGF-Ⅱ protein had the antigenicity to 6×His antibody. The inclusion bodies containing recombinant protein were denaturalized, purified and annealed. The recombinant IGF-Ⅱ protein significantly promoted the proliferation of human breast cancer cells MDA231. Results could provide basic information on the role of IGF-Ⅱ in fish and be helpful to better understand the endocrine mechanism of sex-based dimorphic growth performance of C. semilaevis Günther.