The Study On Cloning And Expression Of Common Carp Insulin-like Growth Factors

Insulin-like growth factors (IGFs), including IGF-Ⅰand IGF-Ⅱ, are single-chainpolypeptide hormones, which possess biological activity of growth stimulating activityand has similar metabolic functions of insulin. At the cellular level, IGFs could stimulatecell proliferation, differentiation, migration, metabolism, etc. Many studies show thatIGFs play very important regulated roles in the development, growth, reproduction,osmoregulation and immune of fishes. Because the concentration of IGFs in the extracellularenvironment is low and most of IGFs are bound to IGF-binding proteins, obtainingsufficient fish IGFs is very difficult.In order to get sufficient quantity of biologically active piseine IGFs, This researchstudied the genetic engineering expression of IGFs in the E. coli and P. pastorisexpression systems at the base of cloning Common carp IGF-Ⅰand IGF-Ⅱ. What havebeen done in this research will be usefull for the continual progress of basic andapplication research of fish IGFs system.1. Common carp IGFs gene was successfully amplified from liver total RNA withRT-PCR method, which was then cloned into vector pMD18-T and identified bysequencing. The sequencing result showed that: IGF-Ⅰwhich was cloned in this researchwas totally the same, compared with the sequences which had been reported in Genebank,and there was a synonymous mutation of 1 bp in IGF-Ⅱ.2. Prokaryotic expression vectors pGEX-KG-IGF-Ⅰm and pET-32a-IGF-Ⅱm wereconstructed. After identified, the vectors were transformed into E. coli BL21 cell, andthen induced by IPTG. SDS-PAGE showed that each kinds of the recombinant stainscould express the fusion proteins (GST-IGF-Ⅰand Trx-IGF-Ⅱ) about 32 and 28 kDa, bothof the recombinant protein expressed in the form of inclusion body. After induced for 3hours by 0.3 mmol/ L IPTG, the yield was the highest, and the recombinant proteinamounted to 30%～35% of the whole protein in the E. coli cell. The method containingwith inclusion body collection-SKL denaturalization-dialysis renaturalization was used topurify these two prokaryotic expression proteins.3. The purified product was used as the antigen to immunize New Zealand rabbit, thenthe rabbit anti-Common carp IGF-Ⅰ/IGF-Ⅱpolyclonal antibody was prepared. The titer ofthe antibody was 3200 and 6400 detected by ELISA. Western-blot suggested that both ofthe antiserums could react with corresponding recombinant protein specifically.4. Yeast expression vector pPIC9K-IGF-Ⅰm was constructed. The vector waslinearizated, then transformed into the Pichia pastorist strain GS115 by LiCl. Theresearchers got recombinant strains with His~+Mut~+ phenotype which could resist2.0mg/mL G418, after phenotype analysis, G418 screening and PCR identification. The recombinant strains were induced by 0.5% methanol in shaking flasks. IGF-Ⅰwasexpressed and secreted into the supematant. When induced for 3 days, the yield was thehighest. Tricine-SDS-PAGE indicated that the recombinant protein was about 7.5 kDawhich was consistent with the expected result. Western-blot proved that the recombinantprotein could be specifically combined with rabbit antibodies to common carp IGF-Ⅰ.