Clonging And Expression Analysis Of Insulin-like Growth Factor I In Tissue Of Cobia(Rachycentron Canadum)and Expression In Escherichia Coli System

In this study, we successfully cloned the insulin-like growth factor I(IGF-I)cDNAs sequence in the cobia Rachycentron canadum, by using Polymerase Chain Reaction(PCR)techniques. The cDNA sequence was analysed by using bioiformatics software, such as BLAST, MEGA. At the same time, we also analysed the expression of IGF-Iin different tissues thought quantitative real-time PCR methods. An E. coli system was then constructed based on the matured peptide of IGF-I to attain the recombinant protein. And these results laid a foundation for the further research on cobia’ growth and metabolism regulation. 1. Cloning and sequence analysis of insulin-like growth factor- IA pair of PCR primers were designed from a consensus analysis of conserved coding regions of known IGF-I ligand sequence from other Perciformes fish. We obttained cDNA sequence from cobia by using PCR methods. The open reading frame of cDNA is 558 nucleotides,encoding a 185 amino acid prepro IGF-I. Moreover,the prepro IGF-I is composed of signal peptide(44 amino acid)and mature peptide(67 amino acid),and matured peptide contains B,C,A and D domains. The mRNA of IGF-I belongs to the Ea-4 subtype. The calculated molecular weight is 8.6kDa and theoretical isoelectric point is 7.9. The deduced mature IGF-Iexhibited higher sequence identities with their corresponding orthologs in ?shes,especially B and A domains,while C and D domains differ significantly. The results of multiple alignment showed that IGF-I of cobia was similar by 74% with Homo sapiens, 99% with Acanthopagrus latus.A phylogenetic tree analysis of IGF-I sequences indicated that the evolutionary relationship of cobia was very closely to marine fish and was far with fresh fish and invertebrate. 2. Escherichia coli expression of IGF-I from cobiaPrimers were designed based on the mature peptide of IGF-I. And The cDNA coding for the mature IGF-I peptide were subcloned into the pEASY vector by using the pEASY seamless cloning technology. Then the correct sequence were inserted to the expression vector pET-21 a to construct the recombinant plasmid. And it was transformed into Rosetta(DE3) pLysS which can produce recombiant protein when induced by IPTG. Tricine SDS-PAGE analysis of protein from IPTG-induced expression demonstrated that the recombinant IGF-I had a molecular weight of 8.6kDa. and from the results of western blotting indicated that the fusion protein had the antigencity to 6×His-tag antibody. All the results show the recombinant protein successfully expression in the E. coli. The production was inclusion body,need denaturalized by guanidine-Hcl, and then it purified using His-Tag Purification column and annealed by sodium acetate, we obtain the recombinant protein solution. Through the real time PCR, we conclude that the highest IGF-I level were observed in liver. The else of other tissue, IGF-I expression is little. 3. Tissue spectic expression of IGF-I in Cobia fed with different carbohydrate levelsThe IGF-I expression in tissue were assessed in cobia fed with distinct dietary carbohydrate levels. Three isonitrogenous diets were formulated to contain 35%(35% carbohydrate, H), 21%(21% carbohydrate, M) and 7%(7% carbohydrate, L). Although fed with different dietary. In group H, the expression in liver is 75 folds higher than the intestine, and in gropu M, the number is 13 folds higher than intestine, and in group L, the number is 470 folds higher than intestine. All data indict that, high and low carbohydrate component content can disturb the normal expression of IGF-I in tissue.