Prokaryotic Expression, Bioactivity Analysis And Tissue Expression Of Insulin-like Growth Factor-I From Hucho Taimen

Insulin like growth factor-I is a kind of polypeptide, which has the function of promoting the growth and development of the fish, and plays an important role in the growth and development of fish. Hucho taimen belongs to Salmoniformes, Salmonidae, Hucho, which is rare cold water fish in china. At present, there are few reports about IGF-I Hucho taimen.In this study, total RNA was isolated from Hucho taimen liver tissue. The cDNA encoding insulin-like growth factor I(IGF-I) peptide was amplified by reverse transcription polymerase chain reaction(RT-PCR) strategy using the isolated total RNA as templates. The IGF-I mRNA level was detected in different tissues using real-time quantitative PCR technique. The results showed that the IGF-I gene cDNA contains an open reading frame(ORF) of 573 bp, encoding a polypeptide with length of 190 amino acids and isoelectric point of 9.21. The polypeptide is composed of signal peptide, B, C, A, D domain and E peptide. The amino acid sequence of Hucho taimen IGF-I was high homological to that of other Salmonidae. Hucho taimen shared the highest identity with Salvelinus alpinus and the homology at nucleotide level of the IGF-I gene were 99.2%. The expression analysis of IGF-I gene with real-time quantitative PCR showed that the highest level of IGF-I mRNA was observed in liver, followed in gill and anterior intestine. There was no obvious expression difference in brain, pronephros, spleen, heart, stomach and muscle.Recombinant expression plasmid(IGF-I/pS) was constructed by prokaryotic expression vector pS and transformed into E.coli Rosetta. The expression of recombinant IGF-I protein of hucho taimen was induced by IPTG. There was a clear target band with expected size between 35~50 kDa observed in SDS-PAGE electrophoresis, and the recombinant protein existed in the form of inclusion body. Purified IGF-I protein was obtained after the inclusion body was denatured/renatured experiment. ELISA results showed that target protein can identify commercial fish IGF-I antibody, indicating that hucho salmon IGF-I protein with immune activity was obtained. Cell proliferation assay(MTT) results showed that recombinant IGF-I protein can significantly promote the propagation of CHSE-214, EPC and RTG-2, indicating that the recombinant IGF-I protein had biological activity. IGF-I fusion protein with biological activity and immunological activity was used to deal with CHSE-214 cells, and the relative expression of IGF-I, GH, IGF-II and IGF-I receptors were detected by real time quantitative PCR. The results showed that the relative expression levels of IGF-I, IGF-II, IGF-I Ra and IGF-I Rb genes in CHSE-214 cells were increased.The study lays a foundation for the research on the function of IGF-I gene, and provides scientific basis for the regulation of the growth and development of hucho taimen.