Clonning And Expression Of A Novel Gene Of Procine Insulin Like Growth Factor-1

In order to construct a novel procine insulin like growth factor-1 gene toimprove the growth performance and metabolism of animal. The experiment wascarried out to reconstruct procine insulin like growth factor-1 gene by use ofoverlapping PCR technique. The enzyme sites of BamHⅠwere introduced to theN-end, BglⅡ, EcoRⅠand termination code inserted to the C-end of the insulin likegrowth factor-1 gene. Two protective bases were added to the outside of enzymesites. Four amino acids of the N-end were deleted and seven amino acids ofGSSSRRA from 32th to 38th were deleted too. The PCR production was cloned intopMD 18-T vector, the sequencing result showed that the cloned cDNA sequence was205bp long, coding 59 amino acids, which accorded with the expected size. Then theIGF-1 cDNA fragment was subcloned into prokaryotic expression vectorpGEX-4T-1, forming the prokaryotic expression plasmid (pGEX-4T-1-IGF-1D). Therecombinant plasmid was digested by BamHⅠand EcoRⅠ, and identified by plasmidPCR. It was showed that the IGF-1 cDNA fragment was successfully inserted intothe plasmid in correct orientation.The recombinant plasmid of pGEX-4T-1-IGF-1D was transformed into theBL21(DE3) competent cells. The transformed clones were incubated with 2xYTAmedium, and the expression of the fusion protein was induced with IPTG. Themolecular weight of the fusion protein was 33kDa detected by SDS-PAGE, which was in accordance with the aim protein. To found the best inducing condition, theexpression of the fusion protein was induced under different incubation times,temperatures and IPTG concentrations. It was found that the expression level of thefusion protein was especially high when induced with 0.5mM IPTG at 37℃after 4hours.The expressed fusion IGF-1 was proved to provoke the singnificantproliferation of procine lymphocytes in vitro compared with the control GST protein(P＜0.01), suggesting the strong bioactivity of the novel IGF-1 to promote the growthand metabolism of eukaryotic cells. These results are of importance for furtherexploration of the biological function of the novel IGF-1 and development ofeffective regulator for the growth and metabolism of animals.