Studies On The Cloing,expression,purification And Characterization Of Insulin-like Growth Factor 1

Insulin-like growth factor 1 (IGF-1) is a multifunctional cell regulating factor. Many studies showed that IGF-l had very wide biological activities and effects on many kinds of tissues and organs. Recently human IGF-1 (hIGF-1) has been used to treat diabetes, insulin-resistant syndrome, dwarfism, some nervous system diseases and other obstinate diseases. The clinic outcomes were rather encouraging. In order to get sufficient quantities of pure biologically active hIGF- 1 to meet the demand of basic research and clinical use, we cloned the gene of hIGF-1 into pRSET B, a prokaryotic expression vector, and recombinant hIGF- 1 (rblGF- 1) was expressed in E. co/i. The characteristics and the purification method of the product have also been discussed. According to the known sequence of nature hIGF-1 gene, we designed and synthesized the gene of hIGF-1 which was suitable for expression in E.coli without changing the amino acid sequence. The gene was then inserted into the plasmid vector pUC 19. We designed a pair of oligonucleotide primers to introduce to the restriction enzyme site. HIGF-1 gene was amplified by PCR from pUCIGF. The PCR product was ligated into the expression plamid pRSET B. The recombinant plasmid pRSET-IOF was identified by PCR, restriction enzyme analysis and DNA sequence assay. The plasmid pRSET-IGF was transformed into the host cells E.coli BL21. SDS- PAGE analysis suggested that the bacteria containing the recombinant plasmid produced the protein of 7.8kDa as it was induced by IPTG, consisting 1O 0% of the total bacterial proteins. After fermentation, the bacteria was splitted by ultrasonic method. The target protein was soluble in the supernatant, and then the supematants were purified by anion chromatography, hydrophobic chromatography and gel- filtration. The purified products were analyzed by SDS-PAGE and HPLC. The result showed that rhIGF-l could be purified up to 98%. Western blotting indicated that the recombinant protein could react with antibodies against anti-hJGF-l. The effect of the protein stimulating NIH3T, proliferation showed that rhIGF- 1 had good biological activity. The results demonstrate that the synthesized gene of hIGF-l could be expressed in soluble form at high level in E.coli. After purification, the pure biologically active rhIGF-1 could be obtained and could be used in basic research and clinical practice.