The Construction, Expression And Purification Of Recombinant Human Insulin-like Growth Factor-1 Precursor

Object IGF-â… Diagnostic kit has been onlyproduced by minority countries, for instance America andGermany, and it's price is too high to limit it's clinical application.Our laboratory had once chosen many carriers to produce IGF-â… protein antigen, but the result was to be disappointed, the yield ofIGF-â… protein was all low, so it brought difficulty to prepareIGF-â… monoclonal antibody and IGF-â… Diagnostic kit. To plomb theblank of Diagnostic kit in China, we plan to construct arecombinant vector containing the cDNA which encodes Humaninsulin-like growth factor-1 precursor and to make it express inE.coli BL21(DE3), then to purify recombinant protein which thestrain expresses, meanwhile, to investigate the antigenicity andapplication of the recombinant protein. Methods 1. By usingPCR technique, we amplified the target fragment which codesHuman insulin-like growth factor-1 precursor cDNA, and theninserted into the expression vector pET32a(+), subsequently. 2. The recombinant vector was transformed into BL21(DE3). Afterinducing with IPTG, the recombinant protein was purified with Ni~+affinity chromatography. 3. we identified its antigenic activity withwestern blot. Result Enzyme digestion analysis and sequencingshowed that the target fragment had been successfully inserted intothe vector pET32a(+).SDS-PAGE showed that the recombinantprotein output the BL21(DE3) expressed accounted for 25% of allprotein the bacteria expressed, what's more important was thatpurified recombinant protein obtained by Ni~+ affinitychromatography was so purified that its purity exceeded 90%.Western blot analysis showed it had immunology specificity.Conclusion The recombinant vector pET32a(+)/hIGF-â… hasbeen constructed successfully, and it could express with highperformance.