Cloning and expression of islet neogenesis associated protein in Pichia yeast / 中华胰腺病杂志

ABSTRACT

Objective To clone the human Islet neogenesis associated protein(rhINGAP)gene,express the gene extraeellulary in Pichia yeast for.further study on biological function and animal test on INGAP.Methods INGAP gene Was amplified with PCR and inserted into the recombinant plasmidα/pUC18.Then,the fusion gene of α and INGAP was digested and inserted into the expression plasmid pPIC9K.The positive recombinant plasmid which integrated INGAP Was confirmed by restriction enzyme digestion and sequencing,and it Was linearized with Sal Ⅰ digestion and transfered into the yeast host strain GS115 through electroporation.The yeast transformants that harbor the desired gene INGAP with high copy were selected by the auxotroph mediam G418,and verified by PCR.The condition of hake-flask culture was optimized,and the recombinant human INGAP Was induced expression with methanol as the only Carbone source.The antigen activity of the desired protein Was detected by Western blotting and ELISA method.Results Recombinant plasmid αINGAP/pPIC9K were successfully constructed and three positive Pichia yeast transformants were obtained.The expressed protein had satisfactory antigen activity,which Was confirmed by the Western blotting and ELISA method.Conclusions Pichia yeast expressing human Islet neogenesis associated protein (rhINGAP)gene was successfully constructed.