The Experimental Study On Excretion Mature Insulin In Hepatocyte By Retroviral-Mediated Transfecting Of Human Proinsulin Mutant Gene

PART ONESite-directed mutagenesis and modification of humanproinsulin geneObjective:To construct a mutant of human proinsulin gene, C peptide is excised by Furin proteolytic enzyme in the non-endocrine cell to excrete mature insulin. Methods:We used the polymerase chain reaction (PCR) for the site-directed mutagenesis. Four sets of primers were designed according to the gene sequence of human proinsulin, and mismatches were introduced into F02 for histidine (CAC) to aspartic acid (GAC) substitution at position B10, F03/R03 for glutamic acid (GAA) to lysine (AAC) substitution at position C3, alanine (GCT) to arginine (CGT) substitution at position C4, F04/R02 for leucine (CTT) to arginine (CGT) substitution at position C32。Mutagenesis was performed in a two-step PCR。The amplified fragments from the second PCR which contain the mutation site, signal peptide and Kozak sequence were subcloned into the T vector and verified by sequencing analysis. Results:The sequencing analysis showed that the mutation sites were correct, and the mutant of human proinsulin was constructed successfully. Conclusion:A mutant human proinsulin has been constructed by PCR-SDM, and it is potential to construct a cell clone with efficiently excreting mature insulin by non-endocrine cell in vitro.