Reconstruction Of The Plasmid Regulating Insulin Expression And The Study Of Its Expressionin In The Hepatic Cells In Vivo And In Vitro

Objective An eukarvotic expression vector was constructed in order to evaluate glucose-regulated mature insulin gene express in hepatocytes. Methods Native human insulin cDNA was derived from fetus pancreas by RT-PCR. Using site-directed mutagenesis(overlap extension PCR), furin consensus cleavage sequence was introduced into proinsulin cDNA while the new sequence was named as INS/furin which may allow us to recognize and processe furin and proteolytic maturation of proinsulin to insulin efficiently within the majority of the cells containing furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid_{p(G1RE)3BP-1Luc}, which contains glucose reaction element G1RE in its promotor. Results The vector p(G1RE)₃BP-11×furin was digested with two enzymes ,Hind â…¢ and EcoRV and then the vector was verified by sequencing to be sure that it contains the target gene at the specific sites. Conclusion The recombined mammalian expression vector p(G1RE)₃BP-11×furin, which contains glucose reaction element G1RE and site-mutated proinsulin ,is a novel candidate vector ideal for diabetic gene therapy.