Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells / 重庆医科大学学报

ABSTRACT

Objective:To construct a mutant of human proinsulin with signal peptide code,so that it may be expressed in eukaryotic system to produce mature human insulin in COS-7 cells.Methods:Human proinsulin gene was mutated by the polymerase chain reaction (PCR) for the site-directed mutagenesis method.Two furin recognized sites of Arg-X-Lys-Arg-motifs were introduced into human proinsulin.Signal peptide,Kozak sequence and restriction enzyme cutting site were added and cloned into pcDNA3.1(+) vector and verified by sequencing analysis.Eukaryotic expression vector was constructed and transfected into COS-7 cells.Insulin were evaluated respectively with RT-PCR,radioimmunoassay and Western blot analysis.Results:The sequencing analysis showed that the mutation sites were correct,and the mutant of human proinsulin was constructed successfully.Human insulin mRNA was highly expressed in transfected COS-7 cells,and secretive recombinant human insulin was detected in the culture supernatant of transfected COS-7 cells by Western blotting.Conclusion:A mutant human proinsulin eukaryotic expression vector is obtained and secretive recombinant human insulin is successfully expressed in COS-7 cells.