Construction Of Eukaryotic Vectors Of Human Insulin Genomic Gene And Their Experimental Application

Insulin is a kind of important hormone used in the therapy of diabetes especially type I in clinical practice. With the increasing of the number of diabetes patients, a large amount of insulin is required. Although the bioactivity of pig insulin is near to that of human's, but as a heteroprotein, immune reaction against pig insulin occurs in diabetes patient leads to reduce the efficiency of it after many times injection. To cope with this problem, it is necessary to produce human-self insulin. Therefore several aspects of research work were reported in this paper. Firstly, Several eukaryotic vectors were constructed. The first intron of human insulin genomic gene was specifically amplified with the method of PCR and cloned into the expression vector pCMV-mLNS constructed formerly to form a new recombinant expression plasmid pCMV-ImINS. The recombinant expression plasmid pEF 1 α -mINS and pEF 1 α -ImINS were also constructed according to the routine method. Then these recombinant expression plasmids were transfected into BI-IK cells by lipofectin reagent respectively. And the expression products of insulin gene were detected by radioimmunoassay (RIA) when the positive clones were passed to 20th generation. The result showed that the expression level of recombinant plasmids containing the first intron was higher than that of which without the first intron in BHK cells. And the EF1 ci promoter was more effective than CMV in BHK cells. These recombinant expression plasmids were then transferred respectively into the new fertillized eggs of housefly by electroporation. The first generation was screened with G4 18 after the founders were incubated to adults. Human insulin was detected by radioimmumoassay, Tricine-SDS-PAGE and Western-blot with samples of maggot of the second generation collected at the time of 48h, 72h, 96h and 1 20h. The results suggested that the housefly maggot has the ability of processing post- translation insulin genomic gene. So housefly can be used as bioreactor to produce human insulin. It was certified once more that the expression level of the human insulin gene containing the first intron was higher than that of the gene without the first intron. And the promoter EF1 a was more effective than CMV. A recombinant transposon vector pFast-mINS was constructed by inserting the 98 Construction of Eukaryotic Vectors of Human Insulin Genomic Gene and Their Experimential Application human insulin gene into the vector pFast Bad. Then it was transformed into E.coli DH 1 OBac which containing shuttle and helper plasmid. Transposition was carried out and the recombinant pBacmid-mINS was constructed in the E.coli DHIOBac. The recombinant plasmid pBacmid-mTh4S was transfected into Sf~ cells by the method of lipofection to generate recombinant baculovirus and expressing human proinsulin in Sf9 cells simultaneously. Human proinsulin was detected in Sf9 cells by Tricine-SDS- PAGE and Western-blot. When the recombinant baculoviruses were used to infect housefly larva, the human insulin was detected by Tricine-SDS-PAGE and Western- blot. The result revealed that baculovirus expression system can be used to express human insulin in housefly. But ifs product was lower than transgenic housefly produced with recombinant expression plasmid pEFI a -ImINS. The transgenic expression products were injected into KM mice intraperitoneally to verify whether it has biological effect or not. The mice were divided into...