Construction Of Mammary Gland-Specific Vector For Human Insulin Gene

In order to construct a high efficient mammary gland-specific vector for using mammary gland bio-reactor to produce human insulin, Using Long PCR technology, we cloned the 5′regulation region of goatβ-casein gene(CSN2) and human insulin gene(INS), base on the human insulin gene we cloned, we constructed a eukaryotic expression vector pBI-GFP which including the 5′and 3′regulation region of bovineβ-casein, the expression sequence of hunan insulin gene, and the report gene (EGFP). These results were expected to lay foundation for the production of human insulin using animal mammary gland bio-reactor. The results of research is as following:1.Human insulin gene was cloned from Human genome with Long-PCR, which was 1624 bp and comprised intact coding regions. Sequence analysis demonstrated that its homology with the relative region of Human insulin gene on GenBank was 99%. Within the sequence we cloned, three site-mutations occurred which could not alter protein's function.2. A pair of primer was designed according to multiple cloning sits of pEGFP-C1 vector and the restriction endonuclease site of theβ-casein promoter sequence. The human insulin was added restriction endonuclease NheⅠand BamHⅠsite.3.Using the strategy of directional clone after double digestion by different endonucleases, while utilizing a eukaryotic vector pEGFP-C1, the non-fusion type of mammary gland expression vector pBI-GFP for human insulin was constructed, which also included antibiotics gene (kana/neo)and report gene (EGFP).4.Two fragments of goatβ-casein gene 5′regulation region was cloned by Long PCR, using which we construct the vector pGGC6.7-GFP.5.The plasmid pGGC6.7-GFP was transfected into mammary epithelial cells cultured in vitro using liposome. After preliminary screening by G418, the expression of report gene EGFP mainly in cytoplasm was observed under fluoroscope.