Construction Of Mammary Gland-Specific Vector For Reconstructed Gene Of Human Insulin

In order to construct a high efficient mammary gland-specific vector for using mammary gland bio-reactor to produce human insulin,we cloned the 5′regulation region of goatβ-casein gene and human insulin gene using Long PCR technology, base on the reconstructed gene of human insulin we cloned, we constructed a eukaryotic expression vector pEBH 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 1696 bp . Three fragments of Human insulin gene were cloned from Human insulin by PCR which included A (262bp),B (218bp),C (928bp),connected C and A, The three fragments comprised intact coding regions. Sequence analysis demonstrated that its himology 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 site.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 pEBH for human insulin was constructed,which also included antibiotics gene (kana/neo)and report gene (EGFP). it could also ensure EGFP and aimed gene express respectively.4. The vector pEBH was transfected into mammary epithelial cells cultured in vitro using liposome. After G418 fastness screening, the expression of report gene EGFP mainly in cytoplasm was observed under fluoroscope. PCR identification demonstrated that the vector had integrated into mammary epithelial cells.5. The fragments of goatβ-casein gene 5′regulation region was cloned by Long PCR, Sequence analysis on NCBI with blastn indicated that the fragments have the homology of 99.0 % . Besides that, goatβ-casein gene 5′regulation region have been cloned. These two fragments comprise beta-casein genomic gene full sequence, which can be to construct high level mammary gland-specific expression vector.