Construction Of Mammary Gland Expression Vector For Human Leptin And Its Initiatory Expression In Cell

In this research, the mammary gland-specific expression vector of human leptin wasconstructed, which was partial consisted of conducting elements and expression sequence.The former includes the 5′and 3′regulation region of bovine β-casein, and the latter is themodified human leptin cDNA. In order to study the rationality of expression vector and theexpressional efficiency of human leptin gene, the vector was transfected into mammaryepithelial cells cultured in vitro. And the results of research is as following:1. The 5′and 3′regulation region of bovine β-casein was respectively cloned by recombinant PCR and common PCR. The 5′regulation region was about 2 826 bp, and consisted of partial upstream region about 1.7 kb, the first exon in 5′untranslated region and partial first intron. The 3′regulation region was 620 bp, and consisted of last intron, the last untranslated exon and the 3′flanking region about 150 bp.2. The two cloned fragments were sequenced, and was compared and analyzed in database of EMBL and Genbank. The result indicated that the homology of the two fragments with the relative region of bovine β-casein gene was 99% and 98% respectively, which proved that the two fragments were elements of bovine β-casein gene. The two fragments was analyzed by several kinds of computer biosoft, and it was found many response elements and many sites bound by transcription and translation factors, such as sites bound by STAT (MGF), C/EBP, milk box and poly A signal for adding the tail. All the above suggested that the cloned regulation regions could direct foreign genes to express specifically in mammary gland epithelia cells at a high level, and it could be used to construct mammary gland – specific expression vector.3. Using the strategy of directional clone after double digestion by different endonucleases, the commonly primary expression vector for mammary gland was constructed, through the fusion of cloned 5′and 3′regulation regions. Utilizing the restriction sites of pMD 18-T vector and peculiar primers, the multi clone sites between 5′and 3′regulation region wasiv 人瘦蛋白乳腺表达载体的构建及细胞表达的初步研究 constructed for the insertion of foreign genes.4. Using the strategy of directional clone after double digestion by different endonucleases, the requisite expressing sequence of human cDNA was merged with the commonly primary expression vector. The fusion vector was sequenced, and the result showed that there were no mutants in expressing sequence of human leptin cDNA, which also had a proper reading frame.5. Using the strategy of directional lone after double digestion by different endonucleases, while utilizing a eukaryotic vector pEGFP-C1, the non-fusion type of mammary gland expression vector pEGFP-BL for human leptin was constructed, which also included antibiotics gene (kana/neo) and report gene (EGFP). The plasmid pEGFP-BL was verified by PCR and endonuclease digestion. And the result showed that the insertion place of leptin expressing sequence in the vector is just right.6. The plasmid pEGFP-BL 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. A pair of detecting primers was designed, and the positive cloned cells were studied through PCR. The result indicated that the whole expression plasmid had been integrated into the chromosome of positive mammary epithelial cells.7. After the primary investigation on the cell expression, it could be proved that the expression vector pEGFP-BL, which had the instructing elements of bovine β-casein regulating region, could successfully transfect into mammary epithelial cells, and the report gene expressed in according with presupposition. In brief, the rationality and the practical value of the constructed mammary gland-specifi...