Construction Of Mammary Gland-Specific Vector For Human Finger-Domain Lacking T-PA And Its Expression In Cells

In this research, the mammary gland-specific expression vector pEBT was constructed, which was consisted of regulating elements regulation region of bovine ?-casein and expression sequence finger-domain lacking t-PA cDNA. Then pEBT was transfected into bovine ear fibroblast cells cultured in vitro and the expression cell strains were abtained. Based on pEBT, BMARs and matrix attachment regions in the human serpin gene(HMARs) were inserted. Therefore, the other two mammary gland-specific expression vector for finger-domain lacking t-PA were constructed. High-level expression cell strains were to be provided for the mammary gland bio-reactor by nuclear transfer. The results of research is as following: 1. Human finger-domain lacking t-PA cDNA was cloned from Human placenta RNA with RT-PCR, which was 1 640 bp and comprised intact coding regions. Sequence analysis demonstrated that its homology with the relative region of Human finger-domain lacking tissue-type plasminogen activator on GenBank was 99 %. Within the open reading frame, three site-mutations occured which could not alter protein's function. 2. By the strategy of directional cloning,　finger-domain lacking t-PA was fused with the commonly primary expression vector pBCP, then was cloned directionally into expression vector pEGFP-C1, the mammary gland-specific expression vecto(rpEBT) for finger-domain lacking t-PA was constructed, which included resistant gene and EGFP reporter gene, it could also ensure EGFP and aimed gene express respectively. 3. The vector pEBT was transfected into bovine ear fibroblast cells cultured in vitro using liposome. After G418 fastness screening, the positive cells low-level expressed stably. PCR identification demonstrated that the vector had integrated into bovine ear fibroblast cellular chromatin. 4. Bovine mammary matrix attachment regions was cloned from bovine genomic DNA, it was about 1 190 bp. Sequence analysis indicated that it involved some important functional regions, such as AT-Richness Pattern,ORI Pattern,ATC Rule and so on. 5. BMARs was inserted into pEGFP-C1 and located in the downstream of EGFP, and the recombination expression vector pBE was constructed. Stop codon TAA was introduced to avoid fusion protein. Then pBE was transfected into bovine ear fibroblast cells. After G418 fastness screening, stable expression cells were observed under fluoroscope. The results indicated that BMAs could improve gene expression level. 6. Based on the vector pBE and pHE(pEGFP-C1-HMARs), by the strategy of directional clone, the other two mammary gland-specific expression vector of finger-domain lacking t-PA were constructed. This purpose of research is to obtain high-level expression cell strain under the regulation of matrix attachment regions after transfection. 7. Three fragments of bovine beta-casein (about 9.7 kb) were cloned by PCR which included 1.7 kb 5' flanking sequence, eight exons and partial the eighth intron. Sequence analysis on NCBI with blastn indicated that the fragments have the homology of 99.0 %, 98.0 %and 98.0 % respectively with the corresponding gene sequence of bovine beta-casein. Besides that, 600 bp polyA of bovine beta-casein have been cloned. These four fragments comprise beta-casein genomic gene full sequence, which can be to construct high level mammary gland-specific expression vector.