The Clone Of BLG Regulation Region And The Research Of Common Expression Vector For Mammary Gland

In order to express foreign gene in mammary gland of mammds, the commonmammary gland expression vector pEBF12 had been constructed, using the elementsof 5' and 3' regnlation of BLG, antibiotics gene (kana/neo) and report gene(EGFP).There are many multi clone sites in pEBF12 between 5' and 3' regulation gene, forinserting the different foreign gene. Through the antibiotics gene (kana/neo) andreport gene, the positive cell could be selected and purified. The expression vectorwas an ideal transgene constructin, and could make convenient the research onmammary gland bioreactor. The related reseach resullts were as following.1. The 5' regulation fragment which including exonⅠ, intron Ⅰand exonⅠ and 3' regulation fragment of BLG were cloned by PCR, the former was 3 572 bp, and the latter was 1 003 bp. After being recovered and pufified, the fragments BF1and BF2 had been cloned into T sites of pMD18-T vector. The recombinant plasmid pBF1,pBF2 were digested by BamHⅠ,which proved the cloning of pBF1 and pBF2 was successive. The fragment BF1 had been sequenced and it was compared with bovine BLG gene variant A,bovine BLG gene rariant B,goat BLG gene and sheep BLG gene.The results indicated the homology were 96.55%, 99.79%,92.70%和 92.09%. Using the same way, the homology of fragment BF1 were 97.45%,99.83%,93.70%和 92.59%. The nucleotid acid sequence was analyzed by compute and it was found many factors or protein binding sites and response slement,sach as RARE, MSBF,NFH, TRE, MGF binding site, MFBF and GRE. All this suggested the clone regulatory 5' and 3' regulatory elenments could direct foreign genes to express specifically in mammary gland,and it could be used to construct mammary gland-specific expression vector.2. After digested by restriction enzyme, the fragment BF1 and BF2 had been organized into pMD 18-T vecotor, and the primary common expression vector (basic common expression vector)pBF12 had been constructed, which was proved successful by AseⅠ. After digested by AseⅠ,the fragment BF12 was separatedAbstract from the primary expression vector pBF12, then was inserted into pEGFP-C1, which was digested by AseⅠ too. After this , the common expression vector pEBF12 of mammary gland-specific was constructed, which was proved successful by digested by AseⅠ. Using constructed common mammary gland expression vector pEBF12, theforeign gene could cloned into pEBF12 conveniently and under the control ofregulating elements, then the mammary gland expression vector for different foreigngene could be constructed. In a short , the common expression vector of mammarygland-specific contructed by this research had an important value for research andappliacation.