The Clone, Expressional Vector Combination And Expression In Mammary Epithelial Cells Of Human Lactoferrin Gene

The key resolution for improving the expressional level of foreign gene in transgenic animals is to select appropriate regulation elements, which can regulate foreign gene specifically and high efficiently expressing in mammary epithelial cells, and the foreign protein should be secreted into milk. Thus the great commercial value of mammary gland bioreactor can be realized. In this research, we used the 5' flanking regulation element of bovine beta-lactoglobulin gene as promoter to regulate human lactoferrin cDNA gene expressing in mammary epithelial cells cultured in vitro for verifying the expressional efficiency of foreign gene and rationality of expressional vector. Positive clone cells were selected and purified by the expression of antibiotics gene (Kana/Neo) and report gene (EGFP). So the cells can be used as sources to create transgenic animals by nuclear transplant techniques. The results of research is following:1. For the purpose of constructing mammary gland specific expressional vector, the bovine 3 -lactoglobulin gene 5' flanking fragment was cloned by PCR amplification. It consisted of part of the gene upstream region about 645bp> the first exon and the first intron about 804bp. After PCR product recovered and purified, the aim fragment was cloned at T site of pMD 18-T Vector. The fragment was sequenced, and it was compared with bovine BLG gene variant A, bovine BLG gene variant B, goat and sheep BLG gene. The results indicated the homology were 98.55%, 99.79%, 90.70% and 90.09% respectively. The nucleotide acid sequence was analyzed by compute, and it was found many factors or protein binding sites and response element such as mammary specific binding factor (MSBF), nuclear factor-1 (NF-1), retinoic acid response element (RARE)and TPA response element (TRE). It suggested the cloned regulatory element should direct foreign genes to express specifically in mammary gland epithelia cells, and it could be used to construct mammary gland-specific expressional vector.2. The ATG which was the translation start code of bovine f3 -lactoglobulin gene 5' flanking fragment consisting in part of the gene upstream region about 645bp, the first exon and the first intron about 804bp was mutated to AAG by PCR amplification. The mutant fragment was marked as MBLG fragment.3. Human lactoferrin (hLF) cDNA gene which consisted of 2259bp nucleotide acids was cloned by RT-PCR from human mammary cancer tissue, and then subcloned into pMD 18-T vector. The fragment was sequenced, and it was compared with lactoferrin cDNA gene of human, mouse, cow, and goat registered in GenBank. The results indicated the homology were 99.7%, 82.19%, 81.79% and 87.28% respectively. Compared with human lactoferrin cDNA sequence showed five allele mutation sites and one unrecognized mutation site. This fragment was marker as LTF fragment.4. MBLG fragment was connected with LTF fragment by recombinant PCR and then were subcloned into pMD 18-T vector. The positive recombination plasmid was marker as pBL vector, ABL fragment was obtained by digesting pBL vector with restrictive enzyme Ase I . pEGFP vector was also digested with restrictive enzyme Ase I . And after the 5' end of digested product was phosphatased, it was connected with ABL fragment using T4 DNA ligase. So the mammary gland-specific expressional vector was constructed, which consisted of report gene (EGFP gene), antibiotics gene (Neo gene), regulatory element (bovine beta-lactoglobulin gene 5' flanking fragment) and foreign aim gene (hLF cDNA gene)5. The recombinant expression vector of human lactoferrin gene was successfully transfected into bovine mammary epithelial cells cultured in vitro using electrotransfection. Positive clone cells were selected and purified by G418, and was observed the expressing of report gene (EGFP) under fluoroscope.6. This research vestified it was feasible that EGFP gene used to construct mammary gland-specific expressional vector.