Primary Studies On The Animal Mammary Gland Bioreactor For Human Lysozyme

Human lysozyme (hLYZ) hydrolyzes the glycosidicβ-l , 4 linkage between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall. Because of the direct exposure of polypeptidoglycan on the outside of the cell wall, hLYZ can directly lyze Gram-positive bacteria and indirectly lyze Gram-negative bacteria in the presence of sIgA or complements. Furthermore,hLYZ has many other activities such as diminishing inflammation,detumescence,repairing putrescence tissues,improving blood supply in local tissues,reducing pus and anti-virus. So it has wide application in medicine and foodstuff industry.To prevent,treat Dairy cow mastitis and improve Dairy cow anti-disease,the expression of human lysozyme in the cow mammary gland is improved by transgenic Dairy cow. Dairy cow mastitis will be decreased. In this research , the 5' regulation region of bovineβ-lactoglobulin was cloned by PCR. We used the cloned 5' regulation region of bovineβ-lactoglobulin as promoter and modified human lysozyme cDNA as the target gene to construct mammary gland specific expressional vector pBEL of human lysozyme gene. In order to study the rationality of expression vector,the vector was transfected into mammary epithelial cells cultured in vitro by means of electroporation. Postive cells were selected and purified by G418. So the cells can be used as sources to create transgenic animal by nuclear transplant techniques. The results of research are following:1 The 5' regulation region of bovineβ-lactoglobulin was cloned by PCR,including of partial upstream region,the first exon and the first intron(about 993bp)in 5'untranslated region. After PCR product was recovered and purified,the aim fragment was cloned at T site of pMD 18-T Vector. It was compared with Bos taurus BLG variant B precursor gene,Bos taurus BLG gene variant B and B.taurus beta-lactoglobulin gene. The results of Sequence analysis indicated the homology were 99%,99% and 98% respectively. Computer Biosoftwire analysis demonstrated that the cloned fragment included some response regulatory elements and sites bound by expression which direct foreign genes to express specifically in mammary gland epithelia cells at a high level. So it could be used to construct mammary gland-specific expression vector.2 By the strategy of directional cloning,the eukaryotic expression vector (pEGFP- hLYZ)was constructed successfully,which consist of resistant gene and EGFP reporter gene and target gene (Human lysozyme). The plasmid pEGFP-hLYZ was verified by PCR and endonuclease digestion. And the result showed that the insertion place of hLYZ in the vector is just right. And no variants happen.3 By the strategy of directional cloning,the cloned 5' regulation region of bovineβ-lactoglobulin as promoter replaced partial CMV of the eukaryotic expression vector (pEGFP- hLYZ)to construct the mammary gland-specific expression vector (pBEL). This vector consisted of antibiotics gene (Ampr/neo) and report gene (EGFP). And it could also ensure EGFP and aimed gene express respectively.4 The constructed mammary gland-specific expression vector (pBEL) was transfected into bovine mammary gland epithelial cells cultured in vitro with electroporation and screened cells by G418. Under UV microscope after 48h we found the EGFP gene expressed,which indicated the constructed vector is useful.