Molecular Cloning And Constructing Mammary Gland-Specific Expression Vector For Trachea Antimicrobial Peptide Gene Of Chinese Holstein Cows

The defensins play an important role in the innate immune system of host defense mechanisms. In the innate immune system of cow mammary gland, mastitis directly induces the expression ofβ-defensins, enhancing the defense of the quarter which suffered mastitis, indicatesβ-defensins play an important role in the innate immune system of mammary gland. Synthesizing protein by the mammary gland bioreactor is the hot research areas in biotechnology, but specifically and efficiently expression of foreign protein in mammary gland is influenced by many factors, needing more research. The study had carried out of the molecular cloning ofβ-defensin tracheal antimicrobial peptide (TAP) gene, constructed the eukaryotic expression vector for TAP gene, and successfully made the bovineβ-lactoglobulin (BLG) gene 5'flanking fragment directing the expression of the aim gene.1.For the purpose of constructing mammary gland special expressional vector, the bovineβ-lactoglobulin (BLG) gene 5'flanking fragment (3 114 bp) was cloned by PCR amplification. It consisted in part of the gene upstream region about 2 100 bp, the first, the second exon and intron about 1000 bp. The aim fragment was cloned into pMD19-T simple vector, and it was compared with bovine BLG gene, the result indicated the semblance was 97%, and found many factor or protein binding sites and response elements which should direct extra genes to specifically express in mammary gland, and it could be used as constructing mammary gland specific expression vector.2.Tracheal antimicrobial peptide (TAP) gene from cow's mammary gland tissue was amplified by RT-PCR and then cloned into pMD19-T simple vector for sequence analysis. The result of DNA sequencing demonstrated that the cDNA sequence contains an open reading frame (ORF) of 195 bp which shares the highest identity of 93.8% with the TAP of bovine and encodes a 64 amino acid prepro-peptide which contains theβ-defensin consensus sequence of six invariantly spaced cysteine residues. Cloning of the complete ORF of TAP may play an important role in the further designing recombinant P-defensins product.3.The cloned BLG gene fragment was cloned into the expression vector pEGFP-Cl to substitute with the virgin promoter and constructed vector pBLG-GFP-C1,and then the TAP gene fragment was respectively inserted into pEGFP-C1 and pBLG-GFP-C1,constructed vector pEGFP-C1-TAP and mammary gland mammary gland specific expression vector pBLG-EGFP-C1-TAP.4.Primary mammary gland epithelial cells of cow was cultured in vitro, pBLG-EGFP-C1-TAP were transfected into mammary gland epithelial cells with liposome, fusionally expressed green fluorescent protein (GFP) was detected by fluorescence microscope. The fusionally expressed GFP appeared in mammary gland epithelial cells.5.The plasmid pEGFP-C1-TAP was ransfected into COS7 cell, the GFP and the mRNA of TAP was detected 72h after transfection, found that GFP gene expressed in COS7, and the mRNA appeared in COS7 cell.6. The plasmids pBLG-EGFP-Cl-TAP and pEGFP-C1-TAP with lipofemint were directly injected into lactating female rabbit mammary gland respectively, the mRNA of TAP was detected by RT-PCR. We found that the TAP mRNA appeared in rabbit mammary gland and up-regulationally expressed in the quarter transfected with pBLG-EGFP-C1-TAP, which indicated the constructed vectors are useful and may play an important role in the research of the mammary gland bioreactor for defensin.