The Prokaryotic Expression And The Construction Of Mammary Gland-specific Vector Of Yak Beta-defensin5

The object of this study was yak beta-defensin5(BNBD5), which prokaryoticand eukaryotic expression was studied respectively. Prokaryotic expression was usedpET-32a (+) as vector, then transformed the constructed prokaryotic expression vectorinto BL21(DE3) and detected the antibacterial activity of recombinant protein in vitro.The mammary gland-specific expressional vector of yak BNBD5, which was partialconsisted of the5′and3′regulation regions of cow β-casein, was constructed withcDNA of yak BNBD5. In order to study the rationality of expression vector，thevector was transfected into mammary epithelial cells. The results of this research asfollowing:1． The mature peptide encoding region and open reading frame of yak BNBD5werecloned from lung tissues by RT PCR. After PCR, products were recovered andpurified, the aim fragments were cloned at T site of pMD19-T Vector. The resultsof sequence analysis indicated the mature peptide encoding region and openreading frame of yak BNBD5were138bp and209bp, respectively. Theencoding region encodes a polypeptide of45amino acids and has six conservedcysteines. Phylogenetic relationships showed that yak BNBD5shared the highestnucleotide homology (86.9%) with Bos taurus.2． The constructed prokaryotic expression vector was transformed into BL21(DE3)and induced to expression by IPTG. The antibacterial activity in vitro ofexpression product was detected after purify. The results showed that anapproximate25kDa expression product of yak BNBD5was obtained afterinduced by IPTG, and0.08mg.mL-1expression product after purify has obviousantimicrobial activity against S.aureus and E.coil.3． The5’ regulation region of cow β-casein was cloned by PCR, including of partialupstream region, the first exon (1947bp) in5’ untranslated region. Moreover, the3’ regulation region of bovine β-casein was cloned also, including of the last intron and the last exon (620bp). By the strategy of directional cloning, theeukaryotic expression vector of mammary gland-specific（pEGFP-BNBD5）was constructed successfully, which consist of resistant gene and EGFP reportergene and target gene (yak BNBD5).4． The constructed mammary gland-specific expression vector (pEGFP-BNBD5)was transfected into yak mammary gland epithelial cells cultured in vitro withliposome2000and screened cells by G418. We found the EGFP gene expressedafter transfect under UV microscope. A pair of specific primers was designed todetect the positive cloned cells through PCR. All above results indicated that theconstructed vector is useful and the vector has been integrated into mammaryepithelial cells.