Isolation And Purification Of Antibacterial Polypeptide From Endometrial Cells, Gene Cloning Of BNBD5 Of Cow And Prokaryotic Expression

Endogenous antimicrobial peptides are an important component in innate immunity of organism defensins are a kind of positive Endogenous antimicrobial peptides, which contain cysteine-rich and distribute abroad in kingdom of animals and plants. Defensins are a large family among cationic endogenous antimicrobial peptide. As a kind of polypeptide,controlled by specific genic code and induced under specific external conditions.Endogenous antimicrobial peptides can resist the invasion of outside microorganism and kill inner mutant cells . According to the differences of position and connect manner of cysteines,the property of precursors,the position of expression, defensins are divided into five types:α-defensins,β-defensins,θ-defensins, insect defensins and plant defensins,β-defensins are mainly proved to be a antimicrobial barrier bacause they distribute in mucosal epithelial cells of mammals and aves.In this study ,we have used cow as experiment. As a pair of primers were designed and synthesized according to the sequence of antibacterial peptide gene published in GenBank. Total RNA was purified from the bovine endometrial cells with the total RNA isolation kit. Then the DNA fragment of bovine antibacterial peptide was amplified by RT-PCR. After gel purification,the RT–PCR product was cloned into a pMD18-T Vector System. The vector mixtures were transformed into competent high DH5a. Some positive clones were selected on the LB agar-plate with X-Gal,Amp and IPTG. Length of the inserted DNA fragment was confirmed using BamHâ… and Hindâ…¢digestion and PCR, then was sequenced. The result showed that the obtained 138bp DNA fragment is identical to the BNBD5 sequence registered in the GenBank. The cDNA fragment code 45 amino acid,which contained six invariantly aminothiopropionic acid residues. Compared to bovine antibacterial peptide,it shared the highest identity of 99.3% with the BNBD5.We analysised ORF sequences of the Endometrial cows and other species BNBD5 antimicrobial peptidesβ-defensin-5 by DNAStar MegAlign of computer sofeware, It indicated that the relationship of cow to Capra hircus(DQ532360) is the highest (80.4%),to theβ-defensins of Mus musculus (NM₀30734))and Rattus norvegicus (NM₀01037549)is middle(56.5%,31.9%),to theβ-defensins of Anopheles gambiae (EU273600),Apis cerana cerana(EU727272) and Gallus gallus (AY621320, AY621307, DQ677636) is the lowest (mostly less than 20%), This shows thatβ-defensins of ruminants are species-specific.And then target genes ,that was fragments of cow which coded the BNBD5 (138bp), were amplified by PCR with a pair of specifically expessive primers and recombinant plasmid as a template respectively, then cloned into pET28a expession vector. We Constructed prokaryotic expression plasmid pET-BNBD5 and transformed into Escherichia coli BL21.Established highly expression target protein inducing by IPTG. Protein molecular weight is about 10 KDa. in coli BL21 with the same size of expection . the best conditions of inducting were detemined that the induction time was six hours,the temperature was 37℃,and the IPTG Concentration was0.5mM. Under these conditions,the expressed recombinant protein existed in the form of inclusion bodies. By detecting the expressed recombinant protein with the method of agar geldiffusion test,theβ-defensins from the endometrial epithelial of cow , expressed in E. coli ,showed that antibacterial activity is not obvious in vitro.The cow was involved in the experiment. Endometrial epithelial was isolated and acid soluble extract of cow was obtained and antibacterial activity was detected by gel overlay technique , Then it was further isolated and purified by HPLC and analyzed antibacterial activity by agarose radial diffusion assay.The molecular weight was determined by Tricine-SDS-PAGE. The result showed that it was able to effectively kill E. coliBL21 and the Staphylococcus bacteria 26003, its molecular weight was about 14KDa.The discovery of BNBD5 of Endometrial of cow provides evidence for understanding the mucosal defense mechanism and laid the foundation of preventing and curing the endometritis of cow .