Isolation And Purification Of Antimicrobial Peptide From PMN, Gene Cloning Of LAP Of Yak And Prokaryotic Expression

Endogenous antimicrobial peptides are an important component in innate immunity of organism and defensins are a kind of positive endogenous antimicrobial peptides,which contain cysteine-rich and distribute abroad in the kingdoms of animal and plant.Defensins are a big family among endogenous antimicrobial peptides.As a kind of polypeptide, controlled by certain genic code and induced under certain outside conditions,ABP can resist the invasion of outside microorganism and kill inner mutant cells.According to the differences of the position and connect manner of cysteines,the property of precursors,the position of expression,defensins are devided into five subfamilies:α-defensin,β-defensin,θ-defensin,insect defensin and plant defensin.β-defensins are proved to be a component of antimicrobial barrier because they distribute prominently in muscosal epithelial cells of mammals and aves.The yak was involved in the experiment.Firstly,this investgation definited the drift density of Yak PMN and others major hemocyte by using cells demixing liquor that was dispensed by definite ratio 40%ficoll and 76%cystografin.Then groped a species Segregation methods of PMN by using 67.5%and 87.5%percoll,namely,percoll gradient Centrifugation,Neutrophils were isolated from yak peripheral blood.The crude extraction from PMN was isolated respectively through a way of extractionion by 5%acetic acid. The results of it's biologic activity detection showed that it's has different biologic activity. As for it's in vitro antibacterial activity,the crude extraction from PMN of yak showed a certain antibacterial activity to Bacterium.The crude extraction showed feeble hemolysis activity to red blood cell of sheep and rabbit.The crude extraction from PMN of yak showed feeble hemoblast anticoagulation activity to blood cell of rabbit,while to blood cell of sheep,they were less strong(+).In the process of extractionion and purification of the crude extraction from PMN of yak,22 apices were collected by Bio-Gel P-10 Polyacrylamide gel filtration and RP-HPLC,and 9 out of 22 apices showed antibacterial activity.9 apices showing antibacterial activity were selected to be analyzed by mass spectrograph.In this study,we have used yak as experiment.Total RNA was extracted from the tougue epithelial tissue of a yak and the cDNA encoding yLAP was amplified by the reverse transcription-PCR(RT-PCR ) with the pair of primers which were designed according to the cDNA conserve sequences of reported ruminents'(cow)β-defensins.The purified RT-PCR product was cloned in pGM-T vector.After the restriction endonuclease pattern analysis of reombinant plasmid,the cDNA was sequenced and the result of cDNA sequencing demonstrated that the yLAP is belong to the family ofβ-defensins because the cDNA contain a open reading frame(ORF) of 192 bases which encoded a 64 amino acid prepro-peptide and the prepro-peptide contained theβ-defensin consensus sequence of six invariantly spaced cysteine residues.We analysised the base pair distribution,amino acid homology by computer software.All this established base for in future research. Allignment results indication that there have highly identical with alexin antimicrobial peptide through protein level in NCBI Blast,The results of pair distances of the cDNA and prepro-peptide sequences of yLAP and otherβ-defensins in other mammals and aves by computer software indicate that the relative relationship of yLAP to tarandus,goat and bubalis is highest(91.7%,88.5%, 93.1%),to theβ-defensins of pig,ass and horse is middle(64.5%～73.3%),to theβ-defensins of canis and rhesus monkey is lowest(mostly less than 30%).Therefore,we can conclude that the relationship of yLAP is closer to tarandus,goat and bubalis'sβ-defensins with the sequences of cDNA and prepro-peptide.The result demonstrates the diversity of defensins evolution.Then target genes,that were CDS fragments of yLAP which coded the yLAP(207bp),were amplified by PCR with a pair of specifically expressive primers and the recombinant plasmid as template respectively,then cloned into pET-28a(+) expression vector,We constructed protein expression vector of yak Lingual antimicrobial peptide and transformation into Escherichia coli(BL21),established highly expression target pretion.Protein molecular weight is 17KDa in BL21.Positive plasmid was identified and transformed into the competent cell BL21(DE3) which was induced by IPTG.The optimal conditions were determined that the induction time was six hours,the temperature was37℃,the pH was 7.2 and the IPTG concentration was 1 mmol/L.Under the conditions,the expressed recombinant protein existed in the form of inclusion bodies,amounting to 31.5%of protein of the induced recombinant bacteria at the presence of IPTG.By detecting the expressed recombinant protein with the method of agar geldiffusion test,the beta-defensins from the tougue of yak,expressed in E.coli,showed antibacterial activity in vitro.The discovery of yLAP provides a powerful evidence for understanding mucosal defense mechanism of yaks.