The Biological Functions Of Sheep Synthetic Antimicrobial Peptide NK-lysin And It’s Treatment Studies In Salmonella Contamination Chicken

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) in the body’s immune defensesystem and neutrophils, macrophages, play a crucial role together. Antimicrobial peptideNK-lysin present in the body cells and NK cells, CTL, and human granulysin (GNLY) arehomologous polypeptide is the major component of the innate immune response, the connectingpins of the adaptive immune response and the immune response ties. In recent years, the abuse ofantibiotics drug residues and the growing problem of bacterial resistance, a serious threat to thedevelopment of animal husbandry and research and development of new antimicrobial drugs forhuman health has become an urgent need. Antimicrobial peptides in the field of research hasbecome a hot spot in the new alternative antimicrobial drugs has broad application prospects onaspects of traditional antibiotics. Study NK-Lysin granulysin homologue has entered clinicalstage and the NK-Lysin research is rare. In this paper, sheep NK-Lysin proteins experimentallystudied prokaryotic expression, chemical synthesis, antimicrobial function and bacterialinfections of animals therapeutic effect, for clinical application of antimicrobial peptides providesa scientific basis.1、prokaryotic expression of sheep antimicrobial peptide NK-LysinE. coli is a first for recombinant protein production host bacteria, which not only has a cleargenetic background, training simple, transformation and high transduction efficiency, growth andreproduction of fast, low-cost, large-scale production quickly. The experiment was based onGenBank (accession number JX559777) in NK-Lysin gene primers were designed, RNA wasextracted from sheep in RT-PCR amplification, the amplified fragments were ligated intopGEM-T vector and the recombinant vector digested sequencing, will verify that the correcttarget gene is inserted into the prokaryotic expression vector, transformed into E. coli DE3competent cells, monoclonal picked positive bacteria, fungus activation shake again, addingIPTG induction. Successfully constructed pGEM-T-NK-Lysin and pGEX-4T-1-NK-Lysinrecombinant vector and E. coli expression system pET series of NK-Lysin(pET28a-NK-Lysin-DE3, pET30a-NK-Lysin-DE3, pET32a-NK-Lysin-DE3), but no expression ofNK-Lysin in the pET series of E. coli expression system.2、the biological function of the chemical synthesis of sheep antimicrobial peptide NK-LysinIn order to study the mechanism of inhibition of sheep antimicrobial peptide NK-Lysinregional structure and its derivative activity and biological function of the polypeptide. Theexperiment was based on the protein secondary structure prediction sheep antimicrobial peptideNK-Lysin and the active region of similar structure derived antimicrobial peptides, using onlinesoftware (http://bioinf.cs.ucl.ac.uk/psipred/and http://swissmodel.expasy.org/) analysis, design,and the amino acid sequence of the antimicrobial peptide selected three polypeptide segments, named NK7652-10, NK559777-18, NK7652-23, while measuring their minimum inhibitoryconcentration of the antibacterial spectrum and by MTT assay their killing activity of tumor cells.The results showed that: a spiral structure, Loop a cyclic structure, such intramolecular disulfidebonds for activity of the cationic antimicrobial peptides is essential, NK55977-18and NK7652-23were gram-negative bacteria (Escherichia coli and Salmonella) have significant antibacterialactivity. Sheep antimicrobial peptide derived peptide NK-Lysin of NK55977-18, NK7652-23isan antibiotic alternative medicine provides a foundation material.3、the synthesis of antimicrobial peptides for treatment of salmonella infection in chickensIn order to verify the synthesis of synthetic sheep sheep NK-Lysin role in the treatment ofchickens, chicks Salmonella infection model antimicrobial peptide treatment. First, a standardcurve and determine the activation of Salmonella absorbance and CFU counts, intranasally,intraperitoneally inoculated chicks quantitative manner, while setting the control group. TheSalmonella infected chickens were randomly divided into A, B, C, D four groups: A group ofuntreated, group B subcutaneous peptide NK559777-18, group C subcutaneous peptideNK7652-23, D group subcutaneous ampicillin sodium, clinical symptoms and death records ofeach group, the final necropsy lesions observed chicks visceral organs and histological changes inthe analysis of synthetic peptides of animal clinical treatment of bacterial infections. The results:A group all died within2h,6h group B in survival after infection was50%in group C survivalrate was100%, D group at6h after infection survival is0, the polypeptide NK559777-18andNK7652-23salmonella in chickens protective effect during the acute infection played much betterthan antibiotic ampicillin, peptides bacterial toxins in the body and in the process played a crucialrole. The experimental results for the study of novel antibiotic alternative medicine provides abasis for addressing the abuse of antibiotics and bacterial resistance issues more likely.