Cloning And Prokaryotic Expression Of Swine Blood Antibacterial Peptide And Mature Peptide Gene PMAP-23 And PG-2

The antibacterial peptide is one part of living things experiences and observes oneself defense the systematic,which is a kind of broad existence and small molecule polypeptide in living things body inner,the characteristic property is that antibiosis broadly,the physics and chemistry character stabilizes.Prokaryote, plant,animal(include an invertebrate and be not a vertebrate) all can produce the a antibacterial peptide being composed of gene code,the ribose body.Since two close structure and positive charge of antibacterial peptide molecule,it can duct boring a hole but forming an ion on bacterium cytoplasm film,bring about bacterium cytoplasm membrane structure destroy,arouse compatriot inner water-solubility matter large amount of effusion,but lead to bacterium death ultimately.This antibiosis distinctive mechanism can't make pathogenic microorganism bacterial strain mutant but produce resistance to drugs,the innocuous side effect,this will resolve two big troublesome difficult problems and provide new approach,which the tradition antibiotic,such as penicillin,strengthens gradually to the bacterium's resistance and medicine that to be left.Being more important is that the antibacterial peptide does not almost act on eukaryote,only,the effect on prokaryote and eukaryote happened examine,have important exploitation as a result applying a prospect.At present,for reducing leaving medicine in the livestock body and improving animal food security,the function carrying out the peptide knowing and designing one kind of the natural antibiosis coming to adjust the animal body inner method's on the antibacterial peptide function that the livestock body produces at ease now just appears most important,among them,pass the genetic engineering means clone and express the antibiosis peptide gene,reform the genetic engineering composing the antibiosis peptide,it is being taken more seriously.PMAP-23 and PG-2 are two kind of antibacterial peptides in the pig blood fluid.PMAP-23 and PG-2 belong to the Cathelicidins family,stems from the marrow cell,the Propeptides as the composited form store up in peripheral PMNs pellet(polymorphonuclear neutrophil white blood cell).When the PMNs activates and escapes the pellet,the endocardial elastic proteinase cut the mature peptide from the propeptide.The PMAP-23 mature peptide is composed of 23 amino acid residues,assumes sends the card structure.PMAP-23 can resist the G~+ and G~-.The PG-2 mature peptide is composed of 16 amino acid residues,not only has the function to the G~+ and G~-,simultaneously also can resist fungus(F) and the pouch membrane virus(V),the PG-2 anti-microorganism activeness maintains at the physiological NaCI density,not suppresses by the extracellular positive ion or theblood serum ingredient.1.pMAP-23 andpG-2 gene cloneThe test is that extracts general RNA in pig marrow,and designs the site according to pig source antimicrobial peptide PIGPMAP23P and the PIGLAP maturity that pMAP-23 and the pG-2 gene by Gen Bank collected.By RT-PCR gain pMAP-23 and the pG-2 gene,the purification is joined linking up with clone carrier pMD18-T,is identifies that again by the fact that pair,the enzyme making the preliminary appraisal by all means,the enzyme cutting success,as a result of measuring order.The array analyses the mature peptide PMAP-23 gene overall length fmally indicating PlGPMAP23P426bp,the array consanguinity sex that the mature PIGLAP peptide PG-2 gene overall length reports among 362 bp and Gen Bank is 100%,the gene has no an abrupt change.2.pMAP-23 and pG-2 gene prokaryotae are expressedThe be sure to reclaim the purpose fragment,good with preparation pET28a of enzyme empty space carrier joins pMAP-23 andpG-2 gene masculine gender clone outcome.The sun character granule that pET28a-pMAP-23 and pET28a-pG.2 structure is shifted to after again,by all means,the enzyme identifies that,links up after the pair successfully,to bacillus coli BL21 feel state,30℃,4 hour of IPTG guidance, classics SDS-PAGE electrophoresis identify that.Result indicates fusion protein,the pMAP-23 gene and the pG-2 gene induce the fusion protein being biggish than 18 KDa a bit out.Again 0-7 hours of classics,30~C guidance,the stopper elects the best guiding condition to be 30℃,6 hour.The experiment studies achievement can be that the experiment provides the experiment basis specifically for pig common illnesses pathogenic microorganism extraneousness.Extracorporeal experiment research result may test that the admeasurement establish a basis for activity and activity strengthen within PMAP-23 and the PG-2 body.