| Defensins,as an important part of the body's natural immune system,widely distributed in epithelial cells of various tissues and organs of mammals.They are a class of cationic endogenous antimicrobial peptides rich in cysteine.Defensins not only directly resist pathogenic microorganisms,but also have immunomodulatory effects.The structure,function,and expression of ?-defensins1 in most mammals have been understood,but the studies on horse,Ass,and mule have rarely been reported.Primers were designed by the conserved sequence of the ?-defensinl cDNA of horse and ass published by NCBI.The cDNA sequences of horse,ass and mule were obtained by RT-PCR.The terminal rapid cloning technique(RACE)was used to amplify the existing sequences of the three animals.In order to understand the expression of p-defensinl in horse ass and mule,the tissues and organs of three animals were detected by RT-qPCR(RealTime Quantitative PCR)technique(tongue,trachea,esophagus,lung,fundus,duodenum,pyloric,jejunum,ileum,rectum,colon,bladder,kidney,spleen,spinal cord).The prokaryotic expression system of p-defensin was established.Through the establishment of expression method in vitro and the study of purification and bacteriostasis of prokaryotic expression products,the bacteriostasis effect of beta defensinl in vitro was preliminarily proved,which laid a theoretical foundation for future defensin bio-industrialization.1.The full-length genes of ?-defensin 1 of ass,horse,and mule are 501 bp?449 bp?and 474 bp respectively.Compared the existing mRNAs in the UniProtKB database,found that horse,ass and mule were in the same evolutionary stage,and the homologous conservation between horse,ass and mule was determined.The evolutionary status of?-defensinl in horse was determined and identified in mammals by comparing the comparison of amino acids and construction of evolutionary trees with other mammals(rats,chimpanzees,brown rat,pig,goat,sheep,dog).An open reading frame(ORF)which consist 207 bases was found,including signal peptide(1-22),pre-fragment(22-33),mature peptide(33-69)and six cysteine residues in the specific site of mature peptide.2.Real-time qPCR method was used to detect the expression of defensin in different tissues and organs of horse,ass and mule.The results showed that defensins were expressed in different degrees in the mucosal layers of various organs in three animals.The expression of pylorus,skin and kidney of donkey was significantly higher than other organs;In horse,the expression of heart,kidney of fundus,esophagus and kidney was the highest in mule.The higher expression of defensin in digestive tract and kidney indicating that feeding pattern may affect the expression of defensin.3.The eBD-1(?-defensingl of horse)prokaryotic expression system was successfully constructed by cofusion expression method with ELP(Elastin-Like Protein,ELP).The fusion recombinant plasmid pET-22b-eBD-1-ELP was obtained by cloning the optimized and synthetic eBD-1-ELP gene to the prokaryotic expression vector pET-22b.Fusion protein expression was induced by IPTG at different temperatures and concentrations.The results of SDS-PAGE showed that the fusion protein could be expressed at 37?,25? and 18?.The expression of fusion protein was highest at 37?,IPTG concentration of 0.5 Mm/L for 6 h,and the protein purification buffer pH was 8.2.The eBD-1 having was successfully obtained after further purification and concentration and had apparent inhibitory effect on Escherichia coli and Staphylococcus aureus. |
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