β-defensin Gene Expression And Characterization In Intestine Of Three Varieties Of Porcine

Defensins is an most important component for mammals’ innate and adaptiveimmunity, which have a quality to kill a broad spectrum of pathogens, and a uniquemechanism to kill bacteria too. It has become a hotpot in the biologic areas research.β-defensins is expressed on the primary barriers of the organism such as mucosalepithelia,preventing the colonization of host tissues by pathogens.In order to explore the defensins gene expression and location in intestinebetween three varieties of porcine breeds in Anhui area, just as Wannan black swine,Wannan spotted swine and Crossed swine also. Firstly, study on the β-defensins geneexpression in intestine between three kinds of porcine by using quantitative Real-timePCR. Secondly, explore the distribution and location of β-defensins in situhybridization. Furthermore, we isolated and purifies antimicrobial peptides which killa broad spectrum of pathogens from small intestine of Wannan black porcine. Theresults are presented as follow:1. Study on the β-defensins gene expression of porcine intestine by Real-timeqPCR.Based on the gene sequence of pBD-1, pBD-2, pBD-3in GenBank, the specificprimers of β-defensins were designed. The fresh tissues which kept in liquid nitrogenwere been for RNA extraction, cDNA synthesis, DNA sequencing, defensinsexpression in different part of porcine small intestines by Real-time qPCR. Thesequencing results of Anhui origin porcine have100%similarity compared with theGenBank published sequences of porcine β-defensins. All three β-defensins have ahigher expression in ileum. The expression of Wannan black porcine has significanthigher than others(p<0.01).2. Study on the distribution and location of β-defensins in intestine in situhybridizationThis paper selects the representatively expressed types: pBD-1and pBD-2,designs a oligonucleotide probes， by using SABC-FITC to detect location ofβ-defensins of intestine in situ.It was found that pBD-1mRNA in the mucosalepithelium and submucosa have hybridization signals, pBD-2mRNA expression inlamina propria and submucosa in situ hybridization.3. The purification and antibacterial test of porcine antimicrobial peptides ofsmall intestine The whole porcine small intestinal tissues and mucosa tissue were collected, andthe tissues were washed with cold sterile saline. The whole intestine tissue andmucosa tissue were broken into small pieces and mixed with ice-cold aqueous5%acetic acid (1:1v/v); the mixtures were then homogenate by using a homogenizer at4℃for overnight. The precipitates were removed by centrifugation and thesupernatant was loaded onto Bio-Rad ion exchange chromatography. The antibacterialactivity was determined by agar dispersion method, Using Tricine-SDS-PAGEelectrophoresis of the purified product was measured for molecular weight, thepurified peptides’ concentration was measured by Bradford method. The resultsshowed that high purity antimicrobial peptides were produced after multiple-step acidextractions, chromatography which possessed high antibacterial activities duringpurification. Protein concentrations were determined as0.15mg/mL.In summary, in order to find the breeds of porcine natural disease resistanceadvantages provides a theoretical basis.In the study, tentatively comparison has been made to explore the breeds ofintestinal β-defensin expression and expression in the intestine in situ detection. In theileum β-defensin expression is the highest,The expression of Wannan black porcinehas higher significant than other breeds(p<0.01). Besides, purifies the antimicrobialpeptide from the intestine of black porcine, which can kill Escherichia coli,Salmonella enteric.