Isolation And Identification Of Porcine Lactobacillus With Inhibition Of PEDV Adhesion And The Effct Of S-layer Protein On PEDV Adhesion

Porcine epidemic diarrhea virus(PEDV)is an important pathogen causing piglet diarrhea.Prevention and control of porcine epidemic diarrhea is usually the use of vaccines,serum,and some comprehensive measures,but the effect of a single vaccine is obviously unstable,it cannot solve the problem of prevention and control of the disease.In this paper,we start from the regulation of nutrition,and isolate and identify Lactobacillus strains which can inhibit the adhesion of porcine epidemic diarrhea virus.The separation of bacteria from feces of piglets using MRS selective culture medium,using conventional morphological observation,biochemical tests and 16S rDNA sequence analysis,the bacterial cell virus co-culture,SDS-PAGE analysis,Real-time PCR and other technologies,andusing porcine small intestine epithelial cells(jejunum)system(IPEC-J2)for in vitro cell model,screening can tolerate the intestinal environment,in order to the adhesion ability and inhibit PEDV of Lactobacillus,Lactobacillus and extract the S-layer protein and explore its role in the inhibition of PEDV.Experiment 1.Isolation and identification of Lactobacillus strains from pigletsIn this study,80 bacterial strains were isolated from 20 fresh fecal samples of weaned piglets by MRS selective medium,and then were cultured for the first time.Then,28 bacterial strains,the gram positive and catalytic test negative,were obtained by preliminary screening of bacteria strains.By 16S rDNA sequence analysis,including 10 strains of Lactobacillus reuteri these 28 strains of bacteria,17 strains of Lactobacillus agilis and 1 strains of Enterococcus faecium.The 28 strains can be divided into 6 types according to the characteristics of the colony and the morphology of the cell.The results of phylogenetic tree can be divided into 4 groups.Based on the above results,7 strains with different characteristics and good growth performance were selected from these 28 strains as the following experimental strains,respectively J3(E.faecium),J14(L.reuteri),J16(L.agilis),J19(L.agilis),J21(L.reuteri),J23(L.agilis)and J26(L.reuteri).Experiment 2.Screening Lactobacillus strains with strong adhesion ability and tolerance to intestinal environmentIn this study,7 strains were screened for acid,bile salt and adhesion properties.The results showed that 7 strains of bacteria,the acid and bile salt tolerance ability differences,in a certain range of pH value(1.5,2 and 2.5)under the condition of 2 h,3 strains with good acid resistance,acid resistance is in the order of J26>J16>J21,J26 under the condition of pH 2.5 survival rate up to 91.4%.J3 and J26 had good bile salt tolerance,and the survival rate was 26.1%and in the bile salt environment with the treatment of 2 h,respectively.After 108 CFU/mL of Lactobacillus and IPEC-J2 cells were cultured for about 2 h,the number of probiotic cells adhered to the cell was 104-106 CFU/mL,and the highest adhesion ability was J26,which reached to 1.72%.Experiment 3.The inhibition of PEDV by Lactobacillus and its S-layer proteinIn this study,we tested the ability of 7 candidate strains and their S-layer proteins to inhibit PEDV.According to SDS-PAGE analysis,the molecular weight of S-layer protein was 50 kDa,and the concentration was about 642.4 ?g/mL.Remove from the bacteria,three aspects of determination,S-layer protein,remove of S-layer protein bacterial andbacterial,inhibition ability of PEDV infected cells,each component using 3 different modes of action,group 1:the prevention group(the bacteria were added to the liquid firstly and incubated with the virus);group 2:the repair group(the virus were added to the liquid firstly and incubated with the bacteria);group 3:the competition group(Adding the mixture of bacteria and virus).Cell viability was detected by MTT when the control group appear CPE.The results show that theremove of S-layer protein bacterial,the bacteria and the S-layer protein all working.S-layer protein of virus inhibition on the whole cell virus inhibition rate was the highest,followed by removal of S-layer protein bacteria,S-layer protein has certain effect;different strain differences in inhibition of PEDV infected cells,the inhibitory effect of different mode of action the virus is also very large,the prevention group>the competition group>the repair group;removal of S-layer protein after virus inhibition rate decreased,play a certain role in the process of S-layer protein could be probiotics inhibit PEDV adhesion,and S-layer protein to inhibit virus related ability and concentration.Using Real-time PCR to detect the proliferation of the virus in the cell was also consistent with the results of the above tests.To sxun up,the inhibition of virus by probiotics is the result of the interaction of various components,and the S-layer protein play an important role.Based on the experimental results,the J26 strain was finally selected and identified as Lactobacillus reuteri.The 16S sequence seek by GenBank number KY863554.This strain not only has strong acid and bile salt resistance,and the adhesion capacity of intestinal epithelial cells.It can be used as a candidate strain for the prevention and treatment of porcine epidemic diarrhea,and lay the foundation for the development of the special probiotics for prevention and treatment of porcine epidemic diarrhea.