Study On An Anti-PEDV Protein And Its Secretory Cells In The Mucus Of Small Intestine Of Pigs

The mucus layer,which is produced by goblet cells of the intestine,presents the first line of defense against virus infection.Mucus is secreted by the intestinal secretory cells,such as goblet cells and Paneth cells.Mucus is a complex secretion and contains a large number of proteins resistant to bacteria and viruses,which plays an important role in innate mucosal immunity.In the current study,the small intestinal mucus of pigs was demonstrated to have the property of defending against porcine epidemic diarrhea virus(PEDV)infection.As the dominating component of intestinal mucus,mucin 2(MUC2)was also shown to exhibit a powerful anti-PEDV property.Subsequently,several proteins with high expression levels were isolated from the small intestinal mucus of pigs and then an anti-PEDV protein with a molecular weight of about 70 KD was identified by plaque reduction neutralization test(PRNT).Then,after preparing the polyclonal antibody targeting anti-PEDV protein,we found that the anti-PEDV protein was secreted by intestinal goblet cells by immunohistochemical(IHC)staining.The expression level of anti-PEDV protein in the intestinal tissue of newborn piglets was lower(P<0.05)than that in the intestinal tissue of adult pigs,which was consistent with a high incidence of PEDV in newborn piglets.Besides,the anti-PEDV protein was analyzed by Liquid chromatography-tandem mass spectrometry(LC-MS/MS),which identified three proteins with high probability.Finally,the expression level of anti-PEDV protein in intestinal tissue was assessed after oral administration of Bacillus subtilis(B.subtilis).The content of this study was divided into three parts:1 Evaluation of the antiviral effect of small intestinal mucus in pigs and screening of antiviral proteinIn the present experiment,firstly,small intestinal mucus of pigs was collected and showed in vitro to exhibit strong antiviral activity against PEDV after plaque reduction neutralization test.The further Western Blot and qRT-PCR assays showed that the principal components of the small intestinal mucus,MUC2,played an important role in defending against PEDV infection.Then,the proteins with a molecular weight of 25-100 KD were collected using Native-PAGE.The proteins in this molecular weight range could effectively inhibit PEDV infection after PNRT.Finally,to identify which protein was involved in the antiviral activity,the proteins with high expression levels were collected.By using PRNT,a protein with a molecular weight of about 70 KD was proved to possess good anti-PEDV property.2 Preparation of polyclonal antibody targeting antiviral protein and distribution of antiviral protein-secreting cells(APSC)Firstly,for the anti-PEDV protein isolated from small intestinal mucus,the rabbitderived polyclonal antibody against anti-PEDV protein was prepared.The agar diffusion test was performed and the antibody titer in serum was 1:32.By colocalization staining of immunohistochemistry(IHC)and periodic acid Schiff(PAS),it was found that anti-PEDV protein was secreted by intestinal goblet cells which were located at the basement of the intestinal villi.And the anti-PEDV protein was concentrated in the central position of the goblet cells.Then,the distribution of anti-PEDV protein in the intestinal tissue was detected.The expression levels of anti-PEDV protein in the intestine between newborn piglets and adult pigs were also analyzed by Western Blot and IHC.Results showed that anti-PEDV protein isolated from intestinal mucus was distributed in all parts of the intestine.The antiPEDV protein had higher expression levels in the rectum than in the small intestine(P<0.05).Also,there were significant differences(P<0.05)in the expression levels of anti-PEDV protein between the newborn piglets and the adult pigs.Finally,by analyzing using LCMS/MS,three proteins with high probability was identified.3 Effect of oral administration of B.subtilis on antiviral protein and its secreting cells of pigsOral administration of B.subtilis could enhance intestinal mucosal immunity of piglets.To furtherly study the effect of B.subtilis on anti-PEDV protein and its secreting cells.In the present study,newborn piglets were administered orally with B.subtilis.Firstly,IHC staining was performed to detect the expression levels of anti-PEDV protein.Then,PAS staining was performed to show PAS positive goblet cells in the intestine.Finally,qRT-PCR test was performed to detect the expression levels of mucus-related genes.Result showed that compared with control group,oral administration of B.subtilis could increase the expression of anti-PEDV protein in the intestine(P<0.05).At the same time,the number of goblet cells had a significant increase(P<0.01).Also,the expression levels of mucus-related genes were significantly increased(P<0.05).In summary,these results provided a new theoretical basis for furtherly exploring the mucus and antiviral protein.