Production of Egg Yolk Antibody (IgY) against Recombinant Porcine Epidemic Diarrhea Virus COE Protein

Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series of clinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of S protein possessing the main B cell epitope, is able to encode proteins with reactogenicity to induce the production of neutralizing antibodies. IgY was found to reduce the mortality in piglets after challenge exposures. Anti-COE IgY antibody has never been reported before, here it is described a method for the production of anti-COE IgY, which could be applied in the treatment for the porcine epidemic diarrhea virus (PEDV) infection. Materials, Methods & Results: A PEDV strain was isolated from a clinical sample. The COE ORF (Open reading frame,ORF) was amplifi ed by PCR and iserted into the pMD18-T clone vector. The isolated was defi ned as Porcine epidemic diarrhea virus strain JS-HZ2012 subtype by sequencing, the clinical sample was defi ned as the nucleic acid sequence has a 99.5% homology with that of PEDV CV777 strain. And then the COE ORF was subcloned into pET-32a by T4 DNA ligase and introduced into the E.coli Bal21 (DE3). COE protein was produced by the induction of the E.coli Bal21 containing pET32a-COE with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant COE protein (rCOE) fused with His-tag was analyzed by SDS-PAGE and detected by western-blotting using anti-His monoclonal antibody. The rCOE was purifi ed by Ni affi nity purifi cation chromatography under denature condition and dialyzed against PBS. The concentration of the rCOE was determined by BCA method. After immunnizing the chickens with rCOE , All animal handling procedures were performed under veterinary supervision and following the recommendations of the local laws and regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and purifi ed by Saturated ammonium sulfate gradient sedimentation, and analyzed by SDS-PAGE. The activity and specifi city of the IgY antibody were analyzed by indirect ELISA and western-blotting. Furthermore the neutralization activity of the IgY antibody was analyzed by virus-neutralization test. SDS-PAGE analysis showed that the rCOE fused with His-tag was a molecular of 37 kDa, and the rCOE could be recognized by the anti-His monoclonal antibody. The IgY antibody isolated by chloroform extraction method and analyzed by SDS-PAGE showed the IgY mainly contained two parts 22 kDa and 66 kDa, which corresponded to light and heavy chain, respctively. In additional, some lower bands around 40 kDa were presented on the gel. The anti-COE IgY reached to 1:12800 after the third immunization, and the antibody levels could last for a long time. The anti-COE-IgY could recognize the COE specially in western blotting, while no reaction was seen with the low molecular. The virus-neutralization test showed the anti-COE-IgY had neutralizing activity, and the neutralization titer reached to 1:12, and the control was less than 1:2. Discussion: The COE protein were expressing with high effi cacy, and the anti-COE IgY could recognize the COE fraction dicated that it possess the satisfi ed immunogenicity. The anti-COE-IgY could neutralize porcine epidemic diarrhea virus partly, and the nuetralization titer reached to 1:12. The result in this study indicated that anti-COE IgY against PEDV could be an alternative way of supplementing prophylactic measures like colostral antibodies from sows. Also, it could be signifi cant at preparation of the products of preventing the porcine epidemic diarrhea.