Production of egg yolk antibody (IgY) against recombinant porcine epidemic diarrhea virus COE protein
Hu, QingsongZhang, XiaofeiLi, LvmuXu, FazhiLv, Yongbiao
Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series ofclinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of Sprotein 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 antibodyhas never been reported before, here it is described a method for the production of anti-COE IgY, which could be appliedin 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 diarrheavirus 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 andintroduced 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 animalhandling procedures were performed under veterinary supervision and following the recommendations of the local lawsand regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and...(AU)
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