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Isolation Of Porcine Epidemic Diarrhea Virus And Preparation Of Monoclonal Antibodies Of PEDV

Posted on:2018-09-02Degree:MasterType:Thesis
Country:ChinaCandidate:Q Q LuFull Text:PDF
GTID:2370330575966975Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Porcine epidemic diarrhea is an important infection disease which can induce porcine diarrhea,especially dehydration and high mortality in piglets.PED is widely spread in EU/Asia/North America,causing devastating effect and colossal economic losses.In this study,we isolated 3 virulent strains of PEDV from clinical specimen and prepared the monoclonal antibodies against PEDV.In addition,three epitopes were identified in N protein.This study laid a foundation in further research in PEDV.The main contents of the study were as following:1.Isolation and phylogenetic analysis of variant PEDV strainsThree strains of PEDV,named MS,YZ and SH-A,were isolated from the clinical samples of small intestine of diseased piglets from Sichuan,Jiangsu and Shanghai.To understand the hereditary variability of the three strains of PEDV,S/N/ORF3 gene were amplified and sequenced.Sequence analysis shows there were sixteen novel amino acid mutation in the S gene of the three isolates,which lead to the mutation of epitope and changes in glycosylation site of S protein;two novel amino acid mutation in the N gene and intersetingly there were twelve successive amino acid deletion in N protein of the SH-A strain;four novel amino acid mutation in the ORF gene.The phylogenetic tree analysis based on S/N/ORF3 gene indicated that the three isolates were variant strains belonged to group G2b and has a close phylogenetic relationship with the isolates of BJ2011-1 and HUA-14PED96.This study laid a foundation of the research of the mechanism of variation,vaccine development and differential diagnosis.2.Preparation and identification of monoclonal antibodies against porcine epidemic diarrhea virusThe splenocytes of BALB/c mice immunized with purified porcine epidemic diarrhea virus(PEDV)were fused with SP2/0 cells.3 monoclonal antibodies(MAbs)against PEDV,designated as 3F10,3H8 and 3H10,were screened and obtained by indirect enzyme-linked immunosorbent assay(iELISA).The titers,subtype and specificity of the monoclonal antibodies were identified.The result showed,the ELISA titers in supernatant were 1:1600?1:3200,and titers in ascites were more than 1:2×1 05.The MAbs 3F10 belongs to IgGl subtype with ? chain,the MAbs 3H8 and MAbs 3H10 both belong to IgG2b subtype with ? chain.Western blot results showed that these MAbs could all reacted specifically with PEDV CV777 strain and the N protein expressed in eukaryotic.Indirect immunofluorescence assay(IFA)showed that these MAbs could combined with the natual CV777 strain and MS strain specially.3.Identification of epitop of monoclonal antibodies against N protein of PEDVTo identify the epitope of McAbs,the truncated PEDV N protein were expressed successfully in prokaryotic expression system and the western blot was done.The results indicated that the McAbs 3F10 specifically recognized the epitope 56QIRWRMRRGERI67;the McAbs 6A11 S318GYAQIASLAPNVAALLFGGNVAVRE342;the McAbs 1C9 398HEEA IYDDV406.Meanwhile,the McAbs 3H8 and 3H10 were the conformational epitope in the N protein of PEDV.Protein sequence alignment indicated that sequence of S318GYAQIAS LAPNVAALLFGGNVAVRE342 were conserved in all the reference strains;sequence of 398HEEAIYDDV406 and sequence of 56QIRWRMRRGERI67 were less conserved in all the reference strains.These results laid the foundation for the analysis of PEDV N protein and facilitate the development of diagnostic method for PEDV infection.
Keywords/Search Tags:Porcine epidemic diarrhea virus(PEDV), Sequence analysis, Isolation, Monoclonal antibodies(McAbs), N protion, Epitops
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