Investigation Of The Infection And Monoclonal Antibody Preparation Of Porcine Epidemic Diarrhea Virus In Shandong Region

Since October 2010, PED(Porcine epidemic diarrhea) outbreak serious in China, after the United States, Mexico, Canada, South Korea, China Taiwan also has erupted PED in different degree, which has caused great economic losses to the global in the pig industry.This research mainly has carried out the following three aspects for PED:1. Infection situation investigation of porcine epidemic diarrhea virus in Shandong regionFor the analysis of prevalence and genetic variation of porcine epidemic diarrhea virus( PEDV) in Shandong region, 510 clinical samples were collected during 2013-2015 and detected by RT-PCR based on N gene, and S1 gene fragments of 16 positive samples were sequenced and analyzed by bioinformatics software. The results showed that the PEDV infection rate increased from 57.8% in 2013 to 77.94% in 2015. The seasonal incidence of PEDV in addition to the winter and spring seasons, the summer also showed a high incidence trend in Shandong region, and the infection rate was various in different regions. Sequence analysis showed that the nucleotide homology of the isolated 16 strains of PEDV S1 gene ranged between 91%-99.8%, and the amino acid homology was 87.8%-99.7%. The homology of nucleotide and amino acid of 16 Shandong isolates was 90.5%~100% and 88.4%~99.9%,respectively, with 15 domestic and foreign reference strains. The molecular epidemiological analysis of S genetic showed that 20 foreign reference strains, 25 domestic reference strains and the 16 Shandong isolates were divided into two different evolutionary branches of G1 and G2. Three isolates of Shandong isolates belonged to G1 branch, which had close distance with CV777 classical strain. The other 13 isolates were in G2 branch, which was given priority to with epidemic strains in recent years. Shandong isolates mainly were in G2 branch,which explained the poor immune protection of CV777 vaccine strains in Shandong region.This study analyzed the molecular epidemiology and variation of PEDV from the aspects of molecular epidemiology which provided a reference to guide the disease prevention of PEDV in region.2. Preparation and identification of monoclonal antibody against PEDVOne of antigen epitopes on S protein(83~276aa) and one of antigen epitope on N protein(126~328aa) of a strain of variant porcine epidemic diarrhea virus were amplified andconnected into the prokaryotic vector pET28 a, and the constructed recombinant plasmid pET28a-Mt S and pET28a-N were transformed in E.coli BL21 respectively. The recombinant protein of Mt S was expressed in soluble form and the recombinant protein of N was expressed in inclusion body form with IPTG. BALB/c mice were immunized by the purified protein Mt S and N of PEDV respectively. Two monoclonal antibodies of Mt S as 1E5 and 5B7 and three monoclonal antibodies of N as 2E1, 2B3 and 2B5 were obtained by lymphocyte hybridoma technique and nominated.The two monoclonal antibodies for S protein, 1E5 and 5B7 were characterized as IgG subtype and ? chain by indirected ELISA, with cell supernatant titres of 1:256 and 1:100,with ascetic titres of 1:12500 and 1:10000, respectively. Western blot results showed that two strains of MAbs could recognize the recombinant Mt S. Indirect immunofluorescence assay(IFA) showed that 5B7 could react with classical strains(CV777) and variant strains of PEDV, while 1E5 only reacted with variant strains, The results showed that the two monoclonal antibodies could identify different antigen sites.The three monoclonal antibodies for N protein, 2E1, 2B3 and 2B5 were characterized as IgG1, IgG2 b and IgG2 a subtype respectively and all for ? chain by indirected ELISA, with cell supernatant titres of 1:256, 1:128 and 1:128 and with ascetic titres of 1:12500, 1:10000and 1:10000, respectively. Western blot and indirect immunofluorescence assay(IFA) showed that three monoclonal antibodies have good reactivity and specificity.3. Development of an indirect ELISA for N proteinAn indirect ELISA for detecting PEDV antibodies was developed using purified recombinant N protein this research. By optimizing the reaction conditions, the indirect ELISA reaction conditions ultimately determined for: The optimal antigen of N protein was set at 1.5?g/mL, and the coated time was overnight at 4?. The best sealing buffer was 5%skimmed milk,and the sealing time was 1.5h at 37?. HRP conjugated anti-pig IgG reaction conditions were set at 1:3000 dilution and incubated for 1h at 37 ?. The dilution of the testing serum was defined as 1:100 and the reaction time was 1.5h at 37?. TMB substrate was used for coloration at room temperature for 15 min. It was judged as positive when the testing serum OD450 ? 0.2008, as negative when the testing serum OD450? 0.1822, and as suspicious between 0.1822 and 0.2008. Tests showed that the indirect ELISA has goodspecificity, repeatability and compliance, which could be used to monitor of PEDV antibody levels in clinical practice.