Preparation Of Monoclonal Antibodies Against N Protein Of Porcine Epidemic Diarrhea Virus And Development Of An Antigen Capture ELISA For Detection Of Antibodies To PEDV

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious disease in swine. The main clinical manifestations are watery diarrhea, vomiting, dehydration and appears to be more severe in piglets, which causes a significantly high mortality of suckling piglets. In China, PED was first reported in 1976, and the prevalence has continuously enhanced recently, causing serious economic losses to pig industry. In this study, we constructed the recombinant N protein of porcine epidemic diarrhea virus by using baculovirus and E.coli expression system, and four McAbs specific to N protein were obtained successfully. By using one of the four McAbs, we developed an antigen capture ELISA method for detection of antibody to PEDV, which provided an important tool for PEDV dignosis and serological survey. The main contents of the research were as following:1. Construction and identification of recombinant baculovirus expressing N protein of PEDVThe virus nucleocapsid protein gene was amplified by RT-PCR and cloned into donor plasmid pFastBac. After identification by PCR, double enzymes digestion and sequencing analysis, the correct recombinant donor plasmid pFastBac-N was transformed into competent DH10Bac cells, which contains a shuttle vector Bacmid. After the recombinant bacmids were screened and identified by two rounds of blue-white plaque assay and PCR, a correct recombinant shuttle was constructed, named Bacmid-N. The Bacmid-N was transfected into Sf9 insect cells, and then recombinant baculoviruses were obtained and confirmed by Western blot. It showed that N proteins of PEDV were expressed in Sf9 cells successfully at a size of about 58KD. The recombinant protein was purified using Ni-NTA affinity chromatography, and the relatively pure protein was obtained. It could be used the development of diagnosis method and subunit vaccine against PEDV.2. Preparation and identification of monoclonal antibodies against the nucleocapsid protein of PEDVIn this study. the virus nucleocapsid (N) protein gene was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a. The recombinant N protein was expressed in E.coli BL21 cells by induction of IPTG, and confirmed by SDS-PAGE and Western Blot assays with the size of about 58KD. Then the recombinant protein N was purified by Ni column. The monoclonal antibodies (MAbs) were prepared by fusing mouse myeloma cells (SP2/0) with the spleen cells from BALB/c mice immunized with the purified recombinant N protein. Four hybridoma cell lines secreting MAbs were screened by indirect enzyme-linked immunosorbent assay (ELISA) and named as 1C9、4C8、4F8 and 6A11, respectively. Western blot and IFA results showed that the four MAbs reacted specifically with the recombinant N protein and PEDV. The isotypes of four MAbs all belong to IgG1 subtype with κ chain. Their ELISA titers in supernatant were 1:1600～1:6400, and titers in ascite were more than 1:1.024 105. The results indicated that these MAbs may be useful for developing the diagnosis methods and studying on the pathogenicity of PEDV in the future.3. Development and preliminary application of an antigen capture ELISA for detection of antibody to PEDVAn antigen capture ELISA method was developed by using an anti-PEDV N protein MAb as coating antigen and purified porcine epidemic diarrhea virus as the capture antigen. The reaction conditions were as follows:The coating MAb concentration was set to 2μg/ ml, and incubated for 2h at 37℃ and overnight at 4℃; the blocking buffer was 5% free-fat milk, and the blocking time was 3h at 37℃; capture antigen concentration was 5 μg/mL, and incubated for 3h at 37℃; pig serum samples for detection were diluted 1:50, and incubated for 1h at 37℃; goat anti-swine IgG-HRP was used at a dilution of 1:2000 and incubated for 45 min at 37℃; The TMB substrate was added and incubated at 37℃ for 10 min before terminated with stop solution. Using S/P value as the criteria by setting positive and negative controls. Serum samples with S/P ratios more than or equal to 0.4 were considered as positive, those with S/P ratios less than 0.3 were considered as negative, and the others were suspicious. The ELISA method was proved to have no cross-reaction with positive serum of PRRSV, PRV, PCV2, CSFV and FMDV. The intra-assay and inter-assay variation coefficient were both less than 10%. The diagnostic sensitivity, specificity and accuracy of the ELISA were 94.6%,91.3% and 93.3%, compared with domestic commercial ELISA kit for detection of antibody to porcine epidemic diarrhea virus. A total number of 308 clinical serum samples were detected using this method. The positive rate was 89.29%. It indicated that this method has good sensitivity, specificity and accuracy, and could be used for antibody detection and epidemiological investigation of PEDV.