Establishment Of ELISA And Quantum Dot Immunochromatographic Assay For Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)is an intestinal coronavirus of pigs.It can cause vomiting,diarrhea,abortion of sows and the decline of feed utilization rate of fattening pigs.In particular,it does serious harm to piglets and the mortality can reach 100%,and has become one of the most serious intestinal pathogens faced by the pig industry.At present,the prevention and control of PEDV still faces problems such as inaccurate clinical diagnosis methods and poor vaccine effect.Establishing a rapid,simple and high-throughput diagnostic method is very important for the prevention and control of PEDV.Firstly,the N protein key antigen epitope of PEDV was analyzed and synthesized by bioinformatics method,and the monoclonal antibody based on this epitope was developed;The recombinant N protein of PEDV was prepared by prokaryotic expression,and its monoclonal antibody was developed;Based on the above monoclonal antibodies,the detection method of double antibody sandwich ELISA and quantum dot immunochromatography test trip were established.The research contents are as follows:(1)The antigenic epitope of N protein of CH/HNQX-3/14 strain was analyzed by bioinformatics method.Balb/c mice were immunized with the synthetic epitope peptide coupled with the vector.A hybridoma cell line(designated as 9C4)which can stably secrete anti PEDV monoclonal antibody was obtained by hybridoma technology.The 9C4 belongs to Ig G2b+?subclass and the antibody titer can reach1?(1.28×10~4),can specifically recognize PEDV.(2)According to the N gene sequence of PEDV CH/HNQX-3/14 strain published by NCBI,specific primers were designed to obtain the amplified fragment.The PCR product was connected to p ET-32a(+)vector,and the recombinant plasmid p ET-32a(+)-N was constructed.The recombinant plasmid was transformed into E.coli BL₂₁(DE3)competent cells.The recombinant protein was induced at 20?by IPTG with a final concentration of 0.4 m M,and purified by Ni-NTA sefinose resin.Balb/c mice were immunized with the purified recombinant N protein,and their spleen cells were fused with NS0 cells.Monoclonal antibodies(designated as 2A6E6)were screened by indirect ELISA,The 9C4 belongs to Ig G1+?subclass and the antibody titer can reach 1?(1.28×10~5).(3)Based on the above two monoclonal antibodies,9C4 was used as the coating antibody and 2A6E6 was HRP labeled as the detection antibody.By optimizing the reaction conditions,a double antibody sandwich ELISA was established.The sensitivity test showed that the minimum detectable amount PEDV is 10^4.7TCID₅₀/m L.And the specificity test shows that this ELISA method can react specifically with PEDV without cross reaction with PRRSV,CSFV,FMDV,PDCoV and PCV.It has good specificity and repeatability and the coefficient of variation is between 1.89%?8.43%.The coincidence rate between the detection results of 90 clinical samples and RT-PCR is 96%.(4)Coupling the water-soluble carboxyl quantum dots(CdSe/ZnS)with 9C4 monoclonal antibody.The coupling of quantum dots and antibody was successfully identified by direct observation,agarose gel electrophoresis and immune layer analysis by optimizing the coupling conditions.Optimize the coating concentration of T line and C line and the detection method of quantum dot immunochromatography test trip was established.The sensitivity test showed that the minimum detectable amount PEDV is 10^4.4TCID₅₀/m L.The specificity test showed that the test trip could specifically detect PEDV without cross reaction with other viruses,and had high repeatability.The coincidence rate between the test trip and RT-PCR was 98%.