Preparation And Identification Of Monoclonal Antibody Against Porcine Epidemic Diarrhea Virus N Protein

Porcine Epidemic Diarrhea Virus(PEDV)is an RNA virus belonging to the genus Coronavirus and has a high similarity with SARS coronavirus and porcine gastroenteritis virus(TGEV).Acute diarrhea could be occured in pigs while infected with PEDV for the first time,The feces are green or gray jets with a fishy smell,and some of sick pigs will vomit.Suckling piglets have a higher mortality rate,whereas adult pigs have a lower mortality rate but a higher morbidity and more contagious.After the sow recovers,it will produce corresponding antibodies to protect the piglets.In PEDV-infected pigs,antibody detection in vivo showed that the level of anti-PEDV N protein was the highest.In addition,the most conserved gene is also the N gene,so the N protein is the preferred target protein for establishing PEDV molecular diagnostic methods.In view of this,E.coli was used to express the N protein of PEDV HUB2018 isolated strain in this study,and the purified PEDV N protein was subcutaneously injected to immunize BALB/c mice three times to preparemonoclonal antibodies.Monoclonal antibodies will lay the foundation for further research on the pathogenic mechanism of PEDV and the establishment of rapid and highthroughput diagnostic detection kits,such as ELISA,IFA and colloidal gold test strips.The main research contents and results are as follows:1.Cloning and prokaryotic expression of N gene of PEDV HUB2018 isolated strainThe N gene of PEDV HUB2018 isolated strain was amplified by RT-PCR,and the length of 1 326 bp target band was amplified.The PEDV-N gene was cloned into the plasmid PET-30a(+),and the recombinant plasmid PET-30a(+)-PEDV-N was constructed.The recombinant plasmid PET-30a(+)-PEDV-N was successfully expressed in the BL-21 E.coli expression strain after induction with 1 m M IPTG.A fusion protein with the same molecular weight as the expected 58 k Da and a His-tag was obtained.After protein purification,the purified PEDV N protein concentration was 7.127mg/m L.The results of Western blot showed that it could also react specific antibody response with His-tag antibody and PEDVpositive sera respectively.2.Establishment of an indirect enzyme-linked immunosorbent assay(ELISA)detection method for PEDV N protein antibodyThe purified and high-purity PEDV N protein was used as an antigen to coat an ELISA plate,and a highly specific indirect ELISA antibody detection method was established.The concentration of each reactant,ambient temperature and action time are optimized and determined as follows: the optimal coating concentration of antigen is 0.5μg/m L,the optimal antigen coating time is 9-12 hours at 4°C;the optimal blocking solution is BSA,the optimal BSA concentration is 1%,and placed in a incubator at 37°C for 2 hours;the optimal dilution of the sample serum is 1:160,andplaced in a incubator at 37°C for 1h;the optimal dilution of the enzyme-labeled secondary antibody is 1:9000,and placed in a incubator at 37 ℃ for1 h.3.Preparation of monoclonal antibody against N protein of PEDV HUB2018 isolated strainThe purified and high-purity PEDV N protein was emulsified with Freund’s complete adjuvant(the first time)and Freund’s incomplete adjuvant(the second and third time),and used as an immunogen after emulsification to immunize mice with subcutaneous injection.The best standard of immunized mice is 6-8 weeks female BALB/c mice Within a month,after three times immunizations,the serum antibody levels of the mice should be detected to ensure that the immunized mice meet the requirements of cell fusion experiments.Under strict sterile conditions in the laboratory,spleen cells were removed from mice immunized three times and subjected to cell fusion experiments with SP2/0 myeloma cells.The positive cell lines were screened by the PEDV N protein indirect ELISA antibody detection method,and after 3 rounds of continuous limiting dilution screening,a positive cell line with a high specific antibody level was obtained.After Western blot verification,a hybridization was obtained.The specific antibody level of this strain of hybridoma cells is high and can be stably secreted,and it is named E5-E5.4.Identification of monoclonal antibody against N protein of PEDV HUB2018 isolated strainThe cultured hybridoma supernatant was collected,and Western blot results showed that the supernatant could specifically react with PEDV N protein.Hybridoma cells were subtyped and chromosomally counted.The isotype identification results showed that the heavy chain type was Ig G1 and the light chain type was Kappa.The hybridoma cells of this strain have about 90-110 pairs of chromosomes.5.Mass preparation of monoclonal antibody against N protein of PEDV HUB2018 isolated strainFemale BALB/ C mice were intraperitoneally injected with E5-E5 hybridoma cells to prepare ascites in large quantities,and antibody titers were determined after ascites samples were collected.The results showed that the titer of the monoclonal antibody was 1:102400.In summary,monoclonal antibodies against PEDV N protein were successfully prepared,which laid the foundation for the research on the pathogenic mechanism of PEDV and the establishment of diagnostic detection kits.