Development Of Monoclonal Antibody Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea is an viral infectious disease caused by Porcine epidemic diarrhea virus(PEDV).The clinical manifestations of the disease are diarrhea,vomiting,dehydration,etc.,and its clinical features are similar to that of porcine transmissible gastroenteritis.The first outbreak in Britain in 1971.Porcine epidemic diarrhea in suckling piglets is the most serious harm,the death rate of piglets infected can be more than 90%,incubation period of 5-8 days,more than in winter.PEDV is the RNA virus of coronavirus family,and the swine epidemic diarrhea caused by PEDV has caused immeasurable loss to the pig industry in most areas of our country.It can find a quick and accurate method to diagnose the disease,which plays an important role in the prevention and control of porcine epidemic diarrhea.Proliferation and purification of PEDV monoclonal antibody antigenhe monoclonal antibody can play an important role in the analysis of the antigenic structure and function,the study of antigenic variation,and the diagnosis and control of porcine infectious diseases.Based on the research of monoclonal antibody,colloidal gold technology is more rapid,simple and efficient.Amplification and purification of porcine epidemic diarrhea virus(PEDV)is the basis for the preparation of monoclonal antibodies against PEDV.Vero cells were used to amplify PEDV cells in this experiment.Virus inoculation after Vero cells can be observed in 36 h lesions,were collected and stored in the Vero cells reached 90%,more than 3 times after repeated freeze thawing,centrifugation supernatant,the supernatant after high-speed centrifugal precipitation,precipitation of liquid collected by the method of PEG concentration of crude extract pure.After concentration,it is the immune antigen used in the experiment.The purified samples were identified by RT-PCR and fluorescent quantitative PCR.Establishment of hybridoma cell line with PEDV monoclonal antibodyBy matrix titration method to establish the indirect ELISA method for detection of PEDV antibody showed that the PEDV package is the most suitable concentration of 2 g/ ml,the best coating time and temperature is 4 DEG C for the optimal enzyme combination diluent for 1%BSA-PBST,horseradish peroxidase labeled Goat anti mouse Ig G(enzyme labeled anti two)the optimum dilution was l: 4000,reaction time was 30 min.The immunized mice were immunized with purified PEDV,and then immunized every 7 days,and the titer was measured by indirect ELISA.The cell fusion can be achieved when the titer of the serum is more than 105.On the third day before the fusion,the immune system with the highest serum titer was selected to enhance the immunity.After three days,the mouse spleen cells were fused with SP2/0 myeloma cellsPreparation of monoclonal antibody against PEDVScreening with PEDV and PEDV antigen purified N protein was subcloned to positive hybridoma cells using limited dilution method,screening,and double positive results showed that: screened 3 stable hybridoma cell strains secreting anti PEDV antibodies(3C10,5G8,5F4).The positive monoclonal antibodies were screened into 96 well plates and then transferred to the 24 hole cell culture plate into 6 cell plate,then to the bottle cells followed by expanding culture,in the process of expanding training in the cryopreservation of positive hybridoma cells.Preparation of monoclonal antibodies against PEDV by ascites induction.Biological identification of the monoclonal antibody against PEDV,successfully obtained 1 strains of hybridoma cell lines stably secreting PEDV antibodies(3C10),chromosome number is higher than that of either parental cell chromosome number;antibody titer of ascites was 1:1×105,the concentration of 4.55 mg/ml.The monoclonal antibody 3C10 was a subtype of Ig G2 b,which was able to bind specifically to PEDV and reacted only with the structural protein N.The results of stability assay showed that 3C10 could secrete antibodies stably.This study laid a foundation for the further establishment of PEDV antibody blocking ELISA and colloidal gold immunochromatographic assay.