Establishment Of Indirect ELISA And The Preparation Of Monoclonal Antibody Against Senecavirus A

Senecavirus A(SVA)can cause a kind of vesicular infectious disease in pigs,which can be infected by pigs of all ages,especially the newborn piglets,whose mortality rate is as high as 30%-70%.The mortality rate of sows is low,but the harm of production performance caused by infection can not be ignored,which will cause certain economic losses.The common clinical symptoms are vesicular lesions of nose,mouth and hoof crown,which lead to ulcer and ulceration,and there are symptoms of claudication and diarrhea.Since 2015,SVA outbreaks have occurred in many provinces of China,so the pig blister disease caused by SVA has attracted the attention of the aquaculture industry.Vaccine immunization is the most effective way to prevent SVA,and detection is the main means of prevention and control.However,there are no commercial vaccines and detection kits available in the domestic market at present,so it is particularly important to develop vaccines and detection reagents for SVA.1.Establishment of indirect ELISA method based on SVA VP2 protein According to the SVA sequence registered in GenBank(GenBank:MN885796),A pair of specific primers of VP2 gene was designed and the target fragment was cloned into pET32a(+)expression vector,prokaryotic expression,purification and activity identification of soluble protein.The concentrated recombinant VP2 protein(rVP2)used as the coating antigen to coat the ELISA reaction plate,and the ELISA reaction conditions optimized to establish an indirect ELISA method based on SVA VP2 protein.The specificity,sensitivity and repeatability of this method were tested,and 60 pig sera were detected and compared with the virus neutralization test.500 clinical pig sera were tested.The results showed that the VP2 gene with a size of 852 bp was amplified in this experiment,and the expressed rVP2 was about 52 kD,which had good reactivity.This method has no cross reaction with the positive serum of common pig disease pathogen,and can specifically detect SVA antibody.The highest dilution ratio of SVA positive serum detected by this method is basically consistent with the neutralization test,and the sensitivity is good.The coefficient of variation of repeatability detection within and between batches is less than 10%,and the repeatability and stability are good.The coincidence rate between 60 sera and virus neutralization test was 93.3%.The positive detection rate of 500 clinical pig sera was 10.6%.This study provides a technical means for serological detection of SVA,and also provides technical support for screening monoclonal antibodies.2.Preparation and identification of monoclonal antibody against SVA VP2 protein Mice were immunized with SVA antigen.The hybridoma technology was used to fuse the spleen cells of mice with higher titer and the myeloma cells cultured in advance to observe the growth state of the hybridoma cells.On about 7-10 days,the supernatant could be taken according to the cell growth state and screened by indirect ELISA.The subclones were detected by limited dilution method,and the subtypes,specificity,stability and indirect immunofluorescence identification were performed.The selected hybridoma cells were injected into the abdominal cavity of mice according to a certain amount,ascites was collected,purified and desalted by automatic protein purifier,and SDS-PAGE,stability,specificity and blocking detection were carried out.The results showed that the selected subtype of 4H5 monoclonal antibody was IgG2a,which had no cross-reactivity with FMD(O/A),PRV(gB/gE),PRRSV,PEDV,CSFV,and pET32a(+)carrier protein,with good specificity.After 20 passages,the antibody can still be secreted,which has good stability.The purity of purified ascites was high,and the titer reached 1:1228800.It did not cross-react with FMDV(O/A),PRV(gE/gB),PRRSV,CFSV,and pET32a(+)carrier protein,but only reacted specifically with SVA with good specificity.After 8 passages,the titer can still reach 1:1228800,which has good stability.After blocking detection,it was effectively blocked by the specific positive serum induced by SVA,with a certain blocking property.This monoclonal antibody lays the foundation for the study of late blocking ELISA method.