| Senecavirus A(SVA)belongs to the genus and family Picornaviridae.Infection with SVA can cause idiopathic blister disease in pigs,with symptoms such as fever,anorexia,claudication,intact or ruptured vesicles in the nose,snout or oral mucosa,ulcerative lesions in the hoof wall and coronal zone,and significantly increase the mortality rate of newborn piglets.Clinical symptoms of SVA are easily confused with other infectious diseases such as foot-and-mouth disease and swine vesicular disease.The VP2 protein of SVA has good immunigenicity and is a critical target protein for clinical diagnosis and vaccine development.To provide technical support for further development of specific diagnostic tools,specific monoclonal antibodies(McAbs)against VP2 protein were prepared using hybridoma technology and a blocking ELISA method for detecting SVA specific antibody was esTablelished based on McAbs.The VP2 structure of SVA was analyzed by Expasy software,and the primary and secondary structure of SVA VP2 protein can be displayed.The B cell epitopes were predicted from linear epitope,β-turn,surface accessibility,flexibility,antigenicity,and hydrophilicity and the result showed that the amino acids 38-58,140-159,164-174,180-194,221-234,and 247-256 are potential epitopes.The soluble VP2 protein was successfully expressed through the prokaryotic expression system.The optimal conditions for protein expression were the culture speed of 120 rpm,the induction time of 8 h,the induction temperature of 30℃,and the inducer IPTG concentration of 0.5 mmol/L.High levels of SVA VP2 specific antibodies could be detected in the serum of mice immunized with purified VP2 protein,and the levels of IFN-y and IL-2 in the serum are significantly increased,which indicated that the VP2 protein has a good immunogenicity.In this study,BALB/c mice were immunized with purified recombinant SVA VP2 protein.By hybridoma technology,two hybridoma cells secreting McAbs against SVA VP2 protein were obtained after 3 to 5 times of subcloningand screening using the esTablelished ligation ELISA method.The number of chromosomes of McAb hybridoma cells is between 98 and 104.According to the subclass identification,the McAbs secreted by the two hybridoma cells are all of the IgG1,which respectively recognize different epitopes.Both McAbs can specifically bind to SVA VP2 protein bywestern blotting and immunofluorescence assays,respectively.The virus neutralization test proved that the two McAb strains 1E9 and 2D4 have neutralizing activity,and the neutralizing titers are 1:20 and 1:40,respectively.The titers of the culture supernatants of the two McAb hybridomas 1E9 and 2D4 were up to 1:2560 and 1:5120,respectively,and the ascites titers were 1:1000×56 and 1:1000×55,respectively.The mice ascites was purified by the caprylic acid-saturated ammonium sulfate method.The affinity constants of the McAb of 1E9 and 2D4 were 1.1×10~8 and 2.6×10~8,respectively,showing high affinity.The high-quality enzyme-labeled antibody 2D4~E,prepared by the glutaraldehyde cross-linking method,was taken as the labeled antibody.A blocking ELISA method for detecting SVA antibodies was esTablelished.Optimized conditions such as a coating concentration of 4 μg/mL,an enzyme-labeled McAb with 3200 times dilution,serum with 4 times diluton,1%BSA blocking solution and dark color for 10min with TMB,and the method showed better specificity,sensitivity and reproducibility.164 serum samples from clinical pigs were detected using the indirect ELISA reported in the literature and the competive ELISA,respectively,and the total coincidence rate was 98.8%(162/164).The blocking ELISA method was used to detect 372 samples in several areas of Hebei province,and the positive rate was 7.8%(29/372)and the incidence rate is relatively high in spring and summer.The results showed that the blocking ELISA method can be applied to clinically detect serum antibodies from pigs infected by SVA,providing technical support for diagnosis and epidemiological investigation of SVA infection. |
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