Establishment Of An Indirect ELISA Method For Detection Of Senecavirus A Antibodies

Senecavirus A(SVA)belongs to the genus Senecavirus of the Picornaviridae family and is a single-stranded,positive-stranded RNA virus without an envelope.The SVA genome is about 7.2 kb in length,including 5’ Untranslated Region,3’ Untranslated Region and an open reading frame.The SVA capsid protein is mainly composed of four structural proteins,VP1,VP2,VP3,and VP4,while the non-structural proteins include2 A,2B,2C,3A,3B,3C,and 3D.Pigs are the natural host of SVA,pigs have clinical symptoms such as blistering lesions on the skin and mucous membranes of the nose,oral epithelium,tongue and hoof crown after infected.Since it was confirmed to spread in my country in 2015,SVA has caused huge economic losses to my country’s pig industry.Because the clinical symptoms of SVA infection are similar to those of vesicular diseases including foot-and-mouth disease,vesicular stomatitis,vesicular dermatitis,and swine vesicular disease,and there is currently no commercial vaccine available for the prevention and control of SVA infection,SVA was investigated for The systematic study of clinicopathological characteristics research,epidemiological investigation,gene signature analysis and establishment of detection methods is of great significance for the prevention and control of SVA.In this study,the genome of a strain of SVA isolated from a pig farm in Chongqing was sequenced and named as SVA/CH-CQ/2019,and the SVA/CH-CQ/2019 VP1 was determined by reference to the VP1 sequences of 11 published SVA strains in GenBank.Genes were subjected to nucleotide sequence alignment analysis,phylogenetic analysis and protein analysis.E.coli BL21(DE3)was transformed with the recombinant plasmid pET-32a(+)-SVA-VP1,and expression of recombinant VP1 protein was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).In this study,BHK-21 cells were used to propagate SVA isolates,and the concentrated virus solution was ground and emulsified with complete Freund’s adjuvant to immunize rabbits to prepare polyclonal antibodies.In order to establish an ELISA method for SVA antibody detection,the recombinant protein was used as the coating antigen,and the polyclonal antibody was used as the positive serum to establish an indirect ELISA detection method based on SVA VP1.The ELSIA reaction conditions were optimized,repeatability.Sequence analysis showed that the nucleotide sequence of SVA/CH-CQ/2019 VP1 was 91.8%-98.8% homologous to the 11 published SVA VP1 sequences in GenBank,among which it was homologous to the domestic SVA/GXHZH-91324/2018 isolate.The sex is 98.8%,the two are in the same branch of the evolutionary tree,and the evolutionary relationship is the closest;the homology with the original isolate SVA/US/SVV-001-P3/2002 is 91.8%,and the evolutionary relationship is the farthest.VP1 is a stable structural protein,containing four hydrophobic clusters,with certain hydrophobicity and no transmembrane domain structure.The recombinant plasmid pET-32a(+)-SVA-VP1 was correctly identified by double digestion.The results of SDS-PAGE and Western Blot experiments showed that IPTG successfully induced the expression of SVA VP1 protein,and the VP1 protein mainly existed in the form of inclusion bodies,and the recombinant protein had good immunogenicity after solubilization and renaturation.Agar diffusion experiments showed that SVA virus condensate successfully induced immune response in rabbits.The recombinant SVA VP1 protein has good immunogenicity and can specifically bind to polyclonal antibodies.In this study,an indirect ELSIA detection method was established based on SVA VP1.By optimizing the reaction conditions,the optimal antigen coating concentration was 0.4 μg/mL,the optimal blocking solution was 2% BSA,and the optimal serum dilution was 1:2 000,the optimal serum incubation time was 1.5 h,the optimal dilution of enzyme-labeled IgG was 1:10 000,the optimal secondary antibody incubation time was 30 min,and the optimal color development time was 15 min.The intra-assay coefficient of variation of repeated experiments was 1.66%-3.30%,and the coefficient of variation of inter-assay replicate experiments was 1.82%-5.59%,with good repeatability;)and Porcine epidemic diarrhea virus(PEDV)positive sera have no cross-reaction and have good specificity;SVA positive sera can still be detected at a dilution factor of 6 400 times,with good sensitivity.The SVA/CH-CQ/2019 strain was isolated in this study,and bioinformatics analysis was performed on it.The analysis showed that the strain was the closest in homology to the domestic SVA/GXHZH-91324/2018 isolate.In this study,the SVA VP1 protein with good immunogenicity was obtained through the prokaryotic expression system,which was used as the coating antigen,and the polyclonal antibody was used as the positive serum to successfully construct SVA with good specificity,sensitivity and reproducibility.Antibody indirect ELISA detection method.This study has important application value for the prevention and treatment of SVA in China,lays the foundation for the development of ELISA detection kits for SVA epidemic strains in China,and provides a theoretical basis for the in-depth study of SVA VP1 protein and the development of subunit vaccines.