| Seneca virus type A（SVA）also known as Seneca valley virus（SVV）,is an emerging infectious virus.The prototype strain of SVA（SVV-001）is nonpathogenic to pigs.However,many studies have shown that SVA is closely related to primary vesicular disease（PIVD）and high mortality of newborn piglets.In 2015,SVA outbreaks were reported in several provinces of China,and showed the trend of spreading to other regions,causing great economic losses to China’s pig industry.Therefore,the development of diagnostic reagents is crucial for the prevention and control of SVA.The aim of this study was to express the structural protein（VP1、VP2、VP3 and VP4）of SVA and prepare theirs polyclonal antibody.The SVA strain and the prepared VP2 polyclonal antibody were used to establish a competitive ELISA method.Based on the established competitive ELISA method,serological investigation of SVA antibody in sera from some large-scale pig farms in Guangxi province was conducted.The structural proteins of SVA VP1,VP2,VP3 and VP4 gene were amplified by RT-PCR using the SVA RNA of SVA strain（Gen Bank No.MK039162）as the template.The amplicon was cloned into the prokaryotic expression vector p ET-30a and p ET-32a,resulting recombinant plasmids p ET-32a-VP1,p ET-30a-VP2,p ET-32a-VP3 and p ET-32a-VP4.The plasmids were transformed into E.coli BL21（DE3）competent cells which were then induced by IPTG.The expressed structural proteins were purified by Ni-NTA agarose from lysate of E.coli competent cells.The purified structural proteins were used to inject the New Zealand White rabbits to prepare their polyclonal antibodies respectively.The protein A affinity chromatography was used to purify polyclonal antibodies from rabbit sera.Four purified polyclonal antibodies were determined by indirect ELISA respectively.The polyclonal antibodies（VP1、VP2、VP3 and VP4）against in BHK-21 cells infected with SVA was analyzed by Western blot and indirect immunofluorescence assay（IFA）.A competitive ELISA method was established by using SVA strain as antigen coated with enzyme label plates.The conditions of specificity test and repeatability test were optimized.Finally,the established competitive ELISA method was used to detect SVA antibody in763 pig clinical serum from 2010 to 2019 in Guangxi region,and some of the serum was tested by IFA.The results are as follows.All the recombinant proteins were induced with 0.5 mmol·L-1final concentration of IPTG at 16℃and expressed as inclusion bodies and soluble solid in E.coli BL21（DE3）cells.Indirect ELISA assay showed that the four purified polyclonal antibodies had a potency of 1:64 000.Results of western blot and indirect immunofluorescence assay showed that the polyclonal antibodies could interact specifically with structural protein of SVA.A competitive ELISA method was developed.Specific test results showed that SVA antigen did not cross-react with the sera positive for type O and type A antibody of foot-and-mouth disease virus,porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,and type 2 porcine virus.Repeatability test results showed that the established competitive ELISA method had good repeatability.The established competitive ELISA method was used to detect SVA antibody in 763 clinical serum samples from large-scale pig farms in Guangxi from 2010 to 2019.369 serum samples were SVA positive with a positive rate of 48.4%.IFA parallel detection was performed on some serum samples with a coincidence rate of 89.6%.The results showed that the established competitive ELISA method had high accuracy and good practicability.It also indicates that high prevalence of SVA infection exists in Guangxi province.The preparation of polyclonal antibodies against SVA structural proteins provided valuable biomaterials for the establishment of serological detection method for SVA.Meanwhile,the established competitive ELISA could be used as a reliable method to detect clinical SVA antibodies. |
