| Senecavirus A(SVA),formerly known as Seneca valley virus(SVV),is the only member of the picornavirus family Senecavirus genus.SVA can cause infection in pigs at different ages,which can lead to blister-like lesions on the nose,mouth,tongue and crown of hoof of pigs,then lead to secondary ulcers and ulceration,and make the sick pigs appear lame and difficult to stand.The most serious harm is the newborn piglets,acute death,mortality as high as 30%～70%.Adult pig infection mortality is low,but the decrease in sow productivity due to infection is not insignificant.In March 2015,the first case of SVA infection was found in Guangdong,followed by SVA outbreaks in other provinces in China.Pig producers need to pay attention to this.Swine blister disease caused by SVA affects the development of pig industry and is a potential threat to pig industry.Vaccine immunization is the most effective and direct method to prevent SVA.Commercial SVA vaccine is not available in the domestic market,and there is no specific drug to treat SVA infection.Therefore,a simple,specific,sensitive,stable and reliable method is needed to detect SVA prevalence and antibody levels in pigs in clinical production.The main contents and results of this study are as follows:1.Cloning and expression of SVA VP2 gene and preparation of polyclonal antibodyIn this study,we used pET-32 a prokaryotic expression vector to construct prokaryotic expression plasmid of VP2 protein gene fragment by referring to VP2 genome sequence of SVA NADC1 strain(GenBank entry number: MZ733980.1)in Gen Bank.The constructed plasmid was transferred into BL21(DE3)receptive cells for expression,and a series of experimental procedures were optimized.Finally,when the induction temperature was 16℃ and the IPTG was 0.9m M,the SDS-PAGE detection showed that there were bands with the size of VP2 protein at 57 KD,which proved the successful expression of VP2 protein.After purification,the concentration of VP2 protein was 1.056mg/m L.The mice were injected intraperitoneally with Freund’s adjuvant mixed with purified protein to produce the required polyclonal antibody.WB analysis showed that the prepared polyclonal antiserum had specific reaction with VP2 protein and could be used as primary antiserum.2.Establishment and preliminary application of SVA indirect ELISA methodVP2 protein was purified and used as the coated antigen.The primary antiserum was obtained by artificially immunizing mice with VP2 protein.The indirect ELISA conditions were optimized and the indirect ELSIA method was established based on VP2 protein.The sensitivity,specificity and repeatability of the proposed method were tested to verify the reliability of the proposed method.The results showed that the optimal indirect ELISA conditions were as follows: antigen coated concentration of 2μg/m L,coated overnight at 4℃,serum of the samples to be tested diluted at 1:100 for 45 min,5% skim milk powder for 2h,enzyme-conjugate secondary antibody 1:20 000 for 1h,and color development time of 10 min.The established method was used to detect SVA,CSFV,PRRSV,PCV2 and PRV positive sera of pigs.Only SVA positive sera were positive,while other sera were negative,indicating that the method has good specificity.SVA positive serum was still positive when diluted 3 200 times,indicating that the established method was sensitive.The coefficient of variation of inter-batch and intra-batch repeatability was less than 10%,and the repeatability was better.The established indirect ELISA method was used to detect 200 serum samples collected from some pig farms in Jilin Province during 2021-2022,and 0 SVA positive samples were detected,with a positive rate of 0%. |
PDF Full Text Request