| For the first time,the Senecavirus A(SVA)was reported in Canada in 2007 and subsequently in the United States,Brazil,Colombia,China,Thailand and Viet Nam.Since 2015,a pig farm in Guangdong province has isolated the SVA,thus determining the prevalence of SVA in China,which has brought huge economic losses to the pig industry.At present,there are many subclinical infections in China,and the lack of approved commercial vaccines can be used,which increases the prevention of Senecavirus disease in China Control difficulty.hence,a novel SVA vaccine with high safety needs to be developed.The purpose of this study was to establish a SYBR Green Ⅰ real-time fluorescence quantitative PCR detection method and apply it to the detection of SVA.The purified SVA VP1 and SVA VP3 protein were combined with adjuvant to evaluate the immune effect of mice.The results are as follows:1.SYBR Green I establishment of real-time fluorescence quantitative PCR method:the copy number of SVA 2C gene standard was in the range of 99×10-99×100 copies/μL Correlation coefficient of 2C gene standard was 0.994,With a slope of 3.736,The sensitivity of the 2C detection method is 1.99×100,10,000 times higher than conventional PCR.The coefficient of variation between 2C groups was between 0.76～0.93%,Coefficient of variation within group was between 0.35～0.66%;Copy number of SVA 3D gene standard is 2.18× 107-2.18×100 within 100 copies/μL,Correlation coefficient of 3D gene standard was 1.000,and the slope is 3.589;The sensitivity of 3D detection method is 2.18×100 copies/μL,1000 times higher than conventional PCR;3D coefficient of variation between groups was between 0.60～1.06%,Coefficient of variation within group was between 0.51～0.89%;The detection methods of both gene construction have good specificity,SVA and PCV2、PCV3、FMDV、PRRSV、PEDV、TGEV do not produce cross-reverses.SYBR Green Ⅰ real-time quantitative PCR was used to detect 49 samples,the positive rate of 2C was 81.6%,the positive rate of 3D was 85.7%.The results show that the established SVA SYBR Green Ⅰ real-time fluorescence quantitative PCR has higher sensitivity and specificity than the common PCR method,and can be considered for rapid detection of SVA.2.Prokaryotic expression of SVA VP1 protein and VP3 protein:The amplified gene fragments of SVA VP1 and VP3 were 792bp and 717bp,respectively,which was consistent with the expectation.SVA VP1 and VP3 were cloned into pET-28a vector.Then,pET-28a-VPl recombinant plasmid and pET-28a-VP3 recombinant plasmid were successfully constructed,Convert to E.coli BL21(DE3)pLySs receptive state,IPTG-induced expression,about 35 KDa、29KDa of target protein;When the temperature was 37℃,0.8mmol/L as the concentration of inducer,and 6h as the induction time,the expression of VP1 protein was the highest.When the temperature was 37℃,0.8mmol/L as inducer concentration,and 4h as induction time,the expression of VP3 protein was the highest.Both recombinant proteins present inclusion bodies.Using His tags Ni2+-NTA metal chelating proteins to purify column purified proteins,The results showed that the purity of VP1 recombinant protein and VP3 recombinant protein were 85%and 84%respectively.3.Immune evaluation of mice with SVA VP1 and VP3 protein combined adjuvant:Mice were immunized with 0.56mg/mL VP1,0.23mg/mL VP3 soluble protein,combined with aluminum hydroxide adjuvant and Frasil adjuvant.The dose of immune ant igen was 50 μg/mouse in experimental group,100 μl for each mouse was injected intramuscular into the leg.14 days after the first immunization,the second immunization was performed.The immune effect of mice was evaluated by humoral immunity and cellular immunity respectively.The results showed that the specific antibody of VP1 subunit vaccine increased at 7 days after the first immunization,and the specific antibody titer of VPI-FCA group reached the highest value(1:163840)at 14 days after the second immunization,while that of PBS group was 1:16.The specific antibody titer of VPl-AL group reached the highest value(1:520)14 days after secondary immunization,while that of PBS group was 1:16.The antibody titer of VP1-FCA group reached the highest value(1:552)at 7 days after secondary immunization,and the antibody titer of PBS group was 1:8.14 days after the second immunization,the antibody titer of VPl-AL group reached the highest value(1:1024),and the antibody titer of PBS group was 1:16.The T lymphocyte subsets CD3+CD4+and CD3+CD8+were significantly increased in VP1-FCA group and extremely significantly increased in VP1-AL group after the second immunization(p<0.01).Lymphocyte proliferation assay showed that both VP1-AL group and VP1-FCA group were significantly increased(p<0.05).The serum cytokines of immunized mice showed that the secretion of IL-4 was significantly increased(p<0.05),while the changes of IL-2 and IFN-γ were not significant.The specific antibody of VP3 subunit vaccine showed an increasing trend 7 days after the first immunization,and the specific antibody titer of VP3 FCA group reached the highest value of 1:163840 at 14 days after the second immunization.The antibody titer of the PBS group was now 1:16.On day 7 after the second immunization,the neutralizing antibody titer was significantly higher than that of the PBS group.On day 14 after the second immunization,the maximum value of the VP3-AL group and the VP3-FCA group was 1:128 and 1:32,respectively.Lymphocyte proliferation assay showed that both VP3-AL group and VP3-FCA group showed a significantly increased trend(p<0.05).The results showed that the secretion of cytokine IL-4 was significantly increased(p<0.05),while the production of IL-2 and IFN-γ was not significantly changed.In this study,SVA VP1 recombinant protein combined with adjuvant AL group could induce immune response in mice with good effect,which laid a foundation for subsequent studies on pig body immunity with this antigen. |
