| 摘要: |
| 【目的】建立基于VP3蛋白检测血清中A型塞内卡病毒(SVA)抗体的间接ELISA方法，为临床上猪群SVA感染及未来疫苗免疫效果评价提供有效手段。【方法】克隆SVA的VP3基因，将其与pQE 30表达载体连接，并进行原核表达，对表达的蛋白进行纯化和鉴定。用纯化的重组VP3蛋白（rVP3）作为包被抗原，通过矩阵法优化ELISA反应条件，建立检测SVA抗体的间接ELISA方法，通过对已知背景的200份SVA阴阳性血清进行检测以确定临界值，并对该方法的特异性、敏感性、重复性和稳定性进行验证。将采自SVA灭活疫苗免疫猪不同时期的160份血清，分别用该试验建立的方法及本课题组前期建立的基于VP1蛋白的间接ELISA方法进行检测，对比两者检测结果。【结果】成功克隆717 bp的VP3基因，表达出分子质量约为32 ku的VP3重组蛋白（rVP3）。Western Blot试验结果表明，纯化的 rVP3 蛋白具有良好的反应原性。建立了基于VP3蛋白的SVA间接ELISA检测方法，当样本OD450(S)/阳性对照OD450（P）＞0.138时判定为SVA抗体阳性。该方法特异性强，与FMDV(O/A型)、PRV、PRRSV、CSFV病原阳性血清均无交叉反应；敏感性较好，检测出的阳性血清最高稀释倍数与中和试验基本一致；重复性和稳定性好，批内变异系数小于4%，批间变异系数小于9%。160份血清检测结果表明，两种方法在不同时期的抗体阳性检出率略有差异。【结论】建立了基于VP3蛋白的SVA间接ELISA检测方法，该方法具有较好的特异性、敏感性、重复性和稳定性。 |
| 关键词: A型塞内卡病毒 VP3蛋白 间接ELISA 病毒检测 |
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| 基金项目:2019年度河南省科技创新领军人才计划项目（204200510012）；动物基因工程疫苗国家重点实验室开放课题“猪塞尼卡病毒病灭活疫苗研究”（SKLGEVV-ZD/ZY-2019） |
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| Establishment of indirect ELISA for detecting antibody based on VP3 protein of senecavirus A |
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SHEN Shuiying,YAN Ruoqian,LEI Congcong,et al
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| Abstract: |
| 【Objective】This study established an indirect ELISA method based on VP3 protein for detection of antibody to senecavirus A (SVA) in serum to provide an effective method for SVA infection in clinical pig population and evaluation of future vaccine immune.【Method】The VP3 gene of SVA was cloned into pQE 30 vector for prokaryotic expression,and the expressed protein was purified and identified.The purified recombinant VP3 protein (rVP3) was used as coating antigen to establish the indirect ELISA method for detection of SVA antibody with optimal reaction conditions obtained by matrix method.The critical value was determined by testing 200 serum samples with known background.The specificity,sensitivity,repeatability and stability of this method were tested.A total of 160 serum samples collected from pigs that were immunized with SVA inactivated vaccine at different stages were tested and compared by the established method in this study and the VP1 protein based indirect ELISA method.【Result】The VP3 gene of 717 bp was cloned successfully,the recombinant VP3 protein (rVP3) of 32 ku was expressed,and Western Blot results showed that the purified rVP3 protein had good reactivity.The SVA indirect ELISA method based on VP3 protein was established.When the ratio of OD450 of sample (S) to the OD450 of positive control (P) was more than 0.138,the sample of SVA antibody was determined to be positive.This method had strong specificity,and showed no cross reactions with the positive sera of FMDV(O/A type),PRV,PRRSV and CSFV.This method had good sensitivity and the highest dilution ratio of positive serum was basically consistent with neutralization test.This method had good repeatability and stability with intra batch variation coefficient of less than 4% and inter-batch variation coefficient of less than 9%.The results of 160 serum samples tests showed that the positive detection rates of antibody in different stages were slightly different between methods.【Conclusion】The established indirect ELISA method based on VP3 protein had good specificity,sensitivity,repeatability and stability. |
| Key words: senecavirus A VP3 protein indirect ELISA virus detecting |
