A型塞内卡病毒VP2蛋白多克隆抗体的制备及间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2024.02.024

摘要/Abstract

摘要： 【目的】制备A型塞内卡病毒(Senecavirus A,SVA)VP2蛋白多克隆抗体,建立检测SVA抗体的间接ELISA方法,以期为SVA致病机制及诊断提供研究基础。【方法】利用同源重组技术将VP2基因克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28a-VP2,并转化大肠杆菌BL21(DE3)感受态细胞进行IPTG诱导表达,表达产物经纯化后皮下多点注射新西兰大白兔制备多克隆抗体。利用间接免疫荧光试验(IFA)、Western blotting和中和试验鉴定多克隆抗体的特异性、反应性和中和活性。以VP2为抗原包被酶标板,通过矩阵优化、临界值确定、特异性鉴定及敏感性和重复性分析建立检测SVA抗体的间接ELISA方法。采集50份临床血清样品,分别用间接ELISA与IFA方法进行检测,分析间接ELISA方法的符合率。【结果】重组VP2蛋白以包涵体形式表达,大小为40 ku。制备的多克隆抗体能与重组VP2蛋白和SVA特异性结合,与其他病毒无交叉反应,且具有较高的中和活性。经对间接ELISA条件的优化,确定VP2包被浓度为4 μg/mL,阳性血清稀释浓度为1∶250,封闭液为5%脱脂乳+5% BSA,血清样品和二抗孵育时间均为90 min,TMB底物反应时间为10 min,临界值为0.182。建立的间接ELISA方法与常见的猪病毒阳性血清不反应,与IFA符合率达94.0%。【结论】原核表达系统表达的VP2蛋白具有良好的免疫原性,制备的多克隆抗体能与SVA和VP2发生特异性反应。建立的间接ELISA方法特异性高,与IFA符合率高,适用于临床SVA抗体检测和疫苗效力评价。

关键词: A型塞内卡病毒(SVA); VP2蛋白; 原核表达; 多克隆抗体; 间接ELISA

Abstract: 【Objective】 This study was aimed to prepare polyclonal antibodies (pAbs) against the VP2 protein of Senecavirus A (SVA), and establish an indirect ELISA method to specifically detect SVA antibodiy, so as to provide a research basis for the pathogenesis and diagnosis of SVA.【Method】 VP2 gene was cloned into the prokaryotic expression vector pET-28a by homologous recombination, the recombinant expression plasmid pET-28a-VP2 were designated and transformed into Escherichia coli BL21 (DE3) competent cell for IPTG induced expression.The New Zealand White rabbits were subcutaneously immunized with the purified recombinant protein to prepare its pAbs.The specificity, reactivity and neutralizing activity of pAbs were analyzed via indirect immunofluorescence assay (IFA), Western blotting and neutralization test.An indirect ELISA based on VP2 coating antigen was established by matrix optimization, determination of cut-off value, specificity identification, sensitivity and repeatability analysis.A total of 50 serum samples were collected, which were tested by indirect ELISA and IFA to analyze the coincidence rate.【Result】 The recombinant VP2 protein was expressed in the form of inclusion body with a size of 40 ku.The pAbs could specifically react with recombinant VP2 protein and SVA, and had no cross-reactivity with other viruses.Moreover, the pAbs had a higher neutralizing activity.After optimizing the conditions of indirect ELISA, the optimal VP2 coating concentration was measured at 4 μg/mL, and optimal serum sample dilution was 1∶250.Furthermore, the best blocking solution was selected as 5% skimmed milk +5% BSA.The optimal reaction time for serum, secondary antibodies and TMB solution were 90, 90 and 10 min, respectively.Finally, the value of cut-off was calculated to 0.182.No cross-reactivity was observed with other virus antibody-positive pig sera, and indirect ELISA showed a 94.0% coincidence rate compared with IFA.【Conclusion】 VP2 protein expressed in the prokaryotic expression system had good immunogenicity, and the prepared pAbs could react specifically with SVA and recombinant VP2 protein.The established indirect ELISA had high specificity and consistency with IFA, which could be suitable for clinical SVA antibody detection and vaccine efficacy evaluation.

Key words: Senecavirus A (SVA); VP2 protein; prokaryotic expression; polyclonal antibody; indirect ELISA

中图分类号:

S852.65⁺9.6

任建乐, 林铱婷, 姬康, 谭姗姗, 陈新新, 晋怡, 王颖, 牛胜, 梁立滨, 李俊平, 赵宇军, 田文霞. A型塞内卡病毒VP2蛋白多克隆抗体的制备及间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2024, 51(2): 678-688.

REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia. Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 678-688.

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