施马伦贝格病毒N蛋白原核表达及间接ELISA抗体检测方法的建立

doi:10.16431/j.cnki.1671-7236.2025.08.036

摘要/Abstract

摘要： 【目的】建立一种具有良好特异性和敏感性的施马伦贝格病毒(Schmallenberg virus,SBV)抗体间接ELISA(indirect ELISA，iELISA)检测方法。【方法】将SBV N蛋白编码基因全长序列克隆至pET-28a(+)质粒中，构建重组质粒pET-28a-SBV，利用大肠杆菌表达系统表达目的蛋白并用His镍柱进行纯化，经SDS-PAGE和Western blotting鉴定后，以纯化的SBV N蛋白作为检测抗原进行包被，建立一种iELISA方法，并对其包被条件、封闭液、封闭时间和血清孵育时间等反应条件进行优化，检测其阴阳性临界值、特异性、灵敏性和重复性。【结果】 SDS-PAGE结果显示，SBV N蛋白分子质量为30 ku；Western blotting结果显示，纯化后SBV N蛋白与SBV阳性血清能产生特异性反应。本研究建立的SBV iELISA检测方法最佳条件为：SBV N抗原包被浓度4 μg/mL、4 ℃包被12 h、1%明胶37 ℃封闭1 h、血清稀释度为1∶200、37 ℃孵育1 h、1∶6 000稀释的酶标二抗37 ℃孵育1 h，其阴阳性临界值为0.371。该iELISA检测方法仅与SBV阳性血清产生特异性反应，与牛副流感病毒3型(BPIV3)、牛轮状病毒(BRV)、牛冠状病毒(BCoV)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)和阿卡斑病毒(AKAV)阳性血清均无明显交叉反应，灵敏性较高，SBV阳性血清稀释度为1∶1 280时仍有明显反应。批间重复试验和批内重复试验变异系数均＜10%；与商品化试剂盒相比，其总符合率为94.57%。【结论】本研究建立了一种以SBV N蛋白为检测抗原的SBV iELISA方法，该方法具有良好的特异性和敏感性，能运用于SBV血清学检测，试验结果为SBV的快速诊断提供更多选择。

关键词: 施马伦贝格病毒(SBV); N蛋白; 间接ELISA(iELISA); 原核表达

Abstract: 【Objective】 The objective of this study was to establish an indirect ELISA (iELISA) method with good specificity and sensitivity for detecting antibodies against Schmallenberg virus (SBV). 【Method】 The full-length sequence of the SBV N protein-coding gene was cloned into pET-28a(+) plasmid to construct recombinant plasmid pET-28a-SBV.The target protein was expressed using the Escherichia coli expression system and purified using a His-tagged nickel column.After identification by SDS-PAGE and Western blotting,the purified SBV N protein was used as the detection antigen for coating to establish an iELISA method.The coating conditions,sealing fluid,sealing time,and incubation time of serum were optimized.The negative and positive critical value,specificity,sensitivity,and repeatability of the method were evaluated. 【Result】 SDS-PAGE results showed that the molecular weight of SBV N protein was 30 ku.Western blotting results demonstrated that the purified SBV N protein specifically reacted with SBV positive serum.The optimal conditions for the established SBV iELISA method were as follows:SBV N antigen coating concentration of 4 μg/mL,coating at 4 ℃ for 12 h,sealing with 1% gelatin at 37 ℃ for 1 h,serum dilution of 1∶200,incubation at 37 ℃ for 1 h,and enzyme-labeled secondary antibody dilution of 1∶6 000 with incubation at 37 ℃ for 1 h.The negative and positive critical value was 0.371.The iELISA method specifically reacted with SBV positive serum and showed no cross-reactivity with positive sera against BPIV3,BRV,BCoV,IBRV,BVDV and AKAV.The method exhibited high sensitivity,with a clear reaction observed even at a serum dilution of 1∶1 280.The coefficient of variation of inter-batch and intra-batch repeated tests was less than 10%.Compared with the commercial kit,the total concordance rate was 94.57%. 【Conclusion】 This study established an SBV iELISA method using SBV N protein as the detection antigen.The method demonstrated good specificity and sensitivity and could be applied for serological detection of SBV,providing an additional option for rapid diagnosis of SBV.

Key words: Schmallenberg virus(SBV); N protein; indirect ELISA(iELISA); prokaryotic expression

中图分类号:

S852.65^＋9.5

李阳, 孙睿雪, 陈天杰, 刘思彤, 曹家慧, 赵建军. 施马伦贝格病毒N蛋白原核表达及间接ELISA抗体检测方法的建立[J]. 中国畜牧兽医, 2025, 52(8): 3877-3887.

LI Yang, SUN Ruixue, CHEN Tianjie, LIU Sitong, CAO Jiahui, ZHAO Jianjun. Prokaryotic Expression of Schmallenberg Virus N Protein and Establishment of Indirect ELISA Antibody Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3877-3887.

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