猪急性腹泻综合征冠状病毒N蛋白间接ELISA抗体检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2025.01.029

摘要/Abstract

摘要：

为建立一种猪急性腹泻综合征冠状病毒(SADS-CoV)血清抗体的快速检测方法，本研究首先经PCR扩增SADS-CoV N基因，纯化鉴定后克隆至表达载体pET-32a(+)，构建重组质粒pET-32a-N。重组N蛋白诱导表达并通过SDS-PAGE和Western blot鉴定后，采用纯化的重组N蛋白作为包被抗原，以兔抗猪HRP- IgG作为二抗，利用方阵滴定法优化各反应条件，建立了一种以SADS-CoV N蛋白为靶标的检测SADS-CoV血清抗体的间接ELISA方法，并对此方法的敏感性、特异性、重复性及临床应用价值进行鉴定。SDS-PAGE及Western blot结果显示，重组表达的N蛋白约为70 ku，且具有良好的反应原性。采用该方法检测SADS-CoV阳性血清抗体效价可达1∶3 200，并且与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪丁型冠状病毒(PDCoV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪轮状病毒(PoRV)和猪圆环病毒(PCV)阳性血清均无交叉反应，具有良好的敏感性和特异性。批内和批间重复性试验的变异系数均小于10%，具有较好的重复性。利用该方法和间接免疫荧光方法(IFA)分别对50份猪临床血清样品进行检测，结果显示二者的总符合率为98%。综上，本研究建立了一种基于SADS-CoV N蛋白的间接ELISA抗体检测方法，可用于临床SADS-CoV血清抗体检测以及流行病学的监测，对防控SADS-CoV的流行具有重要意义。

关键词: 猪急性腹泻综合征冠状病毒(SADS-CoV), N蛋白, 原核表达, 间接ELISA

Abstract:

To establish a rapid detection method for swine acute diarrhea syndrome coronavirus (SADS-CoV) serum antibodies, N gene was amplified by PCR, and then the purified fragment was cloned into pET-32a (+) prokaryotic expression vector to construct the recombinant plasmid pET-32a-N. The expression of recombinant N protein was determined by SDS-PAGE and western blot. The purified recombinant N protein was applied as the coating antigen. Rabbit anti-pig HRP-IgG was used as the secondary antibody. We used the square-array titration to optimize the reaction conditions of the indirect ELISA method targeting SADS-CoV N protein antibodies. In addition, we identified the sensitivity, specificity and repeatability of this method, and evaluated its clinical applicability. SDS-PAGE and western blot results showed that the recombinant N protein was about 70 ku and had good reactivity. The antibody titer of SADS-CoV positive serum detected by this method was 1: 3 200, and there was no cross reaction with PEDV, TGEV, PDCoV, PRRSV, PoRV or PCV positive pig serum, which exhibited good sensitivity and specificity. The coefficient of variation of intra-batch and inter-batch repeatability tests were both less than 10%, demonstrating favorable reproducibility of this method. Fifty randomly selected porcine clinical serum samples were tested by ELISA and immunofluorescence assay, and the coincidence rate of results was 98%. In conclusion, we established an indirect ELISA based on N protein to test SADS-CoV clinical serum antibodies, which is useful for epidemiological surveillance, and is of great significance for prevention and control of SADS-CoV.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), N protein, prokaryotic expression, indirect ELISA

中图分类号:

S852.659.6

曾苗苗, 杨小曼, 张鑫, 刘大凯, 时洪艳, 张记宇, 张燎原, 陈建飞, 冯廷帅, 李修文, 石达, 冯力. 猪急性腹泻综合征冠状病毒N蛋白间接ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2025, 56(1): 319-326.

ZENG Miaomiao, YANG Xiaoman, ZHANG Xin, LIU Dakai, SHI Hongyan, ZHANG Jiyu, ZHANG Liaoyuan, CHEN Jianfei, FENG Tingshuai, LI Xiuwen, SHI Da, FENG Li. Establishment and Preliminary Application of an Indirect ELISA for Swine Acute Diarrhea Syndrome Coronavirus N Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 319-326.

图/表 3

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方法及判定指标 Method and determination index	IFA阳性样品数 Positive samples	IFA阴性样品数 Negative samples	总数 Total	符合率/% Coincidence rate
间接ELISA阳性样品数 Positive samples	4	1	5	100.0
间接ELISA阴性样品数 Negative samples	0	45	45	97.8
总数 Total	4	46	50	98.0

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