猪δ冠状病毒重组M蛋白间接ELISA抗体检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2020.07.023

摘要/Abstract

摘要： 本研究旨在建立一种可靠的检测猪δ冠状病毒（porcine deltacoronavirus，PDCoV）IgG抗体的间接ELISA方法，了解四川地区PDCoV的流行情况。采用原核表达系统表达PDCoV膜蛋白（M）作为检测抗原，通过反应条件的优化，建立了基于PDCoV M蛋白的间接ELISA方法，命名为PDCoV-rM-ELISA，并用该方法对430份临床样品进行检测。成功构建了重组表达菌BL21-pET-32a-M，经诱导表达后获得了重组M蛋白，Western bolt检测表明，重组蛋白具有良好的反应原性。以其作为包被抗原建立的PDCoV-rM-ELISA方法仅对PDCoV血清检测为阳性，与猪流行性腹泻病毒等5种猪常见病原阳性血清均无交叉反应，具有良好的特异性；该方法灵敏性高、重复性好，批间和批内重复性变异系数均小于10%；与成品化PDCoV抗体检测试剂盒同时检测48份临床血清，两种方法的阳性符合率为88.46%，阴性符合率为90.91%，总符合率为89.58%。临床样品检测结果表明，各地样本PDCoV抗体阳性率平均为11.86%，且统计学分析显示，母猪比仔猪和育肥猪高2.25倍的PDCoV感染概率。笔者成功表达了PDCoV M蛋白，建立了PDCoV-rM-ELISA检测方法，该方法具有较高的敏感性、良好的特异性和重复性，可用于临床猪血清样品种PDCoV IgG抗体的检测。

关键词: 猪δ冠状病毒, 重组M蛋白, 原核表达, 间接ELISA

Abstract: This study aimed to establish a reliable indirect ELISA method for detecting PDCoV IgG antibody and to understand the prevalence of PDCoV in Sichuan. The prokaryotic expression system was used to express PDCoV membrane protein (M) as a detection antigen. By optimizing the reaction conditions, an indirect ELISA method named PDCoV-rM-ELISA based on PDCoV M protein was established, and 430 clinical samples were detected by this method. The recombinant expression E.coli strain BL21-pET-32a-M was successfully constructed, and the recombinant M protein was expressed after induction. Western blot assay showed that the recombinant protein had good reactivity. PDCoV-rM-ELISA method could specifically detect PDCoV antibody, and had no cross reaction with five common pathogen positive sera, suggesting good specificity; The sensitivity of this method was high. PDCoV-rM-ELISA was high reproducible with the coefficients of variation were less than 10%; Forty-eight clinic serums were detected using both PDCoV-rM-ELISA and PDCoV antibody detection kits. The results showed that the positive agreement rate was 88.46%, the negative agreement rate was 90.91%, and overall coincidence rate was 89.58%. The results of clinical samples detection showed that the average positive rate of PDCoV antibody was 11.86%, and the statistical analysis showed that the sows were 2.25 times more likely to be infected PDCoV than piglets and fattening pigs. An indirect ELISA was established by using the recombinant M protein of PDCoV express in E. coli BL21. PDCoV-rM-ELISA method had good specificity, reproducibility and stability, and could be used for the detection of PDCoV antibody in clinic.

Key words: PDCoV, recombinant M protein, prokaryotic express, indirect ELISA

中图分类号:

S852.659.6

冯宇, 殷鑫欢, 杨晓宇, 徐雷, 徐志文, 朱玲. 猪δ冠状病毒重组M蛋白间接ELISA抗体检测方法的建立与应用[J]. 畜牧兽医学报, 2020, 51(7): 1710-1718.

FENG Yu, YIN Xinhuan, YANG Xiaoyu, XU Lei, XU Zhiwen, ZHU Ling. Establishment and Application of an Indirect ELISA Based on Recombinant M Protein against Porcine Deltacoronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1710-1718.

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