猪流行性腹泻病毒荧光抗体的制备及应用

doi:10.16431/j.cnki.1671-7236.2018.10.022

摘要/Abstract

摘要：

本研究旨在建立一种可辅助猪流行性腹泻病毒（PEDV）分离鉴定的直接荧光抗体检测方法。借助基因工程技术融合表达PEDV S1基因中和抗原表位区域（COE），层析柱法纯化兔源免疫球蛋白IgG，搅拌法标记荧光素。通过Western blotting和动物免疫试验测定表达融合蛋白的抗原性，间接ELISA方法测定免疫后的抗体效价，直接免疫荧光法测定标记荧光抗体的特异性，梯度稀释方法测定标记抗体的最佳工作浓度。结果显示，本研究成功将423 bp的S1基因中和抗原表位COE进行融合表达，表达蛋白大小约40 ku，蛋白命名为pGEX-6P/COE；Western blotting检测结果表明，融合蛋白具有良好的反应原性；ELISA检测结果显示，免疫后35 d的血清抗体效价达1：25 600；与猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PoRV）、猪瘟病毒（CSFV）及猪细小病毒（PPV）等均无非特异性反应，标记的荧光抗体具有良好的特异性，最佳工作稀释度为1：32~1：64。综上所述，本试验制备的直接免疫荧光抗体可用于细胞平台上的PEDV快速检测，能直观地提供相关检测数据，对于病毒分离过程的取舍具有重要指导意义。

关键词: 猪流行性腹泻病毒(PEDV); COE基因; 原核表达; 直接免疫荧光抗体

Abstract:

The aim of this study was to establish a direct fluorescent antibody assay for the isolation and identification of porcine epidemic diarrhea virus (PEDV).The PEDV S1 gene neutralized antigen epitope region (COE) was successfully fused by genetic engineering technology.Immunoglobulin G (IgG) from rabbit serum was purified by chromatographic column.We used agitation method to combine the antibody with fluorescein isothiocyanate (FITC).The protein antigen and antibody titer were identified by Western blotting and indirect ELISA,respectively.The specificity of labeled fluorescent antibody and the best working concentration were determined by direct immunofluorescence and gradient dilution method,respectively.In this study,the length of S1 gene fragment in the epitope region was 423 bp,the expression protein was named as pGEX-6P/COE fusion protein,and the molecular mass was about 40 ku.Western blotting result showed that the fusion protein had good antigenicity and the titer of serum antibody reached 1:25 600 at 35 d after immunization.According to the experiment results,optimal working concentration of the direct FITC antibody was diluted from 1:32 to 1:64.This method was specific,having no cross-reactivity with other common virus,such as TGEV,PoRV,PPV and CSFV,etc.The results showed that the prepared direct immunofluorescent antibody could be used for rapid detection of PEDV on the cell platform.It could provide the related detection data intuitively,which was of great guiding significance for the selection of the virus separation process.

Key words: porcine epidemic diarrhea virus (PEDV); COE gene; prokaryotic expression; direct immunofluorescence antibody

中图分类号:

S858.28

王金良, 许可军, 陈金龙, 董艳凯, 于智慧, 董林, 魏凤, 沈志强. 猪流行性腹泻病毒荧光抗体的制备及应用[J]. 《中国畜牧兽医》, 2018, 45(10): 2849-2855.

WANG Jinliang, XU Kejun, CHEN Jinlong, DONG Yankai, YU Zhihui, DONG Lin, WEI Feng, SHEN Zhiqiang. Preparation and Application of Fluorescent Antibody Against Porcine Epidemic Diarrhea Virus[J]. , 2018, 45(10): 2849-2855.

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