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

Abstract

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

CLC Number:

S858.28

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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Preparation and Application of Fluorescent Antibody Against Porcine Epidemic Diarrhea Virus

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