Establishment of Neutralizing Antibody Detection Method based on Recombinant Fluorescent Virus of Porcine Epidemic Diarrhea Virus G Ⅱ b Strain
Since 2010,the appearance of the GⅡ variant of porcine epidemic diarrhea virus(PEDV)has significantly increased the mortality of porcine epidemic diarrhea(PED)worldwide.Vaccine immunization has become an important strategy to prevent and control PED,and the neutralizing antibodies level induced by vaccines serves as a vital indicator to evaluate the immune effect and vaccine quality.However,the traditional PEDV neutralization assay on Vero-CCL81 cells depends on the addition of exogenous trypsin,which brings challenges such as unstable and unrepeatable results.This study aims to establish an accurate detection method to evaluate neu-tralizing antibodies in samples by constructing a recombinant viruses stably expressing a green fluorescent protein.In this study,part of ORF1a and ORF1b gene fragments and other structural genes of the GⅡb subtype PEDV-GDU strain were cloned and inserted into the pUC57 vector.Furthermore,ORF3 gene was replaced by EGFP gene by using seamless cloning technology and an infectious cloning plasmid pUC57-PEDV-GDU-dORF3-EGFP was conducted.The recombi-nant virus with EGFP,which named rPEDV-GDU-dORF3-EGFP,was rescued.Furthermore,the biological characteristics of rPEDV-GDU-dORF3-EGFP and its parental virus were compared and evaluated by indirect immunofluorescence assay(IFA),Western blot and single-step growth curve assays.The rPEDV-GDU-dORF3-EGFP was further used to screen a cell line that supports the effective proliferation of PEDV without trypsin,and a neutralization assay was established with this cell line and rPEDV-GDU-dORF3-EGFP.The results demonstrated that the rPEDV-GDU-dORF3-EGFP was successfully rescued,and the rPEDV-GDU-dORF3-EGFP exhibited similar biological characteristics to parental virus in Vero-CCL81 cells.A subcloned Huh7 cells screened by the recombinant reporter virus can support infection and replication of PEDV without the addition of exogenous trypsin.Subsequently,a PEDV neutralization assay was successfully established using Huh7.10 cells and recombinant reporter virus.The analysis of correlation showed the level of correlation between the neutralization assay we established and ELISA was higher than that of traditional PEDV neutralization assay.In our study,the reporter virus rPEDV-GDU-dORF3-EGFP was successfully recued and the reverse genetic operation platform of PEDV-GDU strain has been successfully established,which provides a powerful tool for the rapid preparation of PED vaccine candidates.In addition,the neutralization assay based on the recom-binant reporter virus on Huh7.10 cells can more accurately determine the level of neutralizing an-tibody in serum,which provides effective technical support for the evaluation of vaccine immune effect.
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