基于猪流行性腹泻病毒GⅡb亚型重组荧光病毒中和抗体检测方法的建立

Establishment of Neutralizing Antibody Detection Method based on Recombinant Fluorescent Virus of Porcine Epidemic Diarrhea Virus GⅡb Strain

2010年以来,猪流行性腹泻病毒(PEDV)GⅡ型变异毒株的出现,导致全世界范围内猪流行性腹泻(PED)的病死率显著增加。疫苗免疫是PED有效防控的重要策略之一,疫苗诱导的中和抗体水平是免疫效果评价和疫苗质量评估的重要指标。然而,在Vero-CCL81上开展的PEDV经典中和试验中所添加的胰酶会导致试验结果不稳定等问题。本研究拟在构建稳定表达绿色荧光蛋白的重组病毒基础上,建立一种能准确评估样品中和抗体的检测方法。本研究在pUC57载体上克隆插入GⅡb亚型PEDV-GDU株部分ORF1a和ORF1b基因片段与其他结构基因片段,并通过无缝克隆技术将ORF3基因替换为EGFP基因,构建辅助质粒pUC57-PEDV-GDU-dORF3-EGFP,通过RNA定向重组技术拯救重组荧光病毒rPEDV-GDU-dORF3-EGFP。进一步通过间接免疫荧光试验(IFA)、Western blot和病毒生长曲线测定等方法,比较重组荧光病毒和亲本病毒生物学特性。最终利用重组荧光工具病毒筛选在不添加胰酶的条件下仍能支持PEDV有效增殖的细胞系,并建立中和试验方法。结果表明,成功拯救重组荧光病毒rPEDV-GDU-dORF3-EGFP,且在Vero-CCL81细胞上,重组荧光病毒与亲本病毒呈现相似的生物学特性。利用该重组荧光病毒筛选到一株亚克隆Huh7.10细胞可支持PEDV在无外源胰酶添加条件下的有效感染与复制。并利用Huh7.10细胞和重组荧光病毒成功建立了PEDV中和试验方法,相关性试验分析表明,该方法测定结果与ELISA抗体检测结果相关性高于PEDV经典中和试验。以上试验结果表明,本研究成功建立了PEDV-GDU毒株的反向遗传操作平台,为PED新型疫苗候选毒株的快速制备提供了有力的工具。此外,基于重组荧光病毒在Huh7.10细胞上建立的中和试验方法能更准确地测定血清中和抗体水平,为疫苗免疫效果的评估提供了有效的技术支持手段。

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 neutralizing 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 recombinant 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 can still 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 recombinant reporter virus on Huh7.10 cells can more accurately determine the level of neutralizing antibody in serum,which provides effective technical support for the evaluation of vaccine immune effect.