【摘要】 为构建猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)感染性克隆，进而为解析该病毒尤其当前流行的GⅡ型毒株的基因组复制机制、关键毒力基因功能提供可控的分子工具，本研究根据PEDV CH/GDZH02/1401株基因组反转录的cDNA序列PCR扩增得到9段基因组片段，通过In-Fusion无缝克隆技术将各基因组片段分别与pcDNA3.1质粒进行连接，获得中间质粒pcDNA3.1-PEDV-1～pcDNA3.1-PEDV-9。其中，在构建pcDNA3.1-PEDV-1和pcDNA3.1-PEDV-9前先对质粒pcDNA3.1进行修饰，在质粒pcDNA3.1中依次插入CMV启动子、锤头状核酶序列和PEDV第1段序列构建pcDNA3.1-PEDV-1,在质粒pcDNA3.1依次插入PEDV第9段序列、丁型肝炎核酶和BGH终止子序列，构建pcDNA3.1-PEDV-9。以9个中间质粒为模板，设计9对引物扩增包含至少35 bp同源臂的DNA片段，并将其与酵母载体pYES1L转化到MaV203酵母感受态细胞中，设计检测引物筛选含有pYES1L-PEDV的酵母菌菌落。裂解获得的阳性酵母菌落，电转化至DH10B大肠杆菌中，经PCR鉴定筛选阳性菌落后，提取质粒获得pYES1L-PEDV。将其与辅助质粒pCAGGS-PEDV-N共转染至BHK-21细胞，48 h后收集细胞上清液，将其接种至Vero细胞，通过间接免疫荧光试验(indirect immunofluorescence assay, IFA)以及Western blot进行鉴定。结果显示，Vero细胞接种PEDV病毒液后48 h出现明显细胞病变，IFA结果显示感染细胞出现绿色特异荧光信号，表明成功拯救出rPEDV。Western blot结果显示，rPEDV在Vero细胞进行连续传代后(15代),其感染细胞中仍然可检测到58 kDa的N蛋白。病毒的生物学特性试验结果显示，rPEDV的TCID50、生长曲线与亲本株无明显区别。结果表明，本试验成功构建了PEDV全基因组感染性克隆，并成功拯救出rPEDV,为后续PEDV基因功能的研究和新型疫苗的研发奠定了基础。更多还原

【Abstract】 To construct an infectious cDNA clone of porcine epidemic diarrhea virus（PEDV） and to provide a controllable molecular tool for elucidating the mechanisms of viral genome replication and the functions of key virulence genes—particularly those of currently prevalent genotype GⅡ strains—this study amplified nine genomic fragments of the PEDV CH/GDZH02/1401 strain from reverse-transcribed cDNA by PCR.Using In-Fusion seamless cloning technology, each genomic fragment was ligated individually into the pcDNA3.1 plasmid to generate nine intermediate plasmids, designated pcDNA3.1-PEDV-1 through pcDNA3.1-PEDV-9.Before constructing pcDNA3.1-PEDV-1 and pcDNA3.1-PEDV-9,the pcDNA3.1 vector was modified by sequentially inserting the CMV promoter, hammerhead ribozyme sequence, and the first genomic fragment of PEDV to obtain pcDNA3.1-PEDV-1,and inserting the ninth genomic fragment, hepatitis delta virus ribozyme,and BGH terminator sequence to obtain pcDNA3.1-PEDV-9.Using the nine intermediate plasmids as templates,nine pairs of primers were designed to amplify DNA fragments containing at least35bp homologous arms.These fragments were co-transformed with the yeast vector pYES1Linto Saccharomyces cerevisiae MaV203competent cells.Colonies containing pYES1L-PEDV recombinant plasmids were screened by PCR using specific detection primers.Positive yeast colonies were lysed,and the lysates were electroporated into E.coli DH10Bcells.After PCR identification and screening,positive colonies were selected,and plasmid DNA was extracted to obtain pYES1LPEDV.The recombinant plasmid pYES1L-PEDV and the helper plasmid pCAGGS-PEDV-N were co-transfected into BHK-21cells.At 48 hpost-transfection,cell supernatants were collected and inoculated onto Vero cells for further identification by indirect immunofluorescence assay（IFA）and Western blot analysis.The results showed that obvious cytopathic effects appeared in vero cells at 48 hours post-inoculation with the rescued PEDV.IFA revealed specific green fluorescence in infected cells,indicating successful rescue of recombinant PEDV（rPEDV）.Western blot analysis showed that the N protein（58 kDa）was still detectable in rPEDV-infected Vero cells after 15 serial passages.Biological characterization demonstrated that the TCID₅₀ value and growth curve of rPEDV were comparable to those of the parental strain.These results indicate that an infectious full-length cDNA clone of PEDV was successfully constructed,and rPEDV was successfully rescued,laying a solid foundation for further studies on PEDV gene function and the development of novel vaccines.更多还原