猪痘病毒TK、ORF121、ORF143基因缺失毒株的 构建及其生物学特性的测定

Construction of Swinepox Virus TK,ORF 121 and ORF 143 Genes Deletion Strains and Determination of Their Biological Characteristics

为筛选猪痘病毒(SWPV)复制非必需区并测定其缺失株的生物学特性,本研究根据同源重组原理分别针对SWPV的TK(ORF063)、ORF 121和ORF 143基因设计引物。应用重叠延伸PCR技术拼接同源重组左右臂及EGFP筛选标记表达盒,将拼接片段分别转染到感染SWPV的PK15细胞。利用荧光显微镜观察标记含EGFP的单个蚀斑,筛选获取重组毒株。应用PCR及Western blotting对重组毒株进行鉴定。分别测定各毒株感染PK15细胞的蚀斑大小和皮下接种保育猪致病性。PCR结果表明,目的基因成功整合到相应位点,成功获得重组毒株rSWPV-TK、rSWPV-121和rSWPV-143;Western blotting结果显示,连续传代的重组毒株在PK15细胞上稳定表达外源蛋白。重组毒株在PK15细胞上形成的痘斑均小于亲本毒株,其中ORF121缺失株差异极显著(P<0.01)。各毒株均可导致保育猪痘斑形成,其中ORF121缺失株引起病变时间短,产生痘斑小,毒力较亲本下降。综上所述,本研究筛选到2个SWPV基因组中可供外源蛋白插入的复制非必需区ORF121和ORF143,为构建SWPV基因工程载体奠定了基础。

In order to screen the replication non-essential region of swinepox virus(SWPV)and determine the biological characteristics of the deletion strains,primers were designed and synthesized for TK(ORF 063),ORF 121 and ORF 143 genes of SWPV according to the principle of homologous recombination,and the overlap extension PCR technology was used to splice the gene fragments containing enhanced green fluorescent protein(EGFP)and homologous recombination of the left and right arms.The spliced fragments were transfected into PK15 cells infected with SWPV,respectively.Fluorescence microscope was used to select single plaque with EGFP as the reporter gene,and the recombinant strains were identified by PCR and Western blotting.The plaque size of PK15 cells and the pathogenicity of pigs were measured.The results showed that the gene fragment successfully integrated into recombinant strain genome.These recombinant strains were named as rSWPV-TK,rSWPV-121 and rSWPV-143,respectively.The results of Western blotting showed that the recombinant strains stably expressed the foreign protein on PK15 cells.The formation of pox spots on PK15 cells was smaller than that of the parent strain,and the difference of the ORF 121 gene deletion strain was extremely significant(P<0.01).All the strains could lead to the formation of pox spots on nursery pigs,in which the ORF 121 gene deletion strain caused the lesion for a short time,produced less pox spots,and the virulence was lower than that of wild strains.In summary,in this study,two non-essential replication regions ORF121 and ORF143 in the SWPV genome for foreign protein insertion were selected,laying a foundation for the construction of SWPV gene engineering vectors.