含RGD受体结合位点口蹄疫病毒Asial/JS/China/2005株的拯救及初步鉴定

摘要

[Objective] To construct an infectious full-length cDNA clone of Asial/JS/China/2005 strain with Arg-Gly-Asp (RCD) receptor recognition site. [Methods] We constructed foot-and-mouth disease virus type Asial full-length cDNA clone pFMDV-RGD by using site-directed mutagenesis. The plasmid pFMDV-RGD contained the desired mutation. The plasmids pFMDV-RGD were linearized with Notl enzyme. Linearized plasmid and pcDNAT7P plasmids expressing T7 RNA polymerase cotransfected into BHK-21 cells to rescue FMDV-RGD. [Results] We constructed FMDV Asial/JS/China/2005 strain full-length cDNA clone with Arg-Gly-Asp receptor recognition site by sequence. We obtained rescued virus by plasmid cotransfection. The results of sequencing, indirect immunofluorescence, electron microscope and sulk mice pathogenicity analysis showed foot-and-mouth disease vims containing Arg-Gly-Asp receptor recognition site was successfully rescued. [Conclusion] The results lay a foundation for further study of biology characteristic diversity of rescued virus with Arg-Gly-Asp and Arg-Asp-Asp (RDD) receptor recognition site.%[目的]构建含有精氨酸.甘氨酸.天冬氨酸(RGD)受体结合位点口蹄疫病毒(FmDV)Asial/JS/Chind2005株的全长感染性cDNA克隆.[方法]采用定点突变方法,构建Asial型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD.pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒peDNATIP共转染BHK-21细胞,进行FMDV-RGD病毒拯救.[结果]序列测定结果表明成功构建了FMDV含有RGD受体位点的Asial/JS/China/2005全长cDNA克隆.共转染试验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asial/JS/China/2005株FMDV.[结论]该试验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础.