O型与A型口蹄疫重组标记疫苗病毒株的制备及鉴定

Generation of O and A inter-genotypic chimeric Foot-and-mouth disease virus strain for vaccine production

为研制针对我国边境地区(尤其是西南边境)流行的A型口蹄疫(FMD)的标记疫苗储备病毒株,本研究通过化学合成东南亚地区流行的A型口蹄疫疫毒(FMDV)A/VN/03/2009株的P1基因,将其插入含FMDV编码非结构蛋白3A aa91-aa105位缺失的O/HN/93病毒全长重组质粒p OFS/3A_(91-105)中,构建FMDV A型和O型嵌合的全长重组质粒p O/3A_(91-105)-AP1。该重组质粒经NotⅠ酶切线性化后转染于表达T7 RNA聚合酶的BSR/T7细胞中,拯救得到FMDV重组病毒。间接免疫荧光、RT-PCR和序列测定结果表明拯救的FMDV为正确的A型和O型间嵌合及3A aa91-aa105缺失的重组病毒。病毒蚀斑和一步生长曲线表明拯救病毒的感染性和复制能力稍低于其亲本病毒。该型间嵌合标记病毒的成功拯救为研制防控边境地区A型FMD的储备疫苗奠定了基础。

To develop a O and A inter-genotypic chimeric Foot-and-mouth disease virus（FMDV） as candidate vaccine strain for producing the inactivated vaccine against pandemic serotype A FMDV in border areas,especially in Southwest area,the O and A inter-genotypic chimeric full-length recombinant plasmid of p O/3A91-105-AP1 was constructed by insertion of the P1 gene synthesized according epidemic FMDV A/VN/03/2009 strain in Southeast Asia into the recombinant plasmid of p OFS/3A91-105,which contained FMDV O/HN/93 full-length c DNA with deleted sequence for encoding aa91-aa105 in non-structural protein 3A.Consequently,the O and A inter-genotypic chimeric FMDV was rescued by transfection of BSR/T7 cells expressing T7 RNA polymerase with linearized p O/3A91-105-AP1 which was identified by indirect immunofluorescence assay,RT-PCR and sequencing.Plaque assay and one-step growth curves indicated the rescued virus displayed a slightly reduced infectivity and replication capacity comparing to the parental virus.However,the deletion of aa91-aa105 in 3A could be used as a marker for the O and A inter-genotypic chimeric FMDV for differential detection of the vaccinated and naturally infected animals,The rescued FMDV will lay foundations for development of vaccine against pandemic serotype A FMDV for efficient control the disease in border areas.