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【目的】利用口蹄疫病毒的反向遗传操作技术,构建含不同外源标签口蹄疫病毒的全长克隆,鉴定口蹄疫病毒结构蛋白VP1容忍不同外源标签的能力。【方法】通过融合PCR技术,在FMDV O/HN/93全长感染性克隆的VP1 G-H环分别引入V5、TC12、KT3、3FLAG外源标签,构建全长质粒。全长质粒经Not I线化后转染表达T7 RNA聚合酶的稳定细胞,拯救重组病毒。RT-PCR、序列测定、间接免疫荧光鉴定病毒,噬斑和一步生长曲线分析重组病毒的生物学特性。【结果】成功拯救到表达V5或KT3表位标签的重组病毒,未能拯救到表达TC12或3×FLAG的重组病毒。V5和KT3表位标签的插入均影响了口蹄疫病毒的复制能力。【结论】重组口蹄疫病毒的成功拯救为未来标记疫苗以及口蹄疫病毒作为表达载体等的研究奠定了基础。
【Objective】 The full-length clone containing different exogenous tagged foot-and-mouth disease virus was constructed by reverse genetic manipulation of foot-and-mouth disease virus and the ability of VP1 of different foot-and-mouth disease virus to tolerate different exogenous tags was identified. 【Method】 V5, TC12, KT3 and 3FLAG exogenous tags were introduced into the VP1 G-H loop of FMDV O / HN / 93 full-length infectious clone respectively by fusion PCR and the full-length plasmids were constructed. The full-length plasmid was linearized with Not I and transfected into stable cells expressing T7 RNA polymerase to rescue the recombinant virus. RT-PCR, sequence analysis, indirect immunofluorescence identification of viruses, plaques and one-step growth curve analysis of the biological characteristics of recombinant viruses. [Results] The successful rescue of the recombinant virus expressing the V5 or KT3 epitope tag failed to rescue the recombinant virus expressing TC12 or 3 × FLAG. The insertion of V5 and KT3 epitope tags all affected the replication capacity of foot-and-mouth disease virus. 【Conclusion】 The successful rescue of recombinant foot-and-mouth disease virus laid the foundation for the future research of the tagged vaccine and foot-and-mouth disease virus as the expression vector.