口蹄疫病毒结构蛋白VP1容忍外源标签的能力

Capacity of the structural protein VP1 of foot-and-mouth disease virus potentially accommodating insertion of different foreign tags

摘要:

[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。

Abstract:

[Objective] To identify the capacity that the structural protein VP1 of foot-and-mouth disease virus potentially accommodate insertion of different foreign tags, we constructed recombinant FMDVs containing foreign tags using FMDV reverse genetics system. [Methods] Using overlap extension PCR method, we introduced V5, TC12, KT3, 3×FLAG tag genes into G-H loop of VP1 capsid protein of FMDV. Linearized recombinant plasmids were transfected into BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant viruses. The recombinant viruses were analyzed by RT-PCR, indirect immunofluorescence, plaque phenotype and one-step growth curves. [Results] We successfully rescued the recombinant FMDVs expressing V5 and KT3 tag but could not rescue the recombinant FMDV containing TC12 and 3×FLAG tags. The introduction of V5 and KT3 tags both affected the replication capacity of FMDV. [Conclusion] Our results will lay foundations to study marker vaccine and FMDV vector in future.