3A蛋白104-115位氨基酸缺失口蹄疫A型标记病毒的构建

Construction of type A foot-and-mouth disease marker virus with deletion of 104-115 amino acids of 3A protein

摘要:

[目的]构建一株含3A非结构蛋白104-115位氨基酸缺失的口蹄疫A型标记病毒，分析其生物学特性和发展标记疫苗的潜力。[方法]采用融合PCR技术，在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆pQAHN中引入3A 104-115位氨基酸的缺失，构建全长重组质粒。全长质粒经Not I线化后转染表达T7 RNA聚合酶的稳定细胞系，拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性，并用实验室开发的针对3A优势表位（AEKNPLE）的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。[结果]成功拯救到一株含3A 104-115位氨基酸缺失的口蹄疫A型标记病毒，3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。[结论]本研究构建的3A蛋白104-115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株，用于我国未来口蹄疫A型的有效防控。

关键词:3A蛋白;104-115位氨基酸缺失;口蹄疫A型;标记病毒;构建

Abstract:

[Objective] To structure a type A foot-and-mouth disease (FMD) marker virus with deletion of 104-115 amino acids of the nonstructural protein 3A, then to analyze its biological characteristics and the potential of developing marker vaccine.[Methods] Using overlap extension PCR method, we introduced the deletion of 104-115 amino acids of the nonstructural protein 3A into FMDV A/Sea-97/CHA/2014 full-length infectious clone pQAHN to generate a recombinant full-length plasmid. The marker virus was rescued after transfecion linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase and identified by RT-PCR, sequencing analysis, indirect immunofluorescence and Western blotting. The plaque and one-step growth curves were used to analyze the biological characteristics of the marker virus. A developed block ELISA method targeting to the deleted epitope of 3A was used to analyze the potential of differentiating animals infected with the marker virus and the wild type virus.[Results] We rescued a type A FMD marker virus containing deletion of 104-115 amino acids of 3A protein. The deletion did not affect the plaque phenotype and one-step growth curve of the marker virus. A developed block ELISA method targeted to the deleted epitope could clearly differentiate animals infected with the marker virus and the wild type virus.[Conclusion] The marker virus containing deletion of the dominant epitope of 3A is suitable as a vaccine candidate strain of differentiating infected from vaccinated animals to effectively prevent and control type A FMD in future.

Key words:3A protein;deletion of 104-115 amino acids;type A foot-and-mouth disease;marker virus;construction