表达O型口蹄疫病毒VP1蛋白的重组塞内卡病毒的构建与鉴定

Construction and Identification of Recombinant Senecavirus Expressing VP1 Protein of Foot-and-mouth Disease Virus Type O

A型塞内卡病毒(SVA)是近年来新流行的一种传染性病原,与口蹄疫病毒(FMDV)具有相似的基因组结构,所致临床症状也十分相似。为了研发一种新型的基因工程活载体疫苗,用于预防塞内卡病毒感染和口蹄疫,以SVA全长感染性克隆pSVA-GX01为基础,在SVA 2A与2B基因之间插入O型FMDV的VP1基因,经双酶切及测序鉴定,成功获得了重组质粒pSVA-FMDV-VP1-O。将重组质粒转染BHK-21细胞,拯救获得重组病毒rSVA-FMDV-VP1-O,间接免疫荧光试验鉴定显示,该重组病毒可在细胞中表达O型FMDV的VP1蛋白。对该重组病毒进行体外生长增殖特性以及遗传稳定性分析表明,重组毒株和亲本毒株在BHK-21细胞上具有相似的增殖特性,插入的FMDV VP1基因可在细胞传代过程中稳定存在6代。研究结果为开发以塞内卡病毒为载体构建表达口蹄疫病毒VP1蛋白的重组基因工程活载体疫苗提供了参考。

Senecavirus A(SVA)is an emerging infectious disease pathogen.It has similar viral genome structure and induces similar clinical symptoms in affected pigs with foot-and-mouth disease virus(FMDV).The aim of the present study was to develop a novel genetically engineered live vector vaccine for immune protection against infection of SVA and FMDV.The VP1 gene of FMDV type O was inserted between SVA 2A and 2B genes of SVA based on a full-length infectious clone SVA(pSVA-GX01).The resulting recombinant plasmid pSVA-FMDV-VP1-O was successfully constructed and rescued after transfection of the recombinant plasmid in to BHK-21 cells.The results of indirect immunofluorescence assay(IFA)showed that the recombinant virus can expresse FMDV VP1protein in BHK-21 cells.The analysis of the proliferation of the recombinant virus showed that rSVA-FMDV-VP1-O displayed similar growth kinetics with the parent virus rSVA-GX01 in BHK-21 cells.The analysis of genetic stability of the recombinant virus showed that FMDV VP1 gene in SVA genome is genetically stable during 6 serial passages in BHK-21 cells.This study laid a foundation for the development of genetically engineered live vector vaccine with SVA as vector.