牛Asia 1型口蹄疫病毒感染性克隆的构建

Rescue of bovine Asia 1 serotype foot-and-mouth disease virus from a full-length cDNA clone

摘要:

本研究在完成Asia 1型口蹄疫病毒(FMDV)As01株全基因组测序的基础上, 采用融合PCR扩增得到含有15个C碱基的5￠端片段(约1800 bp), 并利用长片段PCR扩增得到基因组3￠端片段(约6700 bp)。再利用单一的酶切位点将2个片段克隆到pBluescript SK载体中, 从而获得携带As01株基因组全长cDNA的重组质粒pBSAs。将该质粒线性化后作为模板体外转录并转染BHK-21细胞, 可观察到典型的细胞病变。对收获的病毒采用RT-PCR、间接免疫荧光和电镜观察等检测及鉴定, 结果表明, 拯救出了具有感染性的Asia1型FMDV。拯救毒与亲本毒对乳鼠的致病力(LD50)差异不显著, 具有相似的生物学特征。该感染性克隆的构建, 为深入研究FMDV的致病机理及开发新型疫苗提供了有效的反向遗传操作平台。

Abstract:

After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5￠ fragment was 1.8 kb in length including 15Cs, and the 3￠ fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD.