摘要
本研究在完成Asia1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1800bp),并利用长片段PCR扩增得到基因组3′端片段(约6700bp)。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。
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第11期1621-1626,共6页
Chinese Journal of Biotechnology
基金
国家科技支撑计划(No.2006BAD06A17-08)
黑龙江省科技计划(No.GA06B202)
现代农业产业技术体系建设专项资金(No.nycytx-0303)资助~~
关键词
口蹄疫病毒
全长CDNA
感染性克隆
反向遗传
病毒拯救
体外转录
foot-and-mouth disease virus（FMDV）
full-length cDNA
infectious clones
reverse genetics
virus rescue
in vitro transcription
