山羊痘病毒ORF8～ORF18缺失重组毒株的构建和鉴定

Construction and Identification of the Recombinant Goatpox Virus with ORF8—ORF18 Deleted

本研究旨在通过敲除山羊痘病毒（GTPV）大段基因组片段尝试构建安全性更好的基因工程减毒株和病毒表达载体。采用PCR方法,克隆了GTPV AV41株基因组ORF7-ORF8间1 242 bp的基因片段,以及ORF18~ORF19间1 355 bp的片段。接着分别以上述2片段作为左、右侧同源重组臂,构建了包含报道基因EGFP和抗性基因gpt的GTPV重组转移载体pCF-Eg。然后采用脂质体介导法,将重组转移载体pCF-Eg与GTPV AV41株共转染原代羊睾丸细胞,通过空斑纯化筛选阳性重组病毒,并鉴定重组病毒的遗传稳定性和生长特性。结果成功获得1株敲除掉ORF8~ORF18间9个ORF、长达7 kb基因组片段的重组病毒vCF-Eg。该重组病毒能稳定表达绿色荧光蛋白至少10代,并且其增殖滴度与亲本毒株基本相同。表明上述区域是GTPV的复制非必需区。

This experiment was conducted to develop a safer,high effective live goatpox virus（GTPV） attenuated vaccine and viral vector.Genome fragments of GTPV AV41 strain,including ORF7—ORF8 and ORF18—ORF19,were cloned by PCR as flanking sequences for homologous recombinant,respectively.The transfer shuttle plasmid pCF-Eg was constructed by inserting the expression cassettes of EGFP gene and gpt gene.The pCF-Eg and GTPV AV41 were co-transfected into LT cells with lipofectin,and recombinant virus was selected by fluorescence and PCR with EGFP gene primer respectively.Then,resulting recombinant virus was examined by fluorescence,PCR and TCID50 during passaging to 10th in LT cells.A recombinant GTPV with ORF8—ORF18 deleted was obtained and named as vCF-Eg.It was stable,and shared same multiplication ability with its parental virus（AV41） in LT cells.It suggests that genomic region of ORF8—ORF18 may be non-essential regions for replication.