山羊痘病毒serpin基因缺失重组毒株的构建和鉴定

Construction and identification of the recombinant goatpox virus with serpin gene deletion

通过敲除与山羊痘病毒(GTPV)毒力相关的serpin基因以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方法克隆了包含GTPV AV41株serpin基因(ORF149)及其侧翼长为1 745bp的片段,并在serpin基因片段内部分别插入报道基因EGFP和抗性基因gpt表达盒,构建GTPV重组转移载体pSp-Eg。然后将重组转移载体pSp-Eg与GTPV AV41株共转染BHK-21细胞,通过空斑纯化筛选阳性重组病毒,并鉴定其遗传稳定性和生长特性。结果,成功获得一株敲除serpin基因的重组病毒vSp-Eg。该重组病毒在至少10代能稳定表达绿色荧光蛋白,在培养细胞中的生长特性与亲本毒株基本一致,但病毒效价降低1个数量级。结果表明,serpin基因是GTPV的复制非必需基因。

This experiment was to develop a safer,high effective live attenuated GTPV vaccine and viral vector via knock-out viral serpin gene. A 1 745 bp genome fragment of GTPV AV41 containing serpin gene（ORF149） was cloned, and it was employed as flanking sequences for homologous recombinant. The serpin genes was inactivated by inserting the expression cassettes of EGFP gene and gpt gene, resulting in a transfer shuttle plasmid pSp-Eg. The pSp-Eg and GTPV AV41 were co-transfected into BHK-21 cells,and recombinant virus was screened. The resultant recombinant virus was examined by fluorescence observation,PCR detection and tittering during serial culture till the 10th passage in BT cells. A recombinant GT- PV with serpin gene deletion was obtained and named as vSp-Eg. It was stable,and shared similar growth pattern with its parental virus（AV41） in cell culture with a 10-fold lower titer. It suggests that serpin gene could be employed as non-essential replication region for recombinant GTPV construction.