稳定表达非洲猪瘟病毒P54蛋白的Vero细胞系的建立

旨为建立稳定表达非洲猪瘟病毒(ASFV)P54蛋白的Vero细胞系,将ASFV-P54基因与绿色荧光基因Azami Green的融合基因片段,将其克隆至慢病毒载体pLV-puro中构建重组慢病毒质粒pLV-ASFV-P54-AG,将该质粒与慢病毒包装质粒pH1和pH2共转染HEK-293V细胞,包装表达ASFV-P54蛋白的慢病毒。将重组慢病毒在聚凝胺(Polybrene)的介导下感染Vero细胞,筛选出一株稳定表达ASFV-P54蛋白的Vero细胞系,命名为Vero-AG-ASFV-P54。间接免疫荧光试验表明,该细胞系能够与P54多克隆抗体反应;经波兰国家兽医研究所进一步验证,结果显示,该细胞系与ASFV抗体阳性血清也能发生反应,并且与阴性血清无反应。结果表明,Vero-AG-ASFV-P54细胞系能够稳定高效的表达具有生物活性的ASFV-P54蛋白。

Generation of a Vero Cell Line Stably Expressing African Swine Fever Virus P54 Protein

In order to establish the Vero cell lines that may stably express African swine fever virus(ASFV),the fusion gene fragment from the ASFV-P54 gene with green fluorescent gene Azami Green was cloned into the lentivirus vector pLV-puro.The resulting plasmid,pLV-ASFV-P54-AG,was then co-transfected HEK-293V cells together with packaging plasmids pH1 and pH2 for packaging recombinant lentiviruses.The generated lentiviruses were then utilized to infect Vero cells mediated in the presence of polybrene.A Vero cell line,constitutively expressing the ASFV-P54 protein,was screened and designated as Vero-AG-ASFV-P54.Moreover,indirect immunofluorescence assays further demonstrated that the generated cell line reacted with P54 polyclonal antibody.The results further verified by National Veterinary Research Institute in Poland showed that the cell line reacted with the positive sera of ASFV antibody,but did not react with the negative sera.The above results reveal that the Vero-AG-ASFV-P54 cell line generated in this study may stably and efficiently express ASFV-P54 protein with biological activity.