犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。

Non-structural protein NS1 of canine parvovirus induces the apoptosis of cells

[Objective]To investigate the effects of canine parvovirus(CPV) non-structural protein-1(NS1) on the cell apoptosis induced by CPV and preliminarily explore the mechanism of CPV-induced apoptosis.[Methods]First,the NS1 gene was amplified by PCR from CPV genomic DNA and subcloned into pcDNA3.1A vector to generate NS1 eukaryotic expression vector pcDNA-NS1.To verify whether pcDNA-NS1 vector can mediate NS1 expression in eukaryotic cells,the human embryo kideny(HEK) 293FT cells were used to transiently express the recombinant NS1.The effects of NS1 on CPV-induced apoptosis were investigated by infecting the F81 host cells with CPV and transfecting the cells with NS1vector.The apoptosis of the cells was detected by AnnexinV/PI double staining for phosphatidylserine externalization on membrane and by luminescence method for caspase-3/7 activities.[Results]The results show that the sequence of NS1 gene amplified was consistent with the GenBank.The NS1 expression vector was shown to be correct and could mediate NS1 expression in eukaryotic cells.The phosphatidylserine on outside of membrane was detected and the caspase-3/7 activities were increased in both CPV-infected cells and NS1-transfected cells.These results indicate that both CPV and NS1 protein can induce the apoptosis of the cells.[Conclusion]CPV-induced apoptosis was closely related to its non-structural protein NS1.