猪细小病毒NS1蛋白65-133位氨基酸缺失对其核定位的影响  

Effect of amino acid 65-133 deletion of porcine parvovirus NS1 protein on its nuclear localization

作　　者：张驰[1] 徐盟龙 袁一心 王朋涛 靳晓慧[2] 魏战勇[1,2] ZHANG Chi;XU Menglong;YUAN Yixin;WANG Pengtao;JIN Xiaohui;WEI Zhanyong(Engineering College of Animal Husbandry and Veterinary Science,Henan Agricultural University,Zhengzhou 450002;Key Laboratory for Animal-derived Food Safety of Henan Province,Zhengzhou 450002,China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《河南农业大学学报》2020年第4期630-637,共8页Journal of Henan Agricultural University

基　　金：国家自然科学基金面上项目(31772722);河南省高等学校重点科研项目(18A230001)。

摘　　要：为了探究猪细小病毒NS1蛋白入核机制,通过生物信息学网站预测PPV NS1核定位序列,根据GenBank上传的PPV非结构蛋白NS1的序列设计NS1扩增引物和一系列重叠PCR引物,以河南省动物性食品安全重点实验室保存的猪细小病毒DNA为模板,扩增NS1和缺失突变体基因,构建真核表达重组质粒pCAGGS-HA-NS1、pCAGGS-HA-NS165-133aaDel、pCAGGS-HA-NS1207-267aaDel。将其转染至PPV易感猪源细胞系PK-15细胞,通过免疫荧光和Western-blot鉴定NS1和不同的NS1缺失突变体在细胞内的核定位情况。结果表明,pCAGGS-HA-NS1、pCAGGS-HA-NS165-133aaDel、pCAGGS-HA-NS1207-267aaDel编码蛋白均成功表达。免疫荧光和Western-blot显示NS1和NS1207-267aaDel蛋白定位于细胞核,而NS165-133aaDel蛋白的细胞定位较NS1发生改变,由细胞核定位改变为细胞质定位,说明PPV NS1蛋白65-133位氨基酸缺失能改变其在细胞内的核定位,PPV NS1蛋白核定位序列NLS存在于NS1的65-133aa处。In order to explore the nuclear entry mechanism of porcine parvovirus NS1 protein,the bioinformatics website was used to predict the PPV NS1 nuclear localization sequence,and the NS1 amplification primers and a series of overlapping PCR primers were designed based on the sequence of the porcine parvovirus non-structural protein NS1 uploaded by GenBank.Using the porcine parvovirus DNA stored in our laboratory as a template to amplify NS1 and deletion mutant genes,eukaryotic expression recombinant plasmids pCAGGS-HA-NS1,pCAGGS-HA-NS165-133aaDel,and pCAGGS-HA-NS1207-267aaDel were constructed,which was transfected into PK-15 cells of PPV susceptible pig cell line.The nuclear localization of NS1 and different NS1 deletion mutants were identified by immunofluorescence and Western-blot.The results showed that:pCAGGS-HA-NS1,pCAGGS-HA-NS165-133aaDel,pCAGGS-HA-NS1207-267aaDel encoded proteins were all successfully expressed.Immunofluorescence and Western-blot display that NS1 and NS1207-267aaDel proteins were localized in the nucleus.However,the cell localization of NS165-133aaDel protein was changed compared to NS1,which was changed from the nucleus to the cytoplasm,indicating that the deletion of amino acids in PPV NS1 protein at position 65-133 could change its intracellular nuclear localization,and that the nuclear localization sequence NLS of PPV NS1 existed at 65-133aa of NS1.

关 键 词：猪细小病毒 NS1蛋白 核定位 

分 类 号：S855.3[农业科学—临床兽医学] S828[农业科学—兽医学]