荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响  

Impact of fluorescent protein tag on gD envelope protein subcellular localization in BHK-21 cells

作　　者：王世民[1] 张艳楠[2] 胡月[1] 苏艳[1] 冉多良[1] 包晓玮[2] 

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]新疆农业大学食品科学与药学学院,新疆乌鲁木齐830052

出　　处：《微生物学报》2016年第7期1194-1201,共8页Acta Microbiologica Sinica

基　　金：新疆维吾尔自治区高等学校科研计划项目(XJEDU2014S024)~~

摘　　要：【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。[Objective] The fluorescent protein and gD envelope protein of equine herpes virus type 1（EHV-1） were used to study the impact of tags on gD protein subcellular localization in BHK-21 cells.[Methods] With the EHV-1genome as a template,the gD complete gene was amplified by PCR technique.The product of PCR was cloned to p Ac GFP1-C1 and pDsRed2-N1 plasmids.The recombinant plasmids were designated as p Ac-GFP-gD（GFP-gD） and pDs-gD-Red（gD-Red）.The GFP gene was inserted into the posterior position of gD gene signal peptide sequence.The modified gD gene signal peptide sequence was cloned to pVAX-1 plasmid,so that pVAX-S-GFP-gD＇（S-GFPgD＇） recombinant plasmid was constructed.Meanwhile,the flag tag was added to N-terminal of gD sequence and they were cloned to pVAX-1 expression vector for constructing pVAX-Flag-gD recombinant plasmid.The BHK-21 cells were transfected with the 4 different recombinant plasmids and the subcellular localizations of fusion proteins were determined by lasar confocal scan microscopy.[Results] Four eukaryotic expression vectors were constructed successfully.In BHK-21 cells,the vast majority of gD envelope proteins was localized in Golgi,and a small amount of gD was localized in the nucleus.[Conclusion] Our finding reveals that the fluorescent protein of different insertion sites has no significant effects on the subcellular localization of gD,and provides a useful reference for other researchers.

关 键 词：EHV-1 gD囊膜蛋白 亚细胞定位 荧光蛋白标签 

分 类 号：S852.652[农业科学—基础兽医学]