猪瘟病毒NS2、NS3和NS5A与猪Rab25蛋白的亚细胞定位分析  

Subcellular localization analysis of classical swine fever virus NS2,NS3,NS5A protein and porcine Rab25 protein

作　　者：徐盼盼[1] 侯玉凤[1] 王凯[1] 罗弼豪 范昱鑫 郭抗抗[1] XU Pan-pan;HOU Yu-feng;WANG Kai;LUO Bi-hao;FAN Yu-xin;GUO Kang-kang(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2020年第4期673-680,共8页Chinese Journal of Veterinary Science

基　　金：家畜疫病病原生物学国家重点实验室2018年开放基金课题资助项目(SKLVEB2018-KFKT001)。

摘　　要：为了研究NS5A与Rab25的亚细胞定位及作用基因区段,先构建pDsRed1-N1-Rab25红光载体,利用经过改造并加GFP的pcDNA3.1(+)-NS2、pcDNA3.1(+)-NS3和pcDNA3.1(+)-NS5A载体,将pDsRedN1-Rab25红光载体与这3种绿光载体分别共转染于猪血管内皮细胞(SUVEC),通过共聚焦显微镜观察显示Rab25和NS2、NS3共定位现象不明显而与NS5A存在着明显共定位。为了进一步研究NS5A与Rab25作用基因区段,构建NS5A的3个分段基因载体:pEGFP-C1-NS5A-1-84、pEGFP-C1-NS5A-84-804和pEGFP-C1-NS5A-804-1491,转染SUVEC并观察共定位情况,结果显示Rab25与NS5A的804~1491 bp存在明显共定位,而与1~84,84~804 bp的共定位现象不明显。结果说明Rab25与NS5A基因的804~1491 bp存在共定位,这为继续研究Rab25与NS5A的关键作用位点及互作奠定基础。In order to study subcellular localization and functional gene region of non-structural protein 5 A(NS5 A)and Rab25,the pDsRed1-N1-Rab25 was constructed and transfected into swine umbilical vein endothelial cells(SUVEC)along with pcDNA3.1(+)-NS2,pcDNA3.1(+)-NS3 and pcDNA3.1(+)-NS5 A,respectively.Finally,the co-localization of Rab25 and non-structural protein 2(NS2),non-structural protein 3(NS3),NS5 A were observed by laser confocal scanning microscopy.The results showed that the co-localization of Rab25 with NS2 and NS3 were not obvious and there was significant colocalization with NS5 A.In order to further study the functional region of NS5 A and Rab25,the three segmented genes of NS5 A,the pEGFP-C1-NS5 A-1-84,pEGFP-C1-NS5 A-84-804 and pEGFP-C1-NS5 A-804-1491 were constructed.The segmented vectors of NS5 A with pDsRed1-N1-Rab25 were cotransfected into cells respectively,and the colocalization was observed.The results showed that Rab25 colocalized only with the 804-1491 bp of NS5 A.These results indicated that there was a colocalization between Rab25 and the 804-1491 bp of NS5 A,which laid a foundation for further study on the key sites and interactions between Rab25 and NS5 A.

关 键 词：猪瘟病毒 非结构蛋白5A(NS5A) 猪血管内皮细胞(SUVEC) Rab25蛋白 亚细胞定位 

分 类 号：S852.65[农业科学—基础兽医学]