A型禽流感病毒NS1蛋白的表达及其诱导细胞凋亡功能的研究  

Eukaryotic expression and inducing apoptosis of avian influenza A virus NS1 protein

作　　者：孙进华[1,2] 马波[1] 于志丹[1] 霍慧[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030 [2]东北农业大学动物科技学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第4期287-292,共6页Chinese Veterinary Science

基　　金：国家"十五"科技攻关计划项目(2004BA519A)

摘　　要：利用瞬时表达方法表达禽流感病毒(AIV)NS1蛋白,并初步研究了体外表达的NS1蛋白对宿主细胞凋亡的影响。应用脂质体介导法,将含有不同亚型AIV NS1基因的阳性质粒pcDNA3.1(+)-qNS1(H5N1)、pcDNA3.1(+)-rNS1(H9N2)转染Vero细胞系及MDCK细胞系。经SDS-PAGE及Western-blotting检测,qNS1和rNS1基因在Vero细胞及MDCK细胞中均获得了表达。应用AO/EB荧光染色法、Annexin V-PI法分别对转染后的Vero细胞及MDCK细胞进行细胞凋亡检测,结果显示,转染阳性质粒的MDCK细胞凋亡率增高,而转染空载体pcDNA3.1(+)的MDCK细胞及转染的Vero细胞均无明显变化。表明,qNS1、rNS1蛋白在体外培养的MDCK细胞中表达并能够诱导其发生凋亡,而在Vero细胞中却不能发挥此功能。In order to study the effect of transient expression of avian influenza virus（AIV） NS1 protein in vitro on the host cell apoptosis,qNS1 gene of AIV A/Goose/Guangdong/1/96（HSN1） strain and rNS1 gene of AIV A/Chicken/MDJ/2000（HgN2） strain was respectively subcloned into pcDNA3. 1（＋） vector to construct eukaryotic expression vectors pcDNA3. 1 （＋）-qNS1 and pcDNA3. 1 （＋） rNS1. Then the two eukaryotic expression plasmids were respectively transfected into Vero and MDCK cells by Lipofectamine^TM 2000. The SDS-PAGE and Western blot analyses showed that qNS1 and rNS1 genes had been successfully expressed in the Vero and MDCK cells, respectively. Apoptosis in the transfected Vero and MDCK cells were tested by fluorescent microscope analysis and Annexin V-PI, respectively. The results showed that the transfected MDCK cells had an increased apoptosis rate, while the control MDCK cells and the transfected Vero cells had no significant apoptosis. These results indicated that the expressed proteins qNS1 and rNS1 could lead to apoptosis in MDCK cells in vitro ,but not in Vero cells.

关 键 词：禽流感病毒 NS1蛋白 MDCK细胞 VERO细胞 真核表达 细胞凋亡 

分 类 号：S852.659.5[农业科学—基础兽医学] O786[农业科学—兽医学]