鸭肠炎病毒gD基因胞外区的原核表达及在感染鸭胚成纤维细胞中的定位  

Prokaryotic Expression of DEV Extracellular gD Gene and Its Subcellular Localization in Virus-infected DEF

作　　者：刘琰[1] 徐凝[1] 付培芬[1] 母晓宇[1] 董井泉 马波[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《畜牧兽医学报》2012年第6期922-927,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：高等学校博士学科点专项科研基金资助课题(20102325110004);黑龙江省科技攻关项目(GB01B503-02;GB04B504);东北农业大学博士启动基金

摘　　要：以本实验室获得的鸭肠炎病毒(DEV)C-KCE株US区基因序列(GenBank登录号EU714267)为基础PCR扩增出gD基因胞外区(gDw),将其定向克隆至原核表达载体pET-30a(+)并转化至大肠杆菌RosettaTM(DE3)pLys。经IPTG诱导表达,SDS-PAGE分析表明gD基因胞外区在大肠杆菌中获得与预期大小相符的表达蛋白。重组蛋白纯化后免疫家兔制备多克隆抗体,血清琼扩效价达1∶16,噬斑减数中和试验测定血清中和抗体效价达1∶64。通过免疫荧光技术首次对gD进行亚细胞定位检测,结果表明DEV感染鸭胚成纤维细胞(DEF)后4h近核区域细胞质内首先开始出现荧光,12h以后显著增加,明显的点状绿色荧光广泛存在于胞质中。本研究为进一步开展DEV gD的功能研究提供了重要数据和材料。The extracellular gD gene was amplified by PCR from DEV genomic DNA according to our laboratory discovered gene sequence （GenBank accession number EU714267）. The recombi- nant expression plasmid pET-30a-gDw could effectively express gDw fusion protein that was used to immunize rabbits. The titer of antibody tested by agar diffusion reaction was 1：16. Plaque re- duction neutralization test showed that its neutralization antibody titer was up to 1：64. By indi- rected immunofluorescence, the results showed that gD appeared in the cell cytoplasm near the nuclear as early as 4 hours post infection. After 12 hours post infection, the fluorescence in cyto- plasm was increased. Above all, the results afforded significant data for the study on the function of DEV gD.

关 键 词：鸭肠炎病毒 gD 原核表达 噬斑减数中和试验 亚细胞定位 

分 类 号：S852.659.1[农业科学—基础兽医学]