鸭肠炎病毒ul47基因的原核表达及其抗血清中和活性的鉴定  

Prokaryotic expression of ul47 gene of duck enteritis virus and identification of neutralization activity of the antiserum against the expressed product

作　　者：张杨[1] 刘峰源[1] 乌伊罕[1] 刘晓玫[1] 王君伟[1] 马波[1] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2008年第11期946-951,共6页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD06A05-1)

摘　　要：提取鸭肠炎病毒(DEV)基因组DNA,采用PCR方法扩增获得了DEVul47基因,将该基因片段定向克隆到原核表达载体pET-30a,构建重组质粒pET-30a-ul47,并转化受体菌Rosetta(DE3)pLysS中,经IPTG诱导表达,表达产物用SDS-PAGE分析。结果显示,ul47基因在大肠杆菌Rosetta中获得与预期大小相符的表达蛋白。利用Ni-NTA纯化表达蛋白,制备了兔抗DEV UL47蛋白的多克隆抗体,经琼脂双扩散测定,该抗血清效价为1∶16。间接免疫荧光试验和蚀斑减数中和试验结果显示,DEV UL47蛋白主要定位于感染细胞的细胞质和细胞膜,制备的兔抗DEV UL47多克隆抗体对DEV有中和活性。The ul47 gene was amplified by PCR from the extracted duck enteritis virus（DEV） genomic DNA,and then cloned into the prokaryotic expression vector pET 30a,and recombinant expression plasmid pET-30a-ul47 was constructed. The the recombinants were transformed into the host strain Rosetta（DE3） PlysS and induced by IPTG. The specific protein expression was detected by SDS PAGE,and the results indicated that pET 30a-ul47 could effectively express UL47 fusion protein,and the fusion protein was purified by His Purify Kit. The purified protein was used immunize rabbits and produced antibody against UL47. The titer of antibody tested by agar diffusion reaction was 1 ： 16. The UL47 protein was mainly located in the cell membrane and cytoplasm of infected cells with DEV by indirect immunofluorescence and plaque reduction neutralization test, and the anti-rabbit serum possessed neutralization activity against UL47 antigen.

关 键 词：鸭肠炎病毒 ul47基因 原核表达 间接免疫荧光试验 蚀斑减数中和试验 

分 类 号：S852.659.1[农业科学—基础兽医学]