鸭肠炎病毒US10 基因的原核表达及抗血清的制备.pdfVIP

吉林农业大学学报 2013，01 Journal of Jilin Agricultural University E-mail:jlndxb@ * 鸭肠炎病毒 US10 基因的原核表达及抗血清的制备 *∗ ∗* 张 蕾，董井泉，母晓宇，马 波 ，王君伟 东北农业大学动物医学院，哈尔滨 150030 摘 要: 以鸭肠炎病毒 (DEV) C-KCE 株基因组 DNA 为模板，PCR 扩增获得 US10 基因。构建了其原核表达载体 pET-32a-US10 ，经 1. 0 mmol/L IPTG 诱导，目的蛋白在 Rosetta(DE3) pLys 宿主菌中获得了表达。SDS电泳结 果显示目的蛋白以包涵体形式表达，与预期大小相符。利用亲和层析法纯化目的蛋白，以纯化的蛋白为免疫原制备 其抗血清，琼脂扩散法测得抗体效价为 1:4，间接ELISA 法测定抗体效价为 1:102 400。间接免疫荧光试验证实制备 的抗血清可特异性识别鸭肠炎病毒的US10 基因编码产物。 关键词：鸭肠炎病毒；US10 基因；表达 中图分类号：S852.62 文献标识码：A 文章编号：1000-5684(2013) 网络出版地址： Prokaryotic Expression and the Antiserum Preparation of Duck Enteritis Virus US10 Gene 1 1 1 1 1 ZHANG Lei ,DONG Jing-quan ,MU Xiao-yu , MA Bo ,WANG Jun-wei College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030,China Abstract ：The US10 gene was expressed in pET-32a(+)/Rosetta(DE3) pLys system using the DNA of Duck enteritis virus (DEV) C-KCE as temple, US10 gene ORF was amplified by polymerase chain reaction (PCR). Then the recombinant protein of US10 gene induction with IPTG by SDSanalysis showed that the object protein is mainly expressed as inclusion bodies. Using the Ni-NTA Purification System, the recombinant protein immunized to rabbits was purified to make the antiserum. The titer of the antiserum tested by agar diffusion reaction was 1:4. The titer of the antiserum tested by ELISA was 1:102400. The prepared antiserum has a high specificity with the western-blot analysis, and the indirect immunofluorescence (IIF) test proved that the antiserum could react with DE