犬副流感病毒NP基因的克隆与原核表达  

Cloning of the NP Protein Gene of Canine Parainfluenza Virus and Recombined Prokaryotic Expression

作　　者：刘腾飞[1] 马素贞[1] 申卫红[1] 陶玉成[1] 陈胜男[1] 简子健[1] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《新疆农业大学学报》2010年第2期146-150,共5页Journal of Xinjiang Agricultural University

基　　金：新疆维吾尔自治区高新技术项目(200611107)

摘　　要：以犬副流感病毒RNA为模板,采用RT-PCR方法,连接pMD18-T载体获取NP蛋白编码基因核苷酸序列,并应用定向克隆技术,构建原核表达载体pGEX-6P-1-NP。经测序鉴定证实获取的NP蛋白核苷酸序列与GeneBank中标准序列存在两处碱基变异,导致编码的蛋白质出现一个氨基酸的变异。测序结果表明,克隆出的目的基因序列已定向克隆到pGEX-6P-1载体,成功构建了其原核表达载体,并能在大肠杆菌BL21(DE3)中得到表达。Westen-Blotting分析结果显示,表达的GST-pGEX-6P-1-NP重组融合蛋白为82.1 kDa,可被犬副流感阳性血清所识别,表明NP基因所编码的蛋白具有一定的免疫活性。Using the total RNA isolated from canine parainfluenza virus as a template,the cDNA encoding NP was amplified by reverse transcription polymerase chain reaction method. Then, the PCR product was cloned into pMD18-T simple vector and was sequenced. At the same time, the cloned gene was subcloned into expression vector pGEX-6P-1 and the resultant construct was transformed into E. coli BL21（DE3） cells and induced by IPTG for sequencing,identifying and expression. Sequence analysis was showed that there were two mutant base pairs among the nucleotide sequence＇of NP gene which caused one mutant amino acid in its encoded protein,comparing with that of type strain published in GeneBank. Sequence analysis was also indicated that cloned gene in CPIV was ligated into the prokaryotic expression vector pGEX-6P-1 to construct pGEX-6P-1-NP successfully and the fusion protein was expressed smoothly in the E. coli BL21 （DE3）. Western-blotting analysis were shown that a fusion protein of 82. I kDa was expressed which could be recognised by canine antiserum against CPIV,indicating that fusion protein （GST-CPIV NP） possessed strong immunological competence.

关 键 词：犬副流感病毒 NP蛋白 RT-PCR 克隆 原核表达 免疫活性 

分 类 号：S851.659.5[农业科学—预防兽医学]