J亚群禽白血病病毒gp85基因的原核表达及间接ELISA方法建立  

Development of indirect ELISA detecting antibody against avian leukosis virus subgroup J with gp85 as coating antigen

作　　者：刘丽娜[1,2] 邵华斌[1] 程国富[2] 杨峻[1] 张琳[1] 谢华丽[2] 栗绍文[2] 罗青平[1] 

机构地区：[1]湖北省农业科学院畜牧兽医研究所动物胚胎工程及分子育种湖北省重点实验室,湖北武汉430064 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国预防兽医学报》2014年第1期38-41,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：湖北省科技计划研究与开发项目(2010BBB08;CARS-42-G11)

摘　　要：为建立检测J亚群禽白血病病毒(ALV-J)抗体间接ELISA方法,本实验根据已发表的ALV-J gp85基因序列设计一对特异性引物,以该病毒cDNA为模板进行PCR扩增,将目的基因克隆于pET-28a(+)载体中,原核表达的重组蛋白约为38 ku,经western blot分析表明,重组蛋白gp85具有良好的反应原性。以纯化的重组蛋白作为包被抗原建立了间接ELISA抗体检测方法,该方法对其它相关的禽类病原无交叉反应,其组内和组间的变异系数均低于13%,具有良好的重复性。对458份临床血清样品进行检测,同时用IDEXX的同类ELISA抗体检测试剂盒进行对比,总符合率达到96.29%。本研究为ALV-J的流行病学调查提供了一种简便的血清学诊断方法。To establish a indirect ELISA method for detecting antibody against avian leukosis virus subgroup J （ALV-J）, the gp85 gene of ALV-J was amplified by RT-PCR and inserted into pET-28a（＋） vector for expressing in E.coli induced by 1PTG. Western blot showed that the expressed recombinant gp85 protein was about 38 ku and recognized with positive serum. In addition, the indirect ELISA was developed with the purified protein as coating antigen, and the results showed the assay was specificity and repeatability. Furthermore, a total of 458 clinical serum samples were detected by this assay and the agreement was 96.29% with commercial ELISA Kit （IDEXX）. The establishment of the assay could be potentially used for epidemiology investigation of the ALV-J infection.

关 键 词：J亚群禽白血病 GP85基因 蛋白表达 酶联免疫吸附试验 

分 类 号：S852.65[农业科学—基础兽医学]