基于重组E2蛋白的猪瘟病毒抗体间接ELISA检测方法的建立  

Development of an indirect ELISA based on the recombinant E2 protein expressed in baculovirus for detecting antibodies against classical swine fever virus

作　　者：孙元[1] 夏照和[1] 梁冰冰[1] 彭伍平[1] 仇华吉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2008年第4期315-319,共5页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD06A03);国家重点基础研究发展计划(973)项目(2005CB523202)

摘　　要：根据昆虫细胞密码子偏嗜性,对猪瘟病毒E2基因进行密码子优化,并利用昆虫杆状病毒表达系统进行了表达,表达水平约为野生型E2基因的3倍。将表达的重组E2蛋白纯化后作为ELISA包被抗原,建立了一种基于重组E2蛋白的间接ELISA方法,并对此方法的反应条件进行了优化。确定的ELISA最佳条件为:抗原包被浓度为2μg/mL,封闭液为10 g/L聚乙烯醇PBS溶液,被检血清1∶160稀释,辣根过氧化物酶标记的兔抗猪IgG(二抗)1∶5 000稀释。确定的阴性血清临界D450 nm值为0.30,阳性血清临界D450 nm值为0.35。将该ELISA方法与IDEXX公司生产的猪瘟病毒抗体检测试剂盒做相关比较,结果符合率为82.3%。表明,该方法可以用于猪瘟病毒抗体的检测。A classical swine fever virus（CSFV） E2 gene was codon-optimized according to the Sf9 cell codon usage. The codon-optimized E2 gene was expressed in insect baculovirus expression system, with 2 times higher expression level as compared to the wild-type E2 gene. An indirect ELISA was developed and optimized with the purified recombinant E2 protein as coating antigen. The optimal conditions of ELISA were determined as follows. The microplate was coated with 200 ng of the recombinant E2 protein per well,and then blocked with 10 g/L polyvinyl alcohol in PBS buffer overnight at 4 ℃. The serum samples were diluted at 1 ： 160 and rabbit anti-pig HRP-IgG were diluted at 1 ： 5 000. The cutoff values at 450 nm of the negative or the positive sera were 0.30 or 0.35. The coincidence rate between the ELISA and IDEXX CSFV antibody test kit was determined as 82.3% ,and the result indicated that the indirect ELISA could be used for detecting anti-CSFV antibodies.

关 键 词：猪瘟病毒 E2蛋白 密码子优化 杆状病毒表达系统 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学] R446.61[农业科学—兽医学]