犬瘟热病毒N基因在昆虫杆状病毒表达系统中的表达及应用  

Expression of Nucleocapsid Protein of Canine Distemper Virus Base on Bac-to-Bac Baculovirus System

作　　者：冯佩平[1,2] 张蕾[2] 柴秀丽[2] 张海玲[2] 赵建军[2] 胡博[2] 白雪[2] 高晗[2] 邵西群[2] 闫喜军[2] 赵权[1] 徐磊[3] 

机构地区：[1]吉林农业大学,长春130118 [2]中国农业科学院特产研究所预防兽医研究室,长春30122 [3]中国兽医药品监察所,北京100081

出　　处：《中国生物工程杂志》2012年第4期60-66,共7页China Biotechnology

基　　金：科技部星火计划重点项目(2010GA660009)

摘　　要：目的:为检测犬瘟热抗体提供诊断抗原,应用Bac-to-Bac杆状病毒表达系统表达犬瘟热病毒(canine distemper virus,CDV)N蛋白。方法:采用RT-PCR克隆CDV-N基因,构建重组杆粒BacmidCDV-N,并将其转染Sf9昆虫细胞。通过Western blot和间接免疫荧光检测N蛋白的表达。以表达的CDV N蛋白为包被抗原,建立了检测犬瘟热抗体的间接ELISA方法。结果:成功表达了CDV N蛋白。间接ELISA(iELISA)方法中,犬血清最佳稀释度1∶80,N蛋白稀释度1∶40(6.3μg/100μl)。应用该方法共检测了36份犬血清,与中和试验检测方法相比较,其特异性为81.8%,灵敏度为96.0%,符合率为91.7%。结论:表达的CDV-N蛋白具有良好反应原性,建立了快速检测CDV阳性血清的iELISA方法。Objective： The nucleocapsid（N） protein of Canine distemper virus（CDV） was expressed base on Bac-to-Bac baculovirus expression system.CDV-N protein was used as a diagnostic antigen for detecting positive serum of CDV.Methods： N gene fragment was amplified by RT-PCR.Recombinant plasmid Bacmid CDV-N transfected into Sf9 cells was builded.CDV-N protein was expressed and shown by Western blot analysis and indirect immunofluorescence.Results：The N protein was correctly expressed.Thirty six serum were tested at anti-dilution 1∶80,respectivly diluted 1∶40（6.3μg/100μl） with CDV-N protein as the best reaction concentration by indirect ELISA,compared to virus neutralizing sensitivity 96.0%,specificity 81.8% and compliance rate 91.7%.Conclusion：The recombinant CDV-N protein was used as antigen in iELISA and shown to react with CDV antisera as a serological tool for the mass screening of CDV antibodies.

关 键 词：CDV-N蛋白 杆状病毒表达系统 IELISA 

分 类 号：Q786[生物学—分子生物学]