中国兽医科学2017，Vol.47Issue(6)：680-686,7.DOI:10.16656/j.issn.1673-4696.2017.06.002
犬流感病毒NP基因在昆虫细胞中的表达及其抗体间接ELISA检测方法的建立
Expression of NP protein of canine influenza virus in insect cells and development of an indirect ELISA for antibody detection
摘要
Abstract
This study is aimed to establish an indirect ELISA to detect canine influenza virus(CIV)antibodies based on the recombinant NP protein expressed by baculovirus system.Total RNA was extracted from CIV infected MDCK cells,NP gene was amplified by RT-PCR and inserted into vector pFastBacHTA.The recombinant donor plasmid was transformed to competent Escherichia coli DHlOBac.Then the recombinant bacmid NP-DHIO was transfected to insect sf-9 cells and thus obtained the recombinant baculovirus P1-NP.Western-blot and IFA analysis confirmed that the recombinant proteinNPcould be recognized by anti-NP antibody and mice sera against CIV.NP protein was purified using Ni-NTA affinity chromatography and used as a coating antigen to develop an indirect ELISA method to detect CIV antibodies.The reaction conditions were optimized as follows:2 μg/mL coating antigen and 1∶100 dilution of testing serum.Then 136 field serum samples were detected by ELISA and haemagglutination inhibition (HI),and the positive rates were 94.85％(129/136) and 88.23％(120/136),respectively.The coincident rate between the two methods was 93.38％.It suggests that the developed ELISA in this study has a good specificity and stability,and can be used for clinical diagnosis and epidemiological investigation of CIV infection.关键词
犬流感病毒/NP蛋白/杆状病毒表达系统/间接ELISAKey words
canine influenza virus/NP protein/baculovirus expression system/indirect ELISA分类
农业科技引用本文复制引用
楼晶瑶,王永山,刘永杰,王晓丽,欧阳伟,夏兴霞,诸玉梅,潘群兴,王晶宇,安欣,姚凌云..犬流感病毒NP基因在昆虫细胞中的表达及其抗体间接ELISA检测方法的建立[J].中国兽医科学,2017,47(6):680-686,7.基金项目
科技部国际合作专项(2014DFG32770) （2014DFG32770）
国家重点研发计划项目(2016YFD0501000) （2016YFD0501000）
公益性行业(农业)科研专项(201303042) （农业）
江苏省农业科技自主创新资金项目[CX(15)1065] （15）
江苏省优势学科建设项目(PAPD) （PAPD）
