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

旨在利用昆虫细胞表达犬流感病毒(CIV)核蛋白(NP),并以此作为抗原建立检测CIV抗体的间接ELISA方法。从CIV感染的MDCK细胞中提取总RNA,用RT-PCR法扩增NP基因,连接到pFastBacHTA载体上,转化含Bacmid的DH10Bac感受态细胞,获得穿梭质粒NP-DH10,穿梭质粒转染sf-9昆虫细胞,获得重组病毒P1-NP。病毒传至第3代后,用Western-blot及IFA鉴定昆虫细胞表达的蛋白与兔抗犬流感病毒NP蛋白多克隆抗体及鼠抗CIV血清发生特异性反应。NP蛋白纯化后作为抗原,建立检测CIV抗体的间接ELISA方法。优化后确定蛋白最佳包被浓度为2 g/mL,血清最佳稀释度为1∶100。用建立的间接ELISA方法和血凝抑制(HI)同时检测136份血清样品,ELISA法检出129份阳性,检出率为94.85%;HI检出120份阳性,检出率为88.23%,二者符合率为93.38%,ELISA检测方法的阳性率高于血凝抑制。结果表明,利用昆虫细胞表达的NP蛋白做为抗原,建立的检测CIV抗体的间接ELISA方法特异性好,重复性高,可用于犬流感的诊断和流行病学调查。

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 RNiwas extracted from CIV infected MDCK cells,NP gene was amplified by RT-PCR and inserted into vector pFastBacHTi. The recombinant donor plasmid was transformed to competent Escherichia coli DH10Bac. Then the recombinant bacmid NP-DH10 was transfected to insect sf-9 cells and thus obtained the recombinant baculovirus Pi-NP. Western-blot and IFA analysis confirmed that the recombinant protein NP could be recognized by anti-NP antibody and mice sera against CIV. NP protein was purified using Ni-NTA affinity chromatogra- phy and used as a coating antigen to develop an indirect ELISA method to detect CIV antibodies. The reac- tion 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 be- tween the two methods was 93.38%.It ficity and stability,and can be used infection suggests that the developed ELISA in this study has a good speci- for clinical diagnosis and epidemiological investigation of CIV