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

发布时间：2018-01-21 23:56

本文关键词： 犬流感病毒 NP蛋白杆状病毒表达系统间接ELISA　出处：《中国兽医科学》2017年06期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:The aim of this study was to express the nucleoprotein (NPP) of Canine Influenza virus (CIV) using insect cells. It was used as antigen to establish an indirect ELISA method for detection of CIV antibody. Total RNAs were extracted from MDCK cells infected with CIV and NP gene was amplified by RT-PCR method. The shuttle plasmid NP-DH10 was obtained by ligation to pFastBacHTA vector and transformed into DH10Bac receptive cells containing Bacmid. The shuttle plasmid was transfected into sf-9 insect cells. The recombinant virus P1-NP.The virus was transferred to the third generation. Western-blot and IFA were used to identify the specific reaction between the expressed protein of insect cells and rabbit anti-canine influenza virus NP protein polyclonal antibody and mouse anti-#en2# serum. NP protein was purified as. Antigen. An indirect ELISA method for the detection of CIV antibody was established, and the optimal coating concentration of protein was determined to be 2 g / mL. The best dilution of serum was 1: 100. The indirect ELISA method and hemagglutination inhibition assay were used to detect the serum samples. The detection rate was 94.85; The positive rate of HI was 88.23 and the positive rate of Elisa was 93.38. The result showed that the positive rate of Elisa was higher than that of hemagglutination inhibition. Using NP protein expressed in insect cells as antigen, indirect ELISA method for detecting CIV antibody was established, which was specific and reproducible, and could be used in the diagnosis and epidemiological investigation of canine influenza.
【作者单位】：南京农业大学动物医学院;江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心;
【基金】：科技部国际合作专项(2014DFG32770) 国家重点研发计划项目(2016YFD0501000) 公益性行业(农业)科研专项(201303042) 江苏省农业科技自主创新资金项目[CX(15)1065] 江苏省优势学科建设项目(PAPD)
【分类号】：S852.65
【正文快照】： 犬流感(canine influenza,CI)是由正黏病毒科A型流感病毒属的犬流感病毒(canine influenza virus,CIV)引起的一种接触性呼吸道传染病。患犬表现为发热、流鼻涕、食欲不振、嗜睡和精神沉郁[1]。2004年在美国佛罗里达州的一只患有呼吸道症状的赛犬体内首次分离到H3N8犬流感病毒,

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