【作者】 蒲娟；

【导师】 吴清民； 刘金华； 赵继勋；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 近年来,禽流感的暴发与流行给养禽业的发展和公共卫生安全造成了严重的危害,做好禽流感的诊断与监测显得尤为重要。禽流感病毒亚型众多且变异性强,给检测带来了很大困难。因此,本研究制备了一株针对禽流感病毒NP蛋白的型特异性单克隆抗体,经过鉴定及初步应用,用该单抗初步建立了一种快速的针对禽流感抗原的胶体金试纸条检测方法,以期用于禽流感的监控。 1.单抗的制备:蔗糖梯度离心纯化的H9N2亚型AIV免疫小鼠,经PEG法融合,用建立的间接ELISA方法筛选,三次克隆后得到一株分泌针对H9、H7、H5三种亚型禽流感病毒抗体的单克隆杂交瘤细胞株（A1284）,三株仅分泌针对H9亚型流感病毒抗体的单克隆杂交瘤细胞株（G4A3、C9C2、G7A9）。 2.单抗的鉴定:对四株单抗进行了亚类鉴定,仅对A1284株进行了染色体、亲和力等其它特性的鉴定。A1284株、G4A3株、G7A9株、C9C2株单克隆抗体亚类分别为IgM、IgG1、IgG1、IgG2a,轻链均为K链。杂交瘤细胞A1284染色体数目为98条,其单抗针对H9、H7、H5三种亚型禽流感病毒的亲和常数K分别为3.17×10¹⁰、3.17×10¹⁰、5.5×10¹⁰,上清及腹水的ELISA效价分别为1:10⁴、1:10⁶。免疫印迹分析表明,A1284株单抗是针对禽流感NP蛋白的型特异性单抗。 3.单抗的初步应用:A1284株腹水经优球蛋白沉淀法纯化后与胶体金标记,应用于胶体金试纸条检测试验,金标A1284株单抗与H9、H7、H5三种亚型禽流感病毒反应结果呈阳性。 通过制备和初步应用禽流感病毒NP蛋白特异性单克隆抗体,为进一步临床应用打下基础,为禽流感乃至人禽流感的诊断提供一种有用试剂和快速、准确、实用的诊断方法。更多还原

【Abstract】 Outbreaks and prevalence of avian influenza, especially the highly pathogenic form, can be devastating for the poultry industry and representing serious threats to human health. Series of subtypes and rapid variation of AIV result in great difficulty in diagnosis. In this study, a hybridoma cell line which secreted monoclonal antibody against type-specific influenza A nucleoprotein （McAb anti-NP） was prepared and identified. It is suggested that the McAb has the potential to be used in developing a rapid, easy and applied immuno-gold filtration assay for detection of antigen to avian influenza virus from avian species and even from human.1. Preparation of McAbs: the splenocyte of Balb/c mice immunized with the purified avian H₉N₂ influenza virus were fused with sp2/0 myeloma cells. four strain McAbs to avian influenza were established after screening by indirect ELISA and subcloning by limiting-dilution, which were named as A₁2B₄, G₄A₃, C₉C₂ and G₇A₉, respectively. Avian H₉, H₇ and H₅ influenza virus contained the recognized epitopes of the McAb A₁2B₄, and the epitope defined by the others was only found on avian H₉ influenza virus.2. Identification of McAbs: the immunoglobulin subclasses of A₁2B₄C₉C₂, G₄A₃ and G₇A₉ were IgM, IgG2a, IgG1, and IgG1 respectively and the light chains of which were all categorized into chain κ. A₁2B₄McAb had 98 chromosomes. Its affinity constants against avian H9, H7 and H5 influenza virus were 3.17×10¹0,3.17×10¹0 and 5.5×10¹0, respectively. The ELISA titers of culture superatant and ascities were 1:10⁴ and 1:10⁵. The result of western blotting revealed that the McAb A₁2B₄ could react with avian influenza virus nucleoprotein（NP）.3. Application of McAbs: the ascites of McAb A₁2B₄ were purified with euglobulin precipitation, and tagged with colloidal golds, then the immuno-gold filtration assay was developed to examine avian influenza virus. The results showed that the positive outcome was gained with avian H₉, H₇ and H₅ influenza virus.The preparation and preliminary application of the McAb anti-NP will contribute to the farther improvement of the dectection of avian influenza’s antigen and to provide a useful tool and assay for diagnosis of AI, and even of human-avian influenza.更多还原