【作者】 连晓雯；

【导师】 崔燕； 李克生； 杜惠芬；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 禽流感(AIV)是由A型流感病毒感染引起的一种禽类传染性疾病综合征,有重要的公共卫生意义。本研究的目的是以H5N1亚型高致病性禽流感病毒作为免疫原,研制出抗H5N1亚型禽流感病毒的单克隆抗体(McAb),并将其用于H5亚型禽流感的检测中。病毒经差速离心法纯化后,四次免疫8周龄Balb/c雌性小鼠。取免疫小鼠脾细胞与SP2/0细胞融合,采用间接ELISA法检测筛选阳性细胞孔,再经有限稀释法克隆培养,筛选稳定分泌单克隆抗体的细胞株。本研究一共获得5株能够稳定分泌单克隆抗体的细胞株,分别命名为:2C1,4F10,5A5,5C12和6F5,其中4F10的免疫球蛋白亚类为IgM,其余四株单克隆抗体(McAbs)均为IgG1。各株单克隆抗体细胞上清效价依次为:1:640,1:640,1:1280,1:1280,1:2560;各株单克隆抗体腹水效价依次为:1:12800,1:160,1:51200,1:128000,1:256000。染色体计数分析各株杂交瘤细胞的平均染色体数均在98-108之间,符合脾细胞与SP2/0细胞染色体数之和。特异性反应实验结果表明,五株单克隆抗体与SPF鸡胚尿囊液,H9N1型禽流感病毒,鸡新城疫病毒,产蛋下降综合征病毒,鸡传染性支气管炎病毒均无交叉反应。叠加实验表明,5A5、5C12和6F5三株单抗抗原识别位点均不相同。这五株单克隆抗体杂交瘤细胞株将在禽流感的预警预报工作中发挥重要作用。更多还原

【Abstract】 Avian influenza is a group of symptoms caused by influenza virus A. Avian influenza has raised great concerns for its economic importance to poultry industry and potential threat to the public health. In this experiment, a highly pathogenic avian influenza virus (H5N1), was chosen to immunize Balb/c mice in order to product the monoclonal antibodies (McAbs).The 8-week-old female Balb/c mice were immunized with the allantoic fluid, which was purified and concentrated by differential centrifugation. The spleen cells of immunized mice were fused with SP2/0 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridoma cells and limited dilution method was performed to subclone positive clones.Five of McAbs specifically against avian influenza virus (AIV) were obtained, designated as 2C1, 4F10, 5A5, 5C12 and 6F5 respectively. One of these 5 McAbs, 4F10, was of IgM subtype. The others were of IgG1 subtype. The titers of McAbs substrates were: 1:640,1:640,1:1 280,1:1 280,1:2 560. The titers of ascites were: 1:12 800,1:160,1:51 200,1:128 000,1:256 000. The result of chromosome analyse was that the average chromosome number of hybridoma cells were 98 to 108.The results of the cross-reaction test suggest that these McAbs were specific to AIV-H5N1 without detectable cross-reactivity with specific pathogen free (SPF) chick embryo allantoid fluid, AIV-H9, Newcasttle disease virus, Eggs drop syndrome virus and Avian infectious bronchitis virus. The results of superposable test suggest that 5A5, 5C12 and 6F5 were against different antigen epitope.These McAbs against AIV-H5N1 will be very useful in control of avian influenza.更多还原