口蹄疫病毒3B蛋白单克隆抗体制备、抗原位点鉴定及竞争ELISA方法的初步建立

发布时间：2018-06-30 08:19

本文选题：口蹄疫病毒 + 3B蛋白　；参考：《新疆农业大学》2015年硕士论文

【摘要】：为了获得Asia-1口蹄疫病毒非结构蛋白3B的单克隆抗体,以Asia-1型FMDV JS/05毒株RNA为模板,扩增3B蛋白基因,并克隆至pET-30a载体,进行原核表达及纯化。以Asia-1型口蹄疫病毒、纯化的3B蛋白为抗原,免疫4-6周龄的Balb/c雌鼠,经过4次免疫后,取其脾细胞与Sp2/0骨髓瘤细胞按5:1进行融合,用间接ELISA方法对融合后的细胞进行筛选,并进行4次有限稀释,阳性细胞扩大培养后腹腔注射小鼠,制备腹水,并对制取的腹水进行亚型鉴定,反应原性和效价鉴定。将3B蛋白拆分,并构建pGEX-4T1载体,融合GST标签表达,通过间接ELISA验证3B抗体的反应特性,并通过口蹄疫3ABC检测试剂盒筛选临床阳性和阴性血清,利用本实验获得的3B抗体建立竞争ELISA方法,用于检测临床样品中口蹄疫3B蛋白的抗体。结果表明,获得一株稳定分泌抗口蹄疫病毒3B蛋白的细胞株,其抗体亚类为IgM,腹水效价为1:12800,通过Western boltting和间接免疫荧光实验鉴定其能识别口蹄疫病毒3B蛋白。SDS-PAGE结果表明,成功表达和纯化3B截短融合GST标签的重组蛋白,间接ELISA结果表明,本实验获得的抗体和3B1蛋白特异性反应。通过口蹄疫3ABC检测试剂盒,筛选出临床4份阳性血清,和4份阴性血清,利用本实验获得的3B抗体建立竞争ELISA方法,检测口蹄疫3B抗体,结果显示4份阳性血清显示出不同程度的抑制率,且具有剂量依赖性,4份阴性血清无抑制作用,无剂量依赖性,表明此竞争ELISA方法和3ABC检测方法符合率100%。
[Abstract]:In order to obtain the monoclonal antibody against non-structural protein 3B of Asia-1 foot-and-mouth disease virus (FMDV), the 3B protein gene was amplified from Asia-1 FMDV JS05 strain as template and cloned into pET-30a vector for prokaryotic expression and purification. Asia-1 foot-and-mouth disease virus (FMDV) and purified 3B protein were used as antigen to immunize Balb / c female mice aged 4-6 weeks. After 4 times of immunization, the spleen cells were fused with Sp2 / 0 myeloma cells at 5:1, and the fused cells were screened by indirect Elisa. After 4 times of limited dilution, the positive cells were injected intraperitoneally into mice to prepare ascites, and the subtypes of the ascites were identified, and the reactivity and titer of the ascites were identified. The 3B protein was separated, pGEX-4T1 vector was constructed, the expression of GST tag was fused, the reaction characteristics of 3B antibody were verified by indirect Elisa, and the clinical positive and negative sera were screened by 3ABC detection kit. A competitive Elisa was established using the 3B antibody obtained in this study to detect the antibody against foot-and-mouth disease (FMD) 3B protein in clinical samples. The results showed that a cell line secreting 3B protein of foot-and-mouth disease virus (FMDV) was secreted stably. The antibody subclass was IgM, and the ascites titer was 1: 12800. The results of Western boltting and indirect immunofluorescence assay showed that FMDV 3B protein 路SDS-PAGE could recognize foot-and-mouth disease virus. The recombinant protein of 3B truncated fusion GST tag was successfully expressed and purified. The results of indirect Elisa showed that the antibody and 3B1 protein were specific. Four positive sera and four negative sera were screened by 3ABC test kit of foot-and-mouth disease (FMD). A competitive Elisa method was established by using the 3B antibody obtained in this experiment to detect 3B antibody of foot-and-mouth disease (FMD). The results showed that 4 positive sera showed different inhibition rates, and 4 negative sera had no inhibition and no dose dependence, indicating that the competitive Elisa method and the 3ABC assay were in good agreement with each other.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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