【作者】 张立操；

【导师】 金坚；

【作者基本信息】 江南大学 ， 生化与分子生物学， 2008， 硕士

【摘要】 β干扰素(IFNβ)是干扰素家族中的一员,具有抗病毒、抗增殖和免疫调节作用。雷楗勇等构建了人β干扰素与人血清白蛋白融合蛋白(IFNβ-HSA),将其在毕赤酵母系统中进行表达。这种融合的方法增大了蛋白药物的分子量,减少了异源蛋白的免疫原性,从而减少肾小球滤过率和体内清除率,有效提高了IFNβ在体内的半衰期,具有广阔的生产和临床应用前景。当前IFNβ-HSA融合蛋白的研究还处在发酵优化提高产量,摸索蛋白纯化工艺提高得率的研究阶段,并且很快将进入药代动力学和临床应用研究中。在这些研究中迫切需要一种能够从介质中区分IFNβ-HSA融合蛋白及其结构类似物、降解片段等,而定量检测IFNβ-HSA融合蛋白原型药物的分析方法。目前蛋白多肽药物的检测分析方法主要有理化分析法、生物检定法和免疫分析法。酶联免疫吸附测定(ELISA)技术是1971年由Engvall和Perlman建立的一种生物活性物质微量测定技术,以其灵敏度高、特异性好等优点,在生命科学各领域得到广泛应用。本课题旨在建立一种双抗体夹心ELISA定量检测IFNβ-HSA融合蛋白原型药物的分析方法。首先从雷楗勇博士构建的IFNβ-HSA融合蛋白表达菌株出发,通过发酵培养、蛋白纯化得到一定纯度的IFNβ-HSA融合蛋白作为抗原标准参考品。经过定量分析,抗原浓度为166μg/mL。选取了两株针对IFNβ-HSA融合蛋白两端多肽的单克隆抗体,建立了双抗体夹心ELISA定量检测方法,可以定量检测IFNβ-HSA融合蛋白原型药物。经过方法学考核。该方法的灵敏度为10 ng/mL,测量范围51.88 ng/mL～3320 ng/mL,不同时间不同批次对1660 ng/mL、415 ng/mL、103.75 ng/mL三个浓度点测定,批内变异系数为4.86±1.05%,批间变异系数8.03±1.08%。与IFNβ、IFNα、HSA、IFNα2b-HSA等IFNβ-HSA融合蛋白结构类似物基本无交叉反应。发酵液上清液中回收率分别为101.2±9.2%、101.9±8.0%、98.7±6.8%。稳定性试验表明有效期在3个月以上。本方法可以应用到发酵优化和分离纯化研究中,用以跟踪测定IFNβ-HSA融合蛋白的诱导表达,测定纯化得率等。并且为后期药代动力学、临床研究提供了新思路和备选方法。更多还原

【Abstract】 The interferon (IFN) is one member of the potent multifunctional cytokines family,which possesses a wide range of anti-viral, immunoregulatory and growth inhibitory properties. As a member of interferon family, interferonβhas been applied to treat many diseases, such as MS and hepatitis. To prolong the half-life of IFNβin vivo, Lei Jianyong developed a long-acting IFNβ(IFNβ-HSA) by albumin fusion technology. The fused gene IFNβ-HSA was expressed in pichia pastoris.The fusion protein IFNβ-HSA has broad application prospects in the future.At present, the detection analysis of protein peptide drugs mainly adopt physicochemical analysis techniques, bioassay and immunoassay.Enzyme-linked immunosorbent assay (ELISA) , one of immunoassay, developed in 1971 by Engvall and Perlman, was widely used in various fields with its high sensitivity and specificity advantage. This subject’s aim was to establish a method for quantitative analysis of IFNβ-HSA fusion protein drug.We developed a double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IFNβ-HSA by using a anti-IFNβmonoclonal antibody for capture and a HRP-labeled conjugate of another anti-HSA monoclonal antibody for detection antibody to construct a sandwich ELISA. The optimal concentration of the first coating antibody and detection antibody are both 2μg/mL. The standard protein curve regression equation was: y=0.248x-0.861; its linear detection was from 51.88 to 3320 ng/mL (R2=0.991). And recoveries ranged from 91.9 to 110.4% in typical matrixes, while the intra- and inter-assay precisions were 4.86% and 8.08% respectively. The sensitivity of the IFNβ-HSA ELISA with a limit of detection was 10 ng/mL.A sensitive, repeatable double antibody sandwich ELISA is established for quantitative analysis of recombinant fusion protein IFNβ-HSA. It can be used in the research of fermentation , purification and clinical diagnosis.更多还原