多主枝孢霉重组蛋白Cla h8的制备和定量检测

发布时间：2018-06-04 07:56

本文选题：多主枝孢霉 + 定量检测　；参考：《复旦大学》2011年硕士论文

【摘要】：多主枝孢霉(Cladosporium herbarum)是引起过敏性疾病的主要霉菌之一,它不仅是一种主要的室内过敏原,也是夏秋两季主要的室外过敏原,可导致患者出现严重的过敏性哮喘。现已证实,Cla h8是多主枝孢霉的最主要致敏原,它是一种NADP依赖性甘露醇脱氢酶(NADP-dependent MtDH)蛋白,可被57%左右多主枝孢霉过敏患者血清中的IgE抗体特异性识别；临床皮肤点刺实验结果表明Cla h8可以引起患者出现皮肤红肿。为此本研究旨在制备出高纯度的重组蛋白并对其进行免疫鉴定及定量检测,从而为真菌过敏患者提供更准确的诊断及更有效、安全的免疫治疗手段。本研究通过基因工程手段,从多主枝孢霉菌体中提取总RNA,采用RT-PCR的方法扩增Cla h8编码基因,将其连入pET-19b载体。转入大肠杆菌BL21 Star (DE3)pLysS,由于目的蛋白在大肠杆菌BL21 Star (DE3) pLysS中高效表达,蛋白聚积形成不溶性的包涵体蛋白,为此对其进行复性重构,最终纯化制备出的Cla h8蛋白纯度可达98%以上,用Western blotting和Dot-blotting法检测结果表明重组多主枝孢霉变应原Cla h8蛋白可以与多主枝孢霉过敏患者的血清中IgE和IgG抗体特异性结合,与天然蛋白具有相似的免疫活性。据此制备抗Cla h8单克隆抗体(Cla h8 mAb), Western blot筛选出阳性克隆2株即3G10和1F3,并对其纯化、亚型鉴定和标记后行抗体配对实验；通过棋盘滴定法确定包被抗体和酶标抗体的最适工作浓度,初步建立了双单克隆抗体夹心ELISA法检测多主枝孢霉变应原Cla h8含量,此法重复性好,灵敏度高,操作简便。上述多主枝孢霉重组变应原Cla h8的制备和双单克隆抗体夹心ELISA定量检测多主枝孢霉方法的建立,为实现真菌变应原的标准化、特异性诊断与免疫治疗奠定了基础。
[Abstract]:Cladosporium herbarum (Cladosporium herbarum) is one of the main fungi causing allergic diseases. It is not only a major indoor allergen, but also a major outdoor allergen in summer and autumn, which can lead to severe allergic asthma in patients. It has been proved that Cla H8 is the most important allergen of Cladosporium polymorilli, and it is a NADP dependent mannitol dehydrogenase (NADP-dependent MtDH8) protein, which can be specifically recognized by IgE antibodies in about 57% of the sera of allergic patients. The results of clinical skin prick test showed that Cla H 8 could cause skin redness and swelling in patients. The aim of this study was to prepare a recombinant protein with high purity, to identify and detect it quantitatively, and to provide more accurate diagnosis and more effective and safe immunotherapy for fungal allergy patients. In this study, Cla H8 encoding gene was amplified by genetic engineering method and cloned into pET-19b vector. The protein was highly expressed in E. coli BL21 Star DE3 pLysS and the protein accumulated to form insoluble inclusion body protein. The purity of the purified Cla h8 protein was over 98%. The results of Western blotting and Dot-blotting assays showed that the recombinant Cla h8 protein could specifically bind to the antibodies of IgE and IgG in the sera of allergic patients, and had similar immunological activity to the natural proteins. Two positive clones, 3G10 and 1F3, were screened out by Western blot, and purified, identified and labeled with antibody pairing test. The optimum working concentration of coated and enzyme-labeled antibodies was determined by chessboard titration, and a double monoclonal antibody sandwich ELISA method was established for the determination of Cla H8 content. The method has the advantages of good reproducibility, high sensitivity and simple operation. The preparation of the recombinant allergen Cla H8 and the establishment of a double monoclonal antibody sandwich ELISA method for quantitative detection of Cladosporium polychloides have laid a foundation for the standardization, specific diagnosis and immunotherapy of fungal allergens.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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