發(fā)布時間：2018-06-04 07:56

  本文選題：多主枝孢霉 + 定量檢測�。� 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：多主枝孢霉(Cladosporium herbarum)是引起過敏性疾病的主要霉菌之一,它不僅是一種主要的室內(nèi)過敏原,也是夏秋兩季主要的室外過敏原,可導(dǎo)致患者出現(xiàn)嚴(yán)重的過敏性哮喘�，F(xiàn)已證實,Cla h8是多主枝孢霉的最主要致敏原,它是一種NADP依賴性甘露醇脫氫酶(NADP-dependent MtDH)蛋白,可被57%左右多主枝孢霉過敏患者血清中的IgE抗體特異性識別；臨床皮膚點刺實驗結(jié)果表明Cla h8可以引起患者出現(xiàn)皮膚紅腫。為此本研究旨在制備出高純度的重組蛋白并對其進行免疫鑒定及定量檢測,從而為真菌過敏患者提供更準(zhǔn)確的診斷及更有效、安全的免疫治療手段。 本研究通過基因工程手段,從多主枝孢霉菌體中提取總RNA,采用RT-PCR的方法擴增Cla h8編碼基因,將其連入pET-19b載體。轉(zhuǎn)入大腸桿菌BL21 Star (DE3)pLysS,由于目的蛋白在大腸桿菌BL21 Star (DE3) pLysS中高效表達,蛋白聚積形成不溶性的包涵體蛋白,為此對其進行復(fù)性重構(gòu),最終純化制備出的Cla h8蛋白純度可達98%以上,用Western blotting和Dot-blotting法檢測結(jié)果表明重組多主枝孢霉變應(yīng)原Cla h8蛋白可以與多主枝孢霉過敏患者的血清中IgE和IgG抗體特異性結(jié)合,與天然蛋白具有相似的免疫活性。據(jù)此制備抗Cla h8單克隆抗體(Cla h8 mAb), Western blot篩選出陽性克隆2株即3G10和1F3,并對其純化、亞型鑒定和標(biāo)記后行抗體配對實驗；通過棋盤滴定法確定包被抗體和酶標(biāo)抗體的最適工作濃度,初步建立了雙單克隆抗體夾心ELISA法檢測多主枝孢霉變應(yīng)原Cla h8含量,此法重復(fù)性好,靈敏度高,操作簡便。 上述多主枝孢霉重組變應(yīng)原Cla h8的制備和雙單克隆抗體夾心ELISA定量檢測多主枝孢霉方法的建立,為實現(xiàn)真菌變應(yīng)原的標(biāo)準(zhǔn)化、特異性診斷與免疫治療奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前2條

1 王碩;楊澤琳;馬冬月;段素欣;張燕;李細(xì)芬;江月明;房立;;雙抗體夾心ELISA定量檢測牛乳中酪蛋白方法的建立[J];食品科技;2010年09期

2 戶義,劉雪松,朱勇,劉瑩,許曉光,薛江楠,金伯泉;抗Ig融合蛋白Fc段單克隆抗體的制備、鑒定與應(yīng)用研究[J];細(xì)胞與分子免疫學(xué)雜志;2003年02期

相關(guān)碩士學(xué)位論文 前2條

1 沈丹丹;定量檢測人超敏C-反應(yīng)蛋白雙抗體夾心ELISA方法的建立及初步臨床應(yīng)用[D];江蘇大學(xué);2009年

2 王彩紅;阿特拉津與氨芐青霉素單抗制備、鑒定及ELISA方法的初步建立[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年

，

本文編號：1976603