本文關(guān)鍵詞：白色念珠菌熱休克蛋白90單克隆抗體的制備及鑒定　出處：《東北師范大學》2010年碩士論文　論文類型：學位論文

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【摘要】： 白色念珠菌是一種人和動物的常住型寄生菌,棲生于健康宿主的皮膚和黏膜,只有當宿主的免疫力下降時,才會侵入皮膚粘膜內(nèi),進而進入深層組織和血液循環(huán)系統(tǒng),引起系統(tǒng)性感染或稱深層念珠菌感染。白色念珠菌本身能分泌和合成大量的毒性分子和免疫調(diào)節(jié)因子,它們參與真菌的粘附、降解宿主組織蛋白、調(diào)節(jié)白色念珠菌形態(tài)轉(zhuǎn)換等過程。其中某些是白色念珠菌的免疫優(yōu)勢抗原,如熱休克蛋白90、天冬氨酸蛋白酶等,它們可誘導(dǎo)機體產(chǎn)生特異性抗體,并在系統(tǒng)性白色念珠菌感染中起著保護作用。 隨著癌癥患者在腫瘤治療過程中化療藥物的長期使用,導(dǎo)致了患者免疫力下降,這些患者的白色念珠菌的感染也越來越普遍�，F(xiàn)階段人們預(yù)防和治療白色念珠菌感染主要通過兩個環(huán)節(jié)來進行,診斷和抗真菌治療。由于該病沒有明顯的臨床癥狀,加上現(xiàn)階段臨床上人們對真菌的檢測主要通過血培養(yǎng)法,而這種檢測方法敏感性差、周期性長,導(dǎo)致無法對該病進行有效的早期診斷、預(yù)防和治療,病人的死亡率明顯升高,因此如何建立一套白色念珠菌感染的快速檢測體系對患者來說將具有重要意義。但抗真菌治療也存在很多問題,比如真菌的耐藥性,人們把解決這一難題寄希望于藥物的協(xié)同治療。但抗真菌藥物本身的毒性作用再加上它們之間的拮抗作用,使人們把眼光投到了抗體上,單克隆抗體因其特異性高和細胞毒性弱等特點成為抗體中的首選。為了進一步研究白色念珠菌感染的快速檢測及抗真菌治療方法,本研究制備了抗白色念珠菌熱休克蛋白90單克隆抗體。 本實驗利用pGEX-4T-1-Hsp90表達質(zhì)粒,在大腸桿菌BL21中誘導(dǎo)表達白色念珠菌熱休克蛋白90蛋白,再通過親和層析法純化熱休克蛋白90重組蛋白,并以熱休克蛋白90重組蛋白為抗原免疫BALB/c小鼠。利用化學融合法將免疫好的小鼠脾細胞與骨髓瘤細胞融合,經(jīng)過HAT、間接ELISA、有限稀釋法等一系列方法對雜交瘤細胞進行篩選與純化,最后通過ELISA和Western-blot方法對雜交瘤細胞分泌出的抗體進行鑒定,我們得到了兩株分泌抗白色念珠菌熱休克蛋白90雜交瘤細胞系,并制備了抗白色念珠菌熱休克蛋白90單克隆抗體,這為我們進一步研究白色念珠菌感染者的快速檢測和治療具有重要意義。
[Abstract]:Candida albicans is a resident parasite of humans and animals that inhabits the skin and mucosa of healthy hosts and invades the skin and mucosa only when the host's immunity declines. Further into the deep tissue and blood circulatory system, causing systemic infection or deep Candida infection. Candida albicans itself can secrete and synthesize a large number of toxic molecules and immune regulatory factors. They are involved in the adhesion of fungi, the degradation of host tissue proteins, the regulation of Candida albicans morphological transformation and so on. Some of them are immune dominant antigens of Candida albicans, such as heat shock protein 90, aspartate protease and so on. They can induce specific antibodies and play a protective role in systemic Candida albicans infection. With the long-term use of chemotherapeutic drugs in cancer patients, the immunity of cancer patients is reduced. The infection of Candida albicans in these patients is becoming more and more common. At this stage, the prevention and treatment of Candida albicans infection are mainly carried out through two links. Diagnosis and antifungal therapy. Because the disease has no obvious clinical symptoms, and the current clinical detection of fungi is mainly through blood culture, and this method is not sensitive and periodic. As a result of the failure of effective early diagnosis, prevention and treatment of the disease, the mortality of patients significantly increased. Therefore, how to establish a rapid detection system for Candida albicans infection will be of great significance to patients. However, there are many problems in antifungal therapy, such as drug resistance of fungi. People put the hope of solving this problem on the cooperative treatment of drugs. But the toxicity of antifungal drugs and the antagonism between them make people focus on antibodies. Monoclonal antibody is the first choice of antibodies because of its high specificity and weak cytotoxicity. In order to further study the rapid detection of Candida albicans infection and antifungal treatment. In this study, monoclonal antibody against heat shock protein 90 of Candida albicans was prepared. In this study, pGEX-4T-1-Hsp90 expression plasmid was used to induce the expression of heat shock protein 90 (HSP90) of Candida albicans in Escherichia coli BL21. The recombinant protein of heat shock protein 90 was purified by affinity chromatography. Heat shock protein 90 (HSP90) was used as antigen to immunize BALB/c mice. Spleen cells of immunized mice were fused with myeloma cells by chemical fusion method. A series of methods, such as limited dilution method, were used to screen and purify the hybridoma cells. Finally, the antibodies secreted by hybridoma cells were identified by ELISA and Western-blot methods. Two hybridoma cell lines secreting heat shock protein 90 against Candida albicans were obtained and monoclonal antibodies against heat shock protein 90 against candida albicans were prepared. It is of great significance for us to study the rapid detection and treatment of Candida albicans infection.
【學位授予單位】：東北師范大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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