白色念珠菌热休克蛋白90单克隆抗体的制备及鉴定

发布时间：2018-01-17 01:14

本文关键词：白色念珠菌热休克蛋白90单克隆抗体的制备及鉴定　出处：《东北师范大学》2010年硕士论文　论文类型：学位论文

【摘要】： 白色念珠菌是一种人和动物的常住型寄生菌,栖生于健康宿主的皮肤和黏膜,只有当宿主的免疫力下降时,才会侵入皮肤粘膜内,进而进入深层组织和血液循环系统,引起系统性感染或称深层念珠菌感染。白色念珠菌本身能分泌和合成大量的毒性分子和免疫调节因子,它们参与真菌的粘附、降解宿主组织蛋白、调节白色念珠菌形态转换等过程。其中某些是白色念珠菌的免疫优势抗原,如热休克蛋白90、天冬氨酸蛋白酶等,它们可诱导机体产生特异性抗体,并在系统性白色念珠菌感染中起着保护作用。随着癌症患者在肿瘤治疗过程中化疗药物的长期使用,导致了患者免疫力下降,这些患者的白色念珠菌的感染也越来越普遍。现阶段人们预防和治疗白色念珠菌感染主要通过两个环节来进行,诊断和抗真菌治疗。由于该病没有明显的临床症状,加上现阶段临床上人们对真菌的检测主要通过血培养法,而这种检测方法敏感性差、周期性长,导致无法对该病进行有效的早期诊断、预防和治疗,病人的死亡率明显升高,因此如何建立一套白色念珠菌感染的快速检测体系对患者来说将具有重要意义。但抗真菌治疗也存在很多问题,比如真菌的耐药性,人们把解决这一难题寄希望于药物的协同治疗。但抗真菌药物本身的毒性作用再加上它们之间的拮抗作用,使人们把眼光投到了抗体上,单克隆抗体因其特异性高和细胞毒性弱等特点成为抗体中的首选。为了进一步研究白色念珠菌感染的快速检测及抗真菌治疗方法,本研究制备了抗白色念珠菌热休克蛋白90单克隆抗体。本实验利用pGEX-4T-1-Hsp90表达质粒,在大肠杆菌BL21中诱导表达白色念珠菌热休克蛋白90蛋白,再通过亲和层析法纯化热休克蛋白90重组蛋白,并以热休克蛋白90重组蛋白为抗原免疫BALB/c小鼠。利用化学融合法将免疫好的小鼠脾细胞与骨髓瘤细胞融合,经过HAT、间接ELISA、有限稀释法等一系列方法对杂交瘤细胞进行筛选与纯化,最后通过ELISA和Western-blot方法对杂交瘤细胞分泌出的抗体进行鉴定,我们得到了两株分泌抗白色念珠菌热休克蛋白90杂交瘤细胞系,并制备了抗白色念珠菌热休克蛋白90单克隆抗体,这为我们进一步研究白色念珠菌感染者的快速检测和治疗具有重要意义。
[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.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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