噬菌体单链抗体应用于系统性白色念珠菌感染治疗的研究

发布时间：2018-04-09 07:55

  本文选题：噬菌体　切入点：单链抗体　出处：《东北师范大学》2017年硕士论文

【摘要】：在全球,侵袭性真菌感染已经成为导致人类死亡的重要原因。近年来,免疫功能不全的人、HIV感染者、接受化疗或大型手术的患者、接受过器官或者造血干细胞抑制的人在逐渐增加,随之而来的是真菌感染的病人也在逐渐增加。白色念珠菌是常见的医院内感染疾病,是造成系统性念珠菌感染的重要原因,死亡率接近50%。众所周知,系统性白色念珠菌感染治疗的难度很大。目前,临床上允许用于治疗真菌感染的药物主要有四类,分别是核苷酸类似物、唑类、多烯类和棘白菌素类。近年来,虽然抗真菌药物在不断开发,但是真菌感染的发病率和死亡率仍然居高不下。同时不断出现的耐药性菌株也要求我们不断开发新的治疗策略。本研究以白色念珠菌细胞壁蛋白为靶点,研究与其特异性结合的噬菌体单链抗体对系统性白色念珠菌感染治疗的作用。本研究的内容主要是,利用人源噬菌体展示单链抗体文库,筛选出与白色念珠菌孢子体细胞壁蛋白结合能力最强的噬菌体单链抗体,从体外和小鼠系统性白色念珠菌感染模型两方面,评价噬菌体单链抗体的抗真菌作用效果。本研究的主要方法及结果如下:1.人源噬菌体单链抗体的筛选:进行4轮筛选,得到5条与白色念珠菌细胞壁相结合的噬菌体单链抗体,命名为JC1、JC2、JC3、JC4、JC5。2.结合力分析:将5种体单链抗体进行抗体效价分析、Western-blotting、免疫荧光实验,分析其与白色念珠菌的结合能力。结果显示,JC1与白色念珠菌的结合效果最好。3.体外分析JC1的治疗效果:利用细胞活性检测试剂CCK分析了JC1的最佳工作浓度,绘制白色念珠菌的生存曲线,进一步探索JC1的最佳作用时间及温度;利用流式细胞术分析了JC1对白色念珠菌细胞周期进程的影响;利用Hoechst和PI双染法分析了JC1对白色念珠菌细胞凋亡与坏死的影响;利用扫描电镜分析了JC1对白色念珠菌细胞壁表面形态的影响。结果表明,1×109个/ml JC1,30℃下共孵育8小时时,能够显著的抑制白色念珠菌孢子体细胞的增殖,并且能显著的阻滞白色念珠菌的细胞周期,促进其凋亡与坏死,能够使白色念珠菌的细胞壁发生皱缩。4.构建小鼠系统性白色念珠菌感染模型,分析JC1对小鼠肾脏、肝脏、脾脏的白色念珠菌负荷量的影响以及JC1对小鼠存活时间的影响。实验结果显示,JC1能够显著的降低小鼠肾脏、肝脏、脾脏的真菌定植量,并且延长了小鼠的存活时间。综上所述,噬菌体单链抗体JC1,结合了单链抗体与噬菌体的优点,具有显著的治疗系统性白色念珠菌感染的效果。因此,JC1具有用于治疗系统性白色念珠菌感染疾病的潜能。
[Abstract]:Worldwide, invasive fungal infections have become an important cause of death in humans.In recent years, the number of people with HIV infection, chemotherapy or major surgery, organ or hematopoietic stem cell inhibition has been increasing, followed by fungal infection.Candida albicans is a common nosocomial infection and an important cause of systemic Candida infection. The mortality rate is close to 50%.It is well known that systemic Candida albicans infection treatment is very difficult.At present, there are four kinds of drugs that can be used to treat fungal infection. They are nucleotide analogs, azoles, polyenes, and echinacetin.In recent years, although antifungal drugs are being developed, the morbidity and mortality of fungal infections remain high.At the same time, the emergence of drug-resistant strains also requires us to develop new treatment strategies.The purpose of this study was to investigate the effect of phage specific phage scFv on the treatment of systemic Candida albicans infection with Candida albicans cell wall protein as the target.The main content of this study was to screen the phage scFv with the strongest binding ability to the cell wall protein of Candida albicans spore by human phage display scFv library.The antifungal effects of phage scFv were evaluated in vitro and in mice with systemic Candida albicans infection model.The main methods and results of this study are as follows: 1.Screening of human phage scFv: through four rounds of screening, five phage scFv combined with cell wall of Candida albicans were obtained and named as JC1, JC2, JC2, JC3, JC4, JC5.2.Binding ability analysis: the antibody titers of five kinds of single-chain antibodies were analyzed by Western-blotting.Immunofluorescence assay was used to analyze their binding ability with Candida albicans.The results showed that JC1 had the best binding effect with Candida albicans.In vitro analysis of the therapeutic effect of JC1: the best working concentration of JC1 was analyzed by CCK, the survival curve of Candida albicans was drawn, and the optimum time and temperature of JC1 were further explored.The effects of JC1 on cell cycle progression of Candida albicans were analyzed by flow cytometry, and the effects of JC1 on apoptosis and necrosis of Candida albicans were analyzed by Hoechst and Pi double staining.The effect of JC1 on the surface morphology of Candida albicans cell wall was analyzed by SEM.The results showed that after incubated at 30 鈩，

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