展示白色念珠菌热休克蛋白90特异表位的杂合噬菌体抗系统性白色念珠菌感染的免疫保护作用研究

发布时间：2018-05-24 06:46

本文选题：系统性白色念珠菌感染 + HSP90　；参考：《东北师范大学》2007年硕士论文

【摘要】： 白色念珠菌是寄生在健康宿主皮肤、粘膜的条件致病菌,它可以作为机会病原体在人体免疫能力下降和正常菌群失调时,引起浅层感染和系统感染。近年来,随着因治疗扩散性肿瘤以及免疫抑制剂的应用而引起的免疫缺陷患者日益增多,系统性白色念珠菌感染的发病率明显升高,它的危害性极大,死亡率非常高。白色念珠菌得不到控制的主要原因是缺乏有效的早期诊断、治疗和预防的方法。临床上依赖于传统的血培养的方法诊断该类疾病,但存在着检出率低,敏感性差和诊断过程时间长的问题。临床上采用多粘菌素B和吡唑类化学药物对患者进行治疗,但是耐药性的病例在不断增加。为了防止其复发,我们也延长了治疗的周期,但存在着明显的副作用。所以早期检测出白色念珠菌的感染,在患者进入免疫抑制状态前通过疫苗来增强机体免疫力具有重要意义。本研究是利用展示有白色念珠菌热休克蛋白90(HSP90)特异表位(DEPAGE)的杂合噬菌体(以下简称SE-CA-HSP90)作为抗原进行抗系统性白色念珠菌感染的免疫保护作用的研究,以期为真菌预防性疫苗的开发奠定基础。本实验包括以下内容: 1大量制备杂合噬菌体SE-CA-HSP90 在野生噬菌体的帮助下,将含有编码白色念珠菌HSP90特异表位(DEPAGE)DNA序列的pfd8H载体转化大肠杆菌TG1,转化pfd8H的大肠杆菌成功地分泌表达出展示有DEPAGE表位的杂合噬菌体,再用PEG6000回收和纯化培养液中的噬菌体粒子。杂合噬菌体样品经SDS-PAGE电泳后,在凝胶上可见有一条融合蛋白带,明显滞后于野生型噬菌体外壳蛋白带,采用敏感性较高的银染色方法染色,未见其它杂蛋白带,说明达到纯化要求。 2杂合噬菌体SE-CA-HSP90抗系统性白色念珠菌免疫保护研究 (1)建立系统性白色念珠菌感染模型,并筛选最佳感染剂量; (2)以杂合噬菌体SE-CA-HSP90为抗原免疫C57BL/6小鼠,同时注射野生型噬菌体和缓冲液TE作为对照组。数周后尾静脉进行系统性白色念珠菌感染; (3)不同时期进行下列指标的观察和检测: 系统性白色念珠菌感染15天内动物死亡率的统计; 感染15天后,将免疫动物的肾脏研磨成匀浆,在沙堡氏培养基中培养白色念珠菌,检测白色念珠菌菌落数的变化; 制作组织病理切片,进行动物肾脏受损情况的对比。实验结果表明免疫杂合噬菌体SE-CA-HSP90组,与免疫野生噬菌体组和注射TE组相比,对系统性白色念珠菌感染的抵抗力增加,表现在小鼠的存活率增加,寄居在肾中的白色念珠菌数量降低,组织病理学显示肾小管内的白色念珠菌数目显著少于其它组别。结果说明展示外源多肽的杂合噬菌体SE-CA-HSP90有可能发展成为有效的抗真菌疫苗。
[Abstract]:Candida albicans, a conditional pathogen parasitic on the skin and mucous membrane of a healthy host, can be used as an opportunistic pathogen to cause superficial infection and systemic infection when the immune ability of the human body is reduced and the normal flora is unadjusted. In recent years, the immune deficiency patients are increasing with the treatment of the use of diffuse tumor and immunosuppressant. The incidence of systemic Candida albicans infection is significantly higher, its harm is great and the mortality rate is very high. The main reason for the lack of control of Candida albicans is the lack of effective early diagnosis, treatment and prevention. It is clinically dependent on the traditional blood culture method to diagnose the disease, but the detection rate is low, and the sensitivity is poor. The problem of long duration of the diagnosis is the clinical use of polymyxin B and pyrazole chemicals to treat patients, but the cases of drug resistance are increasing. In order to prevent their recurrence, we have also extended the treatment cycle, but there are obvious side effects. So early detection of Candida albicans infection, the patients entered the free. It is of great significance to enhance immunity through vaccination before vaccination.
The present study is to use the heterozygous phage display (HSP90) specific epitope (HSP90) specific epitope (DEPAGE) of Candida albicans (hereinafter referred to as SE-CA-HSP90) as antigen to protect the immune protection of Candida albicans infection, in order to lay the foundation for the development of fungal prophylactic vaccines. This experiment includes the following contents:
1 a large number of heterozygous phage SE-CA-HSP90
With the help of the wild phage, the pfd8H vector containing the HSP90 specific epitope (DEPAGE) DNA sequence of the Candida albicans was transformed into the Escherichia coli TG1. The Escherichia coli transformed from pfd8H was successfully secreted and expressed the heterozygous phage displayed with the DEPAGE epitope, and then the phage particles in the culture liquid were recovered and purified with PEG6000. Heterozygous phage samples were used. After SDS-PAGE electrophoresis, there was a fusion protein band on the gel, which was obviously lagging behind the wild type phage shell protein band, stained with high sensitivity silver staining method and no other white egg white band, indicating that the purification requirement was reached.
Immune protection of 2 heterozygous phage SE-CA-HSP90 against systemic Candida albicans
(1) establish a systemic Candida albicans infection model and screen the best dose of infection.
(2) C57BL/6 mice were immunized with heterozygous phage SE-CA-HSP90 as antigen, and wild type phage and buffer TE were injected into the control group. The systemic Candida albicans infection was carried out in the tail vein for several weeks.
(3) observe and detect the following indicators at different times:
Statistics of animal mortality within 15 days of systemic Candida albicans infection;
After 15 days of infection, the kidneys of the immunized animals were grinded to homogenate, and Candida albicans were cultured in the medium of sand culture, and the colony number of Candida albicans was detected.
The pathological sections were made to compare the damage of animal kidneys.
The experimental results showed that the immune heterozygous phage SE-CA-HSP90 group, compared with the immunized wild phage group and the TE group, increased the resistance to systemic Candida albicans infection, increased the survival rate of the mice, decreased the number of Candida albicans in the kidney, and showed a significant number of Candida albicans in the renal tubules. Less than other groups, the results suggest that heterozygous phage SE-CA-HSP90 displaying exogenous peptides may develop into an effective antifungal vaccine.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R379

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