白色念珠菌热休克蛋白90基因的原核表达及其免疫学活性初探
发布时间：2018-12-28 19:39
【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力下降和正常菌群失调时,引起粘膜性感染和系统性念珠菌感染。真菌感染对机体的危害是很大的,尤其是系统性白色念珠菌感染,据英国科学家最新统计,在使用了抗真菌药物后,该病的死亡率仍高达70%以上。因此,控制白色念珠菌感染的最好办法是预防,而疫苗的开发则是建立有效防范措施的关键环节,研发出一种有效的疫苗以防治白色念珠菌感染是非常重要的。DNA疫苗是继病原体疫苗、亚单位疫苗之后的第三代疫苗,具有许多的优越性。 白色念珠菌的胞浆和细胞壁上存在有一种免疫优势抗原—热休克蛋白90(heat shock protein,Hsp90),该蛋白和它的47kDa、40 kDa降解片段均能够与血清抗体特异性结合。在念珠菌侵染的实验动物和人体中,白色念珠菌Hsp90能够诱导机体产生保护性抗体。Matthews等人利用白色念珠菌感染康复患者的血清,确定了白色念珠菌Hsp90的抗原表位图谱,发现了3个能够与病人血清起反应的常见表位,从而绘制出了Hsp90碳末端的连续表位,即表位C、表位B和表位H,其中只有表位C、H具有免疫原性。 本实验中选择Hsp90基因序列中含有编码C、H表位的一段DNA序列,构建原核重组质粒pET28a(+)/Hsp90,将重组质粒导入大肠杆菌后,在原核细胞中表达出了重组蛋白R-Hsp90。用恶性肿瘤病人血清和正常人血清对R-Hsp90进行Western blot分析,结果表明恶性肿瘤病人血清能够识别该重组蛋白,从而显示了R-Hsp90具有初步的免疫学活性。利用经Ni-NTA Agarose纯化的R-Hsp90免疫ICR小鼠,结果表明它能诱导机体做出相应的免疫应答,产生相应的特异性抗体,进一步验证了该重组蛋白R-Hsp90的免疫学活性。本研究不仅为白色念珠菌感染的早期诊断、治疗和预防奠定了理论基础,更为本课题组进一步进行白色念珠菌DNA疫苗的研究提供了理论依据。
[Abstract]:Candida albicans is a opportunistic pathogen parasitic on the skin and mucosa of healthy host. It can cause mucosal infection and systemic candida infection when the immunity of human body is decreased and the normal flora is disordered. Fungal infection is very harmful to the body, especially systemic Candida albicans. According to the latest statistics of British scientists, the death rate of the disease is still more than 70% after the use of antifungal drugs. Therefore, the best way to control Candida albicans infection is to prevent it, and the development of vaccine is the key to establish effective preventive measures. It is very important to develop an effective vaccine to control Candida albicans infection. DNA vaccine is the third generation vaccine after pathogen vaccine and subunit vaccine which has many advantages. There is an immune dominant antigen-heat shock protein (90 (heat shock protein,Hsp90) on the cytoplasm and cell wall of Candida albicans. This protein and its degradation fragment of 47 kDa 40 kDa can specifically bind to serum antibody. In laboratory animals and human beings infected with Candida albicans, Candida albicans Hsp90 can induce the body to produce protective antibodies. Matthews et al. determined the antigenic epitope of Candida albicans Hsp90 by using the sera of rehabilitated patients infected with Candida albicans. Three common epitopes which can react with patient's serum were found, and the continuous epitopes of Hsp90 carbon terminal, i.e. epitope C, epitope B and epitope H, were plotted, among which only epitope Con H was immunogenicity. In this experiment, we selected a DNA sequence of Hsp90 gene sequence which encodes CnH epitope, and constructed a prokaryotic recombinant plasmid pET28a () / Hsp90, to introduce the recombinant plasmid into Escherichia coli, and then expressed the recombinant protein R-Hsp90 in prokaryotic cells. The Western blot analysis of R-Hsp90 was carried out with the serum of malignant tumor patients and normal people. The results showed that the recombinant protein could be recognized by the serum of patients with malignant tumor, which indicated that R-Hsp90 had preliminary immunological activity. ICR mice were immunized with R-Hsp90 purified by Ni-NTA Agarose. The results showed that ICR mice could be induced to make corresponding immune responses and produce specific antibodies. The immunological activity of the recombinant protein R-Hsp90 was further verified. This study not only laid a theoretical foundation for the early diagnosis, treatment and prevention of Candida albicans infection, but also provided a theoretical basis for the further study of Candida albicans DNA 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】：2010
【分类号】：R379.4
本文编号：2394346
[Abstract]:Candida albicans is a opportunistic pathogen parasitic on the skin and mucosa of healthy host. It can cause mucosal infection and systemic candida infection when the immunity of human body is decreased and the normal flora is disordered. Fungal infection is very harmful to the body, especially systemic Candida albicans. According to the latest statistics of British scientists, the death rate of the disease is still more than 70% after the use of antifungal drugs. Therefore, the best way to control Candida albicans infection is to prevent it, and the development of vaccine is the key to establish effective preventive measures. It is very important to develop an effective vaccine to control Candida albicans infection. DNA vaccine is the third generation vaccine after pathogen vaccine and subunit vaccine which has many advantages. There is an immune dominant antigen-heat shock protein (90 (heat shock protein,Hsp90) on the cytoplasm and cell wall of Candida albicans. This protein and its degradation fragment of 47 kDa 40 kDa can specifically bind to serum antibody. In laboratory animals and human beings infected with Candida albicans, Candida albicans Hsp90 can induce the body to produce protective antibodies. Matthews et al. determined the antigenic epitope of Candida albicans Hsp90 by using the sera of rehabilitated patients infected with Candida albicans. Three common epitopes which can react with patient's serum were found, and the continuous epitopes of Hsp90 carbon terminal, i.e. epitope C, epitope B and epitope H, were plotted, among which only epitope Con H was immunogenicity. In this experiment, we selected a DNA sequence of Hsp90 gene sequence which encodes CnH epitope, and constructed a prokaryotic recombinant plasmid pET28a () / Hsp90, to introduce the recombinant plasmid into Escherichia coli, and then expressed the recombinant protein R-Hsp90 in prokaryotic cells. The Western blot analysis of R-Hsp90 was carried out with the serum of malignant tumor patients and normal people. The results showed that the recombinant protein could be recognized by the serum of patients with malignant tumor, which indicated that R-Hsp90 had preliminary immunological activity. ICR mice were immunized with R-Hsp90 purified by Ni-NTA Agarose. The results showed that ICR mice could be induced to make corresponding immune responses and produce specific antibodies. The immunological activity of the recombinant protein R-Hsp90 was further verified. This study not only laid a theoretical foundation for the early diagnosis, treatment and prevention of Candida albicans infection, but also provided a theoretical basis for the further study of Candida albicans DNA 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】：2010
【分类号】：R379.4
【引证文献】
相关博士学位论文 前1条
1 谢彦海;褶纹冠蚌热休克蛋白基因克隆与表达及2种淡水蚌的血细胞分析[D];南昌大学;2011年
相关硕士学位论文 前1条
1 杨慧敏;锦灯笼果实多糖在核酸疫苗中免疫增强作用研究[D];东北师范大学;2012年
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