【摘要】： 白色念珠菌是寄生在健康宿主皮膚、粘膜的條件致病菌,它可以作為機會病原體在人體免疫力下降和正常菌群失調(diào)時,引起粘膜性感染和系統(tǒng)性念珠菌感染。真菌感染對機體的危害是很大的,尤其是系統(tǒng)性白色念珠菌感染,據(jù)英國科學家最新統(tǒng)計,在使用了抗真菌藥物后,該病的死亡率仍高達70%以上。因此,控制白色念珠菌感染的最好辦法是預防,而疫苗的開發(fā)則是建立有效防范措施的關(guān)鍵環(huán)節(jié),研發(fā)出一種有效的疫苗以防治白色念珠菌感染是非常重要的。DNA疫苗是繼病原體疫苗、亞單位疫苗之后的第三代疫苗,具有許多的優(yōu)越性。 白色念珠菌的胞漿和細胞壁上存在有一種免疫優(yōu)勢抗原—熱休克蛋白90(heat shock protein,Hsp90),該蛋白和它的47kDa、40 kDa降解片段均能夠與血清抗體特異性結(jié)合。在念珠菌侵染的實驗動物和人體中,白色念珠菌Hsp90能夠誘導機體產(chǎn)生保護性抗體。Matthews等人利用白色念珠菌感染康復患者的血清,確定了白色念珠菌Hsp90的抗原表位圖譜,發(fā)現(xiàn)了3個能夠與病人血清起反應的常見表位,從而繪制出了Hsp90碳末端的連續(xù)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。 本實驗中選擇Hsp90基因序列中含有編碼C、H表位的一段DNA序列,構(gòu)建原核重組質(zhì)粒pET28a(+)/Hsp90,將重組質(zhì)粒導入大腸桿菌后,在原核細胞中表達出了重組蛋白R-Hsp90。用惡性腫瘤病人血清和正常人血清對R-Hsp90進行Western blot分析,結(jié)果表明惡性腫瘤病人血清能夠識別該重組蛋白,從而顯示了R-Hsp90具有初步的免疫學活性。利用經(jīng)Ni-NTA Agarose純化的R-Hsp90免疫ICR小鼠,結(jié)果表明它能誘導機體做出相應的免疫應答,產(chǎn)生相應的特異性抗體,進一步驗證了該重組蛋白R-Hsp90的免疫學活性。本研究不僅為白色念珠菌感染的早期診斷、治療和預防奠定了理論基礎(chǔ),更為本課題組進一步進行白色念珠菌DNA疫苗的研究提供了理論依據(jù)。
[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.
【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】：R379.4

【引證文獻】

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