【摘要】： 熱休克蛋白60(hsp60)廣泛存在于生物體內(nèi),它的基本功能是參與蛋白質(zhì)的折疊、轉(zhuǎn)運(yùn)、代謝等。目前,hsp60研究的重點(diǎn)是在感染、腫瘤及風(fēng)濕性疾病等方面的作用及機(jī)制,而其關(guān)于白色念珠菌(Candida albicans)感染的預(yù)防或治療性疫苗方面的研究未見報(bào)道。由于hsp60進(jìn)化高度保守性和在炎癥過(guò)程中的過(guò)度表達(dá),使其成為宿主免疫系統(tǒng)識(shí)別的重要免疫原,也成為真菌疫苗研究的一個(gè)重要靶點(diǎn)。 本研究旨在借助動(dòng)物實(shí)驗(yàn),評(píng)價(jià)通過(guò)基因工程表達(dá)的白色念珠菌hsp60融合蛋白的免疫保護(hù)作用,并初步探討其作用機(jī)制,為開發(fā)真菌疫苗奠定實(shí)驗(yàn)基礎(chǔ)和提供理論根據(jù)。 根據(jù)GenBank中,白色念珠菌hsp60的基因序列設(shè)計(jì)引物,通過(guò)RT-PCR技術(shù)擴(kuò)增其基因片段;將PCR產(chǎn)物連接到pET28b(+)表達(dá)質(zhì)粒中,構(gòu)建pET28b-hsp60重組表達(dá)載體;并將其轉(zhuǎn)入大腸桿菌BL21中誘導(dǎo)表達(dá);按照常規(guī)方法完成通過(guò)SDS-PAGE及Western blot鑒定表達(dá)蛋白的正確性;經(jīng)Ni2+-Saphrose-4金屬螯合親和層析法純化融合蛋白質(zhì)。將重組hsp60蛋白免疫小鼠,通過(guò)ELISA檢測(cè)小鼠血清中抗體效價(jià);淋巴細(xì)胞轉(zhuǎn)化試驗(yàn)檢測(cè)T淋巴細(xì)胞增殖水平;用該菌株的致死量攻擊免疫小鼠,取腎臟進(jìn)行菌落記數(shù),并用非致死量攻擊免疫小鼠,觀察二個(gè)月內(nèi)的存活率,來(lái)評(píng)價(jià)保護(hù)效果。 本研究成功表達(dá)了重組hsp60蛋白,并通過(guò)免疫小鼠,發(fā)現(xiàn)小鼠產(chǎn)生了保護(hù)性的特異性體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答。動(dòng)物保護(hù)試驗(yàn)結(jié)果表明:該重組蛋白免疫后的小鼠對(duì)白色念珠菌的攻擊具有明顯的保護(hù)作用。本研究初步探討了白色念珠菌hsp60蛋白的免疫活性,為開發(fā)白色念珠菌疫苗奠定了理論基礎(chǔ)。
[Abstract]:The heat shock protein 60 (hsp60) is widely present in the living body, and its basic function is to participate in the folding, transport and metabolism of the protein. At present, the focus of the hsp60 study is the role and mechanism of infection, tumor and rheumatic diseases, and the study on the prevention or treatment of Candida albicans infection has not been reported. Because of the high conservation of hsp60 and over-expression in the process of inflammation, it becomes an important immunogen for the identification of the host immune system, and is an important target for the study of the fungal vaccine. The purpose of this study was to evaluate the immune protection of the white Candida hsp60 fusion protein expressed by genetic engineering with the aid of animal experiments, and to explore its mechanism of action to lay a foundation for the development of the fungal vaccine. According to the gene sequence design primer of the candida albicans hsp60 in GenBank, amplifying the gene fragment by the RT-PCR technology, connecting the PCR product to the pET28b (+) expression plasmid, and constructing the pET28b-hsp60 recombinant expression vector. and carrying out SDS-PAGE and Western blot to identify the correctness of the expression protein through SDS-PAGE and Western blot; The fusion protein was purified by e-4 metal affinity chromatography. The recombinant hsp60 protein was immunized with the recombinant hsp60 protein, and the antibody titer in the serum of the mice was detected by ELISA. The lymphocyte transformation test was used to test the level of T-lymphocyte proliferation. The immune mice were challenged with the lethal dose of the strain, and the kidney was taken. The number of colonies was counted and the immune mice were challenged with a non-lethal dose. And the survival rate in two months was observed to evaluate the protective effect. The recombinant hsp60 protein was successfully expressed and the immune response was small. The mice found that the mice produced a protective specific humoral immune response and cellular immune response. The animal protection test results showed that: The mice immunized with the recombinant protein have an obvious protective effect on the attack of Candida albicans.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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