白色念珠菌Hsp60的融合表达及免疫活性的研究
发布时间：2019-04-08 14:26
【摘要】： 热休克蛋白60(hsp60)广泛存在于生物体内,它的基本功能是参与蛋白质的折叠、转运、代谢等。目前,hsp60研究的重点是在感染、肿瘤及风湿性疾病等方面的作用及机制,而其关于白色念珠菌(Candida albicans)感染的预防或治疗性疫苗方面的研究未见报道。由于hsp60进化高度保守性和在炎症过程中的过度表达,使其成为宿主免疫系统识别的重要免疫原,也成为真菌疫苗研究的一个重要靶点。 本研究旨在借助动物实验,评价通过基因工程表达的白色念珠菌hsp60融合蛋白的免疫保护作用,并初步探讨其作用机制,为开发真菌疫苗奠定实验基础和提供理论根据。 根据GenBank中,白色念珠菌hsp60的基因序列设计引物,通过RT-PCR技术扩增其基因片段;将PCR产物连接到pET28b(+)表达质粒中,构建pET28b-hsp60重组表达载体;并将其转入大肠杆菌BL21中诱导表达;按照常规方法完成通过SDS-PAGE及Western blot鉴定表达蛋白的正确性;经Ni2+-Saphrose-4金属螯合亲和层析法纯化融合蛋白质。将重组hsp60蛋白免疫小鼠,通过ELISA检测小鼠血清中抗体效价;淋巴细胞转化试验检测T淋巴细胞增殖水平;用该菌株的致死量攻击免疫小鼠,取肾脏进行菌落记数,并用非致死量攻击免疫小鼠,观察二个月内的存活率,来评价保护效果。 本研究成功表达了重组hsp60蛋白,并通过免疫小鼠,发现小鼠产生了保护性的特异性体液免疫应答和细胞免疫应答。动物保护试验结果表明:该重组蛋白免疫后的小鼠对白色念珠菌的攻击具有明显的保护作用。本研究初步探讨了白色念珠菌hsp60蛋白的免疫活性,为开发白色念珠菌疫苗奠定了理论基础。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2454653
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 黄菁,乔传令,李tD,邢建民;解毒酶基因的克隆及在大肠杆菌中的融合表达[J];遗传学报;2001年06期
2 陈官芝 ,徐秀莲 ,吴延芳 ,王云 ,罗兵;致病性真菌DNA提取方法的实验研究[J];中华皮肤科杂志;2002年05期
3 周村建,郝飞,王鲁,钟白玉;白念珠菌总RNA提取的实验研究[J];中华皮肤科杂志;2004年08期
，本文编号：2454653
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2454653.html
最近更新
教材专著
