白念珠菌蛋白激酶Gin4的功能与结构分析
发布时间：2018-11-11 10:05
【摘要】：白念珠菌是一种条件性致病真菌,寄存在人体内,与健康菌群共生,但在一些情况下,会引起疾病。我们主要研究白念珠菌一些基因功能及其结构。Gin4蛋白属于NIM1蛋白激酶家族,是非常重要的细胞周期调节器。GIN4基因主要分为N端激酶区和C端非激酶区,激酶区功能及结构已被研究非常通透;但对于C端的研究比较少。根据一些研究,发现该基因有三个不同的结合域:脂质结合域(LBD),隔膜蛋白结合域(SBD)和核仁结合域(NAD)。隔膜蛋白结合域(SBD)是Gin4蛋白功能中一项必不可少的结构域,我们本次实验主要研究该区域功能。我们将要研究区域截取几段,与不同标签载体连接,之后利用不同标签特异性吸附达到初步纯化目的,再利用离子交换、分子筛等方法获得纯度较高,均一性较好的蛋白。最后,将得到目的蛋白,利用蛋白晶体初筛试剂盒初筛蛋白晶体,再在此基础上优化,使之得到想要的蛋白结构。蛋白功能方面,本次实验主要涉及了 4个蛋白,分别是Gin4蛋白,Sec15蛋白,Bem1蛋白,Rsr1蛋白。在酿酒酵母中,SEC15基因和众多基因一起组成胞吐复合物结构,在营养吸收,与外界环境沟通,分裂和维护细胞基本形状是至关重要。Rsr1蛋白是一种GTP酶,是一种芽位点蛋白,是一种Ras超家族中一员。Bem1蛋白在细胞极性和出芽功能方面存在作用,Gin4蛋白是一种隔膜蛋白相关蛋白,在细胞分离方面起作用。这几个蛋白在白念珠菌中都有表达,且与极性有关。本次实验主要通过蛋白之间体外相互作用,即利用标签抗体特异性,通过Western Blot方法验证它们之间关系。结果表明,Gin4蛋白C端与Bem1蛋白作用,而Bem1蛋白N端与Sec15蛋白C端作用。Sec15蛋白也与Rsr1蛋白有作用。
[Abstract]:Candida albicans is a kind of conditional pathogenic fungus, which is deposited in human body and symbiotic with healthy bacteria, but in some cases, it can cause disease. We mainly study the function and structure of some genes in Candida albicans. Gin4 protein belongs to NIM1 protein kinase family and is a very important cell cycle regulator. GIN4 gene is mainly divided into N-terminal kinase region and C-terminal non-kinase region. The function and structure of kinase region have been studied very well. But the research on C-terminal is less. According to some studies, the gene has three different binding domains: lipid binding domain (LBD),) membrane protein binding domain (SBD) and nucleolar binding domain (NAD). Membrane protein binding domain (SBD) is an essential domain in the function of Gin4 protein. We will intercept several segments of the study region, connect with different label carriers, then use different label specific adsorption to achieve the primary purification purpose, and then use ion exchange, molecular sieve and other methods to obtain proteins with high purity and good homogeneity. Finally, the target protein was obtained, and the protein crystal was screened using the protein crystal screening kit, and then optimized to obtain the desired protein structure. In terms of protein function, four proteins were involved in this experiment, namely Gin4 protein, Sec15 protein, Bem1 protein and Rsr1 protein. In Saccharomyces cerevisiae, the SEC15 gene, together with many genes, forms the exocytosis complex, which is essential in nutrient absorption, communication with the outside environment, division and maintenance of the basic shape of the cell. Rsr1 protein is a kind of GTP enzyme and a budding site protein. Bem1 protein is a member of Ras superfamily. Bem1 protein plays a role in cell polarity and budding function. Gin4 protein is a membrane protein-associated protein which plays an important role in cell separation. These proteins are expressed in Candida albicans and are related to polarity. In this experiment, the protein interaction in vitro, that is, using the label antibody specificity, Western Blot method to verify the relationship between them. The results showed that C terminal of Gin4 protein interacted with Bem1 protein, while N terminal of Bem1 protein interacted with C terminal of Sec15 protein. Sec15 protein also acted on Rsr1 protein.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379.4
本文编号：2324513
[Abstract]:Candida albicans is a kind of conditional pathogenic fungus, which is deposited in human body and symbiotic with healthy bacteria, but in some cases, it can cause disease. We mainly study the function and structure of some genes in Candida albicans. Gin4 protein belongs to NIM1 protein kinase family and is a very important cell cycle regulator. GIN4 gene is mainly divided into N-terminal kinase region and C-terminal non-kinase region. The function and structure of kinase region have been studied very well. But the research on C-terminal is less. According to some studies, the gene has three different binding domains: lipid binding domain (LBD),) membrane protein binding domain (SBD) and nucleolar binding domain (NAD). Membrane protein binding domain (SBD) is an essential domain in the function of Gin4 protein. We will intercept several segments of the study region, connect with different label carriers, then use different label specific adsorption to achieve the primary purification purpose, and then use ion exchange, molecular sieve and other methods to obtain proteins with high purity and good homogeneity. Finally, the target protein was obtained, and the protein crystal was screened using the protein crystal screening kit, and then optimized to obtain the desired protein structure. In terms of protein function, four proteins were involved in this experiment, namely Gin4 protein, Sec15 protein, Bem1 protein and Rsr1 protein. In Saccharomyces cerevisiae, the SEC15 gene, together with many genes, forms the exocytosis complex, which is essential in nutrient absorption, communication with the outside environment, division and maintenance of the basic shape of the cell. Rsr1 protein is a kind of GTP enzyme and a budding site protein. Bem1 protein is a member of Ras superfamily. Bem1 protein plays a role in cell polarity and budding function. Gin4 protein is a membrane protein-associated protein which plays an important role in cell separation. These proteins are expressed in Candida albicans and are related to polarity. In this experiment, the protein interaction in vitro, that is, using the label antibody specificity, Western Blot method to verify the relationship between them. The results showed that C terminal of Gin4 protein interacted with Bem1 protein, while N terminal of Bem1 protein interacted with C terminal of Sec15 protein. Sec15 protein also acted on Rsr1 protein.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379.4
【参考文献】
相关期刊论文 前1条
1 ;An overview of human protein databases and their application to functional proteomics in health and disease[J];Science China(Life Sciences);2011年11期
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