【摘要】：白念珠菌是一種條件性致病真菌,寄存在人體內(nèi),與健康菌群共生,但在一些情況下,會引起疾病。我們主要研究白念珠菌一些基因功能及其結(jié)構(gòu)。Gin4蛋白屬于NIM1蛋白激酶家族,是非常重要的細(xì)胞周期調(diào)節(jié)器。GIN4基因主要分為N端激酶區(qū)和C端非激酶區(qū),激酶區(qū)功能及結(jié)構(gòu)已被研究非常通透;但對于C端的研究比較少。根據(jù)一些研究,發(fā)現(xiàn)該基因有三個不同的結(jié)合域:脂質(zhì)結(jié)合域(LBD),隔膜蛋白結(jié)合域(SBD)和核仁結(jié)合域(NAD)。隔膜蛋白結(jié)合域(SBD)是Gin4蛋白功能中一項必不可少的結(jié)構(gòu)域,我們本次實驗主要研究該區(qū)域功能。我們將要研究區(qū)域截取幾段,與不同標(biāo)簽載體連接,之后利用不同標(biāo)簽特異性吸附達(dá)到初步純化目的,再利用離子交換、分子篩等方法獲得純度較高,均一性較好的蛋白。最后,將得到目的蛋白,利用蛋白晶體初篩試劑盒初篩蛋白晶體,再在此基礎(chǔ)上優(yōu)化,使之得到想要的蛋白結(jié)構(gòu)。蛋白功能方面,本次實驗主要涉及了 4個蛋白,分別是Gin4蛋白,Sec15蛋白,Bem1蛋白,Rsr1蛋白。在釀酒酵母中,SEC15基因和眾多基因一起組成胞吐復(fù)合物結(jié)構(gòu),在營養(yǎng)吸收,與外界環(huán)境溝通,分裂和維護(hù)細(xì)胞基本形狀是至關(guān)重要。Rsr1蛋白是一種GTP酶,是一種芽位點蛋白,是一種Ras超家族中一員。Bem1蛋白在細(xì)胞極性和出芽功能方面存在作用,Gin4蛋白是一種隔膜蛋白相關(guān)蛋白,在細(xì)胞分離方面起作用。這幾個蛋白在白念珠菌中都有表達(dá),且與極性有關(guān)。本次實驗主要通過蛋白之間體外相互作用,即利用標(biāo)簽抗體特異性,通過Western Blot方法驗證它們之間關(guān)系。結(jié)果表明,Gin4蛋白C端與Bem1蛋白作用,而Bem1蛋白N端與Sec15蛋白C端作用。Sec15蛋白也與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.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R379.4

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相關(guān)期刊論文 前1條

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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本文編號：2324513