發(fā)布時(shí)間：2018-07-14 13:41

【摘要】：研究表明腫瘤細(xì)胞惡性增殖主要是由于細(xì)胞周期的失調(diào)所導(dǎo)致,Cyclin D-CDK4/CDK6是調(diào)控細(xì)胞周期由G1向S期的過(guò)渡關(guān)鍵因子。CDK4基因己被證實(shí)是一種癌基因,在多種腫瘤細(xì)胞和癌變組織中都發(fā)現(xiàn)伴隨有CDK4的過(guò)度表達(dá)和過(guò)度活化現(xiàn)象,因此CDK4可做為腫瘤基因治療中另一個(gè)潛在的靶點(diǎn)。本課題組在前期的工作中,已獲得抗CDK4單鏈抗體并將其轉(zhuǎn)入腫瘤細(xì)胞中,成功的逆轉(zhuǎn)了腫瘤細(xì)胞惡性增殖的表型,但是我們對(duì)于該單鏈抗體究竟以何種方式識(shí)別并作用于抗原分子,抑制靶蛋白功能,進(jìn)而發(fā)揮其抗腫瘤作用的機(jī)制等基因水平上的問(wèn)題還不清楚。 表位代表了抗原分子上的一個(gè)免疫活性區(qū),是抗原抗體識(shí)別的基礎(chǔ),隨著生物學(xué)技術(shù)的發(fā)展,對(duì)于表位的預(yù)測(cè)已經(jīng)發(fā)展了多種方法,其中噬菌體篩選技術(shù)因其高通量、操作簡(jiǎn)單、能同時(shí)預(yù)測(cè)構(gòu)象型表位和線性表位等優(yōu)點(diǎn),現(xiàn)已經(jīng)被廣泛的應(yīng)用于各種研究中,特別是以單鏈抗體為靶蛋白篩選噬菌體庫(kù)來(lái)分析其所對(duì)應(yīng)抗原的表位己成為目前預(yù)測(cè)抗原蛋白表位的重要方法。因?yàn)槊恳环N表位預(yù)測(cè)方法都有一定的局限性,因此采用計(jì)算預(yù)測(cè)(如同源建模、分子對(duì)接等)和生物實(shí)驗(yàn)數(shù)據(jù)結(jié)合的方法進(jìn)行抗原蛋白構(gòu)象表位分析和定位已經(jīng)成了目前的研究熱點(diǎn),并呈現(xiàn)出了快速發(fā)展的趨勢(shì)。 本研究首先分別制備了具有生物活性的CDK4抗原蛋白和抗CDK4的人源單鏈抗體(scFv)蛋白,進(jìn)而以scFv為靶點(diǎn),從噬菌體隨機(jī)環(huán)七肽庫(kù)中篩選得到一組特異性結(jié)合抗CDK4的人源單鏈抗體的環(huán)七肽基因,并用生物學(xué)軟件對(duì)這組環(huán)七肽的序列進(jìn)行了比對(duì)分析。為了能夠獲得更準(zhǔn)確的數(shù)據(jù)支持和更直觀的了解蛋白相互作用的方式,我們接著同源模建了scFv的空間結(jié)構(gòu),用Builter軟件繪制了環(huán)七肽的三維結(jié)構(gòu),然后用分子對(duì)接的計(jì)算機(jī)方法模擬了小肽與scFv相互作用的空間結(jié)構(gòu)。根據(jù)噬菌體篩選和計(jì)算機(jī)模擬的結(jié)果,我們推測(cè)經(jīng)噬菌體篩選的小肽分析獲得的表位可能為CDK4表位的組成部分或者對(duì)CDK4的表位活性空間構(gòu)象的形成起著非常重要的作用。
[Abstract]:The results show that the malignant proliferation of tumor cells is mainly due to the maladjustment of cell cycle. CDK4 gene has been proved to be a kind of oncogene, which is the key factor to regulate the transition from G1 to S phase of cell cycle. The overexpression and over-activation of CDK4 have been found in many kinds of tumor cells and cancerous tissues, so CDK4 can be used as another potential target in tumor gene therapy. In our previous work, anti-CDK4 scFv was obtained and transferred into tumor cells, which successfully reversed the malignant proliferation phenotype of tumor cells. However, it is not clear how the scFv can recognize and act on antigen molecules, inhibit the function of target proteins, and then exert the mechanism of anti-tumor effect. Epitopes represent an immunoreactive region on antigen molecules and are the basis of antigen and antibody recognition. With the development of biological technology, many methods have been developed for epitope prediction, among which phage screening technology has high throughput. It is easy to operate and can predict conformational epitopes and linear epitopes at the same time. In particular, it has become an important method to predict the epitope of antigen by screening phage library with scFv as target protein to analyze the epitopes of its corresponding antigen. Because of the limitations of each epitope prediction method, computational prediction, such as homologous modeling, is used. Molecular docking) and biological experimental data for the analysis and localization of antigen protein epitopes have become the current research focus, and have shown a rapid development trend. In this study, biologically active CDK4 antigen proteins and human scFv anti-CDK4 protein were prepared, and then scFv was used as the target. A group of cyclopeptide genes with specific binding to human single-chain antibody against CDK4 was obtained from phage random cyclic heptapeptide library. The sequence of this group of cyclic heptapeptides was compared and analyzed with biological software. In order to obtain more accurate data support and more intuitively understand the way of protein interaction, we then build the spatial structure of scFv by homologous model, and draw the three-dimensional structure of cycloheptapeptide with build software. Then the spatial structure of the interaction between small peptide and scFv was simulated by the computer method of molecular docking. Based on the results of phage screening and computer simulation, we speculate that the epitopes obtained by phage screening may be part of CDK4 epitopes or play an important role in the formation of epitope active space conformation of CDK4.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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