抗CDK4人源單鏈抗體作用機制的研究
發(fā)布時間：2018-07-14 13:41
【摘要】：研究表明腫瘤細胞惡性增殖主要是由于細胞周期的失調(diào)所導致,Cyclin D-CDK4/CDK6是調(diào)控細胞周期由G1向S期的過渡關鍵因子。CDK4基因己被證實是一種癌基因,在多種腫瘤細胞和癌變組織中都發(fā)現(xiàn)伴隨有CDK4的過度表達和過度活化現(xiàn)象,因此CDK4可做為腫瘤基因治療中另一個潛在的靶點。本課題組在前期的工作中,已獲得抗CDK4單鏈抗體并將其轉入腫瘤細胞中,成功的逆轉了腫瘤細胞惡性增殖的表型,但是我們對于該單鏈抗體究竟以何種方式識別并作用于抗原分子,抑制靶蛋白功能,進而發(fā)揮其抗腫瘤作用的機制等基因水平上的問題還不清楚。 表位代表了抗原分子上的一個免疫活性區(qū),是抗原抗體識別的基礎,隨著生物學技術的發(fā)展,對于表位的預測已經(jīng)發(fā)展了多種方法,其中噬菌體篩選技術因其高通量、操作簡單、能同時預測構象型表位和線性表位等優(yōu)點,現(xiàn)已經(jīng)被廣泛的應用于各種研究中,特別是以單鏈抗體為靶蛋白篩選噬菌體庫來分析其所對應抗原的表位己成為目前預測抗原蛋白表位的重要方法。因為每一種表位預測方法都有一定的局限性,因此采用計算預測(如同源建模、分子對接等)和生物實驗數(shù)據(jù)結合的方法進行抗原蛋白構象表位分析和定位已經(jīng)成了目前的研究熱點,并呈現(xiàn)出了快速發(fā)展的趨勢。 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,進而以scFv為靶點,從噬菌體隨機環(huán)七肽庫中篩選得到一組特異性結合抗CDK4的人源單鏈抗體的環(huán)七肽基因,并用生物學軟件對這組環(huán)七肽的序列進行了比對分析。為了能夠獲得更準確的數(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的空間結構,用Builter軟件繪制了環(huán)七肽的三維結構,然后用分子對接的計算機方法模擬了小肽與scFv相互作用的空間結構。根據(jù)噬菌體篩選和計算機模擬的結果,我們推測經(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CDK4的表位活性空間構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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
本文編號：2121829
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
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