本文選題：CDK4 + 單鏈抗體　； 參考：《吉林大學(xué)》2009年碩士論文

【摘要】： 細(xì)胞周期調(diào)節(jié)失控是造成腫瘤細(xì)胞惡性增殖的主要原因之一。周期蛋白依賴蛋白激酶(cyclin-dependent kinase, CDK)的時(shí)相性激活是細(xì)胞周期調(diào)控機(jī)制的核心。CDK4作為細(xì)胞進(jìn)入增殖周期第一個(gè)被激活的周期蛋白依賴蛋白激酶,是細(xì)胞周期進(jìn)程中G1/S期轉(zhuǎn)換的限速因子。研究發(fā)現(xiàn)在許多腫瘤細(xì)胞和組織中都存在著CDK4的過(guò)度表達(dá)和過(guò)度活化,與腫瘤的發(fā)生和發(fā)展有著密切的關(guān)系。 作為研究和治療腫瘤的重要靶點(diǎn),如何下調(diào)過(guò)度表達(dá)和活化的CDK4已經(jīng)成為研究熱點(diǎn)。單鏈抗體(single chain antibody, scFv)以其分子量小,免疫原性低等優(yōu)點(diǎn),在腫瘤診斷和治療中具有獨(dú)特的優(yōu)勢(shì),目前已經(jīng)有多種基于單鏈抗體的抗腫瘤藥物進(jìn)入臨床試驗(yàn)。 本研究構(gòu)建了pET28a-CDK4表達(dá)載體,獲得有活性的重組人CDK4蛋白后,以其為抗原,從噬菌體庫(kù)中篩選得到抗CDK4人源單鏈抗體基因,將其轉(zhuǎn)化進(jìn)原核細(xì)胞中進(jìn)行了誘導(dǎo)、表達(dá)和純化,并通過(guò)ELISA、Western blot、競(jìng)爭(zhēng)性抑制、親和力測(cè)定、免疫熒光和免疫沉淀等實(shí)驗(yàn)方法對(duì)單鏈抗體進(jìn)行了活性表征。結(jié)果表明純化的單鏈抗體不僅能與重組人CDK4蛋白特異性結(jié)合并具有較高親和力,而且也能與細(xì)胞內(nèi)源性的CDK4蛋白特異性結(jié)合。 本實(shí)驗(yàn)得到的結(jié)果對(duì)后續(xù)開(kāi)展抗CDK4胞內(nèi)抗體腫瘤基因治療的研究奠定了基礎(chǔ)。
[Abstract]:Uncontrolled cell cycle regulation is one of the main causes of malignant proliferation of tumor cells. The chronological activation of cyclin dependent kinases (CDKs) is the core of cell cycle regulation. CDK4 acts as the first cyclin dependent kinase to be activated in cell cycle. It is the limiting factor of G 1 / S phase transition in cell cycle process. It has been found that overexpression and over-activation of CDK4 exist in many tumor cells and tissues, which is closely related to the occurrence and development of tumor. As an important target of tumor research and treatment, how to down-regulate overexpression and activation of CDK4 has become a hot topic. Single chain antibody, scFv) has unique advantages in tumor diagnosis and treatment due to its small molecular weight and low immunogenicity. At present, a variety of anti-tumor drugs based on scFv have been introduced into clinical trials. In this study, the pET28a-CDK4 expression vector was constructed. After the recombinant human CDK4 protein was obtained, the human single-chain antibody gene against CDK4 was screened from the phage library and transformed into prokaryotic cells for induction, expression and purification. The activity of scFv was characterized by Western blot, competitive inhibition, affinity assay, immunofluorescence and immunoprecipitation. The results showed that the purified scFv could not only bind to the recombinant human CDK4 protein with high affinity, but also specifically bind to the intracellular CDK4 protein. The results of this study laid a foundation for further research on tumor gene therapy of anti-CDK4 antibodies.
【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】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王媚娘;抗CDK4人源單鏈抗體作用機(jī)制的研究[D];吉林大學(xué);2011年

，

本文編號(hào)：1837762