本文選題：Cyclin + D1��； 參考：《吉林大學(xué)》2011年碩士論文

【摘要】：細(xì)胞周期是細(xì)胞生命活動(dòng)的基本過程,細(xì)胞周期的紊亂將導(dǎo)致細(xì)胞異常增殖,從而引發(fā)腫瘤形成。周期蛋白Cyclins通過其表達(dá)量在細(xì)胞周期各時(shí)相中的變化調(diào)節(jié)細(xì)胞周期的進(jìn)程,是調(diào)控細(xì)胞周期網(wǎng)絡(luò)系統(tǒng)的關(guān)鍵分子。Cyclin Dl是細(xì)胞進(jìn)入增殖周期合成的第一個(gè)周期蛋白,它能夠特異性的結(jié)合CDK4形成Cyclin Dl/CDK4復(fù)合物,控制細(xì)胞周期進(jìn)入S期。Cyclin Dl在多種人類惡性腫瘤細(xì)胞中呈過度表達(dá),目前人們發(fā)現(xiàn)Cyclin Dl還與腫瘤的發(fā)生、腫瘤細(xì)胞的浸潤和轉(zhuǎn)移都密切相關(guān),因此,開展以Cyclin Dl為靶點(diǎn)的腫瘤基因治療具有巨大的實(shí)際意義和應(yīng)用前景。 胞內(nèi)抗體技術(shù)是近年來剛剛興起的一種能特異性阻斷細(xì)胞內(nèi)重要靶蛋白的生物學(xué)技術(shù),因其具有高效、低毒、作用范圍廣泛、病人不易產(chǎn)生抗藥性等優(yōu)點(diǎn),該技術(shù)已經(jīng)成為利用細(xì)胞內(nèi)免疫機(jī)制來進(jìn)行基因治療的新手段,得到了廣泛的應(yīng)用。尤其是胞內(nèi)單鏈抗體,因其克服了其他工程抗體分子結(jié)構(gòu)復(fù)雜、分子量大、免疫原性強(qiáng),半衰期短等缺點(diǎn),已經(jīng)成為了胞內(nèi)抗體最主要的形式之一。近年來,人們已經(jīng)將單鏈抗體導(dǎo)入多種細(xì)胞內(nèi)實(shí)現(xiàn)了靶蛋白特異性滅活、惡性增殖細(xì)胞表型發(fā)生改變,為腫瘤的基因治療提供了新的思路。 為此,本研究以p3.1-AD載體為模板,PCR擴(kuò)增目的基因AD使其獲得NotⅠ和BssHⅡ的酶切位點(diǎn)及His標(biāo)簽。將該片段正向插入到PDAN5載體的Not I和BssHⅡ區(qū)域。測序鑒定重組質(zhì)粒,將重組質(zhì)粒轉(zhuǎn)化到大腸桿菌HB2151。IPTG,37℃誘導(dǎo)AD基因表達(dá),Wstern-blot鑒定目的蛋白,HisTrap HP Kit柱純化后SDS-PAGE分析純化效果。本研究成功構(gòu)建了pAD質(zhì)粒；并誘導(dǎo)表達(dá)了胞內(nèi)單鏈抗體AD,但是并沒有能夠成功的純化出具有高純度的AD蛋白,我們推測是由于破碎上清中存在過多的雜蛋白,干擾了目的AD蛋白的結(jié)合。綜上所述,以Cyclin Dl為靶點(diǎn)的胞內(nèi)抗體治療作為一種新型腫瘤基因治療方法,有潛在的應(yīng)用價(jià)值,但要獲得抗人細(xì)胞周期蛋白D1胞內(nèi)單鏈抗體AD,還需進(jìn)一步的摸索純化條件。
[Abstract]:Cell cycle is the basic process of cell life activity. The disorder of cell cycle will lead to abnormal proliferation of cells, which will lead to tumor formation. Cyclin Cyclins regulates the process of cell cycle through the changes of the expression of cyclin Cyclins in different phases of cell cycle. It is the key molecule of cell cycle network system. Cyclin D1 is the first cyclin in cell cycle synthesis. It can specifically bind CDK4 to form Cyclin Dl/CDK4 complex, and control the cell cycle into S phase. Cyclin D1 is overexpressed in various human malignant tumor cells. At present, it has been found that Cyclin DL is also associated with tumor genesis. The invasion and metastasis of tumor cells are closely related. Therefore, the development of tumor gene therapy targeting Cyclin D1 has great practical significance and application prospect. Intracellular antibody technology is a new biological technique which can specifically block the important target proteins in cells in recent years, because of its high efficiency, low toxicity, wide range of action and so on, patients are not easy to develop drug resistance. This technique has become a new method for gene therapy using intracellular immune mechanism and has been widely used. In particular, intracellular single-chain antibodies have become one of the most important forms of intracellular antibodies because they overcome the disadvantages of complex molecular structure, high molecular weight, strong immunogenicity and short half-life of other engineering antibodies. In recent years, the introduction of scFv into a variety of cells has achieved target protein specific inactivation, and the phenotype of malignant proliferative cells has changed, which provides a new idea for gene therapy of tumor. In this study, the target gene AD was amplified by using p3.1-AD vector as template to obtain the restriction sites and His tags of Not 鈪，

本文編號(hào)：1927651