發(fā)布時(shí)間：2018-06-28 05:17

  本文選題：GE11 + 靶向性噬菌粒顆粒　； 參考：《復(fù)旦大學(xué)》2005年博士論文

【摘要】：腫瘤基因治療有望成為除手術(shù)、放療和化療等方法外的又一新的抗癌策略。尤其在抗腫瘤轉(zhuǎn)移和復(fù)發(fā)方面將起重要作用。目前還缺乏將基因?qū)肽[瘤細(xì)胞的高效靶向性載體系統(tǒng),這是腫瘤基因治療至今尚未成為臨床常規(guī)治療措施的關(guān)鍵因素之一。表皮生長因子受體(EGFR)由于在很多腫瘤有高表達(dá),因此是一個(gè)很好的靶點(diǎn)。本研究旨在建立一種靶向EGFR受體的基因?qū)胂到y(tǒng)并檢驗(yàn)其有效性,為腫瘤的靶向性基因治療提供可靠的理論依據(jù)與實(shí)踐指導(dǎo)。 第一部分 利用噬菌體多肽展示文庫篩選表皮生長因子受體的配體多肽 【目的】尋求可以與表皮生長因子受體(EGFR)受體特異性結(jié)合的配體多肽�！痉椒ā恳訣GFR蛋白為靶,利用噬菌體展示隨機(jī)12肽庫進(jìn)行篩選;或者構(gòu)建穩(wěn)定高表達(dá)EGFR突變體Ⅲ(EGFRvⅢ)胞外區(qū)的U87細(xì)胞系,以該細(xì)胞系為篩選靶,利用環(huán)7肽庫和12肽庫進(jìn)行篩選。經(jīng)過多輪篩選后,隨機(jī)挑選克隆進(jìn)行測序。把富集的克隆通過免疫組化及噬菌體回收試驗(yàn)鑒定獲得可以與靶蛋白結(jié)合的克隆;進(jìn)一步采用內(nèi)吞實(shí)驗(yàn)研究噬菌體陽性克隆的特征。【結(jié)果】通過EGFR蛋白為靶篩選,經(jīng)過三輪篩選獲得展示GE11(YHWYGYTPQNVI)多肽的噬菌體Phage-GE11。噬菌體回收實(shí)驗(yàn)中,Phage-GE11可以和EGFR有效結(jié)合,而且可以被EGF和GE11多肽所競爭抑制。免疫組化結(jié)果顯示Phage-GE11可以和EGFR表達(dá)陽性的細(xì)胞SMMC-7721,NCI-H460結(jié)合,但和EGFR表達(dá)陰性的細(xì)胞K-562細(xì)胞沒有結(jié)合。此外,免疫熒光結(jié)果顯示Phage-GE11可以被SMMC-7721細(xì)胞所內(nèi)吞。但是,以高表達(dá)EGFR突變體Ⅲ的細(xì)胞系作為篩選靶進(jìn)行篩選并沒有獲得陽性克隆。【結(jié)論】噬菌體文庫展示技術(shù)是一項(xiàng)獲得配體多肽的有效技術(shù),特別是以純化蛋白作為篩選靶可以更有效地獲得陽性克隆。Phage-GE11可以與EGFR特異結(jié)合,并被EGFR表達(dá)陽性的細(xì)胞所內(nèi)吞。
[Abstract]:Tumor gene therapy is expected to be a new anticancer strategy in addition to surgery, radiotherapy and chemotherapy. Especially in anti-tumor metastasis and recurrence will play an important role. At present, there is still a lack of efficient targeting vector system for gene transfer into tumor cells, which is one of the key factors of tumor gene therapy. Epidermal growth factor receptor (EGFR) is a good target because of its high expression in many tumors. The purpose of this study is to establish an EGFR receptor targeting gene transfer system and to test its effectiveness, and to provide a reliable theoretical basis and practical guidance for tumor targeting gene therapy. The first part is to screen the ligand peptide of epidermal growth factor receptor (EGF receptor) by phage display library [objective] to find out whether EGF receptor can be associated with epidermal growth factor receptor (EGF). (EGFR) receptor-specific ligand peptide. [methods] EGFR protein was used as target, Using phage display random 12 peptide library to screen, or to construct U87 cell line with high expression of EGFR mutant 鈪，

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