本文關(guān)鍵詞： 噬菌體肽庫 鼻咽癌 腫瘤標(biāo)記物　出處：《廣西醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的鼻咽癌(Nasopharyngeal Carcinoma,NPC)是一種多基因遺傳性疾病(polygenetic diseases)。目前對鼻咽癌的診斷主要靠組織活檢,因其早期癥狀不明顯,患者的5年生存率僅在50%左右。然而已證明腫瘤患者體內(nèi)可產(chǎn)生相應(yīng)的腫瘤標(biāo)記物,我們可以通過檢測這些腫瘤標(biāo)記物來診斷早期的NPC。通過分子生物學(xué)和免疫學(xué)的方法來找到那些敏感性、特異性比較高的腫瘤標(biāo)記物,可為早期NPC患者的診斷提供新的思路。 噬菌體展示技術(shù)(phage display technique, PDT)是尋找與靶蛋白有特異性結(jié)合關(guān)系的多肽或蛋白的強(qiáng)效生物工具,它的最大優(yōu)點(diǎn)是直接將可呈現(xiàn)的表現(xiàn)型和其基因型聯(lián)系在一起,被展示出來的外源蛋白可保持相對獨(dú)立的空間結(jié)構(gòu)和生物活性,并利用其配體的特異性親和力將所感興趣的蛋白質(zhì)或多肽篩選出來,然后通過序列測定獲得相應(yīng)的結(jié)構(gòu)和功能信息。我們應(yīng)用噬菌體隨機(jī)12肽庫篩選鼻咽癌患者血清,淘選出鼻咽癌患者表達(dá)的特異性腫瘤抗原,期望為鼻咽癌患者的診斷和預(yù)后提供新的標(biāo)記物。 方法1.分別用正常人血清和鼻咽癌患者血清包被微孔板,通過減性篩選的方法,先將噬菌體與正常血清預(yù)吸附,然后將未結(jié)合的噬菌體再與鼻咽癌患者血清進(jìn)行吸附,再把得到與鼻咽癌患者血清結(jié)合的噬菌體擴(kuò)增。重復(fù)篩選三次。2.挑取單個(gè)噬菌斑,用篩選時(shí)的血清通過ELISA方法檢測與鼻咽癌患者血清的結(jié)合情況,選擇差異明顯的噬菌體,擴(kuò)大血清樣本含量再進(jìn)行檢測,最終得到能夠與鼻咽癌患者血清特異性結(jié)合的單個(gè)噬菌體克隆。3.提取并純化單個(gè)噬菌體克隆的DNA,使用引物-96Ⅲ進(jìn)行DNA測序,并根據(jù)短肽插入位點(diǎn)和氨基酸遺傳密碼表得到插入短肽序列。 結(jié)果1.經(jīng)過三輪篩選,挑取單個(gè)噬菌體反復(fù)鑒定后得到兩株特異性較好的噬菌體克隆;2.經(jīng)測序獲這兩個(gè)短肽排列序列;3.這兩株噬菌體克隆ELISA鑒定IgA-VCA陽性鼻咽癌血清及IgA-VCA陰性鼻咽癌血清無顯著差異;4.這兩株噬菌體克隆通過ELISA分別檢測鼻咽癌血清、肝癌血清、卵巢癌血清結(jié)果顯示其與鼻咽癌患者血清的結(jié)合具有特異性。 結(jié)論本實(shí)驗(yàn)通過噬菌體隨機(jī)肽庫篩選出能與鼻咽癌相關(guān)抗原特異性結(jié)合的短肽,為鼻咽癌早期診斷、聯(lián)合檢測以及免疫治療提供了候選標(biāo)記物。
[Abstract]:Background and objective Nasopharyngeal carcinoma (NPC) is a polygenetic disease. At present, the diagnosis of nasopharyngeal carcinoma mainly depends on tissue biopsy, because its early symptoms are not obvious. The 5-year survival rate was only about 50%. However, it has been shown that tumor markers can be produced in patients with cancer. We can detect these tumor markers to diagnose early NPCs. We can find the sensitive and specific tumor markers by molecular biology and immunology, which can provide a new idea for the diagnosis of early NPC patients. Phage display technique (PDT) is a powerful biological tool for searching for peptides or proteins with specific binding to target proteins. Its greatest advantage is that it directly links phenotypes to its genotypes. The extraneous proteins exhibited can maintain relatively independent spatial structures and biological activities and use the specific affinity of their ligands to screen out proteins or peptides of interest. Then we obtained the corresponding structural and functional information by sequencing. We used phage random 12 peptide library to screen the sera of NPC patients and to select the specific tumor antigens expressed in NPC patients by Amoy. It is expected to provide a new marker for the diagnosis and prognosis of nasopharyngeal carcinoma (NPC) patients. Methods 1. The phage was preadsorbed with the normal serum and the normal serum by subtractive screening, and then the unbound phage was adsorbed with the serum of nasopharyngeal carcinoma. Then the phage was amplified from the sera of nasopharyngeal carcinoma patients. The phage was screened three times. 2. Single plaque was picked out. The binding between the sera of nasopharyngeal carcinoma patients and nasopharyngeal carcinoma patients was detected by ELISA method, and the different phages were selected. A single phage clone, which can specifically bind to the serum of nasopharyngeal carcinoma patients, was obtained by enlarging the serum sample content and then detected. The DNA of a single phage clone was extracted and purified. The DNA sequencing was carried out with primer -96 鈪，

本文編號：1530005