噬菌体随机肽库筛选鼻咽癌患者血清肿瘤标记物的实验研究

发布时间：2018-02-24 11:08

  本文关键词： 噬菌体肽库 鼻咽癌 肿瘤标记物　出处：《广西医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景和目的鼻咽癌(Nasopharyngeal Carcinoma,NPC)是一种多基因遗传性疾病(polygenetic diseases)。目前对鼻咽癌的诊断主要靠组织活检,因其早期症状不明显,患者的5年生存率仅在50%左右。然而已证明肿瘤患者体内可产生相应的肿瘤标记物,我们可以通过检测这些肿瘤标记物来诊断早期的NPC。通过分子生物学和免疫学的方法来找到那些敏感性、特异性比较高的肿瘤标记物,可为早期NPC患者的诊断提供新的思路。 噬菌体展示技术(phage display technique, PDT)是寻找与靶蛋白有特异性结合关系的多肽或蛋白的强效生物工具,它的最大优点是直接将可呈现的表现型和其基因型联系在一起,被展示出来的外源蛋白可保持相对独立的空间结构和生物活性,并利用其配体的特异性亲和力将所感兴趣的蛋白质或多肽筛选出来,然后通过序列测定获得相应的结构和功能信息。我们应用噬菌体随机12肽库筛选鼻咽癌患者血清,淘选出鼻咽癌患者表达的特异性肿瘤抗原,期望为鼻咽癌患者的诊断和预后提供新的标记物。 方法1.分别用正常人血清和鼻咽癌患者血清包被微孔板,通过减性筛选的方法,先将噬菌体与正常血清预吸附,然后将未结合的噬菌体再与鼻咽癌患者血清进行吸附,再把得到与鼻咽癌患者血清结合的噬菌体扩增。重复筛选三次。2.挑取单个噬菌斑,用筛选时的血清通过ELISA方法检测与鼻咽癌患者血清的结合情况,选择差异明显的噬菌体,扩大血清样本含量再进行检测,最终得到能够与鼻咽癌患者血清特异性结合的单个噬菌体克隆。3.提取并纯化单个噬菌体克隆的DNA,使用引物-96Ⅲ进行DNA测序,并根据短肽插入位点和氨基酸遗传密码表得到插入短肽序列。 结果1.经过三轮筛选,挑取单个噬菌体反复鉴定后得到两株特异性较好的噬菌体克隆;2.经测序获这两个短肽排列序列;3.这两株噬菌体克隆ELISA鉴定IgA-VCA阳性鼻咽癌血清及IgA-VCA阴性鼻咽癌血清无显著差异;4.这两株噬菌体克隆通过ELISA分别检测鼻咽癌血清、肝癌血清、卵巢癌血清结果显示其与鼻咽癌患者血清的结合具有特异性。 结论本实验通过噬菌体随机肽库筛选出能与鼻咽癌相关抗原特异性结合的短肽,为鼻咽癌早期诊断、联合检测以及免疫治疗提供了候选标记物。
[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，

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