【作者】 吴保平；

【导师】 周殿元； 张亚历；

【作者基本信息】 第一军医大学 ， 消化系病， 2001， 博士

【摘要】 背景、目的和意义 大肠癌（colorectal cancer，CRC）是严重威胁人类生命健康的常见恶性肿瘤之一。目前其已成为西方国家居第二位的高发肿瘤，在我国上海也已上升为第四位的常见癌症。业已表明，大肠癌的预后与早期诊断和治疗密切相关。由于大肠癌的发生发展是一种多基因、多因素、多步骤的复杂过程，目前尚未发现其相当特异的分子病理改变和肿瘤标志物，在其早期诊治研究中普遍面临的问题是缺乏特异、准确、简便的生物学指标和实用方法。自70年代Kohler和Milstein利用细胞融合技术成功制备了杂交瘤单克隆抗体（McAb），其在疾病诊断、治疗、预防和蛋白质提纯等方面显示出了重要作用。然而在体内应用，由于①其为异源性蛋白，易引起人抗鼠抗体（human anti-mouse antibody，HAMA）反应；②其分子量大、组织穿透力较弱，造成组织（尤其是肿瘤组织）对抗体特异性摄取有限。因而影响其疗效。人源性杂交瘤单抗的研制在技术上难以克服融合率低、建株难、不稳定、产量低、人体不能随意免疫等问题。随着基因工程技术及抗体分子遗传学的深入研究，对现有鼠单抗基因进行改造以减少抗体中的鼠源成分，又尽量保留原有的抗体特异性，从而诞生出新型抗体-基因工程抗体。但由于目前大部分基因工程抗体仍是基于小鼠杂交瘤，鼠源蛋白的问题仍未彻底解决。 建立在PCR技术和噬菌体表面呈现（phage display）技术上的噬菌体抗体库技术，从抗体角度为上述难题的解决提供了契机。抗体库技术用细菌克隆取代B细胞克隆表达抗体，不经细胞融合，甚至不经免疫，制备针对任何抗原的抗体分子。其在特异抗体的筛选和制备上独具优势。自90年代初噬菌体抗体库（phage antibody library）技术出现以来，该技术使得人们从应用DNA重组技术改造现有的单抗发展到利用基因工程技术克隆新的抗体分子。在感染性疾病的诊治、自身免疫性疾病与病毒性疾病的鉴别、肿瘤的影像分析和导向治疗或基因治疗，抗原表位分析以指导疫苗分子的设计、新型药物设计、蛋白分子免疫识别机制的研究等多个领域显示出巨大的潜能和广阔的应用前景。作为功能性小分子抗体片段，Fab包括重链VH-CH1（Fd段）和完整的轻链，两者通过一个链间二硫键连接，是完整抗体的三分之一。Fab具有与抗体相近的抗原结合活性，因其分子量小、穿透性强、抗原性低，可在原核系统表达以及易于基因工程操作等优点而受到人们的重视。 目前所知的与大肠癌相关的抗体不仅有限，而且特异性不高。寻找高特异。高亲和性的人肠癌抗体为学者所关注。本课题采用噬菌体抗体库这一前沿生物学技术，在匡呐外首次以大肠癌病人转移淋巳结中的淋巳细胞为源，构建了人大肠癌自然致敏抗体Fab段噬菌体呈现库。旨在从中筛选、富集抗大肠癌相关抗体，为大肠癌的特异性诊断和免疫治疗提供新的载体，并为进一步利用这些抗体筛选大肠癌相关基因及其功能研究提供库源。 材料与方法 1．RT．PCR扩增抗体重链（Fd）和轻链（。）基因片段：手术时剥离 3例大肠癌病人转移淋巴结（肠系膜）。提取组织总RNA。逆转录合成CDNA第一链，以～组不同家族抗体的前导肽和免疫球蛋白Fab段可变区N端第一骨架区厂）DNA序列的相对保守性而设计的5’端引物及按抗体恒定区基因序列而设计的3’端引物配对进行PCR，扩增Fd和K基因片段组合。 2．重链Fd PCR产物重组入噬粒载体pCOmb3：Fd PCR产物经分离纯化，与pComb3分别以适量Spe I／Tho双酶切后进行连接。用纯化的连接产物2011L转化 150 11L感受态 XLI七lllC细胞。以适量（10p，IHI，0．lpl）转化细菌菌液铺板，以测定转化率。挑取单克隆，提质粒后以 PCR和 Spe I／Tho双酶切鉴定是否有目的片段Fd基因的插入。带Fd插段的pComb3，命名为p＋Fd。 3．轻链。PCR产物重组入 p＋Fd及噬菌体抗体Dab）呈现库的构建：。PCR产物及 p＋Fd分别以 Sac ／ha双酶切后进行连接、转化、细菌扩增及转化子测定。挑取单克隆，提质粒后以 Sac ／Xba双酶切，鉴定有无轻链。的插入；经 Xho／Xba酶切鉴定有无 Fab的插入。得到的同时含 Fd和K的质粒，命名为Fd＋。。向上述Fab抗体基因库中加入辅噬菌体VCSM13，超感染后制备成 Fab噬菌体呈现原始库，分别为 K卜 K旷 KC，其等体积混合物为 NMIX。 4．筛选用抗原：将3例病人新鲜大肠癌组织和大肠癌LOV。细胞匀浆并行超声粉碎，离心后收取上清得到大肠癌抗原，分别为AgA，AgB，AgC和LOVO，其等体积混合物为AgMIX。 5．噬菌体Fab呈现原始库的鉴定：采用AP－抗人IgG系统，免疫点印迹法鉴定噬菌体抗体库Fab的呈现表达，以空载体pComb3转化XLlblue并经VCSM13超感染所得上清为对照；ELISA间接法检测噬菌体抗体库呈现Fab与大肠癌抗原的结合活性。 6．Fab噬菌体呈现库的淘选：以 AgMix包被酶标板，加入KMix。特异性 一5一 吸附的噬菌体洗脱后再感染XL lblue更多还原

【Abstract】 Construction and selection of the human naturally immunized Fab antibody phage display library from patients with colorectal cancer ABSTRACT BACKGROUND / OBJECTIVES Nearly 30 years, the occurrence of colorectal cancer (CRC) is rising, and it has becoming one of the most common malignant tumors which threaten the human health. At present, the incidence of CRC has been on the second status in weatern country, and on the fourth in Shanghai, China. It has been clear that the prognosis of CRC is related to early diagnosis and treatment. Since the development and evolution of the CRC is one kind of complicated procedures involving mult-genes, mult-factors and mult-steps, there have been no highly peculiar molecu-pathologic changs and tomor markers. Last decade, the technique of immunology and molecular biology has been extensively applied in diagnosis and treatment of tumor and promoted the study on CRC. In seventies, Kohler and Milstein prepared the monoclonal antibody (McAb) of hybrideoma by means of cell fusion and applied it into prevention, diagnosis and treatment of human diseases. But a major focus of cancer immunology is also on the isolation of antibodies that react selectively with human tumor cells without heterology to human because the antibodies could have important applications for targeting diagnostic and therapeutic agents to tumors and for identifying tumor antigens. The established approach has been to generate large panels of monoclonal antibodies from mice immunized with human tumor cells and to screen the antibodies for reactivity against the tumor. Despite the enormous effort expended on this approach, few antibodies that react preferentially with human tumors, and none that react specifically with one type of tumor (such as colorectal cancer), have been reported. Further attempts to isolate more specific and high affinity antibodies will require improved methods of generating and selecting antibodies against human tumors. One is the introduction of method for synthesizing virtually the entire repertoire of a person抯 antibody genes of variable regions by PCR technique and for expressing the encoded antibodies on the surface of a phage vector. Phage display technique, which has become an increasingly important tool in biotechnology, provides a new way for antibody development. The resulting phage antibody library can be panned to select and clone rare antibodies on the basis of their binding specificities. It can resolve the problems of generating human McAb by hybridoma approach, such as low coefficient of hybridoma cells, difficult establishment of the cells lines, instability and low output of McAb, and no freely immuning to human, et al. Human can be immunized to many antigens (including tumor antigens) but only local lymph nobles, a productive source of antibody producing cells, is readily available. As part of the antibody fragments, Fab is one third of whole antibody made of heavy chain VH-CH1 (Fd fragment) and integrated light chain(K or X). Fab fragment has the similar binding activity as the antibody and has some advantages of low molecule weight, weak heterology, strong infiltration and prokaryotic expression. In this project, by using the leading biotechnology of phage display antibody library, we constructed the naturally immunized phage display libraries expressing Fab antibodies derived from local c更多还原