Identification of Novel Genes Expressed in Bowel Cancer Using Change Mediated Ant

描述（由申请人提供）：体内诱导抗原技术（IVIAT）已经被证明是一种敏感、快速、廉价的方法，用于鉴定在实际感染过程中特异性表达的致病菌的新基因。然而，IVIAT的使用仅限于病原体感染能够产生抗体反应的宿主的疾病分析。在本应用中，我们描述了一种称为变化介导抗原技术（CMAT）的IVIAT的修饰，该技术可以识别在感染过程中特异性表达的病原体和宿主基因。利用油菜黄单胞菌侵染大豆植株完成了原理论证。一般来说，CMAT有可能识别任何细胞在经历任何变化时所表达的任何基因。我们打算扩大CMAT的应用范围，将其作为研究结直肠癌肿瘤发生过程中特异性表达基因的工具。发现的基因将有可能作为研究该病治疗效果的优秀生物标志物，并为新的诊断和疫苗策略提供新的靶点。有3个具体目标。在Specific Aim 1中，将在噬菌体T7中构建人类基因组DNA和结肠癌组织样本cDNA噬菌体展示文库。将获得足够的独立克隆，以确保这些库的完全覆盖。在特异性目标2中，CMAT IgY探针将通过使用来自受试者的结直肠癌组织样本免疫鸡来创建。对免疫原产生的抗体将从鸡蛋中纯化，并用同一受试者的健康组织制成的裂解物吸附。在Specific Aim 3中，CMAT IgY探针将用于对T7文库进行生物筛选，以丰富表达结直肠癌细胞产生的非健康细胞产生的蛋白质的克隆。采用Western blotting的验证步骤将用于消除假阳性。将对经过验证的克隆中的克隆插入体进行测序，并对结果进行基因组和蛋白质组学分析，以生成在结直肠癌的不同阶段由转化细胞表达而不是由健康细胞表达的基因及其蛋白质列表。该清单将为II期工作提供起始材料，这将需要筛选表达蛋白，以确定其作为结直肠癌诊断生物标志物的潜力，并作为早期诊断和疫苗策略的可能靶点。变化介导抗原技术（CMAT）是一种鉴定细胞发生变化时表达的基因的新方法。本项目将使用CMAT来鉴定健康细胞不表达的结直肠癌细胞表达的蛋白质。这些蛋白质可能在监测治疗、诊断和预防这种疾病方面很有用。

DESCRIPTION (provided by applicant): In Vivo Induced Antigen Technology (IVIAT) has been well documented as a sensitive, fast, and inexpensive method for identifying novel genes of pathogenic bacteria that are specifically expressed during an actual infectious process. However, the use of IVIAT is limited to analysis of diseases where the pathogen infects a host that is capable of mounting an antibody response. In this application, we describe a modification of IVIAT called Change Mediated Antigen Technology (CMAT) that allows identification of both pathogen and host genes specifically expressed during infection. Proof of principle has been accomplished using Xanthomonas campestris infection of bean plants. In general, CMAT is potentially capable of identifying any gene that is expressed by any cell when it undergoes any sort of change. We intend to expand the application of CMAT to use it as a tool to study genes that are specifically expressed during oncogenesis in colorectal cancer. Genes that are discovered will potentially serve as excellent biomarkers for studying the efficacy of therapy of this disease and provide novel targets for new diagnostic and vaccine strategies. There are 3 Specific Aims. In Specific Aim 1, phage display libraries of human genomic DNA and cDNA from colorectal cancer tissue samples of subjects will be constructed in bacteriophage T7. Sufficient independent clones will be obtained to assure complete coverage of these libraries. In Specific Aim 2, a CMAT IgY probe will be created by immunizing chickens with colorectal cancer tissue samples from subjects. Antibodies produced in response to the immunogens will be purified from eggs and adsorbed with lysates made from healthy tissue of the same subjects. In Specific Aim 3, the CMAT IgY probe will be used to biopan the T7 libraries to enrich for clones expressing proteins made by colorectal cancer cells that are not made by healthy cells. A verification step employing Western blotting will be used to eliminate false positives. The cloned inserts in verified clones will be sequenced and the results subjected to genomic and proteomic analyses to generate a list of genes and their proteins that are expressed by transformed cells during different stages of colorectal cancer and not by healthy cells. This list will provide the starting material for the Phase II work, which will entail screening of the expressed proteins for their potential to serve as biomarkers in diagnosis of colorectal cancer and as possible targets for early diagnosis and vaccine strategies. Change Mediated Antigen Technology (CMAT) is a new method for identifying genes that are expressed when a cell undergoes a change. This project will use CMAT to identify proteins expressed by colorectal cancer cells that are not expressed by healthy cells. Such proteins are likely to be useful in monitoring treatment, diagnosing this disease, and in preventing it.