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

使用变化介导的蚂蚁鉴定肠癌中表达的新基因

• 批准号: 7266598
• 负责人: Jeffrey D. Hillman
• 金额: $ 10万
• 依托单位: ORAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2007-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7266598
• 关键词: Antibodies; Antibody Formation; Antigens; Ants; Bacteriophage T7; Bacteriophages; Binding; Binding Proteins; Biological Assay; Biological Markers; Capsid; Capsid Proteins; Cells; Chickens; Chromatography; Colorectal Cancer; Complementary DNA; Complex; DNA; DNA Sequence; Development; Diagnosis; Diagnostic; Disease; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Excision; Facility Construction Funding Category; Freezing; Frequencies; Gene Proteins; Genes; Genomics; Heating; Human; Ice; IgY; Immune response; Immunoglobulin G; Infection; Intestinal Cancer; Lesion; Libraries; Liquid substance; Mediating; Metadata; Methods; Modification; Monitor; Monoclonal Antibodies; Mus; Nitrogen; Peptides; Personal Satisfaction; Phage Display; Phase; Phase I Clinical Trials; Plants; Polyethylene Glycols; Polymerase Chain Reaction; Polystyrenes; Preparation; Process; Proteins; Proteomics; Reading Frames; Ribi adjuvant; Sampling; Screening procedure; Serum; Sheep; Site; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Sorting - Cell Movement; Specimen; Staging; Technology; Time; Tissue Sample; Tissues; Treatment Efficacy; Ultracentrifugation; Vaccines; Western Blotting; Work; Xanthomonas campestris; bean; cDNA Library; cancer cell; cell transformation; cesium chloride; egg; genome database; in vivo; novel; novel diagnostics; novel therapeutics; particle; pathogen; pathogenic bacteria; prevent; response; therapeutic target; tool; tumorigenesis

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.

描述（由申请人提供）：体内诱导的抗原技术（IVIAT）已被充分记录为一种敏感，快速且廉价的方法，用于鉴定在实际感染过程中特异性表达的病原细菌的新型基因。但是，艾维亚特的使用仅限于分析病原体感染能够安装抗体反应的宿主的疾病。在此应用中，我们描述了一种称为变化介导的抗原技术（CMAT）的IVIAT修饰，该技术允许鉴定在感染过程中特异性表达的病原体和宿主基因。原理证明是使用豆类植物的xanthomonas campestris感染来实现的。通常，CMAT可能能够识别任何细胞经历任何变化时表达的任何基因。我们打算扩大CMAT的应用，以将其用作研究在结直肠癌肿瘤发生期间特异性表达的基因的工具。被发现的基因将有可能成为研究该疾病治疗功效的出色生物标志物，并为新的诊断和疫苗策略提供新的靶标。有3个具体目标。在特定的目标1中，将在噬菌体T7中构建人类基因组DNA和cDNA的噬菌体显示库。将获得足够的独立克隆，以确保这些库的完全覆盖。在特定的目标2中，将通过从受试者的结直肠癌组织样品中免疫鸡来产生CMAT IGY探针。响应免疫原子产生的抗体将从卵中纯化，并用由同一受试者的健康组织制成的裂解物吸附。在特定的目标3中，CMAT IGY探针将用于Biopan T7文库，以富集表达由不受健康细胞制造的结直肠癌细胞制成的蛋白质的克隆。采用蛋白质印迹的验证步骤将用于消除误报。将对经过验证的克隆中的克隆插入物进行测序，并经过基因组和蛋白质组学分析的结果，以生成在结直肠癌的不同阶段而不是由健康细胞中转化的细胞表达的基因及其蛋白质列表。该列表将为II期工作提供起始材料，该材料将需要筛选表达的蛋白质，因为它们有可能用作结直肠癌诊断的生物标志物，以及作为早期诊断和疫苗策略的可能目标。变化介导的抗原技术（CMAT）是一种新方法，用于识别细胞发生变化时表达的基因。该项目将使用CMAT识别由健康细胞未表达的结直肠癌细胞表达的蛋白质。这种蛋白质可能有助于监测治疗，诊断该疾病并预防。