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）：体内诱导抗原技术（IVIAT）已被充分证明是一种灵敏、快速和廉价的方法，用于鉴定在实际感染过程中特异性表达的病原菌新基因。然而，IVIAT的使用仅限于分析病原体感染能够产生抗体反应的宿主的疾病。在本申请中，我们描述了一种名为变化介导抗原技术（CMAT）的IVIAT修改，该技术可以识别感染期间特异性表达的病原体和宿主基因。原理的证明已经使用菜豆植物的野油菜黄单胞菌感染完成。一般来说，CMAT可能能够识别任何细胞在经历任何变化时表达的任何基因。我们打算扩大CMAT的应用，将其作为研究结直肠癌发生过程中特异性表达基因的工具。所发现的基因将潜在地作为研究该疾病治疗效果的优良生物标志物，并为新的诊断和疫苗策略提供新的靶点。有三个具体目标。在具体目标1中，将在噬菌体T7中构建来自受试者的结肠直肠癌组织样品的人基因组DNA和cDNA的噬菌体展示文库。将获得足够的独立克隆，以确保完全覆盖这些文库。在特定目标2中，将通过用来自受试者的结直肠癌组织样品免疫鸡来产生CMAT IgY探针。将从卵中纯化响应于免疫原产生的抗体，并用从相同受试者的健康组织制备的裂解物吸附。在具体目标3中，CMAT IgY探针将用于生物淘选T7文库，以富集表达由结直肠癌细胞产生的蛋白质的克隆，而不是由健康细胞产生的。采用Western印迹法的验证步骤将用于消除假阳性。将对经验证的克隆中的克隆插入物进行测序，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期间由转化细胞而不是由健康细胞表达的基因及其蛋白质的列表。该列表将为II期工作提供起始材料，这将需要筛选表达的蛋白质，以确定其作为诊断结直肠癌的生物标志物以及作为早期诊断和疫苗策略的可能靶点的潜力。变化介导的抗原技术（CMAT）是一种新的方法，用于识别细胞发生变化时表达的基因。该项目将使用CMAT来鉴定结直肠癌细胞表达的蛋白质，而健康细胞不表达这些蛋白质。这些蛋白质可能有助于监测治疗，诊断这种疾病，并预防它。