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. ? ? ?
