The overall objectives of this project are to designate human cytomegalovirus (CMV)-specific gene functions and to express DMV-specific antigens in prokaryotic and eukaryotic systems for future development of diagnostic antigens and the CMV subunit viral vaccine in order to prevent and control cytomegalovirus infection. The long term objectives will be achieved through the following specific approaches. (1) To continue to construct the detailed restriction enzymes cleavage map of L repeat, S repeat and L-S junction fragments of DMV genome, and to search for and characterize the possible putative """"""""a"""""""" and """"""""c"""""""" sequence in CMV genomes by nucleic acid hybridization and DNA sequencing techniques. The possible origin of DNA replication in putative """"""""c"""""""" sequence will be examined in yeast system and in human fibroblast for its autonomous replication sequence. (2) To map genes coded for the virus-induced DNA polymerase, ribonucleoside reductase, virus-specific structural polypeptides (including 94K, 150K and other glycopolypeptides, etc.) using various expressive vectors of E. coli (pDR540, pDR720, pVE-J001 and Lambdagt11 and of mammalian cell (e.g. pSVOH in COS-1 cell), and various available monoclonal antibodies generated in our laboratory. (3) To complete the classification and characerization of monoclonal antibodies generated against purified virus and infected cell lysate for gene cloning and the development of diagnostic reagent. (4) To express CMV specific non-glycosylated polypeptides in E. coli using expression vectors which carry strong tac and trp promoters (e.g. pDR540, pDR720 and pVEJ1001), and glycosylated (also non-glycosylated) polypeptides in yeast using expression vectors carrying a strong alcohol dehydrogenase promoter (pAAH-5 and pMA56). Finally, (5) the viral polypeptides which are responsible for the induction of a major humoral immune response will be determined by Western blotting, immunoprecipittion, and SDS-PAGE. The antigenic determinants will be studied further by proteolytic digestion and Western blot immunoreaction. The DNA fragments encoding these polypeptides will be engineered and cloned for studying the feasibility of developing the subunit viral vaccine.
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