ANALYSIS OF HUMAN CYTOMEGALOVIRUSES

该项目的总体目标是指定人类 巨细胞病毒（CMV）特异性基因功能，并表达DMV特异性 原核和真核系统中的抗原， 诊断抗原和CMV亚单位病毒疫苗， 控制巨细胞病毒感染。 长期目标是 通过以下具体方法实现。 (1)继续 构建了L重复序列、S重复序列和S重复序列的限制性内切酶酶切图谱 DMV基因组的重复序列和L-S连接片段，并寻找和 描述CMV基因组中可能的推定“a”和“c”序列， 核酸杂交和DNA测序技术。 可能的 假定的“c”序列中的DNA复制起点将在 酵母系统和人成纤维细胞中进行自主复制 顺序 (2)为了绘制编码病毒诱导的DNA聚合酶的基因， 核糖核苷还原酶、病毒特异性结构多肽（包括 94 K、150 K和其他糖多肽等）使用各种表达 E.大肠杆菌（pDR 540、pDR 720、pVE-J 001和Lambdagt 11）中， 哺乳动物细胞（例如COS-1细胞中的pSVOH）和各种可获得的单克隆 在我们实验室产生的抗体。 (3)完成 产生的单克隆抗体的分类和表征 针对纯化的病毒和感染的细胞裂解物进行基因克隆， 诊断试剂的开发。 (4)表达CMV特异性 E.大肠杆菌中使用表达载体， 携带强tac和trp启动子（例如pDR 540、pDR 720和pVEJ 1001），和 糖基化（也包括非糖基化）多肽在酵母中的表达 携带强醇脱氢酶启动子的载体（pAAH-5和 pMA56）。 最后，（5）负责病毒多肽的作用。 主要体液免疫应答的诱导将通过Western blot确定。 印迹、免疫沉淀和SDS-PAGE。 的抗原决定簇 将通过蛋白酶切和Western blot进一步研究 免疫反应 编码这些多肽的DNA片段将被 工程和克隆研究的可行性， 亚单位病毒疫苗

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.