ANALYSIS OF HUMAN CYTOMEGALOVIRUSES

该项目的总体目标是指定人类 巨细胞病毒(CMV)特异性基因的功能及表达 原核和真核系统中的抗原及其未来的发展 诊断抗原和CMV亚单位病毒疫苗，以预防 控制巨细胞病毒感染。长期目标将是 通过以下具体办法实现。(1)继续 构建L、S重复序列的详细限制性内切酶图谱 重复和L-S连接片段的DMV基因组，并搜索和 通过对CMV基因组中可能存在的“a”和“c”序列进行特征分析 核酸杂交和DNA测序技术。可能的 在假定的“c”序列中的DNA复制的起源将在 酵母系统及其在人成纤维细胞中的自主复制 序列。(2)定位病毒诱导的DNA聚合酶的编码基因， 核糖核苷还原酶，病毒特异性结构多肽(包括 94K、150K等糖多肽)使用各种表现力 大肠杆菌(pDR540、pDR720、PvE-J001、Lambdagt11和 哺乳动物细胞(例如COS-1细胞中的pSVOH)和各种可用的单抗 我们实验室产生的抗体。(3)完成 产生的单抗的分类和鉴定 抗纯化病毒和感染细胞裂解物用于基因克隆和 诊断试剂的发展。(4)表达CMV特异性 利用表达载体在大肠杆菌中表达非糖基化多肽 携带强TAC和Trp启动子(例如，pDR540、pDR720和pVEJ1001)，以及 糖基化(也非糖基化)多肽在酵母中的表达 携带强酒精脱氢酶启动子的载体(PAAH-5和 PMA56)。最后，(5)引起病毒感染的病毒多肽 主要体液免疫反应的诱导将由Western 印迹、免疫沉淀和SDS-PAGE。抗原性决定簇 将通过蛋白水解酶和蛋白质印迹进一步研究 免疫反应。编码这些多肽的DNA片段将是 进行基因工程和克隆以研究开发 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.