Development of cytotoxicity test for outbred animals using recombinant viruses.

细胞毒性T淋巴细胞(CTL)在抵抗多种病毒感染的免疫中发挥着重要作用。然而，由于缺乏针对家畜、同伴动物和实验动物(小鼠除外)的CTL检测方法，对这些动物的CTL功能的研究还很少。在这些动物系统中很难产生有用的靶细胞，因为靶细胞需要从感染病毒的同一动物身上获得，以匹配MHC和效应细胞，并且靶细胞在细胞表面表达病毒抗原，直到它们与效应细胞相遇，没有出现CPE。本项目的最终目的是利用改良腺病毒载体，开发和建立对任何动物都适用的新的细胞毒T细胞检测系统。为了开发新的检测系统，我们对每个受试者进行了如下实验并获得了最佳的实验条件：(1)腺病毒感染的宿主动物和细胞范围；(2)腺病毒载体中表达病毒抗原的最佳启动子；(3)被认为是细胞毒性靶标的病毒基因的克隆和鉴定；(4)携带病毒基因的重组腺病毒的构建。(5)重组腺病毒感染后每个动物的外周淋巴细胞产生靶细胞的条件。由于我们测试了几种病毒，所以并不是每个系统都能获得这些条件。但我们成功地构建了含有麻疹和流行性出血热病毒基因的重组腺病毒，并获得了从感染重组腺病毒的猴淋巴细胞中产生靶细胞的最佳条件。此外，我们还确定了腺病毒载体中最好的启动子，以及牛淋巴细胞上特有的MHC分子。这些结果为用腺病毒载体进行动物细胞毒性的基础和应用研究奠定了基础。

The cytotoxic T lymphocytes (CTL) have been considered to play an important role in immunity against the infections of many kinds of viruses. However, the function of CTL in animals including domestic, companion or experimental animals except mice were not well studied because of the lack of CTL assay methods for those animals. It was difficult to produce the useful target cells in those animal systems since target cells should be obtained from the same animal which is infected by viruses to match MHC with the effector cells, and since the target cells should express virus antigens on the cell surface without showing CPE until they meet the effector cells.The final aim of this project is to develop and establish the new cytotoxic T cell assay system which is useful for any kinds of animals by using improved adenovirus vectors. For developing the new assay system, we tested and obtained the optimal conditions in each subjects as follows.(1) Host animal and cell ranges of adenovirus infection.(2) The best promoters to express virus antigens in the adenovirus vector.(3) Cloning and characterizations of virus genes whose products are considered to be the target of cellular cytotoxicity.(4) Construction of recombinant adenoviruses with virus genes.(5) The conditions to produce the target cells from peripheral lymphocytes of each animals after the infection of recombinant adenoviruses.Since we tested several kinds of viruses, not all those conditions have been obtained in each system. But we succeeded to construct recombinant adenoviruses with measles and renderpest virus genes, and obtained the optimal conditions for producing the target cells from monkey lymphocytes infected with the recombinant adenoviruses. In addition, we identified the best promoters in adenovirus vectors, and the unique MHC molecules on bovine lymphocytes. These results are considered to contribute to the basic and applied studies for cellular cytotoxicity of animals using adenovirus vectors.