Development of cytotoxicity test for outbred animals using recombinant viruses.

细胞毒性T淋巴细胞（CTL）在机体抗多种病毒感染的免疫中起重要作用。然而，由于缺乏针对这些动物的CTL检测方法，因此对除小鼠以外的家畜、伴侣动物和实验动物的CTL功能的研究还不够深入。在这些动物系统中很难产生有用的靶细胞，因为靶细胞必须从被病毒感染的同一动物获得，以使MHC与效应细胞匹配，由于靶细胞在接触效应细胞之前，应在细胞表面表达病毒抗原，而不显示CPE，因此本项目的最终目的是开发和建立新的细胞毒性T细胞检测系统，通过使用改进的腺病毒载体可用于任何种类的动物。为了开发新的分析系统，我们在每个主题中测试并获得了如下的最佳条件。(1)腺病毒感染的宿主动物和细胞范围。(2)在腺病毒载体中表达病毒抗原的最佳启动子。(3)其产物被认为是细胞毒性靶标的病毒基因的克隆和表征。(4)病毒基因重组腺病毒的构建。(5)重组腺病毒感染后，从每只动物的外周淋巴细胞中产生靶细胞的条件。由于我们测试了几种病毒，并非所有这些条件都在每个系统中获得。但我们成功地构建了携带麻疹病毒和renderpest病毒基因的重组腺病毒，并获得了重组腺病毒感染猴淋巴细胞产生靶细胞的最佳条件。此外，我们确定了腺病毒载体中最好的启动子，以及牛淋巴细胞上独特的MHC分子。这些结果有助于腺病毒载体对动物细胞毒性的基础和应用研究。

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.