Development of DNA vaccines for fish

利用hirma横纹病毒（hirv）、病毒性出血性败血症（VHS）病毒的g蛋白基因、红鲷虹膜病毒的主要衣壳蛋白基因和附着蛋白基因、鱼氏巴氏杆菌的三个不同的主要抗原蛋白基因和分枝杆菌的抗原85基因构建了鱼DNA疫苗，这些基因的表达在病毒启动子的控制下在鱼细胞中表达。我们还利用牙鲆白细胞介素-1基因构建了一种DNA佐剂。编码KERRY g蛋白基因的DNA疫苗接种1 ~ 2 g比目鱼，接种量为1|jg，对HERRV感染具有较高的保护作用。编码casid蛋白主基因或附着蛋白基因的DNA疫苗对红鲷虹膜病毒也有保护作用。而虹膜病毒DNA疫苗的保护活性不高，RPS值约为50%。为了构建全鱼DNA疫苗载体，我们克隆并鉴定了一些日本比目鱼基因启动子。我们克隆了牙鲆补体成分C3、角蛋白、β-肌动蛋白、明胶酶和绿色荧光（GFP）基因上游的TNF-ct基因启动子。我们构建了转基因斑马鱼来表征启动子的活性。启动子活性分析表明，角蛋白基因启动子最适合用于DNA疫苗，因为它的启动子活性在整个斑马鱼体内都可以检测到。我们开发了从日本比目鱼的防御和免疫相关基因中提取rna的定量技术。

We have constructed fish DNA vaccines using G-protein genes of hirame rhabdovirus (HIRRV), viral haemorrhagic septicemia (VHS) virus, major capsid protein gene and attachment protein gene of red seabream iridovirus, three different major antigenic protein genes of Pasteurdla piscidda, and Antigen85 genes of Mycobacterium sp. The expression of these genes was controlled under a virus promoter for expression in fish cells. We also constructed a DNA adjuvant using Japanese flounder interleukin-1 gene.The DNA vaccine which encoded the G-protein gene of KERRY had high protective activity against HERRV infection when this DNA vaccine was injected over 1|jg to 1 to 2 g Japanese flounder. The DNA vaccines which encoded the major casid protein gene or attachment protein gene also have protective activity against red seabream iridovirus. However, in case of iridovirus DNA vaccine, the protective activities were not so high and the RPS values were about 50 %.We have cloned and characterized some Japanese flounder gene promoters for the construction of an all fish DNA vaccine vector. We cloned the Japanese flounder complement component C3, keratin, β-actin, gelatinase and TNF-ct gene promoters on up stream of the green fluorescent (GFP) gene. We constructed transgenic zebrafish for characterization of the promoter activities. The promoter activity analysis suggest that the keratin gene promoter was most suitable for DNA vaccine because its promoter activity was detected through out the body of zebrafish.We developed techniques for quantification of rnRNA from a number of defense and immune-related gene of Japanese flounder.