Development of a novel method for evaluating immunostimulants in fish based on gene expression.

为了分析免疫刺激剂b-1，3-葡聚糖和海藻酸钠等增强鲤鱼生物防御的作用机制，采用抑制性消减杂交的方法对免疫刺激剂刺激其表达的基因进行了鉴定。结果共鉴定出约50个基因，包括哺乳动物中已知的急性时相蛋白，如五肽和血清淀粉样蛋白A，以及一些趋化因子、趋化因子受体、一个新的C型凝集素、一个新的补体因子B亚型、胶质细胞成熟因子、IL-1β和前B细胞增强因子。它们的完整一级结构也分别从鲤鱼白细胞cDNA文库中分离的全长cDNA克隆中确定。有趣的是，b-1，3-葡聚糖刺激了鲤鱼肾脏和脾中一种新的补体因子B亚型的表达，这表明因子B在多骨鱼类中具有新的作用。另一方面，海藻酸钠诱导了趋化因子和趋化因子受体的表达，这与腹腔注射海藻酸钠刺激白细胞从头肾向腹膜迁移的结果一致。目前的数据表明，每种免疫刺激剂都给出了不同的基因在白细胞中的表达模式，因此，对表达基因的鉴定为深入了解免疫刺激剂介导的硬骨鱼生物防御机制提供了重要的信息。本研究中的方法将有助于评估和筛选多骨鱼类中的免疫刺激剂。

To analyze mode of action of immunostimulants such as b-1,3-glucans and sodium alginate, which have been reported to enhance biodefence of carp (Cyprinus carpio), genes of which expression was stimulated by the immunostimulants were identified by means of the suppression subtractive hybridization. As a result, about 50 genes including acute phase protein known in mammals, such as pentraxin and serum amyloid A, as well as several chemokines, chemokine-receptors, a novel C-type lectin, a novel complement factor B isotypes, glia-maturation factor, IL-1 beta, and pre-B cell enhancing factor were identified. Their complete primary structures were also determined from respective full-length cDNA clones, which were isolated from a carp leukocyte cDNA library. Interestingly, the b-1,3-glucan stimulated expression of a novel complement factor B isotype in carp kidney and spleen, suggesting a new role of factor B in bony fish. On the other hand, sodium alginate induced expression of chemokines and chemokine receptors, in agreement with the results that peritoneal injection of sodium alginate stimulated the migration of leukocytes from head kidney to peritoneal cavity. The present data suggests that each immunostimulant gave distinct pattern of gene expression in leukocytes and therefore, identification of the expressed genes gives an important insight into the mechanism of immunostimulants-mediated biodefense in bony fish. The approach in the present study would be useful to evaluate and to screen immunostimulants in bony fish.