Screening and gene analysis of cell surface molecules that involve in allogeneic recognition in solitary ascidian Halocynthia roretzi.

接触反应被认为是脊椎动物的祖先免疫反应，被认为是由盐生海鞘的FU/HC基因控制的一种异体细胞反应。在本项目中，我们制备了几种能阻断该反应的单抗(CRB1、CRB2和CRB3)。它们都识别细胞表面抗原(CR1、CR2和CR3)，这些抗原主要表达在盐碱鱼体腔液中的非吞噬体腔细胞上。生化和免疫学分析表明，这些抗原是糖蛋白，其抗原性高度依赖于分子上带有N-糖苷键的碳水化合物的存在。CR1和CR2抗原也分别存在于卵母细胞的卵黄膜和卵泡细胞上。卵黄被细胞和卵泡细胞上均有CR3抗原表达。有趣的是，CRB1和CRB2，而不是CRB3，阻止了来自不同盐生植物个体的某些卵和精子组合的受精。由于控制盐碱鱼卵母细胞自交不亲和性的分子位于卵黄膜上，CR1抗原可能既参与了体腔细胞的异基因反应，也参与了盐碱鱼受精过程中的自交不亲和性机制。因此，CR1抗原很可能是控制体腔细胞同种异体和受精过程中自交不亲和的梭梭FU/HC基因的产物。克隆CR1抗原是必要的，也是可能的，以了解脊椎动物原始同种识别系统向发达的脊椎动物免疫系统进化过程中可能发生的事件。

The contact reaction, an allogeneic cellular response in vitro in solitary ascidian Halocynthia roretzi, is regarded as an ancestral immunological reaction of vertebrates and has been believed to be controlled by Fu/Hc genes of Halocynthia. Here, in this project, we prcduced several monoclonal antibodies (CRB1, CRB2 and CRB3) that can block the reaction. They all recognize the cell surface antigens (named CR1, CR2 and CR3) that are expressing mainly on non-phagocytic coelomocytes in coelomic fluid of Halocynthia.Biochemical and immunological analyzes of the antigens revealed that they are glycoproteins and their antigenicity highly depend on the preesnce of carbohdrates with N-glycosidic linkage on the molecules. CR1 and CR2 antigens were demonstrated also on vitelline coat and follicle cells of oocytes, respectively. CR3 antgen was demonstrated on both vitelline coat and follicle cells. Interestingly, CRB1 and CRB2, but not CRB3, blocked the fertilization of certain combinations of eggs and sperms from different individuals of Halocynthia. Since molecules that control self-incompatibility in Halocynthia oocytes are known to be located on vitelline coat, CR1 antigen may involve both in allgeneic response by coelomic cells and the self-incompatibility mechanism in the fertilization in Halocynthia. Thus, it is highly possible that CR1 antigen is the product of the Fu/Hc gene of Halocyntia that controls allogenicity of coelomic cells and self-incompatibility in the fertilization. It is necessary and possible to clone the CR1 antigen to understand the possible events that may occurred during evolution of primitive alloreognition system in prochordates to hoghly developed immune system in vertebrates.