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

接触反应是海鞘（Halocynthia roretzi）的一种同种异体细胞反应，被认为是脊椎动物的一种祖先免疫反应，并被认为是由Halocynthia Fu/Hc基因控制的。本课题制备了几种能阻断该反应的单克隆抗体（CRB 1、CRB 2和CRB 3）。它们都识别主要表达于盐蛙体腔液中非吞噬性体腔细胞表面的细胞表面抗原（CR 1、CR2和CR 3），经生化和免疫学分析表明，它们是糖蛋白，其抗原性高度依赖于分子上是否存在含N-糖苷键的碳水化合物。CR 1和CR2抗原也分别在卵母细胞的卵黄膜和卵泡细胞上表达。CR 3抗原在卵黄被和卵泡细胞上均有表达。有趣的是，CRB 1和CRB 2，而不是CRB 3，阻止了来自不同个体的Halocynthia的卵子和精子的某些组合的受精。由于控制Halocynthia卵母细胞自交不亲和性的分子位于卵黄膜上，因此CR 1抗原可能参与了体腔细胞的异基因反应和Halocynthia受精过程中的自交不亲和性机制。因此，CR 1抗原极有可能是控制体腔细胞异源性和受精过程中自交不亲和性的盐藻Fu/Hc基因的产物。克隆CR 1抗原对了解原索动物原始的异源识别系统向脊椎动物高度发达的免疫系统进化过程中可能发生的事件是必要的和可能的。

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.