Study on metabolism and biosynthesis of paralytic shellfish toxins using toxim-producing bacteria

PSP毒素是由有毒的甲藻如塔玛亚历山大藻产生的毒素。根据甲藻胞内细菌对甲藻毒素的作用，推测甲藻毒素可能来源于胞内细菌。tamarense产生石房蛤毒素，尽管产量很小。本文提出了细菌产生毒素前体物质的假说，并在此基础上尝试寻找细菌中的毒素前体物质。首先，通过利用毒素和硫醇之间的反应，用抗原开发抗石房蛤毒素的特异性抗体，检测具有石房蛤毒素结构的配体的物质。用该抗体对细菌蛋白进行Western印迹分析，显示抗体染色的蛋白条带，表明这些蛋白具有蛤蚌毒素结构的配体。然后，用非特异性蛋白酶消化细菌蛋白级分以获得消化的蛋白片段。酶联免疫吸附试验表明，酶解液中的小分子片段与抗体发生反应。分离ELISA阳性物质的试验表明，由于水解物中污染的许多杂质的污染，难以分离该物质。另一方面，当应用使用抗体的免疫沉淀时，发现仅在一个步骤中有效地分离该物质。正在分离该物质以进行结构鉴定。

PSP toxins are toxins produced by toxic dinoflagellates such as Alexandrium tamarense. On the contrary, we suggested that toxins originate from endocellular bacteria of dinoflagellates, based on the facts that endocellular bacteria of A. tamarense produce saxitoxins, though the productivity is small. We propose here the hypothesis that bacteria produce precursor of toxins which is changed to toxins in dinoflagellates, Based on the hypothesis, we tried to find the precursor substance in bacterial. First of all, the specific antibody against saxitoxins which detects substance(s) possessing ligand with saxitoxin structure, by development of specific antibody against saxitoxin with the antigen utilizing the reaction between toxins and thiols. Western blot analysis of bacterial proteins by the antibody showed the protein bands stained by the antibody, indicating that these proteins possess ligands with saxitoxin structure. Then, the bacterial protein fraction was digested with nonspecific protease to obtain the digested protein fragments. ELISA using the antibody showed that low molecular fragment in the hydrolyzate reacts with the antibody. Trial to isolate the ELISA-positive substance showed the difficulty to isolate the substance, because of contamination of many impurities contaminated in the hydrolyzed. On the other hand, the substance was found to be effectively isolated, just in one step, when immunoprecipitation using the antibody was applied. Isolation of the substance for structure identification is on the way.