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

PSP毒素是由有毒的鞭毛藻如tamarense Alexandrium产生的毒素。相反，我们认为毒素来源于鞭毛藻的胞内细菌，基于A. tamarense的胞内细菌产生石蜡毒素，尽管产量很小。本文提出了细菌产生毒素的前体在鞭毛藻中转化为毒素的假设，并在此假设的基础上，试图寻找细菌的前体物质。首先是针对蛤蚌毒素的特异性抗体，利用毒素与硫醇的反应，与抗原制备针对蛤蚌毒素的特异性抗体，检测具有蛤蚌毒素结构配体的物质。用该抗体对细菌蛋白进行Western blot分析，发现抗体染色的蛋白带表明这些蛋白具有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质片段，得到消化后的蛋白质片段。ELISA检测结果显示，该抗体水解产物中的低分子片段与抗体发生反应。对elisa阳性物质的分离试验表明，由于在水解过程中有许多杂质污染，该物质难以分离。另一方面，当使用抗体进行免疫沉淀时，发现该物质仅在一步中被有效分离。分离该物质用于结构鉴定的工作正在进行中。

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.