论文标题:产河豚毒素菌株RG-3B6、RG-33B的分离纯化及其发酵产物中河豚毒素的检测
Isolation and Characterization of Tetrodotoxin-producing Bacterial Stains RG-3B6 and RG-33B from the Puffer Fish Fugu Rubripes and Detection of Tetrodotoxin in the Fermentation Production
论文作者 武振龙
论文导师 夏国良；张荣庆,论文学位 博士,论文专业 生理学
论文单位 中国农业大学,点击次数 133,论文页数 92页File Size9885k
2005-06-01论文网 http://www.lw23.com/lunwen_2308177/ 河豚毒素;产河豚毒素菌;达松维尔拟诺卡放线菌;梭形芽孢杆菌;红鳍东方豚
Tetrodotoxin; Tetrodotoxin-producing bacteria; Fugu rubripes; Nocardiopsis dassonvillei; Bacillus fusiformis.
河豚毒素(tetrodotoxin,TTX)是一种剧毒的生物碱类天然神经毒素,最初是从豚科鱼的卵巢和肝脏中提取得到的。TTX为典型的Na+通道阻断剂,它能够与肌肉、神经细胞膜上钠离子通道受体结合,抑制动作电位的传导。TTX除用作神经生理学、肌肉生理学的工具药外,还具有相当强的镇痛作用,并极有可能成为新的局部麻醉药和戒除药物依赖性的药物,而且具有不成瘾的特点,这就使TTX的应用范围得到进一步扩展。目前市场上的TTX主要来源于河豚鱼的卵巢和肝脏组织,如果能经微生物发酵生产TTX,不仅可以满足市场的需求,而且对于保护河豚鱼免受因河豚毒素的开发而濒危灭绝有着重要的生态价值。 关于TTX的起源问题一直存在较多争议。从首次从河豚鱼体内分离到TTX以后的很长一段时间内,人们一直认为TTX是河豚鱼自身分泌产生的生物毒素,虽然进行了大量的组织学研究,但在河豚鱼体内并未找到分泌TTX的腺体和导管。研究发现,TTX还广泛分布于河豚鱼之外多种脊椎动物及无脊椎动物的体内或体表。TTX在与河豚鱼无亲缘关系和食物链关系的多种动物体内的发现,使得TTX的起源问题引起研究者的广泛兴趣与关注。尽管研究者已从多种海洋细菌的发酵产物中检测TTX及其类似物,但都没有真正获得微生物来源的TTX的结晶,因此无法从结构与功能上系统地研究和确证其与组织来源的TTX的完全一致性。已经证实,河豚鱼的卵巢和肝脏的毒性高于河豚鱼其它组织与器官,而这种毒性的差异是否与不同组织中的产TTX菌的分布及产TTX能力的不同有关,尚无文献报道。 本论文以采集于我国渤海的红鳍东方豚为研究对象,对其不同组织器官中的微生物进行分离纯化,并对其中两株产TTX的优良菌RG-33B、RG-3B6进行了种属鉴定,对其发酵产物中的TTX进行分离与纯化,并将生物检测与化学检测相结合,对微生物源TTX的结构和生物学功能进行了研究。结果表明:(1)河豚鱼的卵巢、肝脏、肠道中存在可产生TTX的细菌与放线菌,河豚鱼不同组织毒性的差异与其中产TTX菌株的产毒能力有关;(2)培养特征、生理生化特征、16S rDNA序列分析表明,RG-33B为达松维尔拟诺卡氏放线菌,RG-3B6为梭形芽孢杆菌,其中达松维尔拟诺卡氏菌发酵产生TTX的报道在国际上尚属首次;(3)获得微生物源的TTX,并从结构和生物学功能的角度证实,微生物发酵产生的TTX与河豚鱼组织中的TTX是一致的,这一结果为TTX的微生物起源假说提供了有力的实验依据。 总之,本论文首次对河豚鱼体内产TTX菌的分布、微生物源TTX的分离与纯化进行了研究,获得微生物源的TTX,并从结构和功能上证实其与组织来源的TTX相一致,为通过微生物发酵生产TTX提供了一种新思路,同时也为TTX的微生物起源假说奠定了坚实的实验基础。
Tetrodotoxin (TTX) is one of the most potent non-protein neurotoxins that blocks sodium channels and thus inhibits the propagation of action potentials. TTX was widely used as an important pharmacological reagent for neurobiological research. A recent study showed that TTX may be one of the most promising analgesic, detoxification agent and local anesthetic in the future which is currently under phase II clinic trials. All these will increase the market requirement for TTX. Conventionally, TTX is purified from ovaries and livers of puffer fish and the yield is limited. Isolation of TTX-producing bacteria will make it possible to produce large amount of TTX in vitro without destroying the puffer fish resource.The origin of TTX is one of the most controversial topics. Since TTX was originally discovered and isolated from certain species of fish belonging to the order of Tetraodontiforms, for a long time, puffer fish was thought to produce and accumulate TTX within their body. Lot of reserch was conducted but no gland structure was identified. Furthermore, TTX and its analogs were detected in several phyla of vertebrates and invertebrates. The wide distribution of TTX among genetically unrelated animals made the true origin of TTX one of the most interesting and controversial topics. TTX-producing bacteria have been reported to be isolated from different TTX-bearing animals but no bacterial production of TTX was achieved. It has been known for many years that the toxicity of the ovaries and livers of puffer fish is more potent than that of other organs, but whether the anatomical variation of toxicity is due to the different distribution of TTX-producing bacteria is not known.In the present study, three speciemen of the female puffer fish Fugu rubripes were collected from the Bohai Sea of China for the isolating and screening of the TTX-producing bacteria.Our results demonstrated that (1) the distribution of TTX-producing strains and the bacterial production of TTX is close related with the toxicity of each tissue in puffer fish; (2) based on morphological observations, physiological, biochemical characteristics and 16S rDNA alignment, the two TTX-producing strains were identified as Nocardiopsis dassonville and Bacillus Jusiformii, in our knowledge Nocardiopsis dassonville was the first time which was reported to produce TTX; (3) bacterial production of TTX was achieved and idendified by the chemical and biological methods. Our results supported the postulation of bacterial origin of TTX.In summary, the distribution and toxicity of TTX-producing bacteria in puffer fish Fugu rupries was investigated. Bacterial production of TTX was purified and identified by biological and instrument analysis. Our result will make it possible to produce TTX via fermentation and give solid ground on the postulation of bacterial origin of TTX. More research is needed to elucidate the mechanism of TTX synthesis and the role of TTX in bacteria.
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