产河豚毒素菌株的筛选、鉴定及透析培养  

Screening,identification and dialysis culture of a tetrodotoxin-producing strain

作　　者：张宏宇 林涛 武斌 李增鹏 郭文斌 陈明谅 ZHANG Hongyu;LIN Tao;WU Bin;LI Zengpeng;GUO Wenbin;CHEN Mingliang(Third Institute of Oceanography,MNR,Xiamen 361005,China;Hebei Steiner Biotechnology Co.,Ltd.,Shijiazhuang 050011,China)

机构地区：[1]自然资源部第三海洋研究所,福建厦门361005 [2]河北施坦纳生物科技有限公司,河北石家庄050011

出　　处：《应用海洋学学报》2023年第2期210-217,共8页Journal of Applied Oceanography

基　　金：自然资源部第三海洋研究所基本科研业务费专项经费(海三科2018016,2019031);福建省杰出青年科学基金(F180304)。

摘　　要：河豚毒素(Tetrodotoxin, TTX)在镇静、镇痛、麻醉、戒毒等神经性疾病治疗方面效果显著且无致瘾性,因此具有极好的应用前景,但由于其从河豚体内提取的成本过高,所以难以大量推广应用。越来越多的证据表明TTX来源于河豚体内共生的微生物。我们从野生横纹东方鲀(Takifugu oblongus)肝脏中分离得到了一株产TTX较高的芽孢杆菌3G2(Bacillus sp. 3G2),通过ELISA和胶体金的方法检测到其发酵液TTX含量为35 ng/mL。将该菌株接种于专用透析培养器中,不断流加新鲜培养基实现连续培养,同时实现简化分离工艺,对发酵液进行小鼠生物活性检测、TTX试纸条检测,并通过河豚毒素抗体免疫共沉淀纯化得到TTX粗品后进行高效液相色谱鉴定均表明发酵液中含有TTX且含量较高。本研究通过筛选产TTX的优质菌株进一步了证实河豚毒素外源性假说,并初步验证了透析法生产河豚毒素的优越性,为后续商业应用提供实践性基础。Tetrodotoxin(TTX)has an excellent application prospect in the treatment of neuropathic diseases such as sedation,analgesia,anesthesia and detoxification without causing addiction.However,due to its high cost of extraction from puffer fish,it is difficult to be widely applied.There is increasingly evidence that TTX originates from its symbiotic microbes.A strain of TTX-producing Bacillus sp.3G2 was isolated from the liver of wild puffer fish(Takifugu oblongus),and 35 ng/mL TTX in fermentation broth was detected by ELISA and colloidal gold assay.Then,Bacillus sp.3G2 was inoculated in a special dialysis incubator,and continuous culture was achieved by continuous flow and fresh culture medium.The mice biological activity and tetrodotoxin test strips were used to detect TTX in the fermentation broth.The crude TTX was purified by TTX antibody immunoprecipitation and further identified by high performance liquid chromatography(HPLC).Our study further corroborated the exogenous hypothesis of tetrodotoxin by means of screening high-quality strains from puffer fish,and preliminarily verify the superiority of dialysis production of tetrodotoxin.Therefor it provided a practical basis for subsequent commercial application.

关 键 词：海洋生物学 河豚毒素 芽孢杆菌 ELISA检测 透析培养 HPLC 

分 类 号：P735[天文地球—海洋生物学]