海藻希瓦氏菌发酵产生河豚毒素的提取与检测
|
摘要
近年来,河豚毒素的微生物起源越来越得到人们的认可。海洋微生物发酵法产生河豚毒素,具有原料来源广、成本低,不受季节和原料的限制,不破坏生态平衡的特点,因此人们期望通过细菌发酵实现资源的有效利用和河豚毒素的大量生产。国外早在上世纪80年代就已经着手此项研究,并且取得了骄人的成绩。而我国在这方面的研究还很薄弱。
海藻希瓦氏菌最早于1985年由Yuichi Kotati等人从红藻(Jania sp.)表面分离出来,当时被命名为OK-1~T(T=type strain),研究发现OK-1~T与腐败希瓦氏菌有很多相似的生理、生化特征,而且可以产生河豚毒素。本论文目的在于从结构和生物学功能上证实,海藻希瓦氏菌发酵产生的TTX与河豚组织中的TTX是一致的,为TTX微生物起源假说奠定坚实的实验基础。优化海藻希瓦氏菌生产河豚毒素的发酵条件,细致研究海藻希瓦氏菌产生河豚毒素的分离纯化工作,为可持续生产TTX提供新思路。
本文对海藻希瓦氏菌生理生化指标进行了测定。通过测定菌体密度和菌体收获量,研究了部分初始条件及添加不同营养物质对海藻希瓦氏菌生长的影响,采用单因素试验和正交试验对海藻希瓦氏菌生产河豚毒素的发酵条件进行了优化。优化后的培养基最佳配方为:在2216E培养基中添加1.0%葡萄糖、2.5%酵母粉、1.0%磷酸高铁。统计分析,葡萄糖是影响海藻希瓦氏菌菌体收获量的主要因素。采用SCM杯式超滤系统对海藻希瓦氏菌发酵液的过滤除杂进行了研究,考察了操作压力对过滤效果的影响,同时还针对料液的预处理对超滤结果的影响进行了研究,主要措施是调节料液pH值,改变其温度,在其中添加混凝剂。结果表明,操作压力为0.2MP,通量达到最大,截留率也相对较大。料液的pH调至4,加热料液的温度至60℃都能使通量和截留率达到较满意的水平。研究发现在料液中添加50mg/L硫酸铝混凝剂能有效减缓超滤膜污染。本文还考查了分子筛去除分子量较小的杂质的效果,其中蛋白去除率50.8%,有机物去除率82.8%。考查了弱酸性阳离子交换树脂D_(152)对TTX的净化作用,结果显示,除色效果很好,糖类、氨基酸类也有很好的去除效果。本文利用薄层层析对发酵产物经分离得到的TTX进行初步定量,结果与小鼠生物检测法结果有良好的相关性。采用直接竞争抑制性酶联免疫吸附试剂盒对发酵产物进行了进一步的定量测定,并最终运用高效液相色谱法对海藻希瓦氏菌发酵产物中的TTX含量进行精确定性、定量。
Nowadays,people prefer to accept the theory that bacterial is the origin of tetrodotoxin.The method using marine bacteria producing TTX has lot of advantages, such as wide source,low cost,unlimited season and material,never destroying the ecosystem balance.It is supposed to use marine bacteria fermentation to realize the efficient making use of resource and to produce of adequate TTX.It is reported that foreign scientists have done some work on this research and achieve marked success. But it has few report in our country.
Shewanella alga was firstly separated from Jania sp.by Yuichi Kotati,named OK-1~T. Shewanella alga has similar biochemical characteristics with Shewanella putrefaciens. This bacteria can producing TTX.The purpose of this thesis is confirming the TTX origin from bacteria is same as the TTX origin from puffer by mouse assay and HPLC, supporting the postulation of bacteria origin of TTX.The other purpose of this thesis is optimizing the culture medium on Shewanella alga producing tetrodotoxin, optimizing the separation and purification method,offering the new idea for producing TTX.
At this experiment,morphological and biochemical characteristics of Shewanella alga was determined.Different culture media ingredient that have effects on the grow of Shewanella alga were studied by determined cells concentration and cells weight.The fermentation parameters was optimized by single factors experiment and orthogonal experiment.The optimum culture medium for Shewanella alga was in medium of glucose 1.0%,yeast powder 2.5%,phosphatic iron 1.0%.After statistic analysis, glucose was considered to be primary factor.Purification study of fermentation solution of Shewanella alga was carried out adopting SCM cup ultrofiltration system. To improve the ultrofiltration process by change operating pressure,adjust pH, change temperature,add coagulation for the solution.Results showed that 0.2MP operating pressure,pH 4,60℃,100mg/L Al_2(SO_4)_3·18H_2O coagulation can increase flux and removal of impurity effectively.Coagulation can ease membrane pollution. Using molecular sieve chromatography to remove small molecular impurity also studied in this thesis.The rate of removal of protein is 50.8%and the rate of removal of UV_(254) is 82.8%.The purification effect of D_(152) macroreticular cation exchange resin showed that D_(152) is good for getting rid of colour,sugar,amino acid.TLC and direct competitive inhibition enzyme-linked immtmosorbent assay was used to determine TTX quality of fermentation substance of Shewanella alga.The accuracy of TLC and ELISA was compared with the traditional mouse bioassay system.The results showed no significant differences between the two methods.Using HPLC to determine TTX quality and character.
海藻希瓦氏菌最早于1985年由Yuichi Kotati等人从红藻(Jania sp.)表面分离出来,当时被命名为OK-1~T(T=type strain),研究发现OK-1~T与腐败希瓦氏菌有很多相似的生理、生化特征,而且可以产生河豚毒素。本论文目的在于从结构和生物学功能上证实,海藻希瓦氏菌发酵产生的TTX与河豚组织中的TTX是一致的,为TTX微生物起源假说奠定坚实的实验基础。优化海藻希瓦氏菌生产河豚毒素的发酵条件,细致研究海藻希瓦氏菌产生河豚毒素的分离纯化工作,为可持续生产TTX提供新思路。
本文对海藻希瓦氏菌生理生化指标进行了测定。通过测定菌体密度和菌体收获量,研究了部分初始条件及添加不同营养物质对海藻希瓦氏菌生长的影响,采用单因素试验和正交试验对海藻希瓦氏菌生产河豚毒素的发酵条件进行了优化。优化后的培养基最佳配方为:在2216E培养基中添加1.0%葡萄糖、2.5%酵母粉、1.0%磷酸高铁。统计分析,葡萄糖是影响海藻希瓦氏菌菌体收获量的主要因素。采用SCM杯式超滤系统对海藻希瓦氏菌发酵液的过滤除杂进行了研究,考察了操作压力对过滤效果的影响,同时还针对料液的预处理对超滤结果的影响进行了研究,主要措施是调节料液pH值,改变其温度,在其中添加混凝剂。结果表明,操作压力为0.2MP,通量达到最大,截留率也相对较大。料液的pH调至4,加热料液的温度至60℃都能使通量和截留率达到较满意的水平。研究发现在料液中添加50mg/L硫酸铝混凝剂能有效减缓超滤膜污染。本文还考查了分子筛去除分子量较小的杂质的效果,其中蛋白去除率50.8%,有机物去除率82.8%。考查了弱酸性阳离子交换树脂D_(152)对TTX的净化作用,结果显示,除色效果很好,糖类、氨基酸类也有很好的去除效果。本文利用薄层层析对发酵产物经分离得到的TTX进行初步定量,结果与小鼠生物检测法结果有良好的相关性。采用直接竞争抑制性酶联免疫吸附试剂盒对发酵产物进行了进一步的定量测定,并最终运用高效液相色谱法对海藻希瓦氏菌发酵产物中的TTX含量进行精确定性、定量。
Nowadays,people prefer to accept the theory that bacterial is the origin of tetrodotoxin.The method using marine bacteria producing TTX has lot of advantages, such as wide source,low cost,unlimited season and material,never destroying the ecosystem balance.It is supposed to use marine bacteria fermentation to realize the efficient making use of resource and to produce of adequate TTX.It is reported that foreign scientists have done some work on this research and achieve marked success. But it has few report in our country.
Shewanella alga was firstly separated from Jania sp.by Yuichi Kotati,named OK-1~T. Shewanella alga has similar biochemical characteristics with Shewanella putrefaciens. This bacteria can producing TTX.The purpose of this thesis is confirming the TTX origin from bacteria is same as the TTX origin from puffer by mouse assay and HPLC, supporting the postulation of bacteria origin of TTX.The other purpose of this thesis is optimizing the culture medium on Shewanella alga producing tetrodotoxin, optimizing the separation and purification method,offering the new idea for producing TTX.
At this experiment,morphological and biochemical characteristics of Shewanella alga was determined.Different culture media ingredient that have effects on the grow of Shewanella alga were studied by determined cells concentration and cells weight.The fermentation parameters was optimized by single factors experiment and orthogonal experiment.The optimum culture medium for Shewanella alga was in medium of glucose 1.0%,yeast powder 2.5%,phosphatic iron 1.0%.After statistic analysis, glucose was considered to be primary factor.Purification study of fermentation solution of Shewanella alga was carried out adopting SCM cup ultrofiltration system. To improve the ultrofiltration process by change operating pressure,adjust pH, change temperature,add coagulation for the solution.Results showed that 0.2MP operating pressure,pH 4,60℃,100mg/L Al_2(SO_4)_3·18H_2O coagulation can increase flux and removal of impurity effectively.Coagulation can ease membrane pollution. Using molecular sieve chromatography to remove small molecular impurity also studied in this thesis.The rate of removal of protein is 50.8%and the rate of removal of UV_(254) is 82.8%.The purification effect of D_(152) macroreticular cation exchange resin showed that D_(152) is good for getting rid of colour,sugar,amino acid.TLC and direct competitive inhibition enzyme-linked immtmosorbent assay was used to determine TTX quality of fermentation substance of Shewanella alga.The accuracy of TLC and ELISA was compared with the traditional mouse bioassay system.The results showed no significant differences between the two methods.Using HPLC to determine TTX quality and character.
引文
[1]Ragelis E P.J Assoc OFF Chem.1982,65:327-331.
[2]于瑞莲,王琴.河豚毒素的生物合成[J].中国海洋药物,2002,4:40-42.
[3]李晓川,林美娇.河豚鱼及其加工利用[M].北京:中国农业出版社,1998,3194.
[4]张永贵,刘树威,张宇辉,等.河豚毒素(TTX)解毒药的研究[J].脑与神经疾病杂志,1996,4(2):105.
[5]Kao C.Tetrodotoxin,saxitoxin and their significance in the study of excitation phenomena[J].Pharmacol Rev.1996,18:997-1104.
[6]Narahashi T..Chemicals as tools in study of exictable membrances[J].Physiol Rev.1974,54:813-889.
[7]Liu Z,Dai J,Chen Z,Hu W,Xiao Y,Liang S.Isolation and characterization of hainantoxin-Ⅳ,a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider Selenocosmia hainana[J].Cell Mol Life Sci.2003,60:972-978.
[8]Xiao Y C,Liang S P,Purification and characterization of Hainantoxin-Ⅴ,a tetrodotoxin-sensitive sodium channel inhibitor from the venom of spider Selenocosmia hainan[J].Toxin.2003,41:643-650.
[9]赖仞,查宏光,张云.动物离子通道毒素与药物开发[J].动物学研究,2000,21(6):499-506.
[10]梦飞.剧毒的河豚毒素竟有神奇的止痛功效[EB/OL].[2003-月-日].网址?
[11]陈冀胜,潘心富.河豚毒素临床应用简介[J].海洋药物,1986,2:31.
[12]Jim Johnson.Tetrodotoxin-An ancient alkaloid from the sea.2002.
[13]Kotaki.Y,Y.Oshima,T.Yasumoto.Bacterial transformation of paealytic shellfish toxin in coral reef crabs and a marine snail[J].Nippon Suisan Gakkaishi.1985,51:1009-1013.
[14]Yotsu M,Yamazaki T,Meguro Y,et al.Production of tetrodotoxin and its derivatives by Pseudomonas sp,isolated from the skin of pufferfish[J].Toxicon,1987,25(2):225-228.
[15]Usio simidu,Kumiko kita-tsulamoto,Takeshi yasumoto,Mari yotso.Taxonomy of four marine bacterial strains that produce tetrodotoxin[J].International journal of systematic bacteriology,1990,331-336.
[16]杨平,林怡芳.海洋生物毒素研究研究现状[J].国外医学卫生学册,1997,24:2.
[17]Noguchi D,F.Hwang,Vibrio alginolyticus,a tetrodotoxing producing bacterium from puffer[J].Mar.Biol.1987,94:635.
[18]Noguchi T,Jeon J K,Arakawa O,et al.Occurrence of tetrodotoxin and anhydrotetrodotoxin in Vibrio sp.isolated from the intestines of a Xanthid Crab,Ateratisfloridus[J].Biochem.1986,99:311-314.
[19]Narita H,Matsubara S,Miwa N et al.Vibrio alginolyticus,a TTX-producing bacterium isolated from the Starfish Astropecten polyacanthus[J].Nippon Suisan G akkalshi,1987,4:617-621.
[20]Kungsuwan A,Noguchi T,Arakawa O et al.Tetrodotoxin-producing bacteria from the Horseshoe Crab Carcinoscorpius rotundicauda[J].Nippon Sulsan Gakkalshl.1988,10:1799-1802.
[21]Noguchi T,Hwang D F,Arakawa O et al.Vibrio alginolyticus,a tetrodotoxin producing bacterium,in the intestines of the fish Fugu vermicularis vermicularis[J].Marine Biology.1987,94:625-630.
[22]Matsui T,Taketsugu S,Kodama K et al.Production oftetrodotoxin by the intestinal bacteria of a puffer fish Takifugu niphobles[J].Nippon Suisan Gakkalshl.1989,12:2199-2203.
[23]Cheng C A,Hwang D F,Tsai Y H et al.Microflora and Tetrodotoxin- producing bacteria in a Gastropod,Niotha clathrata[J].Fd.Chem.Toxic.1995,33:929-934.
[24]Stuart C,McEvoy E G,Gibson R.The Production of tetrodotoxin like substances by nemertean worms in conjunction with bacteria[J].J Experim Marine Bio Ecol.2003,288:51-63.
[25]Hamasaki K,Ohwada K et al.Presence of tetrodotoxin and tetrodotoxin producing bacteria in freshwater sediments[J].Appl EnviroMicrobiol.1993,99:3934-3937.
[26]Simidu U.Marine bacteria which produce tetrodotoxin[J].Appl.Environ.Microbiol,1987,53:1714.
[27]Do H K,Kogure K,Imada C et al.Tetrodotoxin production of actinomycetes isolated from marine sediment[J].Appl Bacteriol.1991,70:464-468.
[28]Simidu U,Tsukamoto K,Yasumoto T et al.Taxonomy of four marine bacterial strains that produce tetrodotoxin[J].Intern J System Bacteriol.1990,40(4):331-336.
[29]武振龙.产河豚毒素菌株RG-386 RG-33B的分离纯化及其发酵产物中河豚毒素的监测[D].北京:中国农业大学博士学位论文,2005.
[30]Harry S.Mosher and Frederick A.Fuhrman.Occurence and Origin of Tetrodotoxin[J].Seafood toxins.1984,339.
[31]Matsui T,Sato H,Hamada S,Shimizu C.Bull.Japan Soc.Sci.Fish.1982,48:253.
[32]Narita H,Noguchi T,Maruyama J,et al.Occurrence of tetrodotoxin in a trumpet shell," boshubora" Charonia saulinae[J].Bull Jpn Soe Sei Fish,1981,47:935-941.
[33]李秋芬,徐怀恕.河豚毒素及微生物起源[J].海洋通报,1994,13(4).
[34]Kishi Y et al.Amer Chem Soc,1972,94:9219.
[35]Asai M,Nishikawa T,Ohyabu Net al.Stereocontrolled sysnthesis of(-)-5,1-dideoxytetrodotoxin[J].Tetrahedron.2001,57:4543-4558.
[36]Akai S,Nakamura Y,Kajihara Y et al.Crystal structure of(1R,2R,3S,4S,5S,6S,7R,9S)-6-azido-7-cyano-3-C-hydroxymethyl-3,3'-di-O-isopropelydene-9-methoxy-2,4,5-tris(O-methoxy methy 1)-2,3,4,5-tetrahydroxy-8-oxabicyclo[4.3.0]nonane,toward tetrodotoxin.Analytical Sciences.1999,15:501-502.
[37]Akai S,Sugita N,Yoshimura J et al.DL-(1,2,3',4,5,6',/3,6)-6-Asido-5'-O-t-butyldiphenylsilyl-6-(cyano)hydroxymethyl-3,5-di-C-hydroxymethyl-1,2:3,3'-di-O-isopropylidene-4-O-methoxymethyl-1,2,3,4-cyclohexanetetrol,toward(+)- tetrodotoxin[J].Analytical Sciences.1999,15:503-504.
[38]Chun-Fai Yu,Peter Hoi-Fu Yu,Pui-Ling Chan,Qun Yan,Po-Keung Wong.Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes[J].Toxicon.2004,44:641-647.
[39]范延辉,胡江春,马成新,等.渤海红鳍东方豚体内产毒细菌的微生态分布及B3B菌株的生物学特性[J].应用生态学报,2005,16(10):1952-1955.
[40]Wallace R W.Drugs form the sea:harvesting the results of aeon chemical evolution[J].Mol med Today.1997,3(7):291-295.
[41]Fleming L E,Stinn J.Shellfish Poisonings[J].Travel Medicine.1999,3:1-6.
[42]胡卓炎,何松,孙福在,等.黄单胞冰核活性细菌发酵制备的初步研究[J].食品与发酵工业,2001,27(3):5-8.
[43]Gallacher S,Birkbeck T H.Effect of phosphate concentration on production of Tetrodotoxin by Alteromonas tetraodonis[J].Appl environ microbial,1993,11:3981-983.
[44]曲音波,林建强,肖敏.微生物技术开发原理[M].北京:化学工业出版社,2005:102-106.
[45]张青,葛菁萍.微生物学[M].北京:科学技术出版社,2004.
[46]陈启和,何国庆,邬应龙.弹性蛋白酶产生菌的筛选及其发酵条件的初步研究[J].浙江大学学报(农业与生命科学版),2003,29(1):59-64.
[47]钱森和,厉如玉,汤斌,等.紫色非硫光合细菌培养基条件优化的研究[J].安徽工程科技学院学报,2007,22(2):4-8.
[48]吴韶菊.海藻希瓦氏菌产河豚毒素的初步研究[D].青岛:中国海洋大学,2005:14.
[49]Nozue H,Hayashi T,Hashimoto Y et al.Isolation and characterization of Shewanella alga from human clinical specimens and emendation of the description of S.alga Simidu et al.,1990,335[J].Intern J System Bacteriol,1992,628 -634.
[50]李寅,高海军,陈坚.高细胞密度发酵技术[M].北京:化学工业出版社,2005:40.
[51]赵峰梅,赵春贵,赵邑,等.光合细菌ZY2159菌株发酵生产类胡萝卜素研究[J].食品科学,2007,28(6):209-212.
[52]杨博,黄蓉,曾晶,等.BA-25菌株碱性纤维素酶产酶条件优化研究[J].生命科学研究,2006,10(3):92-98
[53]余彬彬,边才苗,吕珈佳.1株四氢呋喃降解细菌基本培养条件的研究[J].化学工程师,2006,5:7-9.
[54]孙军德,赵春燕,黄晓星,等.光合细菌菌数测定数学模型的构建[J].沈阳农业大学学报,2001,32(5):358-359.
[55]杨广.液体培养条件下细菌浓度两种测定方法比较[J].微生物学杂志,2005,25(4):71-72.
[56]董秉直,陈艳,高乃云.有机物的相对分子质量对膜过滤通量的影响[J].同济大学学报,2006,34(12):1643-1645.
[57]高以恒.膜分离技术基础[M].北京:科学出版社,1989.16.
[58]王晓昌,王锦.混凝-超滤去除腐殖酸的试验研究[J].中国给水排水,2002,(3):18-22.
[59]Park Pyung-ju,Lee Chun-hak,Choi San-June,et al.Effect of removal of DOMs on the performance of a coagulation-UF membrane system for drinking water production[J].Desalination,2002,145(1-3):237-245.
[60]赵振业,肖贤明,李丽,等.水体中不同相对分子质量有机质对饮用水消毒的影响[J].环境科学,2002,23(6):45-50.
[61]Ryuji Tachikawa,Kiyoshi Sakai.潘新富等译.从河豚鱼卵巢中分离提取河豚毒素(TTX)[J].中国海洋药物,1982,4:56.
[62]崔建洲,宫庆礼,顾谦群,等.一种高效制备河豚毒素(TTX)方法的研究[J].高技术通讯,2005,15(4):89-94
[63]李霞,王广军.河纯毒素(TTX)的分离、纯化及应用前景[J].水产科技情报,2002,29(4):153-156.
[64]曾漪青.河豚毒素的提取方法[J].科学养鱼,2004,5:64.
[65]郭慧清,李锦文,黎主政,等.联合采用吸附树脂与离子交换树脂提取河豚毒素[J].广州师院学报(自然科学版),1997,2:60-64.
[66]辛剑,孟长功.基础化学实验[M].北京:高等教育出版社,2004:394.
[67]张金艳,王德利.大学化学实验[M].北京:中国农业大学出版社,2004:312.
[68]朱宝泉.生物制药技术[M].北京:化学工业出版社,2004:354-355.
[69]陈瑛,钟俊辉,陶文沂.离子交换法提取茶氨酸的研究[J].氨基酸和生物资源,1998,20(1):31.
[70]周增冉.色谱分析[M].北京:纺织工业出版社,1993:185-186.
[71]林乐明,敖丽娟,张军.河豚毒素的薄层色谱扫描法测定[J].色谱,1991,9(2):113-115.
[72]沈晓书,顾明松,谢剑炜.河豚毒素分析检测方法研究进展[J].军事医学科学院院刊, 2006,30(3):295-298.
[73]岛田和子.食品卫生学杂志[M].1985,26:507-510.
[74]厚生省环境卫生局编.食品卫生检查指针Ⅱ[M].日本食品卫生协会,1978:232-240.
[75]Huang DF,Jeng SS.Chin Biochen Soc,1991,20:80-86.
[76]苏文,薛际深,李维刚.河豚毒素的检测分析[J].防化研究,1992,1:54-64.
[77]D.D.Sheumack,M.E.H.Howden Science,1978:199.
[78]Ikebuchi,J.,Suenagea,K.,Kotoku,s.,Inagaki,O.,Thin-layer chromatography ionization detection for the determination of tetrodotoxin in biological fluids.Chromatopropahy,1988,432:401-406.
[79]杨春,苏秀榕,李太武等.低毒河豚鱼毒素的提取和检测[J].天然产物研究与开发,2003,15(5):398-400.
[80]Chu,F.,Fan,T.,Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish.J Assoc Off Anal Chem 1985,68,13-16.
[81]Watabe,S.,Sato,Y.,Nakaya,M.,Hashimono,K.,Enimoto,A.,Kaminogawa,S.,Yamauchi,K.,Mono clonal antibody raised against tetrodonic acid,a derivative of tetrodotoxin.Toxicon,1989,27,265-268.
[82]Huot,R.I.Armatrong,D.L.,Chanh,T.C.,Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin.J Clin Invest,1989,83,1821-1826.
[83]Raybould,T.J.,Bignami,G..S.,Inouye,L.K.,Simpson,S.B.,Bymen,J.B.,Grothaus,P.G.,Vann,D.C.,A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples.J Clin Lab Anal 1992,6,65-72.
[84]Kawatsu,K.,Hamano,Y.,Yoda,T.,Terano,Y.,Shibata,T.,Rapid and highly sensitive enzyme immuassay for quantitative determination of tetrodotoxin.Jpn Med Sci Biol 1997,50,133-150.
[85]Kawatsu,K.Shibata,T.,Harnano,Y.,Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.Toxicon 1999,37,325-333.
[86]王健伟,罗雪云,计融,等.间接竞争抑制性酶联免疫吸附试验测定豚毒鱼类中河豚毒素的研究.卫生研究,1997,26(2):106-109
[87]计融,王健伟,罗雪云,等.鲀毒鱼类中河豚毒素直接竞争抑制性酶联免疫吸附试验测定方法的研究.中国食品卫生杂志,2002,14(5):7-10
[88]Kao CY.Tetrodotoxin,saxitoxin and their significance in the study of excitat in phenomenta.PharmacolRev,1966,18:997
[89]王晶,王林,黄晓蓉.食品安全快速检测技术[M].北京:化学工业出版社,2002:51.
[90]朱正美,刘辉.简明免疫学技术[M].北京:科学出版社,2002:165.
[91]郭玉莲.农药残留的酶联免疫分析技术及研究进展[J].农机化研究,2006,(2):201-203.
[92]王运浩.食晶农药残留与分析控制技术展望[J].现代科学仪器,2003,87(1):8-12.
[93]苗小飞,宋杰军,高静波.河豚毒素单克隆抗体的制备及特性[J].第二军医大学学报,1993,14:540-544.
[94]Yasumoto T,Nakamura M,Oshima Y,et al.Construction of a continuous tetrodoxin analyzer[J].Bull Japan Soc Fish,1982,48(10):1481-1483.
[95]Nakayama T,Terkawa S.A rapid purification procedure for tetrodotoxin derivatives by high performance liquid chromatography[J].Analytical Biochemistry,1982,126:153-155.
[96]Nagashima Y,Maruyama J,Noguchi T,et al.Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high performance liquid chromatography[J].Nippon Suisan Gakkaishi,1987,53:818-823.
[97]Yotsu M, Endo A, Yasumoto T. An improved tetrodotoxin analyzer[J]. Agric Biol Chem,1988,53(3):893-895.
[98] Onoue Y, Noguchi T, Nagashima Y, et al. Separation of tetrodotoxin and paralytic shellfish poisons by high performance liquid chromatography with a fluorometric detection using o-phthalaldehyde[J]. J Chromatography,1983,257:373-376.
[99] Matugama J, Noguchi T, Matsunaga S, et al. Fast atom bombardment and seconday ion-mass spectrometry of paralytic shellfish poisons and tetrodotoxin[J]. Agric Biol Chem, 1984, 48(11): 2783-2788.
[2]于瑞莲,王琴.河豚毒素的生物合成[J].中国海洋药物,2002,4:40-42.
[3]李晓川,林美娇.河豚鱼及其加工利用[M].北京:中国农业出版社,1998,3194.
[4]张永贵,刘树威,张宇辉,等.河豚毒素(TTX)解毒药的研究[J].脑与神经疾病杂志,1996,4(2):105.
[5]Kao C.Tetrodotoxin,saxitoxin and their significance in the study of excitation phenomena[J].Pharmacol Rev.1996,18:997-1104.
[6]Narahashi T..Chemicals as tools in study of exictable membrances[J].Physiol Rev.1974,54:813-889.
[7]Liu Z,Dai J,Chen Z,Hu W,Xiao Y,Liang S.Isolation and characterization of hainantoxin-Ⅳ,a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider Selenocosmia hainana[J].Cell Mol Life Sci.2003,60:972-978.
[8]Xiao Y C,Liang S P,Purification and characterization of Hainantoxin-Ⅴ,a tetrodotoxin-sensitive sodium channel inhibitor from the venom of spider Selenocosmia hainan[J].Toxin.2003,41:643-650.
[9]赖仞,查宏光,张云.动物离子通道毒素与药物开发[J].动物学研究,2000,21(6):499-506.
[10]梦飞.剧毒的河豚毒素竟有神奇的止痛功效[EB/OL].[2003-月-日].网址?
[11]陈冀胜,潘心富.河豚毒素临床应用简介[J].海洋药物,1986,2:31.
[12]Jim Johnson.Tetrodotoxin-An ancient alkaloid from the sea.2002.
[13]Kotaki.Y,Y.Oshima,T.Yasumoto.Bacterial transformation of paealytic shellfish toxin in coral reef crabs and a marine snail[J].Nippon Suisan Gakkaishi.1985,51:1009-1013.
[14]Yotsu M,Yamazaki T,Meguro Y,et al.Production of tetrodotoxin and its derivatives by Pseudomonas sp,isolated from the skin of pufferfish[J].Toxicon,1987,25(2):225-228.
[15]Usio simidu,Kumiko kita-tsulamoto,Takeshi yasumoto,Mari yotso.Taxonomy of four marine bacterial strains that produce tetrodotoxin[J].International journal of systematic bacteriology,1990,331-336.
[16]杨平,林怡芳.海洋生物毒素研究研究现状[J].国外医学卫生学册,1997,24:2.
[17]Noguchi D,F.Hwang,Vibrio alginolyticus,a tetrodotoxing producing bacterium from puffer[J].Mar.Biol.1987,94:635.
[18]Noguchi T,Jeon J K,Arakawa O,et al.Occurrence of tetrodotoxin and anhydrotetrodotoxin in Vibrio sp.isolated from the intestines of a Xanthid Crab,Ateratisfloridus[J].Biochem.1986,99:311-314.
[19]Narita H,Matsubara S,Miwa N et al.Vibrio alginolyticus,a TTX-producing bacterium isolated from the Starfish Astropecten polyacanthus[J].Nippon Suisan G akkalshi,1987,4:617-621.
[20]Kungsuwan A,Noguchi T,Arakawa O et al.Tetrodotoxin-producing bacteria from the Horseshoe Crab Carcinoscorpius rotundicauda[J].Nippon Sulsan Gakkalshl.1988,10:1799-1802.
[21]Noguchi T,Hwang D F,Arakawa O et al.Vibrio alginolyticus,a tetrodotoxin producing bacterium,in the intestines of the fish Fugu vermicularis vermicularis[J].Marine Biology.1987,94:625-630.
[22]Matsui T,Taketsugu S,Kodama K et al.Production oftetrodotoxin by the intestinal bacteria of a puffer fish Takifugu niphobles[J].Nippon Suisan Gakkalshl.1989,12:2199-2203.
[23]Cheng C A,Hwang D F,Tsai Y H et al.Microflora and Tetrodotoxin- producing bacteria in a Gastropod,Niotha clathrata[J].Fd.Chem.Toxic.1995,33:929-934.
[24]Stuart C,McEvoy E G,Gibson R.The Production of tetrodotoxin like substances by nemertean worms in conjunction with bacteria[J].J Experim Marine Bio Ecol.2003,288:51-63.
[25]Hamasaki K,Ohwada K et al.Presence of tetrodotoxin and tetrodotoxin producing bacteria in freshwater sediments[J].Appl EnviroMicrobiol.1993,99:3934-3937.
[26]Simidu U.Marine bacteria which produce tetrodotoxin[J].Appl.Environ.Microbiol,1987,53:1714.
[27]Do H K,Kogure K,Imada C et al.Tetrodotoxin production of actinomycetes isolated from marine sediment[J].Appl Bacteriol.1991,70:464-468.
[28]Simidu U,Tsukamoto K,Yasumoto T et al.Taxonomy of four marine bacterial strains that produce tetrodotoxin[J].Intern J System Bacteriol.1990,40(4):331-336.
[29]武振龙.产河豚毒素菌株RG-386 RG-33B的分离纯化及其发酵产物中河豚毒素的监测[D].北京:中国农业大学博士学位论文,2005.
[30]Harry S.Mosher and Frederick A.Fuhrman.Occurence and Origin of Tetrodotoxin[J].Seafood toxins.1984,339.
[31]Matsui T,Sato H,Hamada S,Shimizu C.Bull.Japan Soc.Sci.Fish.1982,48:253.
[32]Narita H,Noguchi T,Maruyama J,et al.Occurrence of tetrodotoxin in a trumpet shell," boshubora" Charonia saulinae[J].Bull Jpn Soe Sei Fish,1981,47:935-941.
[33]李秋芬,徐怀恕.河豚毒素及微生物起源[J].海洋通报,1994,13(4).
[34]Kishi Y et al.Amer Chem Soc,1972,94:9219.
[35]Asai M,Nishikawa T,Ohyabu Net al.Stereocontrolled sysnthesis of(-)-5,1-dideoxytetrodotoxin[J].Tetrahedron.2001,57:4543-4558.
[36]Akai S,Nakamura Y,Kajihara Y et al.Crystal structure of(1R,2R,3S,4S,5S,6S,7R,9S)-6-azido-7-cyano-3-C-hydroxymethyl-3,3'-di-O-isopropelydene-9-methoxy-2,4,5-tris(O-methoxy methy 1)-2,3,4,5-tetrahydroxy-8-oxabicyclo[4.3.0]nonane,toward tetrodotoxin.Analytical Sciences.1999,15:501-502.
[37]Akai S,Sugita N,Yoshimura J et al.DL-(1,2,3',4,5,6',/3,6)-6-Asido-5'-O-t-butyldiphenylsilyl-6-(cyano)hydroxymethyl-3,5-di-C-hydroxymethyl-1,2:3,3'-di-O-isopropylidene-4-O-methoxymethyl-1,2,3,4-cyclohexanetetrol,toward(+)- tetrodotoxin[J].Analytical Sciences.1999,15:503-504.
[38]Chun-Fai Yu,Peter Hoi-Fu Yu,Pui-Ling Chan,Qun Yan,Po-Keung Wong.Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes[J].Toxicon.2004,44:641-647.
[39]范延辉,胡江春,马成新,等.渤海红鳍东方豚体内产毒细菌的微生态分布及B3B菌株的生物学特性[J].应用生态学报,2005,16(10):1952-1955.
[40]Wallace R W.Drugs form the sea:harvesting the results of aeon chemical evolution[J].Mol med Today.1997,3(7):291-295.
[41]Fleming L E,Stinn J.Shellfish Poisonings[J].Travel Medicine.1999,3:1-6.
[42]胡卓炎,何松,孙福在,等.黄单胞冰核活性细菌发酵制备的初步研究[J].食品与发酵工业,2001,27(3):5-8.
[43]Gallacher S,Birkbeck T H.Effect of phosphate concentration on production of Tetrodotoxin by Alteromonas tetraodonis[J].Appl environ microbial,1993,11:3981-983.
[44]曲音波,林建强,肖敏.微生物技术开发原理[M].北京:化学工业出版社,2005:102-106.
[45]张青,葛菁萍.微生物学[M].北京:科学技术出版社,2004.
[46]陈启和,何国庆,邬应龙.弹性蛋白酶产生菌的筛选及其发酵条件的初步研究[J].浙江大学学报(农业与生命科学版),2003,29(1):59-64.
[47]钱森和,厉如玉,汤斌,等.紫色非硫光合细菌培养基条件优化的研究[J].安徽工程科技学院学报,2007,22(2):4-8.
[48]吴韶菊.海藻希瓦氏菌产河豚毒素的初步研究[D].青岛:中国海洋大学,2005:14.
[49]Nozue H,Hayashi T,Hashimoto Y et al.Isolation and characterization of Shewanella alga from human clinical specimens and emendation of the description of S.alga Simidu et al.,1990,335[J].Intern J System Bacteriol,1992,628 -634.
[50]李寅,高海军,陈坚.高细胞密度发酵技术[M].北京:化学工业出版社,2005:40.
[51]赵峰梅,赵春贵,赵邑,等.光合细菌ZY2159菌株发酵生产类胡萝卜素研究[J].食品科学,2007,28(6):209-212.
[52]杨博,黄蓉,曾晶,等.BA-25菌株碱性纤维素酶产酶条件优化研究[J].生命科学研究,2006,10(3):92-98
[53]余彬彬,边才苗,吕珈佳.1株四氢呋喃降解细菌基本培养条件的研究[J].化学工程师,2006,5:7-9.
[54]孙军德,赵春燕,黄晓星,等.光合细菌菌数测定数学模型的构建[J].沈阳农业大学学报,2001,32(5):358-359.
[55]杨广.液体培养条件下细菌浓度两种测定方法比较[J].微生物学杂志,2005,25(4):71-72.
[56]董秉直,陈艳,高乃云.有机物的相对分子质量对膜过滤通量的影响[J].同济大学学报,2006,34(12):1643-1645.
[57]高以恒.膜分离技术基础[M].北京:科学出版社,1989.16.
[58]王晓昌,王锦.混凝-超滤去除腐殖酸的试验研究[J].中国给水排水,2002,(3):18-22.
[59]Park Pyung-ju,Lee Chun-hak,Choi San-June,et al.Effect of removal of DOMs on the performance of a coagulation-UF membrane system for drinking water production[J].Desalination,2002,145(1-3):237-245.
[60]赵振业,肖贤明,李丽,等.水体中不同相对分子质量有机质对饮用水消毒的影响[J].环境科学,2002,23(6):45-50.
[61]Ryuji Tachikawa,Kiyoshi Sakai.潘新富等译.从河豚鱼卵巢中分离提取河豚毒素(TTX)[J].中国海洋药物,1982,4:56.
[62]崔建洲,宫庆礼,顾谦群,等.一种高效制备河豚毒素(TTX)方法的研究[J].高技术通讯,2005,15(4):89-94
[63]李霞,王广军.河纯毒素(TTX)的分离、纯化及应用前景[J].水产科技情报,2002,29(4):153-156.
[64]曾漪青.河豚毒素的提取方法[J].科学养鱼,2004,5:64.
[65]郭慧清,李锦文,黎主政,等.联合采用吸附树脂与离子交换树脂提取河豚毒素[J].广州师院学报(自然科学版),1997,2:60-64.
[66]辛剑,孟长功.基础化学实验[M].北京:高等教育出版社,2004:394.
[67]张金艳,王德利.大学化学实验[M].北京:中国农业大学出版社,2004:312.
[68]朱宝泉.生物制药技术[M].北京:化学工业出版社,2004:354-355.
[69]陈瑛,钟俊辉,陶文沂.离子交换法提取茶氨酸的研究[J].氨基酸和生物资源,1998,20(1):31.
[70]周增冉.色谱分析[M].北京:纺织工业出版社,1993:185-186.
[71]林乐明,敖丽娟,张军.河豚毒素的薄层色谱扫描法测定[J].色谱,1991,9(2):113-115.
[72]沈晓书,顾明松,谢剑炜.河豚毒素分析检测方法研究进展[J].军事医学科学院院刊, 2006,30(3):295-298.
[73]岛田和子.食品卫生学杂志[M].1985,26:507-510.
[74]厚生省环境卫生局编.食品卫生检查指针Ⅱ[M].日本食品卫生协会,1978:232-240.
[75]Huang DF,Jeng SS.Chin Biochen Soc,1991,20:80-86.
[76]苏文,薛际深,李维刚.河豚毒素的检测分析[J].防化研究,1992,1:54-64.
[77]D.D.Sheumack,M.E.H.Howden Science,1978:199.
[78]Ikebuchi,J.,Suenagea,K.,Kotoku,s.,Inagaki,O.,Thin-layer chromatography ionization detection for the determination of tetrodotoxin in biological fluids.Chromatopropahy,1988,432:401-406.
[79]杨春,苏秀榕,李太武等.低毒河豚鱼毒素的提取和检测[J].天然产物研究与开发,2003,15(5):398-400.
[80]Chu,F.,Fan,T.,Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish.J Assoc Off Anal Chem 1985,68,13-16.
[81]Watabe,S.,Sato,Y.,Nakaya,M.,Hashimono,K.,Enimoto,A.,Kaminogawa,S.,Yamauchi,K.,Mono clonal antibody raised against tetrodonic acid,a derivative of tetrodotoxin.Toxicon,1989,27,265-268.
[82]Huot,R.I.Armatrong,D.L.,Chanh,T.C.,Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin.J Clin Invest,1989,83,1821-1826.
[83]Raybould,T.J.,Bignami,G..S.,Inouye,L.K.,Simpson,S.B.,Bymen,J.B.,Grothaus,P.G.,Vann,D.C.,A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples.J Clin Lab Anal 1992,6,65-72.
[84]Kawatsu,K.,Hamano,Y.,Yoda,T.,Terano,Y.,Shibata,T.,Rapid and highly sensitive enzyme immuassay for quantitative determination of tetrodotoxin.Jpn Med Sci Biol 1997,50,133-150.
[85]Kawatsu,K.Shibata,T.,Harnano,Y.,Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.Toxicon 1999,37,325-333.
[86]王健伟,罗雪云,计融,等.间接竞争抑制性酶联免疫吸附试验测定豚毒鱼类中河豚毒素的研究.卫生研究,1997,26(2):106-109
[87]计融,王健伟,罗雪云,等.鲀毒鱼类中河豚毒素直接竞争抑制性酶联免疫吸附试验测定方法的研究.中国食品卫生杂志,2002,14(5):7-10
[88]Kao CY.Tetrodotoxin,saxitoxin and their significance in the study of excitat in phenomenta.PharmacolRev,1966,18:997
[89]王晶,王林,黄晓蓉.食品安全快速检测技术[M].北京:化学工业出版社,2002:51.
[90]朱正美,刘辉.简明免疫学技术[M].北京:科学出版社,2002:165.
[91]郭玉莲.农药残留的酶联免疫分析技术及研究进展[J].农机化研究,2006,(2):201-203.
[92]王运浩.食晶农药残留与分析控制技术展望[J].现代科学仪器,2003,87(1):8-12.
[93]苗小飞,宋杰军,高静波.河豚毒素单克隆抗体的制备及特性[J].第二军医大学学报,1993,14:540-544.
[94]Yasumoto T,Nakamura M,Oshima Y,et al.Construction of a continuous tetrodoxin analyzer[J].Bull Japan Soc Fish,1982,48(10):1481-1483.
[95]Nakayama T,Terkawa S.A rapid purification procedure for tetrodotoxin derivatives by high performance liquid chromatography[J].Analytical Biochemistry,1982,126:153-155.
[96]Nagashima Y,Maruyama J,Noguchi T,et al.Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high performance liquid chromatography[J].Nippon Suisan Gakkaishi,1987,53:818-823.
[97]Yotsu M, Endo A, Yasumoto T. An improved tetrodotoxin analyzer[J]. Agric Biol Chem,1988,53(3):893-895.
[98] Onoue Y, Noguchi T, Nagashima Y, et al. Separation of tetrodotoxin and paralytic shellfish poisons by high performance liquid chromatography with a fluorometric detection using o-phthalaldehyde[J]. J Chromatography,1983,257:373-376.
[99] Matugama J, Noguchi T, Matsunaga S, et al. Fast atom bombardment and seconday ion-mass spectrometry of paralytic shellfish poisons and tetrodotoxin[J]. Agric Biol Chem, 1984, 48(11): 2783-2788.