新玫瑰毒鲉毒素马血清抗体制备及抗毒活性研究
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摘要
目的建立毒鲉毒素(俗称石头鱼毒素)马血清抗体的制备方法并研究其抗毒活性,为毒鲉毒素中毒提供治疗手段。方法利用排阻色谱制备毒鲉毒素,免疫马匹制备毒鲉毒素马抗,再制备不含FC的F(ab')2并研究其对小鼠的抗毒活性。结果制备了新玫瑰毒鲉毒素(neoVTX)322.6 mg,纯度为88%,制备的马血清抗体原液中的蛋白含量为41 g/L,F(ab')2的含量为87.05%,IgG含量为1.35%。4 LD50毒鲉粗毒素可被2倍质量的马抗完全中和;3 LD50neoVTX可被3倍质量的马抗完全中和。结论成功制备了毒鲉毒素及其马血清抗体,该抗体具有较高的抗毒活性,为毒鲉毒素中毒提供了治疗手段。
Objective To establish a preparation method of Synanceia(stonefish)horse antibody against neoverrucotoxin(neoVTX)and study its antitoxic activity so as to find a therapy for Synanceia toxin poisoning.Methods NeoVTX was prepared by size exclusion chromatography. F(ab')2 antibody was prepared with horses immunized by Synanceia toxin,and its antitoxic activity was tested with mice.Results 322.6 mg of neoVTX was successfully prepared and the purity was 88%. The content of the horse antibody was 41 g/L.The purity of F(ab')2 was 87.05% and that of IgG was 1.35%.The crude venom of stonefish(4LD_(50))could be completely neutralized by the horse antibody(1∶2,w/w),so could neoVTX(3LD_(50))(1∶3,w/w).Conclusion NeoVTX and its horse antibody are successfully prepared. The antibody has high antitoxic activity,which provides a method for the treatment of stone fish toxin poisoning.
Objective To establish a preparation method of Synanceia(stonefish)horse antibody against neoverrucotoxin(neoVTX)and study its antitoxic activity so as to find a therapy for Synanceia toxin poisoning.Methods NeoVTX was prepared by size exclusion chromatography. F(ab')2 antibody was prepared with horses immunized by Synanceia toxin,and its antitoxic activity was tested with mice.Results 322.6 mg of neoVTX was successfully prepared and the purity was 88%. The content of the horse antibody was 41 g/L.The purity of F(ab')2 was 87.05% and that of IgG was 1.35%.The crude venom of stonefish(4LD_(50))could be completely neutralized by the horse antibody(1∶2,w/w),so could neoVTX(3LD_(50))(1∶3,w/w).Conclusion NeoVTX and its horse antibody are successfully prepared. The antibody has high antitoxic activity,which provides a method for the treatment of stone fish toxin poisoning.
引文
[1]Prentice O,Fernandez WG,Luyber TJ,et al.Stonefish envenomation[J].Am J Emerg Med,2008,26(8):972.e1-e2.
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[3]韩建华,叶志强,邢帮荣,等.17例石头鱼毒鳍刺伤临床分析[J].热带医学杂志,2008,8(7):688-689.
[4]Lee JY,Teoh LC,Leo SP.Stonefish envenomations of the hand--a local marine hazard:a series of 8 cases and review of the literature[J].Ann Acad Med Singapore,2004,33(4):515-520.
[5]Diaz JH.Marine Scorpaenidae envenomation in travelers:epidemiology,management,and prevention[J].J Travel Med,2015,22(4):251-258.
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[7]张龙霄,刘珠果,戴秋云.刺毒鱼毒素研究进展[J].生命的化学,2013,33(5):571-575.
[8]张龙霄.南海石头鱼毒素的分离纯化、毒性测定、分子克隆及原核表达研究[D].湖南衡阳:南华大学,2014.
[9]周志杰,刘株果,戴秋云.双抗体夹心ELISA法检测石头鱼毒素[J].军事医学,2016,40(6):525-534.
[10]国家药典委员会.《中华人民共和国药典》第3部[M].北京:中国医药科技出版社,2015:224-231.
[11]张龙霄,刘珠果,郑兴,等.中国南海石头鱼毒素NeoVTX的分离纯化、基因克隆及表达[J].生物技术通讯,2014,25(3):345-348.
[12]Church JE,Hodgson WC.Stonefish(Synanceia spp.)antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish(Gymnapistes marmoratus)venom[J].Toxicon,2001,39(2-3):319-324.
[13]Gomes HL,Menezes TN,Carnielli JB,et al.Stonefish antivenom neutralizes the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom[J].Toxicon,2011,57(7-8):992-999.
[14]Hoon Eng K.Bioactive proteins from stonefish venom[J].Clin Exp Pharmacol Physiol,2002,29(9):802-806.
[15]Ueda A,Suzuki M,Honma T,et al.Purification,properties and cDNA cloning of neoverrucotoxin(neoVTX),a hemolytic lethal factor from the stonefish Synanceia verrucosa venom[J].Biochim Biophys Acta,2006,1760(11):1713-1722.2018-10-29
[2]Hornbeak KB,Auerbach PS.Marine envenomation[J].Emerg Med Clin North Am,2017,35(2):321-337.
[3]韩建华,叶志强,邢帮荣,等.17例石头鱼毒鳍刺伤临床分析[J].热带医学杂志,2008,8(7):688-689.
[4]Lee JY,Teoh LC,Leo SP.Stonefish envenomations of the hand--a local marine hazard:a series of 8 cases and review of the literature[J].Ann Acad Med Singapore,2004,33(4):515-520.
[5]Diaz JH.Marine Scorpaenidae envenomation in travelers:epidemiology,management,and prevention[J].J Travel Med,2015,22(4):251-258.
[6]Garnier P,Goudey-Perrière F,Breton P,et al.Enzymatic properties of the stonefish(Synanceia verrucosa Bloch and Schneider,1801)venom and purification of a lethal,hypotensive and cytolytic factor[J].Toxicon,1995,33(2):143-155.
[7]张龙霄,刘珠果,戴秋云.刺毒鱼毒素研究进展[J].生命的化学,2013,33(5):571-575.
[8]张龙霄.南海石头鱼毒素的分离纯化、毒性测定、分子克隆及原核表达研究[D].湖南衡阳:南华大学,2014.
[9]周志杰,刘株果,戴秋云.双抗体夹心ELISA法检测石头鱼毒素[J].军事医学,2016,40(6):525-534.
[10]国家药典委员会.《中华人民共和国药典》第3部[M].北京:中国医药科技出版社,2015:224-231.
[11]张龙霄,刘珠果,郑兴,等.中国南海石头鱼毒素NeoVTX的分离纯化、基因克隆及表达[J].生物技术通讯,2014,25(3):345-348.
[12]Church JE,Hodgson WC.Stonefish(Synanceia spp.)antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish(Gymnapistes marmoratus)venom[J].Toxicon,2001,39(2-3):319-324.
[13]Gomes HL,Menezes TN,Carnielli JB,et al.Stonefish antivenom neutralizes the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom[J].Toxicon,2011,57(7-8):992-999.
[14]Hoon Eng K.Bioactive proteins from stonefish venom[J].Clin Exp Pharmacol Physiol,2002,29(9):802-806.
[15]Ueda A,Suzuki M,Honma T,et al.Purification,properties and cDNA cloning of neoverrucotoxin(neoVTX),a hemolytic lethal factor from the stonefish Synanceia verrucosa venom[J].Biochim Biophys Acta,2006,1760(11):1713-1722.2018-10-29