B型肉毒神经毒素的纯化及胰蛋白酶对其作用分析  

Purification of Botulinum Type B Neurotoxin and Effect of Trypsin on Botulinum Neurotoxin

作　　者：张雪平[1] 李小娟[1] 何星[1] 

机构地区：[1]兰州生物制品研究所毒素制剂室,兰州730046

出　　处：《中国生物制品学杂志》2010年第7期744-747,共4页Chinese Journal of Biologicals

摘　　要：目的纯化B型肉毒神经毒素,并比较未激活和用胰蛋白酶激活的B型肉毒神经毒素的分子特性,分析在酸性和碱性条件下B型肉毒毒素复合物的完整性。方法取未激活、胰蛋白酶激活和热处理激活的B型肉毒毒素复合物,采用阴离子交换柱层析纯化神经毒素(分别为A、B和C组)。在酸性条件下溶解激活的B型肉毒毒素复合物(D组)。对各组进行纯度(特异毒性)测定和SDS-PAGE分析。结果柱层析后,神经毒素纯度均比复合物的纯度高,激活的神经毒素比未激活的高2～3倍;在非还原和还原条件下,A组的SDS-PAGE显示神经毒素、重链和轻链3条带;B组显示重链和轻链2条带;C组在非还原条件下显示重链和轻链2条带,在还原条件下只显示轻链1条带。在酸性条件下,B型肉毒毒素复合物由神经毒素与非毒性成分构成,在碱性条件下,神经毒素与非毒性成分分离。结论已成功纯化了B型肉毒神经毒素,经胰蛋白酶处理后,单链裂解为双链,比活性提高。Objective To purify botulinum type B neurotoxin,compare the characteristics of inactive and trypsin-activated botulinum type B neurotoxin and analyze the integrities of botulinum type B neurotoxin complex under acidic and basic conditions.Methods Neurotoxins were purified from inactive,trypsin-activated and heat-activated botulinum type B complexes by anion-exchange column chromatography,and served as groups A,B and C respectively.The trypsin-activated botulinum type B neurotoxin was dissolved under acidic condition and served as group D.The botulinum type B neurotoxins in various groups were determined for purities（specific toxicities）and analyzed by SDS-PAGE.Results The purities of neurotoxins after chromatography were higher than those of complexes before chromatography.However,the purities of activated neurotoxins were 2 ~ 3 times higher than those of inactive ones.Both reduced and non-reduced SDS-PAGE profiles of neurotoxins in group A showed three bands,i.e.neurotoxin,heavy chain and light chain,while those in group B showed only two bands,i.e.heavy and light chains.The neurotoxin in group C showed two bands,i.e.heavy and light chains on non-reduced SDS-PAGE profile,while showed only one band of light chain on reduced SDS-PAGE profile.Botulinum type B neurotoxin complex consisted of neurotoxin and non-toxic components under acidic condition.However,under basic condition,the neurotoxin and non-toxic components were separated.Conclusion Botulinum type B neurotoxin was successfully purified,of which the single chain was cleaved into double chain and the specific activity increased after treatment with trypsin.

关 键 词：肉毒梭菌 肉毒毒素类 纯化 胰蛋白酶 

分 类 号：R378.83[医药卫生—病原生物学]