FUNCTIONAL-CHARACTERIZATION OF BLACK-WIDOW SPIDER NEUROTOXINS SYNTHESIZED IN INSECT CELLS
FUNCTIONAL-CHARACTERIZATION OF BLACK-WIDOW SPIDER NEUROTOXINS SYNTHESIZED IN INSECT CELLS
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DOI：
10.1111/j.1432-1033.1995.tb20628.x
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发表时间：
1995-06-15
期刊：
影响因子：
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通讯作者：
KING, LA
中科院分区：
文献类型：
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作者：
KIYATKIN, NI;KULIKOVSKAYA, IM;KING, LA
利用杆状病毒表达系统在昆虫细胞中合成α-羚羊毒素、α-羚羊毒素和黑寡妇蜘蛛毒液中的低分子质量蛋白。对重组病毒感染细胞的 SDS/PAGE 分析揭示了新的蛋白质，其迁移大小与蜘蛛毒液中的神经毒素相似。通过使用抗（α-羚羊毒素）、抗（α-羚羊毒素）或抗（低分子质量蛋白）IgG 对感染细胞进行免疫印迹分析，证实了这些蛋白为 α-羚羊毒素、α-羚羊毒素或低分子量蛋白。低分子量蛋白和α-河豚毒素在注射到粉纹夜蛾幼虫中或在目标组织的细胞质中直接由病毒衍生合成时均没有毒性。然而，对编码α-腹腔昆虫毒素的重组病毒的生物活性分析表明，它对黑锥虫幼虫具有很强的毒性作用。这些数据表明，天然昆虫毒素的毒性作用可能仅由α-腹腔毒素亚基促进，并且提供了证据表明α-腹腔毒素的作用机制可能是通过部分神经毒素α-亚基分子的内化介导的。
alpha-latrotoxin, alpha-latroinsectotoxin and the low-molecular-mass protein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombinant-virus-infected cells revealed novel proteins that migrated with sizes similar to those of the neurotoxins from spider venom. The identities of these proteins as alpha-latrotoxin, alpha-latroinsectotoxin or the low-molecular-mass protein were confirmed by immunoblot analysis of infected cells with anti-(alpha-latrotoxin), anti-(alpha-latroinsectotoxin) or anti-(low-molecular-mass protein) IgG. Neither the low-molecular-mass protein nor alpha-latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus-derived synthesis directly in the cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding alpha-latroinsectotoxin however, revealed a strong toxic effect on the T. ni larvae. These data indicate that the toxic effect of the native insectotoxin may be promoted by the alpha-latroinsectotoxin subunit alone and provides evidence that the mechanism of action of alpha-latroinsectotoxin may be mediated by internalisation of part of the neurotoxin alpha-subunit molecule.