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目的用计算机辅助分子设计技术,设计河豚毒素(TTX)小分子拮抗肽,分别用免疫学方法和动物实验进行验证。方法利用计算机辅助分子设计技术,对TTX的空间结构进行了优化;用分子对接确定了TTX关键位点;用分子模建设计了3个能与TTX结合的小分子拮抗肽;分别用竞争ELISA进行免疫学筛选;用动物实验进行中和活性测定。结果成功设计了3个针对TTX的小分子拮抗肽并进行了多肽合成,用竞争ELISA筛选到针对TTX的小分子拮抗肽P2,拮抗肽P2浓度与TTX的结合能力成正比。动物实验中,针对TTX的小分子拮抗肽P2对注射2.5倍半数致死剂量TTX昆明小鼠的保护率为25%,起到了一定解毒效果。结论获得了能与TTX特异性结合的中和性小分子拮抗肽,TTX浓度达到4.05μg/mL(LD50的2.5倍),TTX拮抗肽P2对实验组小鼠的保护率为25%。
Objective To design a small molecule antagonist of tetrodotoxin (TTX) using computer-aided molecular design techniques and validate it with immunological methods and animal experiments respectively. Methods The space-structure of TTX was optimized by using computer-aided molecular design techniques. The pivotal sites of TTX were identified by molecular docking. Three small molecule antagonists capable of binding to TTX were designed by molecular modeling. Immunological screening; animal experiments for the determination of neutralization activity. Results Three small-molecule antagonistic peptides against TTX were successfully designed and synthesized. The small-molecule antagonist P2 against TTX was screened by competition ELISA. The concentration of antagonistic peptide P2 was directly proportional to the binding capacity of TTX. Animal experiments, the small molecule antagonist peptide against TTX P2 injection of 2.5 times the lethal dose of TTX Kunming mice was 25% protection, played a certain detoxification effect. Conclusion The neutralizing small molecule antagonist peptide with specific binding to TTX was obtained. The concentration of TTX was 4.05μg / mL (2.5 times of LD50). The protective rate of TTX antagonist peptide P2 to the experimental group was 25%.