金钱白花蛇三指蛋白的表达和纯化
Expression and purification of three-finger toxin family in Bungarus multicinctus
廖雪娇 1徐江 2陈士林3
作者信息
- 1. 山东中医药大学,济南 250355
- 2. 中国中医科学院中药研究所,北京 100700
- 3. 山东中医药大学,济南 250355;成都中医药大学,成都 611137
- 折叠
摘要
目的:银环蛇幼蛇的干燥体是常用的药用动物金钱白花蛇的主要来源,具有祛风通络、定惊止痉的功效.银环蛇毒性大,且毒腺分泌的蛇毒是复杂的具有药用活性的多肽和蛋白质混合物.在了解银环蛇毒素中的三指蛋白家族后,发现其潜在的药用价值,便于探索更好的纯化方法,得到高纯度的三指蛋白.方法:通过质粒的转入,三指蛋白能在大肠杆菌中被诱导表达;将菌液超声破碎后离心,SDS-PAGE验证证明三指蛋白在适宜的温度下能可溶性表达;将上清液上样于纯化柱中,梯度洗脱,得到含有杂质的蛋白;透析、超滤浓缩洗去杂质,得到高纯度的三指蛋白.结果:所建立的三指蛋白诱导表达纯化体系,具有良好的重复性和准确性,应用该方法对三指蛋白进行诱导表达和纯化,测得三指蛋白表达和纯化的SDS-PAGE验证图可得清晰的目的条带;通过透析和超滤浓缩除去杂质蛋白,可显著提高三指蛋白的浓度.结论:本研究所建立的三指蛋白诱导表达和纯化体系操作简便,可获得高浓度和高纯度的三指蛋白,为大批量的生产三指蛋白提供科学有效的依据,同时可为后续三指蛋白相互作用研究提供物质基础.
Abstract
Objective:The dried body of the Bungarus multicinctus is the main source of its medicinal materials.It has the effects of phlegm and blood stasis.It is highly venomous and the venom concealed by the venom glands is a multifaceted mixture of pharmaceutically vigorous peptides and proteins.After a brief understanding of the three-finger toxin(3FTx)family,the papers find its potential medicinal value,and can explore better purification methods to obtain high-purity 3FTx Family.Methods:Through the transfer of plasmid,the 3FTx family could be induced to express in E.coli.The bacterial solution was sonicated and centrifuged,and the 3FTx family was soluble at a suitable temperature by SDS-PAGE.The sample was applied to a purification column and eluted with a gradient to obtain a protein.Dialysis and ultrafiltration were concentrated to wash away the impurity protein for obtaining 3FTx family of high purity.The target protein was collected by plasmid transformation,induced expression,nickel column purification,dialysis,ultrafiltration and concentration.Results:The established three-finger protein induced expression and purification system had good reproducibility and accuracy.The method was used to induce expression and purification of the 3FTx family.The measured 3FTx family expression and purified SDS-PAGE verification map provide a clear target band.The removal of impurity proteins by the established dialysis and ultrafiltration concentration methods could significantly increase the concentration of the 3FTx family.Conclusion:The established 3FTx family induced expression-purification system has good repeatability and accuracy.This system can be used to obtain high concentration of three-finger protein,which provides scientific and effective basis for the production of three-finger protein in large quantities.At the same time,it can provide material bases for the study of subsequent three-finger toxin family interactions.
关键词
金钱白花蛇(银环蛇)/三指蛋白/表达/纯化/相互作用Key words
Bungarus multicinctus/Three-finger toxin famliy/Expression/Purification/Interaction引用本文复制引用
基金项目
中国中医科学院科技创新工程项目(CI2023E002)
出版年
2024
NETL
NSTL
万方数据