生物素化标签真核表达载体的构建及其在蛋白检测中的应用

Construction of eukaryotic biotinylation expression plasmid and its application in Western blotting and pull down

目的构建生物素化标签真核表达载体,利用生物素化蛋白检测快速、灵敏、简便的特点,对传统的Western印迹和pull down技术进行改进和优化。方法利用pCI neo载体骨架构建生物素化真核表达载体。将红色荧光蛋白插入表达载体,用辣根过氧化物酶标记的链亲和素对融合蛋白进行Western印迹检测;将萤火虫荧光素酶插入表达载体,利用pull down技术定量分析生物素化效率;将丙型肝炎病毒NS4B蛋白插入表达载体,通过pulldown技术研究丙型肝炎病毒NS4B蛋白的蛋白质间相互作用。结果构建的生物素标签真核表达载体能使目的蛋白生物素化。报告基因定量分析结果表明目的蛋白生物素化效率达72%。将该载体表达的融合蛋白应用于Western印迹和pull down技术中可实现无需特异性抗体的一步法快速检测。结论将生物素化标签真核表达载体用于Western印迹和pull down技术,建立了快速、灵敏、简便的融合蛋白质检测新方法,为生命科学领域提供了一种有利的研究工具和通用技术平台。

Objective To construct a plasmid for expression of biotinylation tagged fusion protein in mammalian cells using bacterial biotin ligase in order to establish a new sensitive and simple method for detecting fusion protein in pull down and Western blotting.Method A eukaryotic expression biotinylation plasmid carrying His and avidin tag（pHAVI）was established based on the backbone of pCI neo.The biotinylation function of pHAVI was validated using RFP gene.The biotinylation efficiency of pHAVI was quantified by fusion proteins of firefly luciferase and pull down.The protein interaction between hepatitis C virus NS4B was studied using pHAVI and pull down.Results A plasmid expression vector carrying the HAVI and birA genes was established that could obtain high level biotinylated proteins in eukaryotic cells.Seventy-two percent of the fusion protein was biotinylated.We verified the interaction between hepatitis C virus NS4B using HAVI plasmid and pull down.We also established a method of Western blotting by one-step streptavidin capture and tandem anti-streptavidin.Conclusion A new sensitive and simple method of fusion protein detection is established by eukaryotic expression biotinylation plasmid,making Western blotting and pull down easier,simpler and faster.This method will provide a multipurpose platform for protein study.