中国免疫学杂志Issue(5)：655-658,4.DOI:10.3969/j.issn.1000-484X.2015.05.016
基于 Avi-tag技术的双生物素分子位点专一性标记的增强型绿色荧光蛋白
Site-specific biotinylation of EGFP with double biotin molecules based on Avi-tag technology
摘要
Abstract
Objective:To prepare the site-specific biotinylation of enhanced green fluorescence protein with double biotin molecules using Avi-tag technology.Methods:The EGFP gene was prepared by PCR and cloned into pdi-Avitag resulting the vector pEGFP-( Avitag) 2.The fusion protein EGFP-( Avitag ) 2 was expressed in E.coli DH5αand purified by employing IMAC.The site-specific biotinylation was implemented by BirA enzyme in vitro, and then was identified by competitive ELISA and Western blot.Results:The recombinant prokaryotic expression vector pEGFP-(Avitag)2 was correctly constructed,and EGFP-(Avitag)2 fusion was successfully expressed in E.coli DH5α.The results of competitive ELISA and Western blot showed that the EGFP-( Avitag) 2 could be site-specific biotinylation with double biotin molecules based on Avi-tag technology.Conclusion: The site-specific biotinylation of EGFP with double biotin molecules is successfully prepared,and we anticipate that can be used for BAS to improve the sensitivity and specificity of immunosensors.关键词
增强型绿色荧光蛋白/生物素化/Avi-tag/BirA酶Key words
Enhanced green fluorescent protein/Biotinylation/Avi-tag/BirA enzyme分类
生物科学引用本文复制引用
赵爽佳,鲍如梦,唐海科,包雪翠,杨洪鸣,唐金宝..基于 Avi-tag技术的双生物素分子位点专一性标记的增强型绿色荧光蛋白[J].中国免疫学杂志,2015,(5):655-658,4.基金项目
本文受国家自然科学基金（ No.81201346）及山东省自然科学基金（ No.ZR2013HL066）基金资助。 （）
