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

作　　者：赵爽佳 鲍如梦 唐海科 包雪翠 杨洪鸣[1] 唐金宝[1] 

机构地区：[1]潍坊医学院药学院,潍坊261053

出　　处：《中国免疫学杂志》2015年第5期655-658,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.81201346);山东省自然科学基金(No.ZR2013HL066)基金资助

摘　　要：目的:利用Avi-tag技术制备双生物素分子位点专一性标记的增强型绿色荧光蛋白(EGFP)。方法:PCR方式扩增EGFP基因片段,重组至带有双Avi-tag标签的中间质粒载体pdi-Avitag,构建原核表达载体p EGFP-(Avitag)2并转化E-.coli DH5α;表达菌株菌体冻融上清经金属离子亲和色谱纯化目的蛋白EGFP-(Avitag)2,以Bir A酶体外催化生物素分子与目的蛋白在Avi-tag位点的生物连接,并以Western blot和竞争ELISA鉴定生物素化产物EGFP-B2的生物素化效果。结果:成功构建p EGFP-(Avitag)2载体,并在E.coli DH5α中可溶性表达EGFP-(Avitag)2,经Western blot和竞争ELISA鉴定,EGFP-(Avitag)2分子经Bir A酶体外催化可连接两个生物素分子。结论:成功获得双生物素分子位点专一性标记的增强型绿色荧光蛋白,为其在BAS体系中的应用奠定了研究基础。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. coil DH5ot and 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 DH5a. 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 Bir A酶 

分 类 号：R392.11[医药卫生—免疫学]