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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
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
