4-羟基他莫昔芬依赖的内含肽调控绿色荧光蛋白功能研究  

Study on GFP Function Regulated by 4-Hydroxytamoxifen Dependent Intein Splicing

作　　者：赵拯浩 王步森 王玉东 朱丹妮 董铭心 侯利华 陈薇 ZHAO Zheng-Hao;WANG Bu-Sen;WANG Yu-Dong;ZHU Dan-Ni;DONG Ming-Xin;HOU Li-Hua;CHEN Wei(Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所

出　　处：《生物技术通讯》2019年第5期619-623,共5页Letters in Biotechnology

基　　金：国家科技重大专项艾滋病和病毒性肝炎等重大传染病防治（2016ZX10004001-009）

摘　　要：目的:验证小分子4-羟基他莫昔芬(4-HT)依赖的内含肽调控绿色荧光蛋白(eGFP)功能的可行性。方法:根据eGFP的结构与功能,在蛋白上选取7个氨基酸位点,通过同源重组方式将内含肽序列插入不同位点,转染293T细胞系后通过流式细胞术检测eGFP的表达情况,分析4-HT依赖的内含肽调控剪接效率。结果:在选取的7个氨基酸位点中,有5个具有调控效果,其中S148位点的4-HT调控效果最好,4-HT的最适工作浓度为4μmol/L。结论:在eGFP蛋白上发现一个新的高效的4-HT调控的内含肽剪接位点S148,与已报道位点R109相比,剪接效率提高8倍。Objective:To verify the feasibility of small-molecule 4-hydroxytamoxifen(4-HT)dependent intein regulating the function of enhanced green fluorescent protein(eGFP).Methods:According to the structure and function of eGFP,seven splicing sites were selected and the modified interin sequence regulated by 4-HT was inserted into the sites by homologous recombination.293T cells were transfected with these constructs and the splic⁃ing efficiency for intein was analyzed by the expression of eGFP measured by flow cytometry in the presence of 4-HT.Results:The interns in eGFP were spliced in the five insertion sites of the seven sites selected in the presence of 4-HT.The best splicing efficiency appeared in S148.The optimal concentration of 4-HT for efficient splicing is 4μmol/L.Conclusion:A novel and more efficient splicing site for intein in eGFP(S148)was discovered and the splicing efficiency was enhanced 8 fold compared with that reported R109 previously.

关 键 词：内含肽 4-羟基他莫昔芬 小分子依赖 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]