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Tat-GFP融合蛋白的表达纯化及其穿膜活性
引用本文:关新刚,苏维恒,于欣,佟海滨,孙新. Tat-GFP融合蛋白的表达纯化及其穿膜活性[J]. 吉林大学学报(医学版), 2014, 0(4): 725-728. DOI: 10.13481/j.1671-587x.20140406
作者姓名:关新刚  苏维恒  于欣  佟海滨  孙新
作者单位:(1.北华大学生命科学研究中心,吉林 吉林132013;2.吉林大学生命科学学院 艾滋病疫苗国家工程实验室,吉林 长春130012;3.东北师范大学附属中学高中部生物组,吉林 长春130021)
基金项目:国家自然科学基金资助课题(81150015);吉林省科技厅科技发展计划项目资助课题(20111809);吉林省吉林市科技局科技发展计划项目资助课题(项目编号:201233126)
摘    要:目的:获得具备穿膜活性与绿色荧光蛋白(GFP)标记的Tat-GFP融合蛋白,探讨Tat-GFP在MCF-7细胞中的跨膜转运特性。方法:应用pET-24a-Tat-GFP质粒转化大肠杆菌BL21感受态细胞,检测不同异丙基硫代半乳糖苷(IPTG)浓度(0.5和1.0 mmol•L-1)和不同温度(22℃和37℃)诱导融合蛋白的表达情况;利用Ni-IDA树脂亲和纯化Tat-GFP蛋白,利用GFP特异性抗体采用Western blotting法分析洗脱液中的蛋白;激光共聚焦荧光显微镜下检测Tat-GFP融合蛋白的跨膜转运活性。 结果:0.5和1.0 mmol•L-1 IPTG诱导出的细菌总蛋白中所含的Tat-GFP蛋白量无明显差异;低温(22℃)诱导生产的Tat-GFP蛋白量较37℃更高;Western blotting分析,GFP抗体能够特异性识别PVDF膜上的蛋白,条带灰度与Tat-GFP蛋白上样量有关联;细胞穿膜实验,绿色荧光分布于MCF-7细胞的细胞质和细胞核中。 结论:低温诱导时大肠杆菌BL21菌体上清液中Tat-GFP融合蛋白量更高,所生产的Tat-GFP融合蛋白既具备穿膜活性又具有易于检测的绿色荧光。

关 键 词:Tat-GFP  细胞穿膜肽  融合蛋白  穿膜活性  
收稿时间:2013-11-28

Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity
GUAN Xin-gang,SU Wei-heng,YU Xin,TONG Hai-bin,SUN Xin. Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity[J]. Journal of Jilin University: Med Ed, 2014, 0(4): 725-728. DOI: 10.13481/j.1671-587x.20140406
Authors:GUAN Xin-gang  SU Wei-heng  YU Xin  TONG Hai-bin  SUN Xin
Affiliation:1. Life Science Research Center,Beihua University,Jilin 132013,China;2. National Engineering Laboratory for AIDS Vaccine,School of Life Science,Jilin University,Changchun 130012,China;3.Department of Biology,High School Attached to Northeast Normal University,Changchun 130021,China)
Abstract:Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein(GFP),and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7cells.Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations(0.5 and 1.0 mmol· L-1)of isopropyl-β-D-thiogalactopyranoside(IPTG)and cell culture temperatures(22℃and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins.Western blotting analysis was used to identify the Tat-GFP protein,and confocal laser scanning microscope(CLSM)was used to examine the cell penetration of Tat-GFP protein.Results There was no significant difference in the Tat-GFP protein production induced by 0.5and 1.0mmol·L-1IPTG;however,the low temperature(22℃)-induced BL21cells expressed more Tat-GFP proteins than that at37℃induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL21cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.
Keywords:Tat-GFP  cell-penetrating peptides  fusion protein  penetrating activity
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