| 季丙元,程葆华,王春梅,陈京,白波.pB2R-Venus重组真核载体的构建及在HEK293T细胞中的表达[J].济宁医学院学报,2014,(5):309-312 |
| pB2R-Venus重组真核载体的构建及在HEK293T细胞中的表达 |
| Construction of pB2R-Venus eukaryotic expression vectors and its expression in HEK293T cells |
| 投稿时间：2014-07-22 |
| DOI：10.3969/j.issn.1000-9760.2014.05.002 |
| 中文关键词: Venus;B2R;真核表达载体;HEK293T细胞 |
| 英文关键词: Venus;Bradykinin receptor 2;Eukaryotic expression vector;HEK293T cells |
| 基金项目:国家自然科学基金资助项目(No.31271243;No.81070961),山东省自然科学基金资助项目(No.ZR2011CM027;No.ZR2009DZ004),泰山学者建设专项资金 |
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| 中文摘要: |
| 目的 构建带有黄色荧光蛋白突变体Venus标签的人缓激肽2型受体(bradykinin receptor 2,B2R)真核表达载体,用于B2R与相关受体及蛋白的相互作用、B2R受体介导的信号转导机制的研究等。方法 根据人B2R基因序列设计引物,以质粒pcDNA3.1-B2R为模板,PCR扩增目的基因人B2R。EcoRⅠ和BamHⅠ双酶切扩增产物及质粒pVenus-N1,经回收、连接、转化,获取重组质粒。对重组质粒进行酶切、测序鉴定。转染重组质粒至HEK293T细胞,荧光显微镜观察受体B2R的细胞定位,蛋白印迹法检测目的蛋白人B2R蛋白的表达。结果 PCR扩增出了1条长度为1176 bp的基因片段,测序结果与GenBank(AY275465)相同。荧光显示B2R表达于质膜。Western blot结果显示,实验组蛋白印迹条带与目的蛋白大小相同,为44kDa。结论 成功构建了pB2R-Venus重组真核表达载体,瞬时转染成功,获得了转染有重组质粒的HEK293T细胞。成功构建的重组质粒pB2R-Venus可用于后续BRET、FRET等实验研究,有助于B2R介导的信号转导机制的探讨和药物靶点的寻找。 |
| 英文摘要: |
| Objective To investigate the interaction between B2R and other receptors,and signal transduction mechanism,human eukaryotic expression vector that bradykinin receptor 2 fused with Venus was constructed.Methods The primer was designed based on human B2R gene sequence,and B2R gene was then amplified by PCR using plasmid pcDNA3.1-B2R as template.The PCR product was digested by enzyme EcoRⅠand BamH,and cloned into plasmid pVenus-N1.The construct was identified by DNA sequencing.The recombinant plasmid was transiently transfected into HEK293T cells.Cell location and protein expression was detected by confocal microscopy and Western blot,respectively.Results The fragment of 1176bp was amplified by PCR,and its sequence was identical with the gene in Genebank(AY275465).It is shown that the B2R expressed on the membrane by confocal microscopy,and protein band was 44 kd which was identical to target band through Western blot.Conclusion The plasmid pB2R-Venus was successfully constructed and transfected into HEK293T cells.The recombinant plasmid can be used to BRET and FRET experiments,which contribute to investigate the signal transduction mechanism and explore pharmacal targets. |
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