CAJ | 학술논문

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。
목적：구건대유황색형광단백돌변체 Venus표첨적인완격태2형수체（bradykinin receptor 2， B2R）진핵표체재체，용우B2R여상관수체급단백적상호작용、B2R수체개도적신호전도궤제적연구등。방법근거인B2R기인서렬설계인물，이질립pcDNA3．1-B2R위모판，PCR확증목적기인인B2R。EcoRⅠ화BamHⅠ쌍매절확증산물급질립pVenus-N1，경회수、련접、전화，획취중조질립。대중조질립진행매절、측서감정。전염중조질립지 HEK293T세포，형광현미경관찰수체B2R적세포정위，단백인적법검측목적단백인B2R단백적표체。결과 PCR확증출료1조장도위1176 bp적기인편단，측서결과여GenBank （AY275465）상동。형광현시B2R표체우질막。Western blot결과현시，실험조단백인적조대여목적단백대소상동，위44kDa。결론성공구건료pB2R-Venus중조진핵표체재체，순시전염성공，획득료전염유중조질립적HEK293T세포。성공구건적중조질립pB2R-Venus가용우후속BRET、FRET등실험연구，유조우B2R개도적신호전도궤제적탐토화약물파점적심조。
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 pV enus-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 micros-copy ,and protein band was 44 kd which was identical to target band through Western blot .Conclusion The plas-mid pB2R-Venus was successfully constructed and transfected into HEK 293T cells .The recombinant plasmid can be used to BRET and FRET experiments ,which contribute to investigate the signal transduction mechanism and ex-plore pharmacal targets .