大鼠GPR17基因真核表达载体的构建及其功能鉴定

目的:构建大鼠GPR17(rGPR17)基因的真核表达载体pcDNA3.1(+)-rGPR17,并对其功能进行初步研究.方法:从大鼠脑组织提取总RNA,通过RT-PCR扩增rGPR17 cDNA,与载体pcDNA3.1(+)连接,并转化到大肠杆菌DH5α以获得重组载体pcDNA3.1(+)-rGPR17,以PCR、双酶切和测序鉴定;将重组载体pcDNA3.1(+)-rGPR17通过脂质体转染方法,转染HEK293细胞,RT-PCR、免疫荧光法检测rGPR17的表达,Fluo-4测定激动剂LTD_4处理后细胞内钙变化.结果:RT-PCR、双酶切和测序证明,重组的真核表达载体pcDNA3.1(+)-rGPR17构建成功,并在HEK293细胞获得表达,加入外源性激动剂LTD_4可诱导细胞内钙的增加.结论:成功构建了rGPR17的真核表达载体pcDNA3.1(+)-rGPR17,并在HEK293细胞功能性表达,为GPR17受体及其拮抗剂的研究提供了基础.

Construction and identification of eukaryotic expression vector of rat GPR17 gene

Objective: To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells. Methods: Total RNA was extracted from rat brain tissue;full-length GPR17 cDNA was prepared by RT-PCR,and cloned into pcDNA3.1(+) plasmid.The recombinant plasmid was converted into E.coli DH5α and confirmed by PCR,double enzyme digestion analysis and DNA sequencing.The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000.Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining.The exogenous LTD_4 enhanced intracellular calcium was measured using Fluo-4. Results: RT-PCR,double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector,and the recombinant rGPR17 was expressed after transfection in HKE293 cells.LTD_4 increased intracellular calcium release in the transfected HEK293 cells. Conclusions: The eukaryotic expression vector of rGPR17 cDNA has been constructed;it is functionally expressed in HEK293 cells.This work provides a basis for further research of the GPR17 receptor and its antagonists.