大鼠GPR17基因真核表达载体的构建及其功能鉴定  

Construction and identification of eukaryotic expression vector of rat GPR17 gene

作　　者：林卡娜[1] 方三华[1] 蔡蓓蕾[1] 王欣欣[1] 卢韵碧[1] 张纬萍[1] 魏尔清[1] 

机构地区：[1]浙江大学医学院药理学系,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2009年第6期584-590,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(30772561);浙江省科技计划重点资助项目(2005C23041)

摘　　要：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LTD4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LTD4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。Objective： To construct the eukaryotic expression vector of rat GPR17 （rGPR17） cDNA,and to identify its function in HEK293 ceils. 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 DH5a 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 LTD4 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 transfeetion in HKE293 cells. LTD4 increased intraeellular calcium release in the transfeeted HEK293 cells. Conclusions. The eukaryotic expression vector of rGPR17 eDNA has been constructed; it is functionally expressed in HEK293 eells. This work provides a basis for further research of the GPR17 receptor and its antagonists.

关 键 词：半胱氨酸/遗传 受体 白三烯/遗传 遗传载体 GPR17 真核表达载体 HEK293细胞 细胞内钙 

分 类 号：Q78[生物学—分子生物学]