浙江大学学报(医学版)
浙江大學學報(醫學版)
절강대학학보(의학판)
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2009年
6期
584-590
,共7页
林卡娜%方三华%蔡蓓蕾%王欣欣%卢韵碧%张纬萍%魏尔清
林卡娜%方三華%蔡蓓蕾%王訢訢%盧韻碧%張緯萍%魏爾清
림잡나%방삼화%채배뢰%왕흔흔%로운벽%장위평%위이청
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
半胱氨痠/遺傳%受體%白三烯/遺傳%遺傳載體%GPR17%真覈錶達載體%HEK293細胞%細胞內鈣
반광안산/유전%수체%백삼희/유전%유전재체%GPR17%진핵표체재체%HEK293세포%세포내개
Cysteine/genet%Receptors%leukotriene/genet%Genetic vectors%GPR17%Eukaryotic expression vector%HEK293 cell%Intracellular calcium
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.
目的:構建大鼠GPR17(rGPR17)基因的真覈錶達載體pcDNA3.1(+)-rGPR17,併對其功能進行初步研究.方法:從大鼠腦組織提取總RNA,通過RT-PCR擴增rGPR17 cDNA,與載體pcDNA3.1(+)連接,併轉化到大腸桿菌DH5α以穫得重組載體pcDNA3.1(+)-rGPR17,以PCR、雙酶切和測序鑒定;將重組載體pcDNA3.1(+)-rGPR17通過脂質體轉染方法,轉染HEK293細胞,RT-PCR、免疫熒光法檢測rGPR17的錶達,Fluo-4測定激動劑LTD_4處理後細胞內鈣變化.結果:RT-PCR、雙酶切和測序證明,重組的真覈錶達載體pcDNA3.1(+)-rGPR17構建成功,併在HEK293細胞穫得錶達,加入外源性激動劑LTD_4可誘導細胞內鈣的增加.結論:成功構建瞭rGPR17的真覈錶達載體pcDNA3.1(+)-rGPR17,併在HEK293細胞功能性錶達,為GPR17受體及其拮抗劑的研究提供瞭基礎.
목적:구건대서GPR17(rGPR17)기인적진핵표체재체pcDNA3.1(+)-rGPR17,병대기공능진행초보연구.방법:종대서뇌조직제취총RNA,통과RT-PCR확증rGPR17 cDNA,여재체pcDNA3.1(+)련접,병전화도대장간균DH5α이획득중조재체pcDNA3.1(+)-rGPR17,이PCR、쌍매절화측서감정;장중조재체pcDNA3.1(+)-rGPR17통과지질체전염방법,전염HEK293세포,RT-PCR、면역형광법검측rGPR17적표체,Fluo-4측정격동제LTD_4처리후세포내개변화.결과:RT-PCR、쌍매절화측서증명,중조적진핵표체재체pcDNA3.1(+)-rGPR17구건성공,병재HEK293세포획득표체,가입외원성격동제LTD_4가유도세포내개적증가.결론:성공구건료rGPR17적진핵표체재체pcDNA3.1(+)-rGPR17,병재HEK293세포공능성표체,위GPR17수체급기길항제적연구제공료기출.
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.