CAJ | 학술논문

目的：构建含有人二型大麻素受体（CB2）基因的真核表达载体,并实现其在HEK293细胞的表达。方法：首先采用PCR方法从含有CB2基因的质粒pcDNA3.1（＋）-CB2中扩增获得人的CB2基因,再采用基因重组技术将CB2的DNA片段插入真核表达载体pIRES2-EGFP。将获得的pIRES2-EGFP-CB2重组子转染HEK293细胞,应用荧光显微镜观察EGFP的表达情况,RT-PCR和报告基因检测CB2的表达情况。结果：酶切和测序结果验证真核表达载体pIRES2-EGFP-GB2构建成功。可在荧光显微镜下观察到转染HEK293细胞表达的绿色荧光;RT-PCR和双荧光素酶报告基因法检测证明CB2蛋白在真核细胞中成功表达;CB2受体激动剂JWH-015能显著逆转腺苷酸环化酶激动剂forskolin诱发的荧光素酶活性增加。结论：成功构建了真核表达载体pIRES2-EGFP-CB2,为进一步研究CB2基因的生理学和病理生理学功能及调控机制奠定了实验基础。
목적：구건함유인이형대마소수체（CB2）기인적진핵표체재체,병실현기재HEK293세포적표체。방법：수선채용PCR방법종함유CB2기인적질립pcDNA3.1（＋）-CB2중확증획득인적CB2기인,재채용기인중조기술장CB2적DNA편단삽입진핵표체재체pIRES2-EGFP。장획득적pIRES2-EGFP-CB2중조자전염HEK293세포,응용형광현미경관찰EGFP적표체정황,RT-PCR화보고기인검측CB2적표체정황。결과：매절화측서결과험증진핵표체재체pIRES2-EGFP-GB2구건성공。가재형광현미경하관찰도전염HEK293세포표체적록색형광;RT-PCR화쌍형광소매보고기인법검측증명CB2단백재진핵세포중성공표체;CB2수체격동제JWH-015능현저역전선감산배화매격동제forskolin유발적형광소매활성증가。결론：성공구건료진핵표체재체pIRES2-EGFP-CB2,위진일보연구CB2기인적생이학화병리생이학공능급조공궤제전정료실험기출。
Objective：To construct the pIRES2-EGFP-CB2 plasmid and investigate its expression in HEK293 cells.Methods：Full length DNA of CB2 receptor gene amplified from plasmid pcDNA3.1（＋）-CB2 using PCR was inserted into pIRES2-EGFP plasmid by gene recombinantion technology.The recombinant plasmid was transfected into HEK293 cells.The expression of EGFP was examined under fluorescence microscope and the expression of CB2 was examined by RT-PCR and Reporter gene.Results：The specificity of PCR products by DNA scquencing and restriction endonuclease reactions demonstrated that recombinant pIRES2-EGFP-CB2 plasmid was successfully constructed.Green fluorescence expression of the 293 cells can be observed under fluorescence microscope and high expression of CB2 was detected in HEK293 cells by RT-PCR and dual luciferase reporter gene;the CB2 receptor agonist JWH-015 could significantly reverse the luciferase activity enhancement which induced by forskolin.Conclusion：pIRES2-EGFP-CB2 has been successfully cloned,which provided a basis for further investigation of CB2＇s physiological and physiopathological functions and regulatory mechanism.