CAJ | 학술논문

目的：构建 pEGFP - C1-β- arrestin2融合蛋白表达载体，并证实融合蛋白在细胞内的表达及定位。方法：以 pGEX -5X -1-β- arrestin2为模板，PCR 法扩增 hβ- arrestin2编码序列，亚克隆至 pEGFP - C1中， pEGFP - C1-β- arrestin2经双酶切初步鉴定再送测序公司检测正确后瞬时转染人胚肾细胞 HEK293，West-ern blot 检测融合蛋白表达。激光扫描共聚焦显微镜观察融合蛋白在 HEK293细胞中的分布。结果：hβ- ar-restin2编码序列被成功克隆至 pEGFP - C1中，Western blot 检测到融合蛋白表达，分子量约为77kDa，pEGFP
목적：구건 pEGFP - C1-β- arrestin2융합단백표체재체，병증실융합단백재세포내적표체급정위。방법：이 pGEX -5X -1-β- arrestin2위모판，PCR 법확증 hβ- arrestin2편마서렬，아극륭지 pEGFP - C1중， pEGFP - C1-β- arrestin2경쌍매절초보감정재송측서공사검측정학후순시전염인배신세포 HEK293，West-ern blot 검측융합단백표체。격광소묘공취초현미경관찰융합단백재 HEK293세포중적분포。결과：hβ- ar-restin2편마서렬피성공극륭지 pEGFP - C1중，Western blot 검측도융합단백표체，분자량약위77kDa，pEGFP
Objective:To construct the expression plasmid of pEGFP - C1 - β - arrestin2 and identify the expres-sion and localization of its fusion protein. Methods:Using pGEX - 5X - 1 - β - arrestin2 as template,we obtained hu-man β - arrestin2 coding sequence by PCR amplification and cloned it into pEGFP - C1. After the recombinant plas-mid was identified by enzyme digestion and sequencing,the plasmid was transfected into HEK293. The expression of the recombinant plasmid in HEK293 was detected by Western blot. The localization of fusion protein in HEK293 was observed by using laser scanning confocal microscopy. Results:The coding sequence of human β - arrestin2 was cloned into pEGFP - C1. The expression of fusion protein with a molecular weight of 77 kDa was detected by Western blot and localized in the cytoplasm of HEK293. Conclusion:The recombinant plasmid of hβ - arrestin2 gene was suc-cessfully cloned into eukaryotic expression vector,and the fusion protein was localized in the cytoplasm of HEK293.