hβ- arrestin2基因重组质粒构建及蛋白表达和定位

摘要

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.%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