pEGFP-hApelin-R重组真核表达载体的构建及在HEK293细胞中的表达

Construction of pEGFP-hApelin-R Eukaryotic Expression Vector and Its Expression in HEK293 Cells

目的构建与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGEP)融合的人apelin受体(Apelin receptor,apelin-R)真核表达载体。方法以质粒pcDNA3.1-hApelin-R为模板,PCR方法扩增人apelin受体。扩增的人apelin受体用EcoRⅠ和BamHⅠ双酶切,同时用这两种酶双酶切质粒peGFP-C1。然后将两种酶切产物按常规方法连接、转化大肠杆菌Top10。挑取菌落培养,提取质粒,然后进行酶切鉴定,最后进行测序。将测序正确的重组载体用脂质体法转染人胚胎肾(human embryonic kidney293,HEK293)细胞,共聚焦显微镜观察。提取转染细胞的总蛋白,进行Westernblot检测。结果扩增出一条约1200bp的片段,与预期的apelin受体大小相符。酶切结果显示,重组质粒pEGFP-hApelin-R被切成两条片段,其中一条为peGFP-C1载体大小,另一条为目的片段大小。经测序鉴定,序列与GenBank(NM_005161)中的序列高度同源。共聚焦显微镜观察显示,人apelin受体主要在细胞膜上表达。Westernblot结果显示在相对分子质量69000处有一蛋白条带,与预期大小相符。结论构建成功pEGFP-hApelin-R重组表达载体,此表达载体可用于检测apelin受体和κ型阿片受体(kappa opioid receptor,KOR)或与其他受体间的相互作用。

Objective To construct apelin fusion expression vector with enhanced green fluorescent protein （EGFP）. Methods The human apelin receptor （apelin-R） was amplified by PCR using the plasmid pcDNA3.1-apelin-R as template. The PCR product was purified by agarose gel and digested with EcoR I and BamH I , and then was inserted between the EcoR I and BamH I , sites of peGFP-C1 upstream from the 5＇-end of the EGFP eDNA. The construct was identified by sequencing. The recombinant plasmid was transiently transfected into human embryonic kidney 293 （HEK293） cells, and the expression of pEGFP-hApelin-R was detected by confocal microscopy and western blot. Results A fragment of 1 200 bp was amplified by PCR, which is as big as the anticipated apelin receptor, and the recombinant plasmidwas digested into two fragments, one was 1 200 bp, the other was peGFP-C1, and the receptor was identical with the gene in GenBank （NM_005161）. Confocal microscopy sequence of apelin showed that apelin receptor was expressed on the plasma membrane. Western blot showed a 69KDa band. Conclusion The pEGFP-hApelin-R eukaryotic expression vector was successfully constructed, and the expression vector could be used to detect interactions between kappa opioid receptors and other receptors.