Construction and expression of a eukaryotic β_3-adrenoceptor expression vector pcDNA3.1(+)-β_3-AR

Objective To construct the recombinant eukaryotic expression vector pcDNA3.1(+)-β3-AR and express it in HEK293 cells.Methods Human β3-AR cDNA was amplified by PCR from a pENTR-ADRB3-Stop plasmid,and the eukaryotic expression plasmid pcDNA3.1(+)-β3-AR was constructed by inserting the β3-AR cDNA into KpnⅠ/XbaⅠ-digested pcDNA3.1(+).The DNA sequence was confirmed by double digestion,and the pcDNA3.1(+)-β3-AR plasmid was transfected into HEK293 cell line.Human β3-AR mRNA expression on HEK293 cells was detected by RT-PCR,and the protein expression on HEK293 cells was detected by immunofluorescence technique.Results Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the whole coding region of human β3-AR gene.RT-PCR assay indicated the transcription of β3-AR mRNA and the β3-AR protein detectable in the transfected HEK293 cells.Conclusion Recombinant eukaryotic expression vector pcDNA3.1(+)-β3-AR plasmid was successfully constructed by genetic engineering technique and expressed on the surface of HEK293 cells,which will contribute to further studies on the establishment of stable expression cell lines for screening high potent and selective β3-AR agonists.