Construction of pRluc-KOR eukaryotic expression vector and its expression in HEK293 cells
AIM: To construct eukaryotic expression vector pRluc-KOR containing Renilla luciferase gene and kappa opioid receptor and to detect interactions between apelin receptor and kappa opioid receptor by bioluminescence resonance energy transfer (BRET). METHODS: Human kappa opioid receptor(KOR) was amplified by PCR using plasmid pcDNA 3.1- KOR as template. The PCR product was purified by agarose gel extraction and digested with Not Ⅰ and Xho Ⅰ, and then was inserted between the Not Ⅰ and Xho Ⅰ, sites of pRluc (N-terminal) (Hind Ⅲ/EcoRV) upstream from the 5'-end of Rluc cDNA. The construct was identified by sequencing. The recombinant plasmid was transiently transfected into human embryonic kidney 293 (HEK293) cells and the expression of pRluc-KOR was observed by confocal microscopy. RESULTS; A fragment of 1143 bp was amplified by PCR, which was of the same size as the anticipated kappa opioid receptor. The recombinant plasmid was digested into two fragments, one was 1143 bp and the other was pRluc vector, and the sequence of the KOR was identical with the gene in the Gene Bank (NM_000912). Confocal microscopy showed that KOR was expressed on the plasma membrane. CONCLUSION: The pRluc-KOR eukaryotic expression vector is successfully constructed and the expression vector can be used to detect interactions between KOR and other receptors.
kappa opioid receptorseukaryotic expression vector constructionbioluminescence resonance energy transfer
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维普
