利用FRET技术检测缓激肽受体和Apelin受体二聚化的实验分析-生物化学与分子生物学专业论文.docxVIP

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利用FRET技术检测缓激肽受体和Apelin受体二聚化的实验分析-生物化学与分子生物学专业论文.docx

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PAGE PAGE VI Using FRET to monitor the heterodimerization of B1 bradykinin receptor and Apelin receptor Abstract Both B1 bradykinin receptor (BDKRB1/B1R) and Apelin receptor (APJ) both belong to G protein-coupled receptor (GPCR) superfamily. They play a very important role in cardiovascular system and central nervous system. It has not been reported whether B1R and APJ could form heterodimer and produce physiological effects. Here we described the formation of heterodimer of B1R and APJ and the effect of heterodimer on signal transduction. In this study we try to provide experiment basis for studying the molecular mechanism of B1R and APJ and information for related molecular mechanism of disease. It also helps us to search the new breakthrough of related diseases. To explore the possibility that B1R and APJ form heterodimer and to study the effect of B1R and APJ heterodimer on signal transduction, we constructed recombinant plasmid of B1R and APJ. Then, we transfected the recombinant plasmid into human embryonic kidney cells, the expression of the recombinant plasmid was detected by Western blot and laser scanning confocal microscope. We verified the formation of heterodimer of B1R and APJ by laser scanning confocal microscope and FRET, along with the effect of them on signal transduction. Target fragment was amplified, purified and digested and then inserted into the vector that had been digested by restriction enzymes. The recombinant plasmid, pECFP-APJ-Clontech and pEYFP-B1R-pcDNA3.1 were confirmed by DNA sequencing. Using Western blot technology and laser scanning confocal microscope to identify the expression of recombinant plasmid was successful. Based on these previous experiments, we detected the co-localization of B1R and APJ. Our results showed that B1R and APJ were expressed on the cell membrane, and it revealed a high degree of co-localization of them. It was speculated that B1R may interacted with APJ. After 24 hours of the HEK293 cells transfected