Recombinant VPAC2 receptor specific agonist RMBAY, its expression and use
An expression method and agonist technology, applied in the field of genetic engineering, can solve the problems of low synthesis efficiency and high cost, and achieve the effects of low cost, lower blood sugar and high efficiency
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Embodiment 1
[0029] The preparation of embodiment 1 specific agonist RMBAY
[0030] The schematic diagram of the preparation of the specific agonist RMBAY is shown in Figure 1.
[0031] 1. Preparation of RMBAY gene
[0032] Chain extension reaction: Primer F2 (0.1OD / μl) 5μl, F3 (0.1OD / μl) 5μl, 10×TaKaRa LA Buffer 10μl, dNTP 16μl, H 2 O 63μl, 94°C, 10min; lower to 55°C, add TaKaRa LA Taq enzyme 1μl, keep warm for 5min; 72°C, 5min; the result is the extension reaction solution.
[0033] PCR reaction: primer F1 (0.01OD / μl) 1μl, F4 (0.01OD / μl) 1μl, extension reaction solution 1μl, dNTP 10μl, 10×TaKaRa Ex Buffer 10μl, TaKaRaEx Taq enzyme 1μl, H 2 O 76μl, 94°C, 4min; 94°C, 30sec, 55°C, 30sec, 72°C, 30sec, 32 cycles; 72°C, 10min.
[0034] F1: NNNNNN CAT ATG CAT AGC GAT GCG GTG TTT ACC GAA AAC TATACC AAA CTG CGT AAA CAG;
[0035] F2: CTG GCG GCG AAA AAA TAT CTG AAC GAT CTG AAA AAA GGC GGCACC;
[0036] F3: CGC CAG CTG TTT ACG CAG TTT GGT ATA GTT TTC GGT AAA CACCGC ATC GCT ATG CA;
[0037] F...
Embodiment 2
[0042] The receptor selectivity assay of embodiment 2RMBAY
[0043] Using Chinese hamster ovary cells (CHO cells) specifically expressing the three receptors of PACAP, 125 I-labeled ligand and receptor competition binding assay (Competitionbinding assay).
[0044] 1. Membrane protein preparation: collect CHO cells specifically expressing VPAC2 receptors and resuspend in 1mM NaHCO 3 , Immediately freeze in liquid nitrogen for 2-3 hours, take out and thaw, centrifuge at 400g for 10min at 4°C, take the supernatant at 4°C, centrifuge at 20,000 for 10min, and resuspend the pellet in 1mM NaHCO 3 ;, as a crude product of cell membrane protein.
[0045] 2. Take 10μg membrane protein prepared above, add 0.1nmol / 125 I-PACAP38 (1813.14Ci / mmol) and the recombinant polypeptide to be tested, and gradually increase the concentration of the polypeptide (10 -10 -10 -5 mol / L), the reaction was carried out in 100 μl buffer (20mmol / L HEPES, pH7.4, 150mmol / L NaCl, 0.5% BSA, 2mmol / L MgCl2, 0....
Embodiment 3
[0051] Example 3RMBAY promotes the activity measurement of VPAC2-CHO cell cAMP generation
[0052] Using the screened VPAC2-CHO cells highly expressing VAPC2 receptors, the activity of RMBAY in promoting intracellular cAMP synthesis was detected.
[0053] Scrape the VPAC2-CHO cells in the logarithmic growth phase, wash them twice with PBS, and adjust the concentration of the cell suspension to 1×10 6 Each / ml, add the same concentration of RMPACAP or RMBAY, adjust the concentration of the polypeptide from 10 -12 mol / L to 10 -6 mol / L; incubate at 37°C for 5 min, add 2 times the volume of 0.2 mol / L HCl to 100 μl of cell suspension, place at room temperature for 20 min, dissolve the cells by pipetting with a tip and mix well, centrifuge at 1000 g for 10 min, draw the supernatant and press Cyclic AMP Enzyme Immunoassay Kit instructions for measuring cAMP concentration.
[0054] Both RMBAY and RMPACAP can effectively stimulate the generation of intracellular cAMP, and the EC of R...
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