Products for screening modulators of lrpprc and methods of identifying modulators of lrpprc
A regulator, R14 technology, applied in the field of biomedicine, can solve the problems of system instability, easy degradation of RNA, lack of
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Embodiment 1
[0037] Verification of the specific binding of nucleic acid aptamer R14 to LRPPRC
[0038] Co-immunoprecipitation method proves that LRPPRC protein specifically binds to nucleic acid aptamer R14
[0039]1) Collect the cells by centrifugation, and collect the cell lysate by centrifugation at 12000g after lysis;
[0040] 2) Synthesize biotin-labeled AS1411 and R14, AS1411 is a reported specific binding protein nucleolin
[0041] Nucleic acid aptamer; R14 is the nucleic acid aptamer obtained by LRPPRC protein screening, wherein R14:
[0042] GGTGGGTGGGTTGGGTGG;
[0043] AS1411: GGTGGTGGTGGTTGTGGTGGTGGTGG), dissolved in water, made into a 100nM storage liquid;
[0044] 3) with 5mM MgCl 2 The PBS configuration protein-aptamer mixture, protein 500ug, nucleic acid aptamer 200pm, final volume 1ml;
[0045] 4) After the protein-aptamer mixture was reacted at room temperature for 30 minutes, 50ul streptavidin-coated beads were added to recover the biotin-labeled aptamer-protein com...
Embodiment 2
[0049] Fragmentation of LRPPRC protein, proving that aptamer R14 binds to the C-terminus of LRPPRC protein
[0050] 1) According to the sequence characteristics of LPRRC protein, design and cut LRPPRC into 5 fragments, which are respectively denoted as LRP1-LRP5, specifically as figure 2 shown;
[0051] 2) integrating all LRPPRC fragments into the GST expression plasmid, performing prokaryotic expression and purification in Escherichia coli, and dissolving the obtained LRPPRC prokaryotic expression protein in PBS;
[0052] 3) Prepare LRPPRC fragmented protein-biotin-labeled R14 mixture, protein fragment 100ug, biotin-labeled R14 200pmol, final volume 1ml;
[0053] 4) After the LRPPRC fragment protein-R14 mixture was reacted at room temperature for 30 minutes, 50ul streptavidin-coated beads were added to recover the biotinylated R14-LRPPRC fragment protein complex;
[0054] 5) After washing the obtained R14-LRPPRC fragment protein complex with PBS three times, add 30ul prote...
Embodiment 3
[0057] In order to verify whether the nucleic acid aptamer-LRPPRC protein polarization screening system is suitable for a high-throughput drug screening system, this example is verified through the following two aspects:
[0058] 1): Screening system specificity validation
[0059] After FITC-labeling nucleic acids, verify the specificity of the system
[0060] 1) Prokaryotic expression and purification of LRPPRC protein, GST protein as a control protein, and BCA method for quantification;
[0061] 2) Synthesize the LRPPRC-specific nucleic acid aptamer Aptamer (R14, GGTGGGTGGGTTGGGTGG) labeled with fluorescein (including but not limited to fluorescent labeling methods such as HEX, CY3, and CY5) and an irrelevant control sequence (control DNA, AATTTTTAATTATTTATATTA), configured at 100 nM stock solution;
[0062] 2) The complex of configuration protein and nucleic acid sequence, the concentration of LRPPRC-specific nucleic acid aptamer and irrelevant control sequence are all 1...
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