Rad51 inhibitor and application thereof
An inhibitor and protein inhibition technology, applied in the field of biomedicine, can solve the problems of strong toxicity and poor stability, and achieve the effect of broad application prospects.
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Embodiment 1
[0034] This example proves that Nanog can directly interact with Rad51.
[0035] The clones of Nanog (GST tag) and Rad51 (His tag) were respectively constructed on the prokaryotic expression vector, and Rad51 (fused with GST tag) and Nanog (fused with His tag) were respectively constructed into PET30a and pGEX6p-1 expression vectors, and The constructed plasmid was transformed into BL21 protein expression Escherichia coli to induce protein expression and purification, the results were as follows figure 1 As shown (protein bands of corresponding size are marked with "*"), the purified protein was used for GST fusion protein sedimentation (GST pulldown) experiment, and 10 μg of Nanog protein and GST protein (control) fused with GST tag were mixed with 10 μg of Rad5 protein fused with His tag was mixed, and the reaction system was made up to 500 μL, and 1% of the reaction system mixture was taken as Input, and the sample was reserved for detection, and incubated at 16°C for 18 ho...
Embodiment 2
[0038] This example proves that Nanog can inhibit the combination of Rad51 and ssDNA.
[0039] Rad51 is a DNA-binding protein. After being recruited to the damage site, it will replace RPA and combine with ssDNA to form nucleoprotein filaments for homologous recombination-mediated DNA damage repair. In order to clarify that Nanog inhibits the binding of Rad51 and ssDNA, the in vitro The purified protein was subjected to gel shift assay (Electrophoretic Mobility Shift Assay, EMSA). Purified Nanog and Rad51 were taken, and after desalting, the reactants were added according to the system in Table 1, and reacted at 25°C for 20 minutes.
[0040] Table 1
[0041]
[0042]
[0043] EMSA experiment results such as image 3 As shown, "-" means not to add the corresponding components, "+" means to add the corresponding components, and use biotin (biotin) to mark a 90nt ssDNA, Rad51 can bind to ssDNA, so in the non-denaturing polypropylene gel During electrophoresis, the migrati...
Embodiment 3
[0045] This example proves that Nanog can inhibit the function of Rad51 to repair double-strand damage.
[0046]In order to explore whether Nanog can inhibit the repair of Rad51 to double-strand damage, combined with the treatment of CPT (DNA double-strand damage inducer) that causes DNA double-strand damage, the effect of different Nanog expression levels in 293T cells on Rad51 repairing DNA double-strand damage was detected by western blotting experiments. The effect of chain damage activity, overexpressing Rad51 and different concentrations of Nanog (+: 0.1 μg, ++: 0.5 μg, +++: 1 μg) in 293T cells, after 44 hours of expression, treated with 2 μM CPT for 4 hours, collected Cells, histones and whole proteins were extracted to detect the level of γH2AX (DNA double-strand damage marker) and cell expression, respectively.
[0047] The results of Western blotting experiments are as follows: Figure 4 as shown, Figure 5 for Figure 4 Quantitative statistical analysis of the re...
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