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Preparation of TAT-ASPP2 fusion protein and its inhibitory effect against proliferation of glioma cells
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Abstract:
Objective:To prepare the TAT-ASPP2 fusion protein and investigate its inhibitory effect against the proliferation of glioma U-87MG and U251 cells. Methods: TAT-ASPP2 specific primer was designed and recombinant prokaryotic expression vector pET-TAT-ASPP2 was constructed using the In-Fusion cloning technique. After identified by double endonuclease digestion and DNA sequencing, pET-TAT-ASPP2 vector was transformed into E. coli BL21 and TAT-ASPP2 fusion protein was induced by IPTG. TAT-ASPP2 fusion protein was further identified by SDS-PACE and Western blotting analysis. The effects of TAT-ASPP2 fusion protein on proliferation of U-87MG and U251 cells were detected by MTT assay. Results: The prokaryotic expression plasmid pET-TAT-ASPP2 was successfully constructed, and TAT-ASPP2 fusion protein was induced by IPTG in transformed E.coli BL21; the molecular weight of the fusion protein was about 128 000 and it could be specifically recognized by ASPP2 antibody. TAT-ASPP2 fusion protein significantly inhibited the proliferation of U-87MG and U-251 cells, with the inhibitory rates being about (65.0±3.0)% and (64.7±2.5)%, respectively; while ASPP2 protein did not inhibit the proliferation of U-87MG and U-251 cells. Conclusion: TAT-ASPP2 fusion protein has been successfully expressed and purified, and the fusion protein can significantly inhibit the proliferation of glioma cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 30800285), and the Young Scientist Foundation from Health Bureau of Fujian Province (No. 2007-1-20)
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