Preparation of TAT/LMP-3 fusion protein and a primary study of its osteogenic effects
ADM: To express and purify a recombinant fusion protein TAT/LMP-3 with intracellular transduction capability and osteogenic activity and observe the cell transduction activity of TAT/LMP-3 and the efficacy of TAT/LMP-3 to induce osteogenic differentiation of human marrow mesenchymal stem cells (hMSCs). METHODS: Prokaryotic expression vectors pET43. 1a-TAT/LMP-3 and pET43. 1a-LMP-3 were constructed and used to transform E. coli BL21 (DE3) by gene engineering. The expression of the fusion protein was induced by IPTG. The obtained proteins were purified by Ni-NTA affinity chromatography and identified. Meanwhile, polyclonal antibodies against recombinant LMP-3 protein were prepared. The fusion protein was incubated with hMSCs, and intracellular transduction of the fusion protein was analyzed by Western blotting. In order to analyze the effect of the recombinant protein on osteogenic differentiation of hMSCs, the expression of osteoblast markers was detected. RESULTS: The prokaryotic expression vector carrying pET43. 1a-TAT-LMP-3 fusion gene was constructed successfully, and the expression of TAT/LMP-3 in its soluble form was observed. The purity of the purified TAT/LMP-3 was higher than 90%. Rabbit derived polyclonal antibodies against TAT/LMP-3 were successfully prepared. Western blotting analysis demonstrated that TAT-LMP-3 fusion protein can be transduced into hMSCs in a concentration-and time-dependent manner. Meanwhile, TAT/LMP-3 induced osteogenic differentiation of hMSCs successfully. CONCLUSION: Prokaryotic expression, purification and identification of TAT/LMP-3 fusion protein was successful, and TAT/LMP-3 fusion protein exhibited cell transduction capability and osteogenic activity, which provides an experimental basis for utilizing TAT/LMP-3 fusion protein in spinal fusion.
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