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| Expression of Tat-MANF Fusion Protein and Its Bioactivity Analysis |
| LI Chen1, GU Hua1, GUO Jia2, SUN Hui1, HUANG Jing-wei1, JIANG Ming2, JIN Ling-jing3, FANG Jian-min1,2 |
1. Tongji University, Shanghai 200092, China;
2. Tongji University Suzhou Institute, Suzhou 215101, China;
3. Shanghai Tongji Hospital, Shanghai 200065, China |
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Abstract Objective: To express Tat-MANF fusion protein and evaluate its biological activities. Methods: Tat-MANF fusion gene is constructed by PCR method to add the TAT sequence in the N-terminus and a His tag in the C-terminus of the MANF cDNA respectively. The Tat-MANF cDNA is inserted into the pET22b+ expression plasmid and prokaryotic expression system (BL21) is used to express the fusion protein. After purification, Tat-MANF protein is detected by SDS-PAGE and Western blot analyses. In order to evaluate biological activities of Tat-MANF, 6-Hydroxydopamine (6-OHDA) was used to induce apoptosis in human dopaminergic neuroblastoma cells. Tat-MANF is added to the cell-culture medium and the neuroprotection effect is detected by flow cytometry after 24h. B-endo3 cells were used as an in vitro blood-brain barrier model, in which B-endo3 cells are incubated with the FITC-labeled Tat-MANF and examined under microscopy after 4 hours. Results: Tat-MANF-His recombinant protein is successfully expressed in prokaryotic expression system. Western blot revealed that Tat-MANF-His recombinant protein can be detected by both anti-MANF and anti-His antibodies. Flow cytometry demonstrates that Tat-MANF significantly inhibit 6-OHDA-induced neuronal cell apoptosis similar to MANF. After 4 hours co-incubation with Tat-MANF-FITC, intracellular fluorescent signal is observed in the B-endo3 cells, indicating that Tat-MANF is able to cross cell membrane of brain vascular endothelial cells, a key component of BBB(blood-brain barrier). Conclusion: The recombinant Tat-MANF fusion protein is neuroprotective and may be able to cross blood-brain barrier, which may provide a novel therapy to Parkinson's Disease.
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Received: 27 April 2014
Published: 25 June 2014
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