Purpose: To construct recombinant plasmid containing gene of human brain-derived neurotrophic factor variant12 (BDNF variant12)-green fluorescent fusion protein (GFP) and to study the expression and secretion of
BDNF-GFP fusion protein in cell and in media.
Method: BDNF variant 12 genes were cloned by RT-PCR from fresh human retinal tissue and recombined into plasmid, PEGFP-N1 to construct PEGFP-N1-BDNF plasmid with expressing BDNF-GFP fusion protein. Then, the PEGFP-N1-BDNF vectors were transfected into 293T cells, following counting transfection efficiencies by fluorescent microscopy and Image-Pro Plus6.0 Microsoft. The expression and secretion level of BDNF- GFP fusion protein in 293T cells and medium was assayed respectively by Western-blot and ELISA.
Result: 24 hours and 48 hours after transfection, the expression of BDNF-GFP was observed in cytoplasm and nucleus by fluorescent microscopy and laser scanning confocal microscopy. Results of Western-blot showed that pro-BDNF-GFP fusion protein of 52.8KDa size (proBDNF: 25.8KDa+GFP: 27 KDa =52.8KDa) was detected and results of ELISA indicated that the concentration of mature BDNF-GFP fusion protein in media was 12.01ng/ml.
Conclusion: The PEGFP-N1-BDNF plasmid constructed can express and secret human BDNF-GFP fusion protein high level and it would provide potent technique supports for studying the neuroprotection of BDNF for retinal cells and central neuron cells. Meanwhile it could make good preparation for assembling BDNF+CNTF ECT-based intraocular implants.