Construction and expression of the eukaryotic expression plasmid of BDNF-EGFP fusion protein

Objective To construct the eukaryotic expression vector of brain-derived neutrophic factor (BDNF-EGFP) fusion protein and to investigate its expression and location. Methods BDNF DNA was amplified from the genomic DNA isolated from the human leucocytes by PCR with a pair of primers without stop codon, and then subcloned into plasmid pEGFP-N1. The recombinant plasmid was transfected into Hela cells with lipofectamine 2000. Total RNA was isolated, and then expression of BDNF-EGFP gene was detected by RT-PCR. The expression and cellular location of BDNF-EGFP protein were examined by Western blot and fluorescence microscopy. Results By analyzing restriction enzyme digestion and PCR, we found that BDNF gene was correctly inserted into the plasmid vector. RT-PCR results confirmed the expression of BDNF-EGFP gene. The fluorescence microscopy indicated that the BDNF-EGFP fusion protein was mainly distributed in cytoplasm. Western blot results showed that BDNF-EGFP fusion protein existed in the medium of Hela cells. Conclusion We have successfully constructed the eukaryotic expression vector of BDNF-EGFP fusion protein, which could be expressed in Hela cells. BDNF-EGFP fusion protein is mainly located in cytoplasm and could be secreted into the outside.