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.