Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3

Li Bing-nan; Li Wei-dong; Lin Jun-tang; Feng Hui-gen

doi:10.3969/j.issn.2095-4344.2014.15.013

Abstract：BACKGROUND:Human brain-derived neurotrophic factor (BDNF) and neurotrophine-3 (NT-3) are essential genes for celldifferentiation. Viral vector has been used numerously in clinical practice, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question. <br> OBJECTIVE:To construct and identify pIRES2-BDNF-NT-3 bicistronic eukaryotic expression vector. METHODS:BDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-BDNF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-BDNF-EGFP, instead of EGFP, to create plasmid pIRES2-BDNF-NT-3. Subsequently pIRES2-BDNF-NT-3 was used to transfect HEK293 cells, and RT-PCR and western blot analysis were applied to test the co-expression of double genes. <br> RESULTS AND CONCLUSION:The DNA sequencing analysis demonstrated that the BDNF and NT-3 were exactly consistent with the sequence recorded in the GenBank. Enzyme digestion analysis indicated that, in the constructed bicistronic eukaryotic expression vector pIRES2-BDNF-NT-3, BDNF band was obtained by Eco RI/Bam HI digestion, IRES-NT-3 fragment was obtained by Bam HI/Not I digestion, and BDNF-IRES-NT-3 was obtained by Eco RI/Not I digestion. RT-PCR and western blot analysis showed that, after the HEK293 cells were transfected with pIRES2-BDNF-NT-3, double gene was expressed at the mRNA and protein level. Experimental findings suggest that, bicistronic eukaryotic expression vector of BDNF and NT-3 genes can be successful y constructed using IRES sequence.