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

doi:10.3969/j.issn.2095-4344.2014.15.013

Abstract

Abstract:

BACKGROUND: Human brain-derived neurotrophic factor (BDNF) and neurotrophine-3 (NT-3) are essential genes for cell differentiation. 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.
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.
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 successfully constructed using IRES sequence.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: brain-derived neurotrophic factor, nerve growth factors, carrier proteins, tissue engineering

CLC Number:

R318

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen. Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(15): 2369-2376.

Figures/Tables 4

References

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