Construction and identification of pIRES2-LIF-VEGF165 bicistronic eukaryotic expression vector

doi:10.3969/j.issn.2095-4344.2014.24.017

Abstract

Abstract:

BACKGROUND: Human leukemia inhibitory factor (LIF) and vascular endothelial growth factor 165 (VEGF165) play an important role in neuronal survival after spinal cord injuries. The clinical application of viral vector has a hidden danger, and eukaryotic expressing vector is a way to solve this question.
OBJECTIVE: To construct and identify pIRES2-LIF-VEGF165 bicistronic eukaryotic expression vector.
METHODS: LIF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by direct PCR. Then LIF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by twin PCR, then VEGF165 cDNA fragment was cloned into the pIRES2-LIF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid pIRES2-LIF-VEGF165. Then pIRES2-LIF-VEGF165 was applied to transfect HEK293 cells, and the co-expression of double genes was detected using RT-PCR and western blot analysis.
RESULTS AND CONCLUSION: The DNA sequencing analysis demonstrated that the LIF and VEGF165 were exactly consistent with the sequence recorded in the GenBank. The size of LIF gene and VEGF165 gene was respectively was 609 bp and 576 bp. Enzyme digestion analysis indicated that LIF band was found in the double gene co-expressing vector pIRES2-LIF-VEGF165 by EcoRI/BamHI, IRES-VEGF165 fragmet was found by BamHI/NotI, and LIF-IRES-VEGF165 fragment was found by EcoRI/NotI. RT-PCR and western blot analysis showed that, after transfection, both LIF and VEGF165 mRNA and protein were expression in HEK293 cells. LIF and VEGF165 bicistronic eukaryotic expression vector was successfully constructed in this study.

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: leukemia inhibitory factor, vascular endothelial growth factors, transfection, polymerase chain reaction

CLC Number:

R318

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen, Yuan Zhi-qing. Construction and identification of pIRES2-LIF-VEGF165 bicistronic eukaryotic expression vector[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(24): 3870-3877.

Figures/Tables 4

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