title: Construction and identification of pIRES2-VEGF165-NT-3 bicistronic eukaryotic expression vector.
abstract: We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-VEGF165-NT-3. The neurotrophin-3 (NT-3) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NT-3 cDNA fragment was cloned into the pIRES2-VEGF165-EGFP vector in place of enhanced green fluorescent protein (EGFP) to create the plasmid pIRES2-VEGF165-NT-3. Next, pIRES2-VEGF165-NT-3 was transfected into HEK293 cells, and reverse transcription-polymerase chain reaction and Western blotting were used to test co-expression of the double genes. The vascular endothelial growth factor 165 (VEGF165) and NT-3 genes were cloned; DNA sequencing analysis demonstrated that the VEGF165 and NT-3 sequences were the same as those recorded in GenBank. Restriction analysis indicated that the VEGF165 and NT-3 genes were correctly inserted into the expression vector pIRES2-EGFP. The double gene was expressed at both the mRNA and protein levels. The VEGF165 and NT-3 co-expression plasmid was successfully constructed, providing a novel expression system for further study of the functions of the VEGF165 and NT-3 genes.
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China lbnxxmu@163.com.
authors.forename: B N
authors.initials: BN
authors.lastname: Li
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: W D
authors.initials: WD
authors.lastname: Li
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: H G
authors.initials: HG
authors.lastname: Feng
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: J T
authors.initials: JT
authors.lastname: Lin
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Yuan
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins