Construction of a Novel Eukaryotic Expression Plasmid pcDNA6/myc-his-EGFP Band Its Applications in Expression of Recombinant Genes
10.3969/j.issn.1671-8135.2006.12.005
- VernacularTitle:新型真核表达质粒pcDNA6/myc-his-EGFP B的构建及其在重组基因表达中的应用
- Author:
Xin-Jian LI
1
;
Yi-Cheng CAO
;
Zheng-Ping DU
;
Hua-Qiang YANG
;
Zhen-Wu ZHANG
;
Min ZHUO
Author Information
1. 华南理工大学
- Keywords:
Eukaryotic expression plasmid;
pMHES;
EGFP;
myc;
6 × His;
Protein 3-D structure;
simulation
- From:
China Biotechnology
2006;26(12):22-28
- CountryChina
- Language:Chinese
-
Abstract:
Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.
