The construction of recombinant adenovirus expressing bifunctional fusion protein sCAR-EGF and the detection of its activity.
- Author:
Peng-Kang REN
1
;
Feng WANG
;
Hui-Ming LI
;
Zong-Hai LI
;
Qian HUANG
Author Information
1. Central Experimental Laboratory, The First People's Hospital, Shanghai, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
Cell Line;
Coxsackie and Adenovirus Receptor-Like Membrane Protein;
Enzyme-Linked Immunosorbent Assay;
Epidermal Growth Factor;
analysis;
genetics;
Genetic Therapy;
Humans;
Neoplasms;
therapy;
Polymerase Chain Reaction;
Receptors, Virus;
analysis;
genetics;
Recombinant Fusion Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2006;22(5):713-719
- CountryChina
- Language:Chinese
-
Abstract:
To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
