[The construction of recombinant adenovirus expressing bifunctional fusion protein sCAR-EGF and the detection of its activity].
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.
Ren
Peng-Kang
PK
Central Experimental Laboratory, The First People's Hospital, Shanghai, China.
Wang
Feng
F
Li
Hui-Ming
HM
Li
Zong-Hai
ZH
Huang
Qian
Q
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000032:analysis / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D009369:Neoplasms / Q000628:therapy; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor