[A new method for construction of EGFP-labled recombinant adenovirus containing hVEGF(165) and its property in vitro].
By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF(165) was generated quickly and its property was studied in vitro. First, hVEGF(165) coding sequence was subcloned into the shuttle plasmid pAdTrack-CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ(5183). After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacI and transfected into HEK 293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF(165). The further amplified recombinant adenoviruses were purified by CsCl banding at 32,000 rpm for 18 to 24 hours. Electron microscopy and PCR were performed for testing the recombinant adenovirus. The results showed that the purified particles were homogenous hexagon with a high titer up to 2 x 10(12)pfu/ml. An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF(165). Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells. The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad-EGFP/hVEGF(165) was 100. The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour-incubation with Ad-EGFP/hVEGF(165) (MOI = 100), and the expression of hVEGF(165) protein in the conditioned medium was 1385 +/- 332 pg/10(6) cells. It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared high titer of Ad-EGFP/hVEGF(165) is an efficient helpful vector to transfer genes into target cells, all of which make the further in vivo experiments with VEGF(165) possible.
Zhong
Zhao-Dong
ZD
Institute of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zou
Ping
P
Huang
Shi-Ang
SA
Hu
Zhong-Bo
ZB
Liu
Ling-Bo
LB
Lu
Yun-Ping
YP
D000256:Adenoviridae / Q000235:genetics* / Q000648:ultrastructure; D000818:Animals; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008222:Lymphokines / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008222:Lymphokines; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D049452:Green Fluorescent Proteins