[Construction and identification of recombinant adenoviruses containing the gene fragments encoding Her2/neu extracellular and transmembrane domains].
OBJECTIVE
To construct eukaryotic expression vectors using recombinant adenovirus containing the gene fragments encoding Her2/neu extracellular first ligand-binding domain (Her2-ECD), full-length extracellular domain (Her2-ECD), and extracellular and transmembrane domain (Her2-TM).


METHODS
The cDNAs were amplified by RT-PCR and inserted into shuttle pAdTrack-CMV plasmids. Viral plasmids were obtained from homologous recombination in E. coli BJ5183, and transfected into 293 cells via liposome. Formation of viral plaque and expression of green fluorescent protein were observed by fluorescence microscopy, and the target proteins were detected by Western blotting.


RESULTS
The target cDNA fragments were amplified by PCR with expected lengths and the DNA sequences were confirmed against Genbank. Formation of viral plaque, expression of green fluorescent protein and the target proteins were detected in 293 cells transfected by the viral plasmids, which showed elevated expression of Her2/neu protein with the increase of multiplicity of infection (MOI).


CONCLUSIONS
The eukaryotic expression vectors using recombinant adenovirus have been successfully constructed for expression of Her2/neu extracellular and transmembrane domains.
Ma
Shu-dong
SD
Department of Oncology, Nanfang Hospital, Department of Radiation Medicine, Southern Medical University, Guangzhou 501515, China. mashudong@yahoo.com.cn
Luo
Rong-cheng
RC
Ding
Zheng-hua
ZH
Yuan
Chang-qing
CQ
Ding
Xue-mei
XM
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D049452:Green Fluorescent Proteins; D008081:Liposomes; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins