[Construction of recombinant adenovirus vector of hNRAGE gene and its effect on cell cycle of 293 cells].
OBJECTIVE
To construct the recombinant adenovirus vector of hNRAGE gene and study its effect on the cell cycle of 293 cells.


METHODS
hNRAGE gene was amplified by PCR and subcloned into the shuttle vector pAdTrack-CMV to construct a shuttle plasmid pAdTrack-CMV/hNRAGE. After sequencing, it was linearized with Pme I and cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 by electroporation to achieve homologous recombination. After being digested with Pac I, the DNA of identified recombinant plasmid was transfected into QBI-293A cells by calcium phosphate transfection to package adenovirus. With the use of GFP gene expression in pAdTrack-CMV, the appearance of Ad-hNRAGE was observed and its concentration was measured. The expression of the target gene was detected by Western blot and its effect on cell cycle of 293 cells was examined by MTT colorimetry and FCM.


RESULTS
The Ad-hNRAGE was successfully constructed and the expression of hNRAGE gene in 293 cells was proved by Western blot. After harvesting the virus particles, the concentration of Ad-hNRAGE was about 6.5x10(9) Ad/microL. The transfection of Ad-hNRAGE resulted in significantly lower proliferative rate of 293 cells compared with untransfected ones, with cell number of G0-G1 and G2-M phase increasing and that of S phase decreasing.


CONCLUSIONS
hNRAGE gene can inhibit the growth of 293 cells.
Zhou
Li-hua
LH
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, Nanjing 210097, China.
Xue
Bin
B
Guo
Shi-ying
SY
Shi
Yi-huan
YH
Wen
Chuan-jun
CJ
Li
Chao-jun
CJ
D000256:Adenoviridae / Q000235:genetics*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction
D000951:Antigens, Neoplasm; C120897:MAGED1 protein, human; D009363:Neoplasm Proteins; D049452:Green Fluorescent Proteins