[Construction of adenovirus vector for human p27kip1 gene and its expression].
OBJECTIVE
To study the effects of exogenous p27kip1 gene on cell cycle and proliferation.


METHODS
E1 substitutive adenovirus vector pAxlcw containing human p27kip1 cDNA (Cihp27kip1) under the transcriptional control of CMV promoter was constructed and then cotransfected with Ad5 adenovirus DNA-terminal peptide complex EcoT221-digested into 293 cells for preparing p27kip1 recombinant adenovirus Adhp27kip1. After infecting HeLa cells for 24 h, the exogenous p27kip1 protein expression in HeLa cells and its effects on cell cycle and proliferation were determined by Western blot, FACS and MTT assay.


RESULTS
The titers of resultant recombinant adenoviruses was 1.7 x 10(9) pfu/ml. p27kip1 protein was over-expressed in HeLa cells infected with p27kip1 recombinant adenovirus for 24 h, resulting in blockage of cell cycle transition from G1 phase to S phase and inhibition of cell proliferation.


CONCLUSIONS
p27kip1 recombinant adenoviruses prepared in this study can efficiently transfer p27kip1 gene into HeLa cells and over-express p27kip1 protein in the infected cells. Adhp27kip1 can be used as an agent for gene therapy.
Pan
X
X
Department of Cardiology, Changzheng Hospital, Department of Immunology, Second Military Medical University, Shanghai 200433, China.
Zhang
W
W
Wu
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Z
D000256:Adenoviridae / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D014161:Transduction, Genetic; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology
D018797:Cell Cycle Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27