[Construction of a recombinant adenovirus vector containing PTEN and its expression in airway smooth muscle cells].
OBJECTIVE
To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) using the pAdxsi system.


METHODS
PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid pShuttle-GFP-CMV. The shuttle vector was transformed into competent DH5alpha strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively.


RESULTS
GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect (CPE) noted in the cells. After 3 cycles of amplification, the titer of adenovirus containing PTEN reached an appropriate level. The viral titer of pAdxsi-GFP-PTEN was 2x10(10) pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells.


CONCLUSIONS
The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role of PTEN gene in asthma therapy.
Zhong
Hao-hai
HH
Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. zhonghaohaiok@tom.com
Luo
Ya-ling
YL
Lai
Wen-yan
WY
Xu
Jian
J
Chen
Li-li
LL
Ye
Han
H
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D001980:Bronchi / Q000166:cytology*; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human