Construction and identification of recombinant adenovirus containing wild-type human PKGⅠα gene
Journal Title: Di-er Junyi Daxue Xuebao - Year 2009, Vol 30, Issue 1
Abstract
Objective:To clone human PKGⅠα gene and construct a recombinant adenovirus vector containing wild-type PKGⅠα.Methods: RT-PCR was used to amplify the full-length PKGⅠα gene from human pulmonary arterial smooth muscle.After T/A cloning, PKGⅠα cDNA was cloned into shuttle plasmid pAdTrack-CMV to construct pAdTrack-PKGⅠα.The plasmid was linearized by PmeⅠ and transformed into BJ5183 E.coli, where the plasmid was recombined with pAdEasy-1 by homologous recombination.The recombinants were then transfected into Ad293 cells by Lipofectamine2000 for packaging the adenovirus; the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope to determine the titer.Results: PKGⅠα was successfully amplified from human pulmonary arterial smooth muscle by RT-RCR.After 3 cycles of amplification, the titer of adenovirus containing wild-type PKGⅠα reached the indicated level.Conclusion: We have successfully constructed PKGⅠα gene and constructed the PKGⅠα recombinant adenovirus, which provides a foundation for the study of PKGⅠα function and its role in hypoxia pulmonary vessel remodeling.
Authors and Affiliations
Bin YI
Keywords
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