Construction and identification of recombinant adenovirus containing wild-type human PKG I α gene / 第二军医大学学报

ABSTRACT

Objective: To clone human PKG I α gene and construct a recombinant adenovirus vector containing wild-type PKG I α. Methods: RT-PCR was used to amplify the full-length PKG gene from human pulmonary arterial smooth muscle. After T/A cloning, PKG I α cDNA was cloned into shuttle plasmid pAdTrack-CMV to construct pAdTrack-PKG I α. The plasmid was linearized by Pme I 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: PKG I α was successfully amplified from human pulmonary arterial smooth muscle by RT-RCR. After 3 cycles of amplification, the titer of adenovirus containing wild-type PKG I α reached the indicated level. Conclusion: We have successfully constructed PKG I α gene and constructed the PKG I α recombinant adenovirus, which provides a foundation for the study of PKG Iα function and its role in hypoxia pulmonary vessel remodeling.