Recombinant adenovirus-mediated expression of GHS-R1a in HEK 293 cells.

ABSTRACT: To construct the recombinant adenovirus vector carrying human growth hormone secretagogue receptor type 1a (GHS-R1a), for genetic transfection.The full-length human GHS-R1a gene was obtained by PCR amplification and then cloned into the shuttle plasmid pAdTrack-CMV. The linearized plasmid pAdTrack-CMV-GHS-R1a was co-transformed into Escherichia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy1. The HEK293 cells were then infected with adenoviruses. The expression of GHS-R1a was indicated by green fluorescent protein (GFP), and confirmed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot.Enzymatic digestion of pAdGHS-R1a yielded a large fragment (approximately 30 kb) and a small fragment (4.5 kb), indicating the successful construction of recombinant adenovirus expression vector. Expression of GFP was observed by confocal laser scanning microscopy at 24 h after infection. RT-PCR and Western blot further confirmed that GHS-R1a was efficiently expressed in 293 cells.Recombinant adenovirus (AdGHS-R1a) is successfully constructed, and the target gene can be expressed efficiently in 293 cells, which provide a valuable tool for further studying the function of GHS-R1a.

SUBMITTER: Liu L 

PROVIDER: S-EPMC5560300 | biostudies-other | 2010 Jun

REPOSITORIES: biostudies-other