Recombinant adenovirus-mediated expression of GHS-R1a in HEK 293 cells.
OBJECTIVE
To construct the recombinant adenovirus vector carrying human growth hormone secretagogue receptor type 1a (GHS-R1a), for genetic transfection.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Liu
Li
L
Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China.
Xu
Hua-Min
HM
Jiang
Hong
H
Wang
Jun
J
Song
Ning
N
Xie
Jun-Xia
JX
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054440:Receptors, Ghrelin / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D054440:Receptors, Ghrelin; D049452:Green Fluorescent Proteins