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...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 success-ful 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.Conclusion 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.展开更多
基金supported by the grants from the National Basic Research Development Program of China(No.2007CB516701,2006CB500704)the National Natural Science Foundation of China(No.30770757)
文摘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 success-ful 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.Conclusion 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.
