[Construction of a recombinant adenovirus carrying the Hantaan virus glycoprotein G2 gene and its expression in Hela cells].
We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immune-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.
Huyan
Ting
T
Faculty of Life Science, Northwestern Ploytechnical University, Xi'an 710072, China.
Xue
Xiaoping
X
Song
Kai
K
Wang
Hua
H
Yang
Hui
H
Wang
Wei
W
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; C090197:glycoprotein G2, Hantavirus; D049452:Green Fluorescent Proteins