[Construction and identification of a new type of recombinant adenovirus containing S0.7 gene of Hantavirus].
OBJECTIVE
To construct a adenovirus vector containing the 0.7 kb fragment of S gene of Hantavirus, CAG promoter, and WPRE (mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus).


METHODS
The fragments of CAG and WPRE were synthesized according to GenBank, and inserted into the plasmid pShuttle-S0.7 to create a transfer vector pShuttle-S0.7-CAG-WPRE. The S0.7-CAG-WPRE fragment was then cloned into Adeno-X; Viral DNA by PI-Sce I and I-Ceu I digestion. The recombinant adenovirus DNA was linearized by Pac I, transfected into HEK 293 cells via Lipofectamine; 2000, and the titer of the recombinant adenovirus was determined by Adeno-X; Rapid Titer Kit. The expressed product of S0.7-CAG-WPRE fragment was detected by immunofluorescence assay.


RESULTS
The sequence of S0.7-CAG-WPRE fragment was confirmed by sequencing, and the recombinant adenovirus containing S0.7-CAG-WPRE was titered at 10(13); pfu/L. HEK293 cells transfected with recombinant adenoviruses were detected positive by immunofluorescence assay using a specific mAb 1A8 against Hantavirus nucleoprotein.


CONCLUSIONS
The recombinant adenovirus containing the 0.7 kb fragment of S segment of Hantavirus, CAG promoter, WPRE was constructed.
Li
Kai
K
Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
Li
Pu-yuan
PY
Bai
Wen-tao
WT
Hu
Gang
G
Wu
Xing-an
XA
Xu
Zhi-kai
ZK
Zhang
Fang-lin
FL
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006476:Orthohantavirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection
D004274:DNA, Recombinant