[Construction and identification of a new type of recombinant adenovirus containing S0.7 gene of Hantavirus].

AIM: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. CONCLUSION:The recombinant adenovirus containing the 0.7 kb fragment of S segment of Hantavirus, CAG promoter, WPRE was constructed.