Construction And Identification Of GTPV High Expression Vector

Object:Goatpox virus is a member of the Capripox virus genus of family Poxviridae.T-he genome of capripox virus is very large and has strong ability to express foreign genes. This virus is an ideal vector for recombinant live vaccine of some imporant infection diseases in ruminant animals. Recombinant virus vector is one of the most hot spots in virus gene engineering.With the aim,we decide to construct an moreefficient expression goat pox virus (GTPV) vector.Method:This study predicted a bidirectional promoter region of Goatpox virus--Pb56(-), Pb56(+) between ETF-1 and VITF-3,with the aim to construct an more efficient expression goat pox virus(GTPV) vector. Recombinant transfer vectors pUC-TK12-Pb56(+)-EGFP,pUC-TK12-Pb56(-)-EGFP were constructed by fusing the promoter fragment with EGFP reporter gene.These constructs and the standard vector pUC-TK12-P7.5-EGFP, negative vector pUC-TK12-EGFP were transfected into BHK cells with GTPV. The activity of promoters was evaluated by EGFP reporter assay through confocal microscopy,Real-time PCR.As a foreign the Brucella outer membrane protein omp25 gene was inserted into the bidirectional promoter, the constructed vector was named pUC119-TK-Pb56(-)-o-mp25;the activity of foreign gene was evaluated by Western-blotting.Result:The expression of EGFP demonstrated that DNA sequenses are bidirectional promoter fragments of GTPV; the activity of the Pb56(-) and Pb56(+) was stronger than the P7.5 of Vaccinia virus.Foreign gene--omp25 was successfully expressed and immunogenic. Conclusion:We selected an goat pox virus itself bidirectional promoter whose transcriptional activity was higher than the P7.5 promoter of vaccinia virus,providing a theoretical basis for the solution of foreign gene's low expression.