〔Abstract〕 Objective In order to solve the baculovirus as a gene delivery vector transient expression defect, this study intends to construct a sleeping beauty(SB) transposon-mediated baculovirus expression vector, using green fluorescent protein gene EGFP as the target gene, to evaluate the system expression efficiency in mammals.Methods Using the p FastBac DUAL plasmid as the skeleton,the pPh promoter was replaced with the CMVSB100 X-SV40 PA expression element along with the IR/DR-CMV-EGFP-SV40 PA-IR/DR sequence,and the obtained plasmid was named pBacSB-CE. Meanwhile,as a control,pBac-CE was constructed by inserting the CMVEGFP expression element downstream of p Ph promoter. The constructed recombinant plasmids were transformed into DH10 Bac competent cells,and the recombinant bacmids were obtained by blue-white screening. Then the recombinant bacmids were transfected into Sf-9 insect cells by liposome,resulting in recombinant baculovirus BacSB-CE and Bac-CE. U87 cells were transduced with recombinant virus,the cell proliferation and the expression efficiency of EGFP were detected by MTT,inverted fluorescence microscopy and flow cytometry. The subcutaneous glioma tumor model of nude mice was established and the transduction efficiency of recombinant virus in vivo was further observed by immunofluorescence. Results The results showed that the recombinant viruses BacSB-CE and Bac-CE were successfully obtained by PCR identification. MTT results showed that the recombinant virus had no side effect on the proliferation of U87 cells. The results of inverted fluorescence microscopy and flow cytometry showed that EGFP could be expressed for at least 60 d in BacSB-CE transduced U87 cells and 109a. u. total fluorescence intensity could still be detected after 15 d of transduction,while in Bac-CE transduced cells,no fluorescence cells and fluorescence signal could be detected after 15 d. Conclusion In this study,the SB transposon-mediated baculovirus gene delivery system was successfully constructed. The recombinant virus has a good biosafety and can be sustained and stable expressed in vitro and in vivo of mammals.