Abstract: Objective To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH3T3 cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5). Methods HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plas-mid pET32a(+) followed by digestion with BomH Ⅰ and Hind Ⅲ. The recombinant plasmidpET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BomH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E.coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho I was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH3T3 cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR. Results The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 ℃, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfect-ed NIH3T3 cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment. Conclusions The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E.coli and NIH3T3 cells with stable expression of HPV 16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.