Construction of recombinant vaccinia virus rVVJ16/18E7E6 and confirmation of the fusion genes by PCR and western blot analyses. A Schematic diagram of plasmid pneoTKJ16/18E7E6, which contains two expression cassettes in a back-to-back orientation, flanked by vaccinia virus TK region sequences. The expression cassette on the left consists of HPV18E7E6 under the control of the H6 promoter, and the expression cassette on the right is comprised of HPV16E7E6 led by the 7.5 K promoter. Both H6 and 7.5 K promoters are vaccinia-specific promoters. B Schematic diagram of recombinant vaccinia virus rVVJ1175 (top) and rVVJ16/18E7E6 (bottom). Virus rVVJ1175 was a Tiantan strain vaccinia virus with a lacZ gene led by a p11 promoter inserted into the TK region. Virus rVVJ16/18E7E6 was derived from virus rVVJ1175, in which the lacZ expression cassette was replaced by the double expression cassettes for HPV16 and 18 E7E6 fusion proteins. C PCR analyses of the HPV sequences in CEFs infected with rVVJ1175 or rVVJ16/18E7E6 using the primers TKL and P7.5 R and the primers P7.5 and TKR. D Western blot analyses to detect the expression of HPV16E7E6 and HPV18E7E6 fusion proteins in CEFs infected with rVVJ1175 or rVVJ16/18E7E6 using the specific antibodies against HPV16E6, HPV16E7, HPV18E6, or HPV18E7. The marker shown is the low molecular weight protein marker (Institute of Biochemistry and Cell Biology Shanghai Institute for Biological Sciences, China).