Immunological Competence Of DNA Vaccine Of HPV16E2Combined With IL-12

Objective: In order to provide the experimental basis for studying the remedialvaccines on the infections of human papillomavirus type16(HPV16), immunologicalcompetence of BALB/c mice will be analyzed after they are inoculated with theeukaryotic expression vector HPV16E2and IL-12.Methods：(1) The HPV16E2gene was amplified by polymerase chain reaction (PCR) withspecific primers designed by PRIMER5.0. After PCR products were digested withXho I and EcoR I or Not I, the fragment was inserted into pcDNA3.1(+) orpGEX-6P-1, respectively. Eukaryotic expression vector pcDNA3.1(+)/HPV16E2or prokaryotic expression vector pGEX-6P-1/HPV16E2was respectivelyconstructed and identified by DNA sequencing.(2) The plasmid of pGEX-6P-1/HPV16E2was transferred into E.coli BL21.Thefusion protein of GST-HPV16E2was induced with IPTG and purified. Theexpression purifications were analyzed using Western-blot. The plasmid ofpcDNA3.1(+)/HPV16E2was transfected into HeLa cells, and HPV16E2mRNA andits protein expression was detected by RT-PCR or Western-blot.(3) A total of40BALB/c mice were randomly divided into5groups: Group PBS,Group pcDNA3.1(+), Group pcDNA3.1(+)/IL-12, Group pcDNA3.1(+)/HPV16E2and Group pcDNA3.1(+)/HPV16E2+pcDNA3.1(+)/IL-12. The mice were injectedintramuscularly with100g plasmid or100L PBS every two-week for four times.The sera of mice were collected and stored at-20℃in the day before every immunization or the day before execution. The quadriceps femoris of the mice weremade of paraffin section. The spleen cells separated from the mice spleens werecultured.(4) The expressions of HPV16E2in the quadriceps femoris of the immunized micewere detected using immunohistochemistry. The serum specific IgG antibody andIFN-γ or IL-4levels of the cultured supernatant of spleen cells was detect byELISA. The proliferation response of the spleen cells was detected using MTTassay and delegated by stimulation index(SI).Results：(1) DNA sequencing result showed that1098bp HPV16E2gene fragment wascorrectly inserted to plasid pcDNA3.1(+) or pGEX-6P-1. The eukaryoticexpression vector pcDNA3.1(+)/HPV16E2expressed HPV16E2mRNA inHeLa cells by RT-PCR and expressed HPV16E2in the mules of mice, and itsexpressed protein was about42kDa. The prokaryotic expression recombinantpGEX-6P-1/HPV16E2expressed the GST-HPV16E2fusion protein in E.coliBL21, its molecular weight was about68kDa. Western-blot result showed that thefusion protein could bind to antibody of HPV16E2protein.(2) The A450value of IgG in the last immunized sera of the combined DNA vaccinegroup[Group pcDNA3.1(+)/HPV16E2+pcDNA3.1(+)/IL-12] or the single DNAvaccine group[Group pcDNA3.1(+)/HPV16E2]was respectively0.081±0.005or0.084±0.005, which was significantly higher than that of Group pcDNA3.1(+)(0.019±0.009) or Group PBS (0.018±0.005)(P<0.05) respectively; meanwhile, thedifference on the any inoculated time point between the A450value of the combinedDNA vaccine group and the single DNA vaccine group was non-significant (P>0.05).(3) The contents of IFN-γ or IL-4in the supernatant of the spleen lymphocyte ofGroup pcDNA3.1(+)/IL-12(83.98±6.07pg/mL,36.51±2.68pg/mL), the combined DNAvaccine group(116.32±5.15pg/mL,70.08±6.10pg/mL) or the single DNA vaccine group(94.55±5.87pg/mL,68.40±6.13pg/mL) were significantly higher than those fromgroup pcDNA3.1(+)(41.27±5.47pg/mL,36.51±2.68pg/mL) or groupPBS(42.13±6.07pg/mL,36.76±2.12pg/mL)(P<0.05) respectively. Meanwhile, thecontents of IFN-γ from the combined DNA vaccine group was obviously higher thanthat from the single DNA vaccine group(P<0.05), but the difference of IL-4wasnon-significant(P>0.05).(4) The stimulation index(SI) of the spleen cells from Group pcDNA3.1(+)/IL-12(1.25±0.12), the combined DNA vaccine group（2.01±0.07） or the single DNAvaccine group（1.43±0.08）was higher than that from Group pcDNA3.1(+)(1.16±0.16)or Group PBS（1.19±0.07）(P<0.05). And SI of the spleen lymphocyte from thecombined DNA vaccine group proliferated more active than that from the single DNAvaccine group(P>0.05).Conclusions：(1) The eukaryotic expression recombinant pcDNA3.1(+)/HPV16E2can express inHeLa cells or mice’s muscle.(2) The prokaryotic expression recombinant pGEX-6P-1/HPV16E2can expressGST-HPV16E2fusion protein in E.coli BL21, which had a good antigenicity.(3) HPV16E2single gene vaccine or combined with IL-12gene vaccine could raisethe level of specific antibody and IFN-γ.(4) The stimulation index(SI) and the level of IFN-γfrom the combination genevaccine was higher than that from the single gene vaccine.