Immunological Competence Of DNA Vaccine Of Human Papillomavirus Type 16 E5 Combined With IL-12

Objective: In order to provide the experimental basis for studying the Prophylactic and Remedial vaccines on the infections of human papillomavirus type16(HPV16), HPV16 E5 eukaryotic expression vector and prokaryotic expression vector were constructed. After BALB/c mice had been immunized with HPV16 E5 and IL-12 DNA vaccines, the immunological competence was analyzed.Methods:(1) The HPV16 E5 gene was amplified by polymerase chain reaction (PCR) with specific primers designed by PRIMER5.0. After PCR products were digested with BamHI and EcoRI or NotI , the fragment was inserted into pcDNA3.1(+) or pGEX-6P-1, respectively. The inserted fragment was identified by PCR and restriction enzymatic digestion and sequencing. Eukaryotic expression vector pcDNA3.1(+)/E5 or prokaryotic expression vector pGEX-6P-1/E5 was respectively constructed.(2) The plasmid of pGEX-6P-1/E5 was transferred into E.coli BL21. The fusion protein of GST-HPV16 E5 was induced with IPTG and purified. The expression purifications were analyzed using Western blot. The plasmid of pcDNA3.1(+)/E5 was transfected into HeLa cells, and HPV16 E5 mRNA was detected using RT-PCR.(3) A total of 50 BALB/c mice were randomly divided into 5 groups: Group pcDNA3.1(+)/E5, pcDNA3.1(+)/E5+pcDNA3.1(+)/IL-12, pcDNA3.1 (+)/IL-12, pcDNA3.1(+) and PBS. Every group contained ten mice. The mice were injected intramuscularly with 100μg plasmid or 100μl PBS at 2-weeks interval for four times. The sera of mice were collected in the day before every immunization or at the fourteenth day after the last immunization. The spleen cells separated from the mice spleens were cultured. The quadriceps femoris of the mice were made of paraffin section.(4) Indirect ELISA was used to detect quantitatively the specific IgG antibody in sera of the immunized mice. IFN-γor IL-4 levels of the supernatant of the culture medium of mice spleen cells were assayed using Double-antibody sandwich ELISA. The proliferation response of the spleen cells was detected using MTT assay. The expressions of HPV16 E5 in the quadriceps femoris of the immunized mice were detected using immunohistochemistry.Results:(1) DNA sequencing result showed that 252 bp HPV16 E5 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1(+)/E5 expressed HPV16 E5 mRNA in HeLa cells by RT-PCR.(2) The prokaryotic expression recombinant pGEX-6P-1/E5 expressed the GST-HPV16 E5 fusion protein in E.coli BL21, its molecular weight was about 35kDa. Western-blot result showed that the fusion protein could bind to antibody of HPV16 E5 protein.(3) The titers of specific antibody IgG of the sera from the combined DNA vaccine group [pcDNA3.1 (+) / E5 + pcDNA3.1 (+) / IL-12] or the single DNA vaccine group[pcDNA3.1(+)/E5] showed upward trend along with the days of immunization. Their A450 value of IgG in the last immunized sera was respectively 0.771±0.051 or 0.330±0.078, which was significantly higher than that of IgG from group pcDNA3.1(+) (0.080±0.035), group pcDNA3.1(+)/IL-12(0.110±0.015), and group PBS (0.078±0.020) (P<0.01); meanwhile, the value from the combined DNA vaccine group was obviously higher than that from the single DNA vaccine group(P<0.01).(4) The levels of IFN-γor IL-4 in the supernatant of the spleen lymphocyte from the combined DNA vaccine group(352.88±36.76 pg/ml, 680.23.94±36.04 pg/ml) or the single DNA vaccine group(206.53±15.40 pg/ml, 359.94±48.23 pg/ml) were significantly higher than those from group pcDNA3.1(+)(18.04±2.24 pg/ml, 40.41±4.11 pg/ml), group pcDNA3.1(+)/IL-12(42.15±4.89 pg/ml, 177.59±24.33 pg/ml), and group PBS (14.98±2.03 pg/ml, 25.73±2.02 pg/ml) (P<0.01); meanwhile, the levels from the combined DNA vaccine group were obviously higher than those from the single DNA vaccine group(P<0.01). The stimulation index(SI) of the spleen cells from the combined DNA vaccine group or the single DNA vaccine group was respectively 2.14±0.27 or 1.85±0.11, which was higher than that from group pcDNA3.1(+)(1.19±0.07), group pcDNA3.1(+)/IL-12(1.24±0.12), and group PBS (1.13±0.15) (P<0.01); but it was not significantly different between the combined DNA vaccine group and the single DNA vaccine group(P>0.05). HPV16 E5 proteins expressed in the quadriceps femoris of the mice.Conclusions:(1) The eukaryotic expression recombinant pcDNA3.1(+)/E5 was successfully constructed, HPV16 E5 mRNA expressed in HeLa cells.(2) The prokaryotic expression recombinant pGEX-6P-1/E5 was successfully constructed. The GST-HPV16 E5 fusion protein expressed in E.coli BL21 and had a good antigenicity.(3) HPV16 E5 single gene vaccine and combined with IL-12 gene vaccine could induce strong specific cellullar immune response and humoral immune response, and the combination gene vaccine could make stronger immune effect than the single gene vaccine.