HPV16 E2与IL-12联合基因疫苗的免疫效果初步研究

Immunological competence of a DNA vaccine containing human papillomavirus type 16 E2 combined with IL-12

目的将HPV16E2与IL-12真核表达载体联合免疫小鼠,检测小鼠产生的特异性体液免疫和细胞免疫应答水平,为HPV16诱发的宫颈癌治疗性核酸疫苗的研制奠定实验基础。方法40只BALB/c小鼠随机分成PBS组、pcD-NA3.1(+)空质粒组、pcDNA3.1(+)/IL-12组、pcDNA3.1(+)/HPV16E2组以及pcDNA3.1(+)/HPV16E2+pcD-NA3.1(+)/IL-12联合组,每组8只,共免疫4次,每2周1次。于免疫前1d及第8周采血,分离血清,-20℃保存;颈椎脱臼处死小鼠,无菌取脾脏,制备脾细胞悬液。ELISA法测定小鼠血清HPV16E2IgG抗体水平及脾细胞培养上清IFN-γ、IL-4水平;MTT比色法检测脾淋巴细胞的增殖。结果免疫8周后,血清IgG抗体A450值E2组和联合组与PBS组和空质粒组比较差异有统计学意义(P<0.05);但在每个时间点,联合组与E2组比较差异无统计学意义(P>0.05)。IL-12组、E2组、联合组脾淋巴细胞培养上清中的IFN-γ和IL-4含量与PBS组和空质粒组比较差异有统计学意义(P<0.05);联合组与E2组比较IFN-γ含量差异有统计学意义(P<0.05),两组的IL-4含量差异无统计学意义(P>0.05)。IL-12组、E2组、联合组脾淋巴细胞SI值与PBS组和空质粒组比较差异有统计学意义(P<0.05);联合组与E2组比较差异有统计学意义(P<0.05)。结论HPV16E2单基因疫苗及其与IL-12联合基因疫苗能刺激小鼠产生特异性抗体,脾淋巴细胞培养上清IFN-γ含量升高,且联合基因疫苗优于E2单基因疫苗。

Objective In order to provide an experimental basis for studying remedial vaccines to treat infection with human papillomavirus type 16（HPV16）,the immunological competence of BALB/c mice was analyzed after they were inoculated with the eukaryotic expression vectors HPV16 E2 and IL-12.Methods A total of 40 BALB/c mice was randomly divided into 5 groups,the PBS Group,pcDNA3.1（＋） Group,pcDNA3.1（＋）/IL-12 Group,pcDNA3.1（＋）/HPV16 E2 Group（the single DNA vaccine group）,and the pcDNA3.1（＋）/HPV16 E2＋pcDNA3.1（＋）/IL-12 Group（the combined DNA vaccine group）.The mice were injected every four times every two weeks.The sera of mice were collected and stored at-20 ℃the day before every immunization or the day before sacrifice.Spleen cells were cultured from the spleens of the mice.ELISA was used to measure the serum-specific IgG antibody and IFN-γ or IL-4 levels in the supernatant from cultured spleen cells.The proliferative response of the spleen cells was measured with an MTT assay.Results The A450 value for IgG in the final immunized sera from the combined DNA vaccine group and the single DNA vaccine group was significantly higher than that from the pcDNA3.1（＋） Group and PBS Group（P0.05）,respectively.There were no statistically significant differences（P0.05） in the A450 value for the combined DNA vaccine group and the single DNA vaccine group at any of the vaccination times.IFN-γ and IL-4 levels in the supernatant of the spleen lymphocytes from the pcDNA3.1（＋）/IL-12 Group,the combined DNA vaccine group,and the single DNA vaccine group were significantly higher than those from the pcDNA3.1（＋） Group and PBS Group（P0.05）,respectively.IFN-γ levels from the combined DNA vaccine group were markedly higher than those from the single DNA vaccine group（P0.05）,but the difference in IL-4 levels was not statistically significant（P0.05）.The stimulation index（SI） of the spleen cells from the pcDNA3.1（＋）/IL-12 Group,the combined DNA vaccine group,and the single DNA vaccine group was higher than that from the pcDNA3.1（＋） Group and PBS Group（P0.05）.The SI of the spleen lymphocytes from the combined DNA vaccine group was higher than that from the single DNA vaccine group（P0.05）.Conclusion The HPV16 E2 vaccine may stimulate the level of specific antibodies and increase the level of IFN-γ.A combination DNA vaccine may have a more potent immune effect.