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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 To detect the specific humoral and cellular immune response induced by HPV16E2 and IL-12 eukaryotic expression vector and to establish the experimental foundation for the development of HPV16-induced therapeutic nucleic acid vaccine for cervical cancer. Methods 40 BALB / c mice were randomly divided into PBS group, pcDNA3.1 (+) empty plasmid group, pcDNA3.1 (+) / IL-12 group, pcDNA3.1 (+) / HPV16E2 group and pcDNA3.1 (+) / HPV16E2 + pcD-NA3.1 (+) / IL-12 combination group, 8 mice in each group. Blood samples were taken from the first day and the eighth week before immunization and the serum was separated and stored at -20 ℃. The mice were sacrificed by cervical dislocation and the spleen was taken aseptically to prepare spleen cell suspension. The serum levels of HPV16E2 IgG and the levels of IFN-γ and IL-4 in the splenocyte culture supernatant were measured by ELISA. The proliferation of splenic lymphocytes was detected by MTT assay. Results After 8 weeks of immunization, serum IgG antibody A450 E2 group and the combination group with PBS group and empty plasmid group difference was statistically significant (P <0.05); but at each time point, the combination group and E2 group difference Statistical significance (P> 0.05). Compared with PBS group and empty plasmid group, the levels of IFN-γ and IL-4 in spleen lymphocyte culture supernatant of IL-12 group, E2 group and combined group were significantly different (P <0.05); the combination group and E2 group The difference of IFN-γ content was statistically significant (P <0.05). There was no significant difference in IL-4 content between the two groups (P> 0.05). The SI value of spleen lymphocytes in IL-12 group, E2 group and combined group was significantly lower than that in PBS group and empty plasmid group (P <0.05); there was significant difference between combined group and E2 group (P <0.05) . Conclusion HPV16E2 single gene vaccine combined with IL-12 gene vaccine can stimulate mice to produce specific antibodies. The content of IFN-γ in spleen lymphocyte culture supernatant increased, and the combined gene vaccine is better than E2 single-gene vaccine.