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目的研究HPV L1携带EB病毒(EBV)潜伏膜蛋白2(LMP2)多表位DNA联合多肽的免疫效应。方法BALB/c雌性小鼠随机分为4组。pcHPVL1-EBV LMP2DNA免疫组,肌肉注射pcDNA3.1(+)/HPVL1-EBV LMP2多表位DNA,每鼠每次100μg;DNA联合多肽免疫组:先用DNA免疫,每鼠每次50μg,同时皮下注射EBV LMP2多表位肽,每鼠每次5μg;DNA免疫对照组:肌肉免疫空载体pcDNA3.1(+),每鼠每次100μg;多肽免疫对照组:每鼠每次10μg皮下注射不相关多肽。共免疫4次,间隔2周。免疫后第7周收集小鼠血清,采用ELISA法检测小鼠EBV LMP2特异性抗体IgG、IgA以及HPVL1特异性抗体IgG;免疫后第9周测定EBV特异性抗体IgG1与IgG2a亚类,同时采用乳酸脱氢酶(LDH)释放法测定小鼠脾细胞CTL的杀伤活性。结果多肽联合DNA免疫组小鼠血清EBV LMP2特异性抗体IgG、IgA A值分别为1.573±0.025和0.436±0.033,单独DNA免疫组分别为1.282±0.051和0.317±0.022,差异有统计学意义(F=80.393,27.722,P<0.05);两免疫组小鼠血清HPV L1特异性IgG A值别为0.648±0.063和0.702±0.023,差异无统计学意义(F=1.952,P>0.05)。DNA免疫组IgG1和IgG2aA值别为0.723±0.023和0.594±0.084,差异无统计学意义(F=6.643,P>0.05),是混合Th1/Th2免疫反应类型;多肽联合DNA免疫组IgG1和IgG2aA值别为0.897±0.042和0.629±0.035,差异有统计学意义(F=72.306,P<0.05),是偏向Th2免疫反应类型。多肽联合DNA免疫组在效靶比为10︰1时,小鼠脾细胞CTL杀伤效应(杀伤率)为27.70%,DNA免疫组为21.50%,差异有统计学意义(F=51.559,P<0.05)。结论多肽联合DNA免疫可产生有效的CTL杀伤效应,更能发挥有效的体液免疫作用,其免疫策略可为EBV相关肿瘤的免疫治疗提供参考。
Objective To study the immune effect of HPV L1 carrying multiple epitope DNA (EBV) Latent Membrane Protein 2 (LMP2) DNA combined polypeptide. Methods BALB / c female mice were randomly divided into 4 groups. The pcDNA3.1 (+) / HPVL1-EBV LMP2 polyepitope DNA was injected intramuscularly into the pcHPVL1-EBV LMP2 DNA-immunized group, EBV LMP2 multi-epitope peptide injection, each mouse 5μg each; DNA immunization control group: muscle empty vector pcDNA3.1 (+), each mouse each 100μg; peptide immunization control group: each mouse 10μg subcutaneous injection is not relevant Polypeptide. A total of 4 immunizations, an interval of 2 weeks. Serum was collected from the 7th week after immunization, and the IgG, IgA and HPVL1 specific antibodies of EBV LMP2 specific antibodies were detected by ELISA. The EBV specific IgG1 and IgG2a subclasses were determined 9 weeks after immunization, Determination of CTL killing activity of mouse spleen cells by dehydrogenase (LDH) release assay. Results The IgG and IgA A values of serum specific antibody against EBV in combination with DNA vaccine were 1.573 ± 0.025 and 0.436 ± 0.033, respectively, and those in DNA alone group were 1.282 ± 0.051 and 0.317 ± 0.022, respectively, with significant difference (F = 80.393, 27.722, P <0.05). There was no significant difference in the serum IgG L1 specific IgG A between the two immunized groups (0.648 ± 0.063 and 0.702 ± 0.023, F = 1.952, P> 0.05). The DNA immunization group IgG1 and IgG2aA values were 0.723 ± 0.023 and 0.594 ± 0.084, the difference was not statistically significant (F = 6.643, P> 0.05), is a mixed Th1 / Th2 immune response type; (0.897 ± 0.042) and 0.629 ± 0.035 (F = 72.306, P <0.05), which are biased toward Th2 immune response. When the effective target ratio was 10︰1, the cytotoxicity of CTL in the spleen cells was 27.70% and 21.50% in the DNA immunized group, respectively, with statistical significance (F = 51.559, P <0.05) ). Conclusion The combination of peptide and DNA immunization can produce effective CTL killing effect, and can effectively play an effective role in humoral immunity. The immunization strategy can provide a reference for the immunotherapy of EBV-related tumors.