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目的:观察癌胚抗原(CEA)串联表位-HSP70融合基因疫苗诱导的免疫应答,为开发新型肿瘤特异性基因疫苗奠定基础。方法:在已经构建含有变异热休克蛋白(HSP)序列的基础上,插入重组CEA串联表位的编码片段获得CEA串联表位-HSP融合基因疫苗。3次肌肉注射免疫Balb/c小鼠,设立注射生理盐水的阴性对照组、注射氢氧化铝佐剂混悬CEA串联表位的阳性对照组及注射pCITriCEA625-667-mtHSP70的实验组。FCM分析脾脏T细胞亚群;体外培养脾细胞,ELISA检测培养上清中干扰素γ(IFNγ)的相对含量;同时测定小鼠血清CEA特异性抗体的滴度。结果:阴性对照组脾细胞中CD3+和CD4+T细胞分别为55.1%±6.1%和30.2%±4.1%;实验组脾细胞中CD3+和CD4+T细胞分别为78.7%±9.2%和48.9%±4.7%,两组比较差异有显著性(P<0.01)。其体外特异性抗原肽诱导的IFNγ分泌处于本底水平,可视为生理性参数,体外非特异性和特异性的IFNγ分泌分别增加3和6倍(P<0.01)。血清中CEA表位特异性抗体滴度阴性对照组为0,阳性对照组<1:500,而融合基因疫苗免疫组达到1:4000。结论:CEA串联表位-HSP融合基因疫苗在体内诱导以激发辅助性T细胞为特征的免疫应答。
Objective: To observe the immune response induced by CEA tandem epitope-HSP70 fusion gene vaccine and lay a foundation for the development of new tumor-specific gene vaccines. Methods: Based on the constructed heat shock protein (HSP) sequence, a CEA tandem epitope-HSP fusion gene vaccine was inserted into the coding region of recombinant CEA tandem epitopes. Balb / c mice were immunized with 3 intramuscular injections. A negative control group injected with saline, a positive control group injected with aluminum hydroxide adjuvant suspension tandem CEA epitope, and an experimental group injected with pCITriCEA625-667-mtHSP70 were established. FCM was used to analyze the splenic T lymphocyte subsets. The spleen cells were cultured in vitro. The relative content of interferon γ (IFNγ) in the culture supernatant was detected by ELISA. The titer of the serum CEA specific antibody was also determined. Results: In the negative control group, the percentage of CD3 + and CD4 + T cells in spleen cells were 55.1% ± 6.1% and 30.2% ± 4.1%, respectively. The percentage of CD3 + and CD4 + T cells in spleen cells in the control group were 78.7% ± 9.2% and 48.9% ± 4.7%, there was significant difference between the two groups (P <0.01). The in vitro specific antigen peptide-induced IFNγ secretion at the bottom level, can be regarded as physiological parameters, in vitro non-specific and specific IFNγ secretion increased 3 and 6 times (P <0.01). Serum CEA epitope specific antibody titers negative control group was 0, the positive control group <1: 500, and the fusion gene vaccine immunized group reached 1: 4000. CONCLUSIONS: The CEA tandem epitope-HSP fusion gene vaccine induces an immune response characterized by the induction of helper T cells in vivo.