乳腺癌相关抗原ROR1 H-2Kd限制性CTL表位疫苗制备及其抗肿瘤作用实验研究

发布时间：2018-04-28 21:39

本文选题：ROR1 + 胸腺肽α1　；参考：《重庆医科大学》2015年硕士论文

【摘要】：目的：预测鉴定肿瘤抗原ROR1的H02Kd限制性CTL表位,制备胸腺肽α1修饰的表位多肽疫苗,观察多肽疫苗的抗肿瘤作用,为乳腺癌的免疫治疗奠定基础。方法：查询NCBI蛋白质库,获得小鼠肿瘤抗原ROR1的氨基酸序列,利用表位预测软件BIMAS及SYFPEITHI对小鼠肿瘤抗原ROR1的H-2Kd限制性CTL表位进行预测；根据预测结果,合成和纯化候选表位肽。流式细胞仪检测候选表位肽与H-2Kd分子的亲和力。选取亲和力较高的候选多肽与胸腺肽α1偶联制备多肽疫苗ROR1-Tα1。ELISA法检测RORl-Tα1体外刺激小鼠脾细胞产生释放的细胞因子IL-12以及IFN-γ。为研究多肽疫苗对小鼠的免疫保护作用,先用ROR1-Tα免疫BALB/c小鼠三次,每周一次。首次免疫后第7天,在小鼠右侧腋窝接种4T1乳腺癌细胞。末次免疫后3周将小鼠处死,剥离肿瘤,观察肿瘤大小。结果：使用表位预测软件BIMAS及SYFPEITHI进行预测,选取4条表位候选肽ROR1 716-724(DCPPRMYSL), ROR1 269-277 (SNPMILMRL), ROR1 788-796 (NYMFPSQGI), ROR1 591-599(DFLHIAIQI);合成上述4条表位多肽,高效液相色谱法分析显示多肽纯度i95%。肽结合实验表明,随着表位多肽浓度增加,其与MHC-I类分子的亲和力亦逐渐增强,当表位多肽浓度达到30μmol/L的时候,4条表位多肽的荧光系数均1.5,提示具有较高的亲和力。选取两个预测软件分析结果重叠的候选表位肽ROR1 591-599 (DFLHIAIQI)与胸腺肽α1进行偶联合成ROR1-Tα1多肽疫苗。ELISA结果表明ROR1-Tα1疫苗能有效促进小鼠T淋巴细胞的IL-12及IFN-γ的合成分泌,体外具有生物学活性。免疫保护实验结果显示,ROR1-Tα1组肿瘤生长速度较对照组生长慢且肿瘤平均重量明显小于对照组,提示ROR1-Tα1多肽疫苗对荷瘤小鼠具有明显的免疫保护效应。结论：通过预测软件获得与H-2Kd分子具有较高亲和力的候选表位多肽,经胸腺肽α1修饰制备ROR1-Tα1多肽疫苗,体外具有生物学活性,体内对荷瘤小鼠具有明显的免疫保护效应。为进一步研究乳腺癌的免疫治疗奠定了基础。
[Abstract]:Aim: to predict and identify the H02Kd restricted CTL epitope of tumor antigen ROR1, prepare thymosin 伪 1 modified epitope peptide vaccine, observe the antitumor effect of the peptide vaccine, and lay a foundation for the immunotherapy of breast cancer. Methods: the amino acid sequence of mouse tumor antigen ROR1 was obtained by querying the NCBI protein library. The H-2Kd restricted CTL epitopes of mouse tumor antigen ROR1 were predicted by epitope prediction software BIMAS and SYFPEITHI, and candidate epitope peptides were synthesized and purified according to the predicted results. The affinity of candidate epitope peptides to H-2Kd molecules was detected by flow cytometry. ROR1-T 伪 1.ELISA method was used to detect the cytokines IL-12 and IFN- 纬 stimulated by RORl-T 伪 1 in mice spleen cells. In order to study the immune protective effect of polypeptide vaccine on mice, BALB/c mice were immunized with ROR1-T 伪 three times a week. 4T1 breast cancer cells were inoculated in the right armpit of mice on the 7th day after the first immunization. The mice were killed 3 weeks after the last immunization, the tumor was removed and the tumor size was observed. Results: using epitope prediction software BIMAS and SYFPEITHI, four epitope candidate peptides ROR1 716-724 DCPPRMYSLL, ROR1 269-277 SNPMILMRL, ROR1 788-796 NYMFPSQGII, ROR1 591-599 DFLHIAIQIQI were selected, and the four epitope peptides were synthesized, and the purity of the four epitopes was analyzed by high performance liquid chromatography (HPLC). The results of peptide binding test showed that the affinity to MHC-I molecules increased with the increase of epitope peptide concentration. When the concentration of epitope polypeptide reached 30 渭 mol/L, the fluorescence coefficients of the four epitope peptides were all 1.5, which indicated that they had high affinity. Two candidate epitope peptides, ROR1 591-599 (DFLHIAIQI) and thymosin 伪 1, were selected to synthesize ROR1-T 伪 1 peptide vaccine. Elisa results showed that ROR1-T 伪 1 vaccine could effectively promote the synthesis and secretion of IL-12 and IFN- 纬 in T lymphocytes of mice. It has biological activity in vitro. The results of immunological protection test showed that the tumor growth rate of ROR1-T 伪 1 group was slower than that of control group, and the average tumor weight of ROR1-T 伪 1 group was significantly lower than that of control group, suggesting that ROR1-T 伪 1 polypeptide vaccine had obvious immune protective effect on tumor-bearing mice. Conclusion: the candidate epitope peptides with high affinity to H-2Kd molecule were obtained by predictive software, and then modified with thymosin 伪 1 to prepare ROR1-T 伪 1 peptide vaccine. The vaccine has biological activity in vitro and has obvious immune protective effect on tumor-bearing mice in vivo. It lays a foundation for further study on immunotherapy of breast cancer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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