本文選題：ROR1 + 胸腺肽α1�。� 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：預(yù)測鑒定腫瘤抗原ROR1的H02Kd限制性CTL表位,制備胸腺肽α1修飾的表位多肽疫苗,觀察多肽疫苗的抗腫瘤作用,為乳腺癌的免疫治療奠定基礎(chǔ)。方法：查詢NCBI蛋白質(zhì)庫,獲得小鼠腫瘤抗原ROR1的氨基酸序列,利用表位預(yù)測軟件BIMAS及SYFPEITHI對小鼠腫瘤抗原ROR1的H-2Kd限制性CTL表位進行預(yù)測；根據(jù)預(yù)測結(jié)果,合成和純化候選表位肽。流式細胞儀檢測候選表位肽與H-2Kd分子的親和力。選取親和力較高的候選多肽與胸腺肽α1偶聯(lián)制備多肽疫苗ROR1-Tα1。ELISA法檢測RORl-Tα1體外刺激小鼠脾細胞產(chǎn)生釋放的細胞因子IL-12以及IFN-γ。為研究多肽疫苗對小鼠的免疫保護作用,先用ROR1-Tα免疫BALB/c小鼠三次,每周一次。首次免疫后第7天,在小鼠右側(cè)腋窩接種4T1乳腺癌細胞。末次免疫后3周將小鼠處死,剝離腫瘤,觀察腫瘤大小。結(jié)果：使用表位預(yù)測軟件BIMAS及SYFPEITHI進行預(yù)測,選取4條表位候選肽ROR1 716-724(DCPPRMYSL), ROR1 269-277 (SNPMILMRL), ROR1 788-796 (NYMFPSQGI), ROR1 591-599(DFLHIAIQI);合成上述4條表位多肽,高效液相色譜法分析顯示多肽純度i95%。肽結(jié)合實驗表明,隨著表位多肽濃度增加,其與MHC-I類分子的親和力亦逐漸增強,當(dāng)表位多肽濃度達到30μmol/L的時候,4條表位多肽的熒光系數(shù)均1.5,提示具有較高的親和力。選取兩個預(yù)測軟件分析結(jié)果重疊的候選表位肽ROR1 591-599 (DFLHIAIQI)與胸腺肽α1進行偶聯(lián)合成ROR1-Tα1多肽疫苗。ELISA結(jié)果表明ROR1-Tα1疫苗能有效促進小鼠T淋巴細胞的IL-12及IFN-γ的合成分泌,體外具有生物學(xué)活性。免疫保護實驗結(jié)果顯示,ROR1-Tα1組腫瘤生長速度較對照組生長慢且腫瘤平均重量明顯小于對照組,提示ROR1-Tα1多肽疫苗對荷瘤小鼠具有明顯的免疫保護效應(yīng)。結(jié)論：通過預(yù)測軟件獲得與H-2Kd分子具有較高親和力的候選表位多肽,經(jīng)胸腺肽α1修飾制備ROR1-Tα1多肽疫苗,體外具有生物學(xué)活性,體內(nèi)對荷瘤小鼠具有明顯的免疫保護效應(yīng)。為進一步研究乳腺癌的免疫治療奠定了基礎(chǔ)。
[Abstract]:Aim: to predict and identify the H02Kd restricted CTL epitope of tumor antigen ROR1, prepare thymosin 偽 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 偽 1.ELISA method was used to detect the cytokines IL-12 and IFN- 緯 stimulated by RORl-T 偽 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 偽 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 偽 1, were selected to synthesize ROR1-T 偽 1 peptide vaccine. Elisa results showed that ROR1-T 偽 1 vaccine could effectively promote the synthesis and secretion of IL-12 and IFN- 緯 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 偽 1 group was slower than that of control group, and the average tumor weight of ROR1-T 偽 1 group was significantly lower than that of control group, suggesting that ROR1-T 偽 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 偽 1 to prepare ROR1-T 偽 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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1 馬天明;乳腺癌相關(guān)抗原ROR1 H-2Kd限制性CTL表位疫苗制備及其抗腫瘤作用實驗研究[D];重慶醫(yī)科大學(xué);2015年

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本文編號：1817023