Development for CMV vaccine using the cytotoxic peptide after transplantation

使用移植后细胞毒性肽开发巨细胞病毒疫苗

• 批准号: 09671245
• 负责人: TSUNODA Takuya
• 金额: $ 1.92万
• 依托单位: Wakayama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671245/
• 关键词: CMV; CTL; peptide; Vaccination; transplantation

CMV pp65 specific CTL clone was established by limiting dilution method. In order to map the epitope peptide which was restricted by HLA-B35 and specific for CMV pp65, various kinds of truncated pp65 recombinant vaccinia virus were established by homologous recombination. Full (606)-pp65 rVac, 458-pp65 rVac, 383-pp65 rVac, 316-pp65 rVac, 225-pp65 rVac, 122-pp65 rVac and 0-pp65 rVac were established. Mapping was performed by CTL clone against the target infected by various kinds of pp65 truncated recombinant vaccinia virus. It was clarified that the epitope peptide was located from 112 amino acid (aa) to 225 aa from N terminal of pp65 sequence. Furthermore, in order to make sure that epitope peptide was processed on the target cells, a large amounts of target cells were cultured, and were washed by acid buffer (pH2) to remove the peptides from the target cell. By HPLC, this peptides solution was fractionated and applied to LCL (EBV immortalized cell).For analysis of the cytotoxic activity by CTL clone. It was also clarified that epitope peptide was on the cell surface which binds with MHC molecules.

用有限稀释法建立CMV pp 65特异性CTL克隆。为定位CMV pp 65特异性的HLA-B35限制性抗原表位肽，采用同源重组的方法构建了多种pp 65截短型重组痘苗病毒。建立了完整（606）-pp 65 rVac、458-pp 65 rVac、383-pp 65 rVac、316-pp 65 rVac、225-pp 65 rVac、122-pp 65 rVac和0-pp 65 rVac。用不同pp 65截短型重组痘苗病毒感染靶细胞，通过CTL克隆进行定位。结果表明，该表位肽位于pp 65序列N端112 ~ 225个氨基酸之间。此外，为了确保表位肽在靶细胞上被加工，培养大量靶细胞，并用酸性缓冲液（pH 2）洗涤以从靶细胞去除肽。用高效液相色谱法将此多肽溶液分级分离，并应用于LCL（EBV永生化细胞），通过CTL克隆分析其细胞毒活性。还阐明了表位肽位于细胞表面，其与MHC分子结合。

项目成果

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Tsunoda T.：“拓扑异构酶-1 抑制剂 CPT-11 对新鲜分离的人类胃癌和结直肠癌的体外抗肿瘤作用。”Anticancer Res。

Yamaue H.: "Multidisciplinary treatment for gastric cancer patients by chemoimmunotherapy."Hepato-Gastroenterology. 46. 620-625 (1999)

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Terasawa H等：“原位结肠癌模型中白细胞介素2基因修饰的成纤维细胞的抗肿瘤作用”Jpn.J.Cancer Res..90.1000-1006(1999)

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角田卓也他: "グラム陽性球菌の薬剤耐性における最近の動向(第2報)・近畿地区におけるペニシリン耐性肺炎球菌に関するアンケート調査成績"日本感染症学会雑誌. 71. 6-10 (1997)

Takuy​​a Tsunoda等：“革兰氏阳性球菌耐药性的最新趋势（第2次报告）-近畿地区耐青霉素肺炎球菌的问卷调查结果”日本传染病学会杂志71. 6-。 10 (1997)