Development for CMV vaccine using the cytotoxic peptide after transplantation

用有限稀释法建立了CMV pp65特异性CTL克隆。为了定位人类白细胞抗原B35所限制的巨细胞病毒pp65特异性表位多肽，通过同源重组，构建了不同截短的pp65重组痘苗病毒。建立了Full(606)-pp65 rVac、458-pp65 rVac、383-pp65 rVac、316-pp65 rVac、225-pp65 rVac、122-pp65 rVac和0-pp65 rVac。用CTL克隆对不同pp65截短重组痘苗病毒感染的靶标进行定位。结果表明，该表位多肽位于pp65序列N端112个氨基酸(Aa)~225aa之间。此外，为了确保表位多肽在靶细胞上被加工，大量的靶细胞被培养，并用酸性缓冲液(PH2)洗涤以去除靶细胞中的多肽。经高效液相色谱分离后，应用于EBV永生细胞，用CTL克隆法检测其细胞毒活性。表位多肽存在于细胞表面，与MHC分子结合。

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.