Development for CMV vaccine using the cytotoxic peptide after transplantation

用极限稀释法建立CMV pp65特异性CTL克隆。为了定位受HLA-B35限制的CMV pp65特异性表位肽，通过同源重组建立了多种pp65短链重组痘苗病毒。建立了全(606)-pp65 rVac、458-pp65 rVac、383-pp65 rVac、316-pp65 rVac、225-pp65 rVac、122-pp65 rVac和0-pp65 rVac。利用CTL克隆对多种pp65截断型重组痘苗病毒感染的靶标进行定位。结果表明，该表位肽位于pp65序列N端112 ~ 225个氨基酸处。此外，为了确保表位肽在靶细胞上加工，我们培养了大量的靶细胞，用酸性缓冲液（pH2）洗涤以去除靶细胞上的肽。用高效液相色谱法对多肽溶液进行分离，并应用于EBV永生化细胞。用于分析CTL克隆的细胞毒活性。结果表明，表位肽位于细胞表面，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.