Prediction Of HLA-A2 Restricted Cytotoxic T Lymphocyte Epitopes Of TAG-1 And The Expression, Purification Of Fusion Proteins Of These Epitopes And Hsp70

As to a kind of malignant tumor from melanocytes, there is still not an ideal therapy which can cure malignant melanoma (MM). However, with the development of the biological technology, many experiments show that the specific active immunotherapy is an effective method in treating MM, and the core of active immunotherapy lies in the immuno-stimulative function of tumor antigens and their cytotoxic T lymphocyte epitopes (CTL epitopes). Therefore there is no need to say that the tumor antigens and CTL epitopes are very important in treating tumors. TAG-1 is a novel Cancer/Testis antigen (C/T antigen) which is widely expressed in the tissue of tumors and not expressed in the normal tissue except testis and placentas. The expression rate can reach from 48 to 88 percent in MMs. So TAG-1can become the ideal candidate antigen of MM vaccine because of its strong immunogenicity, but its research just started at present. The ideal prediction method of CTL epitopes has become a shortcut of discovering new CTL epitopes and may enhance the efficiency of vaccine development greatly. At the same time, the immunity adjuvants are also essential in tumor non-specific immunotherapy, which can be the potent supplement of vaccine treatment. Among these adjuvants, Heat shock protein 70 (Hsp70) plays an important role in the antigen presentation pathway and can be used as a molecular carrier to enhance the antigenicity of antigens linked to Hsp70. So it is utilized widely in tumor treatment. In this study, the HLA-A2 restricted CTL epitopes of TAG-1 were predicted by the methods of combined prediction, epitope reconstruction and imraunological assay. The expression and purification of the fusion proteins with epitopes and Hsp70 can provide a material base of developing peptide vaccine of MM.1. The prediction and filtration of HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitopes of TAG-1AIM: To predict biological characteristic and HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitopes of TAG-1. METHODS: The amino acid sequence of TAG-1 was acquired from GenBank, then predict biological characteristic of TAG-1 by means of the ANTHEPROT V5. 0 software. Long-distance prediction system SYFPEITHI combined with polynomial method was used to predict the HLA-A2 restricted CTL epitopes of TAG—1. Finally identify and filtrate these epitopesin HLA Ligand/Motif Database. RESULTS: The full sequence of TAG-1 consists of 99 amino acids. The analysis of biological characteristic reveals that TAG-1 antigen has antigenicity and several epitope regions. Three HLA-A2 restricted CTL epitopes of TAG-1 were selected according to their scores and they are bwl: LLLRLECNV (49-57), bw2: TLSRLSNRL (41-49), bw3: FLLLLNSTT (83 -91) . CONCLUSION: TAG-1 is a novel C/T antigen with strong immunogenicity and its HLA-A2 restricted CTL epitopes were predicted and filtrated by the combination of SYFPEITHI prediction method and polynomial method.2. The expression of the linkage gene of Hsp70 to three predicted epitopes in the prokaryotic system and the purification of fusion proteins.AIM: To express and purify the fusion proteins of three predicted epitopes and Hsp70 in prokaryotic system. METHODS: Synthesize and anneal the gene sequences of three predicted epitopes and then subclone them into the expression vector in the responding restriction sites to construct PGEX-bw-Hsp70 which contains the genes of Hsp70 and three predicted epitopes. After sequencing and being induced with IPTG, the expressed fusion proteins were purified with GSTrap FF column of CLOTECH company. RESULTS: The fusion genes PGEX-bw-Hsp70 were constructed and the fusion proteins were acquired successfully. CONCLUSION: The fusion proteins of Hsp70 and three HLA-A2 restricted CTL epitopes were expressed and purified successfully, which will be helpful of...