Prediction of HLA-A2.1 restricted CTL epitope of SSX gene family and affinity assay of these epitopes with MHC-Ⅰ molecule / 第三军医大学学报

Objective To explore the immunotherapeutic strategy based on SSX tumor gene family by the CTL epitope prediction of the members of SSX tumor gene family and affinity assay of the epitope combining with MHCⅠ molecule.Methods The CTL epitope in the members of SSX gene family were predicted with the predictive software of HLA-A2.1 restricted CTL epitope.The 4 predicted epitope peptides were synthesized: P1(AMTKLGFKA),P4 (AMTKLGFNV),P5(AMTKLGFKV) and P6(VMTKLGFKV).After the affinity between these peptides and MHC-Ⅰ molecule were examined respectively,the combining stability between these peptides and the MHC-Ⅰ molecule were investigated respectively.Results As compared with positive control peptide HBcAg18-27,all of the synthesized peptides except P1 showed high affinity with MHC-Ⅰ molecule.The combining stability assay indicated that dissociation complex 50(DC_(50)) for P1,P4 and the positive control HBcAg 18-27 was all above 8.0 h,but DC_(50) for P5 and P6 was between 2 h and 4 h.Conclusion Two ideal peptides were screened out from the predicted CTL epitope peptides by in vitro MHC-Ⅰ combining affinity and stability assay,which provided the experimental data for the further identification of these epitope peptides in vivo.

Cloning,expression and identification of SARS-CoV S1 gene in yeast p.methanolica / 第三军医大学学报

Objective To obtain high-yield and easy-purification severe acute respiratory syndrome coronavirus(SARS-CoV) S1 protein with biological activity and to study the activity of S1 protein and its antibody further.Methods SARS-CoV S1 gene was inserted into yeast expression vector pMET?A by ligation reaction.The recombinant plasmid was verified by enzyme digestion and sequencing,followed by being transformed into yeast host strain PMAD11 with electroporation.After induced with methanol,the S1 gene expression was verified with overlay assay and Western blotting.Results The positive clones of S1 gene into pMET?A were approved by restriction enzyme digestion and sequencing.The expression of S1 protein was confirmed subsequently by overlay assay and Western blotting.Conclusion SARS-CoV S1 gene has been cloned and expressed in yeast p.methanolica,which can provide experimental data for next study on the activity of this protein and its antibody during SARS-CoV infection.