?2m-Linker-HLA A2 Protein And Melan A Peptide Combined To Induce The Activation Of Melan A-specific TCR-transduced CD8~+Jurkat

ObjectTo explore whether?2m-Linker-HLA A2 protein and Melan A peptide?ELAGIGILTV?that can be recognized and presented by HLA A2 can induce Melan A-specific TCR-transduced CD8⁺Jurkat cells which provides a new idea for screening antigen peptide specific CTL in the future.Methods1. ?2m-Linker sequence and Linker-HLA A2-His tag sequence wereamplified from PBMC of HLA A2 positive healthy donor,then?2m-Linker and Linker-HLA A2-His tag were connected through SOE-PCR method to get?2m-Linker-HLA A2-His tag sequence which was cloned into pc DNA3.4 vector.2. The pc DNA3.4/?2m-Linker-HLA A2-His tag plasmid wastransfected into Expi293F cells.The protein of interest was purified through Ni affinity chromatography.3. ?2m-Linker-HLA A2 protein was identified by SDS-PAGE?Western blot?ELISA assay.4.The expression of CD8 on CD8⁺Jurkat cells and Melan A-specific TCR on Melan A-specific TCR-transduced CD8⁺Jurkat cells was detected by flow cytometry.5.The group that?2m-Linker-HLA A2 combined with Melan A peptide stimulated Melan A-specific TCR-transduced CD8⁺Jurkat cells to active specific CTL was named the combined group?S-1?;The group that using Melan A peptide only was called the experimental group?S-2?.The group that using unrelated peptide only was named as negative group?N-1?.The cells used in the control groups were CD8⁺Jurkat cells that were not transfected with Melan A-specific TCR.Then after 24 hours,the expression of CD137 in each group was detected by flow analysis.Results1.The recombinant plasmid structure of pc DNA3.4/?2m-Linker-HLA A2-His tag was successfully confirmed by enzyme digestion and sequencing.2.The recombinant plasmid structure of pc DNA3.4/?2m-Linker-HLA A2-His tag was expressed fusion protein in 293F cells.3. For?2m-Linker-HLA A2 protein,there was a single protein bandbetween molecular weight 40KDa and 55KDa detected by SDS-PAGE,which confirmed that the protein was successfully purified;specific band between molecular weight 40KDa and 55KDa was appeared by Western blot;ELISA results showed that the difference of the OD_450nm value of the experimental group??2m-Linker-HLA A2?and the OD_450nm value of the negative group?Irrelevant protein?and the blank group?PBS?were significant?P<0.05?.So the results of the above experiments confirmed that the purified protein was?2m-Linker-HLA A2 protein.4. CD8⁺Jurkat cells were stained with FITC-conjugated anti-humanCD8 antibody,and Melan A-specific TCR-transduced CD8⁺Jurkat cells were stained by Melan A-Tetramer labeled with BV421 fluorescein.FCM showed that the expression rate of Melan A-specific TCR on Melan A-specific TCR-transduced CD8⁺Jurkat cells was up to 84.2%,and the expression rate of CD8 on CD8⁺Jurkat cells was 69.7%5. After 24 hours,the combined group?S-1?can effectively active MelanA-specific TCR-transduced CD8⁺Jurkat cells,which the expression rate of CD137 was 2.64%.Compared with the experimental group?S-2?,the negative group?N-1?and the control group,there was a significant difference?P<0.05?.ConclusionStimulation of Melan A-specific TCR-transduced CD8⁺Jurkat cells by the combined use of?2m-Linker-HLA A2 protein and Melan A peptide can active tumor-specific CTL.It is beneficial for the cytotoxicity experiment on melanoma tumor cells subsequently.And it provides a new idea and method for the screening of antigen-peptide specific CTL.