Analysis Of CDR3 Spectratyping And Molecular Features Of TCR Beta Chain In The PBL Of CD4~+T And CD8~+T Cells From Individuals Immunized With Hemorrhagic Fever Renal Syndrome Viccine

Objective:1. To detect the CDR3 of TCR (3 chain gene repertoire in the peripheral blood mononuclear cells of Hemorrhagic fever with renal syndrome-vaccine in CD4+T and CD8+T cells after three Vaccination. Analysis of CDR3 molecular features of single peaks.2. Preliminary Discussion of the Relationship Between CDR3 molecular features and HLA Serotyping.3. Analysis of CDR3 molecular features,sequencing the CDR3 of'clonal dominance' TRBV family.Methods:1. Ten individuals who tested negative for anti-hantavirus antibodies were immunized with Hemorrhagic fever with renal syndrome vaccine. Injections were performed according to the manufacturer's instructions at day 0, day 14 and 6 months after the first vaccine administration.PBLwere obtained from each individual by venipuncture,ficolled and separated into CD4+T and CD8+T cells using immunomagnetic beads obtained from Miltenyi Biotec MHC Class-I and class-II typing was performed in Beijing Genomic Center (BGI)2. Synthesis of primers of 26 TCRβchain variable gene, TCRβchain constant gene, Total RNA of CD4+T cells and CD8+T cells (PBL) from 10 HFRS-viccines were transcripted reversely into cDNA.The PCR products were initially analyzed on 1.5% agarose gel by ethidium bromide staining. The cDNA of 26 TRBV families CDR3 was amplified by FQ-PCR. Analysis of the the CDR3 of'clonal dominance'TRBV family by DNA melting curve. Analysis of CDR3 molecular features, Sequence the products on an ABI 377 DNA sequencer. Analyze the molecular features of CDR3.Results:1. Most of TRBV families show more than three peak and NO change between three Vaccination in 10 individuals.but several families show a single peak after Vaccina-tion,This phenomenon may be prompt that:'clonal dominance'. 2. Not the same as'clonal dominance'TRBV families in CD4+T and CD8+T cells between ten individuals.But different individual have common families.3. Obtained the'clonal dominance'CDR3 sequences for The same as HLA-A loci between individual NO.6 and NO.10.although they also had not same cdr3 sequences, but shared many of same motif. For example:GYT,EQY,NEQ,GG,TEAF,GELF.Conclusion:1. After Vaccination, T cell receptor repertoire have change in CD4+T and CD8+T of ten individuals,suggested that vaccination induces alterations in the T cell receptor (TCR) repertoire. The differences in TCR V gene usage may also be due to different HLA loci between individuals2. Not the same as'clonal dominance'of TRBV families in CD4+T and CD8+T cells between ten individuals.But different individual have common families, suggested that The differences in TCR V gene usage may be due to Antigen-diversity and different HLA loci between individuals3. although The same as HLA-A loci between individual NO.6 and NO.10 had not same cdr3 sequences,but shared many of same motif. suggested that'clonal dominance'TRBV families may be Identified the same antigen epitope.