Primary Analysis Of CDR3 Spectratyping And Molecular Features Of TCR Beta Chain In The Peripheral Blood Of CD4~+T And CD8~+T Cells From Individuals Immunized With HBV Vaccine

Objective:The aim of this study was to find the change of spectral patterns in CD4+ and CD8+T cell receptors(TCR) b-chains complementarity determining region 3 (CDR3) before and after immunized with HBV Vaccine. The peripheral blood was obtained from 20 donors (Buyi, Dong, Miao and Han). And to analyse the association between CD8+ T cell CDR3 lineage and HLA-A, so as CD4+ T cell CDR3 lineage and HLA-DR.Furthermore, CDR3 region gene and protein were sequenced to those CD4 and CD8 T cell families which were the monoclonal or oligoclonal expanded after immunized with HBV vaccine.Methods:(1)By drawing 3ml no anticoagulant peripheral blood from the volunteers in the national class of 2008 of zunyi medical college,electricitychemiluminescence screen negative for hepatitis B surface five markers of 20 volunteers hepatitis B vaccine;(2)According to HBV Vaccination procedures and requirements to complete the three HBV vaccine inoculation;(3)Peripheral blood samples were collected (15ml/times) before and after the vaccination;(4)Immunomagnetic beads (Magnetic Activated Cell Sorting. MACS) separate CD4+T cells and CD8+ T cells from the PBL samples, flow cytometry sorting their purity;(5)Extracted CD4+T cells and CD8+T cells total RNA, reverse transcribed cDNA,we design 26 TRBV appropriate primers according to the 26 TCRβ-chain variable region gene family, designed a shared TRBC primer in the constant region,using fluorescence quantitative PCR (fluorescence quantitative polymerase chain reaction, FQ-PCR) amplification of 26 TRBV family CDR3 section contains the complete cDNA; FQ-PCR melting curve analysis of HBV vaccine immunization vaccinated volunteers before and after CD4+T, CD8+T cells in the 26 TCRβchain TRBV drift characteristics of the family pedigree CDR3;(6)Collecting EDTAK2 anticoagulant blood 5ml of 20 cases (Buyi, Dong, Miao and Han) HBV vaccine volunteers vaccinated, genomic DNA, sequencing HLA-A, HLA-DR allele sequence by using PCR-SBT (Sequencing based typing. SBT);(7)Select the part of HBV vaccine volunteers after vaccination CDS+ T and CD4+T cell repertoire map showed a single peak of volunteers TRBV family PCR product of PCR in the same general conditions, the 1.5% agarose gel electrophoresis Gel purified, cloned and sequence. Nucleotide sequences of TCR CDR3 of the PCR products were analyzed by using DNA tools 6.0 and so on.Result:(1)20 volunteers's HBV surface antibodies titre after third HBV vaccine vaccinated are greater than 10IU/L;(2)20 volunteers with HBV vaccine vaccinated after inoculation CD4+T, CD8+T cells 26 TRBV CDR3 of the melting curve family pedigree chart, family and most of those before inoculation did not change, but each volunteer hanve a family or a few TRBV CDR3 advantages;(3)20 volunteer with HBV vaccine before vaccination and after vaccination were vaccinated CD4+T and CD8+T cells showed the advantages used TRBV not the same family;(4)HLA are the same between the different vaccinated volunteers taking advantage of the family together;(5)Significant advantages of the same national volunteers are not found the use of TRBV CDR3 spectrum;Part of the multi-peak into a single peak from the family of HLA same volunteers were sequenced, We have not found the same CDR sequences.Conclusions:(1)The third HBV vaccine HBS-Ab titer of 20 volunteers were greater than 10IU/L, it is prompted to produce the immune response to all volunteer;TCR CDR3 changes in spectrum of HBV vaccinated immunization volunteers before and after inoculation suggesting that HBV vaccination appropriately induces T cell responses of body;(2)CD4+T and CD8+T cells showed the advantages of using TRBV family CDR3 diversity, HBV antigen and individual diversity of molecule HLA between the different T cell responses resulted in the diversification of the body, while T cell response of HBV and the performance of HLA have some associativity;(3)Common TRBV family and CDR3 area were not entirely found among the same natian and HLA epitopes volunteers, it suggestes that HBV T cell response after vaccination may have more cross-reactivity.