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Primary Analysis of the Character of TRBV F&P Gene Family CDR3 in the Peripheral Blood of Mother and Son by Technique of CE&HTS

Author: HeXiaoYan
Tutor: YaoXinSheng
School: Zunyi Medical College,
Course: Immunology
Keywords: complementarity determining region 3 (CDR3) Roche 454 GS FLX high through-put sequencing (HTS) pseudogene/functional gene capillary electrophoresis(CE) T cell receptor repertoire
CLC: R392.1
Type: Master's thesis
Year: 2011
Downloads: 11
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Abstract

Objective(1) Analysis of the 26 TRBV CDR3 length and polymorphism in the peripheral blood of mother and son donors by Capillary Electrophoresis.(2) Analysis of the composition and character of the part of TRBV Functional gene CDR3 and TRBV Pseudogene CDR3 in the healthy donors samples by Cloning and Sequencing.(3) Analysis of the composition and character of TRBV6, TRBV9, TRBV10, TRBV19 CDR3 receptor repertoire with the Roche 454 GS FLX sequencing technology.Method(1) The peripheral blood was collected from ten healthy donors,including mother and son(2 donors sample), mother and twin sons(3 donors sample) and other healthy donors(5 donors sample). Isolating total RNA from PBMC of all samples. Synthesis of the first cDNA by total RNA respectively.(2) Design and synthesis of primers of 26 TRBV family gene and one TRBC gene (labeled with FAM), PCR amplification of CDR3 cDNA.(3) Analysis of the 26 TRBV CDR3 length and polymorphism in the peripheral blood of seven healthy donors by Capillary Electrophoresis technique.(4) Cloning and sequencing the products of 6 healthy donors TRBV6 CDR3 and one donor TRBV10 and TRBV19 CDR3 and analysising the molecular features of CDR3.(5) Design and synthesis of primers of TRBV6/TRBV9/TRBV10/TRBV19 and TRBC with a unique primers in the 5’end for different samples, HTS the CDR3 repertoire by Roche 454 GS FLX. Result(1) There are different length and polymorphism of 26 TRBV CDR3 spectratyping in seven donors and most of the 26 TRBV CDR3 show Gaussian distribution by CE genescan. CDR3 region length and polymorphism in some families have similar trends in the sample of mother and son donors.(2) Cloning and sequencing results show that the TRBJ of TRBV6 family in the mother and son donors have the same or similar motifs and have the frequency of TRBJ dominant identical in some TRBJ families.(3) Roche 454 GS FLX sequencing results show that TRBV6/TRBV9/TRBV10/TRBV19 CDR3 in-frame gene or amino acid overlap rate in the samples of mother and son(or twins) increased significantly larger than the samples of non-genetic relationship.The numbers of out-frame CDR3 of TRBV10/TRBV19 more than in-frame CDR3.Conclusion(1) TCR CDR3 composition and characteristics show some of correlation in the sample of mother and son (or twins), it would be helpful in exploring the"public bias" in TCR CDR3 in some disease with genetic lineage.(2) The in-frame and out-frame CDR3 data of TRBV10 and TRBV19 might be interesting to light on the further study of the functional gene and pseudogenes of TRBV.(3) The Roche 454 GS FLX sequencing technology for analysising the composition and character of the human TCR beta chain CDR3 have been established initially in our laboratory and provides a powerful research tool for the study about the TCR CDR3 repertoire.

Primary Analysis of the Character of TRBV F&P Gene Family CDR3 in the Peripheral Blood of Mother and Son by Technique of CE&HTS

Abstract

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