Use of a Pan-V beta primer permits the amplification and sequencing of TCR beta chains expressed by bovine T-cell clones following a single semi-nested PCR reaction

We report in this Study the design and validation of a Pan-V beta primer that in combination with C beta-specific primers enables the amplification, in a single semi-nested PCR. of TCRP chains expressed by bovine T-cell clones irrespective of the expressed V beta sequence. Using the Pan-V beta primer we examined the TCRP chains expressed by 16 Theileria pan,a-specific clones that had not been previously analysed. TCRP chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRP chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-V beta printer to amplify the TCRP chains expressed by 19 T-cell clones. on which previous analysis using V beta-subfamily-specific primers had failed to do. sequencing of these TCRP chains has identified members of 2 novel bovine V beta subfamilies-V beta 5 and V beta X. This method offers a simple and rapid method of analyzing the TCR beta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses. (C) 2008 Published by Elsevier B.V.