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Title: Understanding chicken BG genes at the RNA and protein levels
Author: Chen, Lei
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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The chicken BG system is a highly polymorphic and polygenic multigene family encoding type I transmembrane proteins, with butryophilins as homologues in mammals, some of which are crucial in T cell regulation. There are three genomic locations where BG genes are found: one singleton BG gene (BG0) on chromosome 2, another singleton gene (BG1) in BF-BL region (the so-called minimal essential chicken MHC) on chromosome 16, and many BG genes arranged tandemly in the BG region just outside the MHC. BG genes in BG region have copy number variation between different chicken haplotypes, so it has been unclear which BG genes are alleles, as very little sequence information has been available for haplotypes other than B12, the best characterized one. Also, the functions of chicken BG genes have been a mystery for half a century, although there is evidence for cytoskeletal regulation and for viral disease resistance. Therefore, the aim of the research was to develop new procedures and reagents to understand the BG system. A novel PCR protocol was established to overcome the difficulty of amplifying full length polymorphic BG transcripts, and then was applied to systematically examine the BG cDNA sequences from T cells and B cells of four different chicken haplotypes. In total 23 BG genes were found, most with alternative splicing isoforms; most strikingly, the transcripts potentially encoding soluble BG proteins were only seen in B cells, indicating functional differences of the same gene in T and B cells. By comparing the dominantly expressed BG genes as 'functional alleles' in these cells, only the cytoplasmic tail region is clearly seen to be under selection, based on the overwhelming preponderance of non-synonymous changes. With many other unexpected findings discovered in this project, a clearer picture of chicken BG genes is presented, and more questions were raised for future study. In order to explore BG functions and further characterize BG proteins, fourteen stable cell lines were developed expressing fusion proteins of the Ig-V domains of the 14 BG genes from the B12 haplotype chicken with the human IgG1 Fc fragment. These BG-Fc fusion proteins were used in sandwich enzyme-linked immunosorbent assays (ELISAs) to screen 290 BG monoclonal antibody (mAb) tissue culture supernatants, and these BG mAbs were further characterized for specificity by western blot using BG-Fc fusion proteins. These solid tools (BG-Fc fusion proteins and BG mAbs) provide the basis to further understand chicken BG functions and answer other interesting questions.
Supervisor: Kaufman, Jim Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: chicken BG genes ; Butyrophilin family ; alternative splicing ; BG-Fc fusion proteins