Recombinant antibodies for the study of livestock infection : from basic genetics to single-chain Fvs

• Main Author: Hosseini-Nohdani, Arsalan
• Published: University of Glasgow 2002
• Subjects: 636; QR Microbiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.513006

Molecular biology has provided new opportunities to understand better the functioning of the immune system and to exploit this information for the construction of specific antibodies against a wide variety of antigens including the pathogens of humans and animals. In spite of the economic importance of cattle, many aspects of the immunology of this animal remain uncharacterised and tools to understand better bovine infections are lacking. This project has addressed aspects of both issues. The bovine immunoglobulin (Ig) system resembles that of other domesticated mammals in some respects, but other properties (eg the length of the third antigen-binding region of the heavy chain) appear unique. The first area for investigation in this project was to characterise the bovine JH locus and to understand why Ig rearrangement apparently favours a single JH segment. PCR was used to recover JH sequence from genomic DNA, either from non-lymphoid tissues or lambda vectors isolated and studied by other investigators. A region of 3200 bp was characterised which included the DQ52 segment, 6 JH segments and the heavy chain enhancer. The bovine DQ52 sequence is longer than those of other species and differs in sequence from a common consensus. For the most part, the JH locus is homologous to that of the sheep. The sixth JH segment identified appears to undergo rearrangement and is expressed in a minority of cattle antibodies. However, none of the segments carried the sequence which is most commonly expressed in bovine Ig. To identify which segment participates in this process, sequence was recovered from the rearranged genomic DNA of isolated bovine B cells using PCR with primers against VH and JH regions. This implicated the rearrangement of the fourth JH segment in the formation of bovine Igs but as the sequence differed between germline and rearranged copies, it appears that a non-conventional process operates in cattle. It is proposed that a gene conversion event or modified rearrangement process introduces sequence to form the fourth framework region of bovine Ig which does not exist at the JH locus in the germline. The mechanism of this modification requires more investigation. The second part of this project aimed to construct a library of recombinant bovine Fab antibodies from the Ig repertoire of a calf vaccinated against Mannheimia haemolytica (previously named Pasteurella haemolyticd).