Title:
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Molecular analysis of ovine CD1 expression
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The aim of the work carried out in this thesis was to further investigate the ovine CD1 family and clarify the existing information at the cellular and molecular level. Initial studies utilised existing anti-CD1 mAbs to clarify the pattern of tissue expression of ovine CD1. Two distinct clusters of mAbs were shown to exist - the majority recognise a molecule with tissue distribution similar to CD1b whilst three mAbs, SBU-T6, CC43 and CC118 demonstrate staining of tissue macrophages, the majority of B cells and monoctyes in addition to thymocytes and dendritic cells. NH2-terminal sequencing was subsequently used to establish the antigens recognised by the mAbs SBU-T6 and CC14. This technique demonstrated that the CC14 antigen was consistent with the predicted sequence of the SCD1B42 cDNA clone whereas the SBU-T6 antigen had closest homology to the predicted amino-acid sequence of the human CD1E gene. This is particularly noteworthy as no protein product of the CD1E gene has yet been described in any species. Subsequent work attempted to isolate the gene encoding the molecule recognised by SBU-T6 using a transient expression system in which COS cells were transfected with a lymph node cDNA library contained within the vector pcDNA3. This was unsuccessful, however a sheep CD1D-like sequence was isolated from this library utilising primers based on the NH2-terminal sequence of the SBU-T6 antigen. Expression of the SCD1D gene was investigated using in situ hybridization and RT-PCR. SCD1D transcripts were demonstrated in thymus, liver, intestine, lymph node and PBLs. A further experiment investigated the expression of the SCD1B52 gene (which contains a precise deletion of exon 4). These studies have extended the knowledge of the ovine CD1 family and establish it as one of the most complex described to date. This work has demonstrated that sheep clearly express multiple CD1 isotypes as in man and rabbits in addition to the multiple CD1B-like genes reported previously.
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