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Title: Cloning expression and characterisation of the globular head regions of mouse CIQ
Authors: Shastri, Abhishek
Advisors: Kishore, U
Keywords: CIQ;Complement;gCIQ;Mouse CIQ
Issue Date: 2014
Abstract: The classical pathway of complement system, activated by immune complexes, involves binding of globular heads of C1q to the Fc regions of aggregated IgG or IgM. At the C-terminal region of C1q, is globular head region (gC1q) where each globular head is composed of one A (ghA), one B (ghB) and one C (ghC) chain. In order to understand modularity of gC1q region of mouse C1q and to generate useful reagents for in vivo studies, we have expressed these recombinant individual heads of mouse C1q in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). This included RNA extraction and cDNA synthesis from mouse spleen, using appropriate primers, full-length mouse C1q A, B and C chains were amplified first. Following this, using the appropriate full length C1q chains, globular heads of mouse C1q A, B and C chains were amplified (mghA, mghB and mghC respectively). We have examined their interaction with heat-aggregated mouse IgG. These fusion proteins (MBP-ghA, -ghB, and -ghC) were found to bind differentially to mouse IgG. ghA and ghB were found to bind highest with IgG. IgG subtypes were purified and their interaction with native human C1q was also studied. Dose-dependent binding was observed. Interaction of recombinant globular heads of C1q with mouse IgM was also examined. ghC was found to bind highest with IgM although ghB and ghA also showed binding to IgM. The ability of these recombinant heads to inhibit complement-mediated lysis was also examined. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG sensitized sheep erythrocytes. Interaction and binding of these globular heads with recombinant prion peptide was also studied. The individual globular heads of mouse C1q were also found to bind to recombinant mouse PrP peptide which will aid in better understanding of pathogenesis of prion disease. Further studies involved interaction of recombinant mouse globular heads of C1q (mghA, mghB and mghC) with pentraxin PTX-3. mghB and mghC showed highest binding (mghB>mghC) to PTX3 as compared to mghA. The study of recombinant gC1q region heads will help in understanding the effects of PTX3 activation on the complement system. Collaborative studies examined the most variable residues in mouse gC1q region as well as alignment of mouse gC1q region with human gC1q region. The expression and functional characterisation of individual mouse gC1q domains has allowed to examine specificity and selectivity of individual globular heads of mouse C1q to known ligands. Production of mouse gC1q domains will be helpful in antibody mapping of anti-C1q autoantibodies recognising gC1q region.
Description: This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University
Appears in Collections:Biological Sciences
Dept of Life Sciences Theses

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