Generation of polyclonal antibodies against Theiler's Murine Encephalomyelitis virus protein 2C, and their use in investigating localisation of the protein in infected cells
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
An investigation of the binding capacities of recombinant domain mutants of the human Polymeric Immunoglobulin Receptor (pIgR)
- Authors: Prinsloo, Earl Adin Gerard
- Date: 2006
- Subjects: Immunoglobulins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10307 , http://hdl.handle.net/10948/403 , Immunoglobulins
- Description: The membrane bound glycoprotein, polymeric immunoglobulin receptor (pIgR) is the primary transport molecule of the polymeric immunoglobulins, dimeric IgA and pentameric IgM, across epithelial cells. This process, known as transcytosis, is essential in order to establish immunity at mucosal surfaces. Typically, pIgR binds to the polymeric immunoglobulin at the basolateral surface of the epithelial cell, via five homologous immunoglobulin-like domains of the ectodomain. Binding is covalent to IgA and non-covalent to IgM; the IgM binding varying among species. The pIgR-bound complex is released at the apical surface of the cell after cleavage of pIgR at Arg585, thereafter referred to as secretory component (SC). SC confers protective and immunologic functions to the polymeric immunoglobulin. Free SC, i.e. not complexed with polymeric immunoglobulins, is also known to be released into mucosal secretions; and binds to pathogenic bacteria and bacterial products. It is known that domain I of the ectodomain is the primary domain in the interaction with polymeric immunoglobulins, while domain V is involved in a covalent linkage with IgA. However, little is known of domains II-IV and their role in immunoglobulin binding, particularly to IgM. This study aimed to characterize the binding of recombinant human pIgR domain mutants to polymeric IgM using immunological, biophysical and cell based techniques; thereby allowing greater insight into the contribution of each of the five domains. The unique domain structure allowed for selective amplification of single and multiple domain mutants from cloned human PIGR ectodomain cDNA. Mutants were cloned and expressed in Esherichia coli BL21 (DE3) as inclusion bodies. Recombinant mutant proteins were refolded in vitro by equilibrium gradient dialysis and purified to homogeneity. Equilibrium binding data show significant contributions to specific binding as a factor of domain presence. Binding kinetics determined by biophysical surface plasmon resonance measurements show the interplay between association and dissociation rates as defined by individual domains. In vitro competitive binding studies using the human intestinal carcinoma, HT29, known to constitutively express pIgR, show that the constructed recombinant domain mutants outcompete native pIgR. The level of competition is shown to be dependant on the domains downstream of domain I. The data also confirm the biological activity of the first in vitro refolded recombinant human SC.
- Full Text:
- Date Issued: 2006
- Authors: Prinsloo, Earl Adin Gerard
- Date: 2006
- Subjects: Immunoglobulins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10307 , http://hdl.handle.net/10948/403 , Immunoglobulins
- Description: The membrane bound glycoprotein, polymeric immunoglobulin receptor (pIgR) is the primary transport molecule of the polymeric immunoglobulins, dimeric IgA and pentameric IgM, across epithelial cells. This process, known as transcytosis, is essential in order to establish immunity at mucosal surfaces. Typically, pIgR binds to the polymeric immunoglobulin at the basolateral surface of the epithelial cell, via five homologous immunoglobulin-like domains of the ectodomain. Binding is covalent to IgA and non-covalent to IgM; the IgM binding varying among species. The pIgR-bound complex is released at the apical surface of the cell after cleavage of pIgR at Arg585, thereafter referred to as secretory component (SC). SC confers protective and immunologic functions to the polymeric immunoglobulin. Free SC, i.e. not complexed with polymeric immunoglobulins, is also known to be released into mucosal secretions; and binds to pathogenic bacteria and bacterial products. It is known that domain I of the ectodomain is the primary domain in the interaction with polymeric immunoglobulins, while domain V is involved in a covalent linkage with IgA. However, little is known of domains II-IV and their role in immunoglobulin binding, particularly to IgM. This study aimed to characterize the binding of recombinant human pIgR domain mutants to polymeric IgM using immunological, biophysical and cell based techniques; thereby allowing greater insight into the contribution of each of the five domains. The unique domain structure allowed for selective amplification of single and multiple domain mutants from cloned human PIGR ectodomain cDNA. Mutants were cloned and expressed in Esherichia coli BL21 (DE3) as inclusion bodies. Recombinant mutant proteins were refolded in vitro by equilibrium gradient dialysis and purified to homogeneity. Equilibrium binding data show significant contributions to specific binding as a factor of domain presence. Binding kinetics determined by biophysical surface plasmon resonance measurements show the interplay between association and dissociation rates as defined by individual domains. In vitro competitive binding studies using the human intestinal carcinoma, HT29, known to constitutively express pIgR, show that the constructed recombinant domain mutants outcompete native pIgR. The level of competition is shown to be dependant on the domains downstream of domain I. The data also confirm the biological activity of the first in vitro refolded recombinant human SC.
- Full Text:
- Date Issued: 2006
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