4 results on '"Dempster, Robert"'
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2. Production of an active recombinant Aspin antigen in Escherichia coli for identifying animals resistant to nematode infection
- Author
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Manderson, Daniel, Dempster, Robert, and Chisti, Yusuf
- Subjects
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ESCHERICHIA coli , *TRICHOSTRONGYLUS , *NEMATODES , *IMMUNOASSAY - Abstract
Abstract: Production of recombinant Aspin, a aspartyl protease inhibitor homologue produced by the parasitic nematode Trichostrongylus colubriformis, in Escherichia coli is reported. Culture conditions were investigated for maximizing the production of Aspin in soluble bioactive form as opposed to inclusion body. High growth and expression rates caused preferential production of inclusion bodies. In fed-batch fermentations, controlling expression at low values by decreasing the bioreactor temperature, dissolved oxygen level and concentration of nutrients, all proved effective in enhancing the production of soluble Aspin. A high volumetric titre of 220mg/L Aspin was attained in batch fermentations induced with 2g/L l-arabinose with the postinduction temperature reduced to 25°C from 37°C. The pH and dissolved oxygen levels were not controlled, as acidic final pH values and low dissolved oxygen levels favored production of soluble Aspin. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
- View/download PDF
3. A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep.
- Author
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Cameron, Timothy C., Cooke, Ira, Faou, Pierre, Toet, Hayley, Piedrafita, David, Young, Neil, Rathinasamy, Vignesh, Beddoe, Travis, Anderson, Glenn, Dempster, Robert, and Spithill, Terry W.
- Subjects
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SHEEP parasites , *FASCIOLA hepatica , *PROTEOMICS , *VETERINARY immunology , *IMMUNOTHERAPY , *FASCIOLIASIS , *THERAPEUTICS - Abstract
A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4 weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica , we hypothesised that antibody from F. gigantica -infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica . Purified IgG from the sera of F. gigantica -infected Indonesian Thin Tail sheep collected pre-infection and 4 weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica , suggesting that the Indonesian Thin Tail week 4 IgG is either recognising individual proteins released from extracellular vesicles or is immunoprecipitating intact exosome-like extracellular vesicles. Five extracellular vesicle membrane proteins were identified including two proteins predicted to be associated with vesicle transport/ exocytosis (VPS4, vacuolar protein sorting-associated protein 4b and the Niemann-Pick C1 protein). RNAseq analysis of the developmental transcription of the 38 immunosloughate proteins showed that the sequences are expressed over a wide abundance range with 21/38 transcripts expressed at a relatively high level from metacercariae to the adult life cycle stage. A notable feature of the immunosloughates was the absence of cytosolic proteins which have been reported to be secreted markers for damage to adult flukes incubated in vitro, suggesting that the proteins observed are not inadvertent contaminants leaking from damaged flukes ex vivo. The identification of tegument protein antigens shared between F. gigantica and F. hepatica is beneficial in terms of the possible development of a dual purpose vaccine effective against both fluke species. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
4. Molecular characterisation and vaccine efficacy of two novel developmentally regulated surface tegument proteins of Fasciola hepatica.
- Author
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McCusker, Paul, Toet, Hayley, Rathinasamy, Vignesh, Young, Neil, Beddoe, Travis, Anderson, Glenn, Dempster, Robert, McVeigh, Paul, McCammick, Erin, Wells, Duncan, Mousley, Angela, Marks, Nikki J, Maule, Aaron G., and Spithill, Terry W
- Subjects
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FASCIOLA hepatica , *VACCINE effectiveness , *OPISTHORCHIS viverrini , *CATTLE vaccination , *SCHISTOSOMA japonicum , *CLONORCHIS sinensis , *FASCIOLIASIS - Abstract
• Two novel sequences encoding F. hepatica tegument proteins were characterized. • Expression of both Fhteg1 and Fhteg5 is upregulated 2–4 days post excystment. • Fhteg1 and Fhteg5 proteins are expressed in cell bodies and at the tegument surface. • RNAi knockdown of these genes did not affect juvenile growth or motility in vitro. • A recombinant Fhteg1 and Fhteg5 vaccine elicited protection in rats but not cattle. The surface tegument of Fasciola hepatica is a crucial tissue due to its key role at the host-parasite interface. We characterised three novel proteins, termed Fhteg1, Fhteg5 and Fhteg8, that are found in the tegument membrane fraction of adult F. hepatica. Bioinformatic analysis of proteomic datasets identified Fhteg5 and Fhteg8 as tegumental glycoproteins and revealed that Fhteg1, Fhteg5 and Fhteg8 are associated with exosomes of adult F. hepatica. Fhteg1, Fhteg5 and Fhteg8 appear to be related to uncharacterised sequences in F. gigantica , Fasciolopsis buski, Echinostoma caproni, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum and S. mansoni , although F. hepatica appears to have expanded this family. Fhteg1 and Fhteg5 were characterised in detail. The Fhteg1 and Fhteg5 gene transcripts each demonstrate significant upregulation in juvenile fluke 2–4 days post-excystment, with transcript levels maintained during development over 3 weeks in vitro. RNAseq data showed that both Fhtegs are expressed in the adult life stage, although the transcript levels were about 8 fold lower than those in juveniles (3 week post infection). Using immunocytochemistry, Fhteg1 and Fhteg5 were each shown to be expressed in cells adjacent to the muscle layer as well as on the surface of 1 week old juveniles, whilst Fhteg5 was also present in cells at the base of the pharynx. RNAi mediated knockdown of Fhteg1 and Fhteg5 transcripts in 4–10 day old juveniles had no effect on parasite survival, movement or growth in vitro. Although no IgG responses were observed for Fhteg1 or Fhteg5 during infection in sheep and cattle, both proteins elicited a low IgG response in a proportion of infected rats. Rats vaccinated with Fhteg1 and Fhteg5 showed good IgG responses to both proteins and a mean 48.2 % reduction in worm burden following parasite challenge. Although vaccination of cattle with both proteins induced a range of IgG responses, no protection was observed against parasite challenge. This is the first study to provide insights into the molecular properties of two novel, developmentally regulated surface tegument proteins in F. hepatica. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
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