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Manipulation of Hawaii Virus Capsid Protein and Gene.

Kuria, Grace Wangui. (1999) Manipulation of Hawaii Virus Capsid Protein and Gene. Doctoral thesis, University of Surrey (United Kingdom)..

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Norwalk viruses cause epidemic diarrhoea in human adults; however, the viruses cannot be grown in the laboratory and this has limited their study. A ready supply of recombinant antigen has partially overcome this, but the immune response to these viruses tends to be type specific. In this study, an attempt has been made to make a more cross reactive antigen for use in diagnosis by removing the presumed immunodominant variable regions of the Hawaii capsid protein using two methods: partial proteolysis and genetic engineering. In the proteolytic approach, Bromelain, Elastase, Chymotrypsin and Papain were used in an attempt to remove projections from the surface of the virus. The 58K protein was mostly broken down to 34K, 41K and 43K. This virus-enzyme reaction was not easily controlled and could not be stopped by ice, aprotinin or a cocktail of protease inhibitors: Thus this method was not suitable for particle production and therefore the genetic engineering approach was pursued. In the genetic approach, the capsid gene of the Norwalk-like virus; Hawaii was manipulated to remove the variable, immunodominant regions. This gene was obtained in a recombinant baculovirus and was re-cloned into a more convenient expression vector adding a histidine tag. The variable region of the capsid gene was removed by PCR to amplify the conserved separately and then ligating these together. Finally, the protein was expressed in Sf21 cells and the expression was confirmed by SDS gel electrophoresis.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Kuria, Grace Wangui.
Date : 1999
Additional Information : Thesis (M.Phil.)--University of Surrey (United Kingdom), 1999.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 12:07
Last Modified : 06 May 2020 12:14

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