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Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Brien, Kayleen Fransiena."

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    Identification of infectious bursal disease virus (IBDV) receptors through the use of recombinant capsid protein, VP2.
    (2014) Brien, Kayleen Fransiena.; Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
    Infectious bursal disease virus (IBDV) is a non-enveloped Birnavirus which infects the immature antibody producing B-cells of the bursa of Fabricius in young chickens. The virus causes infectious bursal disease (IBD) which is highly contagious and immunosuppressive. A compromised immune system in infected chickens leaves them susceptible to other opportunistic pathogens and as a result increases their mortality rate. Major economic losses in the commercial poultry industry are subsequently experienced in affected regions. Currently vaccines are used to control IBDV infection, however, their efficacy is affected by factors such as the presence of maternally derived antibodies in young chickens which reduces vaccine load, the continuous emergence of new virulent IBDV strains and bursal atrophy caused by some vaccines. It is therefore important to consider new ways of controlling the virus such as targeting specific stages in the virus life cycle. Since virus attachment to host cell receptor(s) is the most crucial step in the virus life cycle, developing novel antiviral agents which prevent viral entry represents a good alternative strategy for IBDV control. Identification of receptor binding proteins and receptors of host cell membranes is required for antiviral development. The receptor binding protein and outer capsid of IBDV is VP2, however, the receptor(s) utilised by IBDV to gain entry into host cells have not been conclusively identified. Recombinant VP2 was used to identify possible IBDV receptor(s) on bursal plasma membranes using a virus overlay protein binding assay (VOPBA) and affinity chromatography. Therefore, VP2 was heterologously expressed in an Escherichia coli and a Pichia pastoris expression system as a 64 kDa fusion protein and a 47 kDa protein respectively. In addition, both systems expressed VP2 as high molecular mass proteins which were confirmed by electro-elution and western blotting. Although purification of VP2 expressed in the E. coli system was a challenge because VP2 expressed as inclusion bodies, polyclonal chicken anti-VP2 antibodies were produced using VP2 expressed in this system. Purification of VP2 expressed in P. pastoris was easier and produced a greater yield of VP2 which was used to produce a VP2-coupled affinity matrix for the purification of chicken anti-VP2 antibodies and for the purification of VP2-binding proteins of the bursal plasma membrane. Moreover, peptides were selected from the VP2 amino acid sequence and use to raise polyclonal chicken anti-VP2 peptide antibodies for comparative identification against chicken anti-VP2 antibodies of possible IBDV receptor(s). Two IBDV VP2-binding proteins with molecular masses of 70 and 32 kDa of the bursal plasma membrane were identified in a VOPBA using recombinant VP2 or IBDV and chicken anti-VP2 antibodies. In addition to the VOPBA, four IBDV VP2-binding proteins with molecular masses of 70, 60, 45 and 32 kDa were affinity purified on a VP2-coupled affinity matrix. Analysis of the affinity purified proteins by mass spectrometry identified five proteins which share common peptides which include, the Ig-gamma chain and Ig-lambda chain of Gallus gallus, outer major protein of Serratia marcescens, the 60 kDa chaperonin of Pseudomonas fluorescens and elongation factor-Tu of Yersinia pestis. The results strongly suggest that an Ig-receptor like protein may form part of the IBDV receptor, however, much further work is required in order to establish whether the chicken homologues of the identified bacterial sequences are part of the putative bursal receptor. It is believed that the bacterial proteins contain common peptides with chicken proteins of the chicken genome which has not been fully annotated as yet. Taken together, this study successfully used VP2 to identify possible IBDV receptor(s) on bursal plasma membranes which could ultimately lead to the development of antiviral agents targeted at IBDV entry.