The BVDV genome is a positive sense RNA molecule with one open reading frame (ORF) encoding for a polyprotein which is cleaved into the structural and non-structural proteins. BVDV is a member of the Pestivirus genus within the Flavivirus family. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.īovine viral diarrhoea virus (BVDV) infection of cattle is linked to economically important diseases with losses in the USA being estimated to US$10-40 million per million calves and US$6 million per million calves in the UK. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. coli resulted in predominantly aggregated insoluble IB. The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB). The E2 protein contains 17 cysteine residues creating difficulties in E. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. coli contain endotoxins which need to be removed for in vivo applications. Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies.
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