PCV2b ORF2 Protein Mutation Prevents Nuclear Accumulation
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Solution Overview
Problem
The recombinant expression of PCV2b ORF2 protein in insect cells is hindered by natural nuclear accumulation, leading to reduced expression levels and difficulty in harvesting and purifying the protein, which affects the immunogenicity and availability of PCV2 subunit vaccines.
Innovation Solution
Introducing a specific amino acid substitution, changing Threonine at position 131 to Proline in the PCV2b ORF2 protein, prevents nuclear accumulation, increasing expression levels and facilitating protein harvesting and purification, allowing for the production of effective virus-like particles for vaccines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCV2b ORF2 protein is expressed in insect cells using conventional methods, then the protein can be produced, but it accumulates in the nucleus leading to reduced expression levels and difficulty in harvesting
Solution Approach 1:
The invention changes the amino acid sequence parameter of the ORF2 protein by introducing specific mutations (e.g., substitution of amino acid at position 131 from threonine to proline) to alter the protein's subcellular localization pattern, preventing nuclear accumulation and improving both expression levels and ease of harvesting
2Reliability
If PCV2b ORF2 protein is expressed in insect cells, then vaccine production is enabled, but nuclear accumulation reduces antigenic quality
Solution Approach 1:
By modifying the amino acid sequence parameters of ORF2 protein through site-directed mutagenesis, the invention alters the protein's nuclear localization signal properties, thereby improving both the quantity of protein produced and its antigenic quality for vaccine applications
Data Source
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AI summary
The present invention relates to the field of veterinary vaccines, in particular to porcine vaccines against PCV2 and associated diseases. Specifically the invention relates to the finding that a mutation is required in PCV2b ORF2 protein, to prevent its nuclear accumulation upon expression in insect cells; the mutation introduces a Proline at amino acid position 131. This allows efficient expression in insect cells, easy harvesting, and generates large amounts of virus-like particles. The VLPs are highly effective in vaccines for porcines for reduction of infection by PCV2 or of associated signs of disease.