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Published on: February 10, 2022
Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay
H T Reyburn1, D J Roy, B A Blacklaws
1Department of Veterinary Pathology, University of Edinburgh, Summerhall, U.K.
Abstract:
The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.
Insights
Researchers cloned the maedi-visna virus (MVV) p25 gene into yeast for high-level expression. The resulting p25 protein is authentic and highly antigenic, enabling sensitive detection assays for MVV.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Maedi-visna virus (MVV) is a lentivirus affecting sheep and goats.
- The major core protein, p25, is crucial for viral structure and immunogenicity.
- Efficient production of recombinant p25 is needed for diagnostic and research purposes.
Purpose of the Study:
- To clone and express the MVV p25 gene in a yeast system.
- To produce a soluble, pure, and antigenically authentic p25 protein.
- To develop sensitive detection assays for MVV.
Main Methods:
- Polymerase chain reaction (PCR) was used to amplify the p25 gene.
- The gene was inserted into yeast Ty-VLP expression vectors for fusion protein production.
- Factor Xa protease was used to cleave the p25 protein from the fusion partner.
- Antigenicity was assessed using specific antisera and antibody generation.
Main Results:
- High-level production (50-60 mg/l) of p25 fusion protein in yeast.
- Soluble and highly pure p25 protein was obtained after cleavage.
- The yeast-derived p25 protein was antigenically authentic and elicited antibodies.
- The cleaved, soluble p25 was more antigenic than the VLP form.
- Sensitive MVV p25 detection assays were developed using the generated reagents.
Conclusions:
- Yeast Ty-VLP expression system is effective for producing recombinant MVV p25 protein.
- The soluble, cleaved p25 protein is a valuable reagent for immunological studies.
- Developed assays will aid in studying MVV replication and immune responses.
