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Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line
Published on: September 28, 2022
Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1
Ya A Lomakin1,2, A A Shmidt1, T V Bobik1,2
1M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Abstract:
Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.
Insights
Recombinant Epstein-Barr virus latent membrane protein 1 (LMP1) fragments, CTAR1/2/3, were produced and purified. These fragments demonstrated immunogenicity comparable to full-length LMP1, aiding in functional and diagnostic antibody studies.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) intracellular fragments (CTAR1/2/3) are implicated in viral transformation.
- Understanding LMP1 fragment function and immunogenicity is crucial for EBV research.
Purpose of the Study:
- To generate and purify recombinant LMP1 CTAR1/2/3 fragments for further study.
- To investigate the functional and immunogenic characteristics of these LMP1 fragments.
- To perform epitope mapping of a diagnostic anti-LMP1 antibody using a new fragment library.
Main Methods:
- Development of genetic constructs for prokaryotic expression of LMP1 CTAR fragments.
- Optimization of recombinant protein production and purification protocols.
- Epitope mapping utilizing a library of LMP1 CTAR fragments and a diagnostic antibody (S12).
- Analysis of polyclonal antibodies from immunized mice.
Main Results:
- Successful production and purification of recombinant LMP1 CTAR1/2/3 fragments.
- Epitope mapping of the anti-LMP1 antibody S12 was performed.
- Immunogenicity of CTAR elements was confirmed and found comparable to full-length LMP1.
Conclusions:
- Recombinant LMP1 CTAR fragments can be effectively produced and purified.
- These fragments are immunogenic and suitable for studying LMP1's functional and diagnostic aspects.
- The generated fragment library aids in antibody epitope mapping and understanding EBV pathogenesis.
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