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Purification and properties of the gp340 component of Epstein-Barr virus membrane antigen in an immunogenic form
Abstract:
Preparative SDS-polyacrylamide gel electrophoresis has been used in the purification of the gp340 component of Epstein-Barr (EB) virus-determined membrane antigen (MA), in tractable quantities, from the B95-8 marmoset lymphoblastoid cell line. Successful renaturation of the purified molecule was achieved. This procedure gave a 50-fold increase in the recovery of antigen compared to conventional techniques. The data suggest that the antigenic sites recognized by human sera containing antibodies to MA are largely confined to the protein portion of the molecule. An eightfold improvement in the yield of gp340 was obtained when B95-8 cells were cultured in the presence of 12-O-tetradecanoyl-phorbol-13-acetate. Gel filtration studies indicate that the major polypeptide components of MA are not associated in detergent solution. Immunization of rabbits with purified and renatured gp340 resulted in the generation of high-titre antisera which were specific for gp340, demonstrating that antigen prepared by this procedure is suitable for further evaluation as an experimental vaccine against EB virus infection.
Insights
Researchers purified Epstein-Barr virus (EBV) membrane antigen (MA) using preparative SDS-polyacrylamide gel electrophoresis, achieving a 50-fold increase in recovery. The purified gp340 antigen is suitable for developing an EBV vaccine.
Area of Science:
- Virology
- Immunology
- Biochemistry
Background:
- Epstein-Barr virus (EBV) is a human herpesvirus associated with various malignancies.
- EBV-determined membrane antigen (MA) is a key target for immune responses against EBV infection.
- Efficient purification of MA is crucial for developing diagnostic tools and vaccines.
Purpose of the Study:
- To develop an improved method for purifying the gp340 component of EBV-MA.
- To assess the suitability of the purified gp340 for vaccine development.
Main Methods:
- Preparative SDS-polyacrylamide gel electrophoresis (PAGE) for gp340 purification from B95-8 marmoset lymphoblastoid cells.
- Cell culture optimization using 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to enhance gp340 yield.
- Renaturation of purified gp340.
- Gel filtration chromatography to analyze MA component association.
- Rabbit immunization with purified gp340 to generate specific antisera.
Main Results:
- A 50-fold increase in gp340 recovery was achieved compared to conventional methods.
- Successful renaturation of purified gp340 was accomplished.
- TPA treatment of B95-8 cells resulted in an eightfold improvement in gp340 yield.
- Antigenic sites on MA are primarily located on the protein portion.
- Gel filtration indicated that MA polypeptide components are not associated in detergent solution.
- High-titre, gp340-specific antisera were generated in rabbits.
Conclusions:
- Preparative SDS-PAGE is an effective method for purifying EBV gp340.
- The purified and renatured gp340 antigen is immunogenic and suitable for further evaluation as an experimental EBV vaccine.
- Optimizing cell culture conditions can significantly enhance antigen yield.