Abstract
The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S- transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.
Original language | English |
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Pages (from-to) | 3115-3126 |
Number of pages | 12 |
Journal | Vaccine |
Volume | 24 |
Issue number | 16 |
DOIs | |
State | Published - Apr 12 2006 |
Bibliographical note
Funding Information:Contributors: We are grateful to Drs. Alan G. Wildeman and Mhairi Skinner (Department of Molecular and Cellular Biology, University of Guelph) for providing mammalian cell lines used in this study. We thank Ms. Annette Morrison (Central Animal Facility, University of Guelph) for technical help on mouse injections and bleeding. We also thank Drs. Frances Sharom and Roselynn Stevenson (Department of Molecular and Cellular Biology, University of Guelph) for use of the FluorChem 8900 (Alpha Innotech) and the Fluorescence Microscope (FM), respectively, in their laboratories, Ms. Dilya Kamalova and Mr. Steve Lord for technical assistance. This work was supported by grants to Dr. Peter Krell from NSERC, Canadian Forestry Service, Rohm and Haas Company, Ontario Genomics Institute and Genome Canada, and Biocontrol Canada.
Keywords
- Choristoneura fumiferana
- DNA vaccine
- Ecdysteroid receptor
ASJC Scopus subject areas
- Molecular Medicine
- General Immunology and Microbiology
- General Veterinary
- Public Health, Environmental and Occupational Health
- Infectious Diseases