Related Experiment Videos
High-level expression of the human CB2 cannabinoid receptor using a baculovirus system
K W Nowell1, D A Pettit, W A Cabral
1Department of Microbiology/Immunology, Medical College of Virginia of Virginia Commonwealth University, Richmond 23298-0678, USA.
Biochemical Pharmacology
|August 26, 1998
Summary
A novel baculovirus vector effectively expresses high levels of the human CB2 cannabinoid receptor in insect cells. This recombinant virus is suitable for producing the CB2 receptor for biochemical studies.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The human CB2 cannabinoid receptor plays a crucial role in immune system regulation.
- Efficient expression systems are needed for detailed biochemical and biophysical characterization of the CB2 receptor.
Purpose of the Study:
- To generate a recombinant baculovirus for high-level expression of the human CB2 receptor.
- To characterize the expressed CB2 receptor in Sf9 insect cells.
Main Methods:
- Site-specific transposition to create AcNPV-hCB2 recombinant baculovirus.
- Northern and Western immunoblot analyses to confirm gene and protein expression.
- Immunofluorescence staining and transmission electron microscopy for cellular localization and visualization.
- Radioligand binding assays (Scatchard-Rosenthal) to determine receptor binding kinetics.
Main Results:
- AcNPV-hCB2 successfully expressed a 2.3 kb transcript and immunoreactive human CB2 protein in Sf9 cells.
- Immunofluorescence revealed perinuclear localization suggestive of glycosylation.
- Electron microscopy showed intracellular inclusions consistent with hyperproduction.
- Binding assays yielded a Kd of 2.24 nM and Bmax of 5.24 pmol/mg, with selective binding confirmed by lack of displacement with a CB1 antagonist.
Conclusions:
- The AcNPV-hCB2 system achieves high-level expression of functional human CB2 receptor.
- The expressed receptor retains native binding properties.
- This recombinant baculovirus is a valuable tool for future biochemical and biophysical studies of the CB2 receptor.