High-level expression of the human CB2 cannabinoid receptor using a baculovirus system.
Parole chiave
Astratto
A human CB2 recombinant baculovirus (AcNPV-hCB2) was generated by site-specific transposition and employed to express the human CB2 cannabinoid receptor. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-hCB2 revealed novel expression of a unique 2.3 kb transcript when probed with hCB2 cDNA. This transcript corresponded to the size expected for hCB2 generated from the recombinant virus construct. Western immunoblot analysis of whole cell homogenates of recombinant baculovirus-infected Sf9 cells, using affinity-purified antibody to a human CB2 carboxy terminal domain (anti-hCB2.CV), revealed the presence of novel immunoreactive protein. In addition, when anti-hCB2.CV was employed in immunofluorescence staining, an intense signal was observed within AcNPV-hCB2-infected cells but not within uninfected cells or cells infected with a control beta-galactosidase recombinant baculovirus. The pattern of immunofluorescence at early periods post-infection was in a perinuclear arrangement with a "signet-ring" appearance, suggestive of glycosylation of the expressed recombinant protein. Transmission electron microscopy revealed regions of intranuclear recombinant virus assembly and the presence of numerous intracytoplasmic proteinaceous vesicular inclusions consistent with hyperproduction of hCB2. Scatchard-Rosenthal analysis of [3H]-(-)3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxypro pyl]cyclohexan-1-ol ([3H]CP 55,940) receptor binding indicated a Kd of 2.24 nM and a Bmax equal to 5.24 pmol/mg of protein. The lack of [3H]CP 55,940 displacement with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamidehydrochloride (SR 141716A), the CB1-selective antagonist, confirmed the identity of the receptor as CB2. These data indicate that AcNPV-hCB2 expresses high levels of the human CB2, which retains properties of the native receptor. Thus, this recombinant virus may prove suitable for hyperproduction of receptor for basic biochemical and biophysical characterization studies.
