HIGH-LEVEL EXPRESSION OF THE HUMAN CB2 CANNABINOID RECEPTOR USING A BACULOVIRUS SYSTEM

A human CB2 recombinant baculovirus (AcNPV-hCB2) was generated by site -specific transposition and employed to express the human CB2 cannabin oid receptor. Northern analysis of total RNA from Spodoptera frugiperd a (Sf9) insect cells infected with AcNPV-hCB2 revealed novel expressio n of a unique 2.3 kb transcript when probed with hCB2 cDNA. This trans cript corresponded to the size expected for hCB2 generated from the re combinant virus construct. Western immunoblot analysis of whole cell h omogenates of recombinant baculovirus-infected Sf9 cells, using affini ty-purified antibody to a human CB2 carboxy terminal domain (anti-hCB2 .CV), revealed the presence of novel immunoreactive protein. In additi on, when anti-hCB2.CV was employed in immunofluorescence staining, an intense signal was observed within AcNPV-hCB2-infected cells but not w ithin uninfected cells or cells infected with a control P-galactosidas e recombinant baculovirus. The pattern of immunofluorescence at early periods post-infection was in a perinuclear arrangement with a ''signe t-ring'' appearance, suggestive of glycosylation of the expressed reco mbinant protein. Transmission electron microscopy revealed regions of intranuclear recombinant virus assembly and the presence of numerous i ntracytoplasmic proteinaceous vesicular inclusions consistent with hyp erproduction of hCB2. Scatchard-Rosenthal analysis of lheptyl)phenyl]- 4-[3-hydroxypropyl]cyclohexan-1-ol ([H-3]CP 55,940) receptor binding i ndicated a K-d of 2.24 nM and a B-max equal to 5.24 pmol/mg of protein . The lack of [H-3]CP 55,940 displacement with l)-4-methyl-1H-pyrazole -3-carboxamidehydrochloride (SR 141716A), the CB1-selective antagonist , confirmed the identity of the receptor as CB2. These data indicate t hat AcNPV-hCB2 expresses high levels of the human CB2, which retains p roperties of the native receptor. Thus, this recombinant virus may pro ve suitable for hyperproduction of receptor for basic biochemical and biophysical characterization studies. (C) 1998 Elsevier Science Inc.