High-Level Expression of the Human CB2 Cannabinoid Receptor Using a Baculovirus System

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