The study of physiological function of #O1 late gene involved in morphine dependence

• Published in: Japanese Journal of Pharmacology 1998, Vol.76 (suppl.1), p.171-171
• Main Authors: Ikemoto, Mitsushi, Inoue, Koutarou
• Format: Article
• Language: English
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1016/S0021-5198(19)40795-6

We have previously reported that #O1 gene was isolated by the subtractive cDNA cloning from male ddy mice (6week-old) amygdala and that its mRNA was up-regulated only by long-term morphine administration. This #O1 gene encoded a secreted calcium binding extracellular mairix protein. In the present study, we first investigated the effect of morphine withdrawal on #O1 mRNA level. Quantitative RT-PCR analysis revealed twofold enhancement of #O1 mRNA expression in basolateral amygdala nucleus. This up-regulation was persisted for at least 14 days after withdrawal. Sensitization of #O1 mRNA expression was observed when an additional morphine (100 mg/kg, s.c.) was injected at 1, 7 and 14 days after cessation of morphine, suggesting that the state of neural connections would be changed by repeated morphine administration. Second, to analyze the physiological function in brain, the soluble full coding #O1 protein fused to thioredoxin at Nterminal end (#O1-Trx, 52.2kd) was expressed with pET32 system by AD494 strain. This protein was purified with Ni^2+ affinity chromatography and gel filtration chromatography (>95% estimated by CBB staining). By using this purified protein, we have been trying to make a polyclonal antibody.