| Production and identification of antisera against mu-opioid receptor using synthetic peptide epitope |
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Jang-hern Lee1, Young-bae Kwon1, Ho-jae Han2 |
1Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University
2Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University |
| Synthetic peptide를 이용한 mu-opioid receptor에 대한 항혈청의 생산과 검정 |
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이장헌1, 권영배1, 한호재2 |
1서울대학교 수의과대학 생리학교실
2전남대학교 수의과대학 생리학교실 |
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| Abstract |
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In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398 of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide synthesizer. The specificity and identification of the antisera were tested by analysis of transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR. MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as endoplasmic reticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity was abolished when anti-MOR sera were preincubated with the peptide against which they were raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which was determined to be within the last amino acid, 391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was observed in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain areas showed the mirrored pattern observed in auto radiographic studies of mu-opioid binding as well as a pattern similar to that seen by is situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future. |
| Key Words:
mu-opioid receptor, rabbit antisera, immunohistochemistry, COS-7 cell, confocal microscope |
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