受体－A蛋白融合蛋白制备抗P物质受体血清及其特性

受体－Ａ蛋白融合蛋白制备抗Ｐ物质受体血清及其特性ＷａｌｔｅｒＫ．Ｋ．Ｈｏ（何国强）；ＡｌｅｘｉｓＰ．Ｋ．Ｃｈｅ，郑德枢（香港中文大学；香港生物化学系，香港沙田；广州医学院神经科学研究所，广州５１０１８２）本文报告了用直肠杆菌表达Ｐ物质受体－Ａ蛋白融合...

PRODUCTION AND CHARACTERIZATION OF AN ANTI-SUBSTANCE P RECEPTOR SERUM WITH A RECOMBINANT RECEPTORPROTEIN A FUSION

Substance P is a neuropeptide found widely distributed in the central and peripheral nervous system.Its physiological function is believed to be involved in the transmission and modulation of nerve signals. To characterize the distribution of substance P receptors in the central and peripheral nervous system and to correlate this information with the functional role of the peptide, a well characterized antiserum against the receptor is needed. Inthis study, we have generated a putative antiserum against the substance P, tachykinin-1 receptor by expressingthe receptor as a protein A-fusion protein in E. colt. Although many attempts have been made to prepare a full fusion using various techniques, the best result we have obtained was a protein A-fusion consisting of a truncatedprotein. This fusion protein was purified by affinity chromatography on an IgG column and preparative SDSPAGE. The purified protein was then used as the antigen for immunization. By Western blotting and immunohistochemical studies, the antiserum raised was shown to contain antibodies which could react with tissues or regionsknown to bind substance P. Significantly higher signals were detected in the medial habenular nucleus, hypothalamus, olfactory bulb and layer I and I of the posterior horn of the spinal cord. Furthermore, this antiserum couldpotentiate the action of substance P on the guinea pig ileum contraction bioassay.