GENERATION AND CHARACTERIZATION OF ANTIPEPTIDE ANTIBODIES TO RAT CYTOCHROME-P-450 SIDE-CHAIN CLEAVAGE ENZYME
GENERATION AND CHARACTERIZATION OF ANTIPEPTIDE ANTIBODIES TO RAT CYTOCHROME-P-450 SIDE-CHAIN CLEAVAGE ENZYME
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DOI：
10.1016/0303-7207(91)90090-f
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发表时间：
1991-08-01
影响因子：
4.1
通讯作者：
SOARES, MJ
中科院分区：
文献类型：
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作者：
ROBY, KF;LARSEN, D;SOARES, MJ
在这份报告中，我们描述了大鼠P-450SCC的免疫探针的产生。鉴定了P-450SCC氨基酸序列的两个区域（内部结构域：氨基酸421 - 441;羧基末端结构域：氨基酸509 - 526），化学合成并用作兔中的免疫原。通过酶联免疫测定（EIA）和Western印迹分析监测抗体产生。成功地产生了识别整个49 kDa大鼠P-450SCC蛋白的每个P-450SCC区域的抗血清。针对P-450SCC的内部结构域的抗血清显示出广泛的物种交叉反应性，而针对P-450SCC的羧基末端结构域的抗血清仅与大鼠和小鼠交叉反应。这两种抗血清可用于大鼠P-450SCC表达的Western blot和免疫细胞化学分析。除了识别主要的49 kDa P-450SCC蛋白外，每种抗血清还识别较低分子量的物质。针对P-450SCC的内部结构域的抗血清特异性地识别42kDa的种类，而针对羧基末端结构域的抗血清特异性地识别8kDa的种类。我们推测，这两个较低分子量的免疫反应性物种产生的内部和羧基末端表位之间的大鼠P-450SCC的蛋白水解裂解。
In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.