A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues.
A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues.
复制标题
克隆的嗜铬粒蛋白 A (CgA) cDNA 可检测多种神经内分泌组织中的 2.3Kb mRNA。
DOI：
10.1016/0006-291x(86)91226-x
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发表时间：
1986
影响因子：
3.1
通讯作者：
Mellon,PL
中科院分区：
文献类型：
--
作者：
Deftos,LJ;Murray,SS;Burton,DW;Parmer,RJ;O'Connor,DT;Delegeane,AM;Mellon,PL
CgA是一种未知功能的72 Kd蛋白，存在于许多神经内分泌组织中，并与其固有激素共同分泌。我们准备了一个cDNA文库的表达载体λ gt 11的CgA生产的人甲状腺髓样癌（MTC）细胞的mRNA。用一组针对CgA的一种多克隆抗体和两种单克隆抗体筛选文库。CgA抗体的特异性通过免疫测定、免疫组织学和来自CgA产生组织的mRNA的体外翻译产物的免疫沉淀来证明。显色二抗鉴定了5个免疫反应性克隆。经EcoR Ⅰ酶切和琼脂糖凝胶电泳后，分离它们的cDNA插入片段。这些cDNA经~（32）P标记后作为北方杂交探针。用cDNA探针在显示产生CgA的MTC和肺癌的人细胞系中以及在人嗜铬细胞瘤和牛肾上腺髓质组织中检测到约2.3Kb的mRNA。为了确认其身份，将推定的CgA cDNA之一亚克隆到质粒中，并用于杂交-阻止CgA mRNA的体外翻译。这些研究证明了与来自不同神经内分泌组织的CgA mRNA杂交的cDNA的克隆。
CgA is a 72Kd protein of unknown function that is present in many neuroendocrine tissues and co-secreted with their resident hormones. We prepared a cDNA library to the mRNA from CgA-producing human medullary thyroid carcinoma (MTC) cells in the expression vector lambda gt11. The library was screened with a panel of one polyclonal and two monoclonal antibodies to CgA. The specificity of the antibodies for CgA was demonstrated by immunoassay, immunohistology, and immunoprecipitation of the in vitro translation products of mRNA from CgA-producing tissues. A chromogenic second antibody identified five immunoreactive clones. Their cDNA inserts were isolated after EcoRI digestion and agarose gel electrophoresis. These cDNAs were32P-labelled and used as probes in Northern hybridization studies. An mRNA of approximately 2.3Kb was detected with the cDNA probes in human cell lines from MTC and lung cancers that were shown to produce CgA and in human pheochromocytoma and bovine adrenal medulla tissue. To confirm its identity, one of the putative CgA cDNAs was subcloned into a plasmid and was used to hybridization-arrest the in vitro translation of CgA mRNA. These studies demonstrate the cloning of cDNAs which hybridize with CgA mRNA from diverse neuroendocrine tissues.