CLONING AND SEQUENCE OF A CDNA CODING FOR THE HUMAN BETA-MIGRATING ENDOTHELIAL-CELL-TYPE PLASMINOGEN-ACTIVATOR INHIBITOR
CLONING AND SEQUENCE OF A CDNA CODING FOR THE HUMAN BETA-MIGRATING ENDOTHELIAL-CELL-TYPE PLASMINOGEN-ACTIVATOR INHIBITOR
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DOI：
10.1073/pnas.83.18.6776
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发表时间：
1986-09-01
影响因子：
11.1
通讯作者：
LOSKUTOFF, DJ
中科院分区：
文献类型：
--
作者：
NY, T;SAWDEY, M;LOSKUTOFF, DJ
.lambda。利用抗体探针对从人胎盘mRNA制备的含cDNA插入片段的gt11表达文库进行免疫筛选。从培养的牛主动脉内皮细胞中纯化的迁移型纤溶酶原激活物抑制剂（. β .- pai）。经7次筛选，分离出34个阳性克隆。105噬菌体。三个克隆(.lambda.1.2, .lambda。随机选取。lambda.9.2)进行进一步表征。它们分别包含1.9、3.0和1.9千碱基（kb）长的插入。大肠杆菌对。lambda。9.2有溶原性，但对。lambda无溶原性。gt11产生了180kda的融合蛋白，该融合蛋白被亲和纯化的抗牛主动脉内皮细胞抗体识别。-PAI和。beta。反向纤维蛋白自显影分析pai活性。对最大的cDNA插入片段进行测序，全长2944个碱基对（bp）。它有一个大的3”非翻译区[1788 bp，不包括poly(a) tail]，包含成熟蛋白的整个编码区，但缺乏起始密码子和部分5”末端的信号肽编码区。对携带1.9 kb cDNA插入片段的两个克隆进行了部分测序，结果显示它们与3.0 kb cDNA完全相同，只是它们被截断，缺少大部分3”非翻译区。以3.0 kb cDNA为杂交探针，对人纤维肉瘤细胞系HT-1080的电泳分离RNA进行印迹杂交分析。检测到两个不同的转录本，分别为2.2和3.0 kb，这表明1.9 kb的cDNA可能是从较短的RNA转录本复制而来的。从cDNA中推断出的氨基酸序列与人类. β .- pai的nh2末端序列一致。基于这种排列，成熟的人类。贝塔。-PAI长379个氨基酸，包含一个nh2末端缬氨酸。推导出的氨基酸序列与α具有广泛的同源性（30%）。1-抗胰蛋白酶和凝血酶III，表明。pai是丝氨酸蛋白酶抑制剂（serpin）超家族的成员。
A .lambda.gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the .beta.-migrating plasminogen activator inhibitor (.beta.-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 .times. 105 phages. Three clones (.lambda.1.2, .lambda.3, and .lambda.9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for .lambda.9.2, but not for .lambda.gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell .beta.-PAI and had .beta.-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3'' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5'' terminus. The two clones carryig the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3'' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human .beta.-PAI. Based on this alignment, the mature human .beta.-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with .alpha.1-antitrypsin and antithrombin III, indicating that the .beta.-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.