Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.
Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.
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通过使用逆转录病毒载体插入人 α1-抗胰蛋白酶 cDNA 进行修饰的小鼠成纤维细胞产生糖基化的生理学“正常”人 α1-抗胰蛋白酶。
DOI：
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发表时间：
1987
影响因子：
11.1
通讯作者：
R. G. Crystal
中科院分区：
文献类型：
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作者：
G. Ri;Anna Chytil;Stefan Karlsson;G. Fells;Mark L. Brantly;Michael Courtney;P. Kantoff;A. Nienhuis;W. Anderson;R. G. Crystal
α 1-抗胰蛋白酶（α 1AT）缺乏症是一种遗传性疾病，其特征是血清α 1AT水平降低，导致中性粒细胞弹性酶破坏下呼吸道。作为一种用生理正常的人类α 1AT增加这种疾病中α 1AT水平的方法，我们将全长的正常人类α 1AT cDNA整合到小鼠成纤维细胞的基因组中。为了实现这一点，通过插入猿猴病毒40早期启动子和α 1AT cDNA来修饰逆转录病毒载体N2。Southern分析表明，在转染的小鼠成纤维细胞psi 2和感染的NIH 3T3克隆的基因组中存在完整的cDNA。克隆产生3个含有人α 1AT序列的mRNA转录本（5.8、4.8和2.4千碱基），分泌一个被抗人α 1AT抗体识别的α 1AT分子，其分子质量（52 kDa）与正常人α 1AT相同，并与人中性粒细胞弹性酶络合并抑制人α 1AT。psi2产生的α 1AT是糖基化的，当静脉注射到小鼠体内时，它的血清半衰期与从人血浆中纯化的正常α 1AT相似，明显长于非糖基化的人α 1AT cdna导向酵母产生的α 1AT。这些研究证明了使用逆转录病毒载体将正常人类α 1AT cDNA插入非α 1AT产生细胞的可行性，从而导致生理上“正常”的人类α 1AT的合成和分泌。
Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.