Construction of a transgenicmouse bearing pathogenic antibody to pemphigoid

携带类天疱疮病原性抗体的转基因小鼠的构建

基本信息

批准号：
09670877
负责人：
TANAKA Toshihiro
金额：
$ 2.18万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Bullous pemphigoid is a tissue specific autoimmune disease, characterized with clinical tense blister of the skin and histological subepidermal blistering. Patients have circulating autoantibodies against the component of basement membrane zone of the skin, named BP180 or pemphigoid antigen. We previously reported the establishment of a monoclonal antibody producing cell line. This monoclonal antibody induce subepidermal blister in neonatal mice. The purpose of this Grant-in aid for scientific research is, firstly, the molecular cloning of variable region of this monoclonal antibody and secondary, reconstruct this monoclonal antibody in mice to make an animal model. For this purpose, we fished immunoglobulin variable region cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) by using poly A RNA as a template and primers. Gel electrophoreseis of RT-PCR products revealed single band for heavy and light chain cDNA respectively with expected molecular weight. Each cDNA was sub … More cloned into sequencing vector. The nucleotide sequences of these heavy and light chain cDNA and deduced amino acid sequences of these molecules revealed that they have high homologies to other rat immunoglobuline and that they possess specific sequence at hyper variable regions. These cDNAs were reamplified with primers that contains 5 prime non-coding sequence and 3 prime splicing signals. An expression of immunoglobulin cDNA product was first confirmed by E.coli expression system. A crude extract of E.coli which was transfected with vector was applied to 12.5% SDS-polyacrylamide gel electrophoreses and immunoblot was performed with anti-tag antibody as first antibody. Western blot showed a single band of expected molecular weight, indicating that this cDNA do have an open reading frame and that the open reading frame is in frame to tag sequence. Function of reconstituted cDNA product was assayed by immunoflourescent study. Binding of fusion protein to basement membrane was detected by anti-tag antibody. In summary, experimental procedures are almost fully achieved. Less

大胆的烟碱是一种特异性的自身免疫性疾病，其特征是皮肤的临床时泡和组织学亚表皮泡沫。患者具有针对皮肤基底膜区域的成分的自身抗体，称为BP180或Pemphigoid抗原。我们先前报道了单克隆抗体产生细胞系的建立。这种单克隆抗体在新生儿小鼠中诱导皮下注射水泡。首先，这种对科学研究的授予援助的目的是该单克隆抗体的可变区域的分子克隆和次要的分子克隆重建了小鼠中这种单克隆抗体以形成动物模型。为此，我们通过使用Poly A RNA作为模板和引物，通过逆转录酶 - 聚合酶链反应（RT-PCR）捕获了免疫球蛋白可变区域cDNA。 RT-PCR产物的凝胶电泳揭示了具有预期分子量的重链和轻链cDNA的单条带。每个cDNA均为sub…更多地克隆到测序载体中。这些重链cDNA的核序列以及这些分子的推导氨基酸序列表明它们对其他大鼠免疫球蛋白具有很高的同源性，并且它们在多变区域具有特定的序列。这些cDNA用包含5个原始非编码序列和3个原始剪接信号的引物进行封闭。首先通过大肠杆菌表达系统证实了免疫球蛋白cDNA产物的表达。用载体翻译的大肠杆菌的粗提取物被施加到12.5％的SDS-聚丙烯酰胺凝胶电泳和免疫印迹中，用抗TAG抗体作为第一抗体进行。 Western印迹显示了一个预期分子量的单个带，表明该cDNA确实具有开放的阅读框架，并且开放的阅读框在框架中以标记序列。通过免疫荧光研究分配了重构cDNA产物的功能。通过抗TAG抗体检测到融合蛋白与基底膜的结合。总而言之，实验程序几乎完全实现了。较少的