Construction of a transgenicmouse bearing pathogenic antibody to pemphigoid

大疱性类天疱疮是一种组织特异性自身免疫性疾病，以皮肤临床紧张性水泡和组织学上的真皮下水泡为特征。患者有针对皮肤基底膜区成分的循环自身抗体，称为BP180或类天疱疮抗原。我们先前报道了一株产生单抗的细胞系的建立。该单抗可诱导新生小鼠产生皮下水泡。本次资助的目的是，一是对该单抗可变区进行分子克隆，二是在小鼠体内重建该单抗，制作动物模型。为此，我们以聚A核糖核酸为模板和引物，通过逆转录聚合酶链式反应(RT-PCR)获得了免疫球蛋白可变区基因。RT-PCR产物凝胶电泳法显示重链和轻链均为单一条带，预期分子量较大。每个cDNA都是亚…更多克隆到测序载体中。这些分子的重链和轻链基因的核苷酸序列及其推导的氨基酸序列表明，它们与其他大鼠免疫球蛋白有很高的同源性，并在高变区具有特异性序列。用含有5个碱基非编码序列和3个碱基剪接信号的引物对这些cDNA进行再扩增。用大肠杆菌表达系统首次证实了免疫球蛋白基因产物的表达。将转化载体的大肠杆菌粗提物应用于12.5%的SDS-聚丙烯酰胺凝胶电泳板上，以抗Tag抗体为一抗进行免疫印迹。Western印迹显示一条预期分子量的单一条带，表明该基因确实有一个开放阅读框架，并且该开放阅读框架位于标签序列的框架内。免疫荧光法检测重组表达产物的功能。用抗TAG抗体检测融合蛋白与基底膜的结合。总而言之，实验程序几乎完全完成了。较少

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