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，以临床皮肤紧张性水疱和组织学表皮下水疱为特征。患者循环中有针对皮肤基底膜区成分的自身抗体，称为BP180或类天疱疮抗原。我们之前报道了单克隆抗体产生细胞系的建立。该单克隆抗体诱导新生小鼠表皮下水疱。本次科研资助的目的是，首先对该单克隆抗体可变区进行分子克隆，其次在小鼠体内重建该单克隆抗体，制作动物模型。为此，我们以poly - A RNA为模板和引物，采用逆转录聚合酶链反应（RT-PCR）技术，获得免疫球蛋白可变区cDNA。RT-PCR产物的凝胶电泳结果显示，重链cDNA和轻链cDNA分别为单带，分子量符合预期。每个cDNA被克隆到测序载体上。重链和轻链cDNA的核苷酸序列和推导出的氨基酸序列表明，它们与其他大鼠免疫球蛋白具有高度的同源性，并在高可变区具有特异性序列。这些cdna被包含5 ‘非编码序列和3 ’剪接信号的引物重新扩增。在大肠杆菌表达系统中首次证实了免疫球蛋白cDNA产物的表达。用载体转染大肠杆菌粗提物，用12.5% sds -聚丙烯酰胺凝胶电泳，以抗标签抗体为第一抗体进行免疫印迹。Western blot显示预期分子量为单条带，表明该cDNA确实具有开放阅读框，并且开放阅读框与标记序列在同一帧内。用免疫荧光法检测重组cDNA产物的功能。用抗标签抗体检测融合蛋白与基底膜的结合情况。综上所述，实验程序几乎完全实现。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