Variable regions of a human anti-DNA antibody O-81 possessing lupus nephritis-associated idiotype.
Variable regions of a human anti-DNA antibody O-81 possessing lupus nephritis-associated idiotype.
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具有狼疮肾炎相关独特型的人抗 DNA 抗体 O-81 的可变区。
DOI：
10.1093/nar/20.10.2601
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发表时间：
1992
影响因子：
14.9
通讯作者：
Hiroshi Sano
中科院分区：
文献类型：
--
作者：
Y. Hirabayashi;Y. Munakata;Takeshi Sasaki;Hiroshi Sano
一种抗DNA抗体独特型（Id），称为0-81 Id，存在于人类IgM单克隆抗单链（ss） DNA抗体的抗原结合位点，来源于一例活动性狼疮肾炎患者的0-81克隆（1,2）。0-81 Id在狼疮性肾炎患者的循环免疫复合物IgG和肾免疫沉积物中特异性检测（3,4）。0-81的paratopes负责0-81的独特型表达（未发表数据）。这些结果提示，0-81的序列分析可能有助于了解人类致病性自身抗体的起源。采用Micro Fast Track （Invitrogen, San Diego， CA）技术从0-81克隆细胞中制备Poly (A+) RNA，用oligo-d (T) 12_18引物引物制备ss-cDNA。采用聚合酶链反应（PCR）扩增可变区使用以下引物：vv3 -leader 5' atggagtttgggctgagc3 ', CIL 5' ggagagagaggcacgttcttcttttcttttcttttgcaag 3', vv2 -leader 5' gaagctta - tgaggctccctgctc3 ', Ck 5‘TCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG 3’。产物分别经EcoRI（重链）和HindM-XbaI（轻链）酶切，连接到载体pUC1 18中。用链终止法确定序列。为避免TaqI聚合酶误入，独立进行3次DNA扩增，每次至少获得1个克隆。这些克隆体的序列是相同的。核苷酸序列为VH3-DHQ52-JH4和VK2-JK5。VH片段0-81与VHII种系h1i基因同源性最高(5)，同源性为86。9%匹配（leader-FR3）。是also86。9%与胎儿肝脏cDNA文库中FL2-2基因同源(6)。VK片段与种系V6410基因同源性为98.1%(7)。推测VK片段由V6410基因或其相关基因编码，而VH片段可能来源于未报道的VH3种系基因。
An anti-DNA antibody idiotype (Id), termed 0-81 Id, was in the antigen-binding sites of human IgM monoclonal anti-single stranded (ss) DNA antibodies secreted from the 0-81 clone, derived from a patient with active lupus nephritis (1, 2). The 0-81 Id was specifically detected in circulating immune complex IgG and renal immune deposits of patients with lupus nephritis (3, 4). The paratopes of 0-81 were responsible for the idiotypic expression of 0-81 (unpublished data). These findings suggested that the sequence analysis of 0-81 may contribute to understanding the origin of pathogenic autoantibodies in humans. Poly (A+) RNA was prepared from cells of 0-81 clone by using Micro Fast Track (Invitrogen, San Diego, CA) and used to prepare ss-cDNA which was primed with oligo-d (T) 12_18 primer. For amplification of the variable regions by polymerase chain reaction (PCR) the following primers were used: VH3-leader 5'ATGGAGTTTGGGCTGAGC 3', CIL 5'TGGAAGAGGCACGTTCTTTTC 3', Vk2-leader 5'GAAGCTTA-TGAGGCTCCCTGCTC 3', Ck 5'TCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG 3'. The products were digested by EcoRI (heavy chain) and HindM-XbaI (light chain), respectively, and were ligated into the vector pUC1 18. Sequences were determined by the chain termination method. To avoid misincorporation of TaqI polymerase, DNA amplification was independently performed three times and at least one clone was obtained at each time. The sequences of these clones were identical.The nucleotide sequences revealed VH3-DHQ52-JH4 and VK2-JK5. The VH segment of 0-81 showed the highest homology with the VHII germline H 1I gene (5), with86. 9% matching (leader-FR3). Itwas also86. 9% homologous with the FL2-2 gene obtained from cDNA library of fetal liver (6). The VK segment was 98.1% homologouswith the germline V6410 gene (7). It was suggested that the VK segment was encodedby the V6410 gene or its relative genes but the VH segment might be originated from an unreported VH3 germline gene.