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Molecular interaction between joining chain and immunoglobulin by BIA

BIA 法观察连接链与免疫球蛋白之间的分子相互作用

基本信息

批准号：
11307043
负责人：
MORO Itaru
金额：
$ 23.93万
依托单位：
Nihon-University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11307043/
关键词：
Mucosal immunology Secretory IgA Joining chain Molecular interaction BIA BIA

项目摘要

Immunoglobulin (Ig) Joining (J) chain is required for polymerization of pIgs such as dimeric IgA and pentameric IgM, and has a well-conserved feature among various vertebrate and invertebrate species. Recombinant protein of chicken J chain (rCJ), molecular mass of 25 kDa, was synthesized from E. coli transformed by chicken J chain CDNA. Interaction between rCJ and Igs was analyzed by BIA CORE 3000 system. In result, rCJ has a high binding capacity to α and μ chains but not those of human. The reactivity between anti-human J chain and rCJ was much weaker than that of anti-chicken J chain. In addition, rCJ was made from cDNA substituted by cysteine residue to serine and analyzed the binding capacity to chicken Igs, resulting that no inhibitory effect was detected between α and μ chains. Chicken genomic J chain clones encoding 14 kb were taken from DNA library. The structure of its gene has 4 exons having a high degree of similarities compared to that of human and mouse. However, in 5'-region encoding 3.8 kb, there is no homologous sequence compared to mouse and cow. Luciferase assay was performed to determine the promoter activity among deletion mutants derived from 5'-region, resulting that luciferase activities were observed in all mutans. Futhermore, enhancer region consisted of 0.5 kb was isolated from 3.8 kb to 3.3 kb upstream and several franscriptional binding sites, such as NF-E2, USF-1, MyoD, CdxA and GATA3, were identified using TRANSFUC data base analysis. These results suggest that transcriptional mechanism of the chicken J chain may be different from that of mammals.

免疫球蛋白（IG）连接（J）链是pIg如二聚体伊加和五聚体IgM聚合所必需的，并且在各种脊椎动物和无脊椎动物物种中具有良好保守的特征。从大肠杆菌中合成了鸡J链重组蛋白（rCJ），分子量为25 kDa。鸡J链cDNA转化大肠杆菌。用BIA CORE 3000免疫分析系统分析rCJ与Ig的相互作用。结果表明，rCJ对人α链和μ链具有很强的结合能力，但对人α链和μ链的结合能力不强。抗人J链与rCJ的反应性远弱于抗鸡J链。此外，将半胱氨酸残基替换为丝氨酸的cDNA制备成rCJ，并分析了rCJ与鸡Ig的结合能力，结果表明α和μ链之间没有抑制作用。从DNA文库中获得编码14 kb的鸡基因组J链克隆。其基因结构有4个外显子，与人和小鼠的基因结构具有高度相似性。然而，在编码3.8kb的5 '-区中，与小鼠和牛相比没有同源序列。荧光素酶测定法测定5 ′-区缺失突变体的启动子活性，结果发现所有突变体均具有荧光素酶活性。从上游3.8 kb至3.3 kb的区域中分离出0.5 kb的增强子区域，并利用TRANSFUC数据库分析确定了几个转录结合位点，如NF-E2、USF-1、MyoD、CdxA和GATA 3。这些结果表明，鸡J链的转录机制可能不同于哺乳动物。