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Development of a method to analyze a novel secretion gland specific gene.

开发一种分析新型分泌腺特异性基因的方法。

基本信息

批准号：
16590457
负责人：
TATSUMI Ke-ita
金额：
$ 1.98万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590457/
关键词：
secretion gland novel gene

项目摘要

The novel Pi-a protein was expressed secretion gland, pituitary gland, stomach or pancreas, at mRNA level, but functions or protein expression were unknown. To understand its protein expression, I raised antibodies(Ab) against Pi-a,Firstly, I raised antisera against synthetic peptide and a recombinant protein. Synthesized peptide Pi-a(C+961〜972), common to human and mouse, was immunized, and antiserum showed good reaction to the native antigen. Recombinant protein, mouse Pi-a/His tag, was purified with His tag, immunized, and antiserum showed good reaction to the native antigen. When these antisera were tested by Western blot analyses, both of them failed to show specific signals for secretion glands. Thus I continued to raise further antisera. Three peptides, 154-168, 519-533, 999-101, were synthesized, immunized and antisera showed good reaction to the native antigen after purification by peptide affinity columns. By Western blot analyses, these antisera were tested for their specificity against secretion tissues, such as, pituitary gland, stomach and pancreas, but all of them failed to show specific signals for these tissues. To understand the physical functions of Pi-a, I planned to raise Pi-a deficient mice through gene ablation, I analyzed the nucleotide database elucidated human and mouse Pi-a genome structure with their promoters, and produced recombinant vector for knockout mouse. But further study was abandoned for the lack of antisera for Pi-a for further analyses

新的Pi-a蛋白在分泌腺、垂体、胃或胰腺中以mRNA水平表达，但其功能或蛋白表达尚不清楚。为了了解其蛋白质表达，我制备了针对 Pi-a 的抗体（Ab），首先，我制备了针对合成肽和重组蛋白的抗血清。采用人和小鼠常见的合成肽Pi-a(C+961〜972)进行免疫，抗血清对天然抗原表现出良好的反应。重组蛋白小鼠Pi-a/His标签，用His标签纯化，免疫，抗血清对天然抗原表现出良好的反应。当这些抗血清通过蛋白质印迹分析进行测试时，它们都未能显示出分泌腺的特异性信号。因此我继续进一步筹集抗血清。合成并免疫了三种肽：154-168、519-533、999-101，经肽亲和柱纯化后，抗血清对天然抗原表现出良好的反应。通过蛋白质印迹分析，测试了这些抗血清针对垂体、胃和胰腺等分泌组织的特异性，但均未能显示出针对这些组织的特异性信号。为了了解Pi-a的物理功能，我计划通过基因消融来饲养Pi-a缺陷小鼠，我分析了核苷酸数据库，阐明了人和小鼠Pi-a基因组结构及其启动子，并制作了敲除小鼠的重组载体。但由于缺乏 Pi-a 的抗血清进行进一步分析而放弃了进一步的研究