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

新的Pi-a蛋白在分泌腺、脑垂体腺、胃或胰腺的mRNA水平表达，但功能或蛋白表达未知。为了了解其蛋白表达，我提出了针对PI-a的抗体。首先，我制备了针对合成肽和重组蛋白的抗血清。人工合成的多肽PI-a(C+961~972)免疫人和小鼠，抗血清与天然抗原反应良好。重组蛋白小鼠PI-a/His标签用His标签纯化后免疫，抗血清与天然抗原反应良好。当对这些抗血清进行Western印迹分析时，它们都没有显示出分泌腺的特异性信号。因此，我继续提高更多的抗血清。合成了154-168、519-533、999-101三种多肽，经多肽亲和层析纯化后，获得了与天然抗原反应良好的抗血清。通过Western印迹分析，这些抗血清对脑下垂体、胃和胰腺等分泌组织具有特异性，但它们都没有显示出对这些组织的特异性信号。为了了解pI-a的生理功能，我计划通过基因去除的方法饲养pI-a缺陷小鼠，分析核苷酸数据库，阐明人和小鼠pI-a及其启动子的基因组结构，并制备用于基因敲除小鼠的重组载体。但由于缺乏用于进一步分析的pi-a抗血清，进一步的研究被放弃。

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