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、胃或胰腺中以mRNA水平表达，但功能或蛋白表达未知。为了了解其蛋白的表达，我培养了针对Pi-a的抗体（Ab），首先，我培养了针对合成肽和重组蛋白的抗血清。合成的人、鼠共通肽Pi-a（C+961 ~ 972）经免疫，抗血清对天然抗原反应良好。重组蛋白，小鼠Pi-a/His标签，用His标签纯化，免疫，抗血清对天然抗原反应良好。当这些抗血清通过Western blot分析时，它们都没有显示出分泌腺的特定信号。因此，我继续提出更多的反血清。合成了154 ~ 168、519 ~ 533、999 ~ 101 3条肽段，经肽亲和柱纯化后，抗血清对天然抗原反应良好。通过Western blot分析，检测了这些抗血清对垂体、胃、胰腺等分泌组织的特异性，但均未显示出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培养Pi-a缺陷小鼠，分析核苷酸数据库，阐明人和小鼠Pi-a基因组结构及其启动子，并制作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了

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