| Research Abstract |
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
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