1993 Fiscal Year Final Research Report Summary
Research for noles of oligosaccharide on human prolactin
| Project/Area Number |
04670998
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Nagoya University |
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Principal Investigator |
SUGANUMA Nobuhiko Nagoya University, School of Medicine, Associate Prof., 医学部, 助教授 (30179113)
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| Co-Investigator(Kenkyū-buntansha) |
FUKUGAKI Hiroyuki Nagoya University, School of Medicine, Assistant Prof., 医学部, 助手 (10252248)
KONDO Ikuyo Nagoya University, School of Medicine, Assistant Prof., 医学部, 助手 (40215447)
ASADA Yoshimasa Nagoya University, School of Medicine, Assistant Prof., 医学部, 助手 (70231884)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Prolactin / asparagin-linked oligosaccharide / expression vector / site-directed mutagenesis / gene transfer |
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| Research Abstract |
1. Human prolactin (PRL) cDNA was prepared, and inserted into the expression vector for eukariotic cell. The vector containing PRL cDNA was tranfected into COS-1 cells, and the PRL producing clones were screened by the labeling with ^<35>S-methionine. The clones were labeled with ^<35>S-methionine for 12 hours, and synthesized and secreted PRL in cell lysate or culture medium were immunoprecipitated with anti-PRL antibody. The precipitated PRL was analyzed on SDS-polyacrylamide gel electrophoresis. The PRLs either with or without oligosaccharide were observed in both cell lysate and medium. The amount of PRL without oligosaccharide was larger than that with oligosaccharide both in cell lysate and medium. By these results, we can not clarify the physiological significanses or the oligosaccharide in human PRL on secretion of PRL from cell, and so on.
2. Human PRL cDNA was inserted into MV1190 phage, and single-stranded DNA containing PRL cDNA was inserted into MV1190 phage, and singe-straned DNA containing PRL cDNA was prepared. Site-directed mutagenesis that altered asparagine codons (AAC) to aspartic acid codons (GAC) was performed, and the mutation in nucleotides was confirmed by DNA sequencing. For further experiments, the mutant DNA will be inserted into the expression vector, and purified PRL without oligosaccharide will be prepared. This mutant PRL can be useful for the analysis of the roles of the oligosaccharide on the physiology of PRL.
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