Research for noles of oligosaccharide on human prolactin

低聚糖分子对人催乳素影响的研究

• 批准号: 04670998
• 负责人: SUGANUMA Nobuhiko
• 金额: $ 1.28万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670998/
• 关键词: Prolactin; asparagin-linked oligosaccharide; expression vector; site-directed mutagenesis; gene transfer; 塩基特異的突然変異法

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.

1。制备人催乳素（PRL）cDNA，并插入真皮元细胞的表达载体中。将含有PRL cDNA的载体转染到COS-1细胞中，并通过用 ^<35> s-methionine的标记筛选产生PRL的克隆。将克隆用 ^<35> s-methionine标记12小时，并在细胞裂解物或培养基中合成并分泌的PRL用抗PRL抗体免疫沉淀。在SDS-聚丙烯酰胺凝胶电泳上分析了沉淀的PRL。在细胞裂解物和培养基中都观察到具有或没有寡糖的PRL。没有寡糖的PRL量大于细胞裂解物和培养基中寡糖的量。通过这些结果，我们无法阐明人类PRL中PRL从细胞分泌的生理意义或寡糖，等等。2。将人PRL cDNA插入MV1190噬菌体中，并将​​含有PRL cDNA的单链DNA插入MV1190噬菌体中，并制备了含有PRL cDNA的单粒型DNA。进行了定向的诱变，将天冬酰胺密码子（AAC）改变为天冬酸密码子（GAC），并通过DNA测序证实了核苷酸中的突变。为了进一步的实验，将突变的DNA插入表达载体，并制备无寡糖的纯化PRL。该突变体PRL可用于分析寡糖在PRL生理学上的作用。

项目成果

Suganuma N, Hirooka T, Narita O: "Treatment of hyperprolactinemia (Japanese)" Clin Gynecol Obstet (Japan). 46. 410-412 (1992)

Suganuma N、Hirooka T、Narita O：“高催乳素血症的治疗（日语）”Clin Gynecol Obstet（日本）。

Tsukahara S, Seo H, Kambe F, Kato T, Miyamoto N, Murata Y, Suganuma N, Tomoda Y, Matsui N: "Cloning of POU-box region in human Pit-1. (Japanese)" Annals of The Research Institute of Enveronmenral Medicine, Nagoya University. 43. 160-163 (1992)

Tsukahara S, Seo H, Kambe F, Kato T, Miyamoto N, Murata Y, Suganuma N, Tomoda Y, Matsui N：“克隆人类 Pit-1 中的 POU-box 区域。（日语）” 研究所年鉴

Tsukahara,S.: "Increase in Pit-1 mRNA is not required for the induction of the PRL gene expression by estrogen." Environ.Med.87. 35-38 (1993)

Tsukahara,S.：“雌激素诱导 PRL 基因表达不需要增加 Pit-1 mRNA。”

菅沼信彦: "「不妊症治療のための生殖医学実験マニュアル」第3章-9.核酸実験" (株)南江堂, 24 (1993)

Nobuhiko Suganuma：“不孕症治疗生殖医学实验手册第3-9章核酸实验” Nankodo Co., Ltd.，24（1993）

Tsukahara,S.: "Cloning of the human Pit-1 POU-Box." Environ.Med.36. 55-58 (1992)

Tsukahara,S.：“克隆人类 Pit-1 POU-Box。”