Purification of a new low molecular weight protein and its clinical application

一种新型低分子量蛋白的纯化及其临床应用

• 批准号: 02454499
• 负责人: KAWAI Tadashi
• 金额: $ 2.82万
• 依托单位: JICHI MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454499/
• 关键词: protein 1; ELISA; low molecular weight protein; prostate; Clare cell; renal tubular dysfunction; protein 1; 低分子蛋白; protoin1; Clara cell secretary protein; urine protein1; human clara cell 10kDa Protein; 慢性腎不全

The authors have isolated a new low molecular weight protein (LMWP) (protein 1) from urine, characterized its physicochemical properties, determine it partial amino acid sequences, and thus prepared a monoclonal antibody against this protein. After establishing an enzyme-linked immunosorbent assay (ELISA) for this protein, the significance of this protein in laboratory medicine has been extensively studied.1) Purification and characterization of a new LMWP (protein 1):An unknown protein detected on SDS-PAGE so far proved to be protein 1 after checking the reactivity of rough purified fraction with various antisera (antibody). The present project is thus modified to isolate protein 1 using the antibody as a probe. The established purification procedure comprised of ammonium sulfate precipitation, ConA affinity, gel-filtration, anti-retinol binding protein antibody coupled affinity, and an anion exchange column. Purity was confirmed by SDS-PAGE and its molecular weight was 14kDa and p1 was 4.7. Partial amino acid sequences were determined which proved to be identical with human lung Clara cell 10 kDa secretary protein.2) Preparation of a monoclonal antibody.3) Establishment of an ELISA:A reliable precise assay was initially developed using purified substance as standard and its monoclonal anbitody.4) Clinical application:Protein 1 is distributed in various body fluids, in particular, serum value ranged in normal adults ranged from 2 to 20 mug/l and urine value was varied with marked sex-differences being higher in male than in female. Another extensive studies finally spot another synthetic sites as prostate. Further studies on this protein was both fundamentally and clinically in progress.

作者从尿中分离到一种新的低分子量蛋白（LMWP）（蛋白1），对其理化性质进行了鉴定，测定了部分氨基酸序列，并制备了抗该蛋白的单克隆抗体。在建立了针对该蛋白的酶联免疫吸附试验（ELISA）后，该蛋白在实验医学中的意义已被广泛研究。1）新的LMWP（蛋白1）的纯化和表征：迄今为止在SDS-PAGE上检测到的未知蛋白在检查粗纯化级分与各种抗血清（抗体）的反应性后被证明是蛋白1。因此，本项目修改为使用抗体作为探针分离蛋白1。所建立的纯化程序包括硫酸铵沉淀、ConA亲和、凝胶过滤、抗视黄醇结合蛋白抗体偶联亲和和阴离子交换柱。经SDS-PAGE电泳鉴定其纯度，分子量为14 kDa，p1为4.7。测定了部分氨基酸序列，证明与人肺Clara细胞10 kDa分泌蛋白相同。2）单克隆抗体的制备。3）ELISA的建立：以纯化的物质为标准品及其单克隆抗体，初步建立了一种可靠的精确测定方法。4）临床应用：蛋白1分布于各种体液中，特别是正常成人的血清值范围为2 - 20 μ g/l，尿值不同，性别差异显著，男性高于女性。另一项广泛的研究最终发现了前列腺的另一个合成位点。对这种蛋白质的进一步研究在基础和临床上都在进行中。

项目成果

R.Okutani: "Simple and high-yield purification of urine protein 1 using immuns affinity chromatography" J.chromatography. 577. 25-35 (1992)

R.Okutani：“使用免疫亲和层析对尿蛋白 1 进行简单且高产的纯化”J.层析。

Y.Itoh: "Increased urinary excretion of urine protein1 induad by L-Arginine monohydro chloride" Nephron. 62. 467-468 (1992)

Y.Itoh：“L-精氨酸单盐酸盐增加尿蛋白的尿排泄”肾单位。

y.Itoh et al.: "Increased urinary excretion of urine protein 1 by Lーarginine monohydrochloride" Nephron. (1992)

y.Itoh 等人：“盐酸拉精氨酸增加尿蛋白 1 的尿排泄”Nephron (1992)。

Y.Itoh,et al.: "Purification of urine protein 1 from urine: An initial study toward clinical application."

Y.Itoh 等人：“从尿液中纯化尿蛋白 1：临床应用的初步研究。”

R O Kutani: "simple and high-yield purification of urine protein1 using immuno affinity chro mato graphy" J Chromatogrofhy. 457. 25-35 (1992)

R O Kutani：“使用免疫亲和色谱法简单且高产地纯化尿蛋白1”《J Chromatogrofhy》。