Diagnosis of retinoblastoma by the use of restriction fragment length polymorphism of esterase D gene

酯酶D基因限制性片段长度多态性诊断视网膜母细胞瘤

• 批准号: 62570127
• 负责人: YOSHIIE OKADA
• 金额: $ 1.02万
• 依托单位: Yamanashi Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570127/
• 关键词: Esterase D; Retinoblastoma; Human erythrocytes; Molecular diagnosis; Restriction fragment length polymorphism; cDNA cloning; _<gt> 11; 染色体13番; エステラーゼ D; λgt11; 制限酵素切断断片多型; λgtII

1. Esterases D-1 and D-2 were highly purified from human erythrocytes and were characterized.2. White rabbits were immunized with the purified esterase D-1. The antibodies to esterase D-1 reacted specifically to esterases D.3. The purified esterase D-1 was digested with proteolytic enzymes. The resulting peptides were separated by high performance liquid chromatography. The primary structures of nine purified peptides were analyzed.4. cDNA library was prepared from human placenta and was screened with the antibody against esterase D-1. one positive clone was isolated.5. The insert corresponding to cDNA, approximatelly 300 base paris, was subcloned into pUC 9.6. The base sequence of the cDNA was determined by the dideoxy method. It encodes the amino acid sequence determined in 3.7. The original cDNA library prepared from human placenta was rescreened with the cDNA as a probe. The cDNA clone with 1,200 base pairs as an insert was obtained.8. The base sequence of the longer cDNA showed no methionine codon at the 5'-terminal portion, suggesting that the longer cDNA lacks the 5'-terminal portion encoding approximate 10 amino acids at the amino terminal.

1. 酯酶D-1和D-2从人红细胞中高度纯化，并进行了鉴定。用纯化的酯酶D-1免疫大白兔。酯酶D-1抗体与酯酶d - 3特异性反应。纯化的酯酶D-1用蛋白水解酶消化。所得肽通过高效液相色谱分离。分析了9个纯化肽的一级结构。从人胎盘中制备cDNA文库，并用抗酯酶D-1抗体进行筛选。分离出1个阳性克隆。将cDNA对应的插入片段亚克隆到pUC 9.6中，全长约300碱基。采用双脱氧法测定cDNA的碱基序列。它编码3.7中确定的氨基酸序列。以人胎盘为探针，对原cDNA文库进行重新筛选。获得了以1200个碱基对为插入物的cDNA克隆。较长cDNA的碱基序列在5′端未发现蛋氨酸密码子，表明较长cDNA缺少编码约10个氨基酸的5′端部分。

项目成果

Yoshiie,Okada: Archives of Biochemistry and Biophysics.263. 130-136 (1988)

冈田吉家：生物化学和生物物理学档案.263。

Yoshiie Okada: "Purification and characterization of esterases D-1 and D-2 from human erythrocytes" Archives of Biochemistry and Biophysics. 263. 130-136 (1988)

Yoshiie Okada：“人红细胞中酯酶 D-1 和 D-2 的纯化和表征”生物化学和生物物理学档案。

Yoshiie Okada;Kazuhiro Wakabayashi: Archives of Biochemistry and Biophysics. (1988)

冈田吉家；若林一宏：生物化学和生物物理学档案。