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

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。

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.