Molecular cloning and characterization of cDNA for human myeloperoxidase.
Molecular cloning and characterization of cDNA for human myeloperoxidase.
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人髓过氧化物酶 cDNA 的分子克隆和表征。
DOI：
10.1016/s0021-9258(18)61433-4
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Shigekazu Nagata
中科院分区：
文献类型：
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作者：
Kazuhiro Morishita;Naoki Kubota;Shigetaka Asano;Yoshito Kaziro;Shigekazu Nagata
测定了人髓过氧化物酶的部分氨基酸序列，化学合成了含有4个脱氧肌苷的41个碱基的寡核苷酸。以该寡核苷酸为探针，从人早幼粒白血病HL-60细胞的mRNA文库中克隆了人髓过氧化物酶的基因。对其中一个含有2.6kb碱基插入片段的克隆进行核苷酸序列分析。该序列含有2,235个核苷酸的开放阅读框，编码745个氨基酸的蛋白质，计算的MR为83,868。髓过氧化物酶的重链由467个氨基酸组成，位于蛋白质的COOH末端。用SP6RNA聚合酶制备RNA，翻译到兔网织红细胞裂解液中，用兔抗人髓过氧化物酶抗体进行免疫沉淀，鉴定为人髓过氧化物酶。通过对白血病细胞RNA的Northern杂交分析，发现髓过氧化物酶在人早幼粒细胞HL-60和小鼠髓系白血病细胞NFS-60中高表达。此外，人类基因组DNA的Southern杂交分析结果表明，在人类单倍体基因组中存在一个或两个髓过氧化物酶基因。
Partial amino acid sequence of human myeloperoxidase was determined, and a 41-base oligonucleotide containing deoxyinosines at four positions was chemically synthesized. By using the oligonucleotide as a probe, cDNA clones for human myeloperoxidase were isolated from a cDNA library constructed with mRNA from human promyelocytic leukemia HL-60 cells. One of the clones containing a 2.6-kilobase insert was subjected to nucleotide sequence analysis. The sequence was found to contain an open reading frame, 2,235 nucleotides coding for a protein of 745 amino acids with a calculated Mr of 83,868. The heavy chain of myeloperoxidase, consisting of 467 amino acids, was located on the COOH terminus half of the protein. The RNA specified by the cDNA was prepared using SP6 RNA polymerase and translated in rabbit reticulocyte lysates, and the product was identified as human myeloperoxidase by immunoprecipitation with rabbit anti-human myeloperoxidase antibody. By Northern hybridization analysis of RNA from leukemic cells, it was shown that myeloperoxidase mRNA is abundantly expressed in human promyelocytic HL-60 and mouse myeloid leukemia NFS-60 cells. Furthermore, the results of Southern hybridization analysis of human genomic DNA suggest that there are one or two genes for myeloperoxidase in the human haploid genome.