首页> 外国专利> RECOMBINANT MACROPHAGE MIGRATION INHIBITORY FACTOR; EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF RECOMBINANT PROTEIN

RECOMBINANT MACROPHAGE MIGRATION INHIBITORY FACTOR; EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF RECOMBINANT PROTEIN

机译:重组巨噬细胞迁移抑制因子;大肠杆菌中的表达和纯化重组蛋白。

摘要

According to the known cDNA sequence we used the PCR (polymerase chain reaction) technique to isolate a gene encoding human MIF from uterus endometrium The PCR product was cloned in the Escherichia coli plasmid vector pUC 19 and the nucleotide sequence was confirmed. We were able to successfully express human MIF in the bacteria Escherichia coli by use of the pKP 1500 expression plasmid. The recombinant protein accumulated intracellularly in soluble form, comprising more then 30 % of total cell protein. We have designed an original two step procedure where protein purification was accomplished by gel filtration and ion exchange chromatography. The purified recombinant MIF was used to immunise a rabbit and antibodies obtained were used for MIF detection in human tissues by immunohistochemical techniques.
机译:根据已知的cDNA序列,我们使用PCR(聚合酶链反应)技术从子宫内膜中分离出编码人MIF的基因。将PCR产物克隆到大肠杆菌质粒载体pUC 19中,并确定了核苷酸序列。通过使用pKP 1500表达质粒,我们能够在细菌大肠杆菌中成功表达人MIF。重组蛋白以可溶性形式在细胞内积累,占总细胞蛋白的30%以上。我们设计了一个原始的两步程序,其中蛋白质纯化是通过凝胶过滤和离子交换色谱法完成的。纯化的重组MIF用于免疫兔,获得的抗体用于通过免疫组织化学技术检测人组织中的MIF。

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