人白介素1受体拮抗剂的cDNA克隆和原核表达

Cloning of human interleukin 1 receptor antagonist cDNA and its expression in E coli

从活化的正常人外周血单核细胞中提取总ＲＮＡ，经逆转录多聚酶链反应（ＲＴＰＣＲ）获得了人白介素１受体拮抗剂（ＩＬ１Ｒａ）ｃＤＮＡ。经过ＤＮＡ测序分析，发现该片段和国外发表的ＩＬ１ＲａｃＤＮＡ序列一致。将该目的基因插入ｐＢＶ２２０载体，并转入大肠杆菌ＨＢ１０１中，经热诱导表达重组蛋白。ＳＤＳＰＡＧＥ后发现，菌体超声裂解后，在可溶性上清中有一Ｍｒ１７０００的特异带，约占菌体可溶性总蛋白的８０％以上。反复冻融裂菌后，上清经离子交换纯化获得电泳纯蛋白样品。ＥＬ４和ＣＴＬＬ细胞培养测定表明，得到的重组蛋白具有天然人ＩＬ１Ｒａ的生物活性。

Human IL 1Ra cDNA fragment encoding the mature IL 1Ra was obtained by using RT PCR method from total RNA extracted from adherent IgG stimulated healthy human periferal blood monocytes According to DNA sequencing, the sequence of the hIL 1Ra cDNA thus obtained was the same as reported abroard The expressed protein from B coli HB101 transfered with IL 1Ra cDNA/pBV220 was up to 80% of total soluble bacterial protein with estimated M r 17 000 The protein in supernatant after ultrasonic disruption and bacteria precipitation, was soluble Through freezing and thawing, most of the expressed recombinant protein was released into supernatant in rather high purity which was consequentely purified by anion ionexchange chromatography to homogeneity determined by SDS PAGE EL 4 and CTLL cell culture assay showed that the biological activity of the rhIL 1Ra was similar to that of the natural human IL 1Ra.