【摘要】 目的克隆人白细胞介素 18(IL 18)基因 ,构建重组表达质粒 ,在大肠杆菌中表达重组人白细胞介素 18(rhIL 18) ,并对其生物学活性进行初步鉴定。方法利用RT PCR方法从人外周血单个核细胞中扩增出成熟肽编码区cDNA(496bp) ,克隆到表达载体pBV2 2 0质粒中 ,Western印迹证实rhIL 18在大肠杆菌DH5α中获得表达。经分子筛层析法初步纯化后 ,用溴化四唑蓝 (MTT)比色法测定复性的rhIL 18的体外活性。结果rhIL 18表达量占总菌体蛋白的 2 7%左右 ,经纯化得到纯度达 96 %以上的rhIL 18。实验显示rhIL 18协同IL 2 (10U/ml)诱导NK细胞细胞毒活性。结论rhIL 18具有较好的生物学功能 ,应予进一步的研究更多还原

【Abstract】 PurposeThe aim is to clone human IL 18 cDNA,to construct recombinant human IL 18 expression vector and to perform the bioassay of recombinant human IL 18 (rhIL 18) expressed in E.coli. Methods Human IL 18 cDNA fragment (496 bp) was amplified from PBMC by RT PCR method and sequenced by ABI PRIM TM 377XL DNA Sequencer. The cloned IL 18 cDNA fragment was inserted into pBV220 expression vector, which was then transformed to E.coli. SDS PAGE and Western blot assays showed that rhIL 18 was highly expressed. The bioassay on rhIL 18 was performed by MTT assay in vitro. ResultsThe expression level of recombinant IL 18 protein reached 27% and the purity is over 96% after purification. The refolded rhIL 18 enhanced the cytotoxicity of NK cells in the presence of IL 2(10 U/ml) in vitro. ConclusionThe expressed rhIL 18 is functional and can be applied for further studies.更多还原