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从一名丙型肝炎病人的外周血单个核细胞(PBMC)中分别克隆了前体和成熟人白细胞介素-18(hIL-18)的基因,并应用大肠杆菌表达系统(pQE-30),成功地表达了重组hIL-18的前体和成熟蛋白。从人PBMC中提取总RNA,用poly(T)8-12反转录成cDNA,再以PCR扩增出特异DNA 片段。利用E.coli表达载体pQE-30,构建分别表达hIL-18前体和成熟蛋白的的重组质粒pQEIL18p 和pQEIL18m ,转化E.coliM15后,经IPTG诱导,表达出相对分子质量分别为24KD和19KD的重组蛋白,表达量占菌体蛋白的30% 和35% 。序列测定证实,hIL-18前体蛋白基因长582bp,成熟蛋白基因长477bp。经金属螯合层析纯化,获得了高纯度的表达蛋白。生物学活性鉴定,重组成熟蛋白能显著刺激Con A活化的人PBMC分泌IFN-γ
The genes of precursor and mature human interleukin-18 (hIL-18) were cloned from peripheral blood mononuclear cells (PBMCs) of a Hepatitis C patient and the expression vector pQE-30 was used. The precursor and mature protein of recombinant hIL-18 was successfully expressed. Total RNA was extracted from human PBMC, reverse transcribed into cDNA using poly (T) 8-12, and specific DNA fragments were amplified by PCR. Use E. coli expression vector pQE-30 to construct recombinant plasmids pQEIL18p and pQEIL18m respectively expressing the precursor and mature protein of hIL-18. After induced by IPTG, the recombinant proteins with relative molecular mass of 24KD and 19KD were expressed in E. coliM15 respectively, which accounted for 30% and 35% of the total bacterial proteins. Sequence analysis confirmed that the hIL-18 precursor protein was 582bp in length and the mature protein was 477bp in length. After purification by metal chelate chromatography, high purity of the expressed protein was obtained. Biological activity identification, recombinant mature protein can significantly stimulate Con A-activated human PBMC secretion of IFN-γ