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摘要: 目的克隆人IL-18编码区cDNA,研究其在大肠杆菌中的表达。方法应用RT-PCR方法从人外周血单个核细胞（PBMC）中扩增出成熟（hIL-18）cDNA。利用基因重组技术构建IL-18的原核重组表达质粒pGEX-3X-hIL-18,转化E.coli JM109,以IPTG诱导培养,收集菌体直接进行SDS-PAGE分析。结果经测序证实获得人IL-18的cDNA序列与文献报道的cDNA完全一致;携此重组质粒pGEX-3X-hIL-18的大肠杆菌经IPTG诱导,表达了分子量为43kD的重组融合蛋白。结论克隆了人IL-18cDNA,利用大肠杆菌成功地表达了重组蛋白。为进一步探讨人IL-18的生物学作用及可能的临床应用打下基础。Abstract: Objective To clone mature human interleukim-18 cDNA and express it by E.coli JM109.Methods The cDNA encoding mature human imterleukim-18 was amplified from total RNA of human peripheral blood monooonuclear cells (PBMC) by RT-PCR.The hIL-18 reconbinant prokaryotic expression plasmid was constructed by cloning this cDNA fragment into pGEX-3X, and transform the recombinant plasmid into the competent cells of E.coli JM109.The recombinant IL-18 protein's expression was induced by IPTG, the result was analysed by SDS-PAGE.Results Sequence analysis indicated that this cDNA is 100% homologt to the published hIL-18 cDNA sequence;The recombinant plasmid pGEX-3X-hIL-18 was constructed successfully;By induction of IPTG, the E.coli JM109 harbouring the recombinant plasmid (pGEX-3X-hIL-18) expressed recombinant fusion protein (rhIL-18-GST) with molecular weight 43KD.Conclusion We have cloned human IL-18 cDNA and expressed it by E.coli JM109.It is the basis for the study on biological roles of human IL-18 and for possible application in clinic in the future.
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Key words:
- PBMC /
- Interleukin-18 /
- RT-PCR /
- Gene clone /
- Reconbinant expression
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