人IL-18基因克隆及表达  

Cloning of mature human Interleukim-18 Genes from human PBMC,Expression of the Protein in the E. coli

作　　者：李曼妮[1] 苏先狮[2] 杨春艳[2] 

机构地区：[1]广东省公安边防总队医院二内科,广东深圳518029 [2]中南大学湘雅二医院肝病研究中心,湖南长沙410011

出　　处：《临床肝胆病杂志》2009年第4期264-266,共3页Journal of Clinical Hepatology

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。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 JMI09. 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 re- combinant 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 JMI09. It is the basis for the study on biological roles of human IL - 18 and for possible application in clinic in the future.

关 键 词：PBMC IL-18 RT-PCR 基因克隆 重组表达 

分 类 号：R346[医药卫生—基础医学] Q78[生物学—分子生物学]