PDF下载 ( 301 KB)
-
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.
-
Key words:
- PBMC /
- Interleukin-18 /
- RT-PCR /
- Gene clone /
- Reconbinant expression
-
[1]Okamura H, Tsutsui H, Komatsu T, et al, Cloning of a new cy-tokine that induces IFN-γproduction by T cells[J].Nature, 1995, 378 (2) ∶88~91. [2]Ushio S, Namba M, Okura T, et al.Cloning of the CDNAfor hu-man IFN-γinducing factor, expression in Escherichia coli, andstudies on biological activi ties of the protein[J].J Immunol, 1996, 156 (11) ∶4274~4279. [3]GUY, Kuida K, Tsutsui H, et al.Activation of interferon-γ-inducing factor mediated by interleukin-lβconvertingenzyme[J].Science, 1997, 275 (5297) ∶206~209. [4]GhayurT, Banerjes S, Hugunin M, et al.Caspase-l processesIFN-gamma-inducing factor and regulates LPS-inducing IFN-gamma production[J].Nature, 1997, 386 (6625) ∶619~623. [5]Smith D B, Johnson K S.Single-Step purification of polypeptideexpressed in Escherichia coli as fusions with glutathione S-trans-frase[J].Gene, 1998, 67∶31. [6]胡章勇, 章廉, 文维群, 等.慢性乙型肝炎患者TH1/TH2细胞因子应答状态[J].中华微生物学和免疫学杂志, 2001, (2) ∶12. [7]文维群, 章廉, 肖红, 等.乙型肝炎病毒感染者IL-18基因的转录及表达[J].中华医学杂志, 2001, 81 (11) ∶655~658. -
本文二维码
计量
- 文章访问数: 2459
- HTML全文浏览量: 10
- PDF下载量: 906
- 被引次数: 0
下载:
