烟曲霉硫氧还蛋白还原酶重组蛋白的原核表达及抗原性分析  

Prokaryotic expression and antigenic analysis of recombinant thioredoxin reductase GliT of Aspergillus fumigatus

作　　者：史利宁[1,2] 邵世和[1] 李芳秋[2] 孔小祥[2] 王仕钦[2] 陆静芬[2] 黄梅[2] 邵海枫[2] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013 [2]南京军区南京总医院解放军临床检验医学研究所,南京210002

出　　处：《临床检验杂志》2011年第8期599-601,共3页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省科技支撑计划-社会发展项目(BE2009673);江苏省博士后基金项目(0802032C)

摘　　要：目的克隆烟曲霉硫氧还蛋白还原酶(thioredoxin reductase,TR)基因并构建原核表达载体,获得烟曲霉TR重组蛋白,并鉴定其抗原性。方法用RT-PCR方法从烟曲霉总RNA中扩增出TR cDNA片段,克隆至pMD18-T载体,并转化E.coliJM109。经序列分析证实后,提取重组克隆质粒双酶切获得目的基因,与表达载体pET28a(+)连接,转化E.coli BL21(DE3),筛选重组表达质粒。用异丙基硫代-β-D-半乳糖苷(IPTG)诱导重组融合蛋白表达,用SDS-PAGE及免疫印迹法分析重组蛋白质,并用亲和层析柱进行纯化。结果构建了含TR全长基因的重组表达质粒,IPTG诱导后可高效表达。免疫印迹结果表明该重组蛋白质可被侵袭性曲霉病患者血清识别。结论成功构建了重组表达质粒pET28a(+)/TR,并在大肠埃希菌中获得了高效表达,重组蛋白质具有良好的抗原性,为进一步研究奠定了基础。Objective To clone thioredoxin reductase GliT（TR） gene,construct prokaryotic expression vector,prepare recombinant protein and analyze its antigenicity.Methods The cDNA of TR was amplified from total RNA of Aspergillus fumigatus by reverse transcription-PCR.The amplified fragment was cloned into pMD18-T vector and sequenced.The recombinant plasmid pMD18-T/TR was digested by the restriction enzymes,and the target fragment was inserted into pET-28a（＋） vector which was transformed into E.coli BL21（DE3）.The recombinant TR protein was expressed by IPTG induction.Following the analysis of SDS-PAGE and western blot,the expressed protein was purified by Talon metal affinity resins.Results The recombinant plasmid consisting of full length TR gene was constructed.The recombinant TR protein was expressed richly in E.coli.Western blot showed that the recombinant protein was recognized by the sera from patients with proven invasive aspergillosis.Conclusion The recombinant expression plasmid pET28a（＋）/TR was successfully constructed and expressed richly in E.coli BL21（DE3）.The recombinant protein showed strong anitgenicity and may be used in the further study as the experimental material.

关 键 词：烟曲霉 硫氧还蛋白还原酶 基因克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]