摘要
目的:构建人载脂蛋白O(apolipoprotein O,ApoO)表达质粒,用pET原核表达系统制备重组抗原Trx-ApoO融合蛋白。方法:以人类肝脏cDNA文库为模板,聚合酶链反应(PCR)法扩增ApoODNA,将测序正确的目的基因片段插入质粒pET-32a(+)相应位点构建重组质粒并转化E.coliBL21(DE3),经IPTG诱导表达、Ni-NTA亲和层析制备Trx-ApoO。通过琼脂糖凝胶电泳、DNA测序及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定重组质粒及表达的Trx-ApoO融合蛋白的正确性。结果:PCR扩增产物经琼脂糖凝胶电泳证实在SDS-PAGE上出现1条519 bp的基因片段。DNA测序结果显示插入片段符合GenBank公布的人ApoO基因序列,重组质粒构建成功。ApoOcDNA基因片段经IPTG诱导表达重组蛋白,Ni-NTA柱亲和纯化后SDS-PAGE电泳分析表明在相对分子质量34 kD左右出现新的蛋白条带,与目标分子质量相符。结论:成功克隆出人ApoO基因,重组表达出Trx-ApoO蛋白。
Objective To construct human apolipoprotein O(apolipoprotein O,ApoO) expression vector and obtain recombinant fusion protein thioredoxin(Trx)-ApoO by pET prokaryotic expression system.Methods The ApoO gene fragment from the human liver cDNA library was amplified by PCR.The resulting product was cloned into pET-32a(+) vector and sequenced.The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21(DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside(IPTG).The fusion protein was purified by Ni-NTA resin.Results The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis.The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid.ApoO cDNA gene fragment was induced by IPTG,and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide(SDS-PAGE).Conclusion Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.
出处
《中南大学学报(医学版)》
CAS
CSCD
北大核心
2011年第2期116-120,共5页
Journal of Central South University :Medical Science
关键词
载脂蛋白O
基因克隆
融合蛋白
蛋白纯化
apolipoprotein O
gene cloning
fusion protein
protein purification