CAJ | 학술논문

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ApoO DNA,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.coli BL 21 (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.ApoO cDNA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.
목적:구건인재지단백O (apolipoprotein O, ApoO)표체질립,용pET원핵표체계통제비중조항원Trx-ApoO융합단백.방법:이인류간장cDNA문고위모판,취합매련반응(PCR) 법확증ApoO DNA,장측서정학적목적기인편단삽입질립pET-32a (+)상응위점구건중조질립병전화E.coli BL 21 (DE3),경IPTG유도표체、Ni-NTA 친화층석제비Trx-ApoO.통과경지당응효전영、DNA측서급십이완기류산납-취병희선알응효전영(SDS-PAGE)감정중조질립급표체적Trx-ApoO융합단백적정학성. 결과:PCR 확증산물경경지당응효전영증실재SDS-PAGE상출현1조519 bp 적기인편단.DNA측서결과현시삽입편단부합GenBank 공포적인ApoO기인서렬,중조질립구건성공.ApoO cDNA기인편단경IPTG유도표체중조단백,Ni-NTA주친화순화후SDS-PAGE전영분석표명재상대분자질량34 kD 좌우출현신적단백조대,여목표분자질량상부.결론:성공극륭출인ApoO기인, 중조표체출Trx-ApoO단백.
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.