CAJ | 학술논문

目的:构建弗氏志贺菌 clpB 基因的原核表达质粒,在大肠杆菌中表达后,纯化带 T7标签的 ClpB 蛋白.方法与结果:PCR 扩增得到线性化表达载体 pET24a 与2574 bp 的 clpB 基因片段,利用不依赖连接反应的克隆法(LIC)进行克隆,得到重组质粒 pET-ClpB,转入大肠杆菌 BL21(DE3)中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1 mmol/L IPTG 诱导2 h;利用抗 T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×103的 ClpB-T7融合蛋白.结论:实现了 ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白.
목적:구건불씨지하균 clpB 기인적원핵표체질립,재대장간균중표체후,순화대 T7표첨적 ClpB 단백.방법여결과:PCR 확증득도선성화표체재체 pET24a 여2574 bp 적 clpB 기인편단,이용불의뢰련접반응적극륭법(LIC)진행극륭,득도중조질립 pET-ClpB,전입대장간균 BL21(DE3)중진행유도표체,통과일계렬조건우화,학정가용성표체조건위재30℃하、용1 mmol/L IPTG 유도2 h;이용항 T7단극륭항체경지당주진행친화순화,득도료순도흔고적상대분자질량위95×103적 ClpB-T7융합단백.결론:실현료 ClpB-T7재대장간균중적가용성표체,병순화획득료고순도적융합단백.
Objective: To construct clpB gene of Shigella flexneri 5a M90T strain expression plasmid by liga?tion-independent cloning(LIC) method and express it in E.coli and purify it. Methods & Results: The 2574 bp clpB gene fragment and linearized expression vector pET24a were prepared by PCR amplification, and they were li?gated with LIC method to construct pET-ClpB. The resultant expression vector was transformed into E.coli BL21 (DE3) to express recombinant ClpB-T7. The expression condition and purification procedure were then optimized. At 30℃, by induction of 1 mmol/L IPTG lasted for 2 h, soluble expression protein was obtained. The fusion pro?tein was purified with T7 tag antibody agarose to get the high purity fusion protein ClpB-T7(95 kD). Conclu?sion: High purity of the ClpB-T7 protein was obtained.