重组阳离子抗肿瘤肽AIK的原核表达、纯化及活性测定

发布时间：2018-03-27 09:16

  本文选题：阳离子多肽　切入点：位点特异性重组　出处：《生物工程学报》2015年12期

【摘要】：利用Gateway克隆技术构建重组抗瘤肽AIK的原核表达体系,建立表达及纯化重组AIK的最优条件,为深入研究和利用AIK奠定基础。首先,设计含AttB重组位点的引物,通过重叠PCR技术扩增出Att B-TEV-FLAG-AIK序列,利用BP重组反应将目的序列TEV-FLAG-AIK克隆到供体载体pDONR223中,构建入门载体,再通过LR重组反应,将目的序列转移到目的载体pDEST15中,构建GST-AIK融合蛋白原核表达质粒。随后,在BL21(DE3)工程菌中优化诱导融合蛋白表达的条件。以谷胱甘肽磁珠纯化GST-AIK融合蛋白,再以rTEV酶切除GST,获得FLAG-AIK重组蛋白。最后以MTS法检测FLAG-AIK对白血病细胞HL-60的细胞毒性。菌液PCR验证和测序分析表明成功构建了重组抗瘤肽AIK的入门质粒和原核表达质粒。在BL21(DE3)工程菌中实现了GST-AIK融合蛋白的高效可溶性表达。并测得在37℃下以0.1 mmol/L IPTG诱导工程菌(OD600=1.0)4 h,重组蛋白表达量占菌体总蛋白的30%以上。经GST亲和层析、rTEV酶切除GST标签及二次GST亲和层析获得纯度高于95%的FLAG-AIK蛋白。MTS法测得所制备的FLAG-AIK蛋白抑瘤活性与化学合成的AIK相当。总之,本课题应用Gateway克隆系统成功构建了抗瘤肽AIK的原核表达质粒,实现了GST-AIK融合蛋白的高效可溶性表达,经亲和层析获得了有生物活性的重组AIK多肽,为后续深入研究和大规模制备奠定了基础。
[Abstract]:The prokaryotic expression system of recombinant anti-tumor peptide AIK was constructed by using Gateway cloning technique, and the optimal conditions for expression and purification of recombinant AIK were established, which laid the foundation for further study and utilization of AIK. Firstly, primers containing the recombinant site of AttB were designed. The Att B-TEV-FLAG-AIK sequence was amplified by overlapping PCR technique, the TEV-FLAG-AIK sequence was cloned into donor vector pDONR223 by BP recombination reaction, and the entry vector was constructed. The target sequence was transferred to the target vector pDEST15 by LR recombination reaction. The prokaryotic expression plasmid of GST-AIK fusion protein was constructed. Subsequently, the conditions for inducing the expression of fusion protein were optimized in BL21DDE3 engineering strain. GST-AIK fusion protein was purified by glutathione magnetic beads. Finally, the cytotoxicity of FLAG-AIK to leukemic cell HL-60 was detected by MTS method. The results of PCR verification and sequencing analysis showed that the primer plasmid and prokaryotic expression plasmid of recombinant anti-tumor peptide AIK were successfully constructed. The fusion protein of GST-AIK was expressed in BL21DDE3. The recombinant protein was induced by 0.1 mmol/L IPTG at 37 鈩，

本文编号：1670925