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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。
The construction of prokaryotic expression system of recombinant antitumor peptide AIK by Gateway cloning technology and establishment of optimal conditions for expression and purification of recombinant AIK laid the foundation for further study and utilization of AIK. Firstly, we designed the primers containing AttB recombination sites and amplified the Att B-TEV-FLAG-AIK sequence by overlapping PCR. The target sequence TEV-FLAG-AIK was cloned into the donor vector pDONR223 by BP recombination, Vector, and then by LR recombination reaction, the target sequence was transferred to the destination vector pDEST15 to construct a prokaryotic expression plasmid GST-AIK fusion protein. Subsequently, the conditions for inducing the expression of the fusion protein were optimized in BL21 (DE3) engineered bacteria. GST-AIK fusion protein was purified with glutathione beads, then GST was removed by rTEV enzyme to obtain FLAG-AIK recombinant protein. Finally, the cytotoxicity of FLAG-AIK on HL-60 leukemia cells was tested by MTS assay. Bacterial PCR validation and sequencing analysis showed that the successful construction of the recombinant anti-tumor AIK primer plasmid and prokaryotic expression plasmid. Highly efficient soluble expression of the GST-AIK fusion protein was achieved in BL21 (DE3) engineered bacteria. The results showed that the engineered bacteria (OD600 = 1.0) induced by 0.1 mmol / L IPTG at 37 ℃ for 4 h showed that the recombinant protein accounted for more than 30% of the total bacterial proteins. GST affinity chromatography, rTEV enzyme excision GST tag and secondary GST affinity chromatography to obtain more than 95% purity FLAG-AIK protein. The anti-tumor activity of FLAG-AIK prepared by MTS method was comparable to that of chemically synthesized AIK. In conclusion, we successfully constructed the prokaryotic expression plasmid of AIK with Gateway cloning system to achieve efficient and soluble expression of GST-AIK fusion protein. The recombinant AIK polypeptide with bioactivity was obtained by affinity chromatography, Research and large-scale preparation laid the foundation.