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目的构建CTP-OD-HA融合基因原核表达质粒,表达并纯化融合蛋白,并检测其在人慢性粒细胞白血病(CML)K562细胞株中的转导活性及胞浆定位。方法分别将胞浆转导肽(CTP)、寡聚化区域(OD)基因和流感病毒血凝素抗原表位(HA)片段依次连接入pET32a(+)质粒,构建重组原核表达质粒pCTP-OD-HA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,经亲和纯化、Western blot验证、肠激酶切割、目的蛋白回收和FITC标记后,将FITC-CTP-OD-HA融合蛋白作用于K562细胞,观察其转导活性及细胞定位。结果重组原核表达质粒pCTP-OD-HA经酶切及测序鉴定,证明构建正确。37℃,1 mmol/LIPTG诱导4h,目的蛋白的表达量最高,约占菌体总蛋白的35%,纯化后纯度达95%以上。FITC-CTP-OD-HA融合蛋白在K562细胞中具有高转导活性和显著的胞浆定位特性。结论已成功构建CTP-OD-HA融合基因原核表达质粒,并高效表达了目的蛋白,为进一步研究其在CML信号转导通路中的作用,阐明CML的发病机制及探讨蛋白肽在CML中的治疗策略奠定了基础。
Objective To construct the prokaryotic expression plasmid of CTP-OD-HA fusion gene, express and purify the fusion protein and detect its transduction activity and cytoplasmic localization in human chronic myelogenous leukemia (K562) cell line. Methods CTP, OD gene and influenza virus HA fragment were ligated into pET32a (+) plasmid respectively to construct recombinant prokaryotic expression plasmid pCTP-OD -HA, transformed into competent E. coli BL21 (DE3) and induced by IPTG. FITC-CTP-OD-HA fusion protein was induced by affinity purification, Western blot, enterokinase cleavage, target protein recovery and FITC labeling K562 cells to observe the transduction activity and cell location. Results Recombinant prokaryotic expression plasmid pCTP-OD-HA was confirmed by restriction enzyme digestion and sequencing. 37 ℃, 1 mmol / L IPTG induced 4h, the highest expression of the target protein, accounting for about 35% of the total bacterial protein, purity of more than 95% after purification. FITC-CTP-OD-HA fusion protein has high transduction activity and significant cytoplasmic localization in K562 cells. CONCLUSION: The prokaryotic expression plasmid of CTP-OD-HA fusion gene has been successfully constructed and the target protein is highly expressed. To further investigate its role in the CML signal transduction pathway, clarify the pathogenesis of CML and explore the treatment of protein peptide in CML Strategy laid the foundation.