CTP-OD-HA融合蛋白的原核表达及其活性鉴定

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。

Prokaryotic Expression and Activity of Cytoplasmic Transduction Peptide-Oligomerization Domain-Hemagglutinin Fusion Protein

Objective To construct the prokaryotic expression vector for cytoplasmic transduction peptide(CTP)-oligomerization domain(OD)-hemagglutinin(HA)fusion gene,express and purify the fusion protein and determine its transduction activity and cytoplasmic localization preference in human chronic myeloid leukemia(CML)cell line K562.Methods CTP,OD and HA epitope fragments were sequentially inserted into plasmid pET32a(+),and the constructed recombinant plasmid pCTP-OD-HA was transformed to competent E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by affinity chromatography,identified by Western blot and digested with enteropeptidase,then the target protein was recovered,labeled with FITC,added into K562 cells and observed for transduction activity and cytoplasmic localization.Results Restriction analysis and sequencing proved that recombinant plasmid pCTP-OD-HA was constructed correctly.After induction with 1 mmol / L IPTG at 37℃ for 4 h,the target protein reached a maximum level of about 35% of total somatic protein.The purity of expressed fusion protein was more than 95% after purification and showed high transduction activity and cytoplasmic localization preference in K562 cells.Conclusion The prokaryotic expression vector for CTP-OD-HA fusion gene was successfully constructed,and the target protein was highly expressed,which laid a foundation of further study on the role of CTP-OD-HA fusion protein in CML signal transduction channel,the pathogenic of CML and therapeutic strategy with protein peptide.