CTP-OD-HA融合蛋白的原核表达及其活性鉴定

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.