CTP-OD1-HA和CTP-OD2-HA融合肽在K562细胞中的定位及其与BCR-ABL蛋白的相互作用

Location of CTP-OD1-HA and CTP-OD2-HA fusion peptide in K562 cells and its interaction with BCR-ABL protein

目的研究CTP-OD1-HA和CTP-OD2-HA两种融合肽在慢性粒细胞白血病(CML)K562细胞中的定位及其与BCR-ABL蛋白的相互作用。方法将前期制备的CTP-OD1-HA和CTP-OD2-HA融合肽转导入K562细胞,采用免疫荧光和Western blotting方法检测其入胞情况及胞内定位,His-pull down和CoIP实验检测融合肽与BCR-ABL的相互作用。结果免疫荧光检测显示CTP-OD1-HA和CTP-OD2-HA融合肽均能穿过细胞膜进入细胞并定位于胞质,Western blotting也同时检测到进入细胞质中的两种融合肽。His-pull down实验结果表明,CTP-OD1-HA和CTP-OD2-HA融合肽在细胞外均可直接与BCR-ABL蛋白结合,CoIP实验结果显示两种融合肽在K562细胞内也能与BCR-ABL蛋白结合并形成复合物。结论 CTP-OD1-HA和CTP-OD2-HA融合肽均能成功进入白血病K562细胞并定位于细胞质,且可与BCR-ABL蛋白发生相互作用。

Objective To investigate the intracellular location of cytoplasmic transduction peptide-oligomerization domain 1-hemagglutinin （CTP-OD1-HA） and cytoplasmic transduction peptide-oligomerization domain 2-hemagglutinin （CTP-OD2-HA） fusion peptide and interaction with BCR-ABL protein in chronic myelocytic leukemia （CML） K562 cells. Methods The prepared CTP-OD1-HA and CTP-OD2-HA fusion peptide were transduced into K562 cells, and the cytoplasmic transduction peptide- oligomerization domain-hemagglutinin （CTP-OD-HA） peptide was used as positive control, and cytoplasmic transduction peptide- hemagglutinin （CTP-HA） and PBS were used as negative control. The intracellular location of the fusion peptide was demonstrated by immunotquorescence and Western blotting. The interactions between the fusion peptides and BCR-ABL protein were detected by His-pull down and CoIP test. Results Immunofluorescence assay showed that both fusion peptides CTP-OD 1-HA and CTP-OD2- HA could penetrate the cell membrane, and they were mainly localized in cytoplasm. Western blotting also confirmed the existence of the two fusion peptides in the cytoplasm. His-pull down showed that CTP-OD1-HA and CTP-OD2-HA could directly bind BCR- ABL protein outside the cells, and CoIP experiment showed that both fusion peptides could interact with BCR-ABL protein and form complex in the K562 cells. The negative control CTP-HA fusion peptide and PBS showed no such effects. Conclusion CTP- OD1-HA and CTP-OD2-HA fusion peptides can successfully target into CML K562 cells and locate in cytoplasm, and it interacts with BCR-ABL protein.