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目的构建pCold-TF-CTP-OD2-HA原核表达质粒,在大肠杆菌中表达并纯化融合蛋白。方法以前期构建的质粒p32a(+)CTP-OD-HA为模板,PCR扩增寡聚化结构域(Oligomerization domain,OD)中起关键作用的OD2基因序列,与胞浆转导肽(Cytoplasmic transduction peptide,CTP)和流感病毒血凝素抗原表位(Hemagglutinin,HA)连接后,克隆至pCold-TF-DNA质粒中,构建质粒pCold-TF-CTP-OD2-HA,同时构建质粒pCold-TF-CTP-HA作为对照。将两种质粒分别转化E.coli BL21(DE3),IPTG诱导表达,表达的融合蛋白经Ni-NTA亲和层析纯化后,进行SDS-PAGE和Western blot鉴定。结果质粒pCold-TF-CTP-OD2-HA和pCold-TF-CTP-HA经PCR、双酶切和测序鉴定构建正确;表达的融合蛋白TF-CTP-OD2-HA和TF-CTP-HA相对分子质量分别约为63 000和58 000,两种融合蛋白均主要存在于破菌上清中,表达量分别约占菌体总蛋白的32%和25%,破菌沉淀中有少量表达;纯化的融合蛋白纯度均达95%以上,均可与鼠抗HA-tag多克隆抗体特异性结合。结论成功构建了pCold-TF-CTP-OD2-HA原核表达质粒,在大肠杆菌中表达并纯化了融合蛋白,为后续研究其在CML中的作用机制奠定了基础。
Objective To construct prokaryotic expression plasmid pCold-TF-CTP-OD2-HA and express and purify the fusion protein in E.coli. Methods The plasmid p32a (+) CTP-OD-HA was used as a template to amplify the OD2 gene in the Oligomerization domain (OD) by PCR, and was compared with Cytoplasmic transduction peptide, CTP and Hemagglutinin (HA) were cloned into plasmid pCold-TF-DNA to construct plasmid pCold-TF-CTP-OD2-HA. CTP-HA served as a control. The two plasmids were respectively transformed into E.coli BL21 (DE3) and induced by IPTG. The expressed fusion proteins were purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blot. Results The constructed plasmids pCold-TF-CTP-OD2-HA and pCold-TF-CTP-HA were identified by PCR, double enzyme digestion and sequencing. The relative expression of TF-CTP-OD2-HA and TF- The quality of the fusion protein was about 63 000 and 58 000, respectively. The two fusion proteins were mainly found in the supernatant of the broken bacteria, which accounted for 32% and 25% of the total bacterial proteins, respectively. The purity of the fusion protein reached more than 95%, all can be combined with mouse anti-HA-tag polyclonal antibody. Conclusion The prokaryotic expression plasmid pCold-TF-CTP-OD2-HA was successfully constructed and the fusion protein was expressed and purified in Escherichia coli, which laid the foundation for further study on its mechanism in CML.