TF-CTP-OD_2-HA融合蛋白的原核表达及纯化

Prokaryotic expression and purification of TF-CTP-OD_2-HA fusion protein

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 vector pCold-TF-CTP-ODE-HA, express TF-CTP-OD2-HA fusion protein in E. coil and purify the expressed product. Methods Gene fragment OD2, the key section of gene encoding oligomerization domain （OD）, was amplified by PCR using previously constructed plasmid p32a （＋） CTP-OD-HA as a template, and linked to cytoplasmic transduetion peptide （CTP） and hemagglutinin （HA） antigenic epitope of influenza virus, then cloned into prokaryotic expression vector pCold-TF-DNA to construct recombinant plasmid pCold-TF-CTP-ODrHA. Meanwhile, recombinant plasmid pCold-TF-CTP-HA was constructed as control. Plasmids pCold-TF-CTP-ODz-HA and pCold-TF-CTP- HA were transformed to E. coli BL21 for expression under induction of IPTG. The expressed fusion protein was purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. Results PCR, restriction analysis and sequencing proved that recombinant plasmids pCold-TF-CTP-OD2-HA and pCold-TF-CTP-HA were constructed correctly. The expressed fusion proteins TF-CTP-ODrHA and TF-CTP-HA, with relative molecular masses of about 63 000 and 58 000, mainly existed in the supernatant, a few of which were in precipitate of lysate of E. coli, and contained 32% and 25% of total somatic proteins, respectively. Both the fusion proteins reached purities of more than 95% after purification, and showed specific bindings to mouse polyclonal antibody against HA-tag. Conclusion Prokaryotic expression vector pCold- TF-CTP-OD2-HA was constructed successfully, and fusion protein TF-CTP-ODE-HA was expressed in E. coli and purified, which laid a foundation of further study on action mechanism of the fusion protein in chronic myeloid leukemia （CML）.