吴东,南刚,李佳悦,刘芬玲,崔洪勇.人CD147胞内结构域蛋白的表达与纯化[J].转化医学杂志,2019,8(6):326-328
人CD147胞内结构域蛋白的表达与纯化
Expression and purification of the intracellular domain of human CD147 protein
  
DOI:
中文关键词:  CD147  胞内结构域  表达与纯化
英文关键词:CD147  Intracellular domain  Expression and purification
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作者单位
吴东 陕西 西安空军军医大学基础医学院学员一大队 
南刚 陕西 西安空军军医大学基础医学院细胞生物学教研室 
李佳悦 陕西 西安空军军医大学基础医学院细胞生物学教研室 
刘芬玲 陕西 西安空军军医大学基础医学院细胞生物学教研室 
崔洪勇 陕西 西安空军军医大学基础医学院细胞生物学教研室 
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中文摘要:
      目的 构建人CD147胞内结构域(CD147 intracellular domain,CD147ICD)融合蛋白质粒,表达、提取、纯化CD147ICD蛋白并进行蛋白质互作分析。方法 将CD147ICD的cDNA片段插入pET-32a(+)质粒载体后,转化大肠杆菌Origami B(DE3)感受态细胞,大量培养后用异丙基硫代-β-d-半乳糖苷诱导蛋白表达,超声破碎法裂解细胞,采用Ni-Sepharose FF预装柱纯化TrxA-His6-CD147ICD融合蛋白,经凝血酶酶切、超滤后获得CD147ICD蛋白。结果 CD147ICD融合蛋白质粒经测序比对后表明构建成功;采用Ni-Sepharose FF预装柱纯化的TrxA-His6-CD147ICD融合蛋白纯度和浓度均较高,经凝血酶酶切、超滤后获得的CD147ICD蛋白纯度在95%以上,蛋白浓度为0.74 mg/mL。结论 纯化出了高纯度的、具有生物学活性的CD147ICD蛋白,为CD147ICD互作分子的鉴定及结构解析奠定了基础。
英文摘要:
      Objective To express and purify the recombinant intracellular domain of human CD147 (CD147ICD) and to study the interacting proteins of CD147ICD. Methods cDNA of CD147ICD was ligated into pET-32a(+) plasmid vector, then transformed into E.coli Origami B (DE3) competent cells and the transformants were induced with isopropyl-β-d-thiogalactoside. The fusion proteins were purified with Ni-Sepharose FF column in combination with thrombin digestion and ultrafiltration. Results The recombinant plasmid was confirmed with sequencing. Purified TrxA-His6-CD147ICD protein was obtained with Ni-Sepharose FF column. After thrombin digestion and ultrafiltration, the purity of CD147ICD protein was above 95%, and the protein concentration was 0.74 mg/mL. Conclusion CD147ICD protein was purified with high purity and activity, which would provide a basis for the future studies on characterization of CD147ICD interacting proteins and structure determination.
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