TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定  

Construction of Eukaryotic Expression Vector of TP53BP2 Gene and Its Expression,Purification and Activity Identification in Human Embryonic Kidney Expi293F Cells

作　　者：李全维 高明慧 寇卜心[1] 柴梦音[1] 石英[1] 刘晓霓[1] LI Quanwei;GAO Minghui;KOU Puxin;CHAI Mengyin;SHI Ying;LIU Xiaoni(Beijing Institute of Hepatology/Beijing You’an Hospital,Capital Medical University,Beijing 100069,China)

机构地区：[1]北京肝病研究所/首都医科大学附属北京佑安医院,北京100069

出　　处：《现代检验医学杂志》2024年第6期11-17,共7页Journal of Modern Laboratory Medicine

基　　金：北京市自然科学基金资助项目(编号:7192084);首都卫生发展科研专项(编号:2020-2-1152);北京市属医学研究所公益发展改革试点项目(就医研2021-10)。

摘　　要：目的构建人肿瘤抑制因子p53结合蛋白2(tumor suppressor p53-binding protein 2,TP53BP2)的重组真核表达载体,转染人胚肾Expi293F细胞,获得高纯度的重组人全长TP53BP2蛋白并对其进行生物学活性鉴定。方法利用UniProt网站查询TP53BP2基因序列,并进行Expi293F表达系统序列优化,通过同源重组连接至pcDNA3.1(+)-P2Ae GFP载体并进行双酶切和测序鉴定,通过转染试剂聚乙烯亚胺(polyethylenimine,PEI)将pcDNA3.1(+)-P2A-eGFPTP53BP2质粒瞬时转染至Expi293F细胞,荧光显微镜观察转染效率,收集实验组及对照组细胞,利用免疫印记试验(Western blot,WB)检测TP53BP2重组蛋白表达水平。通过His标签纯化试剂盒及Superdex 20010/300GL层析柱进行蛋白纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDSPAGE)对纯化后重组蛋白进行鉴定。利用免疫共沉淀(Co-immunoprecipitation,Co-IP)检测重组人全长TP53BP2蛋白与p65蛋白结合情况。利用免疫荧光(immunofluorescence,IF)检测重组人全长TP53BP2蛋白与p65蛋白共定位。利用表面等离子体共振(surface-plasmon resonance,SPR)技术,检测纯化后的重组人全长TP53BP2蛋白与TP53BP2抗体的相互作用。结果经测序和双酶切鉴定,重组质粒pcDNA3.1(+)-P2A-eGFP-TP53BP2构建成功。经荧光显微镜观察结果显示转染效率约为60%,WB结果表明TP53BP2蛋白在Expi293F细胞中过表达,证明转染成功。SDS-PAGE结果表明纯化后重组蛋白纯度在90%以上,证明纯化成功。Co-IP结果表明,TP53BP2重组蛋白可与p65蛋白相互作用。IF结果表明,His标签蛋白、TP53BP2蛋白及p65蛋白存在共定位,表明三者之间存在相互作用。SPR结果表明,纯化的重组人TP53BP蛋白与TP53BP2抗体具有较好的结合活性。以上结果均证明重组人全长TP53BP2蛋白具有生物学活性。结论成功构建了TP53BP2基因真核表达载体并在人胚肾Expi293F细胞中成功表达出具有生物�Objective To construct a recombinant eukaryotic expression vector of human tumor suppressor p53-binding protein 2(TP53BP2)and transfect human embryonic kidney Expi293F cells.High-purity recombinant human full-length TP53BP2 protein was obtained and its biological activity was identified.Methods The TP53BP2 gene sequence was queried on the UniProt website,and the Expi293F expression system was optimized.The TP53BP2 gene was connected to pcDNA3.1(+)-P2AeGFP vector by homologous recombination,and identified by double enzyme digestion and sequencing.Transect pcDNA3.1(+)-P2A-eGFP-TP53BP2 plasmid into Expi293F cells of Polyethylenimine(PEI),observe the transfection efficiency with a fluorescence microscope,collected cells from the experiment group and control group.The expression level of TP53BP2 recombinant protein was detected by Western blot(WB).Protein was purified by His label purification kit and Superdex 20010/300 GL chromatographic column.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The purified recombinant protein was identified by SDS-PAGE.Combining recombinant human full-length TP53BP2 protein with p65 protein was investigated for Co-immunoprecipitation(Co-IP)precipitation.Recombinant human full-length TP53BP2 protein was co-localized with p65 protein by Immunofluorescence(IF).The surface plasmon resonance(SPR)technique was used to detect the interaction between purified recombinant human full-length TP53BP2 protein and TP53BP2 antibody.Results The recombinant plasmid pcDNA3.1(+)-P2A-eGFP-TP53BP2 was successfully constructed by sequencing and double digestion.The fluorescence microscopy results showed that the transfection efficiency was about 60%.WB showed that the TP53BP2 protein was overexpressed in Expi293F cells,which proved that transfection was successful.SDS-PAGE results showed that the purity of the purified recombinant protein was above 90%,which proved that the purification was successful.Co-IP results showed that the TP53BP2 could interact with p65 protein.The results of IF showed th

关 键 词：肿瘤抑制因子p53结合蛋白2 人全长肿瘤抑制因子P53结合蛋白2真核表达 人胚肾细胞Expi293 瞬时转染 蛋白纯化 活性鉴定 

分 类 号：Q786[生物学—分子生物学] Q503