再生基因4(REG4)真核表达载体的构建及其蛋白在HEK293T细胞中的表达、纯化  

Construction of Eukaryotic Expression Vector of Regenerating Gene 4(REG4)and Expression and Purification of Its Protein in HEK 293T Cells

作　　者：徐杨[1] 杜惠芬[1] 李克生[1] 柴丹丹[1] 石晓玲 连晓雯[1] 张学良[2] XU Yang;DU Huifen;LI Kesheng;CHAI Dandan;SHI Xiaoling;LIAN Xiaowen;ZHANG Xueliang(Medical Biotechnology Research Center,Gansu Academy of Medical Science,Lanzhou 730050 China;Department of Digestive Oncology,Gansu Provincial Cancer Hospital,Lanzhou 730050,China)

机构地区：[1]甘肃省医学科学研究院医学生物技术研究中心,兰州730050 [2]甘肃省肿瘤医院消化肿瘤内科,兰州730050

出　　处：《现代检验医学杂志》2023年第4期35-39,共5页Journal of Modern Laboratory Medicine

基　　金：兰州市科技发展指导性计划项目(2019-ZD-131):高性能抗RegIV单克隆抗体的制备及鉴定;甘肃省自然科学基金项目(21JR7RA866):PG,MUC1,HIK1083联合检测在胃早癌诊断中的临床研究。

摘　　要：目的构建再生基因4(regenerating gene 4,REG4)的真核表达载体,转染人胚肾细胞293T(human embryonic kidney 293T cells,HEK 293T),获得重组人再生胰岛衍生蛋白IV(regenerating islet-derived protein IV,Reg IV)。方法根据NCBI数据库REG4基因序列进行基因优化、合成,将其克隆至pCDNA3.4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pCDNA3.4-REG4质粒瞬时转染至HEK 293T细胞(实验组),同时以pEGFP-C1质粒作为转染对照组,未转染重组质粒的HEK 293T细胞作为空白对照组。荧光显微镜观察转染对照组转染效率,分别收集实验组及空白对照组细胞和细胞培养液上清,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)与免疫印迹试验(Western-Blot,WB)检测Reg IV蛋白表达水平。通过镍柱及丙烯葡聚糖凝胶S-400(Sephacryl S-400)柱进行蛋白纯化,SDS-PAGE及WB对纯化后重组蛋白进行鉴定。结果经测序和双酶切鉴定,重组质粒pCDNA3.4-REG4构建成功。转染对照组(pEGFP-C1质粒)荧光显微镜观察结果显示转染效率约50%,表明转染成功。WB结果显示仅在实验组(pCDNA3.4-REG4质粒)的细胞中检测到RegIV蛋白。镍柱纯化时目的蛋白无法与镍柱填料有效结合,SephacrylS-400凝胶柱层析纯化获得了此重组蛋白。结论成功构建了REG4基因真核表达载体并在HEK 293T细胞中成功表达,为深入研究Reg IV蛋白的作用机理及开发潜在的抗癌靶向药物奠定了基础。Objective To construct an eukaryotic expression vector of Regenerating gene 4(REG4)and transfection into human embryonic kidney 293T Cells(HEK 293T)to obtain recombinant human regenerating islet-derived protein IV(Reg IV).Methods The REG4 gene sequence was optimized and synthesized according to the NCBI database,and it was cloned into the pCDNA3.4 vector and identified.The pCDNA3.4-REG4 plasmid was transiently transfected into HEK 293T cells by transfection reagent polyethylenimine(PEI)as the experimental group.The pEGFP-C1 plasmid was used as transfection control group,and the HEK 293T cells not transfected with the recombinant plasmid were used as the blank control group.The transfection efficiency of the transfection control group was observed by fluorescence microscope.The cells and cell culture supernatant of the experimental group and the blank control group were collected respectively.The expression level of Reg IV protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western-Blot(WB).The protein was purified by nickel column and Sephacryl S-400 column,and the purified recombinant protein was identified by SDS-PAGE and WB.Results The recombinant plasmid pCDNA3.4-REG4 was successfully constructed by sequencing and double enzyme digestion.The transfection control group(pEGFP-C1 plasmid)showed that the transfection efficiency was about 50%,indicating that the transfection was successful.WB results showed that Reg IV protein was detected only in the cells of the experimental group(pCDNA3.4-REG4 plasmid).The target protein could not effectively bind to the nickel column filler during nickel column purification,and the recombinant protein was purified by Sephacryl S-400 gel column chromatography.Conclusion The eukaryotic expression vector of REG4 gene was successfully constructed and expressed in HEK 293T cells,which laid a foundation for further study of the mechanism of Reg IV protein and the development of potential anticancer targeted drugs.

关 键 词：再生基因4 再生胰岛衍生蛋白Ⅳ 真核表达 人胚肾细胞293T 瞬时转染 蛋白纯化 

分 类 号：Q786[生物学—分子生物学]