人磷酸丙糖异构酶在大肠埃希菌中的表达、纯化及鉴定  

Expression,Purification and Characterization of Human TPI in Escherichia Coli

作　　者：刘洁[1] 金科华[2] LIU Jie;JIN Ke-hua(School of Public Hygiene and Health,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)

机构地区：[1]湖北科技学院医学部公共卫生与健康学院,湖北咸宁437100 [2]湖北科技学院医学部基础医学院生物化学教研室

出　　处：《湖北科技学院学报（医学版）》2023年第4期298-302,共5页Journal of Hubei University of Science and Technology(Medical Sciences)

基　　金：湖北科技学院博士启动项目(2018-20XB015);湖北科技学院糖尿病专项开放课题(L07903/170136)。

摘　　要：目的本研究拟在大肠埃希菌中表达并纯化人磷酸丙糖异构酶(TPI),为其后续研究奠定基础。方法根据TPI编码序列(CDS)设计一对引物,其5′端分别添加Nde I和Xho I位点,PCR扩增TPI CDS。Nde I和Xho I酶切PCR产物和质粒pET-28a,胶回收酶切的质粒和PCR产物,并用DNA连接酶连接,连接产物转化DH5α感受态细胞,用含卡那霉素的抗性平板筛选抗性菌落。将抗性菌落接种于含卡那霉素的LB培养基,于37℃250r/min培养过夜,抽提质粒,双酶切和测序鉴定重组质粒pET-28a-TPI。将重组质粒pET-28a-TPI转化大肠埃希菌BL21(DE3)感受态细胞,于含卡那霉素的抗性平板筛选抗性菌落。将一个抗性菌落接种于100mL含卡那霉素的LB培养基,于37℃摇床中250r/min培养至A600约0.9,加入0.1mmol/L IPTG诱导TPI表达4h,收集菌体,超声裂解细胞,裂解液上清中的TPI用Ni^(2+)亲和层析柱纯化。用SDS-PAGE和Western blot鉴定纯化的表达产物,纯化的TPI经超滤浓缩后,保存于-20℃。结果pET-28a-TPI序列正确,可溶性TPI在大肠埃希菌中获得表达,产量达300mg/L,纯度约90%。结论高产率、高纯度的TPI为后续研究奠定了良好基础。Objective This study aims to express and purify triosephosphate isomerase(TPI)in Escherichia coli(E.coli),laying a foundation for subsequent research.Methods A pair of primers,flanked with Nde I and Xho I sites at their 5′ends,were designed.TPI coding sequence(CDS)was amplified by PCR.The PCR product and plasmid pET-28a were digested by Nde I and Xho I.The resulting products were purified by gel extraction and linked with DNA ligase.The linked products were transformed into DH5αcompetent cells.The resistant colonies were cultured in LB medium containing kanamycin and cultured overnight in 37℃shaker at 250 r/min.The recombinant plasmid pET-28a-TPI was extracted,identified by digestion,and sequenced.Kanamycin resistant colonies were screened in LB plates containing kanamycin.One colony was inoculated into LB medium containing kanamycin,cultured in 37℃at 250 r/min until the A600 reached 0.9.Bacterial solution was induced with 0.1mmol/L of IPTG for 4h,and harvested and sonicated.TPI in supernatant was purified using a Ni^(2+)affinity chromatography column.The purified TPI was identified by SDS-PAGE and western blotting,concentrated by ultrafiltration and stored at-20℃.Results The sequence of recombinant plasmid pET-28a-TPI was correct,and soluble TPI was expressed in pET-28a-TPIBL21(DE3)with a output of 300mg/L and 90%purity.Conclusion The high yield and purity of TPI establishes a good foundation for the further studies.

关 键 词：磷酸丙糖异构酶 可溶性 表达 纯化 超滤 

分 类 号：Q558.9[生物学—生物化学]