人烯醇化酶1在原核细胞中的可溶性表达及纯化  

Expression and Purification of Human ENO1 in Prokaryotic Cell

作　　者：金科华[1] 向念慈 刘洋[2] JIN Ke-hua;XIANG Nian-ci;LIU Yang(School of Basic Medical Sciences,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基础医学院,湖北咸宁437100 [2]湖北科技学院医学部药学院

出　　处：《湖北科技学院学报（医学版）》2022年第5期405-408,412,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：目的用原核表达系统生产人烯醇化酶1(ENO1),为筛选抑制剂奠定基础。方法通过5′分别带Nde I和Xho I位点的一对引物PCR扩增ENO1 cDNA,用Nde I+Xho I酶切cDNA和质粒pET-28a。酶切产物经纯化后用T4 DNA ligase连接,并转化大肠杆菌Top10,用含卡那霉素的LB平板(LBK培养基)筛选转化子。提取转化子质粒,酶切和测序法鉴定。将序列正确的重组子转化大肠杆菌BL21(DE3),用LBK培养基培养抗性菌落至OD_(600)约为0.7,加入终浓度0.2mmol/L IPTG,16℃诱导ENO1表达20h。超声破碎收集的菌体离心取上清,用组氨酸标签蛋白(可溶性)纯化试剂盒纯化ENO1。对纯化的ENO1进行超滤浓缩和缓冲液交换后,BCA法测定浓度,低温保存。结果ENO1正确连入pET-28a,重组菌pET-28a-ENO1-BL21(DE3)经0.2mmol/L IPTG诱导后,从裂解液上清中获得了高纯度的ENO1,产量达427mg/L。结论高产量、高纯度可溶性ENO1奠定了抑制剂筛选模型建立的基础。Objective The prokaryotic expression system was used to produce human enolase 1(ENO1),which laid the foundation for screening inhibitors.Methods A couple of primers,with Nde I and Xho I site flanked at their 5′end,were used to amplified ENO1 cDNA by PCR.The amplified products and plasmid pET-28a were digested by Nde I+Xho I.After purified,digested cDNA and pET-28a were ligated by T4 DNA ligase and transformed into Top10 cells.Kanamycin resistant transformants were screened on LB plate containing kanamycin(LBK plate).One transformant was cultured in LB broth containing kanamycin(LBK broth).The constructs were extracted and identified by digestion and sequencing.The construct of pET-28a ENO1 was delivered into BL21(DE3)cells and screened on LBK plate.One resistant colony was cultured in LBK broth till the OD_(600)reaching about 0.7.Then 0.2mmol/L of IPTG was applied to induce culture to express ENO1 at 16℃for 20h.Bacteria were harvested and cracked by ultrasonication,the lysate was centrifuged,and His tagged protein(soluble)purification kit was applied to purify ENO1 in supernatant.The resultant ENO1 was concentrated by ultrafiltration,the imidazole in ENO1 solution was reduced thoroughly by repeating exchanging buffer.The concentration of ENO1 was determined and cryopreserved.Results ENO1 cDNA was inserted into pET-28a.After induced by 0.2mmol/L of IPTG,recombinant bacteria pET-28a ENO1 BL21(DE3)expressed soluble ENO1 with a yield of 427 mg/L.Conclusion The high yield and purity of ENO1 laid a firm foundation for subsequent establishment of inhibitor screening model.

关 键 词：烯醇化酶1 表达 纯化 原核细胞 可溶性 

分 类 号：Q554.5[生物学—生物化学]