摘要
目的借助新型铁蛋白系统构建pET32a-N-Ferritin-Flag-6His原核表达质粒,优化重组蛋白N-Ferritin诱导表达条件,鉴定其形态结构及稳定性。方法将SARS-CoV-2 N蛋白全长序列连接至Ferritin氮端,碳端连接Flag和6×His标签,基于pET32a载体,构建重组质粒pET32a-N-Ferritin-Flag-6His。通过质粒转化,IPTG诱导、时间及温度优化,采用Western Blot、TEM、DLS对重组蛋白进行鉴定。SDS-PAGE和考马斯亮蓝染色分析重组蛋白在不同冻融次数与不同储存温度下的稳定性。结果成功制备重组N-Ferritin蛋白,其在85 kDa处有目的条带。N-Ferritin重组蛋白呈现均一球形颗粒。重组蛋白粒径为18.23nm。N-Ferritin重组蛋白在反复冻融3~5次及在不同温度条件下(-80℃、-20℃、4℃)储存4周后仍有较好的稳定性。结论成功表达N-Ferritin蛋白并观察到纳米颗粒的形成,且该纳米颗粒蛋白具有良好的稳定性。
Objective To construct the prokaryotic expression vector pET32a-N-Ferritin-Flag-6His by a novel ferritin vaccine platform,optimize the induction expression condition of the recombinant protein N-Ferritin,and characterize its morphological structure and stability in depth.Methods The full-length sequence of the SARS-CoV-2 N(MT108784.1)protein was connected to the N-terminus of the ferritin protein by a flexible linker(G4S)3,and the C-terminus was connected with the Flag and 6×His tags,which was inserted into the pET32a vector,and the recombinant plasmid pET32a-N-Ferritin-Flag-6His of the fusion expression protein N-Ferritin was constructed.The pET32a-N-Ferritin-Flag-6His plasmid was transformed into the BL21(DE3)competent cells,and the optimal induction condition of the recombinant protein N-Ferritin was determined by optimizing the final concentration of IPTG in the induction process(0 mM,0.2 mM,0.4 mM,0.6 mM,0.8 mM,1.0 mM),time and temperature(25℃ induction for 3 h,6 h,9 h;20℃ induction for 6 h,12 h,18 h;15℃ induction for 12 h,24 h,36 h).The induced recombinant protein N-Ferritin was purified by nickel affinity chromatography column,and the recombinant protein was verified and identified by SDS-PAGE,Western blot,transmission electron microscopy,and DLS.The stability of the recombinant protein was analyzed by SDS-PAGE and Coomassie brilliant blue staining under different freezing and thawing times and different storage temperatures.Results The recombinant N-Ferritin protein was successfully prepared,and there was a target band at 90 kDa.Transmission electron microscopy observed that the N-Ferritin recombinant protein showed a uniform spherical particle.The DLS results showed that the particle size of the recombinant protein was 18.23 nm.Repeated freezing and thawing experiments showed that the N-Ferritin recombinant protein still had good stability after repeated freezing and thawing 3-5 times and stored for 4 weeks under different temperatures(80℃,20℃,4℃).Conclusion The N-Ferritin prokaryotic expression vector was successfully constructed,and a large amount of high-purity target protein could be obtained after optimizing the induction conditions,and it has good stability.
作者
曾佑琴
王强
杨凯雯
宋玉洁
黄炼杰
ZENG Youqin;WANG Qiang;YANG Kaiwen;SONG Yujie;HUANG Lianjie(College of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu,611130,China;Sichuan Academy of Medical Sciences-Sichuan Provincial People's Hospital(SAMSPH)Department of Rehabilitation Medicine,Chengdu,610072,China;Sichuan Academy of Medical Sciences-Sichuan Provincial People's Hospital(SAMSPH)Department of Pharmacy,Chengdu 610072,China)
出处
《哈尔滨医药》
2025年第4期1-5,共5页
Harbin Medical Journal
基金
四川省中医药管理局-面上项目(2024MS563)。
