登革病毒铁蛋白纳米颗粒抗原的表达与纯化

Expression and purification of dengue virus ferritin nanoparticles antigen

目的登革病毒(Dengue virus,DENV)在世界范围流行。在我国,登革病毒流行毒株不断发生变化,登革病毒不断变异以及外来输入病例给登革热的预防和控制带来巨大挑战,开发安全有效的疫苗对于该病的预防控制至关重要。本研究将登革病毒包膜蛋白(envelope protein,E)呈递在幽门螺旋杆菌铁蛋白上以形成纳米颗粒,建立一种以铁蛋白纳米颗粒为载体的登革疫苗,表达登革病毒铁蛋白纳米颗粒抗原并进行纯化,以期为新型登革疫苗的研究提供借鉴。方法以质粒pcDNA3.1-DENV2-E-FE为模板扩增得到FE、DENV2-E-FE基因。将目的基因片段通过同源重组的方式分别连接到原核表达载体pET30a上,构建重组质粒pET30a-FE和pET30a-E-FE,经双酶切和测序鉴定后转化入感受态细胞中进行诱导表达,得到重组蛋白,通过SDS-PAGE、非还原性SDS-PAGE、Western blot及透射电镜对目的蛋白表征进行分析鉴定。结果成功扩增出目的基因片段,构建的重组质粒pET30a-FE和pET30a-E-FE转化到感受态细胞后经诱导表达约26 ku和71 ku的可溶性目的蛋白。Western blot检测重组蛋白能被相应抗体识别,透射电镜观察到两种蛋白均可自组装成粒径约13 nm和70 nm的颗粒结构。结论通过大肠埃希菌成功表达可溶性铁蛋白和登革病毒包膜蛋白铁蛋白,两种蛋白均具有反应原性且均能自组装形成纳米结构,可为新型登革纳米颗粒疫苗的研制提供借鉴。

Objective Dengue virus is endemic worldwide.In China,the prevalent strains of dengue virus are constantly changing,the continuous mutation of dengue virus as well as imported cases bring great challenges to the prevention and control of dengue fever,and the development of a safe and effective vaccine is crucial for the prevention and control of this disease.In this study,we presented dengue virus envelope protein on Helicobacter pylori ferritin to form nanoparticles,established a dengue vaccine using ferritin nanoparticles as a carrier,expressed dengue virus ferritin nanoparticle antigens,and purified them,with the aim of contributing to the study of novel dengue vaccines.Methods The FE and DENV2-E-FE genes were amplified by RT-PCR using pcDNA3.1-DENV2-E-FE plasmid as template.The target gene fragments were ligated into the prokaryotic expression vector pET30a by homologous recombination to construct recombinant plasmids pET30a-FE and pET30a-E-FE.After identification by double enzyme digestion and sequencing,the recombinant plasmids were transformed into competent cell BL21(DE3)for expression induction,and the recombinant proteins were obtained.The recombinant protein was purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE,non-reducing SDS-PAGE,Western blot and transmission electron microscopy.Results The target gene fragments were successfully amplified,and the constructed recombinant plasmids pET30a-FE and pET30a-E-FE were transformed into receptor cells and induced to express soluble target proteins of about 26 ku and 71 ku,the induced expression conditions were 37℃and 0.5 mmol/L IPTG for 6h.The recombinant proteins were detected by Western blot and recognized by the corresponding antibodies.The two proteins could self-assemble into granule structures with particle diameters of about 13 and 70 nm by transmission electron microscopy.Conclusion Successful expression of soluble ferritin and dengue virus envelope protein ferritin by Escherichia coli,both of which are reactive and can self-assemble to form nanostructures,may provide a reference for the development of novel dengue nanoparticle vaccines.