Ⅱ型登革病毒铁蛋白纳米颗粒蛋白的真核表达、纯化及免疫效果评价

Eukaryotic expression,purification and immunoefficacy evaluation of ferritin nanoparticles of dengue virus typeⅡ

将Ⅱ型登革病毒E蛋白呈递在幽门螺旋杆菌铁蛋白上,构建一种新型登革纳米颗粒候选疫苗,评价疫苗的免疫学指标,旨在为登革疫苗研发提供新思路。经优化合成E-Ferritin-pcDNA3.1重组质粒后转染HEK-293F细胞,对重组蛋白进行表达、鉴定、纯化和构型分析后使用E-Ferritin纳米颗粒疫苗于0、14、28 d后肢肌肉注射免疫小鼠。使用ELISA、中和试验、流式细胞术和淋巴细胞增殖试验检测免疫小鼠的特异性抗体水平、中和抗体水平、脾细胞中CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞含量和特异性刺激后脾淋巴细胞的增殖情况。结果显示,胞内表达出大小约69 kDa的目的蛋白,条带单一,纯化质量浓度0.407 g/L,纯度达到82.32%。透射电镜观察到E-Ferritin蛋白可重组成粒径约50 nm的颗粒结构。小鼠免疫试验结果表明,E-Ferritin蛋白具有良好的免疫原性,一免后42 d血清中特异性抗体效价平均值为1∶92160,主要表达的抗体亚类为IgG1,倾向于Th2型免疫应答,中和抗体效价达到1∶130。流式细胞术结果表明,重组蛋白E-Ferritin作为免疫原可以诱导机体产生更高水平的CD4^(+)、CD8^(+)T淋巴细胞免疫应答。淋巴细胞增殖试验中疫苗组特异性刺激水平显著高于非特异性刺激组。综上得出结论,本研究构建的登革病毒包膜蛋白铁蛋白纳米颗粒疫苗具有良好的免疫原性,可为新型登革候选疫苗研究提供参考。

The E protein of dengue virus typeⅡwas presented on ferritin of Helicobacter pylori to construct a novel dengue nanoparticle vaccine candidate,and the immunological indexes of the vaccine were evaluated,aiming to provide new ideas for the development of dengue vaccine.The recombinant plasmid of E-Ferritin was optimized and synthesized,and then transfected into HEK-293F cells.The recombinant protein was expressed,identified,purified and analyzed.Mice were immunized with E-Ferritin nanoparticle vaccine by intramuscular injection on the hind limbs on the day 0,14 and 28.ELISA,neutralization test,flow cytometry and lymphocyte proliferation test were used to detect the levels of specific antibodies,neutralizing antibodies,CD3^(+),CD4^(+)and CD8^(+)T lymphocytes in spleen cells and the proliferation of spleen lymphocytes after specific stimulation.The target protein with a size of about 69 kDa was expressed in the cells with a single band.The purified protein concentration was 0.407 g/L,and the purity was 82.32%.The results from transmission electron microscopy showed that E-Ferritin protein could be recombined into a particle structure with a particle size of about 50 nm.The results of mouse immune experiments showed that E-Ferritin protein had good immunogenicity.The average specific antibody titer of Eferritin protein in serum was 1:92160 after immunization 42 d.The main subclass of antibody was IgG1.The results of flow cytometry showed that E-Ferritin as an immunogen could induce higher levels of CD4^(+)and CD8^(+)T lymphocyte immune response.In lymphocyte proliferation test,the level of specific stimulation in the vaccine group was significantly higher than that in the nonspecific stimulation group.In conclusion,the dengue virus envelope protein ferritin nanoparticle vaccine constructed in this study has good immunogenicity,which can provide reference for the development of new dengue vaccine candidates.