基于流感病毒M2蛋白胞外区的双层蛋白质纳米颗粒的制备及免疫学效果评价

Preparation and immunological evaluation of bilayer protein nanoparticles based on influenza virus M2 protein extracellular domain

目的通过两步脱溶剂-交联法制备携带流感病毒血凝素茎部α螺旋区(HAα)、6个串联重复的M2蛋白胞外域(6M2e)和鞭毛蛋白(Flg)的双层蛋白质纳米颗粒, 探究其在小鼠体内的免疫效果。方法采用脱溶剂-交联法将融合蛋白FljB-6M2e-△D2D3-HAα和HAα-6M2e制备成双层蛋白质纳米颗粒。通过透射电镜和纳米颗粒跟踪分析仪进行物理表征后评估其细胞毒性。免疫小鼠后采用ELISA方法进行特异性抗体效价测定, ELISPOT检测细胞免疫水平, CCK-8法检测脾细胞的增殖情况。结果本研究成功制备了粒径为(130.03±2.96) nm的双层蛋白质纳米颗粒FljB-6M2e-△D2D3-HAα/ HAα-6M2e。免疫小鼠后产生的抗M2e-IgG抗体效价为1∶25 600, 抗HAα-IgG抗体效价为1∶12 800, 高于对照组小鼠, 差异均具有统计学意义(t=3.051和3.780, P=0.038和0.019)。与对照组相比, 多肽刺激可以使小鼠脾细胞显著增殖[M2e刺激增殖率:(154.18±2.34)%, HAα刺激增殖率:(151.51±1.56)%](t=12.942和24.591, P均<0.001), 并且诱导产生分泌更多IL-4和IFN-γ的淋巴细胞(M2e刺激:t=5.477和6.571, P=0.005和0.013;HAα刺激:t=9.239和7.671, P=0.001和0.013)。多种细胞因子的mRNA水平显著升高, IFN-γ和IL-4的转录水平均高于对照组(IFN-γ:t=13.253和20.847, P均<0.001;IL-4 :t=6.032和4.824, P=0.004和0.009)。结论本研究所制备的双层蛋白质纳米颗粒可以刺激小鼠产生较高水平的体液和细胞免疫应答, 为开发多表位流感通用疫苗提供了有效依据。

Objective To prepare the double-layer protein nanoparticles carrying influenza virus hemagglutinin stemαhelix region(HAα),6 tandem repeats of M2 protein extracellular domain(6M2e)and flagellin(Flg)by a two-step desolvent-crosslinking method,and to investigate their immune effects in mice.Methods Double-layer protein nanoparticles were prepared from the fusion proteins FljB-6M2e-△D2D3-HAαand HAα-6M2e by desolvent-crosslinking method.The cytotoxicity was evaluated after physical characterization by transmission electron microscopy and nanoparticle tracking analysis.After immunizing mice,the levels of specific antibody titer,cellular immunity and splenic cell proliferation were determined by ELISA,ELISPOT,and CCK-8,respectively.Results The bilayer protein nanoparticles FljB-6M2e-△D2D3-HAα/HAα-6M2e with a particle size of(130.03±2.96)nm were prepared successfully.The titers of anti-M2e-IgG antibody and anti-HAα-IgG antibody produced by immunizing mice were 1∶25600 and 1∶12800,which were significantly higher than those of control mice(t=3.051 and 3.780,P=0.038 and 0.019).Compared with the control mice,polypeptide stimulation significantly increased the proliferation of spleen cells in mice[proliferation rate of M2e:(154.18±2.34)%,proliferation rate of HAα:(151.51±1.56)%](t=12.942 and 24.591,both P<0.001),and induced the production of lymphocytes that secreted more IL-4 and IFN-γ(M2e stimulation:t=5.477 and 6.571,P=0.005 and 0.013;HAαstimulation:t=9.239 and 7.671,P=0.001 and 0.013).The mRNA levels of various cytokines significantly increased,and the transcription levels of IFN-γand IL-4 were higher than those in the control mice(IFN-γ:t=13.253 and 20.847,both P<0.001;IL-4:t=6.032 and 4.824,P=0.004 and 0.009).Conclusions The double-layer protein nanoparticles prepared in this study can stimulate mice to produce higher levels of humoral and cellular immune response,which provide an effective basis for the development of multi-epitope universal influenza vaccine.