Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV- N -lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N -terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV- N -lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV- N -lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N -hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV- N -lys virions, but complexes formed highly organized and extensive network with dense “grains” on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV- N -lys/DHFR-M2e were significantly shifted relative to the control TMV- N -lys virions. The indirect enzymelinked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

中文翻译：

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通过转基因 TMV 颗粒与保守流感抗原的生物偶联形成的复合物：合成和表征

最近，我们获得了转基因烟草花叶病毒 (TMV) 颗粒与携带保守流感抗原（如 M2e 表位）的蛋白质之间的复合物。基于TMV-U1基因组的病毒载体TMV-N-lys是通过将化学活性赖氨酸插入外壳蛋白暴露的N-末端部分来构建的。本氏烟草植物被农杆菌注射并从未接种的叶子中纯化 TMV-N-lys 病毒粒子。通过SDS-PAGE/考马斯染色分析制剂；检测到电泳迁移率为 21 kDa 的主要蛋白质。电子显微镜证实了改性颗粒的稳定性。使用 5 mM 1-乙基-3-(3-二甲氨基丙基)-碳二亚胺和 1 mM N-羟基琥珀酰亚胺进行 TMV-N-lys 病毒粒子和在大肠杆菌中表达的靶流感抗原 M2e 的化学缀合。通过蛋白质印迹证实了化学缀合的效率。对于额外的表征，我们使用了传统的电子显微镜。复合物的直径与最初的 TMV-N-lys 病毒粒子没有显着差异，但复合物形成了高度有组织和广泛的网络，表面上有密集的“颗粒”。动态光散射表明，反映复合物 TMV-N-lys/DHFR-M2e 的单峰相对于对照 TMV-N-lys 病毒粒子显着偏移。使用 TMV 和 DHFR-M2e 特异性抗体进行的间接酶联免疫吸附测定表明，复合物在过夜吸附过程中保持稳定性。因此，结果允许使用这些复合物免疫动物，随后制备针对流感病毒的候选通用疫苗。