RESUMO
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 enzyme-linked 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.
Assuntos
Antígenos Virais/química , Vírus do Mosaico do Tabaco/química , Proteínas da Matriz Viral/química , Agrobacterium tumefaciens/citologia , Agrobacterium tumefaciens/virologia , Antígenos Virais/imunologia , Humanos , Influenza Humana/imunologia , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
Pectin methylesterase (PME), cell wall enzyme, is known as a factor of plant growth and morphogenesis. Recently, PME participation in gene silencing, plant virus reproduction and transgenesis was revealed. Here, the role of PME in antivirus resistance was studied. It has been shown that increasing PME enzymatic activity induced by additional PME gene and even empty vector resulted in suppression of tobacco mosaic virus (TMV) reproduction including short- and long-distance virus movement in plant. We revealed increased PME activity in stably-transformed intact plants. For example, transgenic tobacco and N. benthamiana plants expressing TMV movement protein gene and GFP appeared increased PME activity. The tomato plants co-suppressing polygalacturonase gene have increased PME activity as well. Moreover, light-induced psbO gene activation was accompanied by transcription of PME gene. The introduction of foreign gene in plant cell or excess transcription of own genome resulted in increasing PME activity leading to transcriptome status quo returning.