RESUMO
The epitope presentation system for ectodomain of M2-protein of influenza A virus (M2e) based on Cowpea Mosaic Virus (CPMV) was constructed for expression in plants Vigna unguiculata. CPMV is widely used as a vector for production of immunogenic chimeric virus particles (CVPs) bearing epitopes of different infectious human and animal pathogens. To produce chimeric CPMV virus particles in plants, two binary vectors were constructed bearing modified gene coding for S-coat protein of CPMV with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs obtained were investigated in mice immunization experiments and it was shown that they can induce anti-M2e IgG production and partial protection mice against challenge with low doses of flu virus. However, low infectivity and immunogenicity of CPMV chimeric particles indicate the need for further optimization of plant virus-based systems for M2e-epitopes presentation to use plants as a possible source of flu vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Comovirus/genética , Epitopos/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Comovirus/imunologia , Epitopos/genética , Epitopos/metabolismo , Fabaceae/genética , Fabaceae/imunologia , Fabaceae/metabolismo , Fabaceae/virologia , Humanos , Imunização , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/genética , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/biossíntese , Proteínas da Matriz Viral/genéticaRESUMO
The core antigen of hepatitis B virus (HBcAg) has attracted considerable attention as a carrier for antigenic sequences for various diagnostic and vaccine applications. The hepatitis B core protein has been expressed in different expression systems. At present, for reasons of cost, scale, and safety, the plant-based expression systems are attracting increasing interest. The expression and assembly for the hepatitis B core protein were investigating in N. benthamiana plants using the new expression system based on deleted version of cowpea mosaic virus RNA-2. Analysis of HBcAg expression revealed that the core protein expressed in plants and could self-assemble into virus-like particles. Virus-like particles could be purified by differential and sucrose gradient centrifugation. This expression system has the advantage of biocontainment and can be used for the rapid production of HBcAg virus-like particles for immunological and vaccine applications.
Assuntos
Comovirus/genética , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Nicotiana/metabolismo , Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/genética , RNA Viral/genética , Proteínas Recombinantes/biossínteseRESUMO
Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.
