RESUMEN
The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.
Asunto(s)
ARN Bicatenario , ARN Mensajero , Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Ratones , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón-alfa/biosíntesis , Proteínas Virales/metabolismo , Proteínas Virales/genéticaRESUMEN
An artificial T-cell immunogen consisting of conserved fragments of different proteins of the SARS-CoV-2 virus and its immunogenic properties were studied in BALB/c mice. To create a T-cell immunogen, we used an approach based on the design of artificial antigens that combine many epitopes from the main proteins of the SARS-CoV-2 virus in the one molecule. The gene of the engineered immunogen protein was cloned as part of the pVAX1 plasmid in two versions: with an N-terminal ubiquitin and without it. The obtained plasmids were analyzed for their ability to provide the synthesis of the immunogen protein in vitro and in vivo. It has been shown that protein product of the created artificial genes is actively processed in HEK293T cells and induces cellular immunity in mice.
Asunto(s)
COVID-19 , Linfocitos T , Humanos , Ratones , Animales , Células HEK293 , SARS-CoV-2/genética , EpítoposRESUMEN
During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity.