Development and production of DNA-immunogen based SARS-СоV-2genes

Due to the development of the pandemic and the need for mass vaccination, the development of next-generation vaccines against COVID 19 coronavirus infection is a major challenge for medicine and biotechnology. Due to the prevalence of COVID 19, it is still urgent to develop a safe and protective vaccine, especially one that stimulates T-cell immune response. This work presents the design and production of a DNA immunogen based on plasmid DNA encoding a hybrid protein containing immunogenic fragments of structural proteins of SARS-CoV 2 β-coronavirus. As a result, a vector construct based on plasmid DNA encoding a hybrid protein containing the most immunogenic parts of structural proteins M, S, N, E of SARS-CoV 2 β-coronavirus was created by genetic engineering methods for transient expression in mammalian cells; a strain of E. coli, a producer of DNA-coronavirus β-coronavirus, was created by electroporation. coli, a DNA-immunogen producer; a method of purification of recombinant plasmid DNA was developed on the basis of a sequential step-by-step process: this method allowed to obtain 100 mg of DNA-immunogen pCMV 3Tag 3a-CVVVV3 in a solution with a concentration of 1 mg/mL (100 ml), which corresponds to the concentration established in the State Pharmacopoeia of the Russian Federation (OFS.1.7.1.0013.0013.1). .7.1.0013.18 DNA-vaccines) quality parameters - the obtained result is stably reproduced in laboratory conditions; methods of plasmid DNA quality control were worked out and expression of hybrid protein was tested by Western blotting. The CVVV3 antigen was shown to bind specifically to IgG immunoglobulins from rabbit sera after immunization with pCMV 3Tag 3A-CVVVV3 DNA-immunogen. The proportion of superhelicalized plasmid DNA in the sample of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was 85.64 %, the content of endotoxins was less than 25 U/mg; the concentration of residual proteins of the strain-producer in a solution of 1 mg/ml of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was less than 100ng per 1 ml (mg of total recombinant DNA).