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Preparation method of antisense RNA polyvalent vaccine for CoViD-19

A technology of multivalent vaccine and RNA interference, which is applied in the field of preparation of CoViD-19 antisense RNA multivalent vaccine, to achieve the effect of convenient vaccination, safe use and quick effect

Pending Publication Date: 2020-06-26
翁炳焕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, the existing vaccines are designed and prepared according to the mechanism of antigen-antibody reaction. These traditional vaccines play a preventive role by stimulating the body to produce immune antibodies after vaccination, but so far no vaccines based on the mechanism of RNA interference and SARS-CoV A literature report on the preparation of a CoViD-19 antisense RNA multivalent vaccine that can bind to CoViD-19 homologous mRNA and degrade its homologous mRNA without the participation of antigens and antibodies using the conserved gene sequence of -2

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  • Preparation method of antisense RNA polyvalent vaccine for CoViD-19
  • Preparation method of antisense RNA polyvalent vaccine for CoViD-19

Examples

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Embodiment 1

[0034] Preparation of shRNA Adenovirus Vector Vaccine shRNA / Ad5

[0035] Screen targeted interference gene shRNA from ORF1ab, S, E, M, N genes of SARS-CoV-2, construct interference vector pSilencer-shRNA (pSilencer-ORF1ab / S / E / M / N), and transfer shRNA expression in it Construct the adenovirus shuttle plasmid pDC312-shRNA, and co-transfect HEK293 cells with the adenovirus backbone plasmid pBHGloxAEl, obtain recombinant adenovirus shRNA / Ad5 through homologous recombination, and prepare CoViD by multiple amplification and purification of HEK293 cells -19 shRNA / Ad5 vaccine, after spray vaccination, shRNA is introduced into respiratory epithelial cells by recombinant adenovirus vector (Ad5), and dsRNA, siRNA and antisense RNA are sequentially generated in the cells, and finally SARS-CoV-2 RNA is specifically induced and / or mRNA degradation.

[0036] This example relates to a preparation method of CoViD-19 vaccine, involving but not limited to ORF1ab, S, E, M, N genes and primers th...

Embodiment 2

[0153] Preparation of siRNA / LHNPs for siRNA lipid nanoparticle vaccine

[0154] According to the SARS-CoV-2 genome sequence, using shRNA design software to predict, multiple RNA interference target gene siRNA sequences can be obtained, from which 1 to 3 conserved siRNA sequences with high silencing efficiency and no homology to the human genome can be screened , using a nucleic acid synthesis method to synthesize siRNA from a 4'-C-methyl-modified mononucleotide (AUCG), using a ring-opening reaction to synthesize a lipid-like epoxyalkylamine derivative EAAD, and using a nanoprecipitation method to prepare EAAD Lipid complex nanoparticle carrier siRNA / LHNPs loaded with siRNA, the inner layer is polylactic acid glycolic acid PLGA containing EAAD and nCoV2019 siRNA conjugates, the outer layer is wrapped with liposomes that can improve the biocompatibility of the carrier, and the outermost layer is PEG, which can improve the structural stability of the carrier, and then formulate s...

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Abstract

The invention relates to a preparation method of an antisense RNA polyvalent vaccine for CoViD-19. The preparation method comprises the steps of (1) designing shRNA of 2019-nCoV, constructing pSilencer-shRNA and pDC312-shRNA, performing homologous recombination on the pDC312-shRNA and pBHGloxAEl to obtain shRNA / Ad5 capable of generating dsRNA, siRNA and antisense RNA; (2) synthetizing chemically-modified mononucleotide into the siRNA of 2019-nCoV, and further preparing lipid nanoparticles siRNA / LHNPs loading siRNA; (3) compounding the antisense RNA polyvalent vaccine for pulmonary administration from the shRNA / Ad5, the siRNA / LHNPs and a pressurized spray, wherein the shRNA / Ad5 can degrade target genes after continuously outputting siRNA, siRNA / LHNPs can directly and immediately degrade thetarget genes, when the shRNA / Ad5 and the siRNA / LHNPs are used in a compounding ratio, synergistic effects can be achieved, and immunization failure of a univalent vaccine can be avoided.

Description

technical field [0001] The invention relates to a preparation method of a CoViD-19 antisense RNA polyvalent vaccine, belonging to the field of biological product preparation. Background technique [0002] On January 12, 2020, the World Health Organization named the coronavirus that caused the outbreak of viral pneumonia a new coronavirus (2019-nCoV), and then officially named it SARS-CoV-2, and named it the new coronavirus caused by it. Viral pneumonia is called CoViD-19. Shi Zhengli et al. reported that the basically consistent SARS-CoV-2 genome was obtained from five CoViD-19 patients, and by comparing the seven conserved non-structural proteins, it was found that SARS-CoV-2 belongs to the package of SARSr-CoV. Membrane RNA virus containing 5' untranslated region (UTR), replicase complex (orf1ab), S gene, E gene, M gene, N gene, 3'UTR and several unknown nonstructural open reading frames. For the nucleic acid detection of CoViD-19, the Chinese Center for Disease Control ...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61K9/12A61K9/51A61K47/26A61K47/34A61P31/14A61P37/04A61P31/04
CPCA61K9/12A61K9/5153A61K39/12A61K47/26A61K2039/53A61K2039/542A61K2039/544A61K2039/70A61P31/04A61P31/14A61P37/04C12N2770/20034
Inventor 翁炳焕
Owner 翁炳焕
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