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Preparation method of novel coronavirus pneumonia bivalent vaccine

A coronavirus and vaccine technology, applied in multivalent vaccines, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effects of convenient inoculation, safe use and quick effect

Pending Publication Date: 2020-06-23
翁炳焕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In general, although the method of preparing antigenic vaccines based on pathogen antigens and the preparation methods of existing vaccines based on antigenic vaccination to produce immune antibodies to clear pathogens have been widely used, there is no preparation of dsRNA vaccines based on the conserved genome of pathogens. The method and the literature report and application of the dsRNA vaccine preparation method based on dsRNA vaccination to generate a gene silencing complex so that the homologous mRNA of the pathogen is degraded

Method used

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Examples

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Embodiment 1

[0024] Screen the targeted interference gene shRNA sequence (ORF1ab, S, E, M, N), construct the expression vector pSilencer-shRNA (pSilencer-ORF1ab / S / E / M / N), and transfer the shRNA expression cassette to construct an adenovirus shuttle The plasmid pDC312-shRNA was co-transfected with the adenovirus backbone plasmid pBHGloxAEl into HEK293 cells, and the recombinant adenovirus Ad-shRNA was obtained through homologous recombination, which was amplified and purified by HEK293 cells multiple times to prepare the immunopotentiator as the medium For the dsRNA vaccine against COVID-19, the recombinant adenovirus vector (Ad-shRNA) is used to introduce shRNA into respiratory epithelial cells after spray vaccination, and dsRNA is synthesized in the cells, followed by gene silencing or RNA interference that specifically induces nCoV mRNA degradation.

[0025] This example relates to a preparation method of nCoV2019 vaccine, involving but not limited to nCoV2019ORF1ab, S, E, M, N genes and ...

Embodiment 2

[0141] Screen the protein expression of the new coronavirus nCoV, that is, the antibody production gene, construct the adenovirus shuttle plasmid pShuttle-nCoV (pShuttle-ORF1ab / N) and the recombinant adenovirus backbone plasmid pAd-nCoV (pAd-ORF1ab / N), and package it into a recombinant adenovirus Ad-nCoV (Ad-ORF1ab / N), a recombinant adenovirus Ad-nCoV (Ad-ORF1ab / N) that is non-toxic to the human body, easily enters the respiratory epithelial cells and can express nCoV-mRNA in the cells is prepared in vitro, and then produced A respiratory spray DNA vaccine that can stimulate the body to produce anti-nCoV antibodies to produce specific immunity is used for emergency prevention of new coronavirus pneumonia.

[0142] This example relates to a preparation method of nCoV vaccine, involving but not limited to nCoV ORF1ab, S, E, M, N genes and their primers, involving but not limited to the same experimental methods, cloning vectors and other experimental materials, involving but not ...

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Abstract

The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.

Description

technical field [0001] The invention relates to a preparation method of a novel coronavirus pneumonia bivalent vaccine used in the field of prevention and treatment of infectious diseases, and belongs to the technical field of new vaccine preparation methods. Background technique [0002] Human coronaviruses (HcoV 229E and HcoV OC43) cause up to 30% of colds. Animal coronaviruses, such as porcine gastroenteritis coronavirus (TGEV), mouse hepatitis coronavirus (MHV), avian infectious bronchitis coronavirus (IBV), etc., can infect the respiratory tract, gastrointestinal tract, nervous system and liver of the corresponding host , resulting in corresponding symptoms. The envelope of the coronavirus has petal-shaped protrusions that make the coronavirus look like a crown (Latin, corona), and its nucleocapsid is a variable long helix. The virus particle diameter of the coronavirus is 60nm-140nm, spherical, and the virus genome is a single-stranded positive-sense RNA of 27kb-32kb...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61P31/14C12N15/113C12N15/861
CPCA61K39/12A61K2039/5256A61K2039/53A61K2039/544A61K2039/70A61P31/14C12N15/113C12N15/86C12N2310/14C12N2710/10043C12N2770/20034A61K2039/51C12N15/1131C12N2310/531C12N2710/10343C12N2770/20022
Inventor 翁炳焕李兰娟
Owner 翁炳焕
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