mRNA vaccine and its synthesis method, kit

A technology of vaccines and spike sugars, applied in the direction of positive single-stranded RNA viruses, biochemical equipment and methods, antiviral agents, etc., can solve problems such as undiscovered, and achieve the effects of easy transportation, simple process operation, and wide applicability

Active Publication Date: 2021-06-08
SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In related technologies, no mRNA vaccine capable of dealing with the new coronavirus (2019-nCoV) has been found

Method used

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  • mRNA vaccine and its synthesis method, kit
  • mRNA vaccine and its synthesis method, kit
  • mRNA vaccine and its synthesis method, kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The antigen of embodiment 1 routine mRNA vaccine and long-acting mRNA vaccine

[0063] In this example, conventional mRNA vaccines encoding different antigen fragments and long-acting mRNA vaccines capable of self-replication were transfected into 293T cells, and the expression results of the antigens encoded by the vaccines were detected by Western blot 12 hours later.

[0064] 1) Preparation of transfection reagent. After fully mixing 100ng mRNA and transfection reagent TransIT, add 50uL serum-free medium, and let stand at room temperature for 5min.

[0065] 2) Cell transfection. Remove the medium in the culture plate, wash once with PBS or serum-free medium, add 200uL of fresh medium; then add the transfection reagent prepared in step 1) evenly into the cell culture medium.

[0066] 3) Western blot experiment. After 24 hours, the cells in step 2) were lysed and the lysate was collected for western blotting. The expression levels of conventional mRNA vaccines and ...

Embodiment 2

[0068] Expression yield of embodiment 2mRNA vaccine in cells

[0069] 1. According to Table 1, SEQ ID: 1, SEQ ID: 3, SEQ ID: 5, SEQ ID: 7 and SEQ ID: 9 are connected into a sequence Opti-1 through a connecting peptide, and SEQ ID: 2, SEQ ID : 4, SEQ ID: 6, SEQ ID: 8 and SEQ ID: 10 are connected into a sequence Opti-2 by connecting peptides to synthesize DNA fragments encoding S protein, M protein, E protein, N protein and RBD, and the The DNA fragment and the fragment of the DNA sequence encoding luciferase are simultaneously cloned into a DNA plasmid vector, so that the antigen and luciferase form a fusion protein, and after the DNA plasmid vector is linearized, it is transcribed in vitro to synthesize an mRNA vaccine. At the same time, a control group was set up to search for the optimization scheme (Other1-5) of the novel coronavirus mRNA existing on the market, and to synthesize a control mRNA vaccine. And the mRNA vaccine prepared above was injected into mice to obtain t...

Embodiment 3

[0071] The immune response of embodiment 3mRNA vaccine

[0072] 1. Get vaccinated. Inoculate mice with novel coronavirus 2019-nCoV mRNA vaccine (specifically, inoculate the conventional mRNA vaccine prepared in Example 1 respectively (see Image 6 ) and long-acting mRNA vaccines (see Figure 7 )), intracellular cytokine staining to quantify the production of IFN-g (interferon gamma) or TNF- a (Tumor necrosis factor) percentage of activated T cells. Detection of activated T cells in mouse T cells immunized with luciferase mRNA (n=5 per group) or mRNA vaccine immunized mouse T cells (n=5 per group) at weeks 0, 1 and 2 percentage, get as Image 6 Middle A and Figure 7 Data results shown in A.

[0073] 2. Antibody response induced by mRNA vaccine. The endpoint dilution ELISA titer of novel coronavirus 2019-nCoV antibody in immunized mouse serum was measured by optical density, and obtained as follows: Image 6 Middle B (left) and Figure 7 Data results shown in Center B ...

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Abstract

The invention discloses an mRNA vaccine and its synthesis method and kit. The mRNA vaccine consists of: the epitope antigen gene sequence of trimeric spike glycoprotein S, and / or the epitope of transmembrane protein-envelope E Antigen gene sequence, and / or, epitope antigen gene sequence of membrane glycoprotein M, and / or, epitope antigen gene sequence of nucleocapsid N, and / or, receptor binding in trimeric spike glycoprotein The epitope antigen gene sequence of domain RBD is prepared. The technical solution of the present invention designs an mRNA vaccine through a genetic engineering method to achieve immunity to novel coronaviruses.

Description

[0001] This application claims the priority of the Chinese patent application filed on February 06, 2020, with the application number 202010083614.6, and the title of the invention is "mRNA vaccine and its synthesis method, and kit". technical field [0002] The invention relates to the technical field of biopharmaceuticals, in particular to an mRNA vaccine and its synthesis method and kit. Background technique [0003] Coronavirus (Coronavirus) is a class of RNA viruses that exist widely in nature. It is named after the shape of the virus resembles a crown when observed under an electron microscope. Coronavirus is a large family with many members, which only infects vertebrates and can cause respiratory, digestive and nervous system diseases in humans and animals. The novel coronavirus (2019-nCoV) presents a spherical ellipsoid with a diameter of 80-120nm. Under the electron microscope, there is a club-shaped protrusion on the surface of the virus particle, which is compos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/215A61P31/14
CPCA61K9/0019A61K9/0043A61K39/12A61K2039/523A61K2039/5252A61K2039/54A61K2039/543A61P31/14C12N2770/20034
Inventor 胡勇张苗苗
Owner SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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