mRNA and vaccine for coding a SARS-CoV-2 viral antigen and preparation method of vaccine

A virus antigen, sars-cov-2 technology, applied in the field of vaccines, can solve the problems of complex preparation process, virus production, hidden safety hazards, etc., and achieve the effects of simple preparation process, easy industrialization, and high safety

Active Publication Date: 2020-06-02
LIVERNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no vaccine against new coronary pneumonia, and traditional vaccines are mostly in the form of inactivated viruses. However, regardless of the method used and despite technical improvements, there are usually many shortcomings in its application, especially: (1 ) The preparation process is complicated, and the virus needs to be produced by cell culture, which has certain hidden safety hazards
(2) The requirements for quality control and process amplification are high, and poor control will lead to product quality accidents
(3) The amount of effective antigen produced by the virus is low. In order to ensure the effect, it is required to increase the virus titer, thereby increasing the production cost

Method used

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  • mRNA and vaccine for coding a SARS-CoV-2 viral antigen and preparation method of vaccine
  • mRNA and vaccine for coding a SARS-CoV-2 viral antigen and preparation method of vaccine
  • mRNA and vaccine for coding a SARS-CoV-2 viral antigen and preparation method of vaccine

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preparation example Construction

[0059] The preparation method of the vaccine that uses cationic liposome as delivery formulation to deliver mRNA preferably includes: mixing mRNA and cationic liposome in a working solution to obtain the vaccine. Wherein the working liquid preferably includes deionized water, ultrapure water, sodium chloride solution or PBS. The concentration of mRNA in the working solution is preferably 0.01-0.5 mg / ml. The working solution is preferably PBS, and when the working solution is PBS, the mass ratio of cationic liposomes to mRNA is 1:1-4:1.

[0060] In some preferred embodiments, the preparation method of cationic liposomes includes providing a solution containing neutral helper lipids and cationic lipids, removing the solvent, drying, hydrating and sonicating the solution in sequence to obtain cationic liposomes . Wherein the solvent preferably includes a mixture of chloroform and / or dichloromethane and methanol; the drying time is preferably 1.5 to 2.5 hours; the water splash p...

Embodiment 1

[0063] According to the natural coding region sequence of the SARS-CoV-2 virus S protein, this embodiment designs the full-length sequence of the SARS-CoV-2 virus S protein (SF mRNA sequence). In addition to the coding region, the SF mRNA sequence features also include DNAH2 5 'UTR, HBA23' UTR and 50 polyA, SF-1 and SF-2 mRNA sequences were optimized in the S protein coding region, compared with SF-0 mRNA, the GC content was increased, but kept consistent in the UTR region, the sequence information is as follows Table 1 shows.

[0064] Table 1

[0065]

[0066] The target antigen production detection method (flow cytometry) is as follows:

[0067] 1. In order to detect the expression of SARS-CoV-2 virus S protein mRNA in human cells and the location on the cell membrane, in this embodiment, 293 cells cultured for more than 24 hours were digested and planted in a 6-well plate, and the cell density was controlled at 400,000 per hole.

[0068] 2. After incubating the six-we...

Embodiment 2

[0078] The mRNA encoding the full length of the S protein (SF mRNA is shown in the sequence shown in SEQ ID NO.13), the mRNA encoding the S1 subunit (the sequence shown in SEQ ID NO.14) and the receptor binding region RBD encoding the S protein mRNA (sequence shown in SEQ ID NO.15) transfected cells, detection of S, S1 and RBD protein expression in cells, the results are as follows Figure 4 As indicated, HEK293 cells transfected with each mRNA 24 hours later were lysed, loaded on SDS-PAGE gel with 10 μg of total protein, immunoblotted with anti-SARS-S1 protein antibody, and labeled with goat anti-rabbit-HRP secondary antibody , and then color. Protein expression was quantified using the internal reference b-actin. Cells not transfected with mRNA served as a negative control. The expression of full-length S protein, S1 subunit and RBD protein could be detected.

[0079] Using the target antigen production detection method in Example 1 to detect the expression of the antigen...

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Abstract

The invention provides mRNA and a vaccine for coding a SARS-CoV-2 viral antigen and a preparation method of the vaccine, and relates to the technical field of vaccines. The mRNA for coding the SARS-CoV-2 viral antigen at least contains at least one of S protein and N protein for coding SARS-CoV-2 virus and / or a coding region of a fragment of the at least one protein, and the mRNA is delivered intoa body to enable the body to generate an immune reaction.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to an mRNA encoding a SARS-CoV-2 virus antigen, a vaccine and a preparation method of the vaccine. Background technique [0002] Atypical pneumonia was named COVID-19 by the World Health Organization, and it was finally confirmed that it was caused by a new type of coronavirus (SARS-CoV-2), and the SARS coronavirus (SARS-CoV) in 2003 and the MERS coronavirus in 2012 The virus (MERS-CoV) also belongs to the genus coronavirus β. [0003] Coronaviruses are single-stranded RNA viruses with the longest genome sequences (ranging from 26 to 32 kb) of all RNA viruses. Coronaviruses can infect mammals, birds, and reptiles, including humans, pigs, cows, horses, camels, cats, dogs, bats, and more. Most coronaviruses do not cause clinical symptoms. SARS and MERS coronaviruses are zoonotic pathogens that can cause severe respiratory disease in humans. Compared with the 2003 "SARS" SARS-CoV,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C07K14/165A61K39/215A61P31/14
CPCA61K39/12A61K2039/53A61P31/14C07K14/005C12N2770/20022C12N2770/20034
Inventor 彭育才苏晓晔刘隽向晟楠罗丽平刘琪李爽雷奕欣
Owner LIVERNA THERAPEUTICS INC
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