Recombinant S protein, recombinant plasmid and recombinant bacterium of novel coronavirus and application of recombinant S protein, recombinant plasmid and recombinant bacterium in preparation of exosome drug or exosome vaccine

A coronavirus and exosome technology, applied in the field of vaccines, can solve the problems of poor immunogenicity, enhanced respiratory diseases, and enhanced dependent infection, and achieves a wide range of cell adhesion molecules, simple preparation methods, and enhanced therapeutic effects Effect

Pending Publication Date: 2021-11-05
NANHUA UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the new coronavirus is highly contagious and highly pathogenic, and the vaccine with the whole virus structure cannot guarantee its safety, and may cause ineffective antibodies in the vaccinated subjects
The production of ineffective antibodies often does not have a beneficial effect on the response to viral infection, and may even lead to side effects such as dependent infection enhancement and enhanced respiratory disease, while single-protein vaccines may not have good immunogenicity

Method used

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  • Recombinant S protein, recombinant plasmid and recombinant bacterium of novel coronavirus and application of recombinant S protein, recombinant plasmid and recombinant bacterium in preparation of exosome drug or exosome vaccine
  • Recombinant S protein, recombinant plasmid and recombinant bacterium of novel coronavirus and application of recombinant S protein, recombinant plasmid and recombinant bacterium in preparation of exosome drug or exosome vaccine
  • Recombinant S protein, recombinant plasmid and recombinant bacterium of novel coronavirus and application of recombinant S protein, recombinant plasmid and recombinant bacterium in preparation of exosome drug or exosome vaccine

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] The present invention also provides a preparation method for the above-mentioned exosome drug or exosome vaccine, comprising the following steps: after mixing the above-mentioned recombinant S protein or the recombinant S protein obtained by using the above-mentioned recombinant bacteria with exosomes, performing electrotransduction, The precipitate is collected by centrifugation to obtain the exosome drug or exosome vaccine.

[0042] The electrotransduction described in the present invention preferably includes: taking 10 μg of exosomes and adding them to 200 μl of electrical buffer, putting them in EP tubes, and labeling tube A; adding 10 nmol of the recombinant S protein and 200 μl of electrical buffers into 1.5 ml of EP tubes tube, label tube B; mix the liquids in tubes A and B, transfer to a pre-chilled shock cuvette, and place on ice; then proceed to electrotransduction.

[0043] The parameter of electric transduction described in the present invention preferably ...

Embodiment 1

[0049] 1.1 Find the amino acids of S1 and S2 of the new coronavirus in the Uniprot protein database, use the 5' sequence of the S1 protein and the 3' of the S2 protein to use the flexible (linker) 3 connected.

[0050] 1.2 Digest the 5' and 3' ends of the recombinant S protein coding gene with NheI and EcorV, connect with the pET-28a plasmid using T4 ligase, transform into competent cells, obtain the recombinant plasmid, and obtain stable expression by monoclonal screening Positive colonies of the recombinant S protein.

[0051] 1.3 The above cell lines were expanded and cultured, secreted, expressed and purified to obtain the purified recombinant S protein of the new coronavirus.

[0052] 1.31 Inoculate the cell line of the recombinant S protein in 50 μg / ml ampicillin lysing broth (LB), shake the bacteria at 37°C at 160 rpm overnight, and inoculate the colony of the genetically engineered bacteria in LB / kam according to the inoculation amount of 3%. In the culture medium, s...

Embodiment 2

[0058] 2.1 The exosome-carrying new coronavirus spray vaccine prepared in Example 1 was used for subcutaneous immunization of mice

[0059] Female BALB / c mice aged 6-8 weeks were divided into 2 groups (vaccine particle group and negative control group), with 8 mice in each group, which were inhaled through the nose respectively. Dissolve the vaccine in a volume of 200 µL of PBS. Blood was collected from the tail of the mice every two weeks, and the serum was separated for antibody detection. Serum samples were stored at -20°C until use.

[0060] The level and titer of Delta-specific IgG antibody in serum of immunized mice were detected by indirect ELISA. Coat the ELISA plate with 20 μg / ml S1 or S2 protein, overnight at 4°C. Wash three times with 0.05% PBST, block with 1% BSA-PBST blocking solution, and incubate at room temperature for 1 h. Wash 5 times with 0.05% PBST, add 200μl diluted serum (1:1000 dilution), incubate at 37°C for 2h; wash 5 times with PBST, add HRP-goat ...

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Abstract

The invention provides a recombinant S protein, a recombinant plasmid and a recombinant bacterium of a novel coronavirus and application of the recombinant S protein, the recombinant plasmid and the recombinant bacterium in preparation of an exosome drug or an exosome vaccine, and relates to the technical field of vaccines. According to the invention, the recombinant S protein comprises a subunit S1 and a subunit S2 of a spike protein S; the S1 protein and the S2 protein are connected through utilizing a linker to form the recombinant S protein; all the components are mutually coordinated and supplemented; and the recombinant S protein has good immunogenicity, safety and biological activity and can induce an organism to generate an effective antibody, so that the growth of the novel coronavirus is inhibited, and the infection of the novel coronavirus can be effectively prevented. According to the invention, the recombinant S protein is electrically transferred into an exosome by adopting an artificial exosome carrying form, so that the pharmacokinetic and pharmacodynamic characteristics of a carried therapeutic agent can be improved, thereby enhancing the therapeutic effect and meanwhile, reducing the adverse toxicity and side effects; and the recombinant S protein has wide cell adhesion molecules and is beneficial for penetrating a biological barrier.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to a recombinant S protein, a recombinant plasmid, a recombinant bacterium of a novel coronavirus and the application of preparing exosome medicine or exosome vaccine. Background technique [0002] COVID-19 is a lung disease caused by infection with SARS-CoV-2 (or 2019-nCoV). SARS-COV-2 is highly contagious and is mainly transmitted through mucous membranes. SARS-CoV-2 is similar to SARS virus and belongs to coronavirus, but its base sequence and structure are different from SARS virus. [0003] The existing vaccine production includes traditional vaccines (attenuated live vaccines, inactivated vaccines), subunit vaccines, virus vector vaccines, nucleic acid vaccines, etc. Among them, the subunit vaccine is a vaccine that uses certain surface structural components of microorganisms (antigens) to induce the body to produce antibodies that do not contain nucleic acids. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N1/21A61K39/215A61K47/46A61K9/12A61P31/14C12R1/19
CPCC07K14/005C12N15/70A61K39/12A61K47/46A61K9/12A61P31/14C07K2319/00C12N2770/20022C12N2770/20034A61K2039/541
Inventor 左建宏谢卓熠黄佳璐
Owner NANHUA UNIV
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