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COVID-19 vaccine and preparation method and application thereof

A COVID-19 and vaccine technology, applied in the field of COVID-19 vaccine and its preparation, can solve the problems of complex preparation process, difficult preparation, and low effective antigen amount, and achieve the advantages of wide source of raw materials, low preparation cost and short preparation cycle Effect

Pending Publication Date: 2021-02-23
广东昭泰细胞生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional vaccines are mostly inactivated dead vaccines, attenuated vaccines or recombinant subunit vaccines, and there are many problems in the preparation process: (1) The preparation process is complicated, and cell culture methods are required to produce viruses, which has certain safety hazards; (2) High requirements for quality control and process amplification, poor control will lead to product quality accidents; (3) The amount of effective antigen is low, and production costs need to be increased to increase virus titers
So far, the biological characteristics of COVID-19 are not obvious, and it is highly infectious. Live vaccines lack safety and are difficult to prepare. Inactivated vaccines have the risk of recombination with wild strains and recovery.

Method used

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  • COVID-19 vaccine and preparation method and application thereof
  • COVID-19 vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 constructs the AdV5 adenoviral vector that overexpresses the SARS-CoV-2S protein coding gene

[0046] In this example, molecular cloning technology was first used to obtain the plasmid vector pUC57-S that continuously expresses the SARS-CoV-2 S protein molecule; then PCR was performed on the pUC57-S plasmid to obtain the S protein coding gene (SEQ ID NO: 2), and the homologous Recombinantly connected to the AdV5 adenovirus vector (containing GFP gene) between the Kozak and BGH pA sites, constructed as follows figure 1 The AdV5-S vector shown, the map is as follows figure 2 shown.

Embodiment 2

[0047] Embodiment 2 SARS-CoV-2 pseudovirus packaging

[0048] Cultivate 293T cells in a 10cm culture dish, the medium is DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution); 293T cells in the culture dish When the density reaches 80%, replace the medium with DMEM high glucose medium + 1% FBS + 1% double antibody;

[0049] After 2 hours, prepare the transfection reagent, put 500 μL opti-DMEM into a 15 mL centrifuge tube, add 7.2 μL PEI (linear polyethyleneimine) with a concentration of 10 μg / μL, mix slightly, and let stand for 5 minutes; take 500 μL opti-DMEM - DMEM into a 1.5mL centrifuge tube, take SARS-CoV-2S protein recombinant adenovirus vector 9μg, pMD2.G helper plasmid 3μg and psPAX 12μg, add to the centrifuge tube, mix well, add to the transfection reagent, mix upside down Evenly, let stand for 20min;

[0050] Add all the above mixture to 293T cells, and after incubation for 6 hours, replace with ...

Embodiment 3

[0053] Example 3 Construction of T cells expressing SARS-CoV-2 S protein

[0054] T cell acquisition and sorting: C57 / BL6 mice were dissected in a sterile environment after decapitation, and the complete spleen was isolated; the spleen was ground with a syringe, and filtered through a sieve to form a single-cell suspension; use a mouse Pan T cell isolation kit for T cell sorting, the target is to obtain live T cells ≥ 4×10 7 , survival rate ≥ 70%;

[0055] T cell activation: 24-well plates pre-coated with anti-CD3 / CD28 antibodies were used for T cell activation, and 1×10 7 Resuspend mouse spleen T cells in 2 mL of 1640 medium containing 10% FBS and 300 IU mouse IL-2, add them to a 24-well plate, and place at 37°C, 5% CO 2 Activate in the incubator for 48±3 hours;

[0056] Adenovirus transduction: The activated mouse T cells were collected by centrifugation and resuspended at 1×10 7 The proportion of MOI=200 virus solution was added to each cell / well for AdV transduction, a...

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Abstract

The invention provides a COVID-19 vaccine and a preparation method and application thereof. The COVID-19 vaccine comprises T cells expressing SARS-CoV-2 S protein. The preparation method of the COVID-19 vaccine comprises the following steps that (1), coding genes of the SARS-CoV-2 S protein are inserted into an adenovirus vector to construct a recombinant adenovirus vector; (2), the recombinant adenovirus vector and packaging helper plasmids are used for co-transfecting mammalian cells to prepare recombinant adenovirus; and (3), the recombinant adenovirus is adopted for infecting the T cells to obtain the COVID-19 vaccine. According to the COVID-19 vaccine and the preparation method and application thereof, a virus system is utilized for constructing the recombinant T cells expressing theSARS-CoV-2 S protein, the recombinant T cells are input into a body to continuously express the S protein, the body is induced to generate a specific humoral immune response or a cellular immune response, and the body obtains the protective immunity capacity on SARS-CoV-2.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy, and relates to a COVID-19 vaccine and its preparation method and application. Background technique [0002] The novel coronavirus pneumonia (COVID-19) caused by the 2019 novel coronavirus (SARS-CoV-2) is highly contagious, and researchers at home and abroad are intensively developing vaccines. Studies have found that the infection route of SARS-CoV-2 in the human body is similar to that of SARS-CoV. The virus's spike protein S protein (Spike protein) interacts with angiotensin-converting enzyme 2 (ACE2) to infect respiratory epithelial cells. For SARS, researchers tried to prepare vaccines in various forms such as viroids and protein subunits, and successfully induced neutralizing antibodies in animal experiments, but no vaccine against SARS virus has yet entered the clinical stage. [0003] The S protein of coronavirus plays an important role in virus infection by recognizing hos...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61P31/14C12N15/861C12N15/50C12N5/10
CPCA61K39/12A61P31/14C12N15/86C07K14/005C12N5/0636C12N2710/10343C12N2800/107C12N2770/20022C12N2770/20034A61K2039/5158A61K2039/53Y02A50/30
Inventor 李鹏秦乐汤朝阳蒋治武赵若聪崔元彬姚瑶胡朵
Owner 广东昭泰细胞生物科技有限公司
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