DC vaccine for novel corona viruses, and preparation method and application of DC vaccine

A coronavirus and vaccine technology, applied in the field of biomedicine, can solve problems such as the lack of DC vaccines, and achieve the effects of improving the killing effect, quick start, and shortening the time

Inactive Publication Date: 2020-10-02
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the new coronavirus is a newly discovered...

Method used

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  • DC vaccine for novel corona viruses, and preparation method and application of DC vaccine
  • DC vaccine for novel corona viruses, and preparation method and application of DC vaccine
  • DC vaccine for novel corona viruses, and preparation method and application of DC vaccine

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Embodiment 1

[0033] Embodiment 1, the construction of S protein antigen expression vector

[0034] The nucleic acid sequence of each module of the S protein antigen plasmid construction is as follows:

[0035] (1) Leader CD8 (SEQ ID NO.1)

[0036] (2) 2019nCOV-S (SEQ ID NO.2)

[0037] (3) T2A (SEQ ID NO.3)

[0038] (4) CCL19 (SEQ ID NO.4)

[0039] Beijing Biomed Biotechnology Co., Ltd. was entrusted to sequentially synthesize artificial nucleic acid sequences according to the fusion genes CD8-2019nCOV-S (SEQ ID NO.5) and CD8-2019nCOV-S-T2A-CCL19 (SEQ ID NO.6), and insert them into The adeno-associated virus vector pAAV-IRES-hrGFP was transformed into E.coli (TOP10). After sequencing and identification, the plasmid was extracted and purified using the Qiagen plasmid purification kit to obtain recombinant adeno-associated virus expression vectors pAAV-2019nCOV-S and pAAV - 2019nCOV-S-CCL19 with a plasmid concentration higher than 200ng / ul.

Embodiment 2

[0040] Embodiment 2, packaging and titer determination of adeno-associated virus

[0041] ① Recovery and passage of HEK293 cells

[0042] Take out the frozen HEK293 cells from the liquid nitrogen tank, quickly throw them into a 37°C water bath and shake them quickly, try to completely dissolve the cell solution within 1-2 minutes. Transfer the cell solution to a 50ml centrifuge tube, add 5 ml of fresh complete medium (containing 10% FBS), mix well and centrifuge at 1500 rpm for 5 min. Remove the supernatant, add 1 ml of fresh complete medium to resuspend the cell pellet, transfer it to a T75 bottle, and make up to 10ml of complete medium in each bottle. Place the culture flask steadily at 37°C, 5% CO 2 and cultured in an incubator with 95% relative humidity. The cell viability was observed the next day, and the medium was replaced. Afterwards, the growth of the cells was observed every day, and the cells were passaged when the cells covered 80%-90% of the bottom of the bot...

Embodiment 3

[0049] Embodiment 3, DC vaccine preparation

[0050] Collect 50ml of peripheral venous blood from a healthy donor, add 50ml of normal saline for dilution, first add 20ml of lymphocyte separation solution to a 50ml centrifuge tube, then slowly add 25ml of blood, and centrifuge at 900g for 25min. The liquid surface is divided into four layers, absorb the white blood cell layer in the middle, and add DC medium. 37°C, 5% CO 2 Incubate for 2 hours in an incubator to allow monocytes to adhere to the wall. Discard the culture supernatant, add DC medium (purchased from Dayou) containing recombinant human GM-CSF (500-1000U / ml) and recombinant human IL-4 (500U / ml) to the adherent cells, 37°C, 5 %CO 2 Culture in an incubator, change the medium every other day, and culture for 5 days to obtain imDC (such as figure 2 ).

[0051] Take out the prepared pAAV-2019nCOV-S and pAAV-2019nCOV-S-CCL19 viruses, and infect the above imDCs at MOI=5. After 8-12 hours of infection, wash the cells 2...

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Abstract

The invention provides a DC vaccine for novel corona viruses. The DC vaccine comprises COVID-19 S proteins, chemotactic factors CCL19. The DC vaccine prepared from self peripheral blood is high in safety and does not have exclusive reactions. Compared with other vaccines, the DC vaccine is longer in duration time, can faster recognize antigens, can start up killing and damaging functions of T cells, and can produce antibodies at the same time. During in vitro tests, when the DC vaccine is united with CIK, killing and damaging to target cells can be started within 4h. The 2019nCOV-S-CCL19 DC vaccine prepared by the invention is united with the CIK cells to achieve good killing and damaging experiment effects; in vitro tests, when the 2019nCOV-S-CCL19 DC vaccine is united with the CIK cellsfor use, the 2019nCOV-S-CCL19 DC vaccine, the CIK cells and target cells are mixed for 24h, then detection is performed, when an effector target ratio is 10:1, the cytotoxicity to the target cells isas high as 93.2%, and when the effector target ratio is 5:1, the cytotoxicity to the target cells is as high as 68.6%.

Description

technical field [0001] The invention relates to a DC vaccine against novel coronavirus, a preparation method and application thereof, and belongs to the technical field of biomedicine. Background technique [0002] Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and play an important role in regulating innate and adaptive immune responses. In their immature state, DCs patrol the tissue microenvironment and become activated in the presence of foreign pathogens. This activation occurs following stimulation by exogenous danger signals through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and leads to migration of DCs to draining lymph nodes and presentation of processed epitopes to T cells. During T cell activation, DCs bind to the T cell receptor (TCR), secrete specific cytokines, and stimulate immune responses to TH1, TH2, or Tregs depending on the cytokine environment. [0003] The latest research shows that ...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61K35/17C12N15/864C12N15/62A61P31/14A61P11/00
CPCA61K39/12A61K35/17A61P31/14A61P11/00C12N15/86C07K14/005C07K14/70517C07K14/523A61K2039/57A61K2039/575A61K2039/5156C12N2770/20034C12N2750/14143C12N2770/20022C07K2319/00
Inventor 刘明录冯建海王亮王立新强邦明韩庆梅金海锋张传鹏许淼
Owner SHANDONG XINRUI BIOTECH CO LTD
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