Fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus and preparation methods for fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus

A technology of fusing gene and coronavirus, applied in the field of universal DC vaccine and its preparation, can solve the problems such as universal DC cell vaccine targeting coronavirus that has not been reported

Active Publication Date: 2021-04-02
BEIJING DCTY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, there is no report in the current prior

Method used

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  • Fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus and preparation methods for fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus
  • Fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus and preparation methods for fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus
  • Fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus and preparation methods for fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus

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preparation example Construction

[0059] The present invention also provides a method for preparing the coronavirus universal DC cell vaccine, comprising the following steps: 1) packaging a lentivirus with a recombinant vector comprising the fusion gene of TAX and ST40 to obtain a packaging virus; 2) packaging the packaging Virus-infected DC cells to obtain infected DC cells; 3) The infected DC cells and trophoblasts were first co-cultured for 4-6 weeks, and after CD3+ cells were removed, the second co-culture was performed for 1-2 weeks, and CD3+ cells were removed again After the cells, carry out the third co-culture for 2 to 6 months, collect antigen-presenting cells with limited expansion passages, and obtain an antigen-presenting cell line; 4) Transfer the recombinant vector expressing the fusion gene into the antigen described in step 3). Presenting cell lines to obtain a universal DC cell vaccine targeting coronavirus.

[0060] In the present invention, the recombinant vector including the fusion gene o...

Embodiment 1

[0069] Establishment of antigen-presenting cell lines with limited expansion passages

[0070] 1) synthesizing the fusion gene of TAX and ST40;

[0071] 2) the fusion gene synthesized in step 1) is connected to the expression vector to obtain a recombinant vector;

[0072] 3) According to the instructions of the lentivirus packaging kit (purchased from Beijing Hesheng Gene Technology Co., Ltd.), the vector described in 2) was packaged into a lentivirus to obtain a packaged virus, and the packaged virus was infected with DC cells to obtain an infected DC. cells;

[0073] 4) Inoculate the trophoblasts in the upper culture chamber of the cell culture incubator, inoculate the infected DC cells obtained in the step 3) in the lower culture chamber of the cell culture incubator, cultivate for 4 to 6 weeks, and continue to cultivate after removing the CD3+ cells After 1 to 2 weeks, the CD3+ cells were removed and cultured for more than 2 months to obtain an antigen-presenting cell l...

Embodiment 2

[0106] Example 2 Construction of lentiviral vector expressing ST40-TAX2 fusion gene and transformation of DC

[0107] 1) Packaging lentivirus: 293T cell plating density: 1.8×10 7 , 20mL OPTI-MEM medium was placed at 37°C 5%CO 2 nourish. Dosage of transfected plasmid obtained in Example 1: Dosage of packaging plasmid: 15 μg, P-ST40-TAX2 plasmid (plasmid for preparing immortalized dendritic cells): 15 μg, add the plasmid to the buffer, shake and mix for 5 s, add the transfection Transfection reagent: 60 μL, mix well with a pipette for 5 times, incubate at room temperature for 10 min, distribute the transfection mixture evenly and drop it into the cells, 37°C 5% CO 2 nourish. 3h after transfection, fresh medium was replaced, 37mL OPTI-MEM (6% FBS) medium was added to each T175 culture flask, and the supernatant was harvested at 96h, and virus concentrate (purchased from Beijing Hesheng Gene Technology Co., Ltd.) was added to precipitate. After 24h, centrifuge at 4000g for 30m...

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Abstract

The invention provides a fused gene, fused protein, recombinant vector and general DC vaccine of coronavirus and application of the fused gene, fused protein, recombinant vector and general DC vaccineof the coronavirus and belongs to the technical field of preparation of viral vaccines. The fused gene comprises a gene for encoding a protein N of a COVID-19 virus and a gene for encoding a RBD monomer, dimer or trimer protein of the COVID-19 virus. A general DC cell vaccine of the coronavirus is obtained through expressing the fused gene in an antigen presentation cell line. The general DC vaccine can be used for effectively activating T cells. According to records of embodiments, the general DC vaccine provided by the invention can be used for effectively activating specific antibodies ofmacaca fascicularis to the proteins N and RBD of the COVID-19 virus.

Description

technical field [0001] The invention belongs to the technical field of virus vaccine preparation, and particularly relates to fusion genes, fusion proteins, recombinant vectors, universal DC vaccines of three coronaviruses and preparation methods thereof. Background technique [0002] The coronavirus belongs to the genus Coronaviridae of the family Coronaviridae in the systematic classification. The human diseases caused by coronavirus are mainly respiratory system infections. At present, the types of coronavirus vaccines in the prior art include inactivated coronavirus vaccines, coronavirus spike protein vaccines, and adenovirus vector vaccines. [0003] However, there is currently no report on a universal DC cell vaccine targeting coronaviruses in the prior art. SUMMARY OF THE INVENTION [0004] In view of this, the object of the present invention is to provide a kind of fusion gene of coronavirus, fusion protein, recombinant vector, universal DC vaccine and preparation...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/867A61K39/215A61P31/14
CPCA61K39/12A61K2039/5154A61K2039/5156A61P31/14C07K14/005C07K2319/00C12N15/86C12N2740/10043C12N2770/20022C12N2770/20034
Inventor 焦顺昌张嵘汪坤福
Owner BEIJING DCTY BIOTECH CO LTD
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