2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and method for preparing 2019-nCoV S-antigens

An antigen and antibody technology, applied in biochemical equipment and methods, viruses/bacteriophages, chemical instruments and methods, etc., can solve the problem that antigens cannot produce high and active new coronaviruses, and achieve the effect of enhancing immunity and effective immunity

Inactive Publication Date: 2021-08-13
江西浩然生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention provides a new coronavirus S antigen for producing neutralizing antibodies against the new coronavirus, which is used to solve the technical problem that the antigens in the prior art cannot produce high neutralizing active new coronavirus neutralizing antibodies, and thus cannot effectively immunize animals

Method used

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  • 2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and method for preparing 2019-nCoV S-antigens
  • 2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and method for preparing 2019-nCoV S-antigens
  • 2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and method for preparing 2019-nCoV S-antigens

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Embodiment 1

[0039] To obtain the pseudovirus with the new crown S protein gene, first construct a codon-optimized cDNA (GenBank: NC_045512.2), and based on the cDNA encode the transmembrane motif (TM) and 3xFLAG tag of the full-length S protein ( Such as figure 1shown), cloned into the plasmid pRRL-derived vector (R48) by restriction endonuclease XbaI or XmaI to obtain the constructed plasmid pRRL-19S-FLAG-BSD, and based on the plasmid pRRL-19S-FLAG-BSD, the plasmid SARS- CoV-2s. Lentivirus prepared from plasmid SARS-CoV-2 and other vectors containing packaging elements (pLP-1-Gag / Pol, pLP-2-Rev, and pLP-VSVG) were transfected into 293FT cells. The medium was changed every 12-16 hours after transfection. The pseudovirus stock (named LV-19S) was collected from the supernatant and filtered through a 0.45 µm membrane for later use.

Embodiment 2

[0041] For the expression of S protein, in order to establish a cell line stably expressing Covid-19s, ID8 cells (donated by the Immunotherapy Institute of Fujian Medical University) were seeded at a density of 10,000 cells / well in DMEM (Gibco) + 10% FBS (GeminiBio) Incubate overnight in 6-well culture plates. The next morning, 1 ml of LV-19S (and vehicle control) was added and culture was continued for 24 h before being replaced with selection medium (DMEM + 10% FBS containing 10 µg / ml blasticidin S HCl (Invitrogen)) initial medium. Change the selection medium every 3 days until all non-infected cells have disappeared on day 7. Continue culturing of infected cells, i.e. stable cell lines, maintained in DMEM + 10% FBS complete medium containing 8 µg / ml blasticidin S HCl. In order to verify the expression, structure and function of spike protein, ID8 cells were treated with anti-FLAG-FITC ( figure 2 Middle left panel) or human ACE2-mFc staining, followed by anti-mouse IgG-F...

Embodiment 3

[0043] Immunogen preparation, ID8 cells (ID8 / 19-nCovS) with S antigen were cultured in DMEM+10%FBS complete medium, and the cells were subcultured and expanded every 3 days. Trypsinization was performed when the total number reached 3E+8 in multiple culture dishes, washed 5 times with 1xPBS to remove trypsin and FBS residues, resuspended cells in 20ml PBS, and frozen in a -80°C refrigerator for later use.

[0044] ID8 cells with S antigen were repeatedly frozen and thawed three times, and then prepared with an adjuvant composed of liquid paraffin and lanolin at a volume ratio of 1:1. Therefore, the immunogen includes the new crown S antigen, host cell components, paraffin oil and lanolin.

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Abstract

The invention discloses a 2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and a method for preparing the 2019-nCoV S-antigens. According to the 2019-nCoV S-antigens and the method, the 2019-nCoV S-antigens comprise 2019-nCoV S-antigen trimers combined with a cell membrane based on a Flag tag. The 2019-nCoV S-antigens can generate high-titer neutralizing antibodies so as to more effectively immunize animals.

Description

technical field [0001] This application relates to the technical field of antigen preparation, and more specifically, to the novel coronavirus S antigen and its preparation method for producing neutralizing antibodies against the novel coronavirus. Background technique [0002] The new coronavirus (2019-nCoV) can spread widely among humans, cause severe respiratory diseases, and cause death of patients, and there is no specific drug for treatment. For infectious diseases caused by viruses, it mainly depends on improving the patient's immunity to fight against the virus . [0003] Immunity can be divided into active immunity and passive immunity. As the main treatment method of active immunity, the development of vaccines takes a long time. As passive immunity, exogenous antibodies can be obtained through the following ways, the fastest way is from the serum of recovered people This method has the most abundant types of antibodies against the new coronavirus and has shown go...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K16/10C07K16/06C12N15/50C12N15/867
CPCC07K14/005C07K16/065C07K2317/20C07K2319/43C12N15/86C12N2740/15043C12N2770/20022C12N2800/22
Inventor 胡著阳耿卫锋林学良郑晓伟魏超娟徐富勇
Owner 江西浩然生物制药有限公司
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