Subunit vaccine for novel coronavirus and application of subunit vaccine

A coronavirus, a new type of technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., to achieve the effects of improving stability, increasing immunogenicity, and prolonging plasma half-life

Inactive Publication Date: 2020-08-14
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there are currently no very effective drugs, therapeutic antibodies, vaccines, etc. for the treatment and prevention of SARS-CoV-2 virus infection, the development of vaccines against SARS-CoV-2 virus can protect healthy people and deal with the recurrence of the virus. Outbursts are critical

Method used

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  • Subunit vaccine for novel coronavirus and application of subunit vaccine
  • Subunit vaccine for novel coronavirus and application of subunit vaccine
  • Subunit vaccine for novel coronavirus and application of subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Amplification of SARS2-RBD and SARS2-RBD-Fc fragment genes

[0021] According to the reported clinically isolated SARS-CoV-2 virus (GenBank: QHR63250.2) gene sequence, the forward and reverse primers of the SARS2-RBD gene were designed to amplify the target fragment, and the Fc fragment gene of human IgG1 (GenBank: CAC20454. 1) Gene sequence design primers to amplify the Fc fragment and recombine:

[0022] Forward primer:

[0023]

[0024]

[0025] Note: The horizontal line in SEQ No. 3 is the recombined part on the vector.

[0026] Reverse primer:

[0027]

[0028] Note: The horizontal line in SEQ No. 7 is the recombined part on the vector.

[0029] Use forward primers (1) / (2) and reverse primers (1) to SARS-CoV-2 virus and use forward primers (3) and reverse primers (2) to IgG1 Fc fragment gene bases respectively The sequence was used as a template to perform PCR to obtain SARS2-RBD and Fc gene fragments, and the forward primer (1) and reverse primer (2) were used t...

Embodiment 2

[0032] Example 2: Expression and purification of SARS2-RBD-Fc protein

[0033] Expression using mammalian cell 293F expression system. One day before transfection, 293F cells (control cell density of 5×10 5 A / ml) 40mL was inoculated in a 125mL suspension cell culture flask. Dilute 40μg plasmid (pCAGGS-SARS2-RBD-Fc) in 4mL culture medium and mix gently, then dilute 60μL PEI (polyethylenimine catalog number 24765-1) in the culture medium and mix gently. After incubating at room temperature for 20 minutes, add them dropwise to the cells. Put the cells in a suspension incubator, 250 rpm, 37℃ 8% CO 2 Suspension culture.

[0034] After culturing for 120 hours, the culture supernatant was collected and Goat Anti-human-IgG Fc Antibody was used as an antibody to detect the expression of pCAGGS-SARS2-RBD-Fc by Western Blot ( image 3 ).

[0035] After detecting the expression of pCAGGS-SARS2-RBD-Fc, expand the cell culture and transfection scale to express a large amount of receptor binding...

Embodiment 3

[0037] Example 3: Mouse Immunization of SARS2-RBD-Fc Protein

[0038] The fusion proteins SARS2-RBD-Fc and SARS2-RBD were mixed uniformly with Imject Alum adjuvant (ThermoScientific), and the final protein concentration was 1 mg / ml. Female BABL / c mice aged 4-6 weeks were divided into 4 groups with 4 mice in each group. The first group is to immunize mice with SARS2-RBD+adjuvant 20μl / mouse by nasal drip; the second group is to immunize mice with SARS2-RBD-Fc+adjuvant 20μl / mouse by nasal drip; The group received 20 μl / mouse of SARS2-RBD-Fc+adjuvant by intramuscular injection in the leg; the fourth group was immunized with 20 μl / mouse of PBS+adjuvant by nasal drip. Two weeks later, the same dose was used for the second immunization. Two weeks after the second immunization (28 days in total), a certain amount of blood was taken from the mouse orbital venous plexus, and the specific antibody titer in the serum was measured by ELISA (ELISA coated SARS2-RBD protein). The result is F...

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Abstract

The invention discloses a fusion protein of a novel coronavirus envelope protein and an application of the fusion protein. The fusion protein (SARS2-RBD-Fc) is obtained by fusing an RBD structural domain of a novel coronavirus envelope protein S with an antibody Fc fragment; and as a subunit vaccine, the fusion protein can induce an organism to generate an efficient neutralizing antibody through nasal drip immunization and intramuscular injection. It indicates that the SARS2-RBD-Fc can be used as a candidate vaccine for preventing and treating new coronavirus infection.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and specifically refers to a subunit vaccine against a novel coronavirus and its application. Background technique [0002] The new type of coronavirus (2019-nCoV, SARS-CoV-2) is a type of beta coronavirus. After being infected with the virus, it can cause symptoms such as fever, dry cough, and fatigue; some patients will develop severe pneumonia, and then develop into Acute respiratory distress syndrome, septic shock, coagulopathy and multiple organ failure, and even death. The SARS-CoV-2 virus adsorbs and enters host cells through the envelope protein on its surface, thereby infecting human respiratory epithelial cells and alveolar cells. The envelope protein of SARS-CoV-2, Spike protein (S) is a glycoprotein, which mainly acts on cell adhesion and cell membrane fusion. The S protein is composed of two subunits, S1 and S2. The S1 subunit contains the Receptor Binding Domain (RBD) to mediate ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/215A61K47/68A61P31/14
CPCA61K39/12A61K47/6811A61K2039/543A61P31/14C07K14/005C07K2319/30C12N2770/20022C12N2770/20034
Inventor 龚睿肖庚富张海伟张哲赵少娟张化俊张晓晴潘晓彦詹焱程彭诚高晓霄
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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