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Subunit vaccine for preventing novel coronavirus infection based on bur protein S1 region of novel coronavirus

A subunit vaccine, coronavirus technology, applied in the field of vaccines, to achieve the effect of avoiding ADE risk and low ADE risk

Pending Publication Date: 2020-12-08
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, although full-length S protein combined with aluminum adjuvant has a certain protective effect on virus attack after immunization, it will induce severe immunopathology in the lungs characterized by eosinophil infiltration due to virus infection.

Method used

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  • Subunit vaccine for preventing novel coronavirus infection based on bur protein S1 region of novel coronavirus
  • Subunit vaccine for preventing novel coronavirus infection based on bur protein S1 region of novel coronavirus
  • Subunit vaccine for preventing novel coronavirus infection based on bur protein S1 region of novel coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of antigen

[0025] The gene encoding a new coronary virus S1 region (YP_009724390.1, MET1-TYR695) and RBD regions (YP_009724390.1, ARG328-PRO521) gene is connected to genes encoding the Northern, in accordance with the E. coli codon preferences, by Nanjing Jinsuri synthesis, constructed between BamHI and NOTI-dug sites of plasmid PET-28B, transformed E. coli (BL-21, DE3), 0.4 mM isopropyl-β-D-thiosani Lactoside at room temperature (~ 25 ° C) induces overnight. 4 ° C 8000 rpm was centrifuged for 15 min to collect the bacteria, resuspended in the PBS solution Ultrasound 15 min, 4 ° C 8000 rpm centrifugation 60 min to collect precipitation, ultrasonically cleaning 15 min, 4 ° C 8000 rpm centrifugal 15 min collection inclusion body dissolved in 8 m urea Dialysis of 20 mm Tris-HCl (pH 7.4) solution containing 6 mm, 4 m, 1 m and 0 m was used in 4 ° C. The prepared protein uses a TCA protein concentration test kit (Biyunndian) to determine concentration, 10%...

Embodiment 2

[0027] Example 2 - Animal Immunity

[0028]S1 and RBD protein (Qiqiao) expressed by mammalian cell HEK293, and E. coli expression, E. coli, is diluted with 10 μg / 25 μL, mixed with the same volume of alumina. After the agent (40 mg / ml), the interval was 2 peripheral injection of immunized BALB / C mice (6 weeks old, 14-17 grams, purchased by the Institute of Medicine, Chinese Academy of Sciences, the experimental animal center). After 2 weeks, the heart was centrifuged at 4 ° C after 2 weeks after 3 000 rpm was centrifuged for 10 minutes to take blood, preparing for subsequent immunological analysis.

[0029] Among them, the amino acid sequence of the S1 protein is:

[0030] VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPT...

Embodiment 3

[0032] Example 3 - Antibody titer detection

[0033] S1 and RBD protein 2 μg / ml expressed in PBS were added to S1 and RBD protein 2 μg / ml per well to add 96-pole enzyme board (Corning), 4 ° C overnight package was subjected to TBST (0.05% Tween 20 (Sigma) in PBS 1 time, 200 μl of 5% (w / v) is added to PBS per hole 37 ° C to close 1 h, abandoned TBST was washed 4 times, 100 μL of 100 μL per well was added to 1% milk gradient dilution Example 2 Anti-Ease 37 ° C was incubated for 1 h, TBST was washed 5 times, and 1% milk diluted horseradish peroxidase labeled second anti-sheep anti-mouse IgG / IgA / IgG1 / IgG2A (Saimi fly) 37 ° C After incubation of 1 h, TBST washed 5 times, 100 μL per well was added to 1: 1-based color culture (purchased from BD), and then protected from light at room temperature for 10 minutes, and 100 μl of 2 m sulfuric acid to terminate each well. The light absorption value is detected at 450 nm. Serum dilution concentrations critical in OD450 <0.1 were tak...

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Abstract

The invention provides a subunit vaccine for preventing novel coronavirus infection based on a bur protein S1 region of novel coronavirus and belongs to the field of vaccines. The subunit vaccine comprises a bur protein S1 region antigen of the novel coronavirus and an adjuvant and is used for preventing the novel coronavirus through subcutaneous or intramuscular injection for two or three times.According to the subunit vaccine provided by the invention, potential ADE risk caused by taking a full-length S protein as a novel coronavirus vaccine can be avoided, immunogenicity of RBD can also bereserved without introducing other viral antigens, and an antibody generated after immunization is sufficient to neutralize the novel coronavirus.

Description

Technical field [0001] The present invention belongs to the field of vaccine, and more particularly to a sub-unit vaccine based on new coronary virus-like protein S1 regions to prevent novel coronavirus infections. Background technique [0002] It was found in the new coronavirus (Severe Acute Respiratory Syndromecoronavirus 2, SARS-COV-2) for half a year, as of the end of June 2020, has caused thousands of people infection, and more than 50,000 people die. Including Cleverzhi (Locarwell / Local), hydroxychloroquine, Ruidisir and decetimus, an emergency use of drugs, clinical treatment of neoguan pneumonia (COVID-19) has not shown Satisfaction. Taking into account the strong infectivity and high diseases, lethality, the lethality rate, and the vaccine is still the first choice for COVID-19 other than public defense measures. [0003] A variety of vaccine forms include nucleic acid vaccine (DNA / RNA), viral vector vaccine, attenuated / inactivated vaccine, and subunit vaccines ar...

Claims

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Application Information

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IPC IPC(8): A61K39/215A61K39/39A61P31/14
CPCA61K39/12A61K39/39A61P31/14A61K2039/55505A61K2039/545C12N2770/20034
Inventor 刘存宝曹晗王云飞王丽春
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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