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Recombinant subunit vaccine of novel coronavirus South African mutant strain and application of recombinant subunit vaccine

A technology of coronavirus and mutant strains, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of poor protection of mutant strains in South Africa

Active Publication Date: 2021-11-02
BEIJING HEALTH GUARD BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing vaccines have poor protection against the South African mutant strain, so there is an urgent need to develop a vaccine effective against the South African strain of the new coronavirus

Method used

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  • Recombinant subunit vaccine of novel coronavirus South African mutant strain and application of recombinant subunit vaccine
  • Recombinant subunit vaccine of novel coronavirus South African mutant strain and application of recombinant subunit vaccine
  • Recombinant subunit vaccine of novel coronavirus South African mutant strain and application of recombinant subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, carrier construction

[0019] By chemical synthesis, Beijing Liuhe Huada Gene Technology Co., Ltd. synthesized the fusion protein-related gene sequence of the antigenic epitope of the new coronavirus South African mutant COVID-19 South African mutant vaccine. in,

[0020] The RBD nucleotide sequence of the novel coronavirus South African mutant strain COVID-19 South African mutant strain is as follows (SEQ ID NO: 1):

[0021]

[0022] The amino acid sequence of the encoded RBD is as follows (SEQ ID NO: 2):

[0023]

[0024] The encoded SD1 amino acid sequence is as follows (SEQ ID NO: 3):

[0025]

[0026] The nucleotide sequence of SD1 is as follows (SEQ ID NO: 4):

[0027]

[0028] The encoded hFc amino acid sequence is as follows (SEQ ID NO: 5):

[0029]

[0030] The nucleotide sequence of hFc is as follows (SEQ ID NO: 6):

[0031]

[0032] RBDSD1-hFc amino acid sequence (SEQ ID NO: 7)

[0033]

[0034] The nucleotide sequence...

Embodiment 2

[0038] Example two, cell transient

[0039] ①The day before transfection, according to 2.0×10 6 cells / mL density inoculation, the cell density can reach 4.0×10 on the second day of culture 6 cells / mL or so;

[0040] ② After counting the cells on the second day of culture, the cell viability is >95%, and the viable cell density is ≥4.0×

[0041] 10 6 cells / mL, can be used directly; if the cell density is lower than 4.0×10 6 cells / mL, the cells can be collected by centrifugation (800rpm, 5min), and the cells6 cells / mL density resuspended in Transpro CD01 medium;

[0042] ③According to the optimized transient process, prepare the DNA and PEI mixture;

[0043] ④ Add the mixed solution to the culture medium for cultivation;

[0044] ⑤After 18 hours of culture, it is recommended to add a final concentration of 2mM valproic acid (VPA) and an initial culture volume of 5%+0.5%DN feed 2+DN feed B2, which can further increase the density of viable cells and protein expression. If ...

Embodiment 3

[0046] Embodiment three, protein purification

[0047] The protein extracted in Example 2 was purified by affinity chromatography and then ion exchange as follows.

[0048] 1. For the purification of RBDSD1-Fc, the following Protein A affinity chromatography method is adopted

[0049] One) the overall steps of the Protein A affinity chromatography method are as follows

[0050] Chromatography column: AT ProteinA Diamond affinity chromatography medium, column volume CV: 10ml, flow rate: 4-6ml / min, pressure limit: ≤0.3MPa.

[0051] Pretreatment: first rinse with 0.1M NaOH for at least 3 CVs, and then rinse with purified water for at least 10 CVs.

[0052] Equilibrium: Wash 2 to 3 CVs with washing buffer (0.1M HAc-NaAc, pH3.0), then fully equilibrate at least 10 CVs with binding buffer (0.02M PB, 0.5M NaCl, pH7.0) stand-by.

[0053] Sample loading: Take the filtered CHO suspension cell culture and use 5M NaCl to adjust the conductance to be consistent with the binding buffer,...

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Abstract

The invention discloses a fusion protein containing an antigen epitope of a novel coronavirus South African mutant strain COVID-19 vaccine. The fusion protein is composed of an RBD fragment and an SD1 fragment in an RBD antigen epitope fragment S1 subunit of the novel coronavirus South African mutant strain COVID-19 vaccine and an immune globulin Fc fragment. The research finds that the obtained effect is excellent when the SD1 is fused with the RBD of the novel coronavirus South African mutant strain COVID-19, and particularly the data neutralization effect is outstanding, so the vaccine can be popularized as a vaccine for the novel coronavirus South African mutant strain COVID-19.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, specifically relates to a recombinant subunit vaccine of novel coronavirus and its application, and more specifically relates to the recombinant subunit with immunological activity expressed in eukaryotic cells by viral genes artificially synthesized by genetic engineering means Protein, and the method of using the expressed recombinant protein to develop a vaccine. Background technique [0002] The new coronavirus belongs to the new coronavirus of the genus β, with an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. The structural proteins of the new coronavirus SARS-CoV-2 include S protein, N protein, E protein, and M protein. For SARS-CoV-2, only neutralizing antibodies directed against the S protein can neutralize the virulence of the virus and prevent the virus from infecting the body. The spike protein (Spike protein, S protein) on...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/18C12N15/62A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C07K2319/30C12N2770/20022C12N2770/20034
Inventor 张海江贠炳岭刘永江杨秀芬高文双温鸿研刘洋郭茜王艳伍树明
Owner BEIJING HEALTH GUARD BIOTECH
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