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Novel coronavirus RBD fusion protein subunit vaccine as well as preparation method and application thereof

A technology of subunit vaccine and coronavirus, which is applied in the field of novel coronavirus RBD fusion protein subunit vaccine and its preparation, can solve problems such as unseen results reports, achieve simple process route, high recovery rate, ensure stability and immunity original effect

Pending Publication Date: 2021-01-15
东莞博盛生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direction of research and development of new coronavirus vaccines includes recombinant protein vaccines, inactivated vaccines, DNA vaccines, mRNA vaccines and adenovirus vector vaccines. So far, recombinant new coronavirus (2019-COV) vaccines (adenovirus vectors) have entered the clinical stage. Other unseen results reported

Method used

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  • Novel coronavirus RBD fusion protein subunit vaccine as well as preparation method and application thereof
  • Novel coronavirus RBD fusion protein subunit vaccine as well as preparation method and application thereof
  • Novel coronavirus RBD fusion protein subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of a novel coronavirus RBD fusion protein subunit vaccine:

[0043] 1. Construction of recombinant plasmids

[0044] Select the C-terminal domain RBD, HBM (secretion signal peptide), flag (immune enhancement peptide) and hFc (immune enhancement peptide region) fragments in the S protein sequence of the new coronavirus (COVID-19), and fuse them in series , designed and synthesized on the pfastBac1 vector using codon optimization system, the structure diagram of the constructed recombinant plasmid HBM-flag-RBD-hFc_pfastBac1 is as follows figure 1 shown;

[0045] 2. Construction of recombinant bacmid

[0046] The recombinant plasmid HBM-flag-RBD-hFc_pfastBac1 was transformed into Escherichia coli DH10Bac competent cells, and the blue-white spot screening recombinants were carried out on the LB plate containing X-Gal / IPTG ( figure 2 ), select 5 white colonies for PCR identification, the target fragment size is 1650bp, and the identification results are as fo...

Embodiment 2

[0077] Extraction and purification of new coronavirus RBD fusion protein subunit vaccine insect rod system:

[0078] (1) Sample pretreatment:

[0079] Centrifuge the new coronavirus RBD fusion protein subunit vaccine insect rod system and add imidazole to 500ml of culture supernatant after centrifugation to a final concentration of 30mM. After adding, fully stir until completely dissolved;

[0080] Use NaCl to adjust the conductance to 50ms / cm, adjust the pH to 6.2, filter with a 0.45um membrane, and use the filtrate for chromatographic purification;

[0081] (2) Affinity chromatography

[0082] (1) The affinity chromatography filler is Ni filler resistant to EDTA and alkali; the imidazole concentration, conductivity and pH value of the filler equilibrium solution are the same as the pretreatment solution: 40mM PB, NaCl concentration 0.6M, imidazole concentration 30mM, pH6.2 equilibrium; The height is 15cm, the flow rate is 300cm / h; the equilibrium volume is 20CV;

[0083] ...

Embodiment 3

[0091] Extraction and purification of new coronavirus RBD fusion protein subunit vaccine insect rod system:

[0092] (1) Sample pretreatment:

[0093] Centrifuge the new coronavirus RBD fusion protein subunit vaccine insect rod system and add imidazole to 500ml culture supernatant after centrifugation to a final concentration of 5mM. After adding, stir well until completely dissolved;

[0094] The above treatment solution was adjusted with NaCl to conductance to 20ms / cm, pH to 8.0, filtered with a 0.45um membrane, and the filtrate was used for chromatographic purification.

[0095] (2) Affinity chromatography

[0096] (1) The affinity chromatography filler is Ni filler resistant to EDTA and alkali; the imidazole concentration, conductivity and pH value of the filler equilibrium solution are the same as the pretreatment solution: 0.1M PB, NaCl concentration 0.2M, imidazole concentration 5mM, pH8.0 balance; The column height is 10cm, the flow rate is 100cm / h; the equilibrium vol...

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Abstract

The invention belongs to the technical field of biological vaccines, and particularly relates to a novel coronavirus RBD fusion protein subunit vaccine as well as a preparation method and applicationthereof. RBD fusion protein is formed by fusing an RBD structural domain comprising novel coronavirus S protein, an HBM secretion signal peptide, a flag immunopotentiation peptide and an hFc immunopotentiation peptide, and an amino acid sequence of the fusion protein is as shown in SEQ ID NO.1; the subunit vaccine is obtained by expressing the RBD fusion protein through a baculovirus insect cell expression system, and then purification is conducted; the novel coronavirus RBD fusion protein is helpful for forming a vaccine drug for preventing novel coronavirus, and has important significance for preventing novel coronavirus infection; the high-purity and high-recovery-rate pure novel coronavirus RBD fusion protein subunit vaccine is obtained by extracting and purifying the vaccine; and industrial production can be realized.

Description

technical field [0001] The invention belongs to the technical field of biological vaccines, in particular to a novel coronavirus RBD fusion protein subunit vaccine and its preparation method and application. Background technique [0002] Coronavirus (Coronavirus) is a class of single-stranded positive-sense RNA viruses that can infect a variety of mammals including humans. Coronaviruses can be divided into four families: α, β, γ, and δ. There are 7 known coronaviruses that can infect humans, all of which come from the α and β families, including the low pathogenic HCoV-OC43 , HCoV-229E, HCoV-NL63, HCoV-HKU1 and highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, of which HCoV-229E and HCoV-NL63 belong to the α family of coronaviruses, and SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1 belong to the β family of coronaviruses. SARS CoV-2 is generally spherical, mostly pleom...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/866C07K1/22C07K1/34C07K1/36A61K39/215A61K47/64A61P31/14
CPCA61K39/12A61K47/64A61P31/14C07K14/005C07K2319/02C07K2319/30C07K2319/43C12N15/86C12N2710/14043C12N2770/20022C12N2770/20034C12N2800/107C12N2800/22
Inventor 王弋李静怡郑飞刘昕钟柱培梁燕华陈怡林覃柳斌欧海航
Owner 东莞博盛生物科技有限公司
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