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Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof

A COVID-19 and coronavirus technology, applied in the fields of biotechnology, biomedicine, and immunology technology, can solve the problems of loss of efficacy of biohazardous original vaccines, etc., and achieve easy large-scale mass production, reduce site requirements, and small molecular structure Effect

Pending Publication Date: 2022-02-18
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Traditional vaccines are made by inactivating or attenuating pathogens, and there are problems such as biological hazards and genetic variation that cause the original vaccine to lose efficacy

Method used

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  • Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof
  • Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof
  • Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 The synthetic method of the target polypeptide (the amino acid sequence is the polypeptide shown in SEQ ID NO. 1).

[0040] 1. A polypeptide whose amino acid sequence is shown in SEQ ID NO. 1 is prepared by using a conventional solid-phase method for synthesizing polypeptides, and the specific steps are as follows.

[0041] (1) De-Fmoc protection.

[0042] Commercially available Rink Amide AM resin was placed in a filter, organic solvent-resistant reaction tube and tightly capped with an organic solvent-resistant cap. After washing with DMF (dimethylformamide) for 1 minute, an excess of 20% piperidine / DMF (volume ratio) solution was added, and the reaction tube was gently shaken after capping to make it evenly mixed and kept to remove Fmoc protection for 15 minutes. . After draining, it was washed three times with DMF.

[0043] (2) Peptide bond condensation.

[0044] Fmoc-protected amino acids with 3 times the molar amount of resin amino groups, 3 times th...

Embodiment 2

[0053] Example 2 The preparation method of the synthetic peptide immunogenic conjugate shown in SEQ ID NO.1.

[0054] The synthetic peptides prepared in Example 1 were combined with CRM197, TT, DT, and bacterial outer membrane proteins to form co-conjugated polypeptides through Linker.

[0055] TT tetanus toxin protein and tetanus toxin carrier protein TT can be obtained by tetanus bacillus through fermentation, lysis, centrifugation and chromatography purification.

[0056] DT diphtheria toxin protein and diphtheria toxin carrier protein DT can be obtained by Bacillus diphtheriae through fermentation, lysis, centrifugation and chromatography purification.

[0057] CRM197 recombinant diphtheria toxin protein, CRM197 is a diphtheria bacillus with reconstructed gene sequence, obtained by fermentation, lysis, centrifugation and chromatography purification.

[0058] Meningococcal outer membrane protein: obtained by fermentation, lysis, centrifugation and chromatography purificati...

Embodiment 3

[0071] Example 3 Preparation of vaccine and immune effect.

[0072] 1. Vaccine preparation.

[0073] 1. Materials.

[0074] Polypeptide protein: the amino acid sequence is the polypeptide shown in SEQ ID NO.1.

[0075] Carrier proteins: Diphtheria toxin null mutant (CRM197), Tetanus toxoid (TT), Diphtheria toxoid (DT), Meningococcus OMP.

[0076] Adjuvants: aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), MPL, CpG.

[0077] 2. Preparation method.

[0078] Mix the immunogenic conjugate with adjuvant and PBS solution, shake at room temperature for 1 hour, 30 RPM, the final concentration of which contains polypeptide antigen 100 μg / ml and adjuvant 0.5 mg / ml is the final sample; The sexual conjugate was adsorbed on aluminum adjuvant, shaken at room temperature for 1 hour, 30RPM, then added MPL or CpG adjuvant, shaken at room temperature for 1 hour, 30RPM, the final concentration contained polypeptide antigen 100μg / ml, aluminum adjuvant 0.5mg / ml, MPL or CpG content o...

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Abstract

The invention relates to the fields of immunological technology, biotechnology and biological medicine, in particular to a polypeptide for preventing novel coronavirus pneumonia COVID-19, an immunogenic conjugate and application of the polypeptide and the immunogenic conjugate. The immunogenic conjugate comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, and is used for preparing a vaccine for preventing COVID-19. The immunogenic conjugate is a synthetic peptide vaccine prepared by synthesizing a target polypeptide based on a known or predicted section of antigen epitope amino acid sequence in a pathogen antigen gene, combining the synthesized target polypeptide with a carrier protein to prepare a polypeptide conjugate, and adding an adjuvant. Compared with a traditional attenuated vaccine, an inactivated vaccine and other novel vaccines, the vaccine can quickly cope with virus variation; the field requirement for vaccine production is reduced, and large-scale batch production is easy to realize; the research and development cost and the research and development period are reduced; safety, no toxicity and stability are realized; as the molecular structure is small and simple, the problems of serious complications and iatrogenic infection are rarely caused.

Description

technical field [0001] The present invention relates to the fields of immunology technology, biotechnology and biomedicine, in particular to a polypeptide, an immunogenic conjugate and its use for preventing novel coronavirus pneumonia (COVID-19). Background technique [0002] Coronavirus is a forward enveloped virus with RNA, and its genome size is about 26-32kb, which is the largest known RNA virus. Genomic RNA and phosphorylated nucleocapsid (N) proteins are embedded in phospholipid bilayers and covered by spike glycoprotein trimers (S), membrane (M) proteins (type III transmembrane glycoproteins) and envelope ( E) The protein is located between the S proteins of the viral envelope. Coronaviruses have a variety of hosts, including birds and mammals, especially bats. Coronaviruses are a class of viruses that widely exist in nature and can cause multi-system diseases including respiratory tract, digestive tract and nervous system. Highly pathogenic coronavirus infection h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165G01N33/569A61K39/215A61K39/385A61P31/14
CPCC07K14/005G01N33/56983A61K39/12A61K39/385A61P31/14C12N2770/20022G01N2333/165C12N2770/20034A61K2039/6068A61K2039/627
Inventor 刘刚于文俊张庶民高军周荔葆毛昱孙韦强孙述学车兴华
Owner TSINGHUA UNIV
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