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

A COVID-19, coronavirus technology, applied in the fields of immunology, biotechnology and biomedicine, can solve the problem of loss of efficacy of biohazardous original vaccines, achieve easy mass production, reduce site requirements, and reduce antigenicity effect of loss

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 Synthetic method of the target polypeptide (the amino acid sequence is the polypeptide shown in SEQ ID NO.1).

[0040] 1. The polypeptide whose amino acid sequence is shown in SEQ ID NO.1 was prepared by using the conventional method of solid-phase polypeptide synthesis, and the specific steps are as follows.

[0041] (1) Remove Fmoc protection.

[0042] Fill a commercially available Rink Amide AM resin into an organic-solvent-resistant reaction tube with a filter and cap tightly with an organic-solvent-resistant cap. After washing with DMF (dimethylformamide) for 1 minute, add an excess of 20% piperidine / DMF (volume ratio) solution, cap tightly and shake the reaction tube gently to mix it evenly and keep the Fmoc protection removed for 15 minutes . After draining, wash three times with DMF.

[0043] (2) Peptide bond condensation.

[0044] The Fmoc-protected amino acid of 3 times of resin amino molar weight, the activator HBTU of 3 times of resin amino mol...

Embodiment 2

[0053] Example 2 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 conjugated polypeptides through Linker.

[0055] TT tetanus toxin protein and tetanus toxin carrier protein TT can be obtained from Bacillus tetani through fermentation, lysis, centrifugation and chromatographic purification.

[0056] DT diphtheria toxin protein and diphtheria toxin carrier protein DT can be obtained from diphtheria bacillus through fermentation, cracking, centrifugation and chromatography purification.

[0057] CRM197 Recombinant Diphtheria Toxin Protein, CRM197 is a diphtheria bacillus reconstructed from the gene sequence, which is obtained through fermentation, lysis, centrifugation, and chromatography purification.

[0058] Meningococcal outer membrane protein: obtained from meningococcus through fermentation, lysis, ce...

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 protein: diphtheria toxin null mutant (CRM197), tetanus toxoid (TT), diphtheria toxoid (DT), meningococcus outer membrane protein (Meningococcus OMP).

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

[0077] 2. Preparation method.

[0078] Take the immunogenic conjugate, mix it with adjuvant and PBS solution, shake it at room temperature for 1 hour, 30RPM, the resulting final concentration contains 100μg / ml polypeptide antigen and 0.5mg / ml adjuvant, which is the final sample; or first mix the immunogen Adsorb the conjugate to the aluminum adjuvant, shake at room temperature for 1 hour, 30RPM, then add MPL or CpG adjuvant, shake at room temperature for 1 hour, 30RPM, the resulting final concentration contains 10...

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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 invention relates to the fields of immunology technology, biotechnology and biomedicine, in particular to a polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugates and uses thereof. Background technique [0002] Coronavirus is a positive enveloped virus with RNA, and its genome size is about 26-32kb, which is the largest RNA virus known genome. Genomic RNA and phosphorylated nucleocapsid (N) proteins are buried in the phospholipid bilayer and covered by the spike glycoprotein trimer (S), the membrane (M) protein (type III transmembrane glycoprotein) and the 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. Coronavirus is a kind of virus that widely exists in nature and can cause multi-system diseases including respiratory tract, digestive tract and nervous system. Highly pathogenic coronavirus infec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K1/04A61K39/215A61K39/385A61P31/14A61P11/00
CPCC07K14/005A61K39/12A61K39/385A61P31/14A61P11/00C12N2770/20022C12N2770/20034A61K2039/6037A61K2039/6068
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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