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Polypeptide, immunogenicity conjugate and influenza vaccine

A technology for immunogenicity and influenza vaccines, which is applied in the field of immunogenic conjugates, influenza vaccines, and peptides, can solve the problems of poor immunogenicity and small molecular weight, and achieve the effect of improving survival rate

Active Publication Date: 2017-12-19
华兰生物疫苗股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, synthetic peptide vaccines generally have a small molecular weight and only target a single antigenic site, resulting in poor immunogenicity, better immunity to sequence-dependent antigenic sites, and poorer conformation-dependent sites

Method used

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  • Polypeptide, immunogenicity conjugate and influenza vaccine
  • Polypeptide, immunogenicity conjugate and influenza vaccine
  • Polypeptide, immunogenicity conjugate and influenza vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Design and synthesis of polypeptide M2A6:

[0076] 1. Design of polypeptide M2A6

[0077] Due to the high conservation of influenza virus M2e protein, it has become the research target of general influenza vaccine. The present invention designs a polypeptide M2A6 by comparing the sequences of about 13,000 influenza strains from NCBI, and its amino acid sequence is shown in SEQ ID NO.1, and it is named as polypeptide M2A6.

[0078] 2. Synthesis of polypeptide M2A6

[0079] The present invention adopts polypeptide solid-phase synthesis method to synthesize polypeptide M2A6. Based on Fmoc chemical synthesis, the carboxyl group of the C-terminal amino acid of the target polypeptide to be synthesized is first connected with an insoluble polymer resin in the form of a covalent bond, and then the amino acid is As the starting point of peptide synthesis, the amino group of the amino acid reacts with the activated carboxyl group of other amino acids to form a peptide bond. Thi...

Embodiment 2

[0095] Preparation of immunogen CRM197-M2A6 and its immune effect

[0096] 1. Preparation of immunogen CRM197-M2A6

[0097] 1. Solution preparation:

[0098] (1) Preparation of carrier protein solution: with CRM197 protein (shown in the amino acid sequence of CRM197 protein SEQ ID NO.2) as carrier protein, PBS buffer (20mM phosphate, 150mM sodium chloride, pH7.4) as solvent, Prepare the carrier protein solution to obtain a carrier protein solution with a concentration of 5 g / mL.

[0099] (2) Preparation of SMCC solution: According to 80× excess calculation, 6.69 mg of heterobifunctional cross-linking agent SMCC was weighed and dissolved in 334 μL of DMF solution to obtain a SMCC solution with a concentration of 20 mg / mL;

[0100] (3) Preparation of target peptide solution: Calculated by 10×, weigh 6.81 mg of the polypeptide M2A6 prepared in Example 1 above and dissolve it in 2.27 mL of PBS buffer to obtain a target peptide solution with a concentration of 3 mg / mL.

[0101] ...

Embodiment 3

[0113] Preparation of CRM197-M2A6 conjugate vaccine and its immune effect

[0114] 1. Preparation of CRM197-M2A6 conjugate vaccine

[0115] Dilute the immunogen CRM197-M2A6 prepared in Example 2 with PBS buffer (20 mM phosphate, 150 mM sodium chloride, pH7.4) to a concentration of 250 μg / mL, and then slowly add aluminum hydroxide adjuvant , ensure that the final concentration of aluminum hydroxide adjuvant is 2.5 mg / mL, and shake the sample slowly during the dropping process. After adding aluminum hydroxide, seal the sample well, place it at 2-8°C and slowly shake it for 15-18 hours after adsorption, 15-18 hours (16 hours is the best, according to the adsorption time, 16 hours has the best immune effect). Afterwards, it was diluted to a final antigen concentration of 100 μg / mL to obtain a CRM197-M2A6 conjugate vaccine.

[0116] Second, the detection of antibody titer

[0117] Inject the CRM197-M2A6 conjugate vaccine prepared in Step 1 by intraperitoneal injection. The immu...

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Abstract

The invention discloses a polypeptide, an immunogenicity conjugate and an influenza vaccine, and relates to the bio-technical field. The polypeptide is any one of a), b) or c): a) a polypeptide with the amino acid sequence as shown in SEQ ID NO.1; b) fused protein obtained by connecting a label to the end N and / or the end C of the polypeptide with the amino acid sequence as shown in SEQ ID NO.1; c) a polypeptide which is obtained by substitution and / or deletion and / or adding of one or several amino acid residues for the amino acid sequence as shown in SEQ ID NO.1 and has the same function. The polypeptide can be used for preventing or treating influenza virus, and can be used as an active component for preparing a medicine for preventing or treating the influenza virus. The immunogenicity conjugate disclosed by the invention can improve the immunogenicity and the immune effect on an antigen at a conformational dependency site.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a polypeptide, an immunogenic conjugate and an influenza vaccine. Background technique [0002] Influenza A virus is a common influenza virus. Influenza A virus is the most likely to mutate. The subtype of influenza A virus is called "bird flu". Bird flu (Bird Flu) is caused by bird flu virus. Acute infectious disease, virus gene mutation can infect human beings, the main symptoms after infection are high fever, cough, runny nose, myalgia, etc. Most of them are accompanied by severe pneumonia. In severe cases, heart, kidney and other organ failure lead to death , the fatality rate is high. Influenza A is highly pathogenic to humans and has caused worldwide pandemics many times. Among the influenza A viruses, the subtypes of avian influenza viruses that can directly infect humans have been found so far: A H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8. Among the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C07K19/00A61K39/385A61K39/145A61P31/16
CPCA61K39/12A61K39/385A61K2039/6037C07K14/005C07K2319/55C12N2760/16122C12N2760/16134
Inventor 梁雪爽刘晓刚安文琪张静静郝一楠王满满师方园
Owner 华兰生物疫苗股份有限公司
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