Immunogen HM4-M2e as well as preparation method and application thereof

A sequence and protein technology, applied in biochemical equipment and methods, chemical instruments and methods, pharmaceutical formulations, etc., can solve the problems of poor immunogenicity and small molecular weight, achieve high cross-linking rate, short preparation time, and reduce production costs Effect

Active Publication Date: 2017-01-04
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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  • Immunogen HM4-M2e as well as preparation method and application thereof
  • Immunogen HM4-M2e as well as preparation method and application thereof
  • Immunogen HM4-M2e as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, the design and synthesis of polypeptide M2A6

[0087] 1. Design of polypeptide M2A6

[0088] The present invention designs a polypeptide M2e by comparing the sequences of about 13,000 influenza strains from NCBI, the amino acid sequence of which is shown in Sequence 1, and names it as polypeptide M2A6.

[0089] 2. Synthesis of polypeptide M2A6

[0090] 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. This process is repeated continuously, and the peptide can be obtained by synthesizing from the C-terminus to th...

Embodiment 2

[0105] Embodiment 2, the preparation of immunogen HM4-M2A6

[0106] One, the preparation method of immunogen HM4-M2e

[0107] 1. Solution preparation

[0108] (1) Preparation of carrier protein solution: with HM4 protein (the amino acid sequence of HM4 protein is shown in sequence 2 in the sequence listing) as carrier protein, PBS buffer (20mM phosphate, 150mM sodium chloride, pH7.4) as solvent , preparing a carrier protein solution to obtain a carrier protein solution with a concentration of 1.5 mg / ml;

[0109] (2) Preparation of SMCC solution: According to 30× excess calculation, weigh 3.3 mg of heterobifunctional cross-linking agent SMCC and dissolve it in 165 μL of DMF solution to obtain a SMCC solution with a concentration of 20 mg / mL;

[0110] (3) Preparation of target peptide solution: Calculated at 1×, weigh 0.45 mg of the polypeptide M2A6 prepared in Example 1 above and dissolve it in 150 μL of PBS buffer to obtain a target peptide solution with a concentration of 3...

Embodiment 3

[0119] Example 3, Preparation of HM4-M2A6 conjugate vaccine and its immune effect

[0120] 1. Preparation of HM4-M2A6 conjugate vaccine

[0121] Dilute the immunogen HM4-M2A6 prepared in step 1 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, The final concentration of the aluminum hydroxide adjuvant was 2.5 mg / mL, and the sample was slowly shaken during the dropwise addition. After adding aluminum hydroxide, seal the sample well, place it at 2-8°C for 16 hours with slow shaking and absorb it, then dilute it to a final antigen concentration of 100 μg / mL to obtain the HM4-M2A6 conjugate vaccine.

[0122] Second, the detection of antibody titer

[0123] (1) Immunization method

[0124] Inject the HM4-M2A6 conjugate vaccine prepared in step 1 by intraperitoneal injection. The immunization dose is 50μg / 0.5mL / bird / time. The experimental animal information is shown in Table 1.

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Abstract

The invention discloses an immunogen HM4-M2e as well as a preparation method and an application thereof. Hepatitis B virus core protein HM4 is used as carrier protein and crosslinked with designed and synthesized polypeptide M2e through SMCC, and thus the immunogen HM4-M2e is obtained. Experiments prove that a drug prepared from the prepared immunogen HM4-M2e used as an effective ingredient of an influenza vaccine and a proper immunologic adjuvant used as an auxiliary material has a better immune effect. Besides, the used carrier protein is suitable for mass industrial production, the crosslinking rate is high, the preparation time is short, and the production cost is reduced.

Description

technical field [0001] The invention relates to an immunogen HM4-M2e and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] Synthetic peptide vaccine (Synthetic peptide vaccine) is a virus protective antigen peptide vaccine synthesized by chemical method, which has good safety, will not be polluted by pathogenic microorganisms, and is easy to preserve and control the quality. 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. [0003] Hepatitis B virus core protein HBc (Hepatitis B virus core protein, HBc) has become a hotspot in carrier protein research due to its natural particle assembly ability and specificity in stimulating humoral immunity and cellular immunity against foreign epitopes. In recent years, there have be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C07K19/00A61K39/145A61K39/385A61P31/16
CPCA61K39/12A61K2039/55505A61K2039/6075C07K14/005C07K14/11C07K2319/00C12N2730/10122C12N2760/16122C12N2760/16134
Inventor 安文琪张静静马小伟师方园姚伟利梁雪爽
Owner 华兰生物疫苗股份有限公司
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