Broad spectrum type influenza vaccine and preparation method thereof

A technology of influenza A and vaccine, which is applied in the field of bioengineering, can solve the problem that the virus has no preventive and protective effect, and achieve the effect of high safety, low toxicity and side effects, and good tolerance

Active Publication Date: 2010-11-10
BEIJING JINGYI TAIXIANG TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] At present, the global influenza situation is severe. New influenza virus strains such as avian influenza virus (H5N1) and influenza A virus (H1N1) continue to

Method used

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  • Broad spectrum type influenza vaccine and preparation method thereof
  • Broad spectrum type influenza vaccine and preparation method thereof
  • Broad spectrum type influenza vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the preparation of Neisseria meningitidis outer membrane protein (P64K)

[0083] 1. Cloning of the gene encoding the outer membrane protein (P64K) of Neisseria meningitidis

[0084] According to the sequence of Neisseria meningitidis of GenBank Accession No.X77920.1, primers were designed using Primer5.0 software, upstream primer: 5-CATG CCATGG CTTTAGTTGAATTGAA-3, downstream primer: 5-CCG GAATTC TTATTTTTTCTTTTGCGGAG-3, where the underlined parts are the restriction sites of NcoI and EcoRI respectively. Neisseria meningitidis CMCC 29336 (purchased from China Medical Bacteria Collection Center, referred to as CMCC) was boiled at 100°C for 10 min, and 3 μl was taken as a template for PCR amplification. The PCR reaction system is: 3 μl template, 5 μl 10×PCR buffer, 1 μl 10 mmol / L dNTP, 1 μl Pyrobest high-fidelity DNA polymerase (Shanghai Sangong product), 0.5 μl each of upstream and downstream primers with a final concentration of 0.5 μmol / L, adding Ultra...

Embodiment 2

[0091] Embodiment 2, the preparation of M2e peptide

[0092] 1. Solid phase synthesis of M2e peptide:

[0093] Taking the amino acid Cys at the C-terminus of the M2e peptide sequence SLLTEVETPTRSEWECRCSDSSDC[SEQ ID NO: 1] as the initial reactant, the NH 2 Protect with Fmoc, react with carrier Trityl chloride resin (also Rink Amide resin or Wang resin), the molar ratio is 1:1.5, use DCM or DMF as the reaction solvent, mix under alkaline conditions, and react for about 2 hours; The neutral solvent piperidine removes the protective group Fmoc connected to the first amino acid amino group on the carrier, rinses with DMF to remove excess amino acids, Fmoc and other small molecules; then add the activator DIC / HOBT mixture to activate Cys N-terminal After the free amino group is activated for 15 minutes, the second amino acid (see SEQ ID NO: 1) is added and cycled sequentially to complete the connection of all amino acids on the M2e peptide. After the synthesis is completed, triflu...

Embodiment 3

[0096] Example 3, Preparation of M2e peptide-P64K conjugate

[0097] 1. Activation of P64K:

[0098] Using SMCC (Succinimidyl-4-(N-maleimidomethyl)cydohexane-1-carboxylate) as an activator, mix P64K:SMCC with a molar ratio of 1:20 in phosphate buffer at pH 7.2, stir at room temperature for 2 hours, add An appropriate amount of glycine was used to terminate the reaction, and then the activated P64K was ultrafiltered with an ultrafiltration cup (MW: 30KDa) to remove unreacted SMCC and excess glycine.

[0099] 2. Preparation and purification of conjugates

[0100] Activated P64K was reacted with M2e at a molar ratio of 1:15 at a known concentration at pH 6.5 at 4° C. for 24 h, and the reaction was terminated with mercaptoethanol. Purify the obtained conjugate with an ultrafiltration cup (MW: 30KDa) to remove unreacted M2e and mercaptoethanol.

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Abstract

The invention discloses a broad spectrum type influenza vaccine and a preparation method thereof. The vaccine comprises a conjugate formed by chemically coupling a carrier protein with a highly conserved antigen segment M2e or HA218-72 with a prevention and protection effect in an influenza virus. The broad spectrum type influenza vaccine prepared by the conjugate is easy for large scale production and preparation, has long effective period, can be subjected to bulk stock and overcomes the defect of the development and production technical route of the traditional influenza vaccine, thereby realizing real-time and effective control on epidemic situation caused by various influenza viruses.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a broad-spectrum influenza vaccine and a preparation method thereof. Background technique [0002] Influenza is a respiratory infectious disease caused by influenza virus. It is one of the diseases that still pose a huge threat to human public safety. There have been three world-wide influenzas in human history, resulting in 50 million to 100 million deaths. More than the combined death toll of the two world wars. [0003] The main characteristics of the influenza virus are the rapid mutation of the virus strain, the rapid spread of the epidemic, and the great harm. In nature, humans and other animals are the hosts of influenza virus, and the recombination of virus strains in different hosts makes new influenza virus strains appear continuously, which poses a severe challenge to human prevention and control of influenza epidemics. [0004] So far, preventive influenza va...

Claims

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Application Information

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IPC IPC(8): A61K39/385A61K39/145A61K47/48C07K14/11A61P31/16A61K47/42
CPCA61K39/145C12N2760/16122A61K47/4833C07K14/005C12N2760/16134A61K39/12A61K47/646A61K2039/6068A61P31/16
Inventor 李先钟张琪王鑫孙宇石刘方杰胡品良程虹杨思仪阚伟白先宏
Owner BEIJING JINGYI TAIXIANG TECH DEV
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