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Fusion protein, vaccine containing fusion protein and application of fusion protein

A fusion protein and vaccine technology, applied in the field of genetic engineering, can solve the problems of weak immunogen, insufficient protection of seasonal influenza vaccine, and high production cost, and achieve the effects of short production cycle, good clinical application value, and wide recognition

Active Publication Date: 2021-06-22
LIAONING CHENGDA BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects of insufficient protection, weak immunogen and high production cost of seasonal influenza vaccine produced in the prior art , providing a fusion protein, vaccine containing it and application thereof

Method used

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  • Fusion protein, vaccine containing fusion protein and application of fusion protein
  • Fusion protein, vaccine containing fusion protein and application of fusion protein
  • Fusion protein, vaccine containing fusion protein and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Preparation of fusion protein

[0046] gene synthesis

[0047] 1. The fusion gene nmh encoding the fusion protein NMH has a base sequence as in SEQ ID NO:1 in Table 1, wherein the amino acid sequence of the NP fragment is as shown in SEQ ID NO:3, and its encoding gene np has The base sequence shown in the 1st to 90th positions of the sequence shown in SEQ ID NO:1. The amino acid sequence of M2e is shown in SEQ ID NO:4, and its coding gene m2e has the base sequence shown in the 111th to 248th positions of SEQ ID NO:1. The amino acid sequence of the HA2 fragment is shown in SEQ ID NO:5, and its coding gene ha2 has the base sequence shown in the 270th to 807th positions of SEQ ID NO:1. The base sequence of the DNA linker GSAGSAG (SEQ ID NO:6) encoding the short peptide is located at the 91st to 110th, 249th to 269th, 435th to 455th of the sequence shown in SEQ ID NO:1 bits and bits 621 to 641.

[0048] Table 1

[0049]

[0050] Note: The sequence in bo...

Embodiment 2

[0113] Example 2. Antibody Titer Research

[0114] Serotype study of subunit influenza vaccine immunized mice. Female BALB / c mice of 4-6 weeks were selected as research objects, and the same molar vaccine protein (NMH, NMHC, PBS (control)) was intraperitoneally immunized on day 0, day 14, and day 28 respectively, and the immune volume was 500 μl / mouse, 14 days after the third immunization (Day 42), the serum was collected for antibody research in the serum.

[0115] The hemagglutinin protein (HA) of six influenza subtypes including H1N1, H3N2, H2N2, H5N1, H7N9, and H9N2 (due to the limitation of experimental conditions, only the above six proteins were represented) was coated at a concentration of 2 μg / mL on the On the ELISA plate, wash the plate 4 times with PBST, block with blocking solution, wash the plate 4 times with PBST, and then incubate with the serially diluted sera of each immune group at 37°C for 1 hour, wash the plate 4 times with PBST, and incubate the seconda...

Embodiment 3

[0120] Embodiment 3. Trace neutralization experiment

[0121] On the first day, dilute the sera of different immune groups to the corresponding multiples in a 2-fold ratio, add 50 μl / well to a 96-well plate, and then add 50 μl / well 100TCID 50 The viruses (represented by H1N1 and H3N2 strains) were co-incubated at 37°C for 2 hours, and 100 μl / well MDCK (1.5*10^4, purchased from ATCC CCL-34 (MDCK (NBL-2))) was added to Incubate at 37°C for 20 hours, and set up a control group at the same time, positive control 50 μl virus dilution + 50 μl virus + 100 μl MDCK. Negative control 100 μl virus diluent + 100 μl MDCK. The next day, use ELISA to detect virus-infected cells, first use the mouse monoclonal antibody against the NP protein of influenza A (article number: 11675-T62-50, Beijing Yiqiao Shenzhou Technology Co., Ltd.) to detect the virus in MDCK cells In order to reflect the neutralization effect of the serum sample on a certain virus, the average OD value of the cell positi...

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Abstract

The invention discloses a fusion protein, a vaccine containing the fusion protein and application of the fusion protein, the fusion protein sequentially comprises a nucleoprotein NP fragment of an influenza virus, a matrix protein M2e and a hemagglutinin neck conserved region HA2 fragment from the N end to the C end, and the sequences of the NP fragment are NP1 (335-350aa) and NP2 (380-393aa); the M2e comprises at least two identical copies of type A1 M2e; and the HA2 fragment comprises neck conserved sequences (76-130aa) of three subtypes of H1, H3 and H7. The fusion protein provided by the invention has a wide recognition degree on influenza viruses, can recognize viruses which are different from subtypes of sequences contained in the fusion protein, and can obtain a broad-spectrum immune protection effect on high-variation influenza viruses.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion protein, a vaccine containing it and applications thereof. Background technique [0002] Influenza is an acute respiratory infectious disease caused by influenza virus that seriously endangers human health. Influenza viruses can be divided into three types: A, B, and C according to the different pathogens. Influenza B and C have relatively few mutations and have never caused a worldwide pandemic. Among them, influenza A has many subtypes and great variation, which has become the focus of influenza prevention and vaccine research. The most effective measure to prevent influenza pandemics is vaccination. However, the vaccines produced according to the current influenza A vaccine research and development strategies and technical routes have long production cycles, high costs, and lags, making it difficult to meet the needs of influenza pandemic prevention and control. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/145A61P31/16C12R1/19
CPCC07K14/005C12N15/70A61K39/12A61P31/16C07K2319/00C12N2800/22Y02A50/30
Inventor 徐明恺周荔葆张惠文廖辉李延胜
Owner LIAONING CHENGDA BIOTECH
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