Avian influenza virus trimer subunit vaccine and application thereof

A technology of influenza virus and trimer, which is applied in the field of medicine and can solve the problems of insufficient immunogenicity of monomers

Pending Publication Date: 2022-04-29
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The trimeric form of HA1 of the present invention overcomes the disadvantage of insufficient immunogenicit

Method used

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  • Avian influenza virus trimer subunit vaccine and application thereof
  • Avian influenza virus trimer subunit vaccine and application thereof
  • Avian influenza virus trimer subunit vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of recombinant expression plasmids for H7N9 HA1c-trimer and H7N9 HA1-monomer proteins

[0049] The following two HA1 recombinant expression fragments were designed:

[0050] a.H7N9 HA1c-trimer: 5′-SP+LAH+HA1+6×His+stop codon-3′;

[0051] b. H7N9 HA1-monomer: 5'-SP+HA1+6xHis+stop codon-3'.

[0052] in

[0053] SP is the GP67 signal peptide, and the amino acid sequence is SEQ ID NO: 14;

[0054] LAH is the trimer-forming region LAH (K403-N474) of influenza virus A / California / 07 / 2009 H1N1 HA protein, and its amino acid sequence is SEQ ID NO: 1;

[0055] HA1 is influenza virus A / Shanghai / 2 / 2013 H7N9 HA1 region D18-S340, and its amino acid sequence is SEQ ID NO: 2.

[0056] According to the codon preference of insect cells, the optimized coding nucleic acid sequence of H7N9 HA1c-trimer with SP and 6×His fragments removed is obtained as SEQ ID NO: 19, and then the full-length sequence is linked to the two restriction sites of EcoRI and XhoI. H7N9 ...

Embodiment 2

[0057] Example 2: Trial expression and identification of H7N9 HA1c-trimer and H7N9 HA1-monomer proteins

[0058] DH10Bac competent cells (purchased from Invitrogen) were transformed with the recombinant plasmid obtained in Example 1, cultured at 37°C overnight, and positive clones were identified by blue-white screening and PCR. Recombinant baculovirus DNA (Bacmid) was extracted and the correct recombinants were identified by sequencing. Insect cells Sf9 (Invitrogen) were transfected with Bacmid, and the culture supernatant was collected 3 days after transfection to obtain the first-generation recombinant baculovirus. After continuous expansion for 2 generations, the 3rd generation baculovirus was obtained. Viruses were collected and detected by Western blot (WB) using HRP-labeled Anti-His antibody (MBL).

[0059] After WB detection, it was determined whether H7N9 HA1c-trimer and H7N9 HA1-monomer could be expressed normally, ( figure 1 ), the H7N9 HA1c-trimer construction w...

Embodiment 3

[0060] Example 3: Expression, purification and identification of H7N9 HA1c-trimer and H7N9 HA1-monomer proteins

[0061] After the fourth-generation amplification was performed with the third-generation baculovirus obtained in Example 2, the virus titer was determined. According to the titration results, a suitable virus MOI was selected to infect Sf9 or the insect cell line Hi5 (Invitrogen) for expression, and the cell supernatant was collected by centrifugation 48 hours later.

[0062] The collected supernatant was centrifuged at 5000 rpm for 30 min, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5 mL, GE Healthcare), and non-specifically bound was eluted with 20 mM Tris, 150 mM NaCl, pH 8.0, 20 mM imidazole. protein, the target protein was eluted with 20 mM Tris, 150 mM NaCl, pH 8.0, 100 mM imidazole. The target protein was collected and concentrated and then subjected to molecular sieve chromatography Superdex 200 Increase 10 / 300GL or Superdex 200 Hil...

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Abstract

The invention discloses a subunit avian influenza virus vaccine based on trimerization HA (hemagglutinin). Insect cells are used for in-vitro expression of influenza virus H1N1 HA protein trimer to form fusion trimer proteins H7N9 HA1c-trimer and H5N1 HA1c-trimer in a region LAH at the N terminal and H7N9 HA1 or H5N1 HA1 protein at the C terminal, and compared with respective HA1 monomer proteins, the trimer proteins have better immune effect, and a mouse can generate a higher-titer specific antibody aiming at the influenza virus HA1. The trimer form HA1 provided by the invention overcomes the defect of insufficient immunogenicity of an HA1 monomer, and improves the level of a specific antibody generated by mice and aiming at the influenza virus HA1; the design can be used for improving the immunogenicity of the influenza virus HA1, so that the influenza virus HA1 can be used as a more effective vaccine.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to an avian influenza virus trimer subunit vaccine and its application. Background technique [0002] Influenza virus (influenza virus), referred to as influenza virus. It is a representative species of Orthomyxoviridae, including human influenza virus and animal influenza virus. Human influenza virus is divided into three types: A (A), B (B), and C (C), which are the pathogens of influenza. [0003] Influenza virus is a negative-strand RNA virus containing eight RNA segments encoding at least 12 proteins (PB2, PB1, PB1-F2, PA, PA-X, HA, NA, NP, M1, M2, NS1, and NS2) . Two of these proteins, hemagglutinin (HA) and neuraminidase (NA), are cell surface glycoproteins that enable viruses to enter (through receptor binding and membrane fusion) and escape host cells, respectively; Mutations occur in the process of transmission, resulting in changes in immunogenicity. Therefore, accordin...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N5/10A61K39/145A61P31/16
CPCC07K14/005C12N15/86C12N5/0601A61K39/12A61P31/16C12N2760/16122C12N2710/14043C07K2319/02C12N2760/16134
Inventor 严景华史瑞黄庆瑞
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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