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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 immunogenicity of HA1 monomer, and greatly improves the level of specific antibodies against influenza virus HA1 in mice

Method used

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  • Influenza virus trimer subunit vaccine and application thereof
  • Influenza virus trimer subunit vaccine and application thereof
  • Influenza virus trimer subunit vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Construction of recombinant expression plasmids for H1N1 HA1c-trimer and H1N1 HA1-monomer proteins

[0064] Design the following two HA1 recombinant expression fragments:

[0065] a. H1N1 HA1c-trimer: 5′-SP+LAH+HA1+6×His+stop codon-3′;

[0066] b. H1N1 HA1-monomer: 5′-SP+HA1+6×His+stop codon-3′.

[0067] in

[0068] SP is a GP67 signal peptide, and its amino acid sequence is SEQ ID NO: 14;

[0069] LAH is influenza virus A / California / 07 / 2009H1N1 HA protein trimer forming region LAH (K403-N474), and its amino acid sequence is SEQ ID NO: 1;

[0070] HA1 is influenza virus A / California / 07 / 2009H1N1 HA1 region D18-S340, and its amino acid sequence is SEQ ID NO:2.

[0071] According to the preference of insect cell codons, the nucleic acid sequences encoding the above two amino acid sequences were optimized, and the optimized encoding nucleic acid sequences of the remaining fragments except SP and 6×His were respectively obtained as SEQ ID NO: 29, SEQ ID NO: 40...

Embodiment 2

[0072] Example 2: Expression and identification of H1N1 HA1c-trimer and H1N1 HA1-monomer proteins

[0073] DH10Bac competent cells (purchased from Invitrogen) were transformed with the recombinant plasmid obtained in Example 1, cultured overnight at 37°C, and positive clones were identified by blue-white-white screening and PCR. The recombinant baculovirus DNA (Bacmid) was extracted, and the correct recombinant was identified by sequencing. Insect cells Sf9 (Invitrogen) were transfected with Bacmid, and the culture supernatant was harvested 3 days after transfection to obtain the first-generation recombinant baculovirus. The virus was expanded continuously for 2 generations, and the 3rd generation baculovirus was obtained. Viruses were collected and detected by Western blot (WB) using HR-labeled Anti-His antibody (MBL).

[0074] H1N1 HA1c-trimer and H1N1 HA1-monomer can be expressed normally by WB detection, and the expression level of H1N1 HA1c-trimer is significantly highe...

Embodiment 3

[0075] Example 3: Expression, purification and identification of H1N1 HA1c-trimer and H1N1 HA1-monomer proteins

[0076] After performing the fourth-generation amplification with the obtained third-generation baculovirus obtained in Example 2, the virus titer was determined. According to the titration results, an appropriate virus MOI was selected to infect Sf9 or insect cell line Hi5 (Invitrogen) for expression, and the cell supernatant was collected by centrifugation after 48 hours.

[0077]The collected supernatant was centrifuged at 5000 rpm for 30 minutes, filtered through a 0.22 μm membrane filter, bound to a HisTrap excel column (5 mL, GE Healthcare), and non-specifically bound For protein, the target protein was eluted with 20mM Tris, 150mM NaCl, pH8.0, 100mM imidazole. After the target protein was collected and concentrated, it was subjected to molecular sieve chromatography Superdex 200Increase 10 / 300GL or Superdex 200Hiload 16 / 60 (GE Healthcare). Purpose peak is d...

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Abstract

The invention discloses a subunit influenza virus vaccine based on trimerization HA (hemagglutinin). Insect cells are used for expressing influenza virus H1N1 HA protein trimer in vitro to form fusion trimer proteins H1N1 HA1c-trimer, H3N2 HA1c-trimer or B HA1c-trimer with LAH at the N terminal and H1N1 HA1, H3N2 HA1 or B-type HA1 protein at the C terminal, compared with respective HA1 monomer proteins, the trimer proteins have better immune effect, and mice 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 influenza virus trimeric 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 comprising 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 the virus to enter (via receptor binding and membrane fusion) and escape, respectively, from the host cell; since HA and NA readily Mutations occur during transmission, resulting in changes in immunogenicity. The...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N5/10A61K39/145A61P31/16
CPCC07K14/005C12N15/86C12N5/0601A61K39/12A61P31/16C12N2760/16122C07K2319/00C07K2319/02C07K2319/21C12N2710/14143C12N2800/105C12N2800/22C12N2510/02C12N2760/16134
Inventor 严景华史瑞黄庆瑞
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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