Anti-influenza virus np protein neutralizing nanobody and application thereof

Abstract

The application discloses an anti-influenza virus NP protein neutralizing nanobody and application thereof, and belongs to the technical field of biotechnology, and solves the problems of the existing anti-influenza virus drugs in clinical application, such as insufficient broad spectrum, drug resistance and toxic side effects. The nanobody comprises three complementarity determining regions and four constant regions, and the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively. The application further provides a nucleic acid fragment. The application further provides a biological material. The application further provides an article for preventing or treating influenza virus. The screened nanobody has high specific recognition and binding capacity for influenza virus, has broad spectrum neutralizing effect on different subtypes of influenza virus, and has application prospect in preparation of an article for preventing and treating influenza virus or a detection reagent.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-influenza virus NP protein neutralizing nanobody and its application. Background Technology

[0002] Influenza (flu) is an acute respiratory infectious disease caused by the influenza virus. Influenza viruses can be divided into four types: A, B, C, and D. Types A and B influenza viruses are the main causes of seasonal influenza outbreaks. Influenza viruses are primarily transmitted through respiratory droplets produced by sneezing and coughing, and can also be transmitted through direct or indirect contact with mucous membranes such as the mouth, nose, and eyes. Influenza viruses are antigenically variable and spread rapidly. They are also important zoonotic pathogens, infecting most domestic poultry, wild birds, and waterfowl. In avian influenza, the morbidity and mortality rates are very high in birds.

[0003] Clinically, existing antiviral drugs primarily target viral envelope proteins such as viral hemagglutinin (HA), neuraminidase (NA), or the M2 ion channel. Due to significant differences in HA and NA proteins among different subtypes, mutations are common, resulting in drugs targeting these proteins lacking broad-spectrum efficacy and easily becoming ineffective. While drugs targeting the M2 ion channel have broad-spectrum efficacy, they have been shown to induce severe drug resistance and toxic side effects. Therefore, the development of more effective antiviral drugs with fewer side effects is urgently needed.

[0004] Nucleoprotein (NP) is a protein encoded by the NP gene, containing 498 amino acids. NP protein is the most abundant protein expressed after AIV infection of cells. It possesses RNA polymerase activity and plays a crucial role in the synthesis of viral mRNA, cRNA, and vRNA. It also has important functions such as nuclear importation, nuclear export, and RNA synthesis. NP protein is highly conserved across different subtypes, making it an ideal target for influenza virus prevention and control.

[0005] Naturally occurring antibodies lacking the light chain were accidentally discovered in the serum of camels and sharks. Their variable domain of the heavy chain (VHH) is the smallest known unit of a complete antigen-binding fragment, i.e., nanobodies (Nbs). Nanobodies have a molecular weight of 12–15 kDa, a crystal diameter of 2.5 nm, and a length of 4 nm. They consist of four framework regions (FRs) and three complementarity-determining regions (CDRs).

[0006] Compared to conventional antibodies and antibody fragments, Nbs possess a longer CDR3 region, forming the main part of the antigenic determinant. Due to its unique structure and smaller size, it can recognize target antigens deep within narrow crevices that are inaccessible to conventional antibodies. The disulfide bonds within Nbs give it heat resistance and resistance to proteolysis, maintaining a stable conformation even under extreme temperatures, pH levels, organic solvents, and protease environments. This indicates its potential for oral or nebulized administration, showing promising applications in the treatment of gastrointestinal and respiratory diseases. Furthermore, Nbs are much simpler in structure and chemical composition than conventional antibodies, making them easier to edit and functionally modify. They can be engineered to be multivalent, multi-paraepitope, and multi-specific to meet different needs, facilitating increased affinity, binding to multiple antigens, or in vitro affinity maturation. Their strong hydrophilicity and solubility make them easier to express in large quantities in various expression systems, such as bacteria, yeast, or mammalian cells, making them a potential antibody type suitable for engineered, large-scale production.

[0007] Therefore, NP protein-specific nanobodies with broad-spectrum anti-influenza virus activity are promising candidate antibodies for influenza prevention and drug development. Summary of the Invention

[0008] The purpose of this invention is to provide an anti-influenza virus NP protein neutralizing nanobody to address the problems of insufficient broad-spectrum activity, drug resistance, and toxic side effects of existing anti-influenza drugs in clinical practice. The nanobody provided by this invention can effectively bind to the influenza virus NP protein and possesses stable influenza virus neutralizing activity, providing an effective biomolecular product for influenza prevention and control.

[0009] Another object of the present invention is to provide an application of an anti-influenza virus NP protein neutralizing nanobody.

[0010] The technical solution of the present invention is as follows:

[0011] In a first aspect, the present invention provides an anti-influenza virus NP protein neutralizing nanobody, the nanobody comprising three complementarity-determining regions CDR1, CDR2, and CDR3 and four constant regions FR1, FR2, FR3, and FR4; wherein the amino acid sequence information of the complementarity-determining regions CDR1, CDR2, and CDR3 is as follows:

[0012] CDR1 sequence: GYIFSVDRMG (SEQ ID NO:1);

[0013] CDR2 sequence: DIFESGSLKSENYADFVEG (SEQ ID NO:2);

[0014] CDR3 sequence: RRLRSGTWYDY (SEQ ID NO:3).

[0015] Preferably, the amino acid sequence information of the constant regions FR1, FR2, FR3, and FR4 is as follows:

[0016] FR1: QVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4);

[0017] FR2: WYRQAPGKQRELVA (SEQ ID NO:5);

[0018] FR3: RFTISRENAKNTVYLQMNSLKPEDTAVYYCNL (SEQ ID NO: 6);

[0019] FR4: WGQGTQVTVSS (SEQ ID NO:7).

[0020] Preferably, the amino acid sequence information of the nanobody is as follows:

[0021] QVQLVESGGGLVQPGGSLRLSCAASGYIFSVDRMGWYRQAPGKQRELVADIFESGSLKSENYADFVEGRFTISRENAKNTVYLQMNSLKPEDTAVYYCNLRRLRSGTWYDYWGQGTQVTVSS (SEQ ID NO: 8).

[0022] Secondly, the present invention provides a nucleic acid fragment encoding a neutralizing nanobody that encodes the above-mentioned anti-influenza virus NP protein.

[0023] Preferably, a specific sequence of the nucleic acid fragment is as follows:

[0024] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTAGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGCGCAGCCTCTGGCTACATCTTCAGTGTGGATCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCAGATATCTTCGAAAGTGGTAGCCTGAAGTCTGAGAACTA TGCAGACTTCGTGGAGGGCCGATTCACCATCTCTAGAGAGAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATTTGAGGCGACTTCGATCAGGGACCTGGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA (SEQ IDNO:9).

[0025] Thirdly, the present invention provides a biomaterial related to a neutralizing nanobody against the anti-influenza virus NP protein, wherein the biomaterial is any one of A1) to A12):

[0026] A1) Nucleic acid molecules encoding anti-influenza virus NP protein neutralizing nanobodies;

[0027] A2) Expression cassette containing A1) nucleic acid molecules;

[0028] A3) A recombinant vector containing A1) nucleic acid molecules;

[0029] A4) Recombinant vectors containing A2) expression cassettes;

[0030] A5) Recombinant microorganisms containing A1) nucleic acid molecules;

[0031] A6) Recombinant microorganisms containing the A2) expression cassette;

[0032] A7) Recombinant microorganisms containing A3) recombinant vectors;

[0033] A8) Recombinant microorganisms containing A4) recombinant vectors;

[0034] A9) Transgenic cells containing A1) nucleic acid molecules;

[0035] A10) Transgenic cells containing the A2) expression cassette;

[0036] A11) Transgenic cells containing the A3) recombinant vector;

[0037] A12) Transgenic cells containing the A4) recombinant vector.

[0038] Fourthly, the present invention provides an article for the prevention or treatment of influenza virus, comprising the above-mentioned neutralizing nanoantibody against the anti-influenza virus NP protein, or the above-mentioned nucleic acid fragment, or the above-mentioned biological material.

[0039] The beneficial effects of this invention are: This invention provides a nanobody that can specifically recognize and neutralize influenza virus. The screened nanobody has a highly specific recognition and binding ability to influenza virus and a broad-spectrum neutralizing effect on different subtypes of influenza virus, and has the application prospect of preparing preventive and therapeutic products or detection reagents for influenza virus. Attached Figure Description

[0040] Figure 1 This is an SDS-PAGE image of purified monovalent nanobodies.

[0041] Figure 2 This is a graph showing the results of nanobody affinity testing;

[0042] Figure 3 This is a diagram illustrating the specificity of nanobodies;

[0043] Figure 4 This is a graph showing the results of the neutralizing activity assay for nanobodies;

[0044] Figure 5 This is a graph showing the ability of nanobodies to inhibit the replication of different subtypes of influenza virus;

[0045] Figure 6 This is a graph showing the ability of nanobodies to inhibit influenza virus replication at the RNA level. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] In this invention, the term "nanobody" refers to a naturally occurring antibody that lacks a light chain and contains only a heavy chain (heavy chain antibody). The variable region of this type of antibody is approximately 12-15 kDa, capable of recognizing and binding antigens with extremely high affinity, and is the smallest active antigen-binding fragment. Nanobodies are characterized by small molecular weight, high affinity, high stability, and water solubility.

[0048] The neutralizing nanobody against influenza virus NP protein of this invention comprises a complementarity-determining region (CDR) and a constant region (also called a framework region) (FR). The CDR includes CDR1, CDR2, and CDR3, and the FR includes FR1, FR2, FR3, and FR4. Compared to the CDR, the amino acid sequence of the constant region is more conserved. However, due to amino acid sequence variations in the CDR, different nanobodies exhibit different antibody titers.

[0049] The amino acid sequences of the three complementarity-determining regions CDR1, CDR2, CDR3 and the four constant regions FR1, FR2, FR3, FR4 are SEQ ID NO:1-7, respectively.

[0050] The amino acid sequence of the neutralizing nanobody against the anti-influenza virus NP protein of the present invention is SEQ ID NO:8.

[0051] In an optional embodiment, the amino acid sequence comprises any one of SEQ ID NO: 1-8, or has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with any one of SEQ ID NO: 1-8.

[0052] The present invention also provides a nucleic acid fragment for encoding the above-mentioned nanobody, the sequence of which is shown in SEQ ID NO:9.

[0053] In an optional implementation, the nucleic acid sequence has at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the nucleotide described in SEQ ID NO: 9.

[0054] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0055] Example 1

[0056] Characterization of neutralizing nanobodies against influenza virus NP protein and detection of their ability to inhibit viral replication.

[0057] 1. Experimental Methods

[0058] 1.1 Cloning and Expression of Nanobodies

[0059] The antibody sequence was cloned into the eukaryotic expression vector pcDNA3.1-Fc (containing the CH2 and CH3 gene fragments of human Fc), the plasmid was extracted and transfected into 293F suspension cells, the supernatant was collected after 5 days, dialyzed overnight with PBS buffer, the target antibody was purified using Protein A medium, and SDS-PAGE was performed to analyze the results.

[0060] 1.2 Nanobody Specific Detection

[0061] (1) Inactivated influenza subtypes were coated onto ELISA plates at a concentration of 10 μg / mL, with inactivated Tembusu virus (TMUV) as a control. The plates were incubated overnight. After washing, the plates were blocked with 3% BSA, followed by the addition of 1 μg / well of purified nanobody. The plates were incubated at 37°C for 2 h. After washing, horseradish peroxidase-labeled Fc antibody was added. The OD value at 450 nm was detected after TMB color development.

[0062] (2) Cell plates were formed and infected with different subtypes of influenza virus for 24 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X100, and blocked overnight with 10% skim milk. Purified nanobodies were added and incubated at 37°C for 2 h. After washing, Cy3-labeled Fc fluorescent secondary antibody was added, and the plates were mounted with DAPI and images were acquired by confocal microscopy.

[0063] 1.3 Nanoparticle antibody affinity detection

[0064] The influenza virus NP protein was purified by affinity chromatography. The kinetic curves and stability data of the nanobody and NP protein were collected using biomembrane interference chromatography. The Ka, Kd and affinity KD values ​​were calculated by software.

[0065] Detection of the ability of 1.4 nanoparticle antibodies to inhibit the replication of different subtypes of influenza virus

[0066] A549 cells were seeded in 12-well plates and transfected with recombinant nanobody plasmids when the cell density reached 60%-70%. 24 hours after transfection, cells were infected with different subtypes of influenza virus. Cells and supernatant were collected 24 hours later. The supernatant was serially diluted to infect chicken embryos. 48 hours later, allantoic fluid from the chicken embryos was collected for titration, and the EID was calculated. 50 RNA was extracted from cells, and the viral RNA replication level was determined using NP and M specific primers.

[0067] 2. Experimental Results

[0068] 2.1 Results of Nanobody Cloning and Expression Experiments

[0069] After constructing the nanobodies into the pcDNA3.1-Fc vector, sequencing confirmed the correct sequence recombination into the vector. The constructed plasmid was transfected into 293F cells and cultured for 5 days. The supernatant was collected by centrifugation, and the nanobodies were purified using Protein A affinity chromatography. Results are as follows: Figure 1 As shown, a nanobody with a size of 40 kDa was successfully purified.

[0070] 2.2 Specific detection results of nanobody

[0071] After antigen coating, nanobodies were added, and a negative control was set up. The results are as follows: Figure 2As shown, nanobodies react with different subtypes of influenza virus but not with Tembusu virus (TMUV). Nanobodies were used as primary antibodies to detect cells infected with different subtypes of influenza virus. The results showed that nanobodies could recognize cells infected with different subtypes of influenza, such as... Figure 3 As shown.

[0072] 2.3 Nanobody Affinity Detection

[0073] Affinity tests showed that the Ka value between the nanobody and the NP protein was 6.35 × 10⁻⁶. 4 / Ms, Kd value is 7.08×10 -4 / s, affinity value 11.2 nM, such as Figure 4 As shown.

[0074] 2.4 Results of detection of the ability of nanobodies to inhibit the replication of different subtypes of influenza virus

[0075] A549 cells expressing nanobodies were infected with different influenza virus subtypes for 24 h. Cells were then harvested for qPCR to detect viral replication levels, and the supernatant was used for viral titration. Results showed that the nanobodies significantly inhibited H1N1, H3N2, H6N6, and H9N2 influenza virus subtypes and significantly suppressed viral RNA replication. Figure 5 and Figure 6 As shown.

[0076] The nanobody of this invention can effectively bind to the NP protein of influenza virus and has the property of stably inhibiting the replication of influenza virus. It has a highly specific recognition and binding ability for influenza virus and has the application prospect of preparing influenza virus prevention and treatment drugs or detection reagents.

Claims

1. An anti-influenza virus NP protein neutralizing Nanobody, characterized in that: The nanobody comprises three complementarity determining regions CDR1, CDR2, CDR3 and four constant regions FR1, FR2, FR3, FR4; wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2, CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively.

2. The anti-influenza virus NP protein neutralizing Nanobody according to claim 1, characterized in that: The amino acid sequences of the constant regions FR1, FR2, FR3, FR4 are SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 respectively.

3. A nucleic acid fragment encoding the neutralizing nanobody against the NP protein of the influenza virus according to claim 1 or 2.

4. The nucleic acid fragment of claim 3, wherein: The sequence of the nucleic acid fragment is SEQ ID NO:

9.

5. A neutralizing Nanobody-related biomaterial against the NP protein of the influenza virus according to claim 1 or 2, characterized in that: The biological material is any one of A1) to A12): A1) a nucleic acid molecule encoding the neutralizing nanobody against the NP protein of the influenza virus according to any one of claims 1 to 3; A2) an expression cassette comprising the nucleic acid molecule according to A1); A3) a recombinant vector comprising the nucleic acid molecule according to A1); A4) a recombinant vector comprising the expression cassette according to A2); A5) a recombinant microorganism comprising the nucleic acid molecule according to A1); A6) a recombinant microorganism comprising the expression cassette according to A2); A7) a recombinant microorganism comprising the recombinant vector according to A3); A8) a recombinant microorganism comprising the recombinant vector according to A4); A9) a transgenic cell comprising the nucleic acid molecule according to A1); A10) a transgenic cell comprising the expression cassette according to A2); A11) a transgenic cell comprising the recombinant vector according to A3); A12) a transgenic cell comprising the recombinant vector according to A4).

6. An article of manufacture for the treatment of influenza virus, comprising: The neutralizing nanobody against the NP protein of the influenza virus according to claim 1 or 2.

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