Anti-influenza virus NP protein neutralizing nano antibody and application thereof

Abstract

The invention discloses an anti-influenza virus NP protein neutralizing nano antibody and an application thereof, belongs to the technical field of biology, and solves the problems of insufficient broad spectrum, drug resistance and toxic and side effects of the existing anti-influenza virus drugs clinically. The nano antibody comprises three complementarity determining regions and four constant regions, wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The invention also provides a nucleic acid fragment. The invention also provides a biological material. The invention also provides a product for preventing or treating influenza virus. The nano antibody screened by the invention has high specific recognition and binding capacity on influenza viruses, has broad-spectrum neutralization effect on different subtype influenza viruses, and has application prospect in preparation of products or detection reagents for prevention and treatment of influenza viruses.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-influenza virus NP protein neutralizing nano antibody and an application thereof. Background Art

[0002] Influenza (abbreviated as "flu") is an acute respiratory infectious disease caused by influenza virus. Influenza virus can be divided into four types: A, B, C, and D. The main types of influenza virus that cause seasonal epidemics are influenza A and B viruses. Influenza virus is mainly transmitted through respiratory droplets produced by sneezing and coughing of the infected person, and can also be infected through direct or indirect contact with mucous membranes such as the mouth, nose, and eyes. Influenza virus antigenicity is variable and spreads rapidly. At the same time, influenza virus is also an important zoonosis pathogen. Most poultry, wild birds and waterfowl can be infected. After poultry is infected with avian influenza virus, the morbidity and mortality rates are very high.

[0003] The main targets of existing anti-influenza virus drugs in clinical practice are viral envelope proteins such as viral hemagglutinin (HA), neuraminidase (NA) or ion channel M2. Since HA and NA proteins differ greatly between different subtypes and are prone to mutation, drugs that act on these two proteins are not broad-spectrum and can easily fail. Although drugs that act on the M2 ion channel have a broad spectrum, they have been shown to produce serious drug resistance and toxic side effects. Therefore, the development of more effective anti-influenza drugs with less toxic side effects is particularly urgent.

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

[0005] In the serum of camelids and sharks, antibodies with naturally missing light chains were accidentally discovered. The variable domain of the heavy chain of heavy-chain antibody (VHH) is the smallest unit of known complete antigen-binding fragments, namely 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 are composed of 4 framework regions (FR) and 3 complementarity determining regions (CDR).

[0006] Compared with conventional antibodies and antibody fragments, Nbs have a longer CDR3 region, which constitutes the main part of the antigenic determinant cluster. Due to its unique structure and smaller size, it can recognize the target antigen inside and deep narrow cracks that traditional antibodies cannot reach. The disulfide bonds inside Nbs make it heat-resistant and resistant to proteolysis. It can still maintain a stable conformation in extreme temperatures and pH, organic solvents and protease environments, indicating that it can be administered orally or by atomization, and has good application prospects in the treatment of gastrointestinal and respiratory diseases. In addition, Nbs are much simpler than traditional antibodies in structure and chemical composition, easier to edit and functionally modify, and can be engineered to achieve multivalent, multi-epitope and multi-specific modifications according to different needs, which is convenient for increasing affinity, binding to multiple antigens or in vitro affinity maturation. Its strong hydrophilicity and solubility make it easier to express in large quantities in different expression systems, such as bacteria, yeast or mammalian cells, making it a potential antibody type suitable for engineering and large-scale production.

[0007] Therefore, NP protein-specific nanoantibodies with broad-spectrum anti-influenza virus effects are advantageous candidate antibodies for influenza prevention and treatment and drug development. Summary of the invention

[0008] The purpose of the present invention is to provide an anti-influenza virus NP protein neutralizing nano-antibody to solve the problems of insufficient broad-spectrum, drug resistance and toxic side effects of existing clinical anti-influenza virus drugs. The nano-antibody provided by the present invention can effectively bind to the influenza virus NP protein and has stable influenza virus neutralizing activity, providing an effective biomolecular product for influenza prevention and treatment.

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

[0010] The technical solution of the present invention is as follows: In the first aspect, the present invention provides an anti-influenza virus NP protein neutralizing nanobody, the nanobody comprises three complementary determining regions CDR1, CDR2, CDR3 and four constant regions FR1, FR2, FR3, FR4; wherein the amino acid sequence information of the complementary determining regions CDR1, CDR2, CDR3 is as follows: CDR1 sequence: GYIFSVDRMG (SEQ ID NO: 1); CDR2 sequence: DIFESGSLKSENYADFVEG (SEQ ID NO: 2); CDR3 sequence: RRLRSGTWYDY (SEQ ID NO: 3).

[0011] Preferably, the amino acid sequence information of the constant regions FR1, FR2, FR3, and FR4 is as follows: FR1: QVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4); FR2: WYRQAPGKQRELVA (SEQ ID NO:5); FR3: RFTISRENAKNTVYLQMNSLKPEDTAVYYCNL (SEQ ID NO: 6); FR4: WGQGTQVTVSS (SEQ ID NO:7).

[0012] Preferably, the amino acid sequence information of the Nanobody is as follows: QVQLVESGGGLVQPGGSLRLSCAASGYIFSVDRMGWYRQAPGKQRELVADIFESGSLKSENYADFVEGRFTISRENAKNTVYLQMNSLKPEDTAVYYCNLRRLRSGTWYDYWGQGTQVTVSS (SEQ ID NO: 8).

[0013] In a second aspect, the present invention provides a nucleic acid fragment encoding the above-mentioned neutralizing nano-antibody against influenza virus NP protein.

[0014] Preferably, a specific sequence of the nucleic acid fragment is as follows: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTAGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGCGCAGCCTCTGGCTACATCTTCAGTGTGGATCGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCAGATATCTTCGAAAGTGGTAGCCTGAAGTCTGAGAACTA TGCAGACTTCGTGGAGGGCCGATTCACCATCTCTAGAGAGAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATTTGAGGCGACTTCGATCAGGGACCTGGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA (SEQ IDNO:9).

[0015] In a third aspect, the present invention provides a biomaterial related to a neutralizing nano-antibody against influenza virus NP protein, wherein the biomaterial is any one of A1) to A12): A1) a nucleic acid molecule encoding an anti-influenza virus NP protein neutralizing nanobody; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette of A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette of A2); A7) a recombinant microorganism containing the recombinant vector of A3); A8) a recombinant microorganism containing the recombinant vector of A4); A9) a transgenic cell containing the nucleic acid molecule of A1); A10) a transgenic cell containing the expression cassette of A2); A11) a transgenic cell containing the recombinant vector of A3); A12) Transgenic cells containing the recombinant vector of A4).

[0016] In a fourth aspect, the present invention provides a product for preventing or treating influenza virus, comprising the above-mentioned neutralizing nanoantibody against influenza virus NP protein, or the above-mentioned nucleic acid fragment, or the above-mentioned biological material.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a nano-antibody that can specifically recognize and neutralize influenza viruses, the screened nano-antibody has highly specific recognition and binding capabilities for influenza viruses, and has a broad-spectrum neutralizing effect on different subtypes of influenza viruses, and has application prospects in preparing preventive and therapeutic products or detection reagents for influenza viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS-PAGE identification diagram of the monovalent nanobody purification; Figure 2 This is a graph showing the results of the nanobody affinity test; Figure 3 It is a graph for the specific identification of nanobodies; Figure 4 This is a graph showing the results of the neutralization activity test of the nanobody; Figure 5 This is a test chart of the ability of nano-antibodies to inhibit the replication of different subtypes of influenza viruses; Figure 6 This is a graph showing the ability of nanoantibodies to inhibit influenza virus replication when detected at the RNA level. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0020] The term "nanoantibody" in the present invention refers to an antibody that naturally lacks light chains and contains only heavy chains (heavy chain antibodies). The variable region of this type of antibody is about 12-15 kDa, can recognize and bind antigens, and has extremely high affinity, and is the smallest active antigen-binding fragment. Nanoantibodies have the characteristics of small molecular weight, high affinity, high stability and water solubility.

[0021] The neutralizing nanobody against influenza virus NP protein of the present invention includes a complementary determining region CDR and a constant region (also called a framework region) FR. The complementary determining region CDR includes CDR1, CDR2 and CDR3, and the constant region FR includes FR1, FR2, FR3 and FR4. Compared with the complementary determining region, the constant region has a more conservative amino acid sequence. However, due to the amino acid sequence variation in the complementary determining region, different nanobodies have different antibody titers.

[0022] 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 NOs: 1-7, respectively.

[0023] The amino acid sequence of the neutralizing nanobody against influenza virus NP protein of the present invention is SEQ ID NO:8.

[0024] In an optional embodiment, the amino acid sequence comprises any one of SEQ ID NOs: 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 NOs: 1-8.

[0025] The present invention also provides a nucleic acid fragment encoding the above-mentioned nanobody, whose sequence is shown in SEQ ID NO:9.

[0026] In alternative embodiments, 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 sequence of SEQ ID NO: 9.

[0027] The experimental methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0028] Embodiment 1, Characterization of neutralizing nanoantibodies against influenza virus NP protein and detection of their ability to inhibit viral replication.

[0029] 1. Experimental Methods 1.1 Cloning and expression of nanobodies The antibody sequence was cloned into the pcDNA3.1-Fc (containing the CH2 and CH3 gene fragments of human Fc) eukaryotic expression vector, the plasmid was extracted and transfected into 293F suspension cells, the supernatant was collected after 5 days, dialyzed against PBS buffer overnight, the target antibody was purified using Protein A medium, and the SDS-PAGE test was performed to analyze the results.

[0030] 1.2 Specificity detection of nanoantibodies (1) Inactivated influenza viruses of different subtypes were coated on ELISA plates at 10 μg / mL, and inactivated Tembusu virus (TMUV) was coated as a control and incubated overnight. The plates were washed, blocked with 3% BSA, and then 1 μg / well of purified nanoantibodies were added. The plates were incubated at 37°C for 2 h. After washing, horseradish peroxidase-labeled Fc antibodies were added, and the OD value at 450 nm was detected after TMB color development.

[0031] (2) Cell plates were plated and infected with different subtypes of influenza virus for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X100, and blocked with 10% skim milk overnight. Purified nanoantibodies were added and incubated at 37°C for 2 h. After washing, Cy3-labeled Fc fluorescent secondary antibodies were added, and images were collected using a confocal microscope after DAPI sealing.

[0032] 1.3 Nanoantibody affinity detection The influenza virus NP protein was subjected to affinity chromatography to obtain purified protein. The kinetic curve data and stability data of the nanoantibody and NP protein were collected using biomembrane interference chromatography technology, and the Ka, Kd and affinity KD values ​​were calculated using software.

[0033] 1.4 Detection of the ability of nanoantibodies to inhibit the replication of different subtypes of influenza viruses A549 cells were plated in 12-well plates and transfected with nanoantibody recombinant plasmids when the density reached 60%-70%. Different subtypes of influenza virus were infected 24 hours after transfection. Cells and supernatant were collected 24 hours later. The supernatant was diluted in gradients to infect chicken embryos. Chicken embryo allantoic fluid was collected 48 hours later for titration and EID was calculated. 50 RNA was extracted from the cells, and the viral RNA replication level was determined using NP and M specific primers.

[0034] 2. Experimental Results 2.1 Results of cloning and expression experiments of nanobodies After the nanobody was constructed into the pcDNA3.1-Fc vector, sequencing was performed to confirm that the sequence was correctly recombined into the vector. The constructed plasmid was transfected into 293F cells and cultured for 5 days. The supernatant was collected by centrifugation and purified using Protein A affinity chromatography. The results are shown in Figure 1As shown, a nanobody with a size of 40 kDa was successfully purified.

[0035] 2.2 Nanobody specificity test results After antigen coating, nanoantibodies were added and negative controls were set up. Figure 2 As shown, the nanobody can react with different subtypes of influenza virus, but not with Tembusu virus (TMUV). After cells were infected with different subtypes of influenza virus, the nanobody was used as the primary antibody for detection. The results showed that the nanobody can recognize cells infected with different subtypes of influenza, such as Figure 3 shown.

[0036] 2.3 Nanobody affinity detection The affinity test showed that the Ka value of the nanobody and NP protein was 6.35×10 4 / Ms, Kd value is 7.08×10 -4 / s, the affinity value is 11.2 nM, such as Figure 4 shown.

[0037] 2.4 Results of the test on the ability of nanoantibodies to inhibit the replication of different subtypes of influenza viruses After 24 hours of infection of A549 cells expressing nanobodies with different subtypes of influenza virus, the cells were collected for qPCR detection of viral replication levels, and the supernatant was used for virus titration. The results showed that the nanobodies had a significant inhibitory effect on H1N1, H3N2, H6N6, and H9N2 subtype influenza viruses, and could significantly inhibit the replication of viral RNA. Figure 5 and Figure 6 shown.

[0038] The nanoantibody of the present invention can effectively bind to the influenza virus NP protein, and at the same time has the characteristic of stably inhibiting the replication of influenza virus, has highly specific recognition and binding ability to influenza virus, and has application prospects in 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 complementary determining regions CDR1, CDR2, CDR3 and four constant regions FR1, FR2, FR3, FR4; wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, respectively.

2. An 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, and FR4 are SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

3. An anti-influenza virus NP protein neutralizing nanobody according to claim 1 or 2, characterized in that: The amino acid sequence of the nanobody is SEQ ID NO:

8.

4. A nucleic acid fragment encoding a neutralizing nanobody against the influenza virus NP protein according to any one of claims 1 to 3.

5. A nucleic acid fragment according to claim 4, characterized in that: The sequence of the nucleic acid fragment is SEQ ID NO:

9.

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

7. A product for preventing or treating influenza virus, characterized in that: Contains the neutralizing nanobody against influenza virus NP protein according to any one of claims 1 to 3, or the nucleic acid fragment according to claim 4 or 5, or the biomaterial according to claim 6.

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