CN112175075B

CN112175075B - Broad-spectrum antibody of targeted influenza A virus HA protein

Info

Publication number: CN112175075B
Application number: CN202011075187.3A
Authority: CN
Inventors: 肖鹤; 冯健男; 沈倍奋; 卢星; 陈国江; 王晶; 乔春霞; 罗龙龙; 李新颖; 刘成华
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2020-10-09
Filing date: 2020-10-09
Publication date: 2021-08-20
Anticipated expiration: 2040-10-09
Also published as: CN112175075A

Abstract

The invention discloses a broad-spectrum antibody of targeted influenza A virus HA protein, which HAs amino acid sequences of a heavy chain variable region and a light chain variable region shown in SEQ ID NO.8 and SEQ ID NO. 17. The invention also provides a nucleic acid molecule for encoding the antibody FHA3, an expression vector containing the nucleic acid molecule and a host cell. The present invention provides a monoclonal antibody which can bind to an influenza virus and thus can be used for detecting a split influenza virus and for treating an influenza virus-infected disease.

Description

Broad-spectrum antibody of targeted influenza A virus HA protein

Technical Field

The invention belongs to the fields of cellular immunology and genetic engineering, and relates to a broad-spectrum antibody FHA3 of a targeted influenza A virus HA protein.

Background

Influenza viruses (Influenza viruses) belong to the family of orthomyxoviridae (Ortho myxo viridae) and can be classified into Influenza a (a), Influenza B (B) and Influenza C (C) viruses according to the antigenicity of the Influenza Virus internal Nucleoprotein (NP) and Matrix Protein (Matrix Protein, M1). The recent report suggests the presence of Influenza Virus type D (Collin EA, ethyl. circulation of Two diseases Genetic and Antigenic Lineages of deployed Influenza D Virus in Cattle. journal of Virology 2015, 89(2):1036 + 1042). Influenza c causes only mild upper respiratory infections, while influenza a and b cause a number of serious conditions including lower respiratory infections, pneumonia, meningitis, etc., with a worldwide annual outbreak of influenza of about 6 million people per year. Among them, Influenza A virus poses the most serious health risks to humans (Baigent SJ, McCauley JW: Influenza type A in humans, mammals and birds: determinants of viruses, host-range and integripipes transmission. Bioessays 2003, 25(7): 657. sup. 671.), and besides seasonal outbreaks that infect large numbers of people every year, occasional pandemic is more of a catastrophic consequence.

Influenza a viruses are enveloped, single negative-strand RNA viruses whose genome comprises 8 independent gene segments (segments) with a total length of about 13kb and can encode up to 16 proteins. The Hemagglutinin segment (HA) encodes the glycoprotein HA that influenza viruses recognize host cell surface receptors. The HA protein is the most abundant glycoprotein on the surface of influenza a virus, and is composed of two parts, head and neck, and is responsible for recognizing the entry of receptor into host cell and mediating the membrane fusion process of viral envelope and host cell endosome (endosome), thereby allowing influenza virus to release nucleic acid into host cell. The HA protein is the major surface antigen of influenza virus and is the main target for host immune response.

At present, the detection of influenza virus in China mainly adopts a traditional biological method, a conventional RT-PCR technology combined with a first-generation sequencing technology and a fluorescent quantitative RT-PCR method. The methods are time-consuming and labor-consuming, and cannot meet the requirement of rapid detection; or the cost is too high, and the requirement of large-batch screening cannot be met. And due to the continuous variation of the virus, new technologies and products for systematically screening and identifying the differential diagnosis antigen markers of the epidemic strains and treating diseases caused by influenza virus infection are urgently needed. Therefore, in order to rapidly detect viruses and treat diseases caused by influenza virus infection, the development of the preparation of the anti-virus HA protein monoclonal antibody against influenza viruses is of great significance.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, one of the objects of the present invention is to provide a broad-spectrum antibody against influenza virus.

The second object of the present invention is to provide a means and product for diagnosing influenza virus infection.

The third purpose of the invention is to provide a means and a pharmaceutical composition for treating influenza virus infection diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the first aspect of the present invention provides a broad-spectrum monoclonal antibody against influenza virus, comprising a heavy chain variable region of three CDRs and a light chain variable region of three CDRs; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.10, 11 and 12.

Further, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.4, 5, 6 and 7; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.13, 14, 15 and 16.

Furthermore, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.

Further, the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

In the present invention, the monoclonal antibody is named FHA3 and specifically targets a human antibody of neck conserved sequence of influenza virus HA protein.

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody of the first aspect, wherein the nucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID No.9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 18.

The DNA sequence encoding the antibody molecule of the invention may be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the antibody heavy and light chains can be synthesized from the determined DNA sequences or based on the corresponding amino acid sequences, as desired.

In a third aspect the invention provides an expression vector comprising a nucleic acid molecule according to the second aspect of the invention.

Expression vectors in the present invention include, but are not limited to, MarEx expression vectors produced by Celltrion inc. (korea); a commercially available pCDNA vector; F. r1, RP1, Col, pBR322, ToL and Ti vector; sticking particles; phages such as lambda phage, lambda-shaped phage, M13 phage, Mu phage, P1 phage, P22 phage, Q β phage, T-even phage, T2 phage, T4 phage, T7 phage, and the like; a plant virus. Any of a variety of expression vectors known to those of skill in the art can be used in the present invention, and the choice of expression vector depends on the nature of the host cell chosen. Introduction of the vector into the host cell can be achieved by, but is not limited to, calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection, or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and the host cell used. Preferably, the above-mentioned vector contains one or more selection markers, but is not limited thereto, and a vector not containing a selection marker may also be used. The choice of selectable marker may depend on the host cell chosen (as is well known to those skilled in the art), but is not critical to the invention.

To facilitate purification of the nucleic acid molecules of the invention, a tag (tag) sequence may be inserted into the expression vector. Examples of tags include, but are not limited to, a hexa-histidine tag, a myc tag, or a FLAG tag. Any tag known to those skilled in the art to facilitate purification may be used in the present invention.

In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention, or an expression vector according to the third aspect of the invention.

In the present invention, any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., E.coli) and other microbial systems may be used, or eukaryotic (e.g., mammalian) host cell expression systems may also be used. Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin. As the mammalian cell, one selected from the group consisting of, but not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, a549 cell, HEK293 cell, and HEK293T cell can be preferably used as the host cell. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody according to the first aspect of the invention.

Further, the antibody also comprises a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may also include one or more other therapeutic agents. The therapeutic agent may comprise an antibody, a small molecule, an organic or inorganic compound, an enzyme, a polynucleotide sequence, and the like.

A sixth aspect of the invention provides a use as claimed in any one of the following, comprising:

1) the monoclonal antibody of the first aspect of the invention is used for preparing products for diagnosing influenza virus infection diseases;

2) the use of a monoclonal antibody according to the first aspect of the invention for the preparation of a pharmaceutical composition for the treatment of an influenza virus infection;

3) the monoclonal antibody of the first aspect of the invention is used for preparing a pharmaceutical composition for preventing and treating influenza virus infection;

4) use of a nucleic acid molecule according to the second aspect of the invention in the preparation of a pharmaceutical composition for the treatment of an influenza virus infection;

5) the use of an expression vector according to a third aspect of the invention in the preparation of a pharmaceutical composition for the treatment of an influenza virus infection;

6) use of a host cell according to a fourth aspect of the invention in the preparation of a pharmaceutical composition for the treatment of an influenza virus infection;

7) the application of the pharmaceutical composition of the fifth aspect of the invention in preparing products for treating influenza virus infection diseases.

Further, the influenza virus is an influenza a virus.

Further, the influenza a virus includes H1N1, H3N2, H5N1, H7N 9.

Further, the product in 1) comprises a kit, test paper, a chip and the like. Wherein the chip comprises a protein chip; the protein chip comprises a solid phase carrier and the monoclonal antibody or the fragment thereof fixed on the solid phase carrier; the protein immunoassay kit; the protein immunoassay kit comprises the monoclonal antibody or the fragment thereof.

In the present invention, a monoclonal antibody encompasses a sequence having a certain degree of sequence identity or sequence homology with the amino acid sequence of the antibody or any nucleotide sequence encoding the antibody, and in the present invention, "homology" may be equivalent to "identity".

One skilled in the art will also appreciate that antibodies may be subjected to various post-translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are the deletion of a basic residue at the carboxy terminus (such as lysine or arginine) due to the action of carboxypeptidase.

The invention also includes all antibodies which are obtained by adding, deleting and modifying the amino acid residues of the amino acid sequence of the antibody, comprise human antibodies and non-human antibodies and have the same functions as the FHA3 antibody or are modified and optimized. The deletion, substitution, insertion or addition may occur simultaneously, and the amino acid to be substituted, inserted or added may be of a natural type or a non-natural type.

The CDRs of the invention can include variants, for example, when the CDRs disclosed herein are back mutated to different framework regions. Typically, individual variant CDRs are at least 70% or 80% amino acid identity to the sequences described herein, more typically with increasing identity of preferably at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%.

As used herein, "identity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position of the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine may be substituted with isoleucine or valine. Other amino acids that may be substituted for one another in general include (but are not limited to): phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains), lysine, arginine and histidine (amino acids having basic side chains), aspartic acid and glutamic acid (amino acids having acidic side chains), asparagine and glutamine (amino acids having amide side chains), and cysteine and methionine (amino acids having sulfur-containing side chains).

Generally, modification of one or more amino acids in a protein does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative or variant. Typically, these changes are made over several amino acids to minimize changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, greater variations may be tolerated in some cases. Amino acid substitutions are typically of a single base; insertions will typically be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases, deletions can be much larger.

The antibodies and fragments disclosed herein are expressed at good levels from host cells. Thus, the properties of the antibody and/or binding fragment are suitable for expression on a commercial scale.

In the present invention, the pharmaceutical composition comprises the above-described monoclonal antibody or antibody fragment of the present invention as an active ingredient. A pharmaceutical composition comprising a monoclonal antibody or antibody fragment thereof or binding thereof of the present invention may comprise only the antibody or antibody fragment thereof or binding thereof as an active ingredient. In general, the pharmaceutical compositions are preferably prepared as pharmaceutical formulations produced by suitable methods well known in the art of pharmaceutical technology by admixing them with one or more pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be prepared using various additives, such as buffers, stabilizers, bacteriostats, isotonizing agents, chelating agents, pH controlling agents, and surfactants. The pharmaceutical compositions of the present invention may be administered to a subject by any route.

Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. "Fab" refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded; "Fab'" refers to a Fab fragment comprising part of the hinge region; "F (ab ') 2" refers to a dimer of Fab'; "Fv" refers to the smallest antibody fragment containing the variable regions of the heavy and light chains of an antibody and having all antigen binding sites.

The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus other desired ingredient from a previously sterile-filtered solution thereof. Alternatively, the compositions of the present invention may be in solution, and suitable pharmaceutically acceptable excipients may be added and/or mixed prior to or at the time of delivery to provide injectable unit dosage forms. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain adequate flowability, and delay absorption if necessary.

The choice of the optimal route of administration of the pharmaceutical composition of the invention will be influenced by several factors, including the physicochemical properties of the active molecule in the composition, the urgency of clinical presentation, and the relationship between the plasma concentration of the active molecule and the desired therapeutic effect. For example, monoclonal antibodies of the invention can be prepared with a vehicle that will protect them from rapid release (such as a controlled release formulation), including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used in the present invention. Further, monoclonal antibodies may be coated with, or co-administered with, materials or compounds that prevent inactivation of the antibody. For example, the monoclonal antibody can be administered with a suitable carrier (e.g., a liposome or diluent).

The administration route of the pharmaceutical composition of the present invention can be divided into oral administration and parenteral administration. The preferred route of administration is intravenous injection, but is not limited thereto.

In the present invention, the method for detecting or determining the amount of influenza virus may be any known method. For example, it includes immunodetection or assay methods. The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

In the present invention, there is no limitation on the disease associated with influenza virus as long as it is a disease associated with the expression of influenza virus.

In the present invention, an influenza virus-infected disease can be diagnosed by detecting or assaying cells expressing influenza virus with the monoclonal antibody or antibody fragment of the present invention. The diagnostic agent comprising the monoclonal antibody or antibody fragment thereof or binding substance thereof of the present invention may further comprise a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention has the advantages and beneficial effects that:

the invention provides an anti-influenza virus antibody, which is specifically combined with HA protein, HAs high affinity and good stability, and can be combined with influenza A virus at a lower dose.

The antibody of the invention can effectively inhibit influenza virus and provides a treatment means for relevant virus infection diseases.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of FHA3 antibody;

FIG. 2 is a graph of the antigen binding specificity of the ELISA for detection of FHA3 antibody;

FIG. 3 shows the results of the detection of neutralizing activity of FHA3 antibody against influenza A virus, wherein panel A is H5N1 and panel B is H7N 9.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 screening of anti-HA protein antibodies

1. Construction of phage antibody libraries

Respectively amplifying heavy chain (VH) library genes and light chain (VL) library genes of an antibody variable region by using a computer-designed synthetic antibody library as a template through Polymerase Chain Reaction (PCR); after the phage vector is inserted, the scFv single-chain antibody library is expressed, and the scFv is connected between VH and VL through an artificial joint.

2. Screening of antibodies against influenza viruses H1N1, H3N2, H5N1 and H7N9

(1) The displayed phage library is adhered with H1N1, H3N2, H5N1 and H7N9 proteins in sequence;

(2) repeatedly washing to remove non-specific binding, eluting and sequentially collecting phages bound with H1N1, H3N2, H5N1 and H7N 9;

(3) e.coli was reinfected, single clones were selected and displayed on phage surfaces, and positive clones recognizing H1N1, H3N2, H5N1 and H7N9 antigens simultaneously were screened by ELISA.

3. Determination and analysis of antibody variable region gene sequence

The positive clone is sent to Shanghai Biotechnology Limited company for antibody variable region gene sequence determination, and the sequencing result is searched and analyzed by DNAMAN and a database.

4. Results

The broad-spectrum monoclonal antibody FHA3 which can simultaneously resist H1N1, H3N2, H5N1 and H7N9 is screened out through the steps, and the amino acid sequences of the light chain variable region of the FHA3 monoclonal antibody and the heavy chain variable region of the FHA3 monoclonal antibody are obtained through sequence comparison, wherein the CDR1-3 sequence of the heavy chain is shown as SEQ ID NO. 1-3, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region are shown as SEQ ID NO. 4-7, the heavy chain variable region sequence is shown as SEQ ID NO.8, the CDR1-3 sequence of the light chain is shown as SEQ ID NO. 10-12, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the light chain are shown as SEQ ID NO. 13-16, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.

EXAMPLE 2 preparation of monoclonal antibody FHA3

1. The heavy chain variable region sequence (amino acid sequence is shown as SEQ ID NO.8, and nucleotide sequence is shown as SEQ ID NO. 9) of the antibody FHA3 is amplified by a PCR method, the light chain variable region sequence (amino acid sequence is shown as SEQ ID NO.17, and nucleotide sequence is shown as SEQ ID NO. 18), and the fragment is cloned into an expression vector by a molecular cloning method.

2. The vector containing both the light chain and heavy chain genes of the monoclonal antibody is transfected into a mammalian cell for expression.

3. The expression supernatant was collected and purified by a Protein A FF Protein column from GE.

4. Eluting with citric acid buffer solution of pH3.0, collecting eluate, immediately neutralizing with 1mol/L TRIS-HCL buffer solution of pH 8.5, dialyzing with PBS of pH7.2 and 0.01mol/L for 72h, and filtering with 0.22 μm filter membrane for sterilization.

5. The expression and purification of the antibody are tested by SDS-PAGE and Western Blot experiments, and the concentration of the purified antibody is detected by a BCA method and stored at 4 ℃.

6. As a result, as shown in FIG. 1, it was confirmed that a relatively pure protein was obtained and that light and heavy chains of the antibody were clearly observed after melting.

Example 3 specific detection of monoclonal antibody FHA3

The specificity of the monoclonal antibody FHA3 was determined by ELISA.

1. Coating 1. mu.g/ml of HA antigens of influenza A virus H1N1, H3N2, H5N1 and H7N9 in ELISA plates at 4 ℃ overnight;

2. blocking unbound sites with skim milk powder, then washing 5 times with PBS buffer containing 0.1% tween;

3. preparing antibody FHA3 with different concentrations, adding the antibody FHA3 into the ELISA plate in the step 2, incubating for 1h at 37 ℃, and washing for 5 times by using PBS buffer solution containing 0.1% Tween;

4. adding HRP-labeled goat anti-human antibody, incubating at 37 ℃ for 30min, and washing 5 times with PBS buffer containing 0.1% Tween; color development was performed by adding TMB, and OD was measured after 1mol/L sulfuric acid was terminated₄₅₀The value of (c).

5. Results

As a result, as shown in fig. 2, the antibody FHA3 was able to effectively bind to influenza a virus antigen proteins H1N1, H3N2, H5N1 and H7N9 in a dose-dependent relationship.

Example 4 pseudovirus neutralization assay

The neutralization experiment was carried out by adding FHA3 antibody during infection of MDCK cells with pseudovirus, and using unrelated antibody DGA as control. The method comprises the following specific steps:

1. pseudovirus packaging:

1) culturing HEK293T cells to 80% in 10cm dishes;

2) preparation of influenza A pseudovirus:

HA5, NA1 expression plasmids with pNL-4.3-luc or HA7, NA9 expression plasmids with pNL-4.3-luc, respectively, were ligated at 1: co-transfecting HEK-293T cells with a transfection reagent lipoamine3000 at a ratio of 1: 2;

3) after transfection for 6h, fresh complete DMEM medium was used, after further culture for 66h, cell supernatants were collected, centrifuged at 3000rpm/min for 10min, filtered through a 0.45 μm filter membrane, added with 20% serum, dispensed into 200 μ l/tube, and stored in a low temperature refrigerator at-80 ℃.

2. Infection with pseudovirus

Inoculating MDCK cells into a 96-well cell culture plate, wherein the cell density is 1X10⁵Ml, and left the cell incubator overnight. The supernatant containing the virus-like particles was incubated with the antibody at a corresponding concentration in advance at room temperature for 1 hr. The virus-like particle supernatant + antibody mixture was then used to infect MDCK cells, while virus supernatants without added antibody and those with added irrelevant antibody were used as controls. After 48h, the cells were lysed for luciferase activity (RLU).

3. Results

As shown in fig. 3, FHA3 can effectively neutralize influenza a viruses H5N1 and H7N9, thereby inhibiting infection of target cells by the viruses.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 16

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 17

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Asn Ser Leu Ser Ser Tyr

20 25 30

Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Arg Phe Gly Gln

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 18

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgcc gcaccagcaa tagcctgagc agctataccc attggtatca gcagaaaccg 120

ggcaaagcgc cgaaactgct gatttatgcg gcgagcagcc gcggcagcgg cgtgccgagc 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240

gaagattttg cgacctatta ttgccagcag agccgcaggt ttggccaggg caccaaagtg 300

gaaattaaa 309

Claims

1. A broad-spectrum monoclonal antibody against the HA protein of influenza a virus, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N9, characterized in that said monoclonal antibody comprises a heavy chain variable region of three CDRs and a light chain variable region of three CDRs; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.10, 11 and 12.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.4, 5, 6 and 7; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.13, 14, 15 and 16.

3. The monoclonal antibody according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.8 and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 17.

4. The monoclonal antibody of any one of claims 1-2, wherein the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

5. A nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody of any one of claims 1-4, wherein the nucleotide sequence encoding the variable region of the antibody heavy chain is set forth in SEQ ID No.9 and the nucleotide sequence encoding the variable region of the light chain is set forth in SEQ ID No. 18.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or comprising the expression vector of claim 6.

8. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-4.

9. Use according to any one of the following, comprising:

1) use of the monoclonal antibody of any one of claims 1-4 for the preparation of a product for diagnosing an influenza a virus infection disease, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9;

2) use of the monoclonal antibody of any one of claims 1-4 for the preparation of a pharmaceutical composition for the treatment of an influenza a virus infection disease, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9;

3) use of a monoclonal antibody according to any one of claims 1 to 4 for the preparation of a pharmaceutical composition for the prevention and treatment of an infection with an influenza a virus which is H1N1, H3N2, H5N1 and/or H7N 9;

4) use of the nucleic acid molecule of claim 5 for the preparation of a pharmaceutical composition for the treatment of an influenza a virus infection disease, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9;

5) use of the expression vector of claim 6 for the preparation of a pharmaceutical composition for the treatment of an influenza a virus infection disease, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9;

6) use of the host cell of claim 7 for the preparation of a pharmaceutical composition for the treatment of an influenza a virus infection disease, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9;

7) the use of the pharmaceutical composition of claim 8 in the manufacture of a product for the treatment of an influenza a virus infection, said influenza a virus being H1N1, H3N2, H5N1 and/or H7N 9.