CN114181303B

CN114181303B - Anti-influenza a virus antibodies and kits

Info

Publication number: CN114181303B
Application number: CN202010962515.5A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 唐丽娜; 罗沛
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2022-12-23
Anticipated expiration: 2040-09-14
Also published as: CN114181303A; WO2022052781A1

Abstract

The invention discloses an anti-influenza A virus antibody and a kit, and relates to the technical field of antibodies. The anti-influenza a virus antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has good affinity to influenza A virus antigen, and has good sensitivity and specificity when used for detecting influenza A virus.

Description

Anti-influenza a virus antibodies and kits

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-influenza A virus antibody and a kit.

Background

Influenza viruses (Flu), referred to as Influenza viruses for short, are representative species of the orthomyxoviridae family, including human Influenza viruses, swine Influenza viruses, equine Influenza viruses, avian Influenza viruses, etc., wherein human Influenza viruses can be classified into three types, i.e., a (a), B (B), and C (C), according to the antigenicity of nucleoprotein thereof, and are pathogens of Influenza. Influenza virus can cause infection and morbidity of various animals such as human, poultry, pigs, horses, bats and the like. The human infectable in the medicine is mainly influenza A virus and influenza B virus, mainly causes the infection of the upper respiratory tract, and also causes the infection of the lower respiratory tract of children and adults, mainly pneumonia, and the severe influenza of infants is often accompanied by bronchus and high fever.

Influenza a viruses (Flu-a) are successfully separated in 1933, antigens of the Influenza a viruses are easy to mutate, and the Influenza a viruses can be further divided into subtypes such as H1N1, H3N2, H5N1, H7N9 (H represents hemagglutinin of the Influenza virus and N represents neuraminidase of the Influenza virus), which cause worldwide pandemics for many times and have a peak every year. The degree of the influenza A virus infection is related to personal immunity, typical symptoms mainly comprise intolerance of cold, persistent high fever and headache, and general symptoms such as sore throat, cough, nasal obstruction, general aching all over the body, hypodynamia and the like are accompanied. The literature reports a positive detection rate of 20-40% in the epidemic season and 2-20% in the non-epidemic season. The persistent epidemic of influenza a virus brings great interference and pressure to the public epidemic prevention system of people's health, life and society, and it has become one of the main research objects of epidemiology.

The detection method aiming at the influenza A virus in the market at present mainly comprises a fluorescence PCR method, an immunization method and virus separation culture identification. The fluorescence PCR method is to carry out real-time detection on the PCR process through a fluorescence signal in the PCR amplification process for the purpose of qualitative or quantitative detection, and is a gold standard for pathogen detection; the immunization method is to detect target protein by specific combination of antigen and antibody; the virus isolation and culture method is usually chicken embryo inoculation, animal inoculation, tissue (cell) culture and the like, and the generated results are observed and analyzed. Although the fluorescence PCR method has better sensitivity and specificity, the detection window period is shorter, the time is strived for early diagnosis and early treatment of epidemic diseases, reduction of mortality and control of epidemic situations, and the fluorescence PCR method can be used as a gold standard for diagnosis. However, the method has high requirements on detection personnel, needs professional skill training, and can be used for diagnosis and detection only by using professional instruments and equipment in qualified professional laboratories, so the method is not suitable for quick diagnosis of clinical or epidemic disease monitoring lines. The virus isolation culture identification has long time consumption, high environmental requirement, large infection risk of operators, poor culture effect and very limited application in the aspects of clinical diagnosis, epidemic disease monitoring and the like. The immunoassay detection reagent aims at antigen or antibody in a sample, has high detection speed, high accuracy and low requirement on laboratories and operating personnel, is generally suitable for primary screening of clinical laboratories of hospitals, disease control system laboratories and the like, and has very important functions on initial detection of influenza A, successful outbreak control in hospitals and communities and guidance on treatment.

Currently, immunodiagnostic reagent products for influenza a on the market mainly include enzyme-linked immunosorbent assay (ELISA method), colloidal gold immunochromatography, such as influenza a virus antigen detection reagent of guangzhou boil (colloidal gold method), R & D influenza a ELISA kit, and the like. In the above diagnostic reagent products, specific antibodies against influenza a virus are required, and the specific antibodies against influenza a virus on the market have certain defects in specificity and sensitivity.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an anti-influenza A virus antibody and a kit, wherein the antibody has good affinity to an influenza A virus antigen, and the antibody has good sensitivity and specificity when used for detecting the influenza A virus.

The invention is realized by the following steps:

in one aspect, the invention provides an anti-influenza a virus antibody or functional fragment thereof having the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-X2-X3-W-M-D; wherein: x1 is T or S; x2 is N or D; x3 is G or A;

CDR-VH2: E-X1-R-T-K-X2-S-N-H-A-X3-Y-Y-A-E-S-X4-K-G; wherein: x1 is I, V or L; x2 is T or S; x3 is T or S; x4 is I, V or L;

CDR-VH3: T-X1-Y-D-Y-E-X2; wherein: x1 is I or L; x2 is L, V or I;

CDR-VL1: S-X1-X2-S-S-X3-S-Y-M-H; wherein: x1 is G or A; x2 is T or S; x3 is I, V or L;

CDR-VL2: D-X1-S-K-X2-A-S; wherein: x1 is T or S; x2 is I, V or L;

CDR-VL3: X1-Q-W-S-T-X2-P; wherein: x1 is H or Q; x2 is R, H or N.

The anti-influenza A virus antibody or the functional fragment thereof provided by the invention has the complementarity determining region structure, the complementarity determining region structure can ensure that the antibody or the functional fragment thereof can be specifically combined with an influenza A virus antigen and has better affinity to the influenza A virus antigen, and the antibody or the functional fragment thereof has better specificity and sensitivity when being used for detecting the influenza A virus.

In an alternative embodiment of the method of the invention,

in CDR-VH1, X3 is A;

in CDR-VH2, X3 is T;

in CDR-VH3, X1 is L;

in CDR-VL1, X1 is A;

in CDR-VL2, X1 is T;

in CDR-VL3, X1 is Q.

The present inventors have found that when the mutation site in each complementarity determining region is the amino acid residue, the antibody exhibits better affinity for influenza a virus.

In an alternative embodiment, in CDR-VH1, X1 is T.

In an alternative embodiment, in CDR-VH1, X1 is S.

In an alternative embodiment, in CDR-VH1, X2 is N.

In an alternative embodiment, in CDR-VH1, X2 is D.

In an alternative embodiment, in CDR-VH2, X1 is I.

In an alternative embodiment, in CDR-VH2, X1 is V.

In an alternative embodiment, in CDR-VH2, X1 is L.

In an alternative embodiment, in CDR-VH2, X2 is T.

In an alternative embodiment, in CDR-VH2, X2 is S.

In an alternative embodiment, in CDR-VH2, X4 is I.

In an alternative embodiment, in CDR-VH2, X4 is V.

In an alternative embodiment, in CDR-VH2, X4 is L.

In an alternative embodiment, in CDR-VH3, X2 is L.

In an alternative embodiment, in CDR-VH3, X2 is V.

In an alternative embodiment, in CDR-VH3, X2 is I.

In an alternative embodiment, in CDR-VL1, X2 is T.

In an alternative embodiment, in CDR-VL1, X2 is S.

In an alternative embodiment, in CDR-VL1, X3 is I.

In an alternative embodiment, in CDR-VL1, X3 is V.

In an alternative embodiment, in CDR-VL1, X3 is L.

In an alternative embodiment, in CDR-VL2, X2 is I.

In an alternative embodiment, in CDR-VL2, X2 is V.

In an alternative embodiment, in CDR-VL2, X2 is L.

In an alternative embodiment, in CDR-VL3, X2 is R.

In an alternative embodiment, in CDR-VL3, X2 is H.

In an alternative embodiment, in CDR-VL3, X2 is N.

In alternative embodiments, each complementarity determining region of the antibody, or functional fragment thereof, is selected from any one of the following combinations of mutations 1-64:

in alternative embodiments, the antibody or functional fragment thereof binds influenza a virus with K _D ≤5.48×10 ^-8 Affinity binding in mol/L.

In an alternative embodiment, K _D ≤5×10 ^-8 mol/L, or K _D ≤4×10 ^-8 mol/L, or K _D ≤3×10 ^-8 mol/L, or K _D ≤2×10 ^-8 mol/L, or K _D ≤1×10 ^-8 mol/L, or K _D ≤9×10 ^-9 mol/L, or K _D ≤8×10 ^-9 mol/L, or K _D ≤7×10 ^-9 mol/L, or K _D ≤6×10 ^-9 mol/L, or K _D ≤5×10 ^-9 mol/L, or K _D ≤4×10 ^-9 mol/L, or K _D ≤3×10 ^-9 mol/L, or K _D ≤2×10 ^-9 mol/L。

In an alternative embodiment, 2.07 × 10 ^-9 mol/L≤K _D ≤6.23×10 ^-9 mol/L。

K _D The detection of (2) is carried out with reference to the method in the examples of the present invention.

In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 65-74:

in alternative embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L as set forth in SEQ ID NOS 1-4 in sequence, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H as set forth in SEQ ID NOS 5-8 in sequence.

In general, the variable regions of the heavy chain (VH) and light chain (VL) can be obtained by linking the CDRs and FRs numbered below in a combined arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the corresponding framework region (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8) described above.

In alternative embodiments, the antibody further comprises a constant region.

In alternative embodiments, the constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

In alternative embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO. 10.

In alternative embodiments, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.

Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the skilled person can readily obtain the functional fragments described above.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

In another aspect, the present invention provides a reagent or a kit for detecting influenza a virus, comprising the antibody or the functional fragment thereof according to any one of the above.

In an alternative embodiment, the antibody or functional fragment thereof in the above-described reagent or kit is labeled with a detectable label.

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, one skilled in the art can select a suitable marker according to the detection condition or actual requirement, and whatever marker is used belongs to the protection scope of the present invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or analogs thereof), cy-series dyes and derivatives thereof (e.g., including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc. or analogs thereof), alexa-series dyes and derivatives thereof (e.g., including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, chlorophyll, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (polymetaxanthin), polymetaxanthin-phycoerythrin-protein (cp), etc.).

In alternative embodiments, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

In alternative embodiments, the radioisotope includes, but is not limited to ²¹² Bi、 ¹³¹ I、 ¹¹¹ In、 ⁹⁰ Y、 ¹⁸⁶ Re、 ²¹¹ At、 ¹²⁵ I、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹³ Bi、 ³² P、 ⁹⁴ mTc、 ⁹⁹ mTc、 ²⁰³ Pb、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁴³ Sc、 ⁴⁷ Sc、 ¹¹⁰ mIn、 ⁹⁷ Ru、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁸ Cu、 ⁸⁶ Y、 ⁸⁸ Y、 ¹²¹ Sn、 ¹⁶¹ Tb、 ¹⁶⁶ Ho、 ¹⁰⁵ Rh、 ¹⁷⁷ Lu、 ¹⁷² Lu and ¹⁸ F。

in alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives, dioxetane and its derivatives, lokaline and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In another aspect, the present invention provides a nucleic acid molecule encoding the above antibody or functional fragment thereof.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In another aspect, the present invention provides a recombinant cell comprising the vector described above.

In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing the recombinant cell as described above, and separating and purifying the antibody or functional fragment thereof from the culture product.

Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by separating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above, and this is within the scope of the present invention, regardless of the technique used to prepare the antibody or its functional fragment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is the result of reducing SDS-PAGE of the anti-influenza A virus antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); synthesis of oligonucleotides (oligo Synthesis) (m.j. gate eds., 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; in The Methods of Enzymology (Methods in Enzymology), published by Academic Press, inc.; in Handbook of Experimental Immunology (compiled by D.M.Weir and C.C.Black), in Gene Transfer Vectors for Mammalian Cells (compiled by J.M.Miller and M.P.Calos), in 1987, in Current Protocols in Molecular Biology (compiled by F.M.Ausubel et al, 1987), in PCR, in Polymerase Chain Reaction (compiled by The Polymerase Chain Reaction in Molecular Biology) (compiled by Mullis et al, 1994), in Current Protocols in Molecular Biology (compiled by The same et al, 1987), in vitro immunoassay (compiled by The same et al, in Cologies, in Immunology), in general Methods (compiled by The same et al, in Cologies, in immunologic literature, in E.1991, in each of which is cited.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

1 construction of recombinant plasmid

(1) Antibody Gene preparation

mRNA is extracted from a hybridoma cell strain (6A 10) secreting anti-influenza A virus antigen antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, and after colonies are grown out, the Heavy Chain and Light Chain genes are respectively cloned and respectively sent to a gene sequencing company for sequencing by 4 clones.

(2) Sequence analysis of antibody variable region genes

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 321bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 357bp, belongs to the VH1 gene family, and has a leader peptide sequence of 57bp in front.

(3) Construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the antibody are designed, the two ends of the antibody respectively carry HindIII and EcoRI restriction enzyme cutting sites and protective bases, and a Light Chain gene fragment of 0.73kb and a Heavy Chain gene fragment of 1.43kb are amplified by a PCR amplification method.

The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the gene of the Heavy Chain and the gene of the Light Chain are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.

Coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, each well is 100 ul, 4 ℃ overnight; the next day, washing liquid (main component Na) ₂ HPO ₄ + NaCl) for 2 times, patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing, keeping the temperature at 37 ℃ for 1h, and keeping the volume at 120 mu l per hole; throwing off the liquid in the plate, patting dry, adding diluted influenza A antigen 100 mul per hole, 37 ℃,40min; washing with washing solution for 5 times, and drying; adding 100 mul of influenza A monoclonal antibody marked with HRP into each hole, and performing temperature control at 37 ℃ for 30min; adding a developing solution A (50 mu l/hole), adding a developing solution B (50 mu l/hole), and carrying out 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results showed that the OD of the reaction after the cell supernatant was diluted 1000 times was still greater than 1.0, and the OD of the reaction without the cell supernatant was less than 0.1, indicating that the antibody generated after transient transformation of the plasmid was active against influenza A antigen.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 ug/tube DNA, 10 mul PuvI enzyme and sterile water to 500 mul, and performing enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25; precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water when ethanol is completely volatilized, and finally measuring the concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, transferred into an electric rotating cup, electrically rotated and counted the next day; 25umol/L MSX 96-well pressure culture for about 25 days.

MicroscopeObserving the marked cloning wells with cells, and recording the confluence; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10 ⁶ cells/ml,2.2ml, cell density 0.3X 10 ⁶ cells/ml,2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter, transferring the cell strains to TPP (thermoplastic vulcanizate) for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cell recovery, the cells were first cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at 100% volume, and placed in a shaker at a rotation speed of 120r/min, a temperature of 37 ℃ and a carbon dioxide content of 8%. Culturing for 72h, inoculating and expanding at an inoculation density of 50 ten thousand cells/ml, the expanding volume being calculated according to the production requirements, the medium being 100% Dynamis medium. Then the culture is expanded every 72 h. When the cell quantity meets the production requirement, the seeding density is strictly controlled to be about 50 ten thousand cells/ml for production.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of an external control antibody was used as a control, and the electrophorogram shown in FIG. 1 showed two bands, one of which showed 50KD (heavy chain, SEQ ID NO. 14) and the other of which showed 28KD (light chain, SEQ ID NO. 13) after reducing SDS-PAGE.

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Analyzing the sequence of the antibody (WT) of example 1, wherein the heavy chain variable region is represented by SEQ ID NO:12, wherein the amino acid sequence of each complementarity determining region in the heavy chain variable region is as follows:

CDR-VH1：G-F-S(X1)-F-S-D(X2)-G(X3)-W-M-D；

CDR-VH2：E-L(X1)-R-T-K-S(X2)-S-N-H-A-S(X3)-Y-Y-A-E-S-V(X4)-K-G；

CDR-VH3：T-I(X1)-Y-D-Y-E-V(X2)；

the light chain variable region is shown as SEQ ID NO:11, wherein the amino acid sequences of the complementarity determining regions on the light chain variable region are as follows:

CDR1-VL：S-G(X1)-T(X2)-S-S-V(X3)-S-Y-M-H；

CDR-VL2：D-S(X1)-S-K-I(X2)-A-S；

CDR-VL3：H(X1)-Q-W-S-T-N(X2)-P。

based on the anti-influenza a virus antibody (WT) of example 1, mutations were made in the complementarity determining regions at sites relevant for antibody activity, wherein X1, X2, X3, X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

Antibody binding activity assay in table 1:

coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG1 mug/ml for coating a microplate, wherein each well is 100 mug, and the temperature is 4 ℃ overnight; the next day, washing liquid (main component Na) ₂ HPO ₄ + NaCl) for 2 times, patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding the diluted purified antibody in the table 1, 100 mul/hole, 37 ℃,60min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting dry, adding diluted influenza A antigen 100 mul per hole, 37 ℃,40min; washing with the washing solution for 5 times, and drying; an HRP-labeled influenza A monoclonal antibody (which was paired with the purified antibody and obtained from Fipeng Bio Inc.) was added at 100. Mu.l per well at 37 ℃ for 30min; adding color developing liquid AAdding liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) as color development liquid (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) for 10min; stop solution (50. Mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added ₂ SO ₄ ) (ii) a OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results are shown in Table 2 below.

TABLE 2 Activity data of WT antibodies and mutants thereof

Antibody concentration (ng/ml)	1000	31.25	15.625	7.8125	3.90625	0
							WT	1.864	1.647	1.351	0.824	0.376	0.044
Mutation 1	1.919	1.863	1.526	1.103	0.620	0.045
							Mutation 2	1.821	1.843	1.421	1.024	0.663	0.073
Mutation 3	1.827	1.706	1.545	1.197	0.691	0.056
							Mutation 4	1.752	1.739	1.511	0.989	0.607	0.065
Mutation 5	0.625	0.032	-	-	-	-
							Mutation 6	0.623	0.045	-	-	-	-
Mutation 7	0.645	0.041	-	-	-	-
							Mutation 8	0.613	0.036	-	-	-	-

As can be seen from the data in table 2, the binding activity was better compared to mutations 5-8, wt and mutations 1 to 4, with mutation 1 having the best binding activity.

(2) Affinity detection of antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

Affinity assay

Using AMC sensors, purified antibodies were diluted to 10ug/ml with PBST, influenza a antigen was diluted with PBST in a gradient: 20ug/ml, 6.66ug/ml, 2.22ug/ml, 0.74ug/ml, 0.24ug/ml, 0.082ug/ml, 0.027ug/ml, 0.0091ug/ml;

the operation flow is as follows: equilibrating for 60s in buffer 1 (PBST), immobilizing antibody in antibody solution for 300s, incubating in buffer 2 (PBST) for 180s, binding for 420s in antigen solution, dissociating for 1200s in buffer 2, regenerating the sensor with 10mM pH 1.69GLY solution and buffer 3, and outputting data. K _D Indicating the equilibrium dissociation constant, i.e. affinity. The results are given in Table 4 below.

Table 4 affinity assay data

As can be seen from Table 4, the antibodies obtained by mutation 1 and mutation based on the mutation 1-1 to mutation 1-63 all have better affinity, which indicates that the antibodies obtained by mutation based on the mutation 1 according to the mutation mode in Table 3 all have better affinity.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 affinity assay results for WT antibodies and their mutants

As is clear from Table 6, WT and its mutant also have good affinity, and it is demonstrated that WT and the antibodies obtained by mutation according to the mutation pattern in Table 5 also have good affinity.

(3) Evaluation of stability against naked antibody

Placing the antibody in 4 ℃ (refrigerator), -80 ℃ (refrigerator), 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days, 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the results show that under three examination conditions, no obvious protein state change is seen in 21 days of antibody placement, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the antibody is stable. The following table 7 shows the OD results of the enzyme immunity activity assay for 21 days of the mutation 1 antibody test.

TABLE 7

Sample concentration (ng/ml)	1000	15.625	0
				Samples at 4 ℃ for 21 days	1.826	1.464	0.076
21 days samples at-80 deg.C	1.864	1.471	0.052
				21 day samples at 37 deg.C	1.806	1.432	0.062

Example 3

Application of antibody in colloidal gold detection

1 preparation of colloidal gold test paper

(1) Preparation of nitrocellulose membranes

Preparation of coating buffer: using buffer solution containing 6% methanol and 0.01M PBS (pH7.22PBS) as coating buffer solution, filtering with 0.22 μm membrane, standing at 4 deg.C for one week. 1000ml of 6% methanol in 0.01M pH7.2PBS buffer formulation: naCl 8g, KCl 0.2g, na ₂ HPO ₄ ·12H ₂ O 2.9g、KH ₂ PO ₄ 0.2g, 60ml of methanol and double distilled deionized water to reach the volume of 1000ml.

Preparation of nitrocellulose membrane: diluting an influenza A antibody (obtained from Fipeng biological products, inc.) of the coating strain to 1-5 mg/ml by using a coating buffer solution, adjusting a machine, and marking a T line to be a detection line, wherein the T line is close to and about 5mm away from the end of the gold-labeled pad; diluting the goat anti-mouse IgG antibody to 1-5 mg/ml by using a coating buffer solution, adjusting a machine, and marking to form a C line, namely a control line, wherein the C line is close to the absorption pad and is about 3mm away from the absorption pad. The distance between the two lines is 5-8 mm, and the two lines are uniform. Drying at 37 ℃, and packaging for later use.

(2) Preparation of colloidal gold and gold-labeled monoclonal antibody

(a) Preparation of the solution

(1) Preparing chloroauric acid: dissolving chloroauric acid with double distilled deionized water to prepare 1% solution, standing at 4 deg.C for use, and having validity period of four months. 1000ml 1% chloroauric acid solution formula: 10g of chloroauric acid: double distilled deionized water to 1000ml.

(2) Preparation of trisodium citrate: dissolving sodium citrate with double distilled deionized water to obtain 1% solution, filtering with 0.22 μm membrane, standing at 4 deg.C, and storing in the range of 1000ml.

(3) Preparation of 0.1M potassium carbonate: prepared by double distilled deionized water, filtered by a 0.22 mu m membrane, and placed at 4 ℃ for standby, and the validity period is four months. 1000ml of 0.1M potassium carbonate solution formula: 13.8g of potassium carbonate; double distilled deionized water to 1000ml.

(4) 2% preparation of PEG-20000: prepared by double distilled deionized water, filtered by a 0.22 mu m membrane, and placed at 4 ℃ for standby, and the validity period is four months. 1000ml 2% PEG-20000 solution formulation: 20g PEG-20000; double distilled deionized water to 1000ml.

(5) Preparation of a marking washing preservation solution: 2% Bovine Serum Albumin (BSA), 0.05% sodium azide (NaN 3), 0.01M PBS solution (pH7.2), 0.22 u membrane filtration, placed at 4 ℃ for standby, effective period of four months. 1000ml of marked washing preservation solution formula: 2g BSA,0.5g NaN3, 0.01M pH7.2PBS solution to 1000ml volume.

(b) And (3) preparing colloidal gold.

Diluting 1% chloroauric acid to 0.01% with double-distilled deionized water, boiling in electric furnace, adding 2ml 1% trisodium citrate per 100ml 0.01% chloroauric acid, boiling until the liquid is bright red, stopping heating, cooling to room temperature, and supplementing water. The prepared colloidal gold has the advantages of pure appearance, transparency, no sediment or floating matter and one week of validity.

(c) Preparing colloidal gold labeled antibody.

And (3) adjusting the pH value of the colloidal gold to 8.2 by using 0.1M potassium carbonate, respectively adding the purified antibodies according to the ratio of 8-10 mu g of the antibodies to ml of the colloidal gold, uniformly mixing for 30min by using a magnetic stirrer, adding BSA (bovine serum albumin) until the final concentration is 1% under stirring, and standing for 1 hour. Centrifuging at 13000rpm and 4 ℃ for 30min, discarding the supernatant, washing the precipitate twice with a labeled washing and preserving solution, resuspending the precipitate with the labeled washing and preserving solution with one tenth of the initial volume of the colloidal gold, standing at 4 ℃ for later use, and keeping the validity period for one week.

(3) Preparation of gold label pad

(a) And (4) preparing a sealing liquid.

2% of BSA, 0.1% of TritonX-100, 0.05% of NaN ₃ 0.01M PBS solution with pH7.2, filtering with 0.22 μm membrane, standing at 4 deg.C for use, and prolonging the service life by four months. 1000ml of sealing liquid formula: 2.0g BSA,0.5g NaN3, 1ml TritonX-100, 0.01M pH7.2PBS solution to 1000ml.

(b) Preparation of gold label pad

Soaking the gold label pad in the sealing solution for 30min, and oven drying at 37 deg.C. Then, the prepared gold-labeled antibody is evenly spread on a gold-labeled pad, each milliliter of solution is spread by 20 square centimeters, and the gold-labeled pad is frozen, dried, packaged and placed at 4 ℃ for later use.

(4) Preparation of test paper strip sample pad

(a) And (4) preparing a sealing liquid.

2% BSA, 0.1% TritionX-100, 0.05% NaN ₃ 0.01M PBS solution with pH7.2, and 0.22 μm membrane filtration, and standing at 4 degree for use with validity period of four months. 1000ml of sealing liquid formula: 20g BSA,0.5g NaN ₃ 1ml of TrtioX-100 and 0.01M PBS solution with pH7.2 are added to reach 1000ml.

(b) Preparation of sample pad.

Soaking the sample pad in sealing solution for 30min, oven drying at 37 deg.C, packaging, and standing at 4 deg.C.

(5) Assembly of test paper

Absorbent pads (available from Millipore corporation), nitrocellulose membranes, gold-labeled pads, and sample pads were placed on a non-absorbent support sheet and cut into 3mm wide strips. And packaging every ten small strips with one bag, adding a drying agent, and performing vacuum packaging to obtain the colloidal gold test paper for detecting the influenza A virus.

2 application of antibody in colloidal gold detection

The test strip assembled as described above was used to detect whether or not the test material contained influenza A virus antigen, thereby determining the activity of the antibody obtained in the above example on detection of influenza A virus antigen. The presence or absence of an influenza A virus antigen in a test material is detected by a double antibody sandwich method. During detection, the influenza A virus antigen is combined with the influenza A antibody originally marked by the colloidal gold to form an influenza A antigen-colloidal gold mark-influenza A antibody compound, the influenza A antigen-colloidal gold mark-influenza A antibody compound swims forwards along the nitrocellulose membrane due to capillary action, and when the influenza A antigen-colloidal gold mark-influenza A antibody compound reaches a detection line, the influenza A antigen-colloidal gold mark-influenza A antibody compound is combined with the influenza A antibody obtained in the embodiment to form an influenza A antibody-influenza A antigen-colloidal gold mark-influenza A antibody compound, so that the influenza A antibody-influenza A antigen-colloidal gold mark-influenza A antibody compound is enriched on the detection line, and a red precipitation line is formed. The influenza A antigen-colloidal gold labeled-influenza A antibody compound which is not combined with the influenza A antibody on the detection line passes through the detection line, is captured by goat anti-mouse IgG antibody, and is enriched on the quality control line to form a red precipitation line. And judging as a positive result when the detection line and the quality control line have red precipitation lines simultaneously. If the sample does not contain the influenza A virus antigen, when the colloidal gold-labeled influenza A antibody which is not combined with the influenza A virus antigen reaches the detection line, a compound of the influenza A antibody-influenza A antigen-colloidal gold-labeled influenza A antibody cannot be formed, and the colloidal gold-labeled influenza A antibody compound which is not combined with the influenza A antigen passes through the detection line and is only enriched on the quality control line to form a red precipitation line, and the result is judged to be negative.

The results are shown in Table 8 below.

TABLE 8 antibody detection Activity

Note: the gold label color development is formed by adding a number C, and the smaller the number behind the C is, the stronger the color development is, and the higher the activity is; higher numbers after C indicate weaker color development and lower activity; the numerals have a "+" color slightly stronger than no color development by 0.5-1C, and a "-" color slightly lower than no color development by 0.5-1C. B indicates no activity.

As can be seen from the results in Table 8, the antibodies provided in the examples of the present invention have excellent activity when tested by the double antibody sandwich method on the gold-labeled platform.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Dongguan City of Pengzhi Biotech Co., ltd

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Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

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Asp Ser Ser Lys Ile Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Phe Cys His Gln Trp Ser Thr Asn Pro Pro Thr

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Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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

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Asp Ser Ser Lys Ile Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

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Asp Ala Ala Thr Tyr Phe Cys His Gln Trp Ser Thr Asn Pro Pro Thr

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Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

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Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

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Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

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Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser

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Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr

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Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val

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Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu

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370 375 380

Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu

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Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys

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Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly

435 440 445

Claims

1. An antibody or functional fragment thereof against influenza a virus, comprising the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-X2-X3-W-M-D; wherein: x3 is A;

CDR-VH2: E-X1-R-T-K-X2-S-N-H-A-X3-Y-Y-A-E-S-X4-K-G; wherein: x3 is T;

CDR-VH3: T-X1-Y-D-Y-E-X2; wherein: x1 is L;

CDR-VL1: S-X1-X2-S-S-X3-S-Y-M-H; wherein: x1 is A;

CDR-VL2: D-X1-S-K-X2-A-S; wherein: x1 is T;

CDR-VL3: X1-Q-W-S-T-X2-P; wherein: x1 is Q;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-64:

CDR-VH1 X1/X2 CDR-VH2 X1/X2/X4 CDR-VH3 X2 CDR-VL1 X2/X3 CDR-VL2 X2 CDR-VL3 X2 combination of mutations 1 S/D L/S/V V T/V I N Combination of mutations 2 S/N L/S/L L T/L I H Combination of mutations 3 T/D L/S/I L T/I V R Combination of mutations 4 T/N L/T/V I S/V I H Combination of mutations 5 S/N L/T/L L S/L V R Combination of mutations 6 T/N L/T/I I S/I L N Mutant combination 7 T/D V/S/V V T/I I R Combination of mutations 8 S/D V/S/L V S/V I N Combination of mutations 9 T/N V/S/I V T/L I H Combination of mutations 10 S/N V/T/V I S/L L R Combination of mutations 11 T/D V/T/L I T/V L N Combination of mutations 12 S/D V/T/I I S/I I H Mutant combinations 13 T/N I/T/L L T/L L N Combination of mutations 14 T/D I/T/V L S/V L H Combination of mutations 15 S/N I/T/I L T/V V R Mutant combinations 16 S/D I/S/L V S/I I H Mutant combinations 17 T/D I/S/V V T/I V R Mutant combinations 18 S/D I/S/I I S/L L N Combination of mutations 19 T/N V/S/V V S/V V H Combination of mutations 20 S/N I/T/L V T/I I R Mutant combination 21 S/D L/S/V L S/L V N Mutant combination 22 S/N V/S/L L T/V V R Combination of mutations 23 T/D I/T/V L S/I V N Mutant combinations 24 T/N L/S/L V T/L V H Mutant combinations 25 S/N V/S/I L T/V L N Mutant combinations 26 T/N I/T/I L T/L L H Mutant combinations 27 T/D L/S/I V T/I L R Mutant combinations 28 S/D V/T/V V S/V V N Mutant combinations 29 T/N I/S/L L S/L V H Combination of mutations 30 S/N L/T/V V S/I I R Combination of mutations 31 T/D V/T/L I T/I I H Mutant combinations 32 S/D I/S/V I S/V I R Mutant combinations 33 T/N L/T/L V T/L I N Mutant combinations 34 T/D V/T/I V S/L I R Combination of mutations 35 S/N I/S/I V T/V I N Combination of mutations 36 S/D L/T/I L S/I I H Mutant combinations 37 T/D L/T/I I T/L L R Combination of mutations 38 S/D V/T/I L S/V L N Mutant combinations 39 T/N I/T/L I T/V V H Combination of mutations 40 S/N L/T/L I S/I I N Mutant combination 41 S/D V/T/L V T/I L H Combination of mutations 42 S/N I/T/V V S/V I R Mutant combinations 43 T/D L/T/V I T/I V H Mutant combinations 44 T/N V/T/V L S/L L R Combination of mutations 45 S/N I/T/I I T/V I N Mutant combinations 46 T/N L/S/I L S/I L H Mutant combinations 47 T/D V/S/I I T/L V R Mutant combinations 48 S/D I/S/L L T/V I N Mutant combinations 49 T/N L/S/L L T/L V R Mutant combinations 50 S/N V/S/L L T/I I N Mutant combinations 51 T/D I/S/V I S/V L H Mutant combinations 52 S/D L/S/V V S/L I N Mutant combination 53 T/N V/T/I V S/I L H Mutant combinations 54 T/D I/T/L L T/L V R Mutant combinations 55 S/N V/S/V I S/V L R Mutant combinations 56 S/D I/T/L I T/V L N Mutant combinations 57 T/D L/S/V V S/I L H Mutant combinations 58 S/D V/S/L L T/I L N Mutant combination 59 T/N I/T/V V S/L V H Mutant combinations 60 S/N L/S/L V T/I I R Mutant combinations 61 S/D V/S/I L S/V V H Mutant combinations 62 S/N I/T/I V T/L L R Mutant combinations 63 T/D L/S/I L S/L L N Mutant combinations 64 T/N V/T/V L T/V I H

。

2. An antibody or functional fragment thereof against influenza a virus, comprising the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-X2-X3-W-M-D; wherein: x3 is G;

CDR-VH2: E-X1-R-T-K-X2-S-N-H-A-X3-Y-Y-A-E-S-X4-K-G; wherein: x3 is S;

CDR-VH3: T-X1-Y-D-Y-E-X2; wherein: x1 is I;

CDR-VL1: S-X1-X2-S-S-X3-S-Y-M-H; wherein: x1 is G;

CDR-VL2: D-X1-S-K-X2-A-S; wherein: x1 is S;

CDR-VL3: X1-Q-W-S-T-X2-P; wherein: x1 is H;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 65-74:

CDR-VH1 X1/X2 CDR-VH2 X1/X2/X4 CDR-VH3 X2 CDR-VL1 X2/X3 CDR-VL2 X2 CDR-VL3 X2 mutant combination 65 S/D L/S/V V T/V I N Mutant combinations 66 T/N I/S/L I S/L L N Mutant combinations 67 S/N L/S/L V S/V V H Mutant combinations 68 S/D V/S/L V T/L V N Mutant combinations 69 T/N V/T/V L T/V I R Mutant combinations 70 S/D L/T/L L S/I I R Mutant combinations 71 S/D L/S/I L T/L L R Mutant combinations 72 S/N V/S/V I T/I I R Mutant combinations 73 S/D V/S/L I T/V I H Mutant combinations 74 T/D V/S/V L S/L L N

。

3. The anti-influenza a virus antibody or functional fragment thereof according to any one of claims 1 to 2, wherein the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L having the sequences shown in SEQ ID NOs 1 to 4 in order and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having the sequences shown in SEQ ID NOs 5 to 8 in order.

4. The anti-influenza a virus antibody or functional fragment thereof according to any one of claims 1 to 2, wherein the antibody further comprises a constant region.

5. The anti-influenza A virus antibody or functional fragment thereof according to claim 4, wherein said constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.

6. The anti-influenza a virus antibody or functional fragment thereof according to claim 4, wherein the species source of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

7. The anti-influenza a virus antibody or functional fragment thereof according to claim 6, wherein the species source of the constant region is a bovine.

8. The anti-influenza a virus antibody or functional fragment thereof according to claim 6, wherein the species source of the constant region is turkey or turkey.

9. The anti-influenza a virus antibody or functional fragment thereof according to claim 6, wherein the constant region is derived from a mouse.

10. The anti-influenza a virus antibody or functional fragment thereof of claim 9, wherein the constant region light chain constant region sequence is set forth in SEQ ID No. 9 and the constant region heavy chain constant region sequence is set forth in SEQ ID No. 10.

11. The anti-influenza a virus antibody or functional fragment thereof according to any one of claims 1 to 2, wherein the functional fragment is selected from the group consisting of F (ab') of said antibody ₂ Any one of Fab', fab, fv and scFv.

12. A reagent or kit for detecting influenza a virus comprising the antibody or functional fragment thereof of any one of claims 1 to 11.

13. The reagent or kit of claim 12, wherein the antibody or functional fragment thereof is labeled with a detectable label.

14. The reagent or kit of claim 13, wherein the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

15. The reagent or the kit according to claim 14, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, rhodamine-based dyes and derivatives thereof, cy-based dyes and derivatives thereof, alexa-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.

16. The reagent or kit of claim 14, wherein the enzyme that catalyzes the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

17. The reagent or kit according to claim 14, wherein the radioisotope is selected from the group consisting of ²¹² Bi、 ¹³¹ I、 ¹¹¹ In、 ⁹⁰ Y、 ¹⁸⁶ Re、 ²¹¹ At、 ¹²⁵ I、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹³ Bi、 ³² P、 ⁹⁴ mTc、 ⁹⁹ mTc、 ²⁰³ Pb、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁴³ Sc、 ⁴⁷ Sc、 ¹¹⁰ mIn、 ⁹⁷ Ru、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁸ Cu、 ⁸⁶ Y、 ⁸⁸ Y、 ¹²¹ Sn、 ¹⁶¹ Tb、 ¹⁶⁶ Ho、 ¹⁰⁵ Rh、 ¹⁷⁷ Lu、 ¹⁷² Lu and ¹⁸ F。

18. the reagent or kit according to claim 14, characterized in that said chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, loflunine and its derivatives and peroxyoxalate and its derivatives.

19. The reagent or kit according to claim 14, wherein the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids.

20. The reagent or kit of claim 19, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.

21. The reagent or kit of claim 19, wherein the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, colloidal selenium, and latex.

22. The reagent or kit of claim 21, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.

23. A vector comprising a nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1-11.

24. A recombinant cell comprising the vector of claim 23.

25. A method of producing an antibody or functional fragment thereof according to any one of claims 1 to 11, comprising: culturing the recombinant cell of claim 24, and isolating and purifying the antibody or functional fragment thereof from the culture product.