CN114181303A

CN114181303A - Anti-influenza a virus antibodies and kits

Info

Publication number: CN114181303A
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: 2022-03-15
Anticipated expiration: 2040-09-14
Also published as: CN114181303B; 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 (Influenza a, Flu-a) are successfully separated in 1933, antigens of the Influenza a viruses are easy to change, 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 pandemics all over the world 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 positive detection rate in the circulation period is 20-40% and the positive rate in the non-epidemic season is 2-20% in literature reports. 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, and aims at qualitative or quantitative detection, and the method is a gold standard for pathogen detection; the immunization method is to detect target protein by specific binding 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 and high accuracy, has low requirements on laboratories and operators, 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 of 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 this, the 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-VH 1: 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-VH 2: 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-VH 3: T-X1-Y-D-Y-E-X2; wherein: x1 is I or L; x2 is L, V or I;

CDR-VL 1: 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-VL 2: D-X1-S-K-X2-A-S; wherein: x1 is T or S; x2 is I, V or L;

CDR-VL 3: 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 present 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^-8Affinity binding in mol/L.

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

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

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

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-FR 4.

It is noted that 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 described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8).

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 above-described functional fragments are readily available to those skilled in the art.

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, automated peptide synthesizers, 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, red 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, Cy3, 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, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin (e.g., including, pyrenochrome, and analogs thereof), Allophycocyanin (APC), polymethacrylic flavin-chlorophyll protein (precP), 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 to²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F。

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 ester and its derivatives, dioxane and its derivatives, lotrine 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 metals include, but are 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 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: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

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. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE 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 (6A10) 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 grow out, 4 clones of the Heavy Chain and Light Chain gene clones are respectively taken and sent to a gene sequencing company for sequencing.

(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 a 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 HindIII, BamHI and EcoRI are introduced into the expression vectorThe polyclonal enzyme cutting sites are equal, and are named as pcDNA3.4A expression vectors, and the subsequent expression vectors are called as 3.4A expression vectors for short; 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, two ends of the primers are respectively provided with HindIII and EcoRI restriction 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 Heavy Chain gene and the Light Chain gene 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 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

Coating liquid (main component NaHCO)₃) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, each well is 100 mul, and the temperature is 4 ℃ overnight; the next day, washing liquid (main component Na)₂HPO₄+ NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60 min; 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 ℃, 40 min; 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 30 min; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10 min; 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 630nm) 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 in the wells without the cell supernatant was less than 0.1, indicating that the antibody was produced after transient transformation of the plasmidActive 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 Puv I enzyme, supplementing 500 mul sterile water, and enzyme cutting overnight in 37 deg.C water bath; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring 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 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; 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 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(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

Analysis of the antibody (WT) sequence of example 1 showed that 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, and 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)₂HPO₄+ NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding the diluted purified antibody in the table 1, 100 mul/hole, 37 ℃, 60 min; 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 ℃, 40 min; washing with washing solution for 5 times, and drying; adding HRP-labeled influenza A monoclonal antibody (obtained from Fipeng Bio Inc. by matching with purified antibody) at a concentration of 100 μ l per well at 37 deg.C for 30 min; adding color development liquid A (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) and adding color development 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) for 10 min; stop solution (50. mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added₂SO₄) (ii) a OD readings were taken at 450nm (reference 630nm) 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 for WT and mutations 1 to 4 compared to mutations 5-8, 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, 0.0091 ug/ml;

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

Table 4 affinity assay data

As can be seen from Table 4, the antibodies obtained by mutating 1 and the base thereof, namely, 1-1 to 1-63, have better affinities, which indicates that the antibodies obtained by mutating 1 and the base thereof according to the mutation mode shown in Table 3 have better affinities.

(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, indicating that the antibodies obtained by mutation based on the mutation pattern shown in Table 5 also have good affinity.

(3) Evaluation of stability against naked antibody

Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, 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: coating buffer solution containing 6% methanol and 0.01M buffer solution of pH7.2PBS, filtering with 0.22 μ M 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, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.2g, 60ml of methanol and double distilled deionized water to reach the volume of 1000 ml.

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 obtain 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 is uniform. Drying at 37 ℃, and packaging for later use.

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

(a) Preparation of the solution

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 of 1% chloroauric acid solution formula: 10g of chloroauric acid: double distilled deionized water to 1000 ml.

Preparing trisodium citrate: dissolving sodium citrate with double distilled deionized water to obtain 1% solution, filtering with 0.22 μm membrane, standing for 4 deg.C, and storing for 1000 ml.

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 formulation: 13.8g of potassium carbonate; double distilled deionized water to 1000 ml.

Fourthly, preparing 2 percent 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 1000 ml.

Preparing a marking washing preservation solution: 2% Bovine Serum Albumin (BSA), 0.05% sodium azide (NaN3), 0.01M PBS solution (pH7.2), 0.22 μmembrane filtration, placed at 4 ℃ for use, the effective period of four months. 1000ml mark washing preservation solution formula: 20g BSA, 0.5g NaN3, 0.01M pH7.2PBS solution to volume to 1000 ml.

(b) And (5) 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 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.

Contains 2% BSA, 0.1% TritonX-100, 0.05% NaN₃0.01M PBS solution with pH7.2, 0.22 μ M membrane filtration, standing at 4 deg.C for use, and effective period of four months. 1000ml of sealing liquid formula: 20g BSA, 0.5g NaN3, 1ml TritonX-100, 0.01M pH7.2PBS solution to 1000 ml.

(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.

Contains 2% BSA, 0.1% TrtioX-100, 0.05% NaN₃0.01M PBS solution with pH7.2, 0.22 μ M membrane filtration, standing for 4 degrees for use, and validity period of four months. 1000ml sealThe formula of the occlusive liquid is as follows: 20g BSA, 0.5g NaN₃1ml of TrtioX-100 and 0.01M PBS solution with pH7.2 are added to make the volume reach 1000 ml.

(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. Whether or not the test material contains an influenza A virus antigen 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 is captured by the goat anti-mouse IgG antibody through the detection line 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 the red precipitation line at the same time. 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 mark color development is formed by adding a number C, and the smaller the number behind the number 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 sign with a "+" after the number is slightly stronger than the non-coloration by 0.5-1C, and the sign with a "-" after the number is slightly lower than the non-coloration 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

<120> anti-influenza a virus antibody and kit

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Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

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

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Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

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

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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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Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

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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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Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

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210

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Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

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Ala Glu Leu Arg Thr Lys Ser Ser Asn His Ala Ser Tyr Tyr Ala Glu

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

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Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr

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Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp

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Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

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Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

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Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys

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

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Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp

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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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Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg

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Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val

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Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr

355 360 365

Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr

370 375 380

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

385 390 395 400

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

CDR-VH 3: T-X1-Y-D-Y-E-X2; wherein: x1 is I or L; x2 is L, V or I;

CDR-VL 2: D-X1-S-K-X2-A-S; wherein: x1 is T or S; x2 is I, V or L;

CDR-VL 3: X1-Q-W-S-T-X2-P; wherein: x1 is H or Q; x2 is R, H or N.

2. The anti-influenza A virus antibody or functional fragment thereof according to claim 1,

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;

preferably, in CDR-VH1, X1 is T;

preferably, in CDR-VH1, X1 is S;

preferably, in CDR-VH1, X2 is N;

preferably, in CDR-VH1, X2 is D;

preferably, in CDR-VH2, X1 is I;

preferably, in CDR-VH2, X1 is V;

preferably, in CDR-VH2, X1 is L;

preferably, in CDR-VH2, X2 is T;

preferably, in CDR-VH2, X2 is S;

preferably, in CDR-VH2, X4 is I;

preferably, in CDR-VH2, X4 is V;

preferably, in CDR-VH2, X4 is L;

preferably, in CDR-VH3, X2 is L;

preferably, in CDR-VH3, X2 is V;

preferably, in CDR-VH3, X2 is I;

preferably, in CDR-VL1, X2 is T;

preferably, in CDR-VL1, X2 is S;

preferably, in CDR-VL1, X3 is I;

preferably, in CDR-VL1, X3 is V;

preferably, in CDR-VL1, X3 is L;

preferably, in CDR-VL2, X2 is I;

preferably, in CDR-VL2, X2 is V;

preferably, in CDR-VL2, X2 is L;

preferably, in CDR-VL3, X2 is R;

preferably, in CDR-VL3, X2 is H;

preferably, in CDR-VL3, X2 is N;

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

3. the anti-influenza a virus antibody or functional fragment thereof of claim 2, wherein the antibody or functional fragment thereof binds to an influenza a virus antigen as K_D≤5.48×10^-8Affinity binding of mol/L; preferably, K_D≤6.23×10^-9mol/L。

4. The anti-influenza A virus antibody or functional fragment thereof according to claim 1,

in CDR-VH1, X3 is G;

in CDR-VH2, X3 is S;

in CDR-VH3, X1 is I;

in CDR-VL1, X1 is G;

in CDR-VL2, X1 is S;

in CDR-VL3, X1 is H;

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

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

preferably, the antibody further comprises a constant region;

preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the constant region is from a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fight, or human;

preferably, the constant region is derived from a mouse;

preferably, the light chain constant region sequence of the constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the constant region is shown as SEQ ID NO. 10;

preferably, the functional fragment is selected from any one of VHH, F (ab ') 2, Fab', Fab, Fv and scFv of the antibody.

6. A reagent or kit for detecting influenza a virus comprising the antibody or functional fragment thereof according to any one of claims 1 to 5.

7. The reagent or kit according to claim 6, wherein the antibody or functional fragment thereof is labeled with a detectable label;

preferably, the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label;

preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;

preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;

preferably, the radioisotope is selected from the group consisting of²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and¹⁸F；

preferably, the 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, lowhine and its derivatives, and peroxyoxalate and its derivatives;

preferably, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes;

preferably, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver and colloidal selenium.

8. A vector comprising a nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1-5.

9. A recombinant cell comprising the vector of claim 8.

10. A method for producing an antibody or functional fragment thereof according to any one of claims 1 to 5, comprising: culturing the recombinant cell of claim 9, and isolating and purifying the antibody or functional fragment thereof from the culture product.