CN114276441B

CN114276441B - Anti-influenza B virus antibody, preparation method thereof and detection kit

Info

Publication number: CN114276441B
Application number: CN202011030309.7A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 周全兴; 何雯雯; 罗沛
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2022-12-27
Anticipated expiration: 2040-09-27
Also published as: CN114276441A

Abstract

The invention discloses an anti-influenza B virus antibody, a preparation method thereof and a detection kit, and relates to the technical field of antibodies. The anti-influenza b antibody disclosed herein comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The anti-influenza B virus antibody provided by the invention has better affinity and activity to an influenza B virus antigen, and the antibody has better sensitivity and specificity when being used for detecting the influenza B virus, thereby providing better antibody selection for detecting the influenza B virus.

Description

Anti-influenza B virus antibody, preparation method thereof and detection kit

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-influenza B virus antibody, a preparation method thereof and a detection 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 B is an influenza caused by influenza B (B) virus and is characterized by sudden onset of disease, aversion to cold, fever, rising of body temperature within hours to 24 hours to reach a peak, and 39-40 ℃ or even higher. Headache, general aching pain, hypodynamia and anorexia. The respiratory symptoms are mild, dry throat and sore throat, dry cough and diarrhea. Flush face, conjunctival outer canthus congestion, pharyngeal congestion, and follicular orifices in the soft palate. Can be used for treating with amantadine as M2 ion blocker, or with Chinese medicinal materials.

Influenza b viruses produce many subtypes because Hemagglutinin (HA) and Neuraminidase (NA) antigens of influenza viruses are susceptible to conversion, and their constituent amino acid sites can be varied. After each pandemic of the influenza virus, the novel influenza virus is generated through the amino acid site variation, the human immune system generally lacks resistance to the varied subtype, local epidemics are easily caused, and large-range crowd infection can be caused under special environment. Data monitored by the U.S. and the central center for disease prevention and control (CDC) from 12 months to 1 month in 2017 show that the incidence of influenza b virus is on the rise. Therefore, early and rapid screening of influenza virus is of great importance.

Clinical symptoms after influenza virus infection are atypical, clinical diagnosis mainly depends on laboratory detection, and respiratory viruses capable of infecting human simultaneously are more than 200, so that the sensitivity and specificity of a detection reagent are very important for clinical diagnosis. The screening technology for selecting the influenza virus with short detection time and high detection rate is a technical path and a precondition guarantee for ensuring clinical rapid diagnosis and symptomatic treatment.

The laboratory detection method for influenza virus has various methods, the change of the influenza diagnosis standard of the Ministry of health is known, the early detection depends on chick embryo inoculation, the operation is complex, the technical requirement is high, the clinical application is few, the modern detection technology comprises antigen detection and nucleic acid PCR detection, and the new technology has the characteristics of rapidness, sensitivity and specificity, and provides great help for clinical diagnosis.

The fluorescence PCR amplification technology has the advantages that the sensitivity and the specificity are high, but the requirements of the PCR method on samples, test environment and operators are high, the amplification methodology is suitable for detecting batch samples, the report time is long, and the requirement of clinical rapid diagnosis cannot be well met. The immune colloidal gold technology for detecting the virus antigen can be used as a preferred method for quickly diagnosing the influenza B, has short detection time, can effectively assist clinical diagnosis and greatly help clinical medication for symptoms as early as possible.

The method aims to strengthen the monitoring of the influenza B virus and provide help for the rapid and accurate screening of the influenza B virus infection, and mainly aims to optimize the quality of a quick detection reagent, shorten the sample cracking time, reduce the detection concentration limit of the sample and improve the sensitivity and specificity of the reagent. However, antibodies against influenza B viruses in the current 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 B virus antibody, a preparation method thereof and a detection kit, the anti-influenza B virus antibody provided by the invention has better affinity to an influenza B virus antigen, and the antibody has better sensitivity and specificity when used for detecting the influenza B virus, thereby providing a new antibody selection for detecting the influenza B virus.

The invention is realized in the following way:

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

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

CDR-VH2: T-X1-S-S-G-G-S-Y-X2-Y-Y-P-D-S-X3-K-G, wherein: x1 is I, V or L; x2 is S or T; x3 is I, V or L;

CDR-VH3: T-X1-H-X2-T-X3-A-S-Y, wherein: x1 is K or R; x2 is L, V or I; x3 is T or S;

CDR-VL1: R-A-S-X1-N-X2-Y-T-S-X3-A, wherein: x1 is D or E; x2 is L or I; x3 is I, V or L;

CDR-VL2: Y-X1-A-T-N-X2-A-D, wherein: x1 is G or A; x2 is I, V or L;

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

The anti-influenza B 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 influenza B virus antigen and has better affinity to the influenza B virus antigen, and the antibody or the functional fragment thereof has better specificity and sensitivity when being used for detecting the influenza B virus.

In an alternative embodiment of the method of the present invention,

in CDR-VH1, X3 is M;

in CDR-VH2, X2 is T;

in CDR-VH3, X1 is R;

in CDR-VL1, X2 is I;

in CDR-VL2, X1 is A;

in CDR-VL3, X2 is G.

In alternative embodiments, in CDR-VH1, X1 is T.

In alternative embodiments, in CDR-VH1, X1 is S.

In alternative embodiments, in CDR-VH1, X2 is F.

In alternative embodiments, in CDR-VH1, X2 is W.

In alternative embodiments, in CDR-VH1, X2 is Y.

In alternative embodiments, in CDR-VH2, X1 is I.

In alternative embodiments, in CDR-VH2, X1 is V.

In alternative embodiments, in CDR-VH2, X1 is L.

In alternative embodiments, in CDR-VH2, X3 is I.

In alternative embodiments, in CDR-VH2, X3 is V.

In alternative embodiments, in CDR-VH2, X3 is L.

In alternative embodiments, in CDR-VH3, X2 is L.

In alternative embodiments, in CDR-VH3, X2 is V.

In alternative embodiments, in CDR-VH3, X2 is I.

In alternative embodiments, in CDR-VH3, X3 is T.

In alternative embodiments, in CDR-VH3, X3 is S.

In an alternative embodiment, in CDR-VL1, X1 is D.

In an alternative embodiment, in CDR-VL1, X1 is E.

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

In alternative embodiments, in CDR-VL1, X3 is V.

In alternative embodiments, in CDR-VL1, X3 is L.

In alternative embodiments, in CDR-VL2, X2 is I.

In alternative embodiments, in CDR-VL2, X2 is V.

In alternative embodiments, in CDR-VL2, X2 is L.

In an alternative embodiment, in CDR-VL3, X1 is H.

In an alternative embodiment, in CDR-VL3, X1 is N.

In an alternative embodiment, in CDR-VL3, X1 is Q.

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 1-56:

in alternative embodiments, the antibody or functional fragment thereof binds influenza b virus with K _D ≤4.18×10 ^-8 Affinity binding of mol/L, preferably, K _D ≤7.38×10 ^-9 mol/L。

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

In an alternative embodiment, 1.98 × 10 ^-9 mol/L≤K _D ≤4.18×10 ^-8 mol/L。

In an alternative embodiment, 1.98 × 10 ^-9 mol/L≤K _D ≤7.38×10 ^-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 an alternative embodiment of the method of the invention,

in CDR-VH1, X3 is F;

in CDR-VH2, X2 is S;

in CDR-VH3, X1 is K;

in CDR-VL1, X2 is L;

in CDR-VL2, X1 is G;

in CDR-VL3, X2 is A.

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 57-64:

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

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 present disclosure 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 to cleave 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 b 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, 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, chlorophyll, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, phycoerythrin (PE), allophycocyanin (PC), allophycocyanin (paucin (PC), polymetaxanthin (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 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 a 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 the functional fragment thereof, those skilled in the art can easily think that the antibody or the functional fragment thereof is prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, the antibody or the functional fragment thereof is obtained by separating and purifying from the culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof as described above, which is easily realized by those skilled in the art, and therefore, the antibody or the functional fragment thereof of the present invention is within the protection scope of the present invention regardless of the technique used for preparing the antibody or the functional fragment thereof.

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 B 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); 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), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.

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

Example 1

The restriction enzyme, prime Star DNA polymerase, in this example was purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara. The 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 secreting anti-influenza B virus 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, after a colony grows out, the Heavy Chain and Light Chain genes are respectively taken for cloning, and 4 clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

Putting the gene sequence obtained by sequencing into 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 segment amplified by the Light Chain, the VL gene sequence is 324bp, 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 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, 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 1.42KB 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 mu L of plasmid is mixed with 700 mu L of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on days 3, 5 and 7, and the sampling detection is carried out on day 7.

Coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, each well is 100 μ L,4 deg.C overnight; the next day, the washing solution (main component Na) ₂ HPO ₄ + NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing, wherein each hole is 120 mu L, the temperature is 37 ℃, and the time is 1h; throwing off the liquid in the plate, patting dry, adding diluted influenza B virus antigen 100 muL per hole, 37 ℃,40min; washing with the washing solution for 5 times, and drying; adding anti-influenza B virus antigen monoclonal antibody marked with HRP, wherein each hole is 100 mu L, and the temperature is 37 ℃ for 30min; adding developing solution A (50. Mu.L/hole), adding developing solution B (50. Mu.L/hole), and standing for 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 result shows that the reaction OD is still larger than 1.0 after the cell supernatant is diluted 1000 times, and the reaction OD of the wells without the cell supernatant is smaller than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has the original activity on the influenza B virus.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50 mu L, DNA 100 mu g/tube, puv I enzyme 10 mu L, sterile water supplementing to 500 mu L, water bath enzyme digestion at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume 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 10 ⁷ cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L 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 the cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample 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 cell/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 cells were recovered, they were cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at a culture medium volume of 100%, and placed in a shaker at a rotation speed of 120r/min and a temperature of 37 ℃ with 8% carbon dioxide. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/ml, and calculating the expanding volume according to production requirements, wherein the culture medium accounts for 100 percent. 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 manner: daily feeding was started at 72h in the flask, 3% of the initial culture volume was fed daily by HyCloneTM Cell BoostTM Feed 7a, one thousandth of the initial culture volume was fed daily by Feed 7b, and was continued 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 shows two bands, one of which had an Mr of 50KD (heavy chain, SEQ ID NO: 14) and the other of which had an Mr of 28KD (light chain, SEQ ID NO: 13) after the 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, the heavy chain variable region is shown in 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-S-F(X2)-T-F(X3)-S；

CDR-VH2：T-V(X1)-S-S-G-G-S-Y-S(X2)-Y-Y-P-D-S-I(X3)-K-G；

CDR-VH3：T-K(X1)-H-L(X2)-T-T(X3)-A-S-Y；

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：R-A-S-E(X1)-N-L(X2)-Y-T-S-V(X3)-A；

CDR-VL2：Y-G(X1)-A-T-N-I(X2)-A-D；

CDR-VL3：Q-Q(X1)-F-W-A(X2)-T-P-W-T。

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

TABLE 1 mutation sites related to 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 B virus antigen 100 mul per hole, 37 ℃,40min; washing with the washing solution for 5 times, and drying; adding 100 μ l of HRP-labeled influenza B virus paired monoclonal antibody (obtained from Ficron organism) at 37 deg.C for 30min; 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 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 given in Table 2 below.

TABLE 2 Activity data of WT antibody and its mutants

Antibody concentration (ng/ml)	62.5	31.25	15.625	7.813	1.953	0
							WT	2.045	1.167	0.630	0.446	0.151	0.052
Mutation 1	2.357	1.446	0.969	0.768	0.368	0.073
							Mutation 2	2.217	1.419	0.819	0.689	0.265	0.059
Mutation 3	2.201	1.423	0.842	0.651	0.272	0.047
							Mutation 4	2.218	1.438	0.813	0.698	0.217	0.049
Mutation 5	0.721	0.468	0.221	0.057	-	-
							Mutation 6	0.643	0.456	0.215	0.068	-	-
Mutation 7	0.745	0.543	0.236	0.049	-	-

As can be seen from the data in table 2, WT, mutations 1 to 4 had better binding activity, 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 sensor, the purified antibody was diluted to 10ug/ml with PBST and the influenza B virus antigen was PBST (main component Na) ₂ HPO ₄ + NaCl + TW-20) to carry out gradient dilution;

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. The results are given in Table 4,K _D Indicating the equilibrium dissociation constant, i.e. affinity.

Table 4 affinity assay data

The results in table 4 show that the mutant 1 and its series of mutant antibodies all have high affinity for antigen, which indicates that the antibodies obtained by mutation based on the mutation 1 according to the mutation mode in table 3 all have good 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 antibody and its mutants

	K _D (M)
		WT	3.96E-08
WT1	3.24E-08
		WT2	3.36E-08
WT3	2.78E-08
		WT4	4.18E-08
WT5	2.83E-08
		WT6	2.96E-08
WT7	2.30E-08

The data in Table 6 show that WT and its series of mutants also have good affinity for antigen, indicating that the antibodies obtained by mutation in the manner of the mutations in Table 5 on the basis of WT all have good affinity for antigen.

(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 results of the OD detection of the 21-day assessment of the antibody to mutation 1.

TABLE 7

Antibody concentration (ng/ml)	62.5	15.625	0
				Samples at 4 ℃ for 21 days	2.398	0.686	0.039
21 days samples at-80 deg.C	2.296	0.702	0.078
				21 day samples at 37 deg.C	2.276	0.714	0.002

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 the purified antibodies in the tables 3 and 5 to 1-5 mg/ml by using coating buffer solution, adjusting a machine, and marking to form a T line, namely a detection line, wherein the T line is close to the end of the gold label pad and is 5mm away from the end of the gold label 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

(1) Preparing chloroauric acid: dissolving chloroauric acid with double distilled deionized water to prepare 1% solution, and standing at 4 deg.C for use with a 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 for 4 deg.C, and storing for 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 of PEG-20000; double-distilled deionized water is added to the volume of 1000ml.

(5) Preparation of a marking washing preservation solution: 2% Bovine Serum Albumin (BSA), 0.05% sodium azide (NaN) ₃ ) 0.01M PBS solution with pH7.2, 0.22 mu membrane filtration, and standing at 4 ℃ for use with a validity 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 (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.

Adjusting the pH value of the colloidal gold to 8.2 by 0.1M potassium carbonate, adding another strain of mateable anti-influenza B virus labeled antibody (obtained from Fipeng organism) into the colloidal gold according to the ratio of 8-10 mug antibody/ml, uniformly mixing for 30min by a magnetic stirrer, adding BSA (bovine serum albumin) under stirring until the final concentration is 1%, 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-labeled 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: 20g 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 B virus.

2 application of antibody in colloidal gold detection

The assembled test strip is used for detecting whether the detected material contains the influenza B virus antigen, so that the detection effect of the antibody obtained in the embodiment on the influenza B virus antigen is determined. Detecting whether the material contains influenza B virus antigen by double antibody sandwich method. During detection, the influenza B virus antigen-colloidal gold label-influenza B virus antibody compound is formed by combining the influenza B virus antigen-colloidal gold label-influenza B virus antibody compound with the colloidal gold label, and the influenza B virus antigen-colloidal gold label-influenza B virus antibody compound swims forwards along the nitrocellulose membrane due to capillary action, and when reaching a detection line, the influenza B virus antigen-colloidal gold label-influenza B virus antibody compound is combined with the influenza B virus antibody obtained in the embodiment to form the influenza B virus antibody-influenza B virus antigen-colloidal gold label-influenza B virus antibody compound, so that the influenza B virus antibody-influenza B virus antigen-colloidal gold label-influenza B virus antibody compound is enriched on the detection line to form a red precipitation line. The influenza B virus antigen-colloidal gold label-influenza B virus antibody compound which is not combined with the influenza B virus 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 the red precipitation line at the same time. If the sample does not contain the influenza B virus antigen, when the influenza B virus antibody which is not combined with the influenza B virus antigen and marked by the colloidal gold reaches the detection line, a compound of the influenza B virus antibody-the influenza B virus antigen-the colloidal gold-marked-the influenza B virus antibody cannot be formed, and the influenza B virus antibody compound which is not combined with the influenza B virus antigen is only enriched on the quality control line through the detection line to form a red precipitation line, and the result is judged to be negative.

The results for the partial antibodies are shown in Table 8 below.

TABLE 8

Remarking: 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 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.

The results in the table show that the antibody provided by the embodiment of the invention has good activity when used for double antibody sandwich detection on a 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

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Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser

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450

Claims

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

CDR-VH1: G-F-X1-F-S-S-X2-T-X3-S, wherein: x3 is M;

CDR-VH2: T-X1-S-S-G-G-S-Y-X2-Y-Y-P-D-S-X3-K-G, wherein: x2 is T;

CDR-VH3: T-X1-H-X2-T-X3-A-S-Y, wherein: x1 is R;

CDR-VL1: R-A-S-X1-N-X2-Y-T-S-X3-A, wherein: x2 is I;

CDR-VL2: Y-X1-A-T-N-X2-A-D, wherein: x1 is A;

CDR-VL3: Q-X1-F-W-X2-T-P-W-T, wherein: x2 is G;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-56:

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

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

CDR-VH1: G-F-X1-F-S-S-X2-T-X3-S, wherein: x3 is F;

CDR-VH2: T-X1-S-S-G-G-S-Y-X2-Y-Y-P-D-S-X3-K-G, wherein: x2 is S;

CDR-VH3: T-X1-H-X2-T-X3-A-S-Y, wherein: x1 is K;

CDR-VL1: R-A-S-X1-N-X2-Y-T-S-X3-A, wherein: x2 is L;

CDR-VL2: Y-X1-A-T-N-X2-A-D, wherein: x1 is G;

CDR-VL3: Q-X1-F-W-X2-T-P-W-T, wherein: x2 is A;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 57-64:

CDR-VH1 X1/X2 CDR-VH2 X1/X3 CDR-VH3 X2/X3 CDR-VL1 X1/X3 CDR-VL2 X2 CDR-VL3 X1 mutant combinations 57 S/F V/I L/T E/V I Q Mutant combinations 58 T/W I/I V/T D/V V H Mutant combinations 59 T/Y L/V V/T E/V I Q Mutant combinations 60 T/Y L/I V/S D/V V H Mutant combinations 61 T/Y I/V I/S E/I L Q Mutant combinations 62 S/F I/V L/T E/L L N Mutant combinations 63 T/F V/V I/S E/L I Q Mutant combinations 64 T/F L/L I/S E/V I N

3. An anti-influenza B virus antibody or a functional fragment thereof,

the antibody or functional fragment thereof comprises the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-S-X2-T-X3-S, wherein: x3 is M;

CDR-VH2: T-X1-S-S-G-G-S-Y-X2-Y-Y-P-D-S-X3-K-G, wherein: x2 is T;

CDR-VH3: T-X1-H-X2-T-X3-A-S-Y, wherein: x1 is R;

CDR-VL1: R-A-S-X1-N-X2-Y-T-S-X3-A, wherein: x2 is I;

CDR-VL2: Y-X1-A-T-N-X2-A-D, wherein: x1 is A;

CDR-VL3: Q-X1-F-W-X2-T-P-W-T, wherein: x2 is G;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-56:

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

The antibody also comprises light chain framework regions FR1-L, FR2-L, FR-L and FR4-L with the sequences shown in SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR1-H, FR-H, FR-H and FR4-H with the sequences shown in SEQ ID NO. 5-8 in sequence.

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

CDR-VH1: G-F-X1-F-S-S-X2-T-X3-S, wherein: x3 is F;

CDR-VH2: T-X1-S-S-G-G-S-Y-X2-Y-Y-P-D-S-X3-K-G, wherein: x2 is S;

CDR-VH3: T-X1-H-X2-T-X3-A-S-Y, wherein: x1 is K;

CDR-VL1: R-A-S-X1-N-X2-Y-T-S-X3-A, wherein: x2 is L;

CDR-VL2: Y-X1-A-T-N-X2-A-D, wherein: x1 is G;

CDR-VL3: Q-X1-F-W-X2-T-P-W-T, wherein: x2 is A;

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

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

7. The anti-influenza b virus antibody or functional fragment thereof of claim 5, wherein the constant region is of bovine, equine, porcine, rat, mouse, ovine, caprine, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human species.

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

9. The anti-influenza b virus antibody or functional fragment thereof of claim 7, wherein the species source of the constant region is turkey or turkey.

10. The anti-influenza b virus antibody or functional fragment thereof of claim 7, wherein the constant region is derived from a mouse.

11. The anti-influenza b virus antibody or functional fragment thereof of claim 10, 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.

12. The anti-influenza b virus antibody or functional fragment thereof of any one of claims 1~4, wherein said functional fragment is selected from the group consisting of F (ab') ₂ Any one of Fab', fab, fv and scFv.

13. A reagent or kit for detecting influenza b virus, comprising the antibody or functional fragment thereof according to any one of claims 1 to 12.

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

15. The reagent or kit according to claim 14, wherein the detectable label is selected from the group consisting of fluorescent dyes, enzymes catalyzing the development of a substrate, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

16. The reagent or the kit according to claim 15, wherein the fluorescent dye is selected from the group consisting of 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.

17. The reagent or kit of claim 15, 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.

18. The reagent or kit according to claim 15, 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。

19. the reagent or kit according to claim 15, 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.

20. The reagent or kit according to claim 15, wherein the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids.

21. The reagent or kit of claim 20, wherein the nanoparticle comprises: at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

22. The reagent or kit of claim 20, wherein the colloid is selected from the group consisting of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microspheres.

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

24. A vector comprising a nucleic acid molecule encoding the antibody or functional fragment thereof of any one of claims 1-12.

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

26. A method of producing an antibody or functional fragment thereof according to any one of claims 1 to 12, comprising: culturing the recombinant cell of claim 25, and isolating and purifying the antibody or functional fragment thereof from the culture product.