CN114316032B

CN114316032B - Anti-influenza B virus antibody, reagent and kit for detecting influenza B virus

Info

Publication number: CN114316032B
Application number: CN202011029335.8A
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: CN114316032A

Abstract

The invention discloses an anti-influenza B virus antibody, a reagent and a kit for detecting influenza B virus, and relates to the technical field of antibodies. The anti-influenza B virus antibody disclosed by the invention 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 good affinity to influenza B virus antigen, and the antibody has good sensitivity and specificity when used for detecting influenza B virus, thereby providing a new antibody selection for detecting influenza B virus.

Description

Anti-influenza B virus antibody, reagent and kit for detecting influenza B virus

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-influenza B virus antibody, a reagent for detecting influenza B virus 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 B is an influenza caused by influenza B virus, and is characterized by sudden onset of illness, aversion to cold, fever, body temperature rising to peak within hours to 24 hours, 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, congestion at the outer canthus of conjunctiva, congestion of pharynx, 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 easily converted 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 in 2017 to 1 month in 2018 show that the incidence of influenza b virus is on the rise. Therefore, early and rapid screening of influenza virus is important.

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 the 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 virus 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 reagent and a kit for detecting influenza B virus, the anti-influenza B virus antibody provided by the invention has better affinity to influenza B virus antigen, and the antibody has better sensitivity and specificity for detecting the influenza B virus, thereby providing a new antibody selection for detecting the influenza B virus.

The invention is realized by the following steps:

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

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

CDR-VH3: A-X1-W-X2-N-P-X3-W-Y, wherein: x1 is K or R; x2 is A or G; x3 is L, V or I;

CDR-VL1: K-X1-S-Q-D-X2-S-T-T-X3-A, wherein: x1 is G or A; x2 is I, V or L; x3 is I, V or L;

CDR-VL2: S-A-S-X1-R-X2-T, wherein: x1 is Q, R or N; x2 is Y or S;

CDR-VL3: Q-X1-H-X2-S-X3-P-Y, wherein: x1 is H or Q; x2 is S or Y; x3 is L, V or I.

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

In alternative embodiments, in CDR-VH1, X1 is Y; in CDR-VH2, X1 is I; in CDR-VH3, X2 is G; in CDR-VL1, X1 is A; in CDR-VL2, X2 is Y; in CDR-VL3, X1 is Q.

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

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

In an alternative embodiment, in CDR-VH1, X3 is L.

In an alternative embodiment, in CDR-VH1, X3 is V.

In an alternative embodiment, in CDR-VH1, X3 is I.

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

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

In an alternative embodiment, in CDR-VH2, X3 is N.

In an alternative embodiment, in CDR-VH2, X3 is Q.

In an alternative embodiment, in CDR-VH2, X3 is E.

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

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

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

In an alternative embodiment, in CDR-VH3, X1 is K.

In an alternative embodiment, in CDR-VH3, X1 is R.

In an alternative embodiment, in CDR-VH3, X3 is L.

In an alternative embodiment, in CDR-VH3, X3 is V.

In an alternative embodiment, in CDR-VH3, X3 is I.

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

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

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

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, X1 is Q.

In an alternative embodiment, in CDR-VL2, X1 is R.

In an alternative embodiment, in CDR-VL2, X1 is N.

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

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

In an alternative embodiment, in CDR-VL3, X3 is L.

In an alternative embodiment, in CDR-VL3, X3 is V.

In an alternative embodiment, in CDR-VL3, X3 is I.

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 1-70:

in alternative embodiments, the antibody or functional fragment thereof and influenza b virus are in K _D ≤1.49×10 ^-7 Affinity binding of mol/L, preferably, K _D ≤3.54×10 ^-8 mol/L。

In an alternative embodiment, K _D ≤1×10 ^-7 mol/L、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, 4.14 × 10 ^-9 mol/L≤K _D ≤1.49×10 ^-7 mol/L。

In an alternative embodiment, 4.14 × 10 ^-9 mol/L≤K _D ≤3.54×10 ^-8 mol/L。

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

In an alternative embodiment of the method of the invention,

in CDR-VH1, X1 is F;

in CDR-VH2, X1 is L;

in CDR-VH3, X2 is A;

in CDR-VL1, X1 is G;

in CDR-VL2, X2 is S;

in CDR-VL3, X1 is H.

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 71-81:

in alternative embodiments, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, as shown in sequence in SEQ ID NOS: 1-4, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, as shown in sequence 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 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 directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target 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 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 metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

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

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 required 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 those skilled in the art can also obtain other related drawings based on 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: a Laboratory Manual, second edition (Sambrook et al, 1989); synthesis of oligonucleotides (oligo Synthesis) (m.j. gate eds., 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; 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

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNAamplification 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 done by Invitrogen.

1 construction of recombinant plasmid

(1) Antibody Gene preparation

mRNA is extracted from a hybridoma cell strain (6B 11) 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, the Heavy Chain and Light Chain genes are respectively taken for cloning after colonies are grown, and each 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 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 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 354bp, 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 Heavy Chain and Light Chain gene fragments are respectively subjected to double digestion by HindIII/EcoRI, the 3.4A vector is subjected to double 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 screening

(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 and 700 mu L of cells are mixed, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.

Coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, each well is 100 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, patted dry; adding diluted cell supernatant at a concentration of 100 μ L/well at 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing, wherein each hole is 120 mu L, the temperature is 37 ℃, and the time is 1h;throwing off liquid in the plate, patting dry, adding diluted influenza B virus antigen 100 mu L per hole, 37 ℃,40min; washing with the washing solution for 5 times, and drying; adding monoclonal antibody labeled with HRP for resisting influenza B virus, wherein each well is 100 μ L at 37 deg.C for 30min; adding a developing solution A (50 muL/hole), adding a developing solution B (50 muL/hole), and keeping 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 results show that the reaction OD after the cell supernatant is diluted 1000 times is still larger than 1.0, 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: 50 mu L of Buffer, 100 mu g/tube of DNA, 10 mu L of PuvI enzyme and sterile water are supplemented to 500 mu L, and the mixture is subjected to enzyme digestion in water bath 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 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water when ethanol is completely volatilized, and finally measuring the concentration.

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

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 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 cell recovery, the cells were first cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at 100% volume, and placed in a shaker at a rotation speed of 120r/min, a temperature of 37 ℃ and a carbon dioxide content of 8%. Culturing for 72h, inoculating and expanding at an inoculation density of 50 ten thousand cells/ml, the expanding volume being calculated according to the production requirements, the medium being 100% Dynamis medium. Then carrying out propagation 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 electrophoretogram showed two bands, 1 of which Mr was 50KD (heavy chain, SEQ ID NO: 14) and the other Mr was 28KD (light chain, SEQ ID NO: 13), as shown in FIG. 1 below, after the reducing SDS-PAGE.

Example 2

Detection of antibody Performance

(1) EXAMPLE 1 Activity detection 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(X1)-T-F-A(X2)-S-Y-F-L(X3)-H；

CDR-VH2：Y-L(X1)-N-I(X2)-Y-E(X3)-A-G-T-K-Y-T-Q(X4)-K-F-K-G；

CDR-VH3：A-K(X1)-W-A(X2)-N-P-V(X3)-W-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：K-G(X1)-S-Q-D-I(X2)-S-T-T-L(X3)-A；

CDR-VL2：S-A-S-Q(X1)-R-S(X2)-T；

CDR-VL3：Q-H(X1)-H-Y(X2)-S-L(X3)-P-Y。

based on the anti-influenza b virus antibody (WT) of example 1, mutations were made in the complementarity determining regions at sites involved in 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 1 mu g/ml goat anti-mouse IgG for coating a micropore plate, wherein each micropore is 100 mu l, 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 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, 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 antibodies and mutants thereof

Antibody concentration (ng/ml)	50	25	12.5	6.250	1.563	0
							WT	2.087	1.344	0.752	0.310	0.169	0.092
Mutation 1	2.221	1.585	0.905	0.554	0.218	0.056
							Mutation 2	2.015	1.575	0.962	0.509	0.202	0.042
Mutation 3	2.115	1.584	0.977	0.534	0.233	0.054
							Mutation 4	2.212	1.531	0.931	0.582	0.268	0.051
Mutation 5	0.623	0.465	0.053	-	-	-
							Mutation 6	0.683	0.375	0.047	-	-	-

As can be seen from the data in table 2, WT, mutant 1 to mutant 4 antibodies had higher binding activity to influenza b virus antigen, with the highest binding activity of mutant 1.

(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: equilibration for 60s in buffer 1 (PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69 GLY solution and buffer 3, and data output. The results are shown in Table 4 below. K is _D Indicates the equilibrium dissociation constant, i.e., affinity, in mol/L.

Table 4 affinity assay data

As can be seen from the data in Table 4, the mutant 1 and the series of mutants thereof have better affinity to the influenza B virus antigen, which shows that on the basis of the mutant 1, the antibodies obtained by mutation according to the mutation mode in Table 3 have excellent affinity.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, wherein 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

	K _D (M)
		WT	1.02E-07
WT1	1.49E-07
		WT2	8.12E-08
WT3	6.70E-08
		WT4	1.20E-07
WT5	8.69E-08
		WT6	9.49E-08
WT7	1.10E-07
		WT8	1.16E-07
WT9	9.89E-08
		WT10	1.35E-07

As can be seen from the data in Table 6, WT and its mutant also had good affinity for influenza B virus antigen.

(3) Naked antibody stability assessment

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

TABLE 7

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: 6% methanol, 0.01M PBS buffer solution with pH7.22M as coating buffer solution, filtering with 0.22 μm membrane, standing at 4 deg.C for use, and validity period 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 and 60ml of methanol, and the volume of double-distilled deionized water is up to 1000ml.

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

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

(a) Preparation of the solution

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

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

(3) Preparation of 0.1M potassium carbonate: prepared by double distilled deionized water, filtered by a 0.22 mu m membrane, and placed at 4 ℃ for standby, and the validity period is four months. 1000ml0.1M potassium carbonate solution formula: 13.8g of potassium carbonate; double distilled deionized water to 1000ml.

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

(5) Preparation of a marking washing preservation solution: 2% Bovine Serum Albumin (BSA), 0.05% sodium azide (NaN) ₃ ) 0.01M PBS solution with pH7.2, 0.22 mu membrane filtration, and standing at 4 ℃ for use, and the validity period is four months. 1000ml of marked washing preservation solution formula: 20g BSA,0.5g NaN3, 0.01M pH7.2PBS solution to 1000ml.

(b) And (3) preparing colloidal gold.

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

(c) Preparing colloidal gold labeled antibody.

The pH value of the colloidal gold is adjusted to 8.2 by 0.1M potassium carbonate, the antibodies in the table 3 and the table 5 of the above examples are respectively added into the colloidal gold according to 8-10 mug of the antibodies per ml, the mixture is uniformly mixed for 30min by a magnetic stirrer, and BSA is added into the mixture under stirring until the final concentration is 1 percent and the mixture is kept stand 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.

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

(b) Preparation of gold label pad

Soaking the gold label pad in the sealing solution for 30min, and drying at 37 deg.C. Then, the prepared gold-labeled antibody is evenly spread on a gold-labeled pad, each milliliter of the solution is spread with 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. Packaging every ten small strips with one bag, adding a drying agent, and carrying out 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 red precipitation lines simultaneously. If the sample does not contain the influenza B virus antigen, when the influenza B virus antibody labeled by the colloidal gold which is not combined with the influenza B virus antigen reaches the detection line, a compound of the influenza B virus antibody-the influenza B virus antigen-the colloidal gold labeled-the influenza B virus antibody is not formed, and the influenza B virus antibody compound labeled by the colloidal gold which is not combined with the influenza B virus 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 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

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

<120> anti-influenza B virus antibody, reagent and kit for detecting influenza B virus

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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ile Ile Thr Cys

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Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

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Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Thr Ile Ser Ser Val Gln Thr Glu Asp Leu Ala Val Tyr Phe Cys

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

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

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Val Lys Met Ser Cys Lys Ala Ser

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Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly

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Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

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

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Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

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His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

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Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

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

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

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Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

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Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

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Ser Pro Gly

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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Gln Arg Ser Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Thr

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Glu Asp Leu Ala Val Tyr Phe Cys Gln His His Tyr Ser Leu Pro Tyr

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

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Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Ser Ser Thr Ala Tyr Met

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Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

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Lys Trp Ala Asn Pro Val Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

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Thr Val Thr Val Ser Ser

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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Gln Arg Ser Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Thr

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Glu Asp Leu Ala Val Tyr Phe Cys Gln His His Tyr Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

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

115 120 125

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

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

145 150 155 160

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

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

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Phe Asn Arg Asn Glu Cys

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Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Ala Ser Tyr Phe

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Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly

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Tyr Leu Asn Ile Tyr Glu Ala Gly Thr Lys Tyr Thr Gln Lys Phe Lys

50 55 60

Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Ser Ser Thr Ala Tyr Met

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Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

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Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

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

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

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

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Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

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

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

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Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

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Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

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Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

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Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu

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Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

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His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

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Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

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Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe

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Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

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Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

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Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

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Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

Claims

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

CDR-VH1: G-X1-T-F-X2-S-Y-F-X3-H, wherein: x1 is Y;

CDR-VH2: Y-X1-N-X2-Y-X3-A-G-T-K-Y-T-X4-K-F-K-G, wherein: x1 is I;

CDR-VH3: A-X1-W-X2-N-P-X3-W-Y, wherein: x2 is G;

CDR-VL1: K-X1-S-Q-D-X2-S-T-T-X3-A, wherein: x1 is A;

CDR-VL2: S-A-S-X1-R-X2-T, wherein: x2 is Y;

CDR-VL3: Q-X1-H-X2-S-X3-P-Y, wherein: x1 is Q;

each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-70:

2. an antibody or functional fragment thereof against influenza b virus, comprising the following complementarity determining regions: CDR-VH1: G-X1-T-F-X2-S-Y-F-X3-H, wherein: x1 is F;

CDR-VH2: Y-X1-N-X2-Y-X3-A-G-T-K-Y-T-X4-K-F-K-G, wherein: x1 is L;

CDR-VH3: A-X1-W-X2-N-P-X3-W-Y, wherein: x2 is A;

CDR-VL1: K-X1-S-Q-D-X2-S-T-T-X3-A, wherein: x1 is G;

CDR-VL2: S-A-S-X1-R-X2-T, wherein: x2 is S;

CDR-VL3: Q-X1-H-X2-S-X3-P-Y, wherein: x1 is H; each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 71-81:

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

CDR-VH1: G-X1-T-F-X2-S-Y-F-X3-H, wherein: x1 is Y;

CDR-VH2: Y-X1-N-X2-Y-X3-A-G-T-K-Y-T-X4-K-F-K-G, wherein: x1 is I;

CDR-VH3: A-X1-W-X2-N-P-X3-W-Y, wherein: x2 is G;

CDR-VL1: K-X1-S-Q-D-X2-S-T-T-X3-A, wherein: x1 is A;

CDR-VL2: S-A-S-X1-R-X2-T, wherein: x2 is Y;

CDR-VL3: Q-X1-H-X2-S-X3-P-Y, wherein: x1 is Q;

and the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L with the sequences shown as SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H with the sequences shown as SEQ ID NO. 5-8 in sequence.

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

CDR-VH1: G-X1-T-F-X2-S-Y-F-X3-H, wherein: x1 is F;

CDR-VH2: Y-X1-N-X2-Y-X3-A-G-T-K-Y-T-X4-K-F-K-G, wherein: x1 is L;

CDR-VH3: A-X1-W-X2-N-P-X3-W-Y, wherein: x2 is A;

CDR-VL1: K-X1-S-Q-D-X2-S-T-T-X3-A, wherein: x1 is G;

CDR-VL2: S-A-S-X1-R-X2-T, wherein: x2 is S;

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

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

8. The anti-influenza b virus antibody or functional fragment thereof of claim 5, wherein the species source of the constant region is a bovine.

9. The anti-influenza b virus antibody or functional fragment thereof of claim 5, wherein the species source of the constant region is a chicken fighting or turkey.

10. The anti-influenza b virus antibody or functional fragment thereof of claim 5, 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 the functional fragment thereof according to any one of claims 1 to 4, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.

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

15. The reagent or kit according to claim 13, 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 kit of claim 15, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof.

17. The reagent or the kit according to claim 16, wherein the fluorescein-based dye and the derivative thereof are selected from rhodamine-based dyes and derivatives thereof, cy-series dyes and derivatives thereof, and Alexa-series dyes and derivatives thereof.

18. The reagent or kit according to claim 15, wherein the fluorescent dye is selected from the group consisting of protein dyes and derivatives thereof.

19. 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.

20. 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。

21. 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.

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

23. The reagent or kit of claim 22, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.

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

25. The reagent or kit according to claim 24, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.

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

27. A recombinant cell comprising the vector of claim 26.

28. 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 27, and isolating and purifying the antibody or functional fragment thereof from the culture product.