CN114075278B - anti-Flu-A antibody, preparation method thereof and detection kit - Google Patents

anti-Flu-A antibody, preparation method thereof and detection kit Download PDF

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CN114075278B
CN114075278B CN202010833571.9A CN202010833571A CN114075278B CN 114075278 B CN114075278 B CN 114075278B CN 202010833571 A CN202010833571 A CN 202010833571A CN 114075278 B CN114075278 B CN 114075278B
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CN114075278A (en
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崔鹏
何志强
孟媛
钟冬梅
唐丽娜
何雯雯
罗沛
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Dongguan Pengzhi Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Abstract

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

Description

anti-Flu-A antibody, preparation method thereof and detection kit
Technical Field
The invention relates to the technical field of antibodies, and particularly relates to an anti-Flu-A 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 a viruses (Flu-a) are successfully isolated in 1933, antigens of the Influenza a viruses are easy to change, and the Influenza a viruses can be further divided into subtypes such as H1N1, H3N2, H5N1, H7N9 (H represents hemagglutinin of Influenza virus and N represents neuraminidase of Influenza virus), which cause pandemics many times all over the world, and each year has a peak. The degree of the influenza A virus infection is related to personal immunity, typical symptoms mainly comprise chilliness, persistent high fever and headache, and general symptoms such as sore throat, cough and nasal obstruction, general aching pain, hypodynamia and the like are caused. The literature reports a positive detection rate of 20-40% in the epidemic season and 2-20% in the non-epidemic season. The persistent epidemic of influenza a virus brings great interference and pressure to the public epidemic prevention system of people's health, life and society, and it has become one of the main research objects of epidemiology.
The detection method aiming at the influenza A virus in the market at present mainly comprises a fluorescence PCR method, an immunization method and virus separation culture identification. The fluorescence PCR method is to carry out real-time detection on the PCR process through a fluorescence signal in the PCR amplification process for the purpose of qualitative or quantitative detection, and is a gold standard for pathogen detection; the immunization method is to detect target protein by specific binding of antigen and antibody; the virus isolation and culture method is usually chicken embryo inoculation, animal inoculation, tissue (cell) culture and the like, and the generated results are observed and analyzed. Although the fluorescence PCR method has better sensitivity and specificity, the detection window period is shorter, the time is won for early diagnosis and early treatment of epidemic diseases, reduction of mortality and control of epidemic situations, and the fluorescence PCR method can be used as a gold standard for diagnosis. However, the method has high requirements on detection personnel, needs professional skill training, and can be used for diagnosis and detection only by using professional instruments and equipment in qualified professional laboratories, so the method is not suitable for quick diagnosis of clinical or epidemic disease monitoring lines. The virus isolation culture identification has long time consumption, high environmental requirement, large infection risk of operators, poor culture effect and very limited application in the aspects of clinical diagnosis, epidemic disease monitoring and the like. The immunoassay detection reagent aims at antigen or antibody in a sample, has high detection speed and high accuracy, has low requirements on laboratories and operators, is generally suitable for primary screening of clinical laboratories of hospitals, disease control system laboratories and the like, and has very important functions on initial detection of influenza A, successful outbreak control in hospitals and communities and guidance of treatment.
Currently, immunodiagnostic reagent products for influenza a on the market mainly include enzyme-linked immunosorbent assay (ELISA method), colloidal gold immunochromatography, such as influenza a virus antigen detection reagent of guangzhou boil (colloidal gold method), R & D influenza a ELISA kit, and the like. In the above diagnostic reagent products, specific antibodies against influenza a virus are required, and the specific antibodies against influenza a virus on the market have certain defects in specificity and sensitivity.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an anti-Flu-A antibody, a preparation method thereof and a detection kit, wherein the anti-Flu-A antibody has better affinity to an influenza A virus (Flu-A) antigen, has better sensitivity and specificity when used for detecting the influenza A virus, and provides new antibody selection for detecting the influenza A virus.
The invention is realized in the following way:
in one aspect, the invention provides an anti-Flu-A antibody or functional fragment thereof, said antibody or functional fragment thereof having the following complementarity determining regions:
CDR-VH1: G-X1-S-F-X2-G-Y-Y-X3-H; wherein: x1 is Y or F; x2 is T or S; x3 is L, V or I;
CDR-VH2: R-X1-N-P-Y-X2-G-X3-T-X4-Y-N-Q-D-F-K-G; wherein: x1 is L, V or I; x2 is N or D; x3 is S or G; x4 is S or T;
CDR-VH3: A-R-X1-S-S-X2-D-A-M; wherein: x1 is F or Y; x2 is L, V or I;
CDR-VL1: T-X1-S-S-S-V-X2-S-S-Y-X3-H; wherein: x1 is A or G; x2 is I, V or L; x3 is I, V or L;
CDR-VL2: S-X1-S-X2-X3-A-S; wherein: x1 is T or S; x2 is Q, N or K; x3 is I or L;
CDR-VL3: X1-Q-X2-H-R-S-P; wherein: x1 is H or Q; x2 is W, F or Y.
The anti-Flu-A 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 the influenza A virus antigen and has better affinity to the influenza A virus antigen, and the antibody or the functional fragment thereof has better specificity and sensitivity when used for detecting the influenza A virus.
In an alternative embodiment of the method of the present invention,
in CDR-VH1, X2 is T;
in CDR-VH2, X3 is S;
in CDR-VH3, X1 is F;
in CDR-VL1, X1 is A;
in CDR-VL2, X1 is T;
in CDR-VL3, X1 is H.
In an alternative embodiment, in CDR-VH1, X1 is Y.
In an alternative embodiment, in CDR-VH1, X1 is F.
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, X1 is L.
In an alternative embodiment, in CDR-VH2, X1 is V.
In an alternative embodiment, in CDR-VH2, X1 is I.
In an alternative embodiment, in CDR-VH2, X2 is N.
In an alternative embodiment, in CDR-VH2, X2 is D.
In an alternative embodiment, in CDR-VH2, X4 is S.
In an alternative embodiment, in CDR-VH2, X4 is T.
In an alternative embodiment, in CDR-VH3, X2 is L.
In an alternative embodiment, in CDR-VH3, X2 is V.
In an alternative embodiment, in CDR-VH3, X2 is I.
In an alternative embodiment, in CDR-VL1, X2 is 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 L.
In an alternative embodiment, in CDR-VL1, X3 is V.
In an alternative embodiment, in CDR-VL1, X3 is I.
In an alternative embodiment, in CDR-VL2, X2 is Q.
In an alternative embodiment, in CDR-VL2, X2 is N.
In an alternative embodiment, in CDR-VL2, X2 is K.
In an alternative embodiment, in CDR-VL2, X3 is I.
In an alternative embodiment, in CDR-VL2, X3 is L.
In an alternative embodiment, in CDR-VL3, X2 is W.
In an alternative embodiment, in CDR-VL3, X2 is Y.
In an alternative embodiment, in CDR-VL3, X2 is F.
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 1-48:
Figure BDA0002638864920000031
Figure BDA0002638864920000041
in alternative embodiments, the antibody or functional fragment thereof binds influenza a virus with K D ≤8.74×10 -8 Affinity binding in mol/L, preferably, K D ≤8.44×10 -9 mol/L。
In an alternative embodiment, 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、K D ≤1×10 -8 mol/L、K D ≤9×10 -9 mol/L、K D ≤8×10 -9 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, or K D ≤2×10 -9 mol/L。
In an alternative embodiment, 2.67 × 10 -9 mol/L≤K D ≤8.44×10 -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 present invention,
in CDR-VH1, X2 is S;
in CDR-VH2, X3 is G;
in CDR-VH3, X1 is Y;
in CDR-VL1, X1 is G;
in CDR-VL2, X1 is S;
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 49-55:
Figure BDA0002638864920000042
in alternative embodiments, the antibodies comprise light chain framework regions FR1-L, FR2-L, FR-L and FR4-L, having the sequences shown in SEQ ID NOS 1-4, in order, and/or heavy chain framework regions FR1-H, FR-H, FR-H and FR4-H, having the sequences shown in SEQ ID NOS 5-8, in order.
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 disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.
Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.
In another aspect, the present invention provides a reagent or a kit for detecting Flu-A, which comprises the antibody or the functional fragment thereof as described in 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 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 functional fragment thereof.
In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the present invention provides a recombinant cell comprising the vector described above.
In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing the recombinant cell as described above, and separating and purifying the antibody or functional fragment thereof from the culture product.
Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by separating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above, and this is within the scope of the present invention, regardless of the technique used to prepare the antibody or its functional fragment.
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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 a result of reducing SDS-PAGE of the anti-Flu-A 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
1 construction of recombinant plasmid
(1) Antibody Gene preparation
mRNA is extracted from a hybridoma cell strain secreting anti-influenza A antigen antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, and after colonies grow out, the Heavy Chain and Light Chain genes are cloned and respectively sent to a gene sequencing company for sequencing by 4 clones.
(2) Sequence analysis of antibody variable region genes
Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 327bp, 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
Figure BDA0002638864920000071
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 enzyme cutting sites and protective basic groups, and 0.73KB Light Chai is amplified by a PCR amplification methodn gene fragment and 1.42kb Heavy Chain gene fragment.
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 cell, 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 influenza A antigen to 3. Mu.g/ml, 100. Mu.L per well, overnight at 4 ℃; the next day, washing liquid (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 30min; washing with the washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding color development liquid A (50 muL/hole, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), adding color development liquid B (50 muL/hole, containing citric acid + EDTA & 2Na + TMB + concentrated HCl), 10min; adding stop solution (50. Mu.L/well, EDTA-2 Na + concentrated H) 2 SO 4 ) (ii) a 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 activity on the influenza A antigen.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L,37 ℃ water bath enzyme digestion 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 sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expanding culture
After the cells 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 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 the electrophoretogram shown in FIG. 1 shows two bands after reducing SDS-PAGE, 1 band having an Mr of 50KD (heavy chain, SEQ ID NO: 14) and the other band having an Mr of 28KD (light chain, SEQ ID NO: 13).
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)-S-F-S(X2)-G-Y-Y-L(X3)-H；
CDR-VH2：R-I(X1)-N-P-Y-N(X2)-G-G(X3)-T-T(X4)-Y-N-Q-D-F-K-G；
CDR-VH3：A-R-Y(X1)-S-S-V(X2)-D-A-M；
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：T-G(X1)-S-S-S-V-L(X2)-S-S-Y-L(X3)-H；
CDR-VL2：S-S(X1)-S-N(X2)-L(X3)-A-S；
CDR-VL3：Q(X1)-Q-W(X2)-H-R-S-P。
based on the anti-Flu-A antibody (WT) of example 1, a mutation was made in the complementarity determining region at a site involved in the activity of the antibody, wherein X1, X2, X3, X4 were all mutated sites. See table 1 below.
TABLE 1 mutant sites associated with antibody Activity
Figure BDA0002638864920000081
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) cleaning for 2 times, and 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% of mouse negative blood, sealing, keeping the temperature at 37 ℃ for 1h, and keeping the volume at 120 mu l per hole; throwing off the liquid in the plate, patting dry, adding diluted influenza A antigen 100 mul per hole, 37 ℃,40min; washing with the washing solution for 5 times, and drying; an HRP-labeled influenza A conjugate monoclonal antibody (obtained from Fipeng BioLtd.) was added at 30min and 100. Mu.l/well at 37 ℃; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ 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 results are shown in Table 2 below.
TABLE 2 Activity data of WT antibody and its mutants
Antibody concentration (ng/ml) 5000 2500 1250 625 312.5 0
WT 1.732 1.388 0.946 0.754 0.535 0.060
Mutation 1 1.830 1.480 1.271 0.973 0.734 0.050
Mutation 2 1.849 1.404 1.242 0.946 0.733 0.066
Mutation 3 1.755 1.374 1.153 0.967 0.742 0.054
Mutation 4 1.816 1.361 1.147 0.909 0.619 0.063
Mutation 5 0.742 0.354 0.136 0.051 - -
As can be seen from the data in table 2, WT, as well as mutation 1-mutation 4, had stronger binding activity to influenza a antigen, with the binding activity of mutation 1 being superior.
(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
Figure BDA0002638864920000091
Figure BDA0002638864920000101
Affinity assay
Using AMC sensors, purified antibody was diluted to 10. Mu.g/ml with PBST and influenza A antigen was diluted with PBST (main component Na) 2 HPO 4 + NaCl + TW-20) dilution gradient: 20 μ g/ml, 6.66 μ g/ml, 2.22 μ g/ml, 0.74 μ g/ml, 0.24 μ g/ml, 0.082 μ g/ml, 0.027 μ g/ml, 0.0091 μ g/ml;
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 1.69GLY solution and buffer 3, and data output. The results are given in Table 4 below. K D Represents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate.
Table 4 affinity assay data
Figure BDA0002638864920000102
Figure BDA0002638864920000111
Figure BDA0002638864920000121
As can be seen from the data in Table 4, the mutant 1 antibody and the mutants thereof have higher affinity to influenza A antigen, which indicates that the antibody having higher affinity to influenza A antigen can be obtained by carrying out mutation in the mutation mode in Table 3 on the basis of the mutant 1.
(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
Figure BDA0002638864920000122
TABLE 6 affinity detection results of WT antibody and its mutants
K D (M)
WT 4.05E-08
WT1 3.52E-08
WT2 8.74E-08
WT3 3.28E-08
WT4 7.91E-08
WT5 5.80E-08
WT6 5.15E-08
As can be seen from the data in Table 6, both WT and its mutants also have good affinity for influenza A 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
Antibody concentration (ng/ml) 5000 625 0
Samples at 4 ℃ for 21 days 1.929 0.974 0.054
21 days samples at-80 deg.C 1.902 0.989 0.061
21 day samples at 37 deg.C 1.962 0.976 0.047
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, 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 the two lines are uniform. Drying at 37 ℃, and packaging for later use.
(2) Preparation of colloidal gold and gold-labeled monoclonal antibody
(a) Preparation of the solution
(1) Preparing chloroauric acid: dissolving chloroauric acid with double distilled deionized water to prepare 1% solution, standing at 4 deg.C for use, and having validity period of four months. 1000ml 1% chloroauric acid solution formula: 10g of chloroauric acid: double distilled deionized water to 1000ml.
(2) Preparation of trisodium citrate: dissolving sodium citrate with double distilled deionized water to obtain 1% solution, filtering with 0.22 μm membrane, standing for 4 deg.C, and storing for 1000ml.
(3) Preparation of 0.1M potassium carbonate: prepared by double-steaming 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 to 1000ml.
(5) Preparation of a marking washing preservation solution: 2% Bovine Serum Albumin (BSA), 0.05% sodium azide (NaN 3), 0.01M PBS solution (pH7.2), 0.22 μmembrane filtration, standing at 4 deg.C for use, and effective period of four months. 1000ml mark 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 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, another strain of mateable influenza A labeled antibody (obtained from Fipeng biological products, inc.) is added into the colloidal gold according to 8-10 mug antibody/ml, the mixture is mixed evenly for 30min by a magnetic stirrer, and BSA is added into the mixture under stirring until the final concentration is 1%, and the mixture is kept still 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: 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 solution is spread by 20 square centimeters, and the gold-labeled pad is frozen, dried, packaged and placed at 4 ℃ for later use.
(4) Preparation of test paper strip sample pad
(a) And (4) preparing a sealing liquid.
2% BSA, 0.1% TritionX-100, 0.05% NaN 3 0.01M PBS solution with pH7.2, and 0.22 μm membrane filtration, and standing at 4 degree for use with validity period of four months. 1000ml of sealing liquid formula: 20g BSA,0.5g NaN 3 1ml of TrtioX-100 and 0.01M PBS solution with pH7.2 are added to reach 1000ml.
(b) Preparation of sample pad.
Soaking the sample pad in sealing solution for 30min, oven drying at 37 deg.C, packaging, and standing at 4 deg.C.
(5) Assembly of test paper
Absorbent pads (available from Millipore corporation), nitrocellulose membranes, gold-labeled pads, and sample pads were placed on a non-absorbent support sheet and cut into 3mm wide strips. And packaging every ten small strips with one bag, adding a drying agent, and performing vacuum packaging to obtain the colloidal gold test paper for detecting the influenza A virus.
2 application of antibody in colloidal gold detection
The test strip assembled as described above was used to detect whether or not the test material contained influenza A virus antigen, thereby determining the activity of the antibody obtained in the above example on detection of influenza A virus antigen. Whether or not the test material contains an influenza A virus antigen is detected by a double antibody sandwich method. During detection, the influenza A virus antigen is combined with the influenza A antibody originally marked by the colloidal gold to form an influenza A antigen-colloidal gold mark-influenza A antibody compound, the influenza A antigen-colloidal gold mark-influenza A antibody compound swims forwards along the nitrocellulose membrane due to capillary action, and when the influenza A antigen-colloidal gold mark-influenza A antibody compound reaches a detection line, the influenza A antigen-colloidal gold mark-influenza A antibody compound is combined with the influenza A antibody obtained in the embodiment to form an influenza A antibody-influenza A antigen-colloidal gold mark-influenza A antibody compound, so that the influenza A antibody-influenza A antigen-colloidal gold mark-influenza A antibody compound is enriched on the detection line, and a red precipitation line is formed. The influenza A antigen-colloidal gold labeled-influenza A antibody compound which is not combined with the influenza A antibody on the detection line is captured by the goat anti-mouse IgG antibody through the detection line and is enriched on the quality control line to form a red precipitation line. And judging as a positive result when the detection line and the quality control line have the red precipitation line at the same time. If the sample does not contain the influenza A virus antigen, when the colloidal gold-labeled influenza A antibody which is not combined with the influenza A virus antigen reaches the detection line, a compound of the influenza A antibody-influenza A antigen-colloidal gold-labeled influenza A antibody cannot be formed, and the colloidal gold-labeled influenza A antibody compound which is not combined with the influenza A antigen passes through the detection line and is only enriched on the quality control line to form a red precipitation line, and the result is judged to be negative.
The results for the partial antibodies are shown in Table 8 below.
TABLE 8
Figure BDA0002638864920000141
Remarking: the gold mark color development is formed by adding a number C, and the smaller the number behind the number C is, the stronger the color development is, and the higher the activity is; higher numbers after C indicate weaker color development and lower activity; the sign with a "+" after the number is slightly stronger than the non-coloration by 0.5-1C, and the sign with a "-" after the number is slightly lower than the non-coloration by 0.5-1C. B indicates no activity.
As can be seen from the results in the table above, the antibody provided by the embodiment of the invention has good activity when being 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 Peng Zhi Biotech Co., ltd
<120> anti-Flu-A antibody, preparation method and detection kit thereof
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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
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440

Claims (25)

1. An anti-Flu-a antibody or functional fragment thereof, characterized in that said antibody or functional fragment thereof comprises the complementarity determining regions CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3:
CDR-VH1: G-X1-S-F-X2-G-Y-Y-X3-H; wherein: x2 is T;
CDR-VH2: R-X1-N-P-Y-X2-G-X3-T-X4-Y-N-Q-D-F-K-G; wherein: x3 is S;
CDR-VH3: A-R-X1-S-S-X2-D-A-M; wherein: x1 is F;
CDR-VL1: T-X1-S-S-S-V-X2-S-S-Y-X3-H; wherein: x1 is A;
CDR-VL2: S-X1-S-X2-X3-A-S; wherein: x1 is T;
CDR-VL3: X1-Q-X2-H-R-S-P; wherein: x1 is H;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-48:
CDR-VH1 X1/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2 CDR-VL1 X2/X3 CDR-VL2 X2/X3 CDR-VL3 X2 mutant combination 1 F/L I/N/T V L/L N/L W Combination of mutations 2 Y/L I/N/S V I/I N/I F Combination of mutations 3 F/V V/N/T I V/V K/L Y Combination of mutations 4 Y/V V/N/S I I/V K/I F Combination of mutations 5 F/I L/N/T L I/L Q/L Y Combination of mutations 6 Y/I L/N/S L L/V Q/I W Mutant combination 7 Y/I V/D/S I L/I K/I Y Combination of mutations 8 Y/V V/D/T I V/L Q/I W Combination of mutations 9 Y/L I/D/S L V/I N/I F Combination of mutations 10 F/I I/D/T L I/L K/L Y Combination of mutations 11 F/V L/D/S V V/L Q/L Y Combination of mutations 12 F/L L/D/T V L/L N/L F Mutant combinations 13 Y/I I/N/T L I/V Q/I F Combination of mutations 14 F/V V/D/S L V/V N/L W Combination of mutations 15 Y/L I/N/S V L/V K/L W Mutant combinations 16 F/I V/D/T V I/I N/I F Mutant combinations 17 Y/V V/N/T I V/I Q/L W Mutant combinations 18 F/L I/D/S I L/I K/I Y Combination of mutations 19 Y/V V/N/S V V/L N/L W Combination of mutations 20 Y/V I/D/T L L/I N/I F Mutant combination 21 F/L L/N/T I I/V K/L Y Mutant combination 22 F/L L/D/S L L/V K/I Y Combination of mutations 23 Y/I L/N/S I V/I Q/L Y Mutant combinations 24 Y/I L/D/T V I/L Q/I F Mutant combinations 25 F/V V/D/S I L/L N/I F Mutant combinations 26 F/V V/N/T V V/V K/I W Mutant combinations 27 Y/L I/D/S L I/I Q/I W Mutant combinations 28 Y/L I/N/T V I/V N/L W Mutant combinations 29 F/I L/D/S V I/L K/L F Combination of mutations 30 F/I L/N/T I L/V Q/L Y Combination of mutations 31 Y/I V/D/T I L/I K/I F Mutant combinations 32 Y/V V/N/S L V/L K/L Y Mutant combinations 33 Y/L L/D/T L V/I N/L W Mutant combinations 34 F/I L/N/S I L/L Q/I Y Combination of mutations 35 F/V I/D/T I I/I Q/L W Combination of mutations 36 F/L I/N/S L V/V N/I F Mutant combinations 37 Y/I I/N/T L I/L Q/I F Combination of mutations 38 F/V I/N/S V V/L N/L W Mutant combinations 39 Y/L V/N/T V L/L K/L Y Combination of mutations 40 F/I V/N/S L I/V N/I W Mutant combination 41 Y/V L/N/T L V/V Q/L F Combination of mutations 42 F/L L/N/S V L/V K/I Y Mutant combinations 43 F/L I/N/T V I/I K/I F Mutant combinations 44 Y/L V/D/S I V/I K/L Y Combination of mutations 45 F/V I/N/S I L/I N/L Y Mutant combinations 46 Y/V V/D/T V L/L Q/I F Mutant combinations 47 F/I V/N/T L I/I Q/L F Combination of mutations 48 Y/I I/D/S I V/V N/I W
。
2. An anti-Flu-a antibody or functional fragment thereof, characterized in that said antibody or functional fragment thereof comprises the complementarity determining regions CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3:
CDR-VH1: G-X1-S-F-X2-G-Y-Y-X3-H; wherein: x2 is S;
CDR-VH2: R-X1-N-P-Y-X2-G-X3-T-X4-Y-N-Q-D-F-K-G; wherein: x3 is G;
CDR-VH3: A-R-X1-S-S-X2-D-A-M; wherein: x1 is Y;
CDR-VL1: T-X1-S-S-S-V-X2-S-S-Y-X3-H; wherein: x1 is G;
CDR-VL2: S-X1-S-X2-X3-A-S; wherein: x1 is S;
CDR-VL3: X1-Q-X2-H-R-S-P; wherein: x1 is Q;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 49-55:
CDR-VH1 X1/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2 CDR-VL1 X2/X3 CDR-VL2 X2/X3 CDR-VL3 X2 combination of mutations 49 F/L I/N/T V L/L N/L W Mutant combinations 50 Y/V L/N/S L L/V Q/L Y Mutant combinations 51 F/I L/D/T V V/I K/L F Mutant combinations 52 F/L V/N/S I L/I Q/L Y Mutant combination 53 F/V I/N/T I I/L Q/I Y Mutant combinations 54 F/L L/N/T L V/I Q/I Y Mutant combinations 55 Y/I V/N/T I V/V K/L F
。
3. An anti-Flu-A antibody or a functional fragment thereof according to any one of claims 1 to 2, characterized in that said antibody comprises the light chain framework regions FR1-L, FR-L, FR-L and FR4-L, having the sequence in sequence as shown in SEQ ID NO. 1-4 and/or the heavy chain framework regions FR1-H, FR-H, FR-H and FR4-H, having the sequence in sequence as shown in SEQ ID NO. 5-8.
4. The anti-Flu-A antibody or functional fragment thereof according to any one of claims 1~2, wherein said antibody further comprises a constant region.
5. An anti-Flu-A antibody or a functional fragment thereof, according to claim 4, characterized in that said constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.
6. The anti-Flu-A antibody or functional fragment thereof according to claim 4, wherein the species of said constant region is of sheep, goat, cow, horse, pig, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human origin.
7. An anti-Flu-A antibody or a functional fragment thereof according to claim 6, characterized in that the species origin of said constant region is bovine.
8. The anti-Flu-A antibody or functional fragment thereof according to claim 6, characterized in that the species origin of said constant region is turkey or turkey.
9. An anti-Flu-A antibody or a functional fragment thereof according to claim 6, characterized in that said constant region is of mouse origin.
10. The anti-Flu-A antibody or functional fragment thereof according to claim 9, wherein the light chain constant region sequence of said constant region is as set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of said constant region is as set forth in SEQ ID NO. 10.
11. The anti-Flu-A antibody or functional fragment thereof according to claim 1~2, wherein said functional fragment is selected from the group consisting of F (ab') 2 Any one of Fab', fab, fv and scFv.
12. A reagent or kit for detecting Flu-a, characterized in that it comprises an antibody or functional fragment thereof according to any one of claims 1 to 11.
13. The reagent or kit according to claim 12, wherein the antibody or functional fragment thereof is labeled with a detectable label.
14. The reagent or kit of claim 13, wherein the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
15. The reagent or the kit according to claim 14, wherein the fluorescent dye is selected from the group consisting of fluorescein 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.
16. The reagent or kit of claim 14, wherein the enzyme that catalyzes the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.
17. The reagent or kit of claim 14, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
18. the reagent or kit according to claim 14, characterized in that said chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, loflunine and its derivatives and peroxyoxalate and its derivatives.
19. The reagent or kit according to claim 14, wherein the nanoparticle-based label is at least one selected from the group consisting of nanoparticles and colloids.
20. The reagent or kit of claim 19, wherein the nanoparticles comprise at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
21. The reagent or kit of claim 19, wherein the colloid is selected from the group consisting of latex, colloidal metal, colloidal selenium, disperse dye, dye-labeled microspheres.
22. The reagent or kit according to claim 21, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
23. An isolated nucleic acid molecule encoding the antibody or functional fragment thereof of any one of claims 1-11.
24. A recombinant cell comprising a vector comprising the nucleic acid molecule of claim 23.
25. A method of producing an antibody or functional fragment thereof according to any one of claims 1 to 11, comprising: culturing the recombinant cell of claim 24, and isolating and purifying the antibody or functional fragment thereof from the culture product.
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CN104892753B (en) * 2014-03-07 2019-11-29 神州细胞工程有限公司 A kind of antibody and application thereof for neutralizing people and infecting H7N9 influenza A virus
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KR101561664B1 (en) * 2014-12-02 2015-10-20 대한민국 Specific antibody for ha protein of a/h3n2 influenza virus, and hybridoma producing the same

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