CN117659180B

CN117659180B - Anti-novel coronavirus antibody or functional fragment thereof, reagent for detecting novel coronavirus and kit

Info

Publication number: CN117659180B
Application number: CN202211083230.XA
Authority: CN
Inventors: 刘春艳; 易嘉乐; 张翼
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2024-07-23
Anticipated expiration: 2042-09-06
Also published as: CN117659180A

Abstract

The invention discloses an antibody or a functional fragment thereof for resisting novel coronaviruses, a reagent and a kit for detecting the novel coronaviruses, and relates to the field of antibodies. The anti-novel coronavirus antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, has good reactivity to N protein of the novel coronavirus, and has good sensitivity and detection rate when used for detecting the novel coronavirus.

Description

Anti-novel coronavirus antibody or functional fragment thereof, reagent for detecting novel coronavirus and kit

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-novel coronavirus antibody or a functional fragment thereof, a reagent for detecting novel coronaviruses and a kit.

Background

Structural proteins of the novel coronavirus SARS-CoV-2 are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), which proteins comprise a plurality of epitopes. N protein and viral genome RNA are intertwined to form a viral nucleocapsid, which plays an important role in the synthesis process of viral RNA. Meanwhile, N protein is relatively conserved, the proportion of the N protein in structural proteins of viruses is the largest, and the organism can generate high-level antibodies against the N protein in early infection. Finally, the N protein 15 is an important marker protein of the novel coronavirus, and the antigen can be detected through an N protein monoclonal antibody by utilizing the principle of specific binding of the antigen and the antibody, so that the sample is directly proved to contain the novel coronavirus, and the detection of the novel coronavirus is realized.

Antibodies detected are largely classified into IgM and IgG classes. There is currently no systematic study of the generation and duration of these two classes of antibodies for the novel coronaviruses. In general, igM antibodies are produced early, once infected, and are produced rapidly, the maintenance time is short, the disappearance is rapid, and detection positivity in blood can reflect that the organism is in an acute infection state and can be used as an index of early infection. Compared with the nucleic acid detection method, the antibody 20 detection sample is serum or plasma, is less influenced by sample sampling, is favorable for early diagnosis and suspicious case elimination, and is rapid and convenient to detect and suitable for large-scale screening.

At present, nucleic acid detection and viral gene sequencing are mainly used as etiology diagnosis evidences, and false negative results can appear in the nucleic acid detection due to the influence of various factors such as sampling, operation, reagents and the like. In addition, nucleic acid detection has the disadvantages of high requirements on instruments, detection sites and environmental conditions, long detection time, low flux and the like. Therefore, it is highly desirable to develop a rapid and convenient detection kit for clinical detection, so as to block virus transmission as soon as possible. Thus, antibody detection kits become more important.

At present, SARS-CoV-2N protein monoclonal antibody products are few, and the performances are different.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an antibody or a functional fragment thereof for resisting a novel coronavirus, a reagent and a kit for detecting the novel coronavirus, wherein the antibody can specifically bind to N protein of the novel coronavirus, has higher reactivity to the N protein, and has better sensitivity and detection rate for detecting the novel coronavirus by using the antibody. The invention provides a richer antibody selection for the detection of novel coronaviruses.

The invention is realized in the following way:

In a first aspect, embodiments of the present invention provide an antibody or functional fragment thereof comprising any one of (a) to (c): (a) The amino acid sequences of HCDR 1-3 and LCDR 1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1-3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No. 4-6; (b) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8; (c) The amino acid sequence has a heavy chain variable region and a light chain variable region which are identical to the sequence shown in (b) by more than 80%, and comprises HCDR 1-HCDR 3 and LCDR 1-LCDR 3 of the sequence shown in (a).

In a second aspect, embodiments of the invention provide an antibody conjugate comprising an antibody or functional fragment thereof as described in the previous embodiments.

In a third aspect, embodiments of the invention provide a reagent or kit comprising an antibody or functional fragment thereof as described in the previous embodiments or an antibody conjugate as described in the previous embodiments.

In a fourth aspect, embodiments of the present invention provide a method of detecting a novel coronavirus comprising: mixing the antibody or the functional fragment thereof according to the previous embodiment with a sample to be detected, and contacting the antibody or the functional fragment thereof with the novel coronavirus in the sample to be detected to form an immune complex.

In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding an antibody or functional fragment thereof according to the previous embodiments.

In a sixth aspect, embodiments of the present invention provide a vector comprising the isolated nucleic acid of the previous embodiments.

In a seventh aspect, embodiments of the invention provide a cell comprising an isolated nucleic acid as described in the previous embodiments or a vector as described in the previous embodiments.

In an eighth aspect, embodiments of the present invention provide a method of preparing an antibody or functional fragment thereof according to the previous embodiments, comprising: the cells described in the previous examples were cultured.

Based on the present disclosure of the amino acid sequence of an antibody or functional fragment thereof, it is readily apparent to a person skilled in the art that the preparation of the antibody or functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), e.g., isolation and purification from a culture product of recombinant cells capable of recombinantly expressing an antibody or functional fragment thereof as described in any of the above, is within the scope of the present disclosure, irrespective of the technique used to prepare the antibody or functional fragment thereof.

The invention has the following beneficial effects:

The anti-novel coronavirus antibody disclosed by the invention comprises the heavy chain complementarity determining region and the light chain complementarity determining region, and provides an important raw material source for the detection of novel coronaviruses.

Drawings

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

FIG. 1 shows the result of reducing SDS-PAGE of the anti-novel coronavirus antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Embodiments of the present invention provide an antibody or functional fragment thereof comprising:

(a) The amino acid sequences of HCDR 1-3 and LCDR 1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1-3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No. 4-6;

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is denoted by HCDR, and the 3 CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted by LCDR, and the 3 CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. In the embodiment of the invention, the CDR regions are marked by adopting Kabat annotation standard, but the CDR regions marked by other methods also belong to the protection scope of the invention.

In another aspect, embodiments of the present invention also provide an antibody or functional fragment thereof, comprising:

(b) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

(c) The heavy chain variable region and the light chain variable region having an amino acid sequence having 80% or more identity to the sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a). The antibodies have improved activity.

In alternative embodiments, the antibody or functional fragment thereof comprises a heavy chain framework region in the heavy chain variable region shown in SEQ ID NO. 7 and a light chain framework region in the light chain variable region shown in SEQ ID NO. 8.

In alternative embodiments, the framework region amino acid sequence of the antibody or functional fragment thereof may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the framework region described above.

In alternative embodiments, the antibody or functional fragment thereof further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of any one of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, and human origin.

In an alternative embodiment, the constant region is of murine species origin.

In alternative embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO. 9 or has at least 80% identity thereto.

In alternative embodiments, the light chain constant region sequence is as shown in SEQ ID NO. 10 or has at least 80% identity thereto.

In particular, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above-described constant region (SEQ ID NO:9 or 10).

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

The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

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

In another aspect, embodiments of the present invention also provide an antibody or functional fragment thereof, comprising: a heavy chain with an amino acid sequence shown as SEQ ID NO. 11, and a light chain with an amino acid sequence shown as SEQ ID NO. 12.

In another aspect, embodiments of the invention also provide an antibody conjugate comprising an antibody or functional fragment thereof as described in the previous embodiments.

In alternative embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or functional fragment thereof.

In alternative embodiments, the antibody conjugate further comprises a label conjugated to the antibody or functional fragment thereof.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In an alternative embodiment, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection condition or the actual requirement, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

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

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine 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 colloids include, but are not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

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

In an alternative embodiment, the colloidal metal is colloidal gold.

In alternative embodiments, the antibody conjugate further comprises a solid support coupled to the antibody or functional fragment thereof. In an antibody conjugate, the antibody is conjugated to a solid support.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In an alternative embodiment, the solid support is a nitrocellulose membrane.

In another aspect, embodiments of the invention also provide a reagent or kit comprising an antibody or functional fragment thereof as described in any of the preceding embodiments or an antibody conjugate as described in any of the preceding embodiments.

In another aspect, embodiments of the present invention also provide a method for detecting a novel coronavirus antigen, comprising:

Mixing the antibody or functional fragment thereof according to any of the preceding embodiments or the antibody conjugate according to any of the preceding embodiments or the reagent or kit according to any of the preceding embodiments with a sample to be tested, and contacting the antibody or functional fragment thereof with a novel coronavirus antigen in the sample to be tested to form an immune complex.

In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the novel coronavirus.

On the other hand, the embodiment of the invention also provides application of the antibody or the functional fragment or the antibody conjugate or the reagent or the kit in any embodiment in detection of novel coronaviruses or preparation of products for detection of novel coronaviruses.

In another aspect, embodiments of the present invention also provide the use of an antibody or functional fragment of any of the embodiments described above or an antibody conjugate of any of the embodiments described above or a reagent or kit of any of the embodiments described above in the preparation of a product having at least one of the following uses, including: diagnosing or aiding in diagnosing a disease associated with a novel coronavirus infection, predicting or aiding in predicting at least one of the prognostic efficacy of a disease associated with a novel coronavirus infection.

In alternative embodiments, the novel coronavirus infection-related diseases include at least one of viral pneumonia, secondary bacterial pneumonia, myocarditis, and endocarditis.

In alternative embodiments, the product comprises a reagent or kit.

In another aspect, embodiments of the invention also provide an isolated nucleic acid encoding an antibody or functional fragment thereof according to any of the preceding embodiments.

In another aspect, embodiments of the invention also provide a vector comprising an isolated nucleic acid as described in any of the previous embodiments.

In another aspect, embodiments of the invention also provide a cell comprising an isolated nucleic acid as described in any of the previous embodiments or a vector as described in any of the previous embodiments.

In another aspect, embodiments of the present invention also provide a method of preparing an antibody or functional fragment thereof according to any of the previous embodiments, comprising: culturing the cells of any of the previous examples.

On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.

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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

EXAMPLE 1 preparation of 4F55 monoclonal antibody

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

1. Construction of recombinant plasmids

(1) Preparation of 4F55 monoclonal antibody Gene

MRNA is extracted from hybridoma cell strain secreting antibody against novel coronavirus N protein, DNA product is obtained by RT-PCR method, the product is inserted into pMD-18T vector after adding A reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

The gene sequence obtained by sequencing is placed in a KABAT antibody database for analysis, and VNTI 11.5.5 software is used for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct, wherein in the LIGHT CHAIN amplified gene fragment, the VL gene sequence is 342bp, belongs to VkII gene family, and the front side of the VL gene sequence is 57bp leader peptide sequence; the gene fragment amplified by HEAVY CHAIN primer pair has a VH gene sequence of 336bp, belongs to the VH1 gene family, and has a 57bp leader peptide sequence in front of the VH gene sequence.

(3) Construction of recombinant antibody expression plasmids

PcDNATM3.4vector is a constructed recombinant antibody eukaryotic expression vector, and the expression vector is introduced into HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and is named pcDNA3.4A expression vector, and is subsequently abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI digestion sites and protective bases, and a LIGHT CHAIN gene fragment of 0.72kb and a HEAVY CHAIN gene fragment of 1.41kb are amplified by a PCR amplification method.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.

2. Sample preparation of recombinant antibodies

Recovering HEK293 cells in advance, and subculturing to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml and the cell activity to be more than 95%; cells were washed by centrifugation and reconstituted with medium while the cell density was adjusted to 2.9X10 ⁶ cells/ml as a cell dilution. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding the transfection reagent diluent into the plasmid DNA diluent, uniformly mixing, standing at room temperature for 15min; the mixture is slowly added into cell dilution within 1min, after uniform mixing, sampling and counting are carried out, the activity of the transfected cells is recorded and observed, and the cells are placed in a constant temperature incubator at 35 ℃ for culture, the rotation speed 120rmp and the CO ₂ content of 8 percent, and the cells are centrifugally collected after 13 days. The supernatant was affinity purified using a proteona affinity column. 6. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and the electrophoresis pattern was shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain). The heavy chain and the light chain have the amino acid sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12 respectively.

EXAMPLE 2 Activity assay

Diluting SARS-CoV-2N protein antigen to 1 μg/ml with coating solution (main component NaHCO ₃) to coat microwell plate, 100 μl per well, and standing overnight at 4deg.C; the next day, the washing solution (main component Na ₂HPO₄ +NaCl) is washed 2 times and is patted dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted N protein monoclonal antibody into the mixture at the temperature of 37 ℃ for 30min at the concentration of 100 mu L/hole; washing with washing liquid for 5 times, and drying; adding a developing solution A (50 mu L/hole) and a developing solution B (50 mu L/hole) for 10min; adding a stop solution, 50 mu L/well; OD was read on the microplate reader at 450nm (reference 630 nm).

Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCL); stop solution (EDTA.2Na+ concentration H ₂SO₄).

TABLE 1 Activity data

Concentration (ng/ml)	5000	1000	500	250	125	0.00
							Control antibodies	0.902	0.709	0.405	0.216	0.103	0.024
4F55	1.165	0.962	0.623	0.385	0.206	0.029

Example 3 assessment of antibody stability

The antibody is placed at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, 7 days, 14 days and 21 days are taken for carrying out state observation, and activity detection is carried out on the 21 days, so that the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, the activity is not in a descending trend along with the increase of the examination temperature, and the stability of the expressed antibody is indicated. The following table shows the OD results of the enzyme-free activity assay for 21 days.

Table 2 stability data

Sample concentration (ng/ml)	500	250	0
				4 ℃,21 Days sample	0.652	0.395	0.021
Sample at-80℃for 21 days	0.643	0.372	0.025
				37 ℃ And 21 days of sample	0.661	0.389	0.023

Example 4 test Performance evaluation

The prepared antibody is used as a coating antibody, the other antibody which can be paired is used as a labeling antibody (purchased from Phpeng), an ELISA double-antibody sandwich detection method is adopted, 100 nucleic acid detection positive samples are used as test samples, and the detection rate reaches 97%.

The prepared antibody is used as a coating antibody, the other paired antibody is used as a labeled antibody (purchased from Phpeng), and an ELISA double-antibody sandwich detection method is adopted to detect mutant strains so as to judge whether the detection omission problem exists. The detection result shows that: mutants D103Y and P13L+R21K+G21Rr+G214C were also efficiently detected at concentrations ranging down to 50 pg/ml.

TABLE 3 Table 3

"+" Indicates detected, "-" indicates undetected

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The partial amino acid sequence related to the application is as follows:

Segment(s)	Sequence(s)	SEQ ID No:
			HCDR1	NYGMN	1
HCDR2	WINTYTGEPTYADDFKG	2
			HCDR3	KGNWDEENAMDY	3
LCDR1	RASENIYSNLA	4
			LCDR2	TATDLPD	5
LCDR3	QHFWGTPWT	6

Claims

1. An anti-SARS-CoV-2 antibody or an antigen binding fragment thereof, which is characterized in that the antibody or the antigen binding fragment thereof comprises HCDR 1-3 and LCDR 1-3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID No. 1-3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No. 4-6 in sequence.

2. An anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 7 and a light chain variable region having an amino acid sequence as shown in SEQ ID No. 8;

Or a heavy chain variable region and a light chain variable region which have an amino acid sequence with more than 80% of the sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8, and comprise HCDR 1-HCDR 3 and LCDR 1-LCDR 3 as defined in claim 1.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

7. The antibody or antigen-binding fragment thereof according to claim 4, wherein the constant region is of any one of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, and human origin.

8. The antibody or antigen-binding fragment thereof of claim 4, wherein the constant region is of murine species origin.

9. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region sequence is as shown in or has at least 80% identity to SEQ ID No. 9; the light chain constant region sequence is shown in SEQ ID NO. 10 or has at least 80% identity thereto.

10. An anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID No. 11 and a light chain having an amino acid sequence as shown in SEQ ID No. 12.

11. An antibody conjugate, characterized in that it consists of the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and a label or solid phase carrier conjugated thereto;

The label is selected from at least one of biotin or biotin derivatives, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels;

The enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxygenase.

12. The antibody conjugate of claim 11, wherein the label is colloidal gold.

13. A reagent or kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or an antibody conjugate according to any one of claims 11 to 12.

14. Use of the antibody or antigen binding fragment thereof according to any one of claims 1-10 for the preparation of a product for detecting SARS-CoV-2 antigen, comprising:

Mixing the antibody or the antigen binding fragment thereof according to any one of claims 1-10 with a sample to be detected, and enabling the antibody or the antigen binding fragment thereof to be in contact with SARS-CoV-2 antigen in the sample to be detected to form an immune complex.

15. The use of claim 14, wherein the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof.

16. The use of claim 14, wherein the immune complex further comprises a second antibody that binds to SARS-CoV-2.

17. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-10.

18. A vector comprising the nucleic acid of claim 17.

19. A cell comprising the nucleic acid of claim 17 or the vector of claim 18.

20. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-10, comprising: culturing the cell of claim 19.