CN116444656B - Antibody, reagent and method for identifying novel crown mutant antigen - Google Patents

Antibody, reagent and method for identifying novel crown mutant antigen Download PDF

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CN116444656B
CN116444656B CN202210021930.XA CN202210021930A CN116444656B CN 116444656 B CN116444656 B CN 116444656B CN 202210021930 A CN202210021930 A CN 202210021930A CN 116444656 B CN116444656 B CN 116444656B
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CN116444656A (en
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孟媛
李蔚芝
钟冬梅
游辉
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention discloses an antibody, a reagent and a method for identifying a novel crown mutant antigen. The antibody, the reagent or the method can be used for rapidly and accurately identifying the novel crown mutation type antigen, and has high detection efficiency and low cost.

Description

Antibody, reagent and method for identifying novel crown mutant antigen
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody, a reagent and a method for identifying a novel crown mutant antigen.
Background
Structural proteins of the novel coronavirus 2019-nCoV 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 is an important marker protein of the novel coronavirus, and the antigen can be detected through the 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 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.
A new coronavirus mutant was found in south Africa, 11.2021, and was designated Omicron by the world health organization as a variant of the new coronavirus B.1.1.529. Due to the characteristics of large virus amount and high transmission power. Therefore, early screening for this variant is extremely important. At present, few detection reagents for new crown mutant types, especially detection of omacron mutant strains, are available on the market.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims at providing an antibody, and provides an important raw material source for omacron mutant detection.
In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising the following complementarity determining regions:
HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;
HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;
HCDR3 comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs 3, 21, 32;
LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4 or 22;
LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5 or 23;
LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.
Further, the antibody or functional fragment thereof also has the following framework regions:
the HFR1 amino acid sequence is shown as SEQ ID NO. 7 or has at least 80% homology with the HFR1 amino acid sequence;
the HFR2 amino acid sequence is shown in any one of SEQ ID NO 8, 24 and 33 or has at least 80 percent of homology with the HFR2 amino acid sequence;
the HFR3 amino acid sequence is shown as SEQ ID NO 9 or 25 or has at least 80% homology with the same;
the HFR4 amino acid sequence is shown in SEQ ID NO 10 or 26 or has at least 80% homology with the HFR4 amino acid sequence;
the LFR1 amino acid sequence is shown as SEQ ID NO. 11 or has at least 80% homology with the same;
the LFR2 amino acid sequence is shown as SEQ ID NO. 12 or has at least 80% homology with the same;
The LFR3 amino acid sequence is shown as SEQ ID NO. 13 or has at least 80% homology with the same;
the LFR4 amino acid sequence is shown in SEQ ID NO. 14 or has at least 80% homology therewith.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR 2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, HFR1, LFR2, r3, LFR4 is the amino acid sequence of HFR1, HCDR2, HCDR3, LCDR 4, LFR2, LFR4 as described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody comprising a heavy chain comprising the heavy chain variable region described above and the heavy chain constant region described above and/or a light chain; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there are provided a nucleic acid, a vector, a recombinant cell, and a method for producing the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a reagent or kit for detecting a non-Omicron novel coronavirus or identifying a novel coronamutant, the reagent or kit comprising the above antibody or a functional fragment thereof or the above antibody conjugate.
In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a method for identifying a novel coronal mutant antigen, comprising performing immunodetection on a sample to be detected using antibody 1, antibody 2 and antibody 3, wherein said antibody 1 is capable of binding to M novel coronavirus antigens; the antibody 2 can be combined with M-1 novel coronavirus antigens except for an omacron mutant strain, and the antibody 2 is selected from the antibodies; the antibody 3 can be combined with M novel coronavirus antigens, and M is an integer greater than or equal to 2.
In order to achieve the above object, according to an eighth aspect of the present invention, there is provided an immunochromatographic test strip for identifying a novel crown mutant antigen, the immunochromatographic test strip comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad, wherein a detection line 1 and a detection line 2 are provided on the nitrocellulose membrane, the antibody 3 is provided on the conjugate pad, the antibody 1 is coated on the detection line 1, and the antibody 2 is coated on the detection line 2; the antibody 3 is labeled with a label.
In order to achieve the above object, according to a ninth aspect of the present invention, there is provided the use of the above antibody or a functional fragment thereof, an antibody conjugate, a method, a reagent or a test paper for detecting an Omicron mutant or for preparing an Omicron mutant detection product.
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 19E3 monoclonal antibody of example 1.
Detailed Description
The present invention provides an antibody or functional fragment thereof comprising the following complementarity determining regions:
HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;
HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;
HCDR3 comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs 3, 21, 32;
LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4 or 22;
LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5 or 23;
LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 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 contributing to the binding affinity of an antibody or antigen binding fragment to the antigen or epitope it recognizes. 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 represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and 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, and the 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 Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In alternative embodiments, the antibody or functional fragment thereof comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOS 1-3 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOS 4-6.
In alternative embodiments, the antibodies or functional fragments thereof include HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOS 1, 2, 21 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOS 22, 23, 6.
In alternative embodiments, the antibodies or functional fragments thereof include HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOS 1, 2, 32 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOS 4, 23, 6.
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.
In an alternative embodiment, the antibody or functional fragment thereof further has the following framework regions:
the HFR1 amino acid sequence is shown as SEQ ID NO. 7 or has at least 80% homology with the HFR1 amino acid sequence;
the HFR2 amino acid sequence is shown in any one of SEQ ID NO 8, 24 and 33 or has at least 80 percent of homology with the HFR2 amino acid sequence;
The HFR3 amino acid sequence is shown as SEQ ID NO 9 or 25 or has at least 80% homology with the same;
the HFR4 amino acid sequence is shown in SEQ ID NO 10 or 26 or has at least 80% homology with the HFR4 amino acid sequence;
the LFR1 amino acid sequence is shown as SEQ ID NO. 11 or has at least 80% homology with the same;
the LFR2 amino acid sequence is shown as SEQ ID NO. 12 or has at least 80% homology with the same;
the LFR3 amino acid sequence is shown as SEQ ID NO. 13 or has at least 80% homology with the same;
the LFR4 amino acid sequence is shown in SEQ ID NO. 14 or has at least 80% homology therewith.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided by the present invention 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% homology to the corresponding framework region described above.
In an alternative embodiment, the antibody or functional fragment thereof is in K D ≤10 -7 M affinity binding to non-Omicron novel coronavirus N antigen, e.g. in K D ≤4.90×10 -8 Affinity binding of M.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 10 or less -8 M affinity binding to non-Omicron novel coronavirus N antigen, e.g.with KD.ltoreq.4.02X10 -9 Affinity binding of M.
K D Reference is made to the method in the embodiment of the invention.
In another aspect, embodiments of the present invention provide an antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, HFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR 4.
In an alternative embodiment, the heavy chain variable region amino acid sequence is as set forth in any one of SEQ ID NOs 15, 27, 34;
in alternative embodiments, the light chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 16, 28, 35.
In alternative embodiments, the antibody 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 an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 17 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 18 or 29.
In other embodiments, 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% homology to the constant region described above.
In alternative embodiments, the functional fragment is selected from any one of VHH, 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, the invention provides an antibody comprising a heavy chain and/or a light chain, said heavy chain comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In alternative embodiments, the heavy chain has an amino acid sequence as set forth in any one of SEQ ID NOs 19, 30, 36; the amino acid sequence of the light chain is shown in any one of SEQ ID NO. 20, 31 and 37.
In alternative embodiments, the antibody or functional fragment thereof binds to a non-Omicron novel coronavirus N antigen.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In alternative embodiments, the marker is a substance of a type having a property that can be directly observed by the naked eye or detected by an instrument, such as luminescence, color development, radioactivity, etc., by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, 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 (chlorophyll), 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 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 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, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase 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 phase is a magnetic microsphere.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the present invention provides recombinant cells comprising the above vector.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the recombinant cells as described above were cultured.
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.
In another aspect, the invention provides a reagent or kit for detecting non-omacron novel coronavirus or identifying novel coronamutant, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.
In an alternative embodiment, the novel crown mutant is an omacron mutant.
In another aspect, the present invention provides a method for identifying novel coronal mutant antigens, comprising performing immunodetection on a sample to be detected using antibody 1, antibody 2 and antibody 3, wherein antibody 1 is capable of binding to M novel coronal virus antigens; the antibody 2 can be combined with M-1 novel coronavirus antigens except for an omacron mutant strain, and the antibody 2 is selected from the antibodies; the antibody 3 can be combined with M novel coronavirus antigens, and M is an integer greater than or equal to 2. The M new coronavirus antigens according to the invention include antigens of wild strains and mutant strains of new coronaviruses, wherein the mutant strain is not limited to omacron mutant strain, but may be b.1.1.7 mutant strain, B1.351 mutant strain or b.1.1.28 mutant strain. The "M-1 novel coronavirus antigens other than Omicron mutant" described in the present invention includes wild-type strains, and optionally also includes other mutant strains other than Omicron mutant, such as B.1.1.7 mutant, B1.351 mutant or B.1.1.28 mutant. The relevant detection principle is shown in patent application 202110315361.5.
In alternative embodiments, the antibody 1 and antibody 3 sandwich detect a novel coronavirus antigen; the antibody 2 and the antibody 3 sandwich detect novel coronavirus antigens. The novel coronavirus antigen described herein is not limited to the N antigen, or may be the S antigen, and is not limited to the wild strain, but may be a mutant strain. In some embodiments, the novel coronavirus antigen is a novel coronavirus antigen N antigen.
In alternative embodiments, the novel coronavirus antigen is an N antigen of a novel coronavirus.
In alternative embodiments, the method is selected from immunochromatography, enzyme-linked immunosorbent, chemiluminescence, latex turbidimetry.
On the other hand, the invention provides an immunochromatographic test paper for identifying a novel crown mutant antigen, which comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad, wherein a detection line 1 and a detection line 2 are arranged on the nitrocellulose membrane, the antibody 3 is arranged on the binding pad, the antibody 1 is coated on the detection line 1, and the antibody 2 is coated on the detection line 2; the antibody 3 is labeled with a label. In some embodiments, the immunochromatographic test strip is contacted with the sample to be tested, and when the detection line 1 and the detection line 2 develop color simultaneously or the detection lines 1 and 2 develop colors substantially in agreement (e.g., within 1 color card), the sample is judged to not contain the novel coronavirus Omicron mutant strain; when the detection line 1 develops color, the detection line 2 does not develop color, or when there is a significant difference in the color development of the detection lines 1 and 2 (for example, a difference of 3 color cards or more), it is determined that the sample contains a novel coronavirus omacron mutant.
In an alternative embodiment, a quality control line is also arranged on the nitrocellulose membrane.
In another aspect, the invention provides the use of the above antibody or a functional fragment thereof, an antibody conjugate, a reagent or kit as described above, a method or test paper for detecting an Omicron mutant or for preparing an Omicron mutant detection product.
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.
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 et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (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 Vectors 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, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 preparation of antibodies 19E3, 2F18 or 12H87
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. 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 plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting 19E3, 2F18 or 12H87 monoclonal antibodies, a DNA product is obtained through an RT-PCR method, the product is inserted into a pMD-18T vector after an rTaq DNA polymerase is used for carrying out an A adding reaction, the product is transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain gene clone and the Light Chain gene clone are respectively taken, and 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of antibody variable region genes
The gene sequences obtained by sequencing are placed in a KABAT antibody database for analysis, and VNTI11.5 software is utilized 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 gene fragments amplified by the Light Chain, VL gene sequences are 333bp (19E 3), 333bp (2F 18) and 333bp (12H 87), and a 57bp leader peptide sequence is arranged in front of the VL gene sequences; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 342bp (19E 3), 345bp (2F 18) and 345bp (12H 87), belongs to the VH1 gene family, and a leader peptide sequence of 57bp is arranged in front of the gene fragment. (the heavy and light chain sequences of 19E3 are shown as SEQ ID NO:19 and 20 respectively, the heavy and light chain sequences of 2F18 are shown as SEQ ID NO:30 and 31 respectively, and the heavy and light chain sequences of 12H87 are shown as SEQ ID NO:36 and 37 respectively).
(3) Construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector is a constructed recombinant antibody eukaryotic expression vector, and the expression vector is introduced with HindIII, bamHI, ecoRI and other polyclonal enzymesThe cutting point is named pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.72kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment are amplified by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
2 stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
The coating was diluted with 2019-nCoVN protein (from Figpeng) to 3 μg/ml, 100 μl per well, overnight at 4deg.C; the next day, washing with washing liquid for 2 times, and drying; 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 cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50. Mu.L/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that antibodies produced after transient plasmid transformation were active against 2019-nCoVN protein 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, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 6 Performing seed preservation by using cells/ml and 2 ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(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%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using protein A affinity chromatography columns. 6.6. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and the electrophoresis pattern was shown in FIG. 1.
Example 2
Performance detection of antibodies
(1) Activity detection
Coating liquid (main component NaHCO) 3 ) Microwell plate coating was performed by diluting 2019-nCoVN protein (from Fipeng) to 3 μg/ml, 100 μl per well, overnight at 4deg.C; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody in the example 1, 100 μl/well, 37 ℃ for 30-60 min; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 2 below.
TABLE 2 Activity data
Concentration (ng/ml) 2500.00 1250.00 625.00 312.50 156.25 0.00
19E3 2.372 1.866 0.894 0.311 0.113 0.032
2F18 2.274 1.521 0.831 0.400 0.223 0.043
Concentration (ng/ml) 3000.00 1000.00 333.33 111.11 37.04 0.00
12H87 2.317 2.163 1.918 1.320 0.663 0.038
(2) Affinity detection
Purified antibodies were diluted to 10. Mu.g/mL with PBST using an AMC sensor, and 2019-nCoVN protein (from Phpeng) was gradient diluted with PBST.
The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 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. K (K) D Representing equilibrium dissociation constant, i.e. affinity; kon represents the binding rate; kdis represents the dissociation rate. The results are shown in Table 3 below.
Table 3 affinity assay data
Sample name KD(M) kon(1/Ms) kdis(1/s)
19E3 4.02E-09 3.56E+04 1.43E-04
2F18 2.22E-09 7.43E+04 1.65E-04
12H87 4.90E-08 6.37E+03 3.12E-04
(3) Stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the enzyme-free activity assay OD for 21 days.
TABLE 4 Table 4
(4) Identification of mutant antigens
1. Marking
(1) Preparing colloidal gold: firstly heating chloroauric acid solution to boiling by adopting a traditional sodium citrate reduction method, rapidly adding a certain proportion of trisodium citrate solution, uniformly stirring, stopping heating when the color of the solution is changed into wine red and is not changed any more, and cooling to room temperature to obtain a colloidal gold solution with the concentration of four parts per million;
(2) Marking: adding 0.2. 0.2M K to colloidal gold solution 2 Regulating the pH value of the CO3 solution to 6.0-7.5;
(3) And (3) centrifuging: adding a labeled antibody into the colloidal gold solution with the pH adjusted, uniformly mixing, adding a sealing agent, stopping labeling, centrifuging at 10000rpm/7min/4 ℃, and removing the supernatant;
(4) And (3) re-dissolving: re-suspending to 100uL, and performing ultrasonic treatment for 2-3 times;
(5) Gold paving: the concentrated gold obtained by the re-suspension is diluted and spread on a glass cellulose membrane, and then is put into a freeze dryer for freeze drying (1-2 h) or is put into a drying room at 37 ℃ for drying overnight.
2. Coating
(1) Assembling the nitrocellulose membrane and the colloidal gold PVC base plate for standby;
(2) Diluting the coated antibody to 1.0-1.5mg/ml, and uniformly scribing a T1 line on the NC film by using a metal spraying film drawing instrument; diluting the 19E3, 2F18 or 12H87 antibody to 1.0-1.5mg/ml, and uniformly marking a T2 line on the NC film by using a metal spraying film drawing instrument; drying in a 37 deg.C incubator for at least 45 min. Assembling and cutting strips, and adding samples for detection.
The labeled antibody of this example was 15C8 or Mb117 (from Figpeng), the T1 wire coated antibody was Mb30 (from Figpeng), and 15C8, mb117 or Mb30 each bound to a novel coronavirus N antigen including Omicron mutant; the T2 line coated antibody was the antibody 19E3, 2F18 or 12H87 obtained in example 1.
3. Detection of
(1) And (3) detecting: a novel coronavirus omacron mutant N antigen, and a novel coronavirus wild strain N antigen (2019-ncovin);
(2) The detection method comprises the following steps: and judging the color development reading value by naked eyes according to the color card comparison.
4. Results:
TABLE 5
The above results show that the 19E3, 2F18 or 12H87 antibody does not react with the Omicron mutant strain N antigen and reacts with the novel coronavirus wild strain N antigen, thereby enabling rapid identification of the Omicron mutant strain.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
The partial amino acid sequence related to the application is as follows:
sequence numbering Sequence fragments
SEQ ID NO:1 DYSMH
SEQ ID NO:2 WINTETGEPTYADDFKG
SEQ ID NO:3 GLF
SEQ ID NO:4 KASQSVDYDGDSYMN
SEQ ID NO:5 AASNLES
SEQ ID NO:6 QQSNEDP
SEQ ID NO:7 QIQLVQSGPELKKPGETVKISCKASGYTFT
SEQ ID NO:8 WVKQAPGKGLKWMG
SEQ ID NO:9 RFAFSLETSASTAYLQINNLKNEDTATYFCGR
SEQ ID NO:10 DYWGQGTTLTVSS
SEQ ID NO:11 DIVLTQSPASLAVSLGQRATISC
SEQ ID NO:12 WYQQKPGQPPKLLIY
SEQ ID NO:13 GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC
SEQ ID NO:14 LTFGAGTKLELK
SEQ ID NO:21 GGAS
SEQ ID NO:22 KASQSVDYDGHSYMN
SEQ ID NO:23 TASNLES
SEQ ID NO:24 WVKQAPGKGLRWMG
SEQ ID NO:25 RFAFSLETSASTAYLQINNLKNEDTATYFCAR
SEQ ID NO:26 GYWGQGTTLTVSS
SEQ ID NO:32 GAGT
SEQ ID NO:33 WVKQATGKGLKWMG
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> an antibody, reagent and method for identifying novel crown mutant antigen
<130> P2021103CN01
<160> 37
<170> PatentIn version 3.3
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<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 6
Gln Gln Ser Asn Glu Asp Pro
1 5
<210> 7
<211> 30
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 7
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 8
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 8
Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly
1 5 10
<210> 9
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 9
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln
1 5 10 15
Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Gly Arg
20 25 30
<210> 10
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 10
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
1 5 10
<210> 11
<211> 23
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 11
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys
20
<210> 12
<211> 15
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 12
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 13
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 13
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
20 25 30
<210> 14
<211> 12
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 14
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
1 5 10
<210> 15
<211> 114
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 15
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Gly Arg Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
100 105 110
Ser Ser
<210> 16
<211> 111
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 16
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 17
<211> 324
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 17
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 18
<211> 107
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 18
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 19
<211> 438
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 19
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Gly Arg Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
100 105 110
Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
115 120 125
Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys
130 135 140
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu
145 150 155 160
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
165 170 175
Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu
180 185 190
Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
195 200 205
Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr
210 215 220
Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
225 230 235 240
Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp
245 250 255
Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
260 265 270
Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn
275 280 285
Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp
290 295 300
Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro
305 310 315 320
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala
325 330 335
Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp
340 345 350
Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile
355 360 365
Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
370 375 380
Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys
385 390 395 400
Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys
405 410 415
Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu
420 425 430
Ser His Ser Pro Gly Lys
435
<210> 20
<211> 218
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 20
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 21
<211> 4
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 21
Gly Gly Ala Ser
1
<210> 22
<211> 15
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 22
Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly His Ser Tyr Met Asn
1 5 10 15
<210> 23
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 23
Thr Ala Ser Asn Leu Glu Ser
1 5
<210> 24
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 24
Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met Gly
1 5 10
<210> 25
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 25
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln
1 5 10 15
Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
20 25 30
<210> 26
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 26
Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
1 5 10
<210> 27
<211> 115
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 27
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Ala Ser Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser
115
<210> 28
<211> 111
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 28
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly His Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 29
<211> 107
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 29
Arg Ala Glu Gly Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 30
<211> 439
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 30
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Ala Ser Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 31
<211> 218
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 31
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly His Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 110
Ala Glu Gly Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 32
<211> 4
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 32
Gly Ala Gly Thr
1
<210> 33
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 33
Trp Val Lys Gln Ala Thr Gly Lys Gly Leu Lys Trp Met Gly
1 5 10
<210> 34
<211> 115
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 34
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Thr Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gly Ala Gly Thr Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser
115
<210> 35
<211> 112
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 35
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 36
<211> 439
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 36
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Thr Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gly Ala Gly Thr Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 37
<211> 218
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 37
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (41)

1. An antibody or functional fragment thereof against a novel coronavirus 2019-nCoV, wherein the antibody or functional fragment thereof comprises a complementarity determining region of any one of the following groups:
the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 1, 2, 3, 4, 5 and 6;
The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 1, 2, 21, 22, 23 and 6;
the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 1, 2, 32, 4, 23 and 6.
2. The antibody or functional fragment thereof according to claim 1, further having a framework region of any one of the group:
HFR1 amino acid sequence is shown in SEQ ID NO. 7 or has at least 80% homology with it, HFR2 amino acid sequence is shown in SEQ ID NO. 8 or has at least 80% homology with it, HFR3 amino acid sequence is shown in SEQ ID NO. 9 or has at least 80% homology with it, HFR4 amino acid sequence is shown in SEQ ID NO. 10 or has at least 80% homology with it, LFR1 amino acid sequence is shown in SEQ ID NO. 11 or has at least 80% homology with it, LFR2 amino acid sequence is shown in SEQ ID NO. 12 or has at least 80% homology with it, LFR3 amino acid sequence is shown in SEQ ID NO. 13 or has at least 80% homology with it, LFR4 amino acid sequence is shown in SEQ ID NO. 14 or has at least 80% homology with it;
HFR1 amino acid sequence as shown in SEQ ID NO. 7 or has at least 80% homology therewith, HFR2 amino acid sequence as shown in SEQ ID NO. 24 or has at least 80% homology therewith, HFR3 amino acid sequence as shown in SEQ ID NO. 25 or has at least 80% homology therewith, HFR4 amino acid sequence as shown in SEQ ID NO. 26 or has at least 80% homology therewith, LFR1 amino acid sequence as shown in SEQ ID NO. 11 or has at least 80% homology therewith, LFR2 amino acid sequence as shown in SEQ ID NO. 12 or has at least 80% homology therewith, LFR3 amino acid sequence as shown in SEQ ID NO. 13 or has at least 80% homology therewith, LFR4 amino acid sequence as shown in SEQ ID NO. 14 or has at least 80% homology therewith;
HFR1 amino acid sequence is shown in SEQ ID NO. 7 or has at least 80% homology with it, HFR2 amino acid sequence is shown in SEQ ID NO. 33 or has at least 80% homology with it, HFR3 amino acid sequence is shown in SEQ ID NO. 25 or has at least 80% homology with it, HFR4 amino acid sequence is shown in SEQ ID NO. 26 or has at least 80% homology with it, LFR1 amino acid sequence is shown in SEQ ID NO. 11 or has at least 80% homology with it, LFR2 amino acid sequence is shown in SEQ ID NO. 12 or has at least 80% homology with it, LFR3 amino acid sequence is shown in SEQ ID NO. 13 or has at least 80% homology with it, LFR4 amino acid sequence is shown in SEQ ID NO. 14 or has at least 80% homology with it.
3. The antibody or functional fragment thereof according to claim 2, wherein the antibody or functional fragment thereof has a KD.ltoreq.10 -7 The affinity of M binds to the wild-type novel coronavirus N antigen.
4. An antibody or functional fragment thereof against a novel coronavirus 2019-nCoV, wherein the antibody or functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, HFR1, LFR2, r3, LFR4 is the amino acid sequence of HFR1, HCDR2, HCDR3, LCDR3, LFR4 of the amino acid sequence of HFR1, HCDR2, LCDR3, LCDR 4 of claim 1, HFR2, HFR3, LFR 4.
5. An antibody or functional fragment thereof against a novel coronavirus 2019-nCoV, wherein said antibody or functional fragment thereof comprises a heavy chain variable region and a light chain variable region,
the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 16; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 27 and SEQ ID NO. 28; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 34 and SEQ ID NO. 35.
6. The antibody or functional fragment thereof of any one of claims 1 to 5, wherein the antibody or functional fragment thereof further comprises a constant region.
7. The antibody or functional fragment thereof of claim 6, wherein the constant region comprises a heavy chain constant region and/or a light chain constant region.
8. The antibody or functional fragment thereof of claim 7, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
9. The antibody or functional fragment thereof according to claim 7, wherein the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose or human origin.
10. The antibody or functional fragment thereof according to claim 9, wherein the constant region is of murine origin.
11. The antibody or functional fragment thereof according to claim 7, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 17 or has at least 80% homology thereto and the light chain constant region sequence is as shown in SEQ ID No. 18 or 29 or has at least 80% homology thereto.
12. The antibody or functional fragment thereof according to any one of claims 1 to 5, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
13. An antibody against a novel coronavirus 2019-nCoV comprising a heavy chain and a light chain, characterized in that the heavy chain comprises a heavy chain variable region as defined in claim 4 or 5 and a heavy chain constant region as defined in any one of claims 7 to 11; the light chain comprises the light chain variable region of claim 4 or 5 and the light chain constant region of any one of claims 7 to 11.
14. An antibody for resisting novel coronavirus 2019-nCoV comprises a heavy chain and a light chain, and is characterized in that the amino acid sequence of the heavy chain is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain is shown as SEQ ID NO. 20; or the heavy chain amino acid sequence is shown as SEQ ID NO. 30, and the light chain amino acid sequence is shown as SEQ ID NO. 31; or the heavy chain amino acid sequence is shown as SEQ ID NO. 36, and the light chain amino acid sequence is shown as SEQ ID NO. 37.
15. The antibody or functional fragment thereof of any one of claims 4, 5, 13 and 14, wherein the antibody or functional fragment thereof binds to wild-type novel coronavirus N antigen.
16. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1-15.
17. The antibody conjugate of claim 16, wherein the antibody or functional fragment thereof is labeled with a label.
18. The antibody conjugate of claim 17, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
19. The antibody conjugate according to claim 18, characterized in that the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, rhodamine-based dyes and derivatives thereof, cy-based dyes and derivatives thereof, alexa-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.
20. The antibody conjugate of claim 18, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
21. The antibody conjugate of claim 18, wherein the radioisotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110 msin, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.
22. The antibody conjugate of claim 18, wherein the chemiluminescent reagent is selected from the group consisting of luminol and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, ruthenium bipyridine and derivatives thereof, acridinium esters and derivatives thereof, dioxane and derivatives thereof, lotensine and derivatives thereof, and peroxyoxalate and derivatives thereof.
23. The antibody conjugate of claim 18, wherein the nanoparticle-based label is selected from the group consisting of a nanoparticle and a colloid.
24. The antibody conjugate of claim 23, wherein the nanoparticle-based label is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
25. The antibody conjugate of claim 23, wherein the colloid is selected from the group consisting of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microsphere.
26. The antibody conjugate of claim 25, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
27. The antibody conjugate of claim 16, wherein the antibody or functional fragment thereof is coated onto a solid phase.
28. The antibody conjugate of claim 27, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
29. The antibody conjugate of claim 27, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
30. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1-15.
31. A vector comprising a nucleic acid fragment encoding the antibody or functional fragment thereof of any one of claims 1-15.
32. A recombinant cell comprising the vector of claim 31.
33. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 15, comprising: culturing the recombinant cell of claim 32.
34. A reagent or kit for detecting a wild-type novel coronavirus or for identifying a novel coronamutant, wherein the reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 15 or the antibody conjugate according to any one of claims 16 to 29, and wherein the novel coronavirus is an omacron mutant.
35. Use of an antibody or functional fragment thereof according to any one of claims 1 to 15 for the preparation of a product for identifying novel coronal mutant antigens, characterized in that the sample to be tested is subjected to an immunoassay using antibody 1, antibody 2 and antibody 3, said antibody 1 being capable of binding to M novel coronavirus antigens; said antibody 2 being capable of binding to M-1 novel coronavirus antigens other than omacron mutant, said antibody 2 being selected from the group consisting of the antibodies of any one of claims 1 to 15; the antibody 3 can be combined with M novel coronavirus antigens, M is an integer greater than or equal to 2, and the novel coronavirus mutant is an omacron mutant.
36. The use according to claim 35, wherein the antibodies 1 and 3 sandwich detect a novel coronavirus antigen; the antibody 2 and the antibody 3 sandwich detect novel coronavirus antigens.
37. The use according to claim 35, wherein the novel coronavirus antigen is a novel coronavirus N antigen.
38. The use according to claim 35, wherein the method for identifying a neocrown mutant antigen in said use is selected from the group consisting of immunochromatography, enzyme-linked immunosorbent, chemiluminescence, latex turbidimetry.
39. An immunochromatographic test paper for identifying a novel crown mutant antigen, which is characterized by comprising a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad, wherein a detection line 1 and a detection line 2 are arranged on the nitrocellulose membrane, an antibody 3 according to any one of claims 35-38 is arranged on the binding pad, the antibody 1 according to any one of claims 35-38 is coated on the detection line 1, and the antibody 2 according to any one of claims 35-38 is coated on the detection line 2; the antibody 3 was labeled with a marker, and the novel crown mutant was an omacron mutant.
40. The immunochromatographic test strip as defined in claim 39, wherein the nitrocellulose membrane is further provided with a quality control line.
41. Use of an antibody or functional fragment thereof according to any one of claims 1 to 15, an antibody conjugate according to any one of claims 16 to 29, a reagent or kit according to claim 34 or a test paper according to claim 39 or 40 for the preparation of omacron mutant detection products.
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