Antibodies against rotavirus and uses thereof

Abstract

The application discloses an antibody against rotavirus and application thereof, and relates to the field of antibodies. The antibody against rotavirus disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of rotavirus, and has good activity and sensitivity.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202311417547.7, filed on October 27, 2023, entitled "An Antibody Against Rotavirus and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to an antibody against rotavirus and its application. Background Technology

[0004] Rotavirus (RV) is a double-stranded RNA virus belonging to the Reoviridae family. Its genome consists of 11 unique double-stranded RNA molecules, totaling 18,555 nucleoside base pairs. Each helix or segment is a gene, numbered 1 to 11 from largest to smallest. Rotavirus is the most common cause of diarrhea in infants and young children; almost every child experiences at least one rotavirus infection by age five. However, immunity gradually strengthens after each infection, thus reducing the impact of subsequent infections, and rotavirus rarely affects adults. Rotavirus infects the intestinal mucosal cells of the small intestine and produces enterotoxins, which cause gastroenteritis, leading to severe diarrhea and sometimes even death due to dehydration. Besides its effects on human health, rotavirus also infects animals, being one of the pathogens affecting livestock.

[0005] Rotavirus is transmitted via the fecal-oral route, through contact with contaminated hands, surfaces, and objects, and can also be transmitted through the respiratory tract. The feces of an infected person can contain more than 10 trillion infectious viral particles per gram; just 10 to 100 of these particles can infect another person. Rotavirus is stable in the natural environment and can also be found in estuarine samples, where approximately 1 to 5 infectious rotavirus particles can be found per US gallon. Sanitation facilities designed to eliminate bacteria and parasites appear ineffective in controlling rotavirus, as the incidence of rotavirus infection is similar in countries with high and low sanitation levels.

[0006] Currently, the main method for detecting rotavirus is colloidal gold immunochromatography assay, which is an immunological detection method based on specific reaction of antigen and antibody. The basic principle is as follows: an antigen or antibody is labeled with colloidal gold, and the corresponding paired antigen or antibody is coated on a nitrocellulose membrane. When detecting a sample, the colloidal gold label and the ligand in the sample combine to form a complex, and then move upward by chromatography, combine with the coated antigen or antibody, and agglomerate to develop color, so as to realize the determination of the detection result of the sample. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence method, etc.

[0007] The above immunological detection methods all need to prepare antibodies against rotavirus, and therefore, there is a strong demand in the art for rotavirus antibodies with good performance. SUMMARY

[0008] The present application provides an antibody against rotavirus, which provides an important raw material source for the detection of rotavirus and has good activity or sensitivity.

[0009] In order to achieve the above-mentioned purpose, according to one aspect of the present application, an antibody against rotavirus is provided, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 20 and 21.

[0010] In order to achieve the above-mentioned purpose, according to a second aspect of the present application, an antibody against rotavirus is provided, which comprises the following complementarity determining regions:

[0011] HCDR1 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 1 (SYVMH);

[0012] HCDR2 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2 (YISPNNEGAMYNEKFKG);

[0013] HCDR3 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 3 (GAYYRFEDFDY);

[0014] LCDR1 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 4 (RASQDIGNYLN);

[0015] LCDR2 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 5 (YTSRLHS); and

[0016] LCDR3 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 6 (QQGTTLPWT).

[0017] To achieve the above object, according to a third aspect of the present application, there is provided an antibody against rotavirus, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as set forth in SEQ ID NO: 18; the amino acid sequence of the light chain variable region being as set forth in any one of SEQ ID NO: 20, 21.

[0018] To achieve the above object, according to a fourth aspect of the present application, there is provided an antibody against rotavirus, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 19; the amino acid sequence of the light chain being as set forth in any one of SEQ ID NO: 22, 23.

[0019] To achieve the above object, according to a fifth aspect of the present application, there is provided an antibody conjugate, comprising the antibody as described above.

[0020] To achieve the above object, according to a sixth aspect of the present application, there is provided a reagent or kit, comprising the antibody as described above or the antibody conjugate as described above.

[0021] To achieve the above object, according to an eighth aspect of the present application, there is provided a use of the antibody, the antibody conjugate, the reagent or the kit as described above in the preparation of a product for detecting rotavirus.

[0022] To achieve the above object, the present application further provides a nucleic acid, a vector, a cell and a method for preparing the antibody as described above. BRIEF DESCRIPTION OF DRAWINGS

[0023] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as a limitation to the scope, and for those skilled in the art, other related drawings can also be obtained without paying creative labor on the basis of these drawings.

[0024] Figure 1 Results of the reducing SDS-PAGE of Anti-RV 11G8 Rmb1~Anti-RV 11G8 Rmb2. DETAILED DESCRIPTION

[0025] In a first aspect, the embodiments of the present application provide an antibody against rotavirus, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 20, 21.

[0026] It should be noted that the HCDR1, HCDR2 and HCDR3 are the same amino acid sequences of HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody of the first aspect, and the LCDR1, LCDR2 and LCDR3 are the same amino acid sequences of LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody of the first aspect.

[0027] For example, the HCDR1, HCDR2 and HCDR3 are the same amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 18, and the LCDR1, LCDR2 and LCDR3 are the same amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 20.

[0028] In the present application, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity.

[0029] The antigen-binding fragment of the antibody generally has the same binding specificity as the antibody from which it is derived, and can be selected from any one of F(ab')2, Fab', Fab, Fv and scFv, and those skilled in the art can easily understand from the content disclosed in the present application that the above-mentioned antigen-binding fragment can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structural basis of the complete antibody disclosed in the present application, those skilled in the art can easily obtain the antigen-binding fragment of the above-mentioned antibody.

[0030] The antigen-binding fragment of the antibody can also be obtained by recombinant genetics technology or by synthesis, for example, by an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems and the like.

[0031] In the present application, the term "complementarity determining region," "CDR," or "CDRs" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, referring to the regions that contain one or more, or even all, of the amino acid residues that play a major role in the binding of the antibody or antigen-binding fragment to its recognized antigen or epitope. In the detailed description of the application, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0032] In the present application, the heavy chain complementarity determining regions are denoted as HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted as LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0033] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). See also Chothia et al., J Mol Biol 196:901-917 (1987) for the "Chothia definition". Other methods of defining CDRs can not strictly follow one of the above schemes, but will still overlap at least a portion of the CDR regions defined by Kabat, although they can be shortened or lengthened according to prediction or experimental results for particular residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, with slight variations in the definitions in different literature. Given the variable region amino acid sequence of a given antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It is noted that CDRs defined by other methods than those in Table 1 are also within the scope of the present disclosure.

[0034] Table 1: CDR definitions 1

[0035]

[0036]

[0037] 1The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with "H+ numbers" for amino acid positions on the heavy chain and "L+ numbers" for amino acid positions on the light chain. One of ordinary skill in the art can unambiguously correlate this Kabat numbering system to any variable region sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 "AbM" as used in Table 1 with a lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0039] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0040] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0041] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0042] According to embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by any one or a combination of Kabat, Chothia, IMGT, AbM, or Contact.

[0043] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0044] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0045] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0046] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0047] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0048] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM or Contact system.

[0049] According to embodiments of the present application, the Kabat, Chothia, AbM or IMGT system defined HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 amino acid sequence corresponding Kabat numbering position is as follows:

[0050] CDR Kabat AbM IMGT Chothia HCDR1 H31 ~ H35 H26 ~ H35 H26 ~ H33 H26 ~ H32 HCDR2 H50 ~ H65 H50 ~ H58 H51 ~ H57 H52 ~ H56 HCDR3 H95 ~ H102 H95 ~ H102 H93 ~ H102 H95 ~ H102 LCDR1 L24 ~ L34 L24 ~ L34 L27 ~ L32 L24 ~ L34 LCDR2 L50 ~ L56 L50 ~ L56 L50 ~ L51 L50 ~ L56 LCDR3 L89 ~ L97 L89 ~ L97 L89 ~ L97 L89 ~ L97

[0051] In a second aspect, embodiments of the present application provide an antibody against rotavirus, the antibody comprising the following complementarity determining regions:

[0052] HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 (SYVMH);

[0053] HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 (YISPNNEGAMYNEKFKG);

[0054] HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3 (GAYYRFEDFDY);

[0055] LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 (RASQDIGNYLN);

[0056] LCDR2, which comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5 (YTSRLHS); and

[0057] LCDR3, which comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 (QQGTTLPWT).

[0058] According to embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0059] In the present application, the "framework region" or "FR" region, which includes both heavy chain framework region and light chain framework region, refers to the region of the antibody heavy chain variable region and light chain variable region except for CDR; wherein the heavy chain framework region can be further subdivided into regions adjacent to CDR separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into regions adjacent to CDR separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0060] In the present application, the heavy chain variable region is obtained by connecting the CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; and the light chain variable region is obtained by connecting the CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0061] In optional embodiments, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0062] In optional embodiments, the HFR1 comprises / consists of an amino acid sequence of SEQ ID NO: 7 or having at least 80% identity thereto;

[0063] the HFR2 comprises / consists of an amino acid sequence of SEQ ID NO: 8 or having at least 80% identity thereto;

[0064] the HFR3 comprises / consists of an amino acid sequence of SEQ ID NO: 9 or having at least 80% identity thereto;

[0065] the HFR4 comprises / consists of an amino acid sequence of SEQ ID NO: 10 or having at least 80% identity thereto;

[0066] the LFR1 comprises / consists of an amino acid sequence of SEQ ID NO: 11 or having at least 80% identity thereto;

[0067] the LFR2 comprises / is an amino acid sequence of SEQ ID NO: 12 or has at least 80% identity thereto;

[0068] the LFR3 comprises / is an amino acid sequence of SEQ ID NO: 13 or has at least 80% identity thereto; and

[0069] the LFR4 comprises / is an amino acid sequence of SEQ ID NO: 14 or has at least 80% identity thereto.

[0070] It should be noted that in other embodiments, each of the framework regions of the anti-rotavirus antibody provided by the present application can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) described above.

[0071] In an alternative embodiment, the LFR3 comprises / is an amino acid sequence of SEQ ID NO: 17.

[0072] In a third aspect, an embodiment of the present application provides an anti-rotavirus antibody, comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprising an amino acid sequence of any one of SEQ ID NO: 20 or 21.

[0073] In an alternative embodiment, the antibody of the first aspect, the second aspect, or the third aspect described above further comprises a constant region.

[0074] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0075] In an alternative embodiment, the heavy chain constant region is selected from a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD, or a combination of multiple constant region segments.

[0076] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0077] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0078] In an alternative embodiment, the light chain constant region is selected from a kappa or lambda light chain constant region.

[0079] In an alternative embodiment, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock or human.

[0080] In an alternative embodiment, the species origin of the constant region is mouse.

[0081] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0082] It should be noted that in other embodiments, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO: 15 or 16).

[0083] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0084] In a fourth aspect, the present application provides an antibody against rotavirus, including a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22 or 23.

[0085] In a fifth aspect, the present application provides an antibody conjugate, including the above-mentioned antibody.

[0086] In an alternative embodiment, the above-mentioned antibody conjugate further includes biotin or a biotin derivative coupled with the antibody.

[0087] In an alternative embodiment, the above-mentioned antibody conjugate further includes a label coupled with the antibody.

[0088] In an alternative embodiment, the above-mentioned label refers to a kind of substance having a characteristic such as luminescence, color development, radioactivity, etc. that can be directly observed by naked eye or detected or probed by an instrument, through which qualitative or quantitative detection of the corresponding target can be realized.

[0089] In an alternative embodiment, the above-mentioned label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

[0090] In actual use, the skilled in the art can select appropriate labels according to the detection conditions or actual needs, and no matter what kind of labels are used, they all belong to the protection scope of the present application.

[0091] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to, fluorescein isothiocyanate (FITC), hydroxyfluorine (FAM), tetrafluorophorin (TET) and the like or analogs thereof), rhodamine dyes and derivatives thereof (for example, including but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC) and the like or analogs thereof), Cy series dyes and derivatives thereof (for example, including but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3 and the like or analogs thereof), Alexa series dyes and derivatives thereof (for example, including but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and the like or analogs thereof), and protein dyes and derivatives thereof (for example, including but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP) and the like).

[0092] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0093] In optional embodiments, the radioisotopes include, but are 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.

[0094] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, ruthenium bipyridine and derivatives thereof, acridinium ester and derivatives thereof, dioxetane and derivatives thereof, lucigenin and derivatives thereof, and peroxymalate and derivatives thereof.

[0095] In alternative embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0096] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0097] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0098] In alternative embodiments, the colloidal metal is colloidal gold.

[0099] In alternative embodiments, the antibody conjugate further includes a solid support coupled to the antibody.

[0100] In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

[0101] In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, micro-well plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0102] In a sixth aspect, the present application provides a reagent or a kit, which includes the antibody or the antibody conjugate as described above.

[0103] As described above, the antibody in some embodiments or examples of the present application can effectively bind to rotavirus, and thus the reagent or the kit including the rotavirus antibody can effectively detect rotavirus qualitatively or quantitatively. The reagent or the kit provided by the present application can be used in, for example, immunoblotting, immunoprecipitation, and other detection methods involving the specific binding of rotavirus and its antibody. As described above, the antibody in some embodiments or examples of the present application has higher binding activity or affinity to rotavirus, and thus the reagent or the kit including the antibody has higher detection sensitivity or specificity.

[0104] In a seventh aspect, the present application provides a method for detecting rotavirus, which includes: a) contacting the antibody, the antibody conjugate, the reagent or the kit as described above with rotavirus in a test sample under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0105] In alternative embodiments, the immunocomplex further includes a second antibody that binds to the antibody.

[0106] In an alternative embodiment, the immune complex further comprises a second antibody that binds to rotavirus.

[0107] In an eighth aspect, the present application provides use of the above-mentioned anti-rotavirus antibody, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting rotavirus.

[0108] In a ninth aspect, the present application provides a nucleic acid molecule encoding the above-mentioned antibody.

[0109] In a tenth aspect, the present application provides a vector containing the above-mentioned nucleic acid molecule.

[0110] In an eleventh aspect, the present application provides a cell containing the above-mentioned vector.

[0111] In a twelfth aspect, the present application provides a method for preparing an anti-rotavirus antibody, comprising: culturing the above-mentioned cell.

[0112] On the basis of the amino acid sequences of the anti-rotavirus antibodies disclosed in the present application, it is easy for those skilled in the art to conceive of preparing the anti-rotavirus antibodies by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, isolating and purifying the antibodies from the culture products of recombinant cells capable of recombinantly expressing the antibodies as described in any one of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technique is used to prepare the anti-rotavirus antibodies of the present application, it all falls within the protection scope of the present application.

[0113] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Unless otherwise specified, the specific conditions in the embodiments are carried out according to conventional conditions or manufacturer's recommended conditions. Unless otherwise specified, the reagents or instruments used are all conventional products that can be purchased on the market.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. Materials, methods, and examples are illustrative only and not limiting.

[0115] The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir and C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller and M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0116] The features and advantages of the present application are further illustrated in the following examples.

[0117] Example 1 Preparation of Anti-RV 11G8 Monoclonal Antibody

[0118] Restriction enzymes and Prime Star DNA polymerase used in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. Hybridoma cell line secreting Anti-RV 11G8 monoclonal antibody was prepared in the laboratory.

[0119] (1) Antibody gene preparation

[0120] The mRNA was extracted from the hybridoma cell line secreting Anti-RV 11G8 monoclonal antibody, and the DNA product was obtained by RT-PCR method. The product was inserted into the pMD-18T vector after A reaction with rTaq DNA polymerase, and transformed into DH5a competent cells. After the colonies were grown, the Heavy Chain and Light Chain gene clones were taken respectively, and each 4 clones were sent to the gene sequencing company for sequencing.

[0121] (2) Sequence analysis of Anti-RV 11G8 antibody variable region gene

[0122] The gene sequence obtained by the above sequencing was placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the amplified genes of the heavy chain and light chain primer pairs were correct. Among them, the VL gene sequence in the Light Chain amplified gene fragment was 321 bp, and there was a 57 bp leader peptide sequence in front of it; the VH gene sequence in the Heavy Chain primer pair amplified gene fragment was 360 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0123] (3) Construction of recombinant antibody expression plasmid

[0124] pcDNA TM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector. The expression vector has introduced HindIII, BamHI, EcoRI and other multiple enzyme digestion sites, and is named pcDNA3.4A expression vector, which is referred to as 3.4A expression vector hereinafter; according to the antibody variable region gene sequencing results in the above pMD-18T, the VL and VH gene specific primers of the antibody were designed, which had HindIII, EcoRI enzyme digestion sites and protection bases at both ends. The 0.70 kb Light Chain gene fragment and the 1.41 kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0125] The Heavy Chain and Light Chain gene fragments were digested by Hind III / Eco RI, and the 3.4A vector was digested by Hind III / Eco RI. After purification and recovery of the fragments and the vector, the Heavy Chain gene and the Light Chain gene were connected to the 3.4A expression vector, respectively, to obtain the recombinant expression plasmids of the Heavy Chain and the Light Chain.

[0126] 2. Production of recombinant antibody

[0127] The HEK293 cells were recovered in advance and subcultured in a 200 ml system to make the cell density reach 3-5 x 10 6 cells / ml. The cell density reached the concentration of the selected antibody, and the cell viability was >95%. The cells were centrifuged and washed, and the cells were resuspended with the culture medium, while the cell density was adjusted to 2.9 x 10 6 cells / ml. The cells were washed and resuspended with the culture medium, which was used as a cell diluent. The plasmid DNA and the transfection reagent diluent were prepared with the culture medium, respectively. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and then placed at room temperature for 15 min. The mixture was slowly added to the cell diluent within 1 min, mixed, sampled, counted, and recorded. The cell viability after transfection was observed, and the mixture was placed in a 35℃ constant temperature incubator for culture at a speed of 120 rpm and a CO2 content of 8%. After 13 days, the sample was collected by centrifugation. The supernatant was subjected to affinity purification with a protein A affinity chromatography column. 6 ug of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain), and the other with Mr of 28 KD (light chain).

[0128] The obtained antibody was named Anti-RV 11G8Rmb1. The mutation was performed on Anti-RV 11G8Rmb1 to obtain a mutant antibody. The sequences of the heavy chain (H) and the light chain (L) of the above-mentioned antibody are shown in the following table.

[0129] Table 2: Antibody sequence

[0130] Antibody name Heavy chain Light chain Anti-RV 11G8Rmb1 SEQ ID NO: 19 SEQ ID NO: 22 Anti-RV 11G8Rmb2 SEQ ID NO: 19 SEQ ID NO: 23

[0131] Performance detection of the antibody of Example 2

[0132] 1. Activity identification

[0133] Coating solution (main component NaHCO3) dilute goat anti-mouse IgG 1 ug / ml to coat microplate, 100 uL per well, 4°C overnight; the next day, wash 2 times with washing solution (main components Na2HPO4+NaCl), pat dry; add blocking solution (20% BSA+80% PBS), 120 uL per well, 37°C, 1 h, pat dry; add diluted purified antibody, 100 uL per well, 37°C, 60 min; shake off the liquid in the plate, pat dry, add 20% mouse negative blood blocking, 120 uL per well, 37°C, 1 h; shake off the liquid in the plate, pat dry, add diluted positive sample, 100 uL per well, 37°C, 40 min; wash 5 times with washing solution, pat dry; add HRP-labeled anti-rotavirus monoclonal antibody (from Fepeng Biological, matched with purified antibody), 100 uL per well, 37°C, 30 min; add color developing solution A liquid (50 uL per well), add color developing solution B liquid (50 uL per well), 10 min; add stop solution, 50 uL per well; read OD value at 450 nm (reference 630 nm) on a microplate reader.

[0134] Note: A liquid (main components citric acid+NaAc+acetanilide+urea peroxide); B liquid (main components citric acid+EDTA·2Na+TMB+concentrated HCl); stop solution (EDTA·2Na+concentrated H2SO4)

[0135] Table 3: Activity data

[0136] Concentration (ng / ml) 125 62.50 31.25 15.63 7.81 0.00 Control 1.502 0.998 0.536 0.329 0.124 0.023 Anti-RV 11G8Rmb1 2.174 1.745 1.217 0.654 0.398 0.014 Anti-RV 11G8Rmb2 2.023 1.744 1.240 0.715 0.412 0.015

[0137] 2. Stability evaluation

[0138] The above antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples taken at 7 days, 14 days, and 21 days were observed for state, and the 21-day sample was detected for activity. The results showed that under the three evaluation conditions, the antibody placed for 21 days did not show obvious changes in protein state, and the activity did not show a downward trend with the increase of evaluation temperature, indicating that the above antibody was stable. Table 4 below shows the OD results of enzyme immunoactivity detection of the antibody Anti-RV 11G8Rmb2 evaluated for 21 days.

[0139] Table 4: Stability data

[0140] Sample concentration (ng / ml) 31.25 15.63 0 4°C, 21-day sample 1.233 0.776 0.031 -80°C, 21-day sample 1.271 0.754 0.033 37°C, 21-day sample 1.265 0.724 0.034

[0141] 3. Colloidal gold platform performance detection

[0142] 3.1 Antibody labeling process

[0143] (1) Adjust the labeling pH: take 4 / 10,000 colloidal gold 10 ml, centrifuge, add 0.2M K2CO3 150 ul, stir and mix for 2 min;

[0144] (2)Coupling antibody: 200 ug of antibody Anti-RV-C (from Feipeng Biological) was added respectively, and the reaction was stirred for 15 min;

[0145] (3) Blocking: 10% (mass / volume) BSA was added, and the reaction was stirred for 15 min;

[0146] (4) Centrifugal preservation: 10000 rpm / 7 min / room temperature, supernatant was removed, and the sample was resuspended to 1 ml with gold standard solution and stored at 4°C for standby;

[0147] 3.2 Coating

[0148] (1) The nitrocellulose membrane was assembled with the colloidal gold PVC bottom plate for standby;

[0149] (2) The antibodies Anti-RV 11G8Rmb1, Anti-RV 11G8Rmb2 and the control antibody Anti-RV-D (from Feipeng Biological) were diluted to 1.0-2.0 mg / ml respectively, and a gold spraying film instrument was used to draw lines on the NC membrane uniformly, and then the sample was placed in a 50°C constant temperature box for drying, and the drying time was at least 4 h or more. The strip was assembled, and the sample was detected.

[0150] 3.3 Detection

[0151] (1) Sample: RV quality control containing different concentrations

[0152] (2) Detection method: colloidal gold detection, the intensity of the detection line was observed by naked eye, and the result was determined. According to the intensity of the color of the displayed strip, the activity of the antigen and antibody combination in the sample can be indicated. The color of the T line strip of the colloidal gold test paper reaction was compared with the standard color card, and the color number closest to the color was selected to mark the activity of the product,

[0153] The smaller the number, the stronger the color development and the higher the activity; the higher the number, the weaker the color development and the lower the activity; the number after the "+" represents slightly strong, and the number after the "-" represents slightly weak, and "B" represents negative.

[0154] 3.4 Detection results

[0155] The detection results are shown in Table 5, and the results show that the detection sensitivity of the antibodies Anti-RV 11G8Rmb1 and Anti-RV 11G8Rmb2 used in the colloidal gold platform is better than that of the control antibody.

[0156] Table 5: Colloidal gold platform detection results

[0157] Quality control Anti-RV-D Anti-RV 11G8Rmb1 Anti-RV 11G8Rmb1 Rotavirus antigen 1 / 400 C3 C2 C2 Rotavirus antigen 1 / 4K C5 C4 C4 Rotavirus antigen 1 / 40K C9 C8+ C8 Dilution B B B

[0158] Part of the amino acid sequences involved in the present application are shown in Table 6:

[0159] Table 6: Amino acid sequence table

[0160]

[0161] The above descriptions are only the preferred embodiments of the present application and are not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibody against rotavirus, characterized in that, The antibody comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18 and three complementarity determining regions of a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 20, 21. The complementarity determining regions of the variable region are defined by any one of Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against rotavirus, characterized in that, The antibody comprises the following complementarity determining regions: HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1; HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2; HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3; LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4; LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5; and LCDR3 having an amino acid sequence as set forth in SEQ ID NO:

6.

3. The antibody according to claim 1 or 2, characterized in that, The antibody further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

4. The antibody of claim 3, wherein: the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; the HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; the HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; the HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; the LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; the LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; the LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and the LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

5. An antibody against rotavirus comprising a heavy chain variable region and a light chain variable region, characterized in that, the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO: 18; and the light chain variable region amino acid sequence is as set forth in any one of SEQ ID NOs: 20, 21.

6. The antibody of any one of claims 1, 2, 4, 5, wherein, The antibody further comprises a constant region.

7. The antibody of claim 6, wherein The constant region comprises a heavy chain constant region and / or a light chain constant region.

8. The antibody of claim 7, wherein The heavy chain constant region is selected from a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD, or a combination of multiple constant region segments.

9. The antibody of claim 7, wherein The heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

10. The antibody of claim 6, wherein The species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

11. The antibody of claim 6, wherein The species origin of the constant region is mouse.

12. The antibody of claim 7, wherein the heavy chain constant region sequence is as set forth in SEQ ID NO: 15 or has at least 80% identity thereto.

13. The antibody of claim 7, wherein The light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

14. An antibody against rotavirus comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 1, and the light chain comprises SEQ ID NO:

2. The amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in either SEQ ID NO:22 or 23.

15. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody as described in any one of claims 1 to 14 and biotin conjugated to the antibody.

16. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody as described in any one of claims 1 to 14 and a label or solid-phase carrier conjugated to the antibody.

17. The antibody conjugate of claim 16, wherein, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

18. A reagent or kit characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 14 or the antibody conjugate as described in any one of claims 15 to 17.

19. Use of the antibody according to any one of claims 1 to 14, the antibody conjugate according to any one of claims 15 to 17, or the reagent or kit according to claim 18 in the preparation of products for detecting rotavirus.

20. Use according to claim 19, characterized in that, The uses include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the antibody of any one of claims 1 to 14, the antibody-drug conjugate of any one of claims 15 to 17, or the reagent or kit of claim 18 is contacted with rotavirus in the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of rotavirus in the sample to be tested.

21. Use according to claim 20, characterized in that, The immune complex further includes a second antibody, which binds to the antibody.

22. The use according to claim 20, characterized in that, The immune complex also includes a second antibody that binds to rotavirus.

23. A nucleic acid, comprising: It encodes the antibody as described in any one of claims 1 to 14.

24. A vector, comprising: It contains the nucleic acid as described in claim 23.

25. A cell, comprising: It contains the nucleic acid as described in claim 23 or the vector as described in claim 24.

26. A method of producing an antibody according to any one of claims 1 to 14, characterized in that, It includes: Culture the cells as described in claim 25.

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