Anti-rotavirus antibody and application thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application entitled “Anti-rotavirus antibody and its application” with application number 202311417515.7 filed with the China Patent Office on October 27, 2023, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the technical field of antibodies, and in particular to an anti-rotavirus antibody and an application thereof. Background Art

[0004] Rotavirus (RV) is a double-stranded RNA virus belonging to the Reoviridae family. Its genome consists of 11 unique RNA double helix molecules, with a total of 18,555 nucleotide base pairs. Each helix or segment is a gene, and is numbered from 1 to 11 in descending order according to the size of the molecule. Rotavirus is the most common cause of diarrhea in infants and young children. Almost every child experiences at least one rotavirus infection by the age of about five years. However, after each infection, the human body's immunity gradually increases, so the impact of subsequent infections will be reduced, and it is rarely affected in adults. Rotavirus infects the intestinal mucosal cells on the wall of the small intestine and produces enterotoxins, which can cause gastroenteritis, severe diarrhea, and sometimes even death due to dehydration. In addition to its impact on human health, rotavirus can also infect animals and is one of the pathogens of livestock.

[0005] Rotavirus is transmitted by the fecal-oral route, through contact with soiled hands, surfaces, and objects, and possibly by the respiratory route. Feces of an infected person may contain more than 10 trillion infectious virus particles per gram; only 10 to 100 of these particles can be transmitted to infect another person. Rotavirus is stable in the environment and has been found in samples from estuaries at concentrations of 1 to 5 infectious rotavirus particles per US gallon. Sanitation measures that eliminate bacteria and parasites appear to be ineffective in controlling rotavirus, as the incidence of rotavirus infection is similar in countries with high and low sanitation levels.

[0006] At present, the main method for detecting rotavirus is colloidal gold immunochromatography (Immunochromatography Assay), which is an immunological detection method based on antigen-antibody specific reaction. Its basic principle is: using colloidal gold to label an antigen or antibody, the corresponding paired antigen or antibody is coated on a nitrocellulose membrane, and when the sample is detected, the colloidal gold marker combines with the ligand in the sample to form a complex, and then moves upward through chromatography, combines with the coated antigen or antibody to condense and develop color, thereby realizing the determination of the sample detection result. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc.

[0007] The above immunological detection methods all require the preparation of antibodies against rotavirus. Therefore, there is a strong demand in this field for anti-rotavirus antibodies with good performance. Summary of the invention

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

[0009] In order to achieve the above-mentioned object, according to one aspect of the present invention, an anti-rotavirus antibody is provided, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 19 and 20, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23 and 24.

[0010] In order to achieve the above object, according to a second aspect of the present invention, an anti-rotavirus antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0011] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1 (NYWMH);

[0012] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2 (NIYPDSGVTNYAEKFKN);

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

[0014] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4 (KSSQSLFDSRTRKNYLA);

[0015] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5 (WASTRES); and

[0016] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 (KESYNLYT).

[0017] In order to achieve the above-mentioned object, according to the third aspect of the present invention, an anti-rotavirus antibody is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24.

[0018] In order to achieve the above-mentioned object, according to the fourth aspect of the present invention, an anti-rotavirus antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

[0019] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0020] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above antibody or the above antibody conjugate.

[0021] In order to achieve the above object, according to the eighth aspect of the present invention, there is provided a use of the above-mentioned antibody, antibody conjugate, reagent or kit in the preparation of a product for detecting rotavirus.

[0022] In order to achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The results of reducing SDS-PAGE of Anti-RV 10D5 Rmb1 to Anti-RV 10D5 Rmb3 are shown. DETAILED DESCRIPTION

[0025] In the first aspect, an embodiment of the present invention provides an anti-rotavirus antibody, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 19 and 20, and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23 and 24.

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

[0027] For example, the HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:19; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:23.

[0028] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific antibodies, 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 an 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. A person skilled in the art can easily understand, based on the contents described in the present invention, that the antigen-binding fragment of the above-mentioned antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the antigen-binding fragment of the above-mentioned antibody.

[0030] Antigen-binding fragments of antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0031] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0032] 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 region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0033] The definition methods of CDR are well known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also belong to the scope of protection of the present disclosure.

[0034] Table 1: CDR Definition 1

[0035]

[0036]

[0037] 1The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying 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. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0039] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0040] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0041] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0042] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

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

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

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

[0049] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are 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, an embodiment of the present invention provides an anti-rotavirus antibody, wherein the antibody comprises the following complementary determining regions:

[0052] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1 (NYWMH);

[0053] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2 (NIYPDSGVTNYAEKFKN);

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

[0055] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4 (KSSQSLFDSRTRKNYLA);

[0056] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5 (WASTRES); and

[0057] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 (KESYNLYT).

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

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

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

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

[0062] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

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

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

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

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

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

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

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

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

[0071] In an optional embodiment, the HFR3 comprises / is represented by the amino acid sequence shown in SEQ ID NO:17.

[0072] In an optional embodiment, the LFR1 comprises / is represented by the amino acid sequence shown in SEQ ID NO:18.

[0073] In a third aspect, an embodiment of the present invention provides an anti-rotavirus antibody, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24.

[0074] In an optional embodiment, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect are selected from any combination of the following:

[0075] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:19 SEQ ID NO:23 2 SEQ ID NO:20 SEQ ID NO:23 3 SEQ ID NO:19 SEQ ID NO:24 .

[0076] In an optional embodiment, the antibody described in the first aspect, the second aspect, and the third aspect further comprises a constant region.

[0077] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0078] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0079] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0080] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0081] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0082] In an alternative embodiment, the species of origin of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0083] In an alternative embodiment, the species origin of the constant region is mouse.

[0084] In an optional 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.

[0085] It should be noted that, 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% identity with the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0086] In an alternative embodiment, the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0087] In a fourth aspect, the present invention provides an anti-rotavirus antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

[0088] In an optional embodiment, the antibody of the first aspect, the second aspect, the third aspect or the fourth aspect comprises any combination of the following heavy chains and light chains:

[0089] combination Heavy chain Light chain 1 SEQ ID NO:21 SEQ ID NO:25 2 SEQ ID NO:22 SEQ ID NO:25 3 SEQ ID NO:21 SEQ ID NO:26

[0090] In a fifth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody.

[0091] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0092] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody.

[0093] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0094] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0095] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0096] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0097] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0098] In an optional embodiment, the radioactive isotopes 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.

[0099] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0100] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0101] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0102] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0103] In an optional embodiment, the colloidal metal is colloidal gold.

[0104] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0105] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0106] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0107] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0108] As mentioned above, the antibodies in some embodiments or examples of the present invention can effectively bind to rotavirus, so the reagent or kit containing the rotavirus antibody can effectively detect rotavirus qualitatively or quantitatively. The reagent or kit provided by the present invention can be used, for example, for detection involving the specific binding properties of rotavirus and its antibody, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with rotavirus, so the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0109] In a seventh aspect, the present invention provides a method for detecting rotavirus, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with rotavirus in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0111] In an alternative embodiment, the immune complex further comprises a second antibody that binds to rotavirus.

[0112] In an eighth aspect, the present invention 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.

[0113] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0114] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0115] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0116] In a twelfth aspect, the present invention provides a method for preparing anti-rotavirus antibodies, comprising: culturing the cells as described above.

[0117] Based on the amino acid sequence of the anti-rotavirus antibody disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-rotavirus antibody, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-rotavirus antibody of the present invention, it belongs to the protection scope of the present invention.

[0118] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0120] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0121] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0122] Example 1 Preparation of Anti-RV 10D5 Monoclonal Antibody

[0123] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-RV 10D5 monoclonal antibody was prepared in this laboratory and revived for future use.

[0124] (1) Antibody gene preparation

[0125] mRNA was extracted from the hybridoma cell line secreting Anti-RV 10D5 monoclonal antibody, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0126] (2) Sequence analysis of the variable region gene of Anti-RV 10D5 antibody

[0127] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 336bp, preceded by a 57bp leader peptide sequence; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 366bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.

[0128] (3) Construction of recombinant antibody expression plasmid

[0129] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.71kb Light Chain gene fragment and the 1.41kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0130] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0131] 2. Recombinant Antibody Production

[0132] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a proteinA affinity chromatography column. Take 6ug layer of purified antibodies for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0133] The obtained antibody was named Anti-RV 10D5Rmb1. Anti-RV 10D5Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0134] Table 2: Antibody sequences

[0135] Antibody Name Heavy chain Light chain Anti-RV 10D5Rmb1 SEQ ID NO:21 SEQ ID NO:25 Anti-RV 10D5Rmb2 SEQ ID NO:22 SEQ ID NO:25 Anti-RV 10D5Rmb3 SEQ ID NO:21 SEQ ID NO:26

[0136] Example 2 Performance testing of antibodies

[0137] 1. Activity identification

[0138] Coating solution (main component NaHCO3) diluted goat anti-mouse IgG 1ug / ml for microplate coating, 100uL per well, 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+NaCl), pat dry; add blocking solution (20% BSA+80% PBS), 120uL per well, 37℃, 1h, pat dry; add diluted purified antibody, 100uL / well, 37℃, 60min; shake off the liquid in the plate, pat dry, add 20% mouse negative blood for blocking, 120ul per well, 37℃, 1h; shake off the liquid in the plate , pat dry, add diluted positive specimen, 100uL per well, 37℃, 40min; wash 5 times with detergent, pat dry; add HRP-labeled anti-rotavirus monoclonal antibody (from Feipeng Bio, paired with purified antibody), 100uL per well, 37℃, 30min; add colorimetric solution A (50uL / well), add colorimetric solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on the microplate reader.

[0139] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0140] Table 3: Activity data

[0141] Concentration (ng / ml) 250 125 62.50 31.25 15.63 0.00 Comparison 1.129 0.652 0.337 0.128 0.057 0.023 Anti-RV 10D5Rmb1 1.749 1.230 0.773 0.432 0.223 0.082 Anti-RV 10D5Rmb2 1.896 1.389 0.959 0.574 0.292 0.067 Anti-RV 10D5Rmb3 1.772 1.471 1.098 0.701 0.371 0.033

[0142] 2. Stability assessment

[0143] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. Table 4 below shows the OD results of the enzyme immunoassay activity test of the antibody Anti-RV 10D5Rmb3 for 21 days.

[0144] Table 4: Stability data

[0145] Sample concentration (ng / ml) 125 62.50 0 4℃, 21-day samples 1.428 1.007 0.028 -80℃, 21 days sample 1.453 1.089 0.022 37℃, 21 days sample 1.466 1.023 0.031

[0146] 3. Colloidal gold platform performance testing

[0147] 3.1 Antibody labeling process

[0148] (1) Adjust the pH of the label: Take 10 ml of 4 / 10,000 colloidal gold, centrifuge, add 150 μl of 0.2 M K2CO3, and stir for 2 min;

[0149] (2) Conjugated antibody: add 200 μg of antibody Anti-RV-A (from Feipeng Bio) and stir for 15 min;

[0150] (3) Blocking: Add 10% (mass-volume ratio) BSA and stir for 15 min;

[0151] (4) Centrifuge and store at 10,000 rpm / 7 min / room temperature, remove the supernatant, resuspend to 1 ml with gold labeling solution, and store at 4°C for later use;

[0152] 3.2 Coating

[0153] (1) Assemble the nitrocellulose membrane and the colloidal gold PVC base plate and set aside;

[0154] (2) Antibodies Anti-RV 10D5Rmb1, Anti-RV 10D5Rmb2, Anti-RV 10D5Rmb3 and control antibody Anti-RV-B (from Feipeng Biotechnology) were diluted to 1.0-2.0 mg / ml, and lines were evenly drawn on the NC membrane using a gold spraying film drawing instrument, and then placed in a 50°C constant temperature oven for drying for at least 4 hours. The strips were assembled and tested with samples.

[0155] 3.3 Detection

[0156] (1) Sample: RV quality control products with different concentrations

[0157] (2) Detection method: Colloidal gold detection, observe the depth of the test line with the naked eye to determine the result. The depth of the color of the displayed strip can indicate the activity of the antigen and antibody binding in the sample. Compare the color of the T-line strip developed by the reaction of the colloidal gold test paper with the standard color card, select the closest color, and mark the activity of the product with the color number corresponding to the color.

[0158] The smaller the number, the stronger the color and the higher the activity; the higher the number, the weaker the color and the lower the activity; a “+” after the number means slightly stronger, a “-” after the number means slightly weaker, and “B” represents negative.

[0159] 3.4 Test results

[0160] The test results are shown in Table 5. The results show that the detection sensitivity of the antibodies Anti-RV 10D5Rmb1, Anti-RV 10D5Rmb2, and Anti-RV 10D5Rmb3 on the colloidal gold platform is better than that of the control antibody.

[0161] Table 5: Colloidal gold platform test results

[0162]

[0163] Some of the amino acid sequences involved in this application are shown in Table 6:

[0164] Table 6: Amino Acid Sequence Listing

[0165]

[0166]

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-rotavirus antibody, comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 19 and 20 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 23 and 24.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-rotavirus antibody, characterized in that: The antibody comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1 (NYWMH); HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2 (NIYPDSGVTNYAEKFKN); HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3 (EDYYGSSPAWFAY); LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4 (KSSQSLFDSRTRKNYLA); LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5 (WASTRES); and LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6 (KESYNLYT); Optionally, the antibody further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, 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.

4. An anti-rotavirus antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: ； Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

5. An anti-rotavirus antibody comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of a product for detecting rotavirus; Optionally, the use includes: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with the rotavirus in the sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to rotavirus.

9. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 5, wherein the nucleic acid encodes the antibody according to any one of claims 1 to 5; the vector contains a nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

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