Anti-influenza A virus nano antibody and application thereof

Abstract

The invention provides an anti-influenza A virus nano antibody and application thereof, the nano antibody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the CDR3 of the heavy chain variable region is selected from an amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 16. The anti-influenza A virus nano antibody can be combined with various influenza A viruses, including subtype influenza viruses such as H1N1, H2N2, H3N2 and H7N9, has strong broad spectrum, has different binding epitopes of the two nano antibodies, and can be applied to double antibody sandwich method detection, enzyme-linked immunosorbent assay and colloidal gold detection. According to the invention, multivalent transformation is also carried out on the nano antibody to obtain a dodecavalent hexamer cyclic structure nano antibody, so that the application scene of the anti-influenza A virus nano antibody can be further extended.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a nanobody against influenza A virus and its application. Background Art

[0002] Influenza (referred to as flu) is an acute respiratory infectious disease caused by influenza virus infection. According to the differences in viral nucleoprotein (NP) and matrix protein (MP), it is divided into four categories, namely influenza A virus (also known as type A influenza virus), influenza B virus (type B influenza virus), influenza C virus (type C influenza virus), and influenza D virus (type D influenza virus). Among them, influenza A and B viruses are more common.

[0003] All influenza A viruses are negative-sense single-stranded RNA viruses, and their genomes include 8 single-stranded RNA segments located within the virus particles. They encode RNA polymerase subunits, viral glycoprotein - haemagglutinin (HA), neuraminidase (NA), viral nucleoprotein (N protein), matrix protein (M1), membrane protein (M2), non-structural protein NS1, and nuclear export protein (NEP).

[0004] Influenza virus is a virus that mutates very easily, and the characteristics of its surface antigens also change with gene changes. Currently, 16 antigenically different haemagglutinin HA subtypes and 9 antigenically different neuraminidase NA subtypes have been identified in the laboratory, that is, there are 16 subtypes of H (H1 - H16) and 9 subtypes of N (N1 - N9). All known influenza A virus strains are composed of different combinations of HA and NA subtypes. Different subtypes of influenza A virus are usually named in the form of HxNx, such as H1N1, H3N2, H7N9, etc. Influenza A has a high pathogenicity to humans, and H1N1, H2N2, H3N2, etc. have caused worldwide pandemics many times. In addition, the H7N9 avian influenza virus has gradually attracted people's attention in recent years. H7N9 belongs to a new type of avian influenza, which is prone to break out in the poultry breeding industry, causing economic losses to the breeding industry. In recent years, it has been found that it can infect humans and cause respiratory diseases. Preventing and detecting influenza virus is of great significance to human health and the economic development of the livestock and poultry breeding industry. The HA and NA of influenza virus vary greatly among different subtypes and are not suitable as molecular targets for detecting and diagnosing influenza virus, while the N protein has relatively small variation and good conservation among subtypes. Developing specific antibodies against influenza N protein can be used for its detection and diagnosis.

[0005] Nanobodies are a novel type of antibody that has gradually attracted the attention of researchers in recent years for the development of therapeutic drugs and diagnostic antibodies. Nanobodies (Nb) mainly come from the variable domain VHH of heavy-chain antibodies in camelid animals. Nb consists of four conserved framework regions (FR) and three complementarity-determining regions (CDR). The number of amino acids in its CDR region is generally longer than that of the CDR3 of traditional human or murine antibodies, and can form a convex loop structure (traditional antibodies usually have concave or flat antigen-binding sites). There are a large number of CDR loops exposed in the solvent, which improves the specificity and affinity for antigen binding. Four hydrophilic residues in the FR2 region of Nb replace the four hydrophobic residues of the FR2 of traditional antibodies, resulting in higher solubility; the internal disulfide bonds make it more heat-resistant, acid- and alkali-resistant than traditional antibodies, with reduced polymerization. It still has biological activity after long-term placement in a high-temperature environment or under strong denaturing conditions, and has higher in vitro stability. It can be amplified and expressed in large quantities using prokaryotic and yeast systems, and the production cost is relatively lower. Nanobodies have a small molecular weight and do not require light-chain pairing. They are single-domain antibodies of the smallest antigen-binding fragments that exist naturally, which makes it extremely easy to structurally transform nanobodies into multivalent antibodies.

[0006] Most existing N antibodies against influenza A virus are obtained through hybridoma or rabbit single-cell technology, and most are traditional monoclonal antibodies. The production cost of this type of traditional antibody is relatively high, and the price is expensive, which is not conducive to large-scale promotion and application in pandemic infectious diseases.

[0007] Currently, most influenza A virus antibodies in patents are antibodies against a single subtype of N antigen. For example, patent CN103936852B is specifically against the H3N2 subtype, patent CN106188283A is specifically against H7N2, patent CN108303529A is specifically against the H7 subtype, and patent CN112079917A is specifically against the H7N9 subtype. There is no specific antibody that can simultaneously detect influenza virus subtypes such as H1N1, H2N2, H3N2, and H7N9. Therefore, developing a broad-spectrum influenza A virus N antibody with high specificity, high sensitivity, and high affinity has extremely high application value in the detection of influenza A virus. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an anti-influenza A virus nanobody and its application. The nanobody can specifically detect influenza virus subtypes such as H1N1, H2N2, H3N2, and H7N9, effectively avoiding phenomena such as "missed detection" in the detection of different subtypes of viruses. The anti-influenza A virus nanobody of the present invention is a broad-spectrum influenza A virus N antibody with high specificity, high sensitivity, and high affinity, and has extremely high application value in the detection of influenza A virus.

[0009] To achieve the purpose of this invention, the following technical solutions are adopted in the present invention:

[0010] In the first aspect, the present invention provides an anti-influenza A virus nanobody, the nanobody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2, and CDR3, and the CDR3 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:8 or SEQ ID NO:16.

[0011] The nanobody in the present invention can specifically bind to multiple subtypes of influenza viruses (including H1N1, H2N2, H3N2, H7N9, etc.), has broad-spectrum properties, and is conducive to detecting multiple subtypes of influenza viruses.

[0012] The antibody type in the present invention is a nanobody. Nanobody (Nb) is a novel antibody, which is the variable domain VHH of the heavy chain antibody naturally lacking the light chain in camelids. Due to the natural lack of the light chain and its small molecular weight, it can be amplified and expressed in large quantities using prokaryotic and yeast systems, and the production cost is relatively lower, which is conducive to popularization and application in infectious diseases such as pandemic viruses.

[0013] Four hydrophilic residues in the FR2 region of Nb replace four hydrophobic residues of the traditional antibody FR2, resulting in higher water solubility; the internal disulfide bonds make its heat resistance, acid and alkali resistance stronger than those of traditional antibodies, the polymerization is reduced, and it still has biological activity after long-term placement under high-temperature environments or strong denaturing conditions, with higher in vitro stability, which is more conducive to the development of antigen detection products that require room temperature conditions.

[0014] Preferably, the CDR1 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:6 or SEQ ID NO:14;

[0015] and / or, the CDR2 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:7 or SEQ ID NO:15.

[0016] Preferably, the heavy chain variable region further comprises FR1, FR2, FR3, and FR4.

[0017] Preferably, the FR1 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:2 or SEQ ID NO:10.

[0018] Preferably, the FR2 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:3 or SEQ ID NO:11.

[0019] Preferably, the FR3 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:4 or SEQ ID NO:12.

[0020] Preferably, the FR4 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:5 or SEQ ID NO:13.

[0021] Preferably, the CDR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:6, CDR2 is as shown in SEQ ID NO:7, and CDR3 is as shown in SEQ ID NO:8; the FR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:2, FR2 is as shown in SEQ ID NO:3, FR3 is as shown in SEQ ID NO:4, and FR4 is as shown in SEQ ID NO:5;

[0022] Or, the CDR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:14, CDR2 is as shown in SEQ ID NO:15, and CDR3 is as shown in SEQ ID NO:16; the FR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:10, FR2 is as shown in SEQ ID NO:11, FR3 is as shown in SEQ ID NO:12, and FR4 is as shown in SEQ ID NO:13.

[0023] Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO:1 or SEQ ID NO:9.

[0024] In a second aspect, the present invention provides a multimeric antibody structure, which is composed of the anti-influenza A virus nanobody described in the first aspect, an hIgG-FC fragment, and a μtp amino acid sequence, and the amino acid sequence of the multimeric antibody structure is as shown in any one of SEQ ID NO:17-22.

[0025] Preferably, the multimeric antibody structure includes a dimeric antibody structure and a hexameric antibody structure.

[0026] The present invention performs multivalent modification on nanobodies to obtain a new multivalent nanobody structure, which has a structure similar to the hexamer-like structure of IgM antibodies and is suitable for some application scenarios that require the detection of large structural molecules; at the same time, it has the structural characteristics of the Fc region of IgG antibodies, is easier to express and prepare than traditional IgM antibodies, and is applicable to most scenarios where IgG antibodies are used.

[0027] The nanobodies involved in the present invention, in whatever form of genetic engineering modification or derivative form they appear, include but are not limited to the following modifications: amino acid mutation, small molecule structure modification, humanization, bispecific nanobody modification, bivalent or multivalent nanobody modification, coupling with other structural proteins or chemical reagents, etc. Derivatives related thereto are all within the protection scope of the present invention.

[0028] The amino acid sequence of the nanobody VHH1 against influenza A virus is SEQ ID NO:1:

[0029] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXS TYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYW GQGTQVTVSS.

[0030] The specific amino acid sequences of framework regions 1-4 (FR1-4) and complementarity-determining regions (CDR1-3) of the nanobody VHH1 against influenza A virus are shown as follows:

[0031] The sequence of FR1 is SEQ ID NO:2: QVQLQESGGGLVQPGGSLRLSCVAS.

[0032] The sequence of FR2 is SEQ ID NO:3: MGWYRQAPGKQRELVAR.

[0033] The sequence of FR3 is SEQ ID NO:4:

[0034] TYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYC.

[0035] The sequence of FR4 is SEQ ID NO:5: WGQGTQVTVSS.

[0036] The sequence of CDR1 is SEQ ID NO:6: RXISSRXV.

[0037] The sequence of CDR2 is SEQ ID NO:7: ITGXGXS.

[0038] The sequence of CDR3 is SEQ ID NO:8: NAXDYYTDXDDPLRDXRDY.

[0039] The amino acid sequence of the influenza A virus nanobody VHH2 is SEQ ID NO:9:

[0040] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVT VSS.

[0041] The specific amino acid sequences of framework regions 1-4 (FR1-4) and complementarity-determining regions (CDR1-3) of the influenza A virus nanobody VHH2 are shown below:

[0042] The sequence of FR1 is SEQ ID NO:10: QVQLQESGGGLAQPGGSLRLSCTAS.

[0043] The sequence of FR2 is SEQ ID NO:11: MGWYRQAPGKQRELVAY.

[0044] The sequence of FR3 is SEQ ID NO:12:

[0045] EYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYC.

[0046] The sequence of FR4 is SEQ ID NO:13: WGRGTQVTVSS.

[0047] The sequence of CDR1 is SEQ ID NO:14: XSIFDXYH.

[0048] The sequence of CDR2 is SEQ ID NO:15: IXSSGXT.

[0049] The sequence of CDR3 is SEQ ID NO:16: SXPAGGVXYDSAXVY.

[0050] The amino acid sequences of the hexameric dodecavalent cyclic nanobody are shown in VHH1-IgGμtp and VHH2-IgGμtp.

[0051] The amino acid sequences corresponding to VHH1-IgGμtp include 3 types, including SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0052] Mutation of the 140th amino acid C to amino acid S in SEQ ID NO:17:

[0053] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPTLYNVSLVMSDTAGTCY。

[0054] Mutation of the 365th amino acid P to T in SEQ ID NO:18:

[0055] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0056] The 140th amino acid C of SEQ ID NO:19 is mutated to amino acid S, and the 365th amino acid P is mutated to T:

[0057] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0058] The amino acid sequences corresponding to VHH2-IgGμtp include three types, including SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0059] The 136th amino acid C of SEQ ID NO:20 is mutated to amino acid S:

[0060] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPTLYNVSLVMSDTAGTCY。

[0061] SEQ ID NO:21 (amino acid 361) P mutated to T:

[0062] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0063] SEQ ID NO:22 (amino acid 136) C mutated to amino acid S; (amino acid 361) P mutated to T:

[0064] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0065] The present invention uses N proteins of multiple different subtypes of influenza A virus to cross - immunize alpacas, and detects and evaluates the antibody titers of alpaca sera. After the immunization, peripheral blood of alpacas is collected, and plasma and peripheral blood lymphocytes (PBMCs) are isolated. Total RNA of PBMCs is extracted and reverse - transcribed into cDNA, and then alpaca nanobodies (VHHs) are amplified by multiplex PCR, and a nanobody phage library is constructed. Then, through the method of multi - antigen cross - panning, nanobodies against influenza A virus N protein with high affinity and broad - spectrum binding activity are screened. The nanobodies are modified into multivalent forms, and the binding specificity, broad - spectrum binding, binding affinity and colloidal gold detection application are evaluated.

[0066] In a third aspect, the present invention provides a nucleic acid molecule that encodes the anti - influenza A virus nanobody described in the first aspect, or encodes the multimeric - structure antibody described in the second aspect.

[0067] In a fourth aspect, the present invention provides a composition for detecting influenza A virus, which composition comprises the anti - influenza A virus nanobody described in the first aspect, or the multimeric - structure antibody described in the second aspect.

[0068] In a fifth aspect, the present invention provides a colloidal gold test strip for detecting influenza A virus, and the colloidal gold test strip contains the multimeric - structure antibody described in the second aspect.

[0069] Preferably, the multimeric - structure antibody is a hexameric - structure antibody.

[0070] Preferably, the hexameric - structure antibody is used as a capture antibody and a detection antibody respectively for double - antibody sandwich detection.

[0071] In a sixth aspect, the present invention provides a kit for detecting influenza A virus, and the kit comprises any one or a combination of at least two of the anti - influenza A virus nanobody described in the first aspect, the multimeric - structure antibody described in the second aspect, or the colloidal gold test strip for detecting influenza A virus described in the fifth aspect.

[0072] The present invention also provides the biological activity characteristics of the above-mentioned nanobodies, including data such as antibody binding specificity, binding broad-spectrum, binding affinity, epitope detection, colloidal gold and enzyme-linked immunosorbent assay applications, etc. The above-mentioned nanobodies have potential application value in the detection and diagnosis of influenza A, epidemiological research and the detection and diagnosis of avian influenza.

[0073] In the seventh aspect, the present invention provides the use of any one or at least two combinations of the anti-influenza A virus nanobody described in the first aspect, the multimeric antibody structure described in the second aspect, the nucleic acid molecule described in the third aspect, the composition for detecting influenza A virus described in the fourth aspect, and the colloidal gold test strip for detecting influenza A virus described in the fifth aspect in the preparation of products for influenza A detection and diagnosis.

[0074] The numerical ranges described in the present invention not only include the above-listed point values, but also include any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] (1) The nanobodies of the present invention are screened by the method of multi-antigen cross-immunization and panning. The influenza A virus N antigen nanobodies have specific and broad-spectrum binding activities and can specifically bind to multiple subtypes of influenza A virus (not limited to the following subtypes: H1N1, H2N2, H3N2, and H7N9, etc.), avoiding phenomena such as missed detection in clinical tests. It has an application theoretical basis both in scientific research and clinical industrial applications.

[0077] (2) The nanobodies of the present invention can be highly expressed through prokaryotic cells. Compared with the expression of traditional antibodies in eukaryotic mammalian cells, their production cost is lower, which is more conducive to popularization and application in the detection of such infectious diseases.

[0078] (3) The present invention provides a dodecavalent hexameric nanobody and a method for its eukaryotic preparation. The dodecavalent hexameric nanobody in the present invention has both the properties of traditional IgM antibody multimerization and retains the functions of the Fc fragment of traditional IgG antibody. It can be simply prepared by protein A or Protein G purification methods, and the preparation and purification are simpler, increasing the potential application scenarios of nanobodies, such as IgM antibody detection, colloidal gold IgM antibody detection and diagnosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is the SDS-PAGE electrophoresis detection and purification result of the nanobody.

[0080] Figure 2 These are the results of ELISA for detecting the binding affinity of nanobodies.

[0081] Figure 3 These are the results of analysis on binding specificity and broad spectrum.

[0082] Figure 4 These are the results of electrophoresis detection.

[0083] Figure 5 These are the results of SEC separation.

[0084] Figure 6 These are the results of Dot-blot detection.

[0085] Figure 7 These are the schematic diagrams of the structure of colloidal gold test strips.

[0086] Figure 8 These are the results of colloidal gold detection applications. Specific Embodiments

[0087] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0088] For those technical or conditions not specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0089] Example 1 Multiple Antigen Cross-Immunization

[0090] Take 200 μg of H7N9 and 2000 μg of H1N1 N full-length antigens (Sino Biological) and mix them with an equal volume of Freund's complete adjuvant (Sigma). Inject them subcutaneously at multiple points near the front limbs of the alpaca's neck for the first immunization. Then immunize once every 2 weeks for a total of 4 times. Freund's incomplete adjuvant (Sigma) is used for the 2nd - 4th immunizations. On the 7th day after the end of immunization, collect 20 - 30 mL of peripheral blood from the alpaca's vein, and separate the plasma and PBMC samples for subsequent construction of the immune library.

[0091] Example 2 Detection of Plasma Titer

[0092] Take 100 ng of the equally mixed influenza A virus H1N1 and H7N9 N antigens to coat the ELISA plate and incubate overnight at 4°C. Wash 3 times with PBST (0.05%). Add 200 μL of 2% BSA to each well and incubate at room temperature for 2 hours. Wash 3 times with PBST. Dilute the plasma before and after immunization in Example 1 by 10 4 、10 5 、106 , 10 7 and 10 8 times, and then add it to the corresponding ELISA plate and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 100 μL of anti-alpaca H&L IgG HRP (Abcam) diluted 2500 times to each well, and place it in the dark at room temperature for 1 hour. Wash 5 times with PBST, add 100 μL of TMB chromogenic solution (abcam), develop color for 10 minutes, add an equal volume of TMB stop buffer (abcam) to terminate the color development, and read the OD450 value.

[0093] The ELISA test results are shown in (Table 1). After immunization, the plasma titer increased significantly. When the plasma was diluted 10 7 times, the OD450 value of the antibody detected after immunization was 2.93 times that before immunization, and the immunized plasma titer reached 10 7 , indicating that after immunization, the alpaca has obtained abundant antibodies against the N antigen of influenza A virus in its body.

[0094] Table 1

[0095]

[0096]

[0097] Note: Positive judgment: Average OD450 after immunization / Average OD450 before immunization ≥ 2.1.

[0098] Example 3 Obtaining Nanobodies

[0099] Take the blood sample of the immunized alpaca, and isolate the lymphocytes in the peripheral blood of the alpaca according to lymphocyte separation medium (GE, 17-1440-02) and density gradient centrifugation method. Extract RNA from the lymphocytes with reference to the instructions of Trizol reagent (INVITROGEN, 15596-018). Then take 20 μg of RNA, and according to the kit (the commodity name of the kit is SuperScript TMIII First-Strand Synthesis SuperMix (Invitrogen)) and the process to synthesize cDNA. Then, using the reverse transcription product cDNA as a template, construct a library according to the phage library construction and screening process (refer to the phage library construction and screening process of researchers such as Vincke C, Generation of singledomain antibody fragments derived from camelids and generation of manifold constructs (doi:10.1007 / 978-1-61779-974-7_8.PMID:22907350)), and obtain a clone library with a library capacity of about 10 7 , and a clone library with an abundance greater than 93%. Use the N antigens of influenza H7N9 and H1N1 to perform 2-3 rounds of panning enrichment on the phage library. Select clones from the affinity-enriched library for antigen affinity clone ELISA detection, and screen the positive reaction well clone bacteria for Sanger sequencing (Sanger sequencing) to identify 2 specific nanobodies (VHH1 and VHH2).

[0100] The amino acid sequence of the influenza A virus nanobody VHH1 is SEQ ID NO:1:

[0101] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSS.

[0102] The amino acid sequence of the influenza A virus nanobody VHH2 is SEQ ID NO:9:

[0103] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSS.

[0104] Example 4 Induced Expression and Purification of Nanobodies

[0105] (1) Induced Expression of Nanobodies

[0106] Separate monoclonal nanobodies from Example 3 were inoculated into 10 mL of ampicillin-containing medium and cultured overnight at 37°C with 220 rpm. The next day, 2 mL of the overnight culture was inoculated into 200 mL of ampicillin-containing medium and cultured at 37°C with 220 rpm until the logarithmic phase (OD600 was 0.6 - 0.8), and IPTG was added for overnight induction of nanobody expression. The next day, the cell pellet was collected. After disrupting the cells by the hypotonic method, the supernatant was collected by high-speed centrifugation for subsequent protein purification.

[0107] (2) Purification of nanobodies

[0108] Affinity purification using His-nickel filler (abbreviated as Ni filler, BioRad) was used to obtain purified nanobodies. The Ni filler was packed into a column, first washed with ultrapure water, and then washed with the equilibration buffer PBS; the above-mentioned disrupted supernatant was added to the purification column at a flow rate of 1 mL / min; an appropriate volume of PBS was used to wash away the miscellaneous proteins until the OD280 was below 0.0001; then the target protein was eluted with 10 volumes of the elution buffer (150 mM imidazole). For the purified target protein, an equal volume was taken and detected by 12% SDS-PAGE for the expression and purification of nanobodies. Figure 1 For the SDS-PAGE electrophoresis detection and purification results of nanobodies, in the figure, Lane 1: VHH1 nanobody; Lane 2: VHH2 nanobody; Lane M: protein ladder. From Figure 1 It can be seen that the size of the nanobody band is about 15 KD and the purity > 95%.

[0109] Example 5 ELISA analysis of the binding affinity of nanobodies to influenza A virus N antigen

[0110] 100 ng of H1N1 N antigen was used to coat the ELISA plate at 4°C overnight. Washed 3 times with PBST (0.05%). 200 μL of 2% BSA was added to each well and incubated at room temperature for 2 hours. Washed 3 times with PBST. The nanobodies expressed and prepared in Example 4 and a COVID-19 nanobody (NC) were serially diluted. The starting concentration was 3 μg / mL (~200 nM), and serially diluted 5-fold gradient, with a total of 8 concentration gradients, added to the wells, and each concentration gradient was repeated in 3 wells, 100 μL per well, and incubated at room temperature for 1 hour. Washed 6 times with PBST, added 100 μL of anti HA, HRP (abcam) diluted 2000 - 3000 times per well, and placed at room temperature for 1 hour. Washed 6 times with PBST, added 100 μL of TMB chromogenic solution (abcam), developed color for 10 minutes, and added an equal volume of TMB stop buffer (abcam) to stop color development and read the OD450 value.

[0111] Figure 2This is the detection result of the binding affinity of nanobodies by ELISA. In the figure, NC: negative control. The ELISA detection results show that both VHH1 and VHH2 nanobodies have good affinity activities for the N antigen of influenza A virus. The half-maximal effective concentration (EC50) of VHH1 and VHH2 are 0.009127 μg / mL (0.6085 nM) and 0.006305 μg / mL (0.4204 nM) respectively, and their affinities are both at the sub-nanomolar level.

[0112] Example 6 ELISA analysis of the binding specificity and broad-spectrum binding of influenza nanobodies

[0113] Add 50 ng of N antigens of four subtypes of influenza A (Sino Biological) and 2 types of N antigens of influenza B to each well of the ELISA plate, 50 μL per well. Wash 3 times with PBST, add 2% BSA for blocking, at room temperature for 2 hours. Wash 3 times with PBST, add 0.75 μg / mL of the 2 nanobodies in Example 4, 50 μL per well, repeat 2 wells; 1 type of nanobody against SARS-CoV-2 as a negative control; PBST as a blank well control. Incubate at room temperature for 1 hour. Wash 5 times with PBST, add 100 μL of anti-HA, HRP (abcam) diluted 2000 - 3000 times per well, and place at room temperature for 1 hour. Wash 6 times with PBST, add 100 μL of TMB chromogenic solution (abcam), develop color for 10 minutes, and add an equal volume of TMB stop buffer (abcam) to stop color development. Finally, place the ELISA plate in a microplate reader (BioTek) for reading.

[0114] The analysis results of binding specificity and broad-spectrum binding are as Figure 3 shown, Figure 3 where NC is the negative control; PBST is the blank well control. Figure 3 The results show that the 2 nanobodies in this example can specifically bind to the N antigens of 4 subtypes of influenza (H1N1, H2N2, H3N2 and H7N9), with strong broad-spectrum binding; at the same time, the 2 nanobodies do not bind to the N antigen of influenza B, with high binding specificity.

[0115] Example 7 Monovalent nanobody multivalent modification, eukaryotic expression and purification

[0116] In this embodiment, the nanobody is transformed into a dodecavalent hexameric cyclic structure, and the antibody is composed of a VHH sequence, a human hIgG-FC, and a μtp amino acid sequence. After expression in 293 cells, the fusion protein composed of these three parts forms a hexameric dodecavalent cyclic nanobody structure. The amino acid sequences are shown in VHH1-IgGμtp and VHH2-IgGμtp. Among them, the sequences of the nanobody monomer, dimer, and hexamer structures are all VHH1-IgGμtp and VHH2-IgGμtp. That is, after these two sequences are expressed, hexamers (mostly), dimers, and monomers (a small part) will be produced, and the aggregation form is the result of the interaction of disulfide bonds between antibody molecules.

[0117] There are 3 amino acid sequences corresponding to VHH1-IgGμtp, including SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

[0118] SEQ ID NO:17 contains one mutation, where the (140th amino acid) C is mutated to the amino acid S.

[0119] SEQ ID NO:18 contains one mutation, where the (365th amino acid) P is mutated to T.

[0120] SEQ ID NO:19 contains two mutations, where the (140th amino acid) C is mutated to the amino acid S, and the (365th amino acid) P is mutated to T.

[0121] The mutation of sequence SEQ ID NO:17 is C140S. Its advantage is that the C amino acid residue at position 140 in the hinge region normally forms a disulfide bond with the C amino acid at the end of the light chain of a traditional antibody. Since there is no light chain structure in this antibody structure, mutating the amino acid residue C at position 140 to S can prevent the appearance of the C amino acid residue in the absence of the light chain and prevent non-specific aggregation caused by the formation of disulfide bonds between antibodies.

[0122] The mutation of SEQ ID NO:18 is P365T. Its advantage is that after mutating the P amino acid at position 365 to the T amino acid, the last 3 amino acids TCY of this antibody sequence are the same as the last 3 amino acids TCY of the IgM antibody sequence, which is beneficial to the stable formation of a natural multimeric antibody structure like IgM.

[0123] The mutations of SEQ ID NO:19 are C140S and P365T. Its advantage is that both mutations are beneficial to the formation of a multimeric structure like IgM and avoid the unstable state of non-specific aggregation caused by the appearance of free C amino acids in the antibody hinge region in the absence of a light chain structure.

[0124] The (140th amino acid) C of SEQ ID NO:17 is mutated to the amino acid S:

[0125] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPTLYNVSLVMSDTAGTCY。

[0126] Mutation of P to T at amino acid 365 (SEQ ID NO:18):

[0127] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0128] Mutation of C to S at amino acid 140 and mutation of P to T at amino acid 365 (SEQ ID NO:19):

[0129] QVQLQESGGGLVQPGGSLRLSCVASRXISSRXVMGWYRQAPGKQRELVARITGXGXSTYADSVKGRFFVSRDNNKSTVYLQMNNLSPEDTAVYYCNAXDYYTDXDDPLRDXRDYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0130] The amino acid sequences corresponding to VHH2-IgGμtp include three kinds, including SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0131] SEQ ID NO:20 contains one mutation, where C (the 136th amino acid) mutates to amino acid S.

[0132] SEQ ID NO:21 contains one mutation, where P (the 361st amino acid) mutates to T.

[0133] SEQ ID NO:22 contains two mutations, where C (the 136th amino acid) mutates to amino acid S; P (the 361st amino acid) mutates to T.

[0134] SEQ ID NO:20, C (the 136th amino acid) mutates to amino acid S:

[0135] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPTLYNVSLVMSDTAGTCY。

[0136] SEQ ID NO:21 (amino acid 361) P mutated to T:

[0137] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0138] SEQ ID NO:22 (amino acid 136) C mutated to amino acid S; (amino acid 361) P mutated to T:

[0139] QVQLQESGGGLAQPGGSLRLSCTASXSIFDXYHMGWYRQAPGKQRELVAYIXSSGXTEYGDAVKGRFTISRDTAKNTVYLQMDSLKPEDTAVYYCSXPAGGVXYDSAXVYWGRGTQVTVSSASNTKVDKKVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0140] The nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:22 were gene-synthesized into the expression vector pcDNA3.1 vector. The recombinant plasmid was diluted with OPM-293-CD05 medium (OPM) and slowly added dropwise to the PEI (polyethylenimine) solution required for transformation. After mixing by inverting up and down, it was incubated for 15 minutes, and then the mixture was slowly added dropwise to HEK293 cells for expression. It was cultured in an incubator under the culture conditions of 37 °C, 8% CO2, relative humidity ≥80%, and 110 rpm. After 24 hours, 4% by volume of the SMS293-SUPI medium additive (Sino Biological) was added to the cultured cell solution. After 72 hours, 0.5% was added continuously. After culturing for 5 days, the transient expression culture supernatant was collected. The supernatant was used for subsequent protein purification. The supernatant was first purified by a protein A affinity chromatography column to obtain the eluates of VHH1-IgGμtp and VHH2-IgGμtp. The purified eluates were detected by non-reducing electrophoresis under the electrophoresis conditions of 120 V and 90 minutes. The electrophoresis detection results are as Figure 4 shown, Figure 4 in which lane 1 is VHH1-IgGμtp; lane 2 is VHH-IgGμtp, and lane M is the protein ladder. The results showed that the purified antibody had three components, namely dodecavalent hexameric nanobody molecules, dimeric nanobody molecules, and monomeric nanobody molecules. Among them, the hexameric nanobody molecules accounted for the main part.

[0141] After that, the hexameric nanobody molecules without other polymers were obtained by molecular sieve separation and purification. The SEC separation results are as Figure 5As shown, the molecular sieve results showed that the separation positions of the two purified hexameric antibody molecules were at 9.43 and 9.41 mL respectively, corresponding to a molecular weight of approximately 480 KD, which was consistent with the theoretical size, indicating that the separated products were hexameric antibodies (VHH1-IgGμtp and VHH2-IgGμtp); the elution positions of the dimers and monomers were near 12.35 mL (VHH1-FC and VHH2-FC). The final transient transfection expression yields of the two antibodies were approximately 86 mg / L.

[0142] Example 8 Dot-blot Detection of the Epitopes Bound by Nanobodies

[0143] After activating two PVDF membranes in methanol, 10 μL of the monomeric antibody molecules VHH1-FC and VHH2-FC separated by SEC in Example 7 were respectively added dropwise onto the two membranes, and then they were respectively placed in separate containers containing 2% BSA solution and incubated for 2 hours for blocking. The membranes were washed 3 times with PBST. Influenza A virus H1N1 N antigen (abbreviated as influenza N antigen) with a dilution concentration of 3 μg / mL was added to both containers and incubated at room temperature for 2 hours. After washing 3 times with PBST, VHH1 and VHH2 nanobodies prepared by prokaryotic expression from Example 4 with a concentration of 3 μg / mL were respectively added to the two containers and incubated at room temperature for 2 hours. After washing 3 - 5 times with PBST, anti HA, HRP (abcam) diluted 1000-fold was added and incubated in the dark at room temperature for 2 hours. The membranes were washed 5 times with PBST, the water on the surface of the membranes was drained, developing solution (BIO-RAD) was added and developed for photography. The Dot-blot detection results are as Figure 6 shown. In the figure, "+" indicates a positive development; " / " indicates the antibody self-binding control. The results showed that after the influenza N antigen bound to VHH1-FC or VHH2-FC on the membrane, the epitopes bound by the influenza N antigen were respectively occupied by VHH1-FC and VHH2-FC. After adding VHH1 and VHH2 nanobodies respectively for the second time, the VHH1-FC-N antigen complex could be bound by the VHH2 nanobody again, showing a black developed print, and the print was significantly stronger than the binding to its own VHH1; the VHH2-FC-N antigen complex could be bound by the VHH1 nanobody again, indicating that VHH1 and VHH2 nanobodies could bind to different epitopes of the influenza N antigen. The two antibodies can be applied to the sandwich ELISA detection.

[0144] Example 9 Application of Colloidal Gold Test Strips

[0145] According to the results of Example 8, since VHH1 and VHH2 bind to different epitopes of the influenza N antigen, the principle of the sandwich ELISA method can be combined with the colloidal gold detection method to detect influenza virus proteins.

[0146] First, take VHH1-IgGμtp and super C+ (Biosino) from Example 7, dilute them to 1 mg / mL, and draw lines on a CN110 nitrocellulose membrane (Baisui Kang) using an XYZ scribing instrument (Goldmark Biotech). The parameter is 1 μL / cm. Coat the T line with VHH1-IgGμtp and the C line with super C+. The two antibodies correspond to the T line and the C line respectively. Place the NC membrane in an oven and dry it at 45 °C for 1 - 2 hours.

[0147] Take an appropriate amount of CG40 colloidal gold solution (Shenbaiao), add 13 mL of 0.1 M K2CO3 per milliliter to adjust its pH, and then add an appropriate amount of VHH2-IgGμtp to make the final concentration 10 μg / mL. After thorough mixing, let it stand for 10 - 15 minutes. Then add 40 - 50 μL of 10% BSA solution with pH = 7.0 per milliliter for blocking, and let it stand at room temperature for 10 minutes. Centrifuge at 8000 g for 7 minutes. Carefully discard the supernatant, taking care not to touch the gold precipitate, and then add an appropriate amount of colloidal gold washing buffer, 1 - 2 mL, mix well to wash the gold precipitate. Centrifuge at 8000 g for 7 min, carefully discard the supernatant, and then add half of the starting volume of the colloidal gold resuspension solution to resuspend the gold precipitate. Finally, dot the gold resuspension solution on the glass fiber membrane and air-dry it at room temperature.

[0148] Add the prepared NC membrane and gold conjugate pad, add the sample pad and absorbent pad, and prepare a colloidal gold test strip according to the Figure 7 form combination.

[0149] Dilute the H1N1 virus N protein to 500 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 5 ng / mL, and 0 ng / mL, and dot them on the sample pad of the colloidal gold test strip in sequence. After 15 minutes, observe the color development of the C line and T line on the NC membrane. The application results of the colloidal gold detection are as Figure 8 shown. The results show that the C line shows normal color development. Using 0 ng / mL as a blank control, no non-specific binding occurred, indicating that the experimental results are normal and the data are accurate; the T line shows that the colloidal gold test strip can detect as low as 5 ng / mL.

[0150] In summary, the present invention uses N proteins of multiple different subtypes of influenza virus to cross-immunize alpacas, and detects and evaluates the antibody titers of alpaca sera. After the immunization, the peripheral blood of the alpaca is collected, and plasma and peripheral blood lymphocytes are isolated. The total RNA of the peripheral blood lymphocytes is extracted and reverse-transcribed into cDNA, and then the alpaca nanobodies are amplified by multiplex PCR, and the nanobody phage library is constructed. Then, by the method of multi-antigen cross-selection, the N nanobodies of influenza A virus with high affinity and broad-spectrum binding activity are screened. And the multivalent modification, binding specificity, binding broad-spectrum, binding affinity and colloidal gold detection application evaluation of the nanobodies are carried out. The production cost of the nanobodies is low, and they have extremely high application value in the detection of influenza A virus.

[0151] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An anti-influenza A virus nanobody, characterized in that, The nanobody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the CDR3 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:8 or SEQ ID NO:

16.

2. The nanobody against influenza A virus according to claim 1, wherein The CDR1 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:6 or SEQ ID NO:14; and / or, the CDR2 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:7 or SEQ ID NO:

15.

3. The nanobody against influenza A virus according to claim 1 or 2, characterized in that, The heavy chain variable region further comprises FR1, FR2, FR3 and FR4; Preferably, the FR1 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:2 or SEQ ID NO:10; Preferably, the FR2 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:3 or SEQ ID NO:11; Preferably, the FR3 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:4 or SEQ ID NO:12; Preferably, the FR4 of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO:5 or SEQ ID NO:

13.

4. The nanobody against influenza A virus according to any one of claims 1-3, characterized in that, The CDR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:6, CDR2 is as shown in SEQ ID NO:7, and CDR3 is as shown in SEQ ID NO:8; the FR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:2, FR2 is as shown in SEQ ID NO:3, FR3 is as shown in SEQ ID NO:4, and FR4 is as shown in SEQ ID NO:5; or, the CDR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:14, CDR2 is as shown in SEQ ID NO:15, and CDR3 is as shown in SEQ ID NO:16; the FR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:10, FR2 is as shown in SEQ ID NO:11, FR3 is as shown in SEQ ID NO:12, and FR4 is as shown in SEQ ID NO:

13.

5. The nanobody against influenza A virus according to any one of claims 1-4, characterized in that, The amino acid sequence of the nanobody is as shown in SEQ ID NO:1 or SEQ ID NO:

9.

6. A polymeric structure antibody, characterized in that, The multimeric antibody is composed of the anti-influenza A virus nanobody according to any one of claims 1-5, an hIgG-FC fragment and a μtp amino acid sequence, and the amino acid sequence of the multimeric antibody is as shown in any one of SEQ ID NO:17-22; Preferably, the multimeric antibody comprises a dimeric antibody and a hexameric antibody.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-influenza A virus nanobody according to any one of claims 1-5, or encodes the multimeric antibody according to claim 6.

8. A composition for detecting influenza A virus, characterized in that, The composition comprises the anti-influenza A virus nanobody according to any one of claims 1-5 and / or the multimeric antibody according to claim 6.

9. A colloidal gold test strip for detecting influenza A virus, characterized in that, The colloidal gold test strip contains the antibody with a multimeric structure as claimed in claim 6; Preferably, the antibody with a multimeric structure is an antibody with a hexameric structure; Preferably, the antibody with a hexameric structure is used as a capture antibody and a detection antibody respectively for double antibody sandwich assay.

10. A kit for detecting influenza A virus, characterized in that, The kit contains any one or a combination of at least two of the anti-influenza A virus nanobody as claimed in any one of claims 1-5, the antibody with a multimeric structure as claimed in claim 6, or the colloidal gold test strip for detecting influenza A virus as claimed in claim 9.

11. Use of any one or a combination of at least two of the anti-influenza A virus nanobody as claimed in any one of claims 1-5, the antibody with a multimeric structure as claimed in claim 6, the nucleic acid molecule as claimed in claim 7, the composition for detecting influenza A virus as claimed in claim 8, or the colloidal gold test strip for detecting influenza A virus as claimed in claim 9 in the preparation of a product for influenza A detection and diagnosis.

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