Anti-influenza B virus nano antibody and application thereof

Abstract

The invention provides an anti-influenza B 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 nano antibody is obtained through a multi-antigen cross immunization and panning method, the nano antibody can be specifically combined with Colorado / 06 / 2017 and Florida / 4 / 2006 influenza B viruses, the sensitivity is high, the specificity is high, and the phenomenon of missing detection in clinical detection can be avoided in clinical application. The nano antibody can be efficiently expressed through prokaryotic cells, and compared with eukaryotic mammalian cell expression of a traditional antibody, the nano antibody is lower in production cost and more beneficial to popularization and application in detection of infectious diseases.

Description

Technical Field

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

[0002] Influenza (abbreviated 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] Influenza B virus is a negative-sense single-stranded RNA virus, and its genome includes 8 single-stranded RNA segments located within the virus particle. They encode RNA polymerase subunits, viral glycoprotein - haemagglutinin (HA), neuraminidase (NA), viral nucleoprotein (nucleoprotein, N protein), matrix protein (M1), membrane protein (M2), non-structural protein NS1, and nuclear export protein (NEP). The outer layer of the influenza virus is composed of two different glycoproteins to form radial protrusions, namely haemagglutinin HA and neuraminidase NA. Mutations in two genes encoding this antigenic structure cause antigenic structure variation, thus leading to the variation of influenza virus and the emergence of new subtypes. Influenza A virus mutates relatively fast, and many subtypes have been discovered so far. Influenza B virus also has mutations, but has not been divided into subtype transitions. Both influenza A and B viruses can cause human respiratory system diseases. Compared with influenza A virus, the infectivity of influenza B virus is relatively small and has not caused a worldwide pandemic yet, but the prevention and detection of this virus still cannot be ignored. The N protein of influenza B virus has good conservation and relatively small mutations, and is an important target molecule for specific detection and diagnosis of influenza B virus.

[0004] In the field of antibody detection and diagnosis, IgG - structured antibodies developed based on traditional antibody Fab fragments occupy a mainstream position. Nanobodies are a type of novel antibody that have gradually attracted the attention of researchers in recent years in the development of therapeutic drugs and detection - diagnostic antibodies. Nanobodies (Nb) mainly come from the variable domain VHH of heavy - chain antibodies in camelid animals. Nb is composed 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, do not require light - chain pairing, and are single - domain antibodies that are the smallest naturally occurring antigen - binding fragments, which makes it extremely easy to be structurally modified into multivalent - structured antibodies.

[0005] Based on the above characteristics of nanobodies, the development of a high - affinity nanobody against the N antigen of influenza B virus has important application value for influenza virus detection. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an anti - influenza B virus nanobody and its application. The nanobody can detect two similar strains of influenza B virus, Colorado / 06 / 2017 (Victoria lineage) and Florida / 4 / 2006 (Yamagata lineage), effectively avoiding the phenomenon of "missed detection" in the detection of different types of influenza B virus. In the application of colloidal gold detection, the influenza B virus nanobody of the present invention shows better sensitivity than commercially available antibodies and has the prospect of industrial application.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides an anti - influenza B 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 sequence shown in SEQ ID NO:8 or SEQ ID NO:16.

[0009] In the present invention, alpacas were immunized with two different types of influenza B virus N proteins, and the serum antibody titers of the alpacas were detected and evaluated. After the immunization, the peripheral blood of the alpacas was collected, and plasma and peripheral blood lymphocytes (PBMCs) were isolated. Total RNA of PBMCs was extracted and reverse-transcribed into cDNA, and then alpaca nanobodies (VHHs) were amplified by multiplex PCR, and a nanobody phage library was constructed. Then, by the method of multi-antigen cross-selection, a nanobody against influenza B virus with high affinity and broad-spectrum binding activity was screened and obtained.

[0010] Preferably, 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;

[0011] and / or, 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.

[0012] Preferably, the heavy-chain variable region further comprises FR1, FR2, FR3 and FR4.

[0013] Preferably, 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.

[0014] Preferably, 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.

[0015] Preferably, 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.

[0016] Preferably, 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.

[0017] Preferably, 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; FR1 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;

[0018] Alternatively, the CDR1 of the heavy chain variable region of the nanobody is as shown in SEQ ID NO: 14, the CDR2 is as shown in SEQ ID NO: 15, and the CDR3 is as shown in SEQ ID NO: 16; the FR1 of the nanobody is as shown in SEQ ID NO: 10, the FR2 is as shown in SEQ ID NO: 11, the FR3 is as shown in SEQ ID NO: 12, and the FR4 is as shown in SEQ ID NO: 13.

[0019] Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO: 1 or SEQ ID NO: 9.

[0020] The nanobody against the N antigen of influenza B virus of the present invention can detect two or more influenza B viruses (including but not limited to Colorado / 06 / 2017, Florida / 4 / 2006, etc.).

[0021] The antibody type of the present invention is a nanobody. A nanobody (Nb) is a novel antibody, which is the variable domain VHH of a heavy chain antibody that is naturally lacking a light chain in camelids. Due to the natural lack of a 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 its popularization and application in infectious diseases such as pandemic viruses.

[0022] 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, reduce polymerization, and still have biological activity after long-term placement in a high-temperature environment or under strong denaturing conditions, with higher in vitro stability, which is more conducive to the development of antigen detection products that require room temperature conditions.

[0023] In the second aspect, the present invention provides a bivalent structure antibody, which is composed of the anti-influenza B virus nanobody described in the first aspect and rabbit IgG-FC. The amino acid sequence of the bivalent structure antibody is as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0024] In the third aspect, the present invention provides a hexameric multivalent structure antibody, which is composed of the anti-influenza B virus nanobody described in the first aspect, an hIgG-FC fragment, and a μtp amino acid sequence. The amino acid sequence of the hexameric multivalent structure antibody is as shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0025] The present invention performs multivalent modification on the nanobody to obtain various stable derivative structure molecules, including multivalent nanobodies such as bivalent and dodecavalent (hexamer), meeting various antibody application scenarios.

[0026] The nanobodies involved in the present invention, in any form of genetic engineering modification or derivative form, 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 within the protection scope of the present invention.

[0027] The amino acid sequence of the nanobody VHH1 against influenza B virus is SEQ ID NO:1:

[0028] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSS.

[0029] The specific amino acid sequences of framework regions 1-4 (FR1-4) and complementarity-determining regions (CDR1-3) of the nanobody VHH1 against influenza B virus are shown as follows:

[0030] The sequence of FR1 is SEQ ID NO:2: QVQLQESGGGLVQPGGSLKLSCAAS.

[0031] The sequence of FR2 is SEQ ID NO:3: MSWYRQAPGKEREFVAT.

[0032] The sequence of FR3 is SEQ ID NO:4:

[0033] NYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYC,

[0034] The sequence of FR4 is SEQ ID NO:5: WGQGTQVTVSS.

[0035] The sequence of CDR1 is SEQ ID NO:6: GFTXSXYA.

[0036] The sequence of CDR2 is SEQ ID NO:7: IXSFGGXP.

[0037] The sequence of CDR3 is SEQ ID NO:8: NXGYRXTGXFDY.

[0038] The amino acid sequence of the nanobody VHH2 against influenza B virus is SEQ ID NO:9:

[0039] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQVTVSS。

[0040] The specific amino acid sequences of framework regions 1-4 (FR1-4) and complementarity-determining regions (CDR1-3) of the nanobody VHH2 against influenza B virus are shown as follows:

[0041] The sequence of FR1 is SEQ ID NO:10: QVQLQESGGGLVQPGGSLRLSCVTS,

[0042] The sequence of FR2 is SEQ ID NO:11: MSWYRQASGKERELVAS,

[0043] The sequence of FR3 is SEQ ID NO:12:

[0044] TYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYC。

[0045] The sequence of FR4 is SEQ ID NO:13: WGQGTQVTVSS。

[0046] The sequence of CDR1 is SEQ ID NO:14: GFTXSXFI。

[0047] The sequence of CDR2 is SEQ ID NO:15: IXTGGAXT。

[0048] The sequence of CDR3 is SEQ ID NO:16: KAXNIWXGDEXVEY。

[0049] The amino acid sequence of VHH1-FC is SEQ ID NO:17:

[0050] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSSPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0051] VHH2-FC amino acid sequence SEQ ID NO:18:

[0052] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQVTVSSPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0053] VHH1-IgGμtp amino acid sequence SEQ ID NO:19:

[0054] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSSASNTKVDKKVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0055] Amino acid sequence of VHH2-IgGμtp SEQ ID NO:20:

[0056] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0057] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the anti-influenza B virus nanobody described in the first aspect, or encoding the bivalent structured antibody described in the second aspect, or encoding the hexameric multivalent structured antibody described in the third aspect.

[0058] In a fifth aspect, the present invention provides a composition for detecting influenza B virus, which composition comprises any one or a combination of at least two of the anti-influenza B virus nanobody described in the first aspect, the bivalent structured antibody described in the second aspect, or the hexameric multivalent structured antibody described in the third aspect.

[0059] Sixth aspect, the present invention provides a colloidal gold test strip for detecting influenza B virus, and the colloidal gold test strip contains the hexameric multivalent structure antibody described in the third aspect.

[0060] The hexameric multivalent structure antibody is used as a capture antibody and a detection antibody respectively for double antibody sandwich assay.

[0061] In the present invention, the colloidal gold test strip combines the principle of double antibody sandwich assay with the colloidal gold detection method to detect influenza virus protein. The test strip consists of a sample pad, a gold conjugate pad, an NC membrane and an absorbent pad attached to a bottom plate in sequence. The detection antibody and the capture antibody on the colloidal gold test strip are VHH1-IgGμtp and VHH2-IgGμtp respectively.

[0062] In the present invention, the colloidal gold test strip can detect two types of influenza B virus, Colorado / 06 / 2017 and Florida / 4 / 2006. In the application of colloidal gold detection, the detection sensitivity is as low as 2.5 ng / mL, and it has the potential for industrial application.

[0063] Seventh aspect, the present invention provides a kit for detecting influenza B virus, and the kit contains any one or at least two combinations of the anti-influenza B virus nanobody described in the first aspect, the bivalent structure antibody described in the second aspect, the hexameric multivalent structure antibody described in the third aspect or the colloidal gold test strip for detecting influenza B virus described in the sixth aspect.

[0064] Eighth aspect, the present invention provides the application of any one or at least two combinations of the anti-influenza B virus nanobody described in the first aspect, the bivalent structure antibody described in the second aspect, the hexameric multivalent structure antibody described in the third aspect, the nucleic acid molecule described in the fourth aspect, the composition for detecting influenza B virus described in the fifth aspect, and the colloidal gold test strip for detecting influenza B virus described in the sixth aspect in the preparation of products for influenza B detection and diagnosis.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] (1) The nanobody of the present invention is obtained by the method of multi-antigen cross-immunization and panning. The nanobody can specifically bind to influenza B virus Colorado / 06 / 2017 and Florida / 4 / 2006, and can avoid the missed detection phenomenon in clinical detection in clinical applications.

[0067] (2) The nanobody of the present invention can be highly expressed in prokaryotic cells. Compared with the eukaryotic mammalian cell expression of traditional antibodies, its production cost is lower, which is more conducive to popularization and application in the detection of such infectious diseases.

[0068] (3) The nanobody of the present invention is verified by colloidal gold detection, with better sensitivity than commercially available antibodies and has the prospect of industrial application. Description of the Drawings

[0069] Figure 1 It is the SDS-PAGE electrophoresis detection result of the purified nanobody.

[0070] Figure 2 It is the ELISA detection result of the binding affinity of the nanobody.

[0071] Figure 3 It is the analysis result of the binding specificity and broad-spectrum property of the nanobody.

[0072] Figure 4 It is the electrophoresis detection result of the antibody after multivalent modification.

[0073] Figure 5 It is the result of ELISA detecting the antibody binding epitope of influenza B virus.

[0074] Figure 6 It is the schematic diagram of the colloidal gold test strip.

[0075] Figure 7 It is the application result of colloidal gold detection. Detailed Embodiments

[0076] 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.

[0077] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0078] Example 1 Multi-Antigen Cross-Immunization

[0079] Take 200 μg of the full-length antigen of influenza B virus N from Colorado / 06 / 2017 and Florida / 4 / 2006 (Sino Biological) and mix it with an equal volume of Freund's complete adjuvant (sigma). Inject it subcutaneously at multiple points near the forelimbs of the alpaca's neck for the first immunization, and then immunize once every 2 weeks for a total of 4 times. Freund's incomplete adjuvant (sigma) is used for the 2nd - 4th immunizations. 7 days after the end of immunization, collect 20 - 30 mL of the alpaca's peripheral venous blood, and separate the plasma and PBMC samples for subsequent construction of the immune library.

[0080] Example 2 Detection of Plasma Titer

[0081] Coat an ELISA plate with 100 ng of an equal mixture of the N antigens of the Colorado / 06 / 2017 and Florida / 4 / 2006 influenza B viruses 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 h. Wash 3 times with PBST. Dilute the pre-immunization and post-immunization plasma in Example 1 10 4 -, 10 5 -, 10 6 -, 10 7 - and 10 8 -fold, then add to the corresponding ELISA plate and incubate at room temperature for 1 h. Wash 5 times with PBST. Add 100 μL of anti-alpaca H&L IgG HRP (Abcam) diluted 2500-fold to each well and incubate in the dark at room temperature for 1 h. Wash 5 times with PBST. Add 100 μL of TMB chromogenic solution (Abcam) and develop color for 10 minutes. Add an equal volume of TMB stop buffer (Abcam) to terminate the color development and read the OD450 value.

[0082] The ELISA test results are shown in (Table 1). The titer of the post-immunization plasma increased significantly. At a plasma dilution of 10 6 -, the OD450 of the antibody detection after immunization was 4.7 times that before immunization, and the titer of the immunized plasma reached 10 6 -, indicating that after immunization, the alpaca had obtained abundant antibodies against the N antigen of influenza B virus in its body.

[0083] Table 1

[0084]

[0085] Note: Positive judgment: Average OD450 after immunization / Average OD450 before immunization ≥ 2.1.

[0086] Example 3 Obtaining Nanobodies

[0087] Collect a blood sample from the immunized alpaca and isolate the lymphocytes in the peripheral blood of the alpaca using lymphocyte separation medium (GE, 17-1440-02) and density gradient centrifugation. Extract RNA from the lymphocytes according to the instructions of Trizol reagent (INVITROGEN, 15596-018). Then take 20 μg of RNA and according to SuperScript TMIII Use the First-Strand Synthesis SuperMix (Invitrogen) kit and 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 of researchers such as Vincke C, Generation of single domain 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 8 , and a clone library with an abundance greater than 93.4%. Use the N antigen of influenza B virus Colorado / 06 / 2017 and Florida / 4 / 2006 to perform 3 rounds of panning enrichment on the phage library. Select clones from the affinity-enriched library for antigen affinity clone ELISA detection, screen the positive reaction well clone bacteria for sanger sequencing, and identify 2 specific nanobodies (VHH1 and VHH2).

[0088] The amino acid sequence of the influenza B virus nanobody VHH1 is SEQ ID NO:1:

[0089] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSS.

[0090] The amino acid sequence of the influenza B virus nanobody VHH2 is SEQ ID NO:9:

[0091] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQVTVSS.

[0092] Example 4 Induced expression and purification of nanobodies

[0093] (1) Induced expression of nanobodies

[0094] 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.

[0095] (2) Purification of nanobodies

[0096] Affinity purification using His-nickel filler (referred to 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 times the volume 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 ). Figure 1 Figure Figure 1 shows the SDS-PAGE electrophoresis detection results of the purified nanobodies. In

[0097] which, lane 1: VHH1 nanobody; lane 2: VHH2 nanobody; lane M: protein ladder. Figure 1 It can be seen that the size of the nanobody band is about 15 KD and the purity > 95%.

[0098] Example 5 ELISA analysis of the binding affinity of nanobodies to the N antigen of influenza B virus

[0099] Coat an ELISA plate with 100 ng of the N antigen of the influenza B virus Colorado / 06 / 2017 at 4°C overnight. Wash 3 times with PBST (0.05%). Add 200 μL of 2% BSA to each well and incubate at room temperature for 2 h. Wash 3 times with PBST. Gradient dilute the nanobody expressed and prepared in Example 4 and a COVID-19 nanobody (NC). The starting concentration is 3 μg / mL (~200 nM), and it is serially diluted 5-fold, with a total of 8 concentration gradients. Add to the wells, with 3 replicates for each concentration gradient, 100 μL per well, and incubate at room temperature for 1 h. Wash 6 times with PBST, add 100 μL of anti-HA, HRP (abcam) diluted 2000 - 3000 times per well, and place at room temperature for 1 h. 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 terminate color development. Read the OD450 value.

[0100] The ELISA test results are shown in ( Figure 2 ). Figure 2 These are the ELISA test results for the binding affinity of the nanobodies. Figure 2 In the figure, NC: negative control. Both the VHH1 and VHH2 nanobodies have good affinity activities for the N antigen of the influenza B virus. The half-maximal effective concentrations (EC50) of VHH1 and VHH2 are 0.01062 μg / mL (0.8333 nM) and 0.005604 μg / mL (0.3737 nM) respectively, and their affinities are both at the sub-nanomolar level.

[0101] Example 6 ELISA analysis of the binding specificity and broad-spectrum properties of influenza nanobodies

[0102] Add 50 ng of the N antigens of four subtypes of influenza A virus (Sino Biological) and 2 types of N antigens of influenza B virus to each well of the ELISA plate for coating, 50 μL per well. Wash 3 times with PBST, add 2% BSA for blocking, and incubate at room temperature for 2 h. Wash 3 times with PBST, add 0.75 μg / mL of the 2 nanobodies in Example 4, 50 μL per well, with 2 replicates; 1 COVID-19 nanobody as the negative control; PBST as the blank well control. Incubate at room temperature for 1 h. Wash 5 times with PBST, then wash 5 more times with PBST, add 100 μL of anti-HA, HRP (abcam) diluted 2000 - 3000 times per well, and place at room temperature for 1 h. 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 terminate color development. Finally, place the ELISA plate in a microplate reader (BioTek) to read the value.

[0103] Figure 3Results of the binding specificity and broad-spectrum analysis of the nanobody Figure 3 In Figure 3 , NC: negative control; PBST is the blank well control. Figure 3 The results showed that the nanobody of 2 in this example could specifically bind to the N antigens of two similar strains of influenza B virus, Colorado / 06 / 2017 (Victoria lineage) and Florida / 4 / 2006 (Yamagata lineage), and did not bind to the N antigens of influenza A virus subtypes, with strong specificity.

[0104] Example 7 Multivalent modification, eukaryotic expression and purification of monovalent nanobody

[0105] In this example, the nanobody can be modified into a bivalent and dodecavalent hexameric ring structure. Both antibody molecules can be stably and highly expressed, and can adapt to different application scenarios. The bivalent structure antibody is composed of VHH fused with rabbit IgG-FC. The amino acid sequences are shown in VHH1-FC (SEQ ID NO:17) and VHH2-FC (SEQ ID NO:18). The hexameric multivalent structure antibody is composed of VHH sequence, hIgG-FC fragment, and a μtp amino acid sequence. The amino acid sequences are shown in VHH1-IgGμtp (SEQ ID NO:19) and VHH2-IgGμtp (SEQ ID NO:20).

[0106] Amino acid sequence of VHH1-FC SEQ ID NO:17:

[0107] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSSPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK.

[0108] Amino acid sequence of VHH2-FC SEQ ID NO:18:

[0109] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQ VTVSSPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0110] The amino acid sequence of VHH1-IgGμtp SEQ ID NO:19:

[0111] QVQLQESGGGLVQPGGSLKLSCAASGFTXSXYAMSWYRQAPGKEREFVATIXSFGGXPNYSNSVKGRFTISRDNVKNMLYLEMNSLKPEDTAVYYCNXGYRXTGXFDYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0112] Note: (the 134th amino acid) C is mutated to amino acid S; (the 359th amino acid) P is mutated to T. The above mutations are beneficial to the formation of a multimeric structure like IgM and avoid the non-specific aggregation and instability caused by the appearance of free C amino acids in the antibody hinge region in the absence of a light chain structure.

[0113] Amino acid sequence of VHH2-IgGμtp SEQ ID NO:20:

[0114] QVQLQESGGGLVQPGGSLRLSCVTSGFTXSXFIMSWYRQASGKERELVASIXTGGAXTTYRDSVKGRFTISRDNDKDTVDLQMDNLKSEDTAVYYCKAXNIWXGDEXVEYWGQGTQVTVSSASNTKVDKKVEPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS T GKPTLYNVSLVMSDTAGTCY。

[0115] The above amino acid sequence was synthesized into the expression vector pcDNA3.1 by gene synthesis. 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 mixed solution was slowly added dropwise to HEK293 cells for expression. It was cultured in an incubator at 37°C, 8% CO2, relative humidity ≥80%, and cultured at 110 rpm. After 24 hours, 4% volume of 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 protein A affinity chromatography column to obtain the eluates of VHH1-FC, VHH2-FC, VHH1-IgGμtp and VHH2-IgGμtp. The purified eluate was detected by electrophoresis, and the electrophoresis conditions were 120V, 90min.

[0116] Figure 4 The electrophoresis detection results of the multi-valent modified antibody Figure 4In it, lane 1 is the flow-through solution after VHH1-FC binds to the column; lane 2 is the purification wash solution of VHH1-FC; 3 is VHH1-FC under non-reducing conditions; 4 is VHH1-FC under reducing conditions; lane 5 is the flow-through solution after VHH2-FC binds to the column; lane 6 is the purification wash solution of VHH2-FC; lane 7 is VHH2-FC under non-reducing conditions; lane 8 is VHH2-FC under reducing conditions; lane 9 is VHH1-IgGμtp; lane 10 is VHH2-IgGμtp, and lane M is the protein ladder. The results show that the purity of all four antibodies is >90%. The molecular weights of VHH1-FC and VHH2-FC are around 80 KD, and the molecular weights of VHH1-IgGμtp and VHH2-IgGμtp are about 480 KD, which are dodecavalent hexameric nanobody molecules.

[0117] Example 8 ELISA Detection of Nanobody Binding Epitopes

[0118] Dilute the VHH2-FC antibody in Example 6 to 1.5 μg / mL and coat it on an ELISA plate, 50 μL per well, overnight at 4°C; wash 3 times with PBST, add 2% BSA for blocking, 2 hours at room temperature, and wash 3 times with PBST. Dilute the N antigen of influenza B virus Colorado / 06 / 2017 to a concentration of 1 μg / mL, 50 μL per well, and incubate at room temperature for 1 hour. After washing 3 times with PBST, dilute the nanobodies VHH1 and VHH2 in Example 4 to 1.5 μg / mL, and use PBST as a blank well sample control, and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 50 μL of anti-HA HRP (abcam) diluted 3000-fold, and incubate at room temperature in the dark for 1 hour. Wash 5 times with PBST, add 50 μL of TMB solution and develop color in the dark for 10 minutes, and then add 50 μL of TMB stop buffer (abcam) to stop color development. Finally, place the ELISA plate in a microplate reader (BioTek) for reading.

[0119] Figure 5 For ELISA detection of the binding epitope of influenza B virus antibodies, Figure 5 The results show that VHH1 can bind to the N antigen of influenza B virus that has already bound VHH2-FC, indicating that VHH1 and VHH2 have different binding sites.

[0120] Example 9 Application of Colloidal Gold Test Strip Detection

[0121] According to the results of Example 8, VHH1 and VHH2 have different binding epitopes to the influenza N antigen. The principle of the sandwich immunoassay can be combined with the colloidal gold detection method to detect influenza virus proteins.

[0122] First, take VHH1-IgGμtp and super C+ (Bioeasy) from Example 7, dilute them to 1 mg / mL, and draw lines on a CN110 nitrocellulose membrane (Baisui Kang) using an XYZ scribing instrument (Gold Label Biotech) with a parameter of 1 μL / cm for 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.

[0123] Take an appropriate amount of CG40 colloidal gold solution (Shenbaiao), add 12 μL 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, 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 minutes, 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.

[0124] Add the prepared NC membrane and the gold-labeled pad, the sample pad, and the absorbent pad, and assemble them in the form of Figure 6 to prepare a colloidal gold test strip.

[0125] Dilute the N antigens of influenza B virus from Colorado / 06 / 2017 and Florida / 4 / 2006 to 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 5 ng / mL, 2.5 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 the T line on the NC membrane. The results are as Figure 7 shown, Figure 7 for the commercially available antibody (Huakui Gold Label Antibody Colloidal Gold Test Strip), the results show that: the C line shows normal color development, with 0 ng / mL as the blank control, no non-specific binding occurs, 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 2.5 ng / mL.

[0126] In summary, the present invention utilizes in vitro passive cross-immunization of camelid animals and combines phage display technology to develop high-affinity nanobodies against the N antigen of influenza B virus. The said antibodies can detect two similar strains of influenza B virus, namely Colorado / 06 / 2017 (Victoria lineage) and Florida / 4 / 2006 (Yamagata lineage), effectively avoiding the phenomenon of "undetected cases" in the detection of different types of influenza B virus. In the colloidal gold detection application of the influenza B nanobodies of the present invention, the sensitivity is superior to that of commercially available antibodies, showing the prospect of industrial application.

[0127] The applicant declares that the above description 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 B 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 B virus according to claim 1, characterized in that, 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 B 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 B 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 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 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 B 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 bivalent structured antibody, characterized in that, The bivalent structured antibody is composed of the anti-influenza B virus nanobody described in any one of claims 1-5 and rabbit IgG-FC, and the amino acid sequence of the bivalent structured antibody is as shown in SEQ ID NO:17 or SEQ ID NO:

18.

7. A hexameric multivalent structured antibody, characterized in that, The hexameric multivalent structured antibody is composed of the anti-influenza B virus nanobody described in any one of claims 1-5, an hIgG-FC fragment and a μtp amino acid sequence, and the amino acid sequence of the hexameric multivalent structured antibody is as shown in SEQ ID NO:19 or SEQ ID NO:

20.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-influenza B virus nanobody described in any one of claims 1-5, or encodes the bivalent structured antibody described in claim 6, or encodes the hexameric multivalent structured antibody described in claim 7.

9. A composition for detecting influenza B virus, characterized in that, The composition comprises any one or a combination of at least two of the anti-influenza B virus nanobodies described in any one of claims 1-5, the bivalent structured antibody described in claim 6, or the hexameric multivalent structured antibody described in claim 7.

10. A colloidal gold test strip for detecting influenza B virus, characterized in that, The colloidal gold test strip contains the hexameric multivalent structured antibody described in claim 7; Preferably, the hexameric multivalent structured antibody is used for double antibody sandwich assay as a capture antibody and a detection antibody respectively.

11. A kit for detecting influenza B virus, characterized in that, The kit comprises any one or a combination of at least two of the anti-influenza B virus nanobodies described in any one of claims 1-5, the bivalent structured antibody described in claim 6, the hexameric multivalent structured antibody described in claim 7, or the colloidal gold test strip for detecting influenza B virus described in claim 10.

12. Use of any one or a combination of at least two of the anti-influenza B virus nanobody described in any one of claims 1-5, the bivalent structured antibody described in claim 6, the hexameric multivalent structured antibody described in claim 7, the nucleic acid molecule described in claim 8, the composition for detecting influenza B virus described in claim 9, or the colloidal gold test strip for detecting influenza B virus described in claim 10 in the preparation of a product for detecting and diagnosing influenza B.

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