Anti-h5 subtype avian influenza nanobody protein and application thereof

Abstract

This invention belongs to the field of biomedical technology and discloses an anti-H5 subtype avian influenza nanobody protein and its application. Specifically, it discloses an anti-H5 subtype avian influenza virus nanobody, which comprises three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. This invention develops nanobodies against the H5 subtype avian influenza virus and screens for a nanobody fusion protein 5B2 that specifically binds to the H5-Re14 strain with high affinity. This nanobody fusion protein 5B2 is a nanobody with high binding affinity against the H5 subtype avian influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-H5 subtype avian influenza nanobody protein and its application. Background Technology

[0002] Influenza viruses are widespread globally, with many people contracting them each year during the winter-spring flu season. Furthermore, the influenza virus has a very broad host spectrum, currently being isolated from various animals including bats, birds, humans, pigs, horses, dolphins, and seals, posing a significant threat to public health. Influenza viruses belong to the Orthomyxoviridae family and the Influenza virus genus. Based on the different NP and M types, they can be divided into three types: A, B, and C. Avian influenza virus (AIV) belongs to type A influenza viruses. Based on their pathogenicity, it can be divided into low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI). Highly pathogenic avian influenza (HPAI) is caused by H5 or H7 subtype strains (represented by H5N1 and H7N7), and can infect almost all domestic and wild birds. Infection results in severe clinical symptoms, with a mortality rate of up to 100%. Currently, cases of highly pathogenic avian influenza virus infecting humans occur frequently and are widely distributed. Through antigenic drift and antigenic shift, it is possible for new subtypes of the virus to evolve and cause a human influenza pandemic, and its potential threat cannot be ignored.

[0003] The structure of avian influenza virus (AIV) can be divided into three main parts from the inside out: the core, matrix proteins, and the envelope. Its genetic material is single-stranded negative-sense RNA, composed of eight discontinuous segments, encoding at least 10 proteins (PB1, PB2, PA, HA, NP, NA, M1, M2, NS1, NS2). Hemagglutinin (HA) is one of the main components of the AIV envelope spikes. It exists as a trimer on the surface of the envelope, with a molecular weight of approximately 75 kDa. Its primary structure has four domains: a signal peptide that recognizes the endoplasmic reticulum membrane, a cytoplasmic domain, a transmembrane domain, and an extracellular domain. The HA trimer head has five antigenic determinants: A, B, C, D, and E. These can induce the production of protective neutralizing antibodies and are the most important protective antigen of AIV. They stimulate the body to produce corresponding antibodies, thereby generating immunity. Therefore, HA is of great significance for the development and research of AIV antibodies.

[0004] Based on the different functions and antigenic characteristics of various structural proteins of AIV, various experimental methods for detecting AIV have been established both domestically and internationally. These include serological tests such as hemagglutination assay (HA), hemagglutination inhibition assay (HI), enzyme-linked immunosorbent assay (ELISA), virus neutralization assay (VN), and agar diffusion assay; and molecular biological tests such as polymerase chain reaction (PCR) and real-time reverse transcription-polymerase chain reaction (RT-PCR). Among these various AIV detection methods, the HI test is the most commonly used due to its speed, simplicity, and low cost. However, the preparation of polyclonal antiserum is relatively complicated, resulting in poor sensitivity and specificity, and significant quality variations between different batches of antibodies. Monoclonal antibodies still face challenges in practical applications, such as high cost, complex processes, and ethical issues related to antibody production through immunogenic induction in ascites fluid in experimental animals. While polyclonal antibodies are relatively inexpensive, they also suffer from drawbacks such as the susceptibility to cross-reactivity leading to misidentification of subtypes and batch-to-batch variability. Nanobody (Nb) has made rapid progress in the field of immunoassay in recent years due to its small molecular weight, good stability, affinity, high specificity and ease of high-quality production. It avoids many of the disadvantages mentioned above. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a nanobody against H5 subtype avian influenza virus and its application. The nanobody exhibits good stability and biological activity, can be applied to the diagnosis and detection of H5 subtype avian influenza virus, and possesses excellent detection sensitivity and accuracy.

[0006] The first objective of this invention is to provide a nanobody against H5 subtype avian influenza virus.

[0007] A second aspect of the present invention is to provide a fusion protein.

[0008] A third aspect of the present invention is to provide a nucleic acid molecule.

[0009] The fourth aspect of this invention aims to provide biomaterials related to nucleic acid molecules in the third aspect of this invention.

[0010] The fifth aspect of this invention aims to provide the application of the nanobody of the first aspect of this invention, the fusion protein of the second aspect of this invention, the nucleic acid molecule of the third aspect of this invention, or the biomaterial of the fourth aspect of this invention in the preparation of products.

[0011] The sixth aspect of this invention aims to provide a product.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a nanobody against H5 subtype avian influenza virus, the nanobody comprising three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0014] In some embodiments of the present invention, the nanobody further comprises framework regions FR1, FR2, FR3, and FR4, wherein the amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

[0015] In some embodiments of the present invention, the amino acid sequence of the nanobody is shown in SEQ ID NO:1.

[0016] This nanobody was selected from a phage library and targets the H5 subtype of avian influenza virus.

[0017] A second aspect of the present invention provides a fusion protein containing the nanobody of the first aspect of the present invention.

[0018] In some embodiments of the present invention, the fusion protein further includes a histidine-binding protein.

[0019] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:11.

[0020] A third aspect of the present invention provides a nucleic acid molecule comprising the following components:

[0021] (1) A nucleic acid fragment for encoding the nanobody of the first aspect of the present invention; or

[0022] (2) Nucleic acid fragments for use in the fusion protein of the second aspect of the present invention.

[0023] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:9 or SEQ ID NO:10.

[0024] A fourth aspect of the present invention provides a biomaterial related to the nucleic acid molecule of the third aspect of the present invention, said biomaterial comprising at least one of a1) to a3):

[0025] a1) An expression cassette containing the nucleic acid molecule as described in claim 5 or 6;

[0026] a2) A recombinant vector containing the nucleic acid molecule as described in claim 5 or 6 or the expression cassette in a1);

[0027] a3) Recombinant cells containing the nucleic acid molecule as described in claim 5 or 6, the expression cassette in a1) or the recombinant vector in a2).

[0028] In some embodiments of the present invention, the recombinant vector is a plasmid vector, a viral vector, or a cell vector.

[0029] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cell vector does not include propagation material.

[0030] In some preferred embodiments of the present invention, the carrier is a PET series carrier.

[0031] In some preferred embodiments of the present invention, the carrier is pET-9a, pET-28a(+), pET-22b(+), pET-26b(+) or pET-31b(+).

[0032] In some more preferred embodiments of the present invention, the carrier is pET-28a(+).

[0033] In some embodiments of the present invention, the expression vector is specifically obtained by inserting the nucleic acid molecule described in the third aspect of the present invention into pET28a(+) through the restriction enzyme sites XhoI and NcoI.

[0034] In some embodiments of the present invention, the cells are prokaryotic cells.

[0035] In some embodiments of the present invention, the recombinant cells are bacteria, yeast, or fungi.

[0036] In some embodiments of the present invention, the recombinant cells are Escherichia coli.

[0037] In some embodiments of the present invention, the recombinant Escherichia coli BL21(DE3) is used.

[0038] The fifth aspect of the present invention is to provide the use of the nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, or the biomaterial of the fourth aspect of the present invention in the preparation of products.

[0039] In some embodiments of the present invention, the function of the product is any one of c1) to c5):

[0040] c1) Prepare reagents for detecting and / or diagnosing avian influenza viruses;

[0041] c2) Detection and / or diagnosis of avian influenza virus;

[0042] c3) Prepare drugs for the prevention and / or treatment of diseases related to avian influenza virus infection;

[0043] c4) Prevention and / or treatment of diseases related to avian influenza virus infection;

[0044] c5) triggers or enhances the body's immune response.

[0045] In some embodiments of the present invention, the product includes, but is not limited to, reagents, kits, and drugs.

[0046] In some embodiments of the present invention, the drug includes a vaccine.

[0047] In some embodiments of the present invention, the drug may be in the form of a nasal spray, an oral preparation, a suppository, or a parenteral preparation.

[0048] In some embodiments of the present invention, the avian influenza virus is the H5 subtype avian influenza virus.

[0049] A sixth aspect of the present invention is to provide a product comprising the nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, or the biomaterial of the fourth aspect of the present invention.

[0050] In some embodiments of the present invention, the products include, but are not limited to, pharmaceuticals, reagents, and kits, and the pharmaceuticals include vaccines.

[0051] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable excipients.

[0052] In some embodiments of the present invention, the excipients include carriers, diluents, excipients, preservatives, antibacterial agents, and / or immune adjuvants.

[0053] The present invention also provides a method for preparing the fusion protein described in the second aspect of the present invention, specifically, cloning the gene of the fusion protein described in the third aspect of the present invention into an expression vector to obtain a recombinant expression vector, then transforming the recombinant expression vector into Escherichia coli to obtain recombinant expression cells and expressing them, collecting the expression products, and performing disruption, separation and purification.

[0054] In some embodiments of the present invention, the separation and purification includes a step of processing with nickel column affinity chromatography.

[0055] In some embodiments of the present invention, the method of obtaining recombinant expression cells and expressing them is induced expression.

[0056] In some embodiments of the present invention, the inducer for induced expression is IPTG.

[0057] In some embodiments of the present invention, the final concentration of IPTG is 0.05 to 0.15 mmol / L.

[0058] In some embodiments of the present invention, the final concentration of IPTG is 0.1 mmol / L.

[0059] In some embodiments of the present invention, the conditions for inducing expression are 27–29°C for 14–18 hours.

[0060] In some embodiments of the present invention, the recombinant expression strain is cultured to the bacterial culture OD... 600 Induction was performed by adding an inducing agent when the concentration was between 0.6 and 0.8.

[0061] This invention uses a prokaryotic expression system to construct recombinant bacteria expressing nanobodies against H5 subtype avian influenza virus, which effectively reduces the production cost of nanobodies and improves accuracy and reproducibility. Moreover, the produced nanobodies have high purity and can realize the large-scale production of anti-H5 avian influenza virus nanobodies.

[0062] The beneficial effects of this invention are:

[0063] This invention relates to the development of nanobodies targeting the H5 subtype avian influenza virus, and the identification of nanobody fusion protein 5B2, which specifically binds to the H5-Re14 strain with high affinity. The inventors verified its ability to specifically inhibit viral agglutination of erythrocytes through hemagglutination inhibition assays, and further demonstrated its ability to bind to the H5 subtype (H5-Re14 strain) with high binding affinity in enzyme-linked immunosorbent assays. These findings indicate that nanobody fusion protein 5B2 is a highly binding nanobody targeting the H5 subtype avian influenza virus.

[0064] The method for producing anti-H5 avian influenza virus nanobody protein provided by this invention is simple to operate and low in cost, and can realize the large-scale production of anti-H5 avian influenza virus nanobody.

[0065] The anti-H5 avian influenza virus nanobody fusion protein provided by this invention exhibits high purity, high sensitivity during detection, good stability, and biological activity, providing a potential nanobody reagent for the clinical prevention, treatment, and detection of avian influenza virus. It can be used to develop new immunoassay methods for VHH antibodies against avian influenza virus, addressing the problems of low antibody affinity, low purity during production, and low sensitivity and efficiency during detection encountered in previous research and development processes. Attached Figure Description

[0066] Figure 1 This is a recombinant expression vector for the anti-H5 avian influenza virus nanobody fusion protein 5B2.

[0067] Figure 2The image shows the PCR validation results. Lane 1 is the Yeasen 2000 Marker, lanes 2-5 are the PCR amplification results of multiple single colonies after transformation, and lane 6 is the negative control.

[0068] Figure 3 The figure shows the results of protein analysis by SDS-PAGE electrophoresis. In the figure, lane 1 is the VazymeMP102 Maker and lane 2 is the expression result of the fusion protein 5B2.

[0069] Figure 4 The graph shows the results of hemagglutination inhibition (HI). In the graph, the first row of antigens is H5-Re14, the second row of antigens is H7-Re4, the third row of antigens is H9 subtype (SD696 strain), the 11th column is the negative control, and the 12th column is the blank control.

[0070] Figure 5 Line graph showing the ELISA reaction between fusion protein 5B2 and inactivated antigen H5-Re14 strain. Detailed Implementation

[0071] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0072] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available. For example: Ex Taq enzyme, 4× Protein SDS PAGE Loading Buffer, DL 2000 Marker, DL5000 Marker, restriction endonucleases Nco I and Xho I were purchased from TAKARA; protein marker, 5× CEⅡ Buffer, Exnase II, Loading Buffer terminator and nucleic acid dye were purchased from Vazyme; murine anti-His monoclonal antibody and HighAffinity Ni-NTA medium were purchased from Genscript Biotech; some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. For example, E. coli competent cells DH5α were purchased from Vazyme; E. coli protein expression vector pET-28a was purchased from Solarbio; some synthetic biological materials, such as primers and sequences, which require artificial synthesis, were all commissioned to synthetic companies. For example, the primers in this invention were synthesized by Aiji Biotechnology Co., Ltd.

[0073] Example 1: Construction of a phage nanobody library

[0074] Bactrian camels were immunized with a recombinant AIV inactivated vaccine (H5N8, H5-Re14) every three weeks for a total of four immunizations. Approximately 50 mL of peripheral blood was collected from the donors after immunization, and peripheral blood lymphocytes (PBMCs) were isolated. Total RNA was extracted according to the manufacturer's instructions, and cDNA was obtained through reverse transcription. This cDNA was used as a template for two nested PCR amplifications to obtain the VHH fragment. The target band was excised and recovered. The purified DNA was used as a template for nested PCR amplification of the VHH fragment, which was then ligated into the pcantab-5E phage vector. The ligation product was transformed into XL-Blue bacteria to construct an antigen-specific nanobody library. The host bacteriophage XL-Blue was overstained with helper phage VCSM13, and the supernatant was collected after overnight culture to obtain the phage nanobody library.

[0075] Example 2: Screening of phage nanobody libraries

[0076] The H5 subtype avian influenza inactivated antigen (H5-Re14 strain) was coated onto an ELISA plate using sodium carbonate-bicarbonate buffer (0.05 mol / L, pH 9.6), 100 μL per well (10% inactivated antigen), and incubated at 37°C for 2 h. The liquid in the wells was discarded, and the plate was washed once with 100 μL PBS, followed by 300 μL of 3% BSA and incubated at room temperature for 1 h. The blocking solution was discarded, and 100 μL of phage antibody solution was added, followed by incubation at room temperature for 1 h. The liquid in the wells and any unbound phages were discarded, and the plate was washed five times with 0.05% PBST for 5 min each time. 100 μL of glycine-hydrochloric acid elution buffer (pH 2.2) was added, and the elution buffer was collected and mixed with an appropriate amount of 2M... Tris base is used to neutralize the liquid containing the phage, thus obtaining the phage for the first round of enrichment screening. The phage is then further amplified and enters the next round of screening. After five rounds of "adsorption-elution-amplification" screening process, a phage clone with high specificity can finally be obtained (the first round of screening uses non-specific conditions, while the subsequent rounds of screening improve specificity by changing the antigen coating concentration).

[0077] Single colonies were selected for ELISA screening of positive clones: Single colonies were coated onto microplates using the method described above, followed by blocking. A blank microplate (uncoated) served as a control. 100 μL / well of HRP-M13 was added, and the plates were incubated at 37°C for 1 hour. The plates were washed five times with 0.05% PBST for 5 minutes each time. 100 μL / well of TMB substrate was added, and the plates were incubated at room temperature in the dark for 20 minutes. The OD value at 620 nm was measured using a microplate reader. A positive result was defined as a P / N ratio (the ratio of OD reading in positive wells to OD reading in control wells) greater than or equal to 3. Positive clones were then sent for sequencing to obtain the anti-H5 subtype avian influenza virus nanobody sequences.

[0078] The amino acid sequence of the anti-H5 subtype avian influenza virus nanobody is shown in SEQ ID NO:1, including a framework region (FR) and an antibody gene complementarity-determining region (CDR). The framework region is divided into four parts, defined as FR1, FR2, FR3, and FR4, with amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively. The complementarity-determining region is divided into three parts, defined as CDR1, CDR2, and CDR3, with amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0079] Among them, SEQ ID NO:1:

[0080] MDNQVQLVESGGGSVQAGGSLLRLSCLISAYDYFKAMAWFRQAPGKEREGVASI YGGNAYYADSVQGRVTISSRDNAKATLYLQMNSLKPEDTAMYYCAASTRYVPTTQILH EFQYTDWGQGTQVTVSS.

[0081] SEQ ID NO:2: MDNQVQLVESGGGSVQAGGSLLRLSCLIS.

[0082] SEQ ID NO:3: WFRQAPGKEREGVAS.

[0083] SEQ ID NO: 4: YYADSVQGRVTISRDNAKATLYLQMNSLKPEDTAMYYC.

[0084] SEQ ID NO:5: WGQGTQVTVSS.

[0085] SEQ ID NO:6: AYDYFKAMA.

[0086] SEQ ID NO:7: IYGGNA.

[0087] SEQ ID NO:8: AASTRYVPTTQILHEFQYTD.

[0088] The gene encoding the aforementioned anti-H5 subtype avian influenza virus nanobody protein has the nucleotide sequence shown in SEQ ID NO:9, where SEQ ID NO:9:

[0089] ATGGACAATCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTTAATCTCTGCATACGACTACTTTAAGGCAATGGCCTGGTTCCGCCAAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCTTCTATCTATGGTGGTAACGCATACTATGCGGACTCCGT GCAGGGCCGCGTCACCATCTCCCGAGACAACGCCAAGGCCACGCTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCCAGTACACGCTATGTACCTACTCAGATCCTGCATGAATTTCAATATACCGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0090] Example 3: Construction of nanobody fusion protein particle 5B2 in pET-28a(+) against H5 avian influenza virus

[0091] (1) Based on the gene sequence of the anti-H5 avian influenza nanobody (SEQ ID NO.9), its C-terminus was linked to a histidine tag, and the nucleotide sequence of the fusion expression is shown in SEQ ID NO:10. The amino acid sequence of the anti-H5 avian influenza virus nanobody fusion protein is shown in SEQ ID NO:11.

[0092] Among them, SEQ ID NO.10:

[0093] ATGGACAATCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTTAATCTCTGCATACGACTACTTTAAGGCAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCTTCTATCTATGGTGGTAACGCATACTATGCGGACTCCGTGCAGGGCCGC GTCACCATCTCCCGAGACAACGCCAAGGCCACGCTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCCAGTACACGCTATGTACCTACTCAGATCCTGCATGAATTTCAATATACCGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCACATCATCATCATCATCATTAA.

[0094] SEQ ID NO.11:

[0095] MDNQVQLVESGGGSVQAGGSLLRLSCLISAYDYFKAMAWFRQAPGKEREGVASI YGGNAYYADSVQGRVTISSRDNAKATLYLQMNSLKPEDTAMYYCAASTRYVPTTQILH EFQYTDWGQGTQVTVSSHHHHHH.

[0096] XhoI and NcoI restriction sites were added to both ends of the fusion gene during gene synthesis, and then ligated into the pET-28a(+) vector that had undergone double digestion with XhoI and NcoI, named 5B2 in pET-28a(+). The results are as follows: Figure 1 As shown, the gene for the anti-H5 avian influenza nanobody and a gene with a 6-histidine tag are sequentially linked. The 5B2 gene is the gene for the anti-H5 avian influenza nanobody. The nucleotide sequence of the resulting linker fragment was optimized and synthesized by General Biotechnology (Anhui) Co., Ltd.

[0097] (2) Plasmid extraction: The synthetic plasmid 5B2 inpET-28a(+) obtained in step (1) was transformed into the recipient bacterium DH5α. The bacterial solution was plated on LB plates containing kanamycin (containing 30 mg / L kanamycin (Kan)) for resuscitation and activation. After incubation at 37°C for 16 h, several single colonies were picked for transformant PCR confirmation. Single colonies with correct PCR band size results were re-transferred and plated. After transfection, a portion of the colonies were transferred to 15 mL of liquid LB medium and shaken on a shaker at 37°C and 200 rpm for 16 h. A portion of the bacterial solution was temporarily aliquoted and stored at 4°C. Another portion of the bacterial solution was collected in 15 mL centrifuge tubes, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and plasmid extraction was performed.

[0098] Example 4: Induced expression of anti-H5 avian influenza virus nanobody fusion protein

[0099] (1) The plasmid expressing the anti-H5 avian influenza virus nanobody fusion protein, preserved in the above examples, was transformed into the recipient *Escherichia coli* BL21(DE3). After being placed on ice for 30 min, it was heat-shocked in a 42℃ water bath for 90 s, and then 1 mL of LB broth was added. After revival and activation on a shaker at 37℃ and 220 rpm, it was centrifuged at 6000 rpm for 1 min. After discarding 90% of the supernatant, the bacterial culture was plated on an LB plate containing kanamycin (containing 30 mg / L kanamycin) for revival and activation. After incubation at 37℃ for 16 h, multiple single colonies were picked and transformed by PCR using T7 primers (T7-F: 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 12); T7-R: 5'-TGCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 13)). The results are as follows. Figure 2 As shown. The cloned colonies of the target band, approximately 600 bp in size, were re-transferred to a plate and stored; the specific steps are as follows:

[0100] Amplification system: PCR Master Mix 25 μL, forward primer (10 pmol / μL) 1 μL, reverse primer (10 pmol / μL) 1 μL, gene template 3 μL, ddH2O to make up to 50 μL.

[0101] Amplification reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 58℃ for 20 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min.

[0102] After the PCR reaction was completed, 1% agarose gel electrophoresis was performed. The gel electrophoresis showed a target band of approximately 600 bp in size.

[0103] (2) After transfer, a portion of the colonies was transferred to 100 mL of liquid LB medium and shaken on a shaker at 37°C and 200 rpm; wait for the bacterial culture to OD 600 When the value is 0.6, IPTG is used for induction, and the final concentration of IPTG is 0.1 mM. After 16 h of induction, the bacterial culture is collected in 50 mL centrifuge tubes, centrifuged at 8000 rpm for 10 min, and the supernatant is discarded.

[0104] (3) Use protein lysis buffer (H2PO4·H2O (MW137.99g / mol) 6.9g, i.e. 0.05M / L, NaCl (MW58.44g / mol) 17.54g, i.e. 0.3M / L, imidazole (MW68.08g / mol) 0.68g, i.e. 0.01mM / L), add about 900mL of deionized water, stir to dissolve, add NaOH to adjust the pH of the solution to 8.0, add deionized water to make up to 1000mL), take about 30mL to resuspend the preserved bacterial culture, and use an ultrasonic disruptor to disrupt it. The ultrasonic program is 5s disruption, 5s interval, and 30min ultrasonic disruption.

[0105] (4) The product after ultrasonic disruption was centrifuged at 8000 rpm for 20 min, the supernatant was collected, and nickel column affinity chromatography was performed. The supernatant was concentrated and purified to obtain purified anti-H5 avian influenza virus nanobody protein. The specific operation method is as follows: Take 0.5 mL of HisTrap affinity columns purchased from GE Healthcare into Poly-Prep Chromatography Columns (purchased from BIO-RAD), add 7 mL of the above protein lysis buffer three times each time, for a total of 21 mL to balance the column, then slowly add the lysed supernatant and let it drip slowly into the waste liquid tank. Finally, add elution buffer (H2PO4·H2O (MW 137.99 g / mol) 6.9 g, i.e. 0.05 M / L, NaCl (MW 58.44 g / mol) 17.54 g, i.e. 0.3 M / L, imidazole ... 68.08 g / mol (0.68 g, i.e., 0.25 mM / L) was added to approximately 900 mL of deionized water. After stirring and dissolving, NaOH was added to adjust the pH of the solution to 8.0. Deionized water was then added to bring the volume to 1000 mL. The eluent was collected, and the solution was dialyzed overnight at 4°C with PBS. The liquid collected after dialyzing was the purified protein. The target peak was determined by SDS-PAGE. The results are as follows: Figure 3As shown, purified nanobody fusion protein 5B2 (i.e., anti-H5 avian influenza virus nanobody fusion protein) was obtained; 20% (v / v) of glycerol was added, and it was stored at -20°C. Example 5: Functional Verification of Anti-H5 Avian Influenza Virus Nanobody Fusion Protein (Nanobody Fusion Protein 5B2)

[0106] To investigate whether the anti-H5 avian influenza virus nanobody fusion protein has the function of inhibiting hemagglutination, this embodiment uses a hemagglutination inhibition (HI) test to examine the antibody. The procedure is as follows: First, the inactivated virus titer is determined by the hemagglutination (HA) test, four units are prepared, and then the HI test is performed. The hemagglutination (HA) test in this embodiment is performed according to GB / T14926.53-2001 standard, and the hemagglutination inhibition test is performed according to GB / T14926.54-2001 standard. Details are as follows:

[0107] (1) HA experiment:

[0108] ① Add 25 μL of PBS to wells 1-11 of a 96-well plate, and add 50 μL of PBS to well 12;

[0109] ② Add 25 μL of inactivated H5-Re14 strain HI test antigen (purchased from Harbin Weike Biotechnology Development Co., Ltd.) to well 1, mix well and aspirate to well 2, then serially dilute to well 11, mix well and discard 25 μL, do not add to well 12;

[0110] ③ Add 25 μL of diluted PBS to each well from 1 to 11;

[0111] ④ Gently shake to mix 1% red blood cells, add 25 μL to each well (wells 1-12); shake, incubate at room temperature (24-25℃) for 40 min, and observe the results. Preparation of 1% chicken red blood cell suspension: Draw anticoagulated chicken blood, centrifuge at 700 rpm for 5 min; wash with PBS. Centrifuge again, remove white blood cells from the surface of the precipitated red blood cells, and continue washing until no white blood cells are visible on the surface of the red blood cells and the PBS washing solution is clear and colorless. Gently mix with PBS at a ratio of 99:1 to obtain a 1% chicken red blood cell suspension.

[0112] Four-unit preparation: After the HA titer is measured, prepare four units of antigen (4HAU) by diluting the stock solution 2n-2 times. After preparation, perform the HA step to verify the four units. If blood coagulation occurs in the first two wells during verification, the four units are considered to be prepared successfully.

[0113] (2) HI experiment:

[0114] ① Add 25 μL of PBS to wells 1-11 of a 96-well plate, and add 50 μL of PBS to well 12;

[0115] ② Add 25 μL of anti-H5 avian influenza virus nanobody protein to well 1, mix well and aspirate to well 2, then serially dilute to well 10, mix well and discard, do not add to wells 11 and 12;

[0116] ③ Add 25 μL of diluted AIV H5 subtype Re-14 strain 4 units antigen suspension to each well from well 1 to well 11, and let stand at room temperature (24-25℃) for at least 30 min. Do not add to well 12.

[0117] ④ Gently shake and mix 1% red blood cells, add 25 μL to each well from 1 to 12; shake, let stand at room temperature (24 to 25°C) for 40 min, and then observe the results.

[0118] The hemagglutination inhibition test procedures for H7 and H9 strains are the same as those for H5-Re14 strain. The hemagglutination inhibition test antigens for H5, H7, and H9 avian influenza are H5-Re14 strain, H7-Re4 strain, and H9 subtype (SD696 strain), respectively, all of which were purchased from Harbin Weike Biotechnology Co., Ltd.

[0119] The HI results showed that the titer of the anti-H5 avian influenza nanobody fusion protein antibody against the H5 antigen was 6 log2, and the IC100 was 3.13 μg / mL. IC100 refers to the concentration at which the nanobody fusion protein 5B2 completely inhibited the highest dilution of 4 HAU units of antigen-agglutinated erythrocytes. Furthermore, the nanobody fusion protein 5B2 showed no hemagglutination inhibition against H7 and H9 subtypes, indicating that it is a specifically binding anti-H5 avian influenza virus nanobody antibody. Figure 4 ).

[0120] Example 6: Application of anti-H5 avian influenza virus nanobody fusion protein in antigen specificity detection

[0121] The anti-H5 avian influenza virus nanobody fusion protein is a specific antibody against H5 avian influenza and can be used to screen expressed nanobodies for detection of coated antigens.

[0122] This embodiment provides a method for detecting H5 avian influenza virus, including the following steps:

[0123] (1) Antigen coating: Take 30 μL of H5 avian influenza hemagglutination inhibition test antigen (H5-Re14 strain) and dilute the antigen according to antigen: carbonate coating buffer = 3:7. Add 100 μL of diluted AIV H5-Re14 strain antigen suspension to each well from 1 to 12. Incubate at 37°C for 2 h or at 4°C overnight. Then discard the liquid in the plate.

[0124] (2) Blocking: Add 300 μL of 3% BSA and block for 1 h;

[0125] (3) Primary antibody: The anti-H5 avian influenza virus nanobody fusion protein 5B2 (prepared in Example 4) is the primary antibody. The nanobody fusion antibody 5B2 is serially diluted 2 times to well 11, with 3 replicate wells for each gradient, 100 μL per well; well 12 is only added with an equal amount of PBS as a blank control.

[0126] (3) Washing: Wash 3 to 5 times with PBS washing solution containing 0.05% Tween-20, soaking time 5 minutes each time, 5 washes;

[0127] (4) Secondary antibody: The mouse anti-his polyclonal antibody from Genscript was diluted with PBS at a ratio of 1:3000. 100 μL of diluted antibody was added to each well and incubated for 1 h.

[0128] (5) Washing: Wash 3 to 5 times with PBS washing solution containing 0.05% Tween-20, soaking time 5 minutes each time, for a total of 5 washes;

[0129] (6) Triple antibody: The goat anti-mouse-HRP antibody from Shanghai Sangon Biotech Co., Ltd. was diluted with PBS at a ratio of 1:5000. 100 μL of the diluted antibody was added to each well and incubated for 1 h.

[0130] (7) Washing: Wash 3 to 5 times with PBS washing solution containing 0.05% Tween-20, soaking time 5 minutes each time, for a total of 5 washes;

[0131] (8) Color development: Add HRP substrate color development solution and develop color for 20 min.

[0132] (9) Termination: Add 100 μL of stop solution to each well and take a reading using a microplate reader at a wavelength of 650 nm.

[0133] ELISA results showed that the P / N ratio decreased gradually with decreasing levels of the nano-fusion antibody 5B2, with the mean blank control value being 0.04 and the maximum P / N ratio exceeding 3. The half-maximal effective concentration (COP) of this nano-fusion antibody against the H5 avian influenza hemagglutination inhibition assay antigen (H5-Re14 strain) was calculated using GraphPad Prism 7.0. 50 The concentration was 187.9 ng / mL. This demonstrates that the prokaryotically expressed nano-fusion antibody against H5 subtype avian influenza virus possesses good biological activity. Figure 5 ).

[0134] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nanobody against H5 subtype avian influenza virus, characterized in that, The nanobody contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

2. The nanobody according to claim 1, characterized in that, The nanobody further comprises framework regions FR1, FR2, FR3, and FR4, wherein the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

3. The nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

4. A fusion protein, the amino acid sequence of which is shown in SEQ ID NO:

11.

5. A nucleic acid molecule, said nucleic acid molecule comprising the following components: (1) A nucleic acid fragment for encoding the nanobody according to any one of claims 1 to 3; or (2) A nucleic acid fragment for encoding the fusion protein of claim 4.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:9 or SEQ ID NO:

10.

7. A biomaterial associated with the nucleic acid molecule of claim 5 or 6, wherein the biomaterial is selected from at least one of a1) to a3): a1) An expression cassette containing the nucleic acid molecule as described in claim 5 or 6; a2) A recombinant vector containing the nucleic acid molecule as described in claim 5 or 6 or the expression cassette in a1); a3) Recombinant cells containing the nucleic acid molecule as described in claim 5 or 6, the expression cassette in a1) or the recombinant vector in a2).

8. The use of the nanobody according to any one of claims 1 to 3, the fusion protein according to claim 4, the nucleic acid molecule according to claim 5 or 6, or the biomaterial according to claim 7 in the preparation of a product, wherein the product is a reagent, a kit, or a drug; The function of the product is c1) or c2). c1) Detection and / or diagnosis of avian influenza virus; c2) Prevention and / or treatment of diseases related to avian influenza virus infection; The avian influenza virus in question is the H5 subtype of avian influenza virus.

9. A product characterized in that, The product includes the nanobody according to any one of claims 1 to 3, the fusion protein according to claim 4, the nucleic acid molecule according to claim 5 or 6, or the biomaterial according to claim 7, and the product is a reagent, kit, or drug.

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