Anti-H5 subtype avian influenza virus nano antibody as well as construction method and application thereof

Abstract

The invention relates to the technical field of biology, and discloses an anti-H5 subtype avian influenza virus nano antibody, the amino acid sequence is shown as SEQ ID NO: 1, as antigen evolution of H5AIV forms a great challenge for development of efficient and lasting vaccines or antiviral drugs, the nano antibody targeting conservative epitopes disclosed by the invention can be used as a prevention or treatment reagent, and has good application prospects. The invention discloses an anti-H5 subtype avian influenza virus nano antibody and a construction method of the anti-H5 subtype avian influenza virus nano antibody, and further discloses application of the anti-H5 subtype avian influenza virus nano antibody, an anti-H5 subtype avian influenza virus drug and the anti-H5 subtype avian influenza virus nano antibody.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to nanobodies against H5 subtype avian influenza virus, their construction methods and uses. Background Art

[0002] Among the highly pathogenic avian influenza viruses (HPAIV) of subtype H5 that break out in poultry, according to the phylogenetic analysis of hemagglutinin (HA), H5 avian influenza virus (AIV) can be divided into 9 different evolutionary branches (evolutionary branches 0-9), and some of these evolutionary branches are further subdivided into sub-evolutionary branches.

[0003] Currently, the main methods used to combat avian influenza virus infection are vaccination or administration of antiviral drugs. Inactivated vaccines Re5, Re6, Re8, Re10, Re11, Re12, Re13 and Re14 have been produced and used to reduce the impact of H5 HPAIV on the poultry industry according to the prevailing strains at that time. However, the production of vaccines against newly emerging viruses is expected to take at least several months, and the effectiveness of antiviral drugs is limited due to the emergence of viral drug resistance. Therefore, antibody therapy remains an important and viable treatment option against the virus. To date, many monoclonal antibodies (mAbs) that neutralize AIV and target ancestral H5Nx lineage viruses have been reported, but only a few antibodies can target circulating evolutionary branches 2.3.4.4 or 2.3.2.1.

[0004] Nanobodies have been used as diagnostic and therapeutic agents because of their ideal properties such as high solubility, good thermal stability, easy production and small size. Nanobodies, also known as variable domains of heavy-chain antibodies (VHH), are the antigen-binding components of heavy-chain antibodies (HCAb) produced by camelids, with a molecular weight of about 15 kDa. Due to their longer and smaller complementarity-determining region 3 (CDR3), nanobodies can penetrate the cavities of immunogens. This indicates that VHH may be able to target certain epitopes that are inaccessible to conventional antibodies; in turn, this provides valuable information for structure-based vaccine design. However, the number of studies on nanobodies against subtype H5 or currently prevailing strains is limited. In addition, most mAbs studied in animal studies are usually administered via the intraperitoneal or intravenous route. In contrast, equal amounts of antibodies or nanobodies delivered via the intratracheal or intranasal route have been shown to directly reach the respiratory epithelium compared to mAbs administered via the intraperitoneal or intravenous route, resulting in higher survival rates and significant weight recovery. Therefore, intratracheal or intranasal delivery of antibodies or nanobodies may enhance their neutralizing potency and reduce the required dose, thereby reducing costs and increasing the feasibility of clinical applications.

[0005] HA is initially produced as a single polypeptide that assembles into a trimeric structure called HA0. During infection, host proteases cleave HA0 into its subunits HA1 and HA2. The receptor-binding site (RBS) is part of subunit HA1 and is located in the globular head domain of the HA molecule; it is important for attachment to host receptors and facilitates virus entry during influenza infection. Subunit HA2, together with several residues of subunit HA1, constitutes the conserved stem region of HA, which enables the virus and cell membrane to fuse after activation in the acidic environment in vivo.

[0006] As recorded in "Research Progress on Nanobody Screening and Expression Technology" published by Zhu Guang, Wang Yichen, Song Shasha, Zhang Maishou, Wang Jiacai, etc., the currently commonly used nanobody screening technologies include phage display technology, yeast two-hybrid technology, mRNA display technology, high-throughput sequencing and mass spectrometry analysis. In yeast two-hybrid technology, in the yeast two-hybrid system, a recombinant vector with a known protein gene sequence and BD domain sequence needs to be constructed, called "bait"; a vector composed of an unknown protein gene sequence and AD domain sequence is constructed, called "prey". If there is no interaction between the bait and the prey, BD and AD will not be activated, and the reporter gene cannot be activated and expressed. In host cells, if the bait and the prey interact and are expressed, then BD and AD will bind to the activation sequence, and the downstream reporter gene will thus be activated and expressed;

[0007] Thus, the antibodies screened by the yeast two-hybrid system are highly specific relative to their bait.

[0008] The problem to be solved in this scheme: How to provide a nanobody targeting the circulating clade 2.3.4.4 or 2.3.2.1 virus. Summary of the Invention

[0009] The purpose of this application is to provide a nanobody targeting the circulating clade 2.3.4.4 or 2.3.2.1 virus to enrich the current abundance of nanobodies against the H5 subtype clade 2.3.4.4 or 2.3.2.1.

[0010] To achieve the above purpose, this scheme provides a nanobody against H5 subtype avian influenza virus, and its amino acid sequence is as shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence is as shown in SEQ ID NO: 2.

[0012] Preferably, the nanobody against H5 subtype avian influenza virus is designed according to the H5-Re8 virus.

[0013] In addition, the present application also discloses the use of the anti-H5 subtype avian influenza virus nanobody as described above in the preparation of an anti-H5 subtype avian influenza virus drug.

[0014] Preferably, the anti-H5 subtype avian influenza virus drug is specifically a drug against the 2.3.2.1 branch and / or 2.3.4.4 branch of the anti-H5 subtype avian influenza virus.

[0015] Preferably, the anti-H5 subtype avian influenza virus drug is specifically an anti-H5-Re14 virus drug.

[0016] In addition, the present application also discloses an anti-H5 subtype avian influenza virus drug containing the above-mentioned anti-H5 subtype avian influenza virus nanobody.

[0017] In addition, the present application also discloses a construction method for constructing the above-mentioned anti-Hx subtype avian influenza virus nanobody, including the following steps:

[0018] Step 1: Immunize a camelid with an inactivated vaccine or a live attenuated vaccine of the H5 subtype avian influenza virus to induce the camelid to produce antibodies;

[0019] Step 2: Use the yeast two-hybrid system and use HA1 of the H5 subtype avian influenza virus as a bait to screen the antibodies to obtain the target antibody;

[0020] Step 3: Sequence and express the target antibody to obtain an anti-H5 subtype avian influenza virus nanobody.

[0021] Preferably, Step 1 is specifically: immunize a camelid with an inactivated vaccine of the H5 subtype avian influenza virus to induce the camelid to produce antibodies.

[0022] The beneficial effects of the present application are as follows: The antigenic evolution of H5 AIV poses a major challenge to the development of highly effective and long-lasting vaccines or antiviral drugs. Nanobodies targeting conserved epitopes can be used as preventive or therapeutic reagents and are candidates for the design of universal vaccines with broad and long-lasting protection. A broad-spectrum neutralizing nanobody Nb10 against the H5 subtype avian influenza virus was screened in the present application, and its good neutralizing activity was demonstrated by in vivo and in vitro experiments. Description of the Drawings

[0023] Figure 1 Schematic diagram of blue colonies growing on a QDO / X / A plate after pGBKT7-Re8-HA1 was transformed into competent cells;

[0024] Figure 2 Page diagram after purification of the nanobody Nb10 protein;

[0025] Figure 3 Survival curve of Re6 / PR8 in mice;

[0026] Figure 4 Weight curve of Re6 / PR8 in mice;

[0027] Figure 5 Survival curve of Re8 / PR8 in mice;

[0028] Figure 6 Weight curve of Re8 / PR8 in mice;

[0029] Figure 7 Survival curve of Re10 / PR8 in mice;

[0030] Figure 8 Weight curve of Re10 / PR8 in mice;

[0031] Figure 9 Survival curve of Re11 / PR8 in mice;

[0032] Figure 10 Weight curve of Re11 / PR8 in mice;

[0033] Figure 11 Survival curve of Re14 / PR8 in mice;

[0034] Figure 12 Weight curve of Re14 / PR8 in mice. Detailed implementation manners

[0035] The present application will be clearly and completely described below in conjunction with the embodiments of the present application. In the description of the present application, it should be noted that for those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.

[0036] Example 1

[0037] Construct a yeast nanobody library against H5 subtype avian influenza virus

[0038] A healthy two-year-old male Bactrian camel was inoculated with 3 ml of inactivated virus vaccine H5-Re8 (Harbin Weike Biotechnology Development Company, China). This procedure was carried out five times at three-week intervals. Three weeks after the injection of the last booster dose, 200 ml of blood was drawn to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were separated by density gradient separation and total RNA was extracted from PBMCs. The gene fragment encoding VHH was amplified by two-step nested PCR. Reverse transcription PCR was used to amplify the VH–CH1–CH2 and VHH–CH2 regions, using forward and reverse primers CALL001 and CALL002, respectively. Then, the amplified fragment of approximately 750 bp in size was purified and used as a template for the second PCR. The VHH-encoding sequence of approximately 400 bp in size was amplified by the second PCR using nested primers VHH-Forward and VHH-Reverse. Subsequently, the VHH cDNA was ligated to the linearized vector pGADT7-Rec using primers pGADT7-Rec-F and pGADT7-Rec-R. Then, the plasmid was introduced into the competent cells of yeast Y187 by the lithium acetate transformation technique. After culturing at 28 °C for 3 days, all were collected and stored in a -80 °C refrigerator for standby, and the Y2H library was obtained. The primers are shown in Table 1:

[0039] Table 1

[0040]

[0041] Example 2

[0042] Screening of nanobodies by yeast two-hybrid system

[0043] The coding sequence of the bait protein Re8-HA1 was amplified using primers bait-HA1-F and bait-HA1-R, and it was ligated to the linearized pGBKT7 using primers pGBKT7-F and pGBKT7-R to construct the recombinant bait vector pGBKT7-Re8-HA1. The competent cells of Y2HGold were transformed by the chemical transformation method, and then the cells were inoculated on SD medium lacking tryptophan (SD / -Trp) agar and cultured at 30 °C for 3 - 5 days. Single colonies were picked for colony PCR verification, and the inserted sequence was confirmed by sequencing using primers GAL4AD-F and 3AD-R. For the screening process, the OD of the newly cultured 600≈1.0 The bait strain in the logarithmic growth phase was concentrated and mixed with 1 ml of the yeast strain Y187 (pGADT7 - preys) of the nanobody library, and cultured with shaking at 30 - 50 rpm for 20 hours to promote diploid formation. Then the cells were spread on DDO / X / A (lacking leucine and tryptophan, SD / –Leu / –Trp, supplemented with 40 μg / ml X - alpha(α)-Gal and 200 ng / ml aureobasidin A) plates for primary screening. As Figure 1 , they were incubated at 30 °C for 3 - 5 days, and the formation of blue colonies was observed. The blue colonies growing on the DDO / X / A plates were transferred to the more stringent QDO / X / A (lacking adenine, histidine, tryptophan and leucine, SD / –Ade / –His / –Leu / –Trp, with X - α - Gal and aureobasidin A added on the plates) agar plates and cultured at 30 °C for 3 - 5 days for more stringent screening, and the well - growing blue colonies were selected. DNA sequencing was performed on the potential positive clones thus obtained. The primers are shown in Table 2:

[0044] Table 2

[0045]

[0046] One broad - spectrum neutralizing nanobody against H5 subtype avian influenza virus was obtained by screening, and its amino acid sequence is as shown in SEQ ID NO: 1; QVQLQESGGGSVQAGGSLRLSCSASEYTRSMAWFRQVPGKEREGVAAIDTGNGNTYYPTVDGRFIISRGNAKNSVDLEMNSLTPDDTAIYYCAATQGPLWPTLGTQFSTESYNYWGQGTLVTVSS;

[0047] The nucleotide sequence encoding the above - mentioned broad - spectrum neutralizing nanobody against H5 subtype avian influenza virus is as follows (SEQ ID NO: 2):

[0048] Caagttcagttacaggaatctgggggaggctcggtgcaggctggagggtctctgagactctcctgttcagcctctgaatacactcgaagtatggcctggttccgccaggttccagggaaggagcgcgagggggtcgcagctatcgatactggtaatgggaacacatactatcccaccgtagacggccgattcatcatctcccgaggcaacgccaagaactccgtagatctggaaatgaacagcctgacacctgacgacactgccatctactactgtgcggctacgcagggccccctctggcctactttagggactcagttttcgactgaaagttataattactggggccaggggaccctggtcaccgtctcctca。

[0049] Among them, the nanobody protein against H5 subtype avian influenza virus includes a framework region (FR) and a complementarity-determining region (CDR) of the antibody gene. The framework region is divided into four parts, which are sequentially defined as FR1, FR2, FR3, and FR4, and their amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; the complementarity-determining region is divided into three parts, which can be sequentially defined as CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9;

[0050] SEQ ID NO: 3: QVQLQESGGGSVQAGGSLRLSCSAS;

[0051] SEQ ID NO: 4: WFRQVPGKEREGVAA;

[0052] SEQ ID NO: 5: YYPTVDGRFIISRGNAKNSVDLEMNSLTPDDTAIYYC;

[0053] SEQ ID NO: 6: WGQGTLVTVSS;

[0054] SEQ ID NO: 7: EYTRSMA;

[0055] SEQ ID NO: 8: IDTGNGNT;

[0056] SEQ ID NO: 9: AATQGPLWPTLGTQFSTESYNY.

[0057] Example 3

[0058] Expression of nanobody

[0059] The full-length Nb10 sequence was synthesized by General Biosystems and an 8-His-tag was introduced at the C-terminus. This sequence was cloned into the EcoRI and XbaI sites of the expression vector pPICZαA to construct the recombinant expression plasmid pPICZαA-Nb10. The recombinant plasmid was introduced into P. pastoris X33 competent cells by chemical transformation. The transformed products were spread on YPD plates containing 100 μg / ml bleomycin and cultured at 30 °C for 3 - 5 days. Positive clones were screened by PCR using primers 5α-Factor-F and 3AOX1-R. The positive clones were inoculated into BMGY medium and cultured with shaking at 30 °C and 250 rpm. OD 600 was monitored until it reached 2 - 6 to ensure the logarithmic growth phase. The cells were resuspended in BMMY medium and protein expression was induced by adding 1% (v / v) methanol. The culture was continuously incubated at 28 - 30 °C and 250 - 300 rpm for 3 days, and methanol was replenished every 24 hours to a final concentration of 0.5%. After three days, the supernatant was collected by centrifugation at 4 °C and 5000 g for 10 minutes, and the culture supernatant was purified using a Ni-NTA affinity chromatography column. Observation Figure 2 showed that the PAGE pattern of the purified nanobody Nb10 protein indicated that its molecular weight was approximately 16 KDa. After dialysis to remove imidazole, the protein concentration was determined using the BCA method. The primers are shown in Table 3:

[0060] Table 3

[0061] Primer Sequence (5′-3′) 5α-Factor-F TACTATTGCCAGCATTGCTGC 3AOX1-R GGCAAATGGCATTCTGACAT

[0062] Example 4 Verification of in vitro biological activity of broad-spectrum neutralizing nanobody

[0063] In this study, the hemagglutination inhibition assay (HI) and microneutralization assays (MN) were used to verify the biological activity of the broad-spectrum neutralizing nanobody.

[0064] In the HI assay, the purified Nb10 was serially diluted 2-fold in a V-bottom 96-well plate and mixed with 4 HA units of H5 AIV. The plate was incubated at room temperature for 30 minutes, and 1% chicken red blood cells were added to each well. HI-IC 50Defined as the nanobody concentration that inhibits 50% of the hemagglutination of red blood cells. The evaluation was carried out by repeating the measurement three times. The preparation of 4 HA units requires the determination of the virus titer first, and the operation is as follows: Dilute 25 μL of the allantoic fluid containing the virus in a 96-well V-bottom plate with PBS at a ratio of 1:2. Next, add 25 μL of PBS and add 25 μL of chicken red blood cells diluted to 1% to the virus dilution. Incubate the plate at 4 °C for 40 minutes. The HA titer refers to the dilution multiple of the allantoic fluid that can completely agglutinate red blood cells.

[0065] Maintain MDCK cells in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1% l-glutamine at 37 °C and 5% CO2. One day before the experiment, add 25,000 MDCK cells to each well of a 96-well plate. Mix Nb10 with an equal volume of 100 TCID 50 virus for 1 hour, then add it to MDCK cells at 37 °C for 1 hour. Remove the mixture and culture the cells in DMEM medium supplemented with 1 μg / ml of TPCK-treated trypsin and the appropriate concentration of Nb10 at 37 °C for 20 hours. Wash the cells twice with PBS, fix them with 80% ice-cold acetone at -20 °C for at least 1 hour, wash them 3 times with PBS, block them with 3% BSA-PBS for 30 minutes, and then treat them with 2% H2O2 for 30 minutes. Incubate the cells with mouse anti-NP antibody (1:3000) in 3% BSA-PBS at room temperature for 1 hour, and then incubate with 488-conjugated goat anti-mouse IgG (1:5000) in 3% BSA-PBS at room temperature for 1 hour. Visualize using a fluorescence microscope. The signal from the uninfected wells represents 100% inhibition on average, and the signal from the wells of the infected wells that have not been incubated with the antibody represents 100% infection on average. All experiments were carried out in triplicate. MN-IC 50 Represents a 50% reduction in the fluorescence signal to the level obtained in the 100% infection control group of the virus.

[0066] The broad-spectrum neutralizing activity of nanobody Nb10 was verified by using multiple recombinant H5 viruses, including clades 2.3.2.1 (Re6 / PR8, Re10 / PR8, and Re12 / PR8) and 2.3.4.4 (Re8 / PR8, Re11 / PR8, and Re14 / PR8). All the viruses used above were reverse recombinant influenza viruses. The plasmid containing HA and the remaining seven influenza virus genes derived from A / Puerto Rico / 8 / 34 (H1N1) were transfected into co-cultured 293T and MDCK cells by using Lipofectamine 2000, thereby rescuing the rearranged viruses. 72 hours after transfection, the medium was inoculated into chicken embryos or MDCK cells. The original virus was propagated in the allantoic cavity of 10-day-old embryonated eggs, and the allantoic fluid containing the virus was harvested and stored in aliquots at -80 °C. The virus titer was determined by standard hemagglutination assay. The HA gene of the rearranged virus from the second passage was sequenced to confirm the introduced HA gene. Re6 / PR8, Re8 / PR8, Re10 / PR8, Re11 / PR8, Re12 / PR8, and Re14 / PR8 were constructed, and their HA genes were derived from viruses such as A / duck / Guangdong / S1322 / 2010, A / chicken / Guizhou / 4 / 2013, A / duck / Anhui / S1246 / 2014, A / duck / Guizhou / S4184 / 2017, A / chicken / Liaoning / SD007 / 2017, and A / whooperswan / Shanxi / 4-1 / 2020;

[0067] Test results of the hemagglutination inhibition titer and neutralization concentration of nanobody Nb10 against each recombinant virus

[0068] The specific test results are shown in Table 4-5:

[0069] Table 4

[0070]

[0071]

[0072] \nTable 5

[0073]

[0074] The Re6 / PR8, Re10 / PR8, and Re12 / PR8 viruses belong to the 2.3.2.1 clade, and the Re8 / PR8, Re11 / PR8, and Re14 / PR8 viruses belong to the 2.3.4.4 clade. Overall, Nb10 has slightly better neutralizing activity against the 2.3.4.4 clade of H5AIV than the 2.3.2.1 clade, and has the best hemagglutination inhibition and neutralizing activities against the Re14 / PR8 virus, with HI-IC 50 and MN-IC 50 values of 0.05 and 0.01 μg / ml, respectively;

[0075] Meanwhile, to our surprise, during the yeast two-hybrid screening process, we screened for antibodies using the sequence of the HA1 of the Re8 virus as a bait. At the same time, the characteristic of the yeast two-hybrid system is that the antibodies obtained by screening are highly specific to the bait used for screening;

[0076] On this basis, when we tested HI-IC 50 and MN-IC 50 , we found that although it has extremely excellent hemagglutination inhibition titers and neutralizing concentrations against the Re8 / PR8 virus, its hemagglutination inhibition titers and neutralizing concentrations are even more excellent against the Re14 / PR8 virus. At the same time, its hemagglutination inhibition titers and neutralizing concentrations against the Re11 virus, which is in the same clade as Re8 and Re14, are far from those of the two, and its hemagglutination inhibition titer and neutralizing concentration against the Re11 virus are almost 20 times that of the Re8 virus;

[0077] And its hemagglutination inhibition titer and neutralizing concentration against the Re14 virus are almost about 50% of the Re8 virus;

[0078] This result exceeded our expectations. On the one hand, we screened for antibodies using the HA1 sequence of the Re8 virus, and in the yeast two-hybrid system, the antibody and the bait have extremely high specificity. In theory, we should have obtained an antibody that is particularly suitable for the Re8 virus, but this antibody has a more significant neutralizing ability against Re14. It is speculated that the reason for this phenomenon may be that the recognition site of the nanobody is not hindered by glycosylation. The nanobody identified by yeast two-hybrid may interact with Re8-HA1 in the absence of glycosylation, but the HA1 protein contains multiple glycosylation sites. The RBS region of Re8-HA1 recognized by Nb10 has an N-glycosylation modification site, while the RBS region of Re14-HA1 lacks this feature; this leads to stronger neutralization of Nb10 against Re14 / PR8 than Re8 / PR8.

[0079] Example 5 In vivo biological activity verification of broad-spectrum neutralizing nanobody

[0080] To further evaluate the protective potential of Nb10 against H5 subtype AIV-infected mice, we infected female mice (n = 5) aged 6 to 8 weeks with Re6 / PR8, Re8 / PR8, Re10 / PR8, Re11 / PR8, and Re14 / PR8 viruses at a dose of 10 ml D 50 After 24 hours of virus infection, the Re8 / PR8 and Re14 / PR8 infection groups were nebulized with Nb10 at a dose of 2.5 mg / kg or 3.5 mg / kg via intratracheal administration, and the Re6 / PR8, Re10 / PR8, and Re11 / PR8 groups were treated with a dose of 4 mg / kg or 8 mg / kg via intratracheal administration. Animals with an initial weight loss of more than 25% were immediately euthanized by CO2 asphyxiation and recorded as non-survivors. The control group was nebulized with PBS (placebo) via intratracheal administration. The mortality of the animals was observed daily, and the weight fluctuations up to 14 days after infection were recorded. Referring to Figure 3-12 , the protective effect of Nb10 was dose-dependent: 2.5 mg / kg of Nb10 protected 80% of the mice infected with Re8 / PR8 from death and prevented significant weight loss in the mice under a lethal challenge. 3.5 mg / kg of Nb10 provided 100% protection, and the body weight increased significantly ( Figure 5-6 ). Nb10 at a dose of 2.5 mg / kg or 3.5 mg / kg could provide complete protection for the mice infected with Re14 / PR8, and the body weight of the mice began to show an increasing trend on the fourth day after infection ( Figure 11-12 ). In the in vivo protection experiment, Nb10 had the best neutralizing protection effect against Re14 / PR8. 4 mg / kg of Nb10 could provide a 60% protection rate for the mice infected with Re10 / PR8, and 8 mg / kg could reach an 80% protection rate, with significant fluctuations in the body weight of the mice ( Figure 7-8 ). 4 mg / kg provided only 20% and 40% protection rates for the mice infected with Re6 / PR8 and Re11 / PR8, respectively, but the protection rates of 8 mg / kg could reach 60% and 80%, and the body weight of the mice increased less in the later stage of the experiment ( Figure 3-4 and 9-10). All PBS-treated control animals showed continuous weight loss after virus challenge and died within 8 days after infection. These results indicate that passive immunization with Nb10 provides good neutralizing protection for mice against avian influenza viruses of the 2.3.2.1 and 2.3.4.4 branches of the H5 subtype.

Claims

1. A nanobody against H5 subtype avian influenza virus, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. The anti-H5 subtype avian influenza virus nanobody according to claim 1, characterized in that The nucleotide sequence is shown in SEQ ID NO:

2.

3. The anti-H5 subtype avian influenza virus nanobody according to claim 1, characterized in that The anti-H5 subtype avian influenza virus nanobody is designed based on the H5-Re8 virus.

4. Use of the anti-H5 subtype avian influenza virus nanobody according to any one of claims 1 to 3 in preparing anti-H5 subtype avian influenza virus drugs.

5. The use according to claim 4, characterized in that The anti-H5 subtype avian influenza virus drug is specifically a drug against the 2.3.2.1 branch and / or the 2.3.4.4 branch of the H5 subtype avian influenza virus.

6. The use according to claim 4, characterized in that The anti-H5 subtype avian influenza virus drug is specifically an anti-H5-Re14 virus drug.

7. An anti-H5 subtype avian influenza virus drug, characterized in that: Contains the anti-H5 subtype avian influenza virus nanobody according to any one of claims 1-3.

8. A method for constructing the anti-H5 subtype avian influenza virus nanobody according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: Immunize camelids with an inactivated or attenuated vaccine of the H5 subtype avian influenza virus to induce the camelids to produce antibodies; Step 2: Using the yeast two-hybrid system and HA1 of the H5 subtype avian influenza virus as bait, the antibodies were screened to obtain the target antibodies; Step 3: Sequence and express the target antibody to obtain anti-H5 subtype avian influenza virus nanoantibodies.

9. The method for constructing an anti-H5 subtype avian influenza virus nanobody according to claim 8, characterized in that: The step 1 specifically comprises: immunizing camelids with an inactivated vaccine of the H5 subtype avian influenza virus to induce the camelids to produce antibodies.

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