Recombinant nano antibody for resisting H9 subtype avian influenza virus HA protein and application of recombinant nano antibody

Abstract

The invention discloses a recombinant nano antibody for resisting H9 subtype avian influenza virus HA protein and application of the recombinant nano antibody, and relates to the field of bioengineering, and the amino acid sequence of the recombinant nano antibody is SEQ ID NO: 2; the nucleotide sequence for coding the amino acid sequence of the recombinant nano antibody is SEQ ID NO: 1. The yield of the recombinant nano-antibody is 1.84 mg / mL, the recombinant nano-antibody is only specifically combined with H9 virus and HA protein, has hemagglutination inhibition activity and HI titer of 8.3 log2, is the first nano-antibody with hemagglutination inhibition activity reported at present, has half effective inhibition concentration IC50 of 16.31 mu g / mL, can significantly reduce the detoxification level of the virus in chicks, has high biological activity, and can be used for preparing the recombinant nano-antibody. The strain can be applied to prevention and / or treatment of H9 subtype avian influenza, provides a novel, efficient and customizable biological preparation platform for prevention and control of avian influenza, and has important scientific research value and industrialization prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a recombinant nanobody against HA protein of H9 subtype avian influenza virus and application thereof. BACKGROUND

[0002] H9 subtype avian influenza virus (H9 AIV) infection can cause respiratory symptoms and production performance decline in chicken flocks, and is prone to secondary bacterial infection, etc., which brings serious economic losses to poultry farming. The genome of avian influenza virus is composed of 8 segments, which encode PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, and NS2 proteins, respectively. These proteins play an important role in virus infection, replication, and immune escape, etc. HA protein and NA protein are the most important surface glycoproteins of influenza virus, and are also the basis for the classification of influenza virus subtypes, which are involved in virus adsorption, internalization, and release of progeny virus. In recent years, the widespread use of H9 subtype avian influenza vaccine has significantly reduced the outbreak and prevalence of the disease, but new features such as atypical symptoms of “old disease and new outbreak” have appeared. Poultry immunization with virus, immunization failure, regional prevalence, and mixed infection are common, and prevention and control are facing new challenges. Therefore, it is necessary to develop new prevention and control technologies and products.

[0003] According to the genetic variation of HA gene, H9 subtype can be further divided into h9.4.2.1, h9.4.2.2, h9.4.2.3, h9.4.2.4, h9.4.2.5, and h9.4.2.6. At present, the h9.4.2.5 branch is the main epidemic strain. H9 AIV mutates rapidly, and the development speed of vaccine cannot keep up with the mutation speed of the pathogen. In addition, the registration period of vaccine development is long. These present situations are not conducive to the prevention and control of H9 AIV, and the strategy of vaccine prevention and control has limitations.

[0004] Nanobody (Nb) is a heavy chain antibody (HcAb) derived from camelids or cartilaginous sharks, which lacks light chains. The variable domain of heavy chain antibody (VHH) has a molecular weight of about 15 kDa, and is the smallest antibody fragment known to have the smallest binding activity. At present, nanobody technology has played an important role in the diagnosis and treatment of immunological diseases and tumor diseases. However, there are few reports on recombinant nanobody against HA protein of H9 subtype avian influenza virus. SUMMARY

[0005] The application aims to provide a recombinant nanobody against HA protein of H9 subtype avian influenza virus and an application thereof.

[0006] The technical solution adopted by the application to solve the technical problems is as follows:

[0007] Firstly, the application provides a recombinant nanobody against HA protein of H9 subtype avian influenza virus.

[0008] According to the application, the amino acid sequence of the recombinant nanobody is shown as SEQ ID NO: 2, and the nucleotide sequence of the amino acid sequence encoding the recombinant nanobody is shown as SEQ ID NO: 1.

[0009] Secondly, the application provides a preparation method of the recombinant nanobody against HA protein of H9 subtype avian influenza virus.

[0010] According to the application, the preparation method comprises the following steps:

[0011] A VHH phage library of immunized alpaca is established, and an antibody library solid-phase screening technology is used to screen VHH sequences capable of binding to HA protein of H9 subtype avian influenza virus, i.e., the sequence of the recombinant nanobody against HA protein of H9 subtype avian influenza virus.

[0012] Thirdly, the application provides a recombinant expression vector pPIC9K-HA-Nb2.

[0013] According to the application, the recombinant expression vector pPIC9K-HA-Nb2 comprises the coding gene of the recombinant nanobody against HA protein of H9 subtype avian influenza virus.

[0014] According to the application, the nucleotide sequence of the recombinant expression vector pPIC9K-HA-Nb2 is shown as SEQ ID NO: 3.

[0015] Fourthly, the application provides a recombinant yeast engineering strain.

[0016] According to the application, the recombinant yeast engineering strain is prepared from the recombinant expression vector pPIC9K-HA-Nb2.

[0017] According to the application, the recombinant yeast engineering strain is used to express the recombinant nanobody against HA protein of H9 subtype avian influenza virus.

[0018] Fifthly, the application provides an application of the recombinant nanobody against HA protein of H9 subtype avian influenza virus in preparation of a medicine against H9 subtype avian influenza virus.

[0019] According to the application, the medicine is a neutralizing antibody medicine, a diagnostic reagent, or a vaccine adjuvant, etc.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] The present application provides a recombinant nanobody HA-Nb2 with high specificity and high affinity, which is directed against H9 subtype avian influenza virus HA protein, has a clear amino acid sequence (SEQ ID NO: 2) and encoding nucleotide sequence (SEQ ID NO: 1), and clear structure, easy to direct modification and large-scale production. Experimental data show that the recombinant nanobody HA-Nb2 is only specifically combined with H9 subtype avian influenza virus and HA protein, and has no cross reaction with H1, H3, H5, H7 and other subtype influenza viruses and newcastle disease virus, showing good specificity.

[0022] The recombinant nanobody HA-Nb2 of the present application has good neutralizing activity and diagnostic potential: the recombinant nanobody HA-Nb2 shows significant neutralizing activity in chicken embryo neutralization test, the half effective inhibition concentration IC 50 is 16.31 μg / mL, and the HI titer is 8.3 log2, showing good virus inhibition capacity. Therefore, the recombinant nanobody HA-Nb2 of the present application can be used for developing neutralizing antibody drugs, diagnostic reagents or vaccine adjuvants, and providing new tools for prevention and control of H9 subtype avian influenza.

[0023] The present application constructs a recombinant expression vector pPIC9K-HA-Nb2 (SEQ ID NO: 3) and a recombinant yeast engineering strain, realizes efficient expression and purification of nanobody in Pichia pastoris system, the molecular weight of the expression product is about 15 kDa, which is consistent with the theoretical value, the purity is up to 60%, and the yield is 1.84 mg / mL.

[0024] In addition, the present application also establishes an efficient nanobody screening and expression system: a VHH phage library is constructed by using immunized llama, the library capacity is up to 3.5x10 5 CFU / mL, the insertion rate is 100%, and the sequence diversity is rich (consistency 18.43%~93.8%), which lays a foundation for screening high-performance nanobody.

[0025] The recombinant nanobody HA-Nb2 of the present application has the advantages of small molecule, easy modification and short production cycle, and is more suitable for dealing with rapidly mutating virus strains, overcoming the limitations of long development cycle of traditional vaccines and not keeping up with virus mutation. The present application first reports the recombinant nanobody against H9 subtype avian influenza virus HA protein, and provides a complete preparation and application scheme, filling the blank of anti-H9 subtype avian influenza virus HA protein nanobody research. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1The results of detecting the antibody titer of the serum of the immunized lama.

[0027] Figure 2 The results of electrophoresis analysis. In the figure, A is the band of the first round of nested PCR amplification product; B is the band of the second round of nested PCR amplification product; C is the band of the target gene in the double enzyme digestion product; D is the band of the vector in the double enzyme digestion product.

[0028] Figure 3 The results of detecting the VHH fragment insertion rate of the VHH phage library of the immunized lama and analyzing the diversity of VHH sequences. In the figure, A is the result of positive colony PCR amplification; B is the result of diversity analysis.

[0029] Figure 4 The results of Phage-ELISA detection.

[0030] Figure 5 The map of the recombinant expression vector pPIC9K-HA-Nb2.

[0031] Figure 6 The results of identifying the recombinant nanobody HA-Nb2. In the figure, A is the result of SDS-PAGE detection; B is the result of Western Blot detection.

[0032] Figure 7 The results of specificity detection of the recombinant nanobody HA-Nb2.

[0033] Figure 8 The results of IFA identification of the recombinant nanobody HA-Nb2 and the HA protein of the H9 subtype avian influenza virus.

[0034] Figure 9 The hemagglutination inhibition titer of the recombinant nanobody HA-Nb2.

[0035] Figure 10 The results of determination of the half maximal inhibitory concentration of the recombinant nanobody HA-Nb2. DETAILED DESCRIPTION

[0036] The advantages and features of the present application will become more apparent with the description of the specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation on the protection scope of the present application.

[0037] I. Source of materials

[0038] H9 subtype avian influenza virus A / chicken / China / NJ150 / 2020 (NJ150 strain, GenBank accession number: OP804327.1, EID 50 / 0.1 mL is 1.0×10 13, 1 HAU about 1.84 μg], inactivated H1 subtype influenza virus (H1 IV, 1 HAU about 2.29 μg], H3 subtype influenza virus (H3 IV, 1 HAU about 2.41 μg], recombinant baculovirus rBV-HA (expressing H9 subtype avian influenza virus HA protein, titer 2.5×10 5 PFU / mL), and insect Sf9 cells were preserved by Jiangsu Academy of Agricultural Sciences Institute of Veterinary Medicine.

[0039] Four-year-old female alpacas were purchased from Jiangsu Dengyuanhe Biological Technology Co., Ltd.

[0040] Nine-day-old SPF chicken embryos were purchased from Jiangsu Boehringer Ingelheim Vito Biological Technology Co., Ltd.

[0041] Avian influenza virus H5 subtype (Re-14 strain) hemagglutination inhibition test antigen (H5 AIV, 1 HAU about 3.95 μg], avian influenza virus H7 subtype (Re-4 strain) hemagglutination inhibition test antigen (H7 AIV, 1 HAU about 3.75 μg], and Newcastle disease (LaSota strain) hemagglutination inhibition test antigen (NDV, 1 HAU about 3.60 μg] were purchased from Harbin Weike Biological Technology Co., Ltd.

[0042] H9 subtype avian influenza virus HA protein was purchased from Beijing Yiqiao Godz Technology Co., Ltd.

[0043] Escherichia coli TG1 competent cells and Pichia pastoris GS115 competent cells were purchased from Shanghai Weidi Biological Technology Co., Ltd.

[0044] II. Animal immunization and determination of serum antibody titer

[0045] (1) 2 mL of inactivated NJ150 strain (containing 1 mg of purified virus) was mixed and emulsified with 2 mL of Freund's complete adjuvant, and Freund's incomplete adjuvant was used for booster immunization. Multiple point injection was performed on the neck muscles of 4-year-old healthy female alpacas, booster immunization was performed every 2 weeks, a total of 6 times, and serum was collected and separated before each immunization for determination of antibody titer. Animal experiments were strictly in accordance with the standards of animal welfare and ethics of Jiangsu Academy of Agricultural Sciences Experimental Animal Management Committee (approval number: IACUC-OE-2023-07-005).

[0046] (2) The purified NJ150 strain was coated at 5 HAU / well, and 5% BSA was used for blocking treatment. The llama serum was diluted at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000 as a primary antibody. Rabbit anti-camel HRP-labeled antibody was used as a secondary antibody to detect the antibody titer of the llama serum. The D450 nm value was collected, and P / N>2.1 was judged as positive. According to the published “GB / T 18936-2025 Avian Influenza Diagnosis Technology”, four hemagglutination units of antigen (4 HAU) of the NJ150 strain were prepared. 25 μL PBS was added to wells 1-11, and 50 μL PBS was added to well 12. 25 μL of llama serum was added to well 1, and it was diluted by two-fold to well 11, and 25 μL was discarded. 25 μL of 4 HAU was added to wells 1-11, and incubated at room temperature for 30 min. 25 μL of 1% chicken red blood cells was added to each well, and incubated at room temperature. The results were judged by the appearance of button-like red blood cells in well 12. The results are shown in Figure 1 As shown in FIGS. 1A and 1B, the antibody titer of the serum showed an upward trend with the number of immunizations, and reached a peak after the fifth immunization. The ELISA antibody titer reached 1:64000, and the HI titer reached 11 log2. The peripheral blood of the llama was collected for the construction of a nanobody library.

[0047] III. Construction of a VHH phage library of the immunized llama

[0048] (1) The peripheral blood of the llama was collected, and the lymphocytes were separated using a camel peripheral blood lymphocyte separation kit, RNA extraction kit, and cDNA first-strand synthesis kit according to the instructions. The RNA was extracted and reverse transcribed into cDNA. The cDNA was used as a template, and the VHH-P1-F primer and the VHH-P1-R primer were used for first-round nested PCR amplification. The expected band was recovered as a second-round amplification template, and the VHH-P2-F primer and the VHH-P2-R primer were used for second-round nested PCR amplification to obtain the expected VHH sequence. The two-round nested PCR products were analyzed by electrophoresis, and the results are shown in FIGS. 2A and 2B. Figure 2 As shown in FIGS. 2A and 2B, expected bands of about 700 bp and about 400 bp can be seen, respectively.

[0049] The first-round nested PCR amplification primer sequences were VHH-P1-F and VHH-P1-R, and the sequence information is as follows:

[0050] VHH-P1-F: 5'-GTCCTGGCTGCTCTTCTACAAGG-3';

[0051] VHH-P1-R: 5′-GGTACGTGCTGTTGAACTGTTCC-3′.

[0052] The primer sequences for the second round of nested PCR amplification are VHH-P2-F and VHH-P2-R, and their sequence information is as follows:

[0053] VHH-P2-F: 5′-GAGCTCATGGATGTGCAGCTGGTGGA-3′;

[0054] VHH-P2-R: 5′-ACTAGTTGAGGAGACGGTGACCT-3′.

[0055] The first round of nested PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 1 minute, this step is repeated for 35 cycles; extension at 72℃ for another 7 minutes.

[0056] The second round of nested PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, this step is performed for 35 cycles; extension at 72℃ for another 7 minutes.

[0057] (2) The VHH recovered product and pComb3XSS vector were digested, recovered, ligated, and desalted by restriction endonucleases Sac I and Spe I. The mixture was then electroporated into TG1 competent cells and plated on 2×YTA solid plates (1% Trypton, 1.6% Yeast Extract, 5 g NaCl, and 2% Agar, containing 100 μg / mL Amp). The double-digested products were analyzed by electrophoresis. The results are as follows: Figure 2 As shown in C and D, a target gene band of approximately 400 bp and a vector band of approximately 3300 bp are visible. The following day, the library size was calculated based on the dilution factor. Twenty-three randomly selected single-clone colonies were identified by PCR using VHH-P2-F and VHH-P2-R primers to determine the VHH insertion rate. Sequencing results were processed using BLAST and MEGA software to calculate the nucleotide sequence identity of each VHH phage, and a homology matrix was constructed to analyze library sequence diversity. Thus, the VHH phage library for immunizing alpacas was successfully constructed. The calculated library size is approximately 3.5 × 10⁻⁶. 5 CFU / mL, positive colony PCR amplification results are as follows Figure 3 As shown in Figure A, the insertion rate of the VHH fragment is 100%. The diversity analysis results are as follows: Figure 3 As shown in B, the nucleotide sequence identity of the VHH library ranged from 18.43% to 93.8%, indicating rich sequence diversity in the library.

[0058] IV. Screening and refining of specific nanobodies;

[0059] (1) Take the VHH phage library of the immunized alpaca and inoculate it into 200 mL of 2xYTA liquid medium for culture until the D600 nm is about 0.5, and then perform superinfection with a MOI of 25, add M13KO7 helper phage to rescue the VHH phage library, use PEG6000 solution to purify the recombinant phage, calculate the titer of the recombinant phage, and use it for subsequent tests.

[0060] (2) Dilute the purified NJ150 strain with the antigen coating solution, and coat it at 5 HAU / well in the first round, set up 3 duplicate wells and a negative control, block with 5% BSA, and then add 1.0x10 11 Incubate the recombinant phage for 2 h, wash it 10 times with 0.5% PBST solution, shake and elute each well with 120 μL of Gly-HCl solution (pH=2.2), immediately neutralize it with 30 μL of Tris-HCl solution (pH=8.0), and determine the titer of the eluted recombinant phage. Collect the output bacterial solution the next day, prepare the recombinant phage according to the test scheme of step (1) above, coat it at a concentration of 3 HAU / well in the second round, and follow the same steps as above. Refer to the published “GB / T 18936-2025 Avian Influenza Diagnosis Technology” to determine the hemagglutination inhibition titer of the recombinant phage.

[0061] The results are shown in Table 1. Figure 4 Among the 60 positive recombinant phages obtained by screening, the VHH sequence numbered as HA-Nb2 has the highest P / N value (23.92), the amino acid sequence thereof is shown as SEQ ID NO: 2, and the nucleotide sequence encoding the amino acid sequence is shown as SEQ ID NO: 1.

[0062] V. Expression and purification of recombinant nanobodies

[0063] (1) According to the preference of Pichia pastoris codons, the codons are optimized without changing the amino acid sequence. An EcoR I restriction site is introduced at the 5' end of the sequence, a (G4S) flexible Linker sequence, a 6xHis tag, a Not I restriction site, and a stop codon (TGA) are introduced at the 3' end. The synthesized gene sequence (synthesized by General Biosystems Co., Ltd.) is ligated to the Pichia pastoris recombinant shuttle vector pPIC9K using T4 DNA ligase (purchased from Baori Biotechnology Co., Ltd.), transformed into E. coli competent cells, and a strain containing the recombinant expression vector pPIC9K-HA-Nb2 is obtained. The map of the recombinant expression vector pPIC9K-HA-Nb2 is shown in FIG. 1, and the nucleotide sequence thereof is shown as SEQ ID NO: 3. Figure 5

[0064] ​(2) Linearize the recombinant expression vector pPIC9K-HA-Nb2 with restriction enzyme Sal I, add to the competent cells of Pichia pastoris GS115, mix, then transfer to a pre-cooled electroporation cup, ice bath, then transfer to an electroporator for electroporation. After electroporation, add 1 mL of pre-cooled 1 M sorbitol solution, mix, then transfer to a centrifuge tube, and incubate at 30°C for 2 h. Centrifuge at 3000 r / min for 3 min at room temperature, collect the bacteria, and resuspend in YPG medium. Plate on YPG solid medium containing geneticin, and incubate at 30°C for 2 d. Pick single colonies for PCR identification. After correct identification, obtain the recombinant yeast engineering strain for expression of the recombinant nanobody.

[0065] (3) Inoculate the positive strain in BMGY liquid medium (1% Yeast extract, 2% Peptone, 100 mmol / L K3PO4, 1.34% YNB, and 1% Glycerol), and culture until D450 nm is 2.0. Centrifuge to discard the supernatant, and replace with BMMY medium (1% Yeast extract, 2% Peptone, 100 mmol / L K3PO4, 1.34% YNB, and 0.5% methanol) for induction culture. Supplement with a 0.5% methanol solution every 24 h, and induce culture for 120 h. Analyze the expression of the recombinant protein by SDS-PAGE and Western Blot, purify the recombinant protein by referring to the Ni-NTA column affinity chromatography kit, and determine the concentration of the recombinant protein by using a BCA kit. The identification results are shown in Figure 6 . The obtained recombinant protein has a relative molecular mass of about 15 kDa, which is consistent with the expected value, indicating that the recombinant protein is the recombinant nanobody HA-Nb2, with a concentration of 1.84 mg / mL and a purity of 60%.

[0066] Six, determination of the binding activity of the recombinant nanobody;

[0067] (1) Verify the binding activity of the recombinant nanobody HA-Nb2 to the H9 subtype avian influenza virus by ELISA detection. The detection steps are as follows: coat NJ150 strain at 5 HAU / well, set H1 IV, H3 IV, H5 AIV H5, H7 AIV, NDV, and 10 μg / well of chicken embryo allantoic fluid at 5 HAU units, block with 5% BSA, use the recombinant nanobody as a primary antibody (set PBS as a control), use a mouse anti 6×His Tag HRP-labeled antibody as a secondary antibody, determine the D450 nm value, and set P / N=2.1 as the positive critical value.

[0068] The ELISA results are shown in Figure 7As shown, the recombinant nanobody HA-Nb2 showed no cross-reactivity with H1 IV, H3 IV, H5 AIV, H7 AIV, NDV, and chicken embryo allantoic fluid (CEAF), and specifically bound only to the H9 subtype avian influenza virus, indicating that the recombinant nanobody HA-Nb2 has good specificity.

[0069] (2) The binding activity of recombinant nanobody HA-Nb2 to H9 subtype avian influenza virus HA was verified by IFA assay. The detection steps are as follows: Recombinant baculovirus rBV-HA (expressing H9 subtype avian influenza virus HA protein, titer of 2.5 × 10⁻⁶) was used to verify the binding activity of recombinant nanobody HA-Nb2 to H9 subtype avian influenza virus HA protein. 5 Sf9 cells grown to 80% coverage were inoculated with PFU / mL at a MOI of approximately 10. After 48 h of infection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and blocked with 5% BSA solution. Recombinant nanobody was used as the primary antibody, and Mouse anti 6×His FITC-tagged polyclonal antibody was used as the secondary antibody. After DAPI staining, the cells were observed under a fluorescence microscope.

[0070] IFA results are as follows Figure 8 As shown, green fluorescence signals were observed in all Sf9 cells of the nanobody group, indicating that the nanobody can react with the HA protein of H9 subtype avian influenza virus.

[0071] VII. Determination of the neutralizing activity of recombinant nanobodies;

[0072] Take 50 μL of recombinant nanobodies of different concentrations (50 μg / mL, 25 μg / mL, 12.5 μg / mL, 5 μg / mL, 0.5 μg / mL and 0.05 μg / mL) and mix them with an equal volume of 2000 EID. 50 NJ150 strain was co-incubated at 37℃ for 2 h, and then inoculated with 9-day-old SPF chicken embryos, with 3 embryos per group. A positive control group was set up (inoculated with 50 μL of 2000 EID). 50 Chicken embryos (NJ150 strain) and a negative control group (100 μL PBS) were observed for mortality every 12 h. Allantoic fluid was harvested from the embryos on day 4 post-inoculation, and the HA titer was measured. The lowest concentration of recombinant nanobody with no HA titer was used as the neutralization endpoint. The neutralization percentage was calculated as [1 - (average HA titer of the positive group - average HA titer of the experimental group) / (average HA titer of the positive group - average HA titer of the negative group)] × 100%. Data processing and analysis were performed using GraphPad Prism (Ver. 10.1.2). Hemagglutination inhibition activity results are as follows: Figure 9 As shown, the HI titer was 8.3 log2, and the results of the chicken embryo neutralization test were as follows. Figure 10 As shown, the HA-Nb2 group exhibited significant neutralizing activity, with an IC50 value of [missing information]. 50was 16.31 μg / mL.

[0073] The present application verifies the specificity, purity, activity and the ability of inhibiting virus replication of the recombinant nanobody HA-Nb2 by various methods such as ELISA, Western Blot, SDS-PAGE, neutralization experiment, etc. The data is reliable. The present application combines the advantages of nanobody and the efficient yeast expression system, successfully develops a kind of recombinant nanobody HA-Nb2 against H9 subtype avian influenza virus HA protein which is efficient, specific and can be produced on a large scale, has important scientific research value and clinical application prospect, especially provides a new strategy for the prevention and treatment of H9 avian influenza.

[0074] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant nanobody against the HA protein of H9 subtype avian influenza virus, characterized in that, The amino acid sequence of the recombinant nanobody is shown in SEQ ID NO:

2.

2. The recombinant nanobody against the HA protein of H9 subtype avian influenza virus according to claim 1, characterized in that, The nucleotide sequence encoding the amino acid sequence of the recombinant nanobody is shown in SEQ ID NO:

1.

3. The method for preparing a recombinant nanobody against the HA protein of H9 subtype avian influenza virus as described in claim 1, characterized in that, Includes the following steps: A VHH phage library for immunizing alpacas was established. Antibody library solid-phase screening technology was used to screen for VHH sequences that can bind to the HA protein of H9 subtype avian influenza virus, which are the recombinant nanobody sequences against the HA protein of H9 subtype avian influenza virus.

4. A recombinant expression vector pPIC9K-HA-Nb2, characterized in that, The recombinant expression vector pPIC9K-HA-Nb2 contains the encoding gene of a recombinant nanobody against the HA protein of H9 subtype avian influenza virus as described in claim 1.

5. The recombinant expression vector pPIC9K-HA-Nb2 according to claim 4, characterized in that, The nucleotide sequence of the recombinant expression vector pPIC9K-HA-Nb2 is shown in SEQ ID NO:

3.

6. A recombinant yeast strain, characterized in that, The recombinant yeast strain was prepared from the recombinant expression vector pPIC9K-HA-Nb2 described in claim 4.

7. The recombinant yeast strain according to claim 4, characterized in that, The recombinant yeast strain is used to express the recombinant nanobody against the HA protein of H9 subtype avian influenza virus as described in claim 1.

8. The application of the recombinant nanobody against the HA protein of H9 subtype avian influenza virus as described in claim 1 in the preparation of drugs against H9 subtype avian influenza virus.

9. The application according to claim 8, characterized in that, The drug is a neutralizing antibody drug, a diagnostic reagent, or a vaccine adjuvant.

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