A nano antibody targeting influenza A virus H1N1 subtype NA protein and its application

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a nano antibody targeting the NA protein of the H1N1 subtype of influenza A virus and its application. The nano antibody comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1. The nano antibody has high affinity, and experimental results show that the antibody can effectively inhibit the proliferation of PR8 H1N1 virus in vivo and resist the infection of mice by a lethal dose of the virus. At the same time, it is the first nano antibody with preventive and therapeutic effects targeting the N1 subtype NA protein, filling a gap in the market and can be applied to laboratory research, biosensor development, clinical diagnosis, infection prevention and treatment and other fields. The nano antibody provided by the present invention has many advantages such as small molecular weight, high stability, and low production cost. Compared with traditional IgG antibodies, it has a wider application potential and market competitive advantage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a nano antibody targeting influenza A virus H1N1 subtype NA protein and its application. Background Art

[0002] Influenza A virus is a highly contagious RNA virus. The hemagglutinin (HA) and neuraminidase (NA) proteins on its surface are key factors for virus invasion and release. Since the first outbreak in 1918, the H1N1 subtype virus has caused global influenza pandemics many times, resulting in millions of infections and hundreds of thousands of deaths. In recent years, with the continuous mutation of viral genes, new influenza virus strains have continued to emerge, posing a serious threat to global public health security.

[0003] At present, drugs for influenza A virus mainly include small molecule antiviral drugs including neuraminidase inhibitors (NAI), M2 ion channel inhibitors and viral RNA polymerase inhibitors. However, existing drugs have obvious shortcomings in anti-influenza virus treatment:

[0004] (1) Drug resistance: As viruses continue to mutate, some viruses have become resistant to the above-mentioned drugs, resulting in reduced therapeutic efficacy. Since influenza viruses are widely resistant to M2 ion channel inhibitors, this type of drug is no longer recommended for the treatment of clinical influenza.

[0005] (2) Side effects: Some drugs may cause side effects during treatment, such as nausea, vomiting, headache, etc., which may affect patient compliance.

[0006] (3) Usage restrictions: Some drugs are only effective against specific types of influenza viruses and need to be used in the early stages of viral infection to achieve the best effect. For example, NAI's representative drug oseltamivir is only effective if used within 48 hours after the onset of symptoms, and some viral mutations have also shown resistance to it.

[0007] It can be seen that although existing drugs have played a certain role in the treatment of influenza, there are still problems with the high variability of influenza viruses and the limitations of existing drugs. Nanobodies refer to the heavy chain variable region of heavy chain antibodies. Compared with traditional monoclonal antibodies, nanobodies are only 1 / 10 of the size of traditional monoclonal antibodies, can recognize hidden epitopes that traditional monoclonal antibodies cannot reach, and have higher stability, low large-scale production costs, and easy modification. They show broad application prospects in the field of pathogen diagnosis and new anti-influenza drug research. Therefore, there is an urgent need to develop new nanoantibodies for the diagnosis and treatment of influenza A virus. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nano antibody with small molecular weight, high stability and strong affinity for NA protein of influenza A virus H1N1 subtype and its application.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a nanobody against NA protein of H1N1 subtype of influenza A virus, wherein the nanobody comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0011] As a preferred technical solution, the complementary determining regions of the Nanobody include CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 4.

[0012] In a second aspect, the present invention provides a DNA molecule encoding the above-mentioned nanobody against the NA protein of the H1N1 subtype of influenza A virus.

[0013] As a preferred technical solution, the sequence of the DNA molecule is shown as SEQ ID NO:5.

[0014] As a preferred technical solution, the primer pair used to amplify the DNA molecule includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 7.

[0015] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned DNA molecule.

[0016] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises the above-mentioned DNA molecule or the above-mentioned recombinant vector.

[0017] In a fifth aspect, the present invention provides the use of the above-mentioned nanobody in the preparation of a detection kit for detecting influenza A virus H1N1 subtype NA protein or its content in a sample.

[0018] In a sixth aspect, the present invention also provides the use of the above-mentioned nanoantibodies in the preparation of drugs for preventing and treating influenza A virus.

[0019] The present invention has the following advantages: The present invention provides a high-affinity specific nano antibody for the N1 subtype NA protein derived from H1N1. The results of in vivo experiments in mice show that the antibody can effectively inhibit the proliferation of PR8 H1N1 virus in vivo and resist the infection of mice by lethal doses of the virus. At the same time, this is the first nano antibody with preventive and therapeutic effects against the N1 subtype NA protein, which fills the gap in the market and provides more and better options for the diagnosis and treatment of subtype influenza H1N1. It has many application potentials and advantages and can be applied to laboratory research, biosensor development, clinical diagnosis, infection prevention and treatment, and other fields. The nano antibody provided by the present invention has many advantages such as small molecular weight, high stability, and low production cost. Compared with traditional IgG antibodies, it has a wider application potential and market competitive advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the titer of serum antibodies after four immunizations with alpaca NA protein.

[0021] Figure 2 This is the electrophoresis result of the first step PCR product of amplifying the VHH fragment using the nested PCR method.

[0022] Figure 3 This is the electrophoresis result of the second step PCR product of the VHH fragment amplified by the nested PCR method.

[0023] Figure 4 This is a graph showing the affinity determination results of N1E4 for N1-NA.

[0024] Figure 5 The figure is a curve of body weight changes of mice after injection of N1E4.

[0025] Figure 6 The figure shows the survival rate of mice after injection of N1E4. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments, and the protection scope of the present invention is not limited to the following:

[0027] Example 1: NA protein alpaca immunization

[0028] H1N1-NA protein was purchased from Beijing Sino Biological Technology Co., Ltd., catalog number: 11058-V07B, corresponding to the strain A / California / 04 / 2009. For the first immunization, 0.5 mg of NA protein was emulsified with an equal volume of complete Freund's adjuvant and immunized alpacas at multiple points under the neck; for the next three immunizations, 0.25 mg of NA protein was emulsified with an equal volume of incomplete Freund's adjuvant, with an immunization interval of two weeks. On the 7th day after the fourth immunization, 2 mL of peripheral blood was collected to separate serum; at the same time, 40 mL of peripheral anticoagulant blood was collected to separate peripheral blood lymphocytes.

[0029] Serum antibody titer was tested by ELISA:

[0030] 1) Coat the ELISA plate with H1N1-NA protein (200 ng / well) at 4°C overnight;

[0031] 2) The next day, remove the coating solution, wash with PBS, add 5% skim milk, and block at room temperature for 1 hour;

[0032] 3) Add 100 μL of 2-fold serially diluted serum (negative control is pre-immune alpaca serum) to each well and incubate at room temperature for 1 hour;

[0033] 4) Wash 6 times with PBST, add 1:10000 diluted horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody to each well, and incubate at room temperature for 30 min;

[0034] 5) Wash three times with PBST and PBS respectively;

[0035] 6) Add 50 μL TMB substrate to each well, incubate at room temperature, add 50 μL ELISA stop solution after a clear color change is observed, and measure the OD450nm reading.

[0036] The ELISA test serum titer is defined as a positive well value that is more than twice the negative well value of the corresponding dilution. The specific results are as follows: Figure 1 As shown, the results showed that the antibody titer against H1N1-NA protein in plasma was 1:64000.

[0037] Example 2: Isolation of alpaca peripheral blood lymphocytes (PBMC)

[0038] On the 7th day after the fourth immunization, 50 mL of anticoagulated blood was collected for PBMC separation. The specific operation was as follows:

[0039] 1) Use a pipette to carefully add the density gradient centrifugation solution (Tianjin Haoyang Biological Products) into the SepMate™ test tube through the central hole of the SepMate™ insert, so that it fills the bottom insert;

[0040] 2) Dilute the sample with an equal volume of PBS + 2% FBS and mix gently;

[0041] 3) Keep the SepMate™ tube vertical and add the diluted sample along the tube wall with a pipette;

[0042] 4) Centrifuge at room temperature, 1200 g for 10 min;

[0043] 5) Pipette the upper plasma into a clean centrifuge tube for antibody titer detection;

[0044] 6) Pour off the upper layer of liquid into a new tube, which contains the enriched PBMCs;

[0045] 7) Wash PBMCs twice with PBS + 2% FBS;

[0046] 8) Calculate the total number of cells using a cell counter and dispense into clean 1.5 ml centrifuge tubes (about 1-2 × 10 cells per tube). 7 cells);

[0047] 9) Remove the supernatant by centrifugation, add 1 mL of RNAisoplus (TaKaRa, 9109) to each tube, mix thoroughly, lyse the cells, and freeze at -80°C for later use.

[0048] Example 3: Construction of phage display antibody library

[0049] The PBMC obtained in Example 2 was used as raw material, total RNA was extracted, cDNA fragments were obtained by reverse transcription reaction, VHH gene fragments were amplified by nested PCR, and then the VHH fragments were cloned into phagemid vectors, and then competent cells were transformed to construct a phage library.

[0050] 3.1 Extraction of total cell RNA

[0051] 1) Take the lysed PBMCs in Example 2, add 200 μl of chloroform to each tube, mix well, and centrifuge at 4°C, 12,000×g for 15 min;

[0052] 2) The sample is divided into two layers, the upper transparent liquid layer (containing RNA) and the lower organic phase layer. The upper transparent supernatant is transferred to a new centrifuge tube;

[0053] 3) Add an equal volume of isopropanol, mix well, leave at room temperature for 10 min, and centrifuge at 12,000 × g for 10 min at 4°C;

[0054] 4) Discard the supernatant, add 1 ml of 75% ethanol (pre-cooled with DEPC water) at 4°C or in an ice bath, mix well (gently pipette a few times), and centrifuge at 4°C, 10,000 × g for 5 min to wash the RNA precipitate;

[0055] 5) Dry in a clean bench for a few minutes and dissolve the precipitate with 50 μl RNase-free water / DEPC water;

[0056] 6) Determine the concentration and purity of RNA (OD260 / 280, OD260 / OD230).

[0057] 3.2 Reverse transcription to obtain cDNA

[0058] Reverse transcription synthesis of cDNA was performed using a reverse transcription kit (TaKaRa, 6210A). The specific steps are as follows:

[0059] 1) Prepare the following mixture in a Microtube, see Table 1 for details:

[0060] Table 1: cDNA synthesis reaction premix

[0061] Reagents Usage Random6mers (50 μM) 2μl dNTP Mixture (10 mM each) 1μl Total RNA (586ng / μl, ~3μg) 5.1μl <![CDATA[RNaseFreeddH2O]]> Make up to 10 μl

[0062] 2) Keep at 65℃ for 5 min and then cool rapidly on ice;

[0063] 3) Prepare the following reverse transcription reaction solution in the above Microtube, with a total volume of 20 μl, as shown in Table 2:

[0064] Table 2: cDNA synthesis reaction mixture

[0065] Reagents Usage The above denatured reaction solution 10μl 5*PrimerScriptⅡ Buffer 4μl RNase Inhibitor (40 U / μl) 0.5 μl (20 U) PrimeScriptⅡRTase (200 U / μl) 1μl (200U) <![CDATA[RNaseFreeddH2O]]> Make up to 20 μl

[0066] 4) Slowly mix the above components and perform reverse transcription reaction according to the following conditions:

[0067] 30℃, 10min;

[0068] 42°C, 60 min;

[0069] 95℃, 5min;

[0070] Chill on ice.

[0071] 5) Determine the concentration of cDNA using a micro-spectrophotometer and store at -20°C to avoid repeated freezing and thawing.

[0072] 3.3 VHH fragment amplification

[0073] A two-step method was used to construct the library, and the VHH fragment was amplified by the nested PCR method. The first step of the PCR reaction is shown in Table 3:

[0074] Table 3: First step PCR reaction

[0075]

[0076] The primer sequences in Table 3 are as follows:

[0077] AlpVH-F: 5'-GTCCTGGCTGCTCTTCTACA-3' SEQ ID NO: 8

[0078] AlpVH-R: 5'-GCCATCAATRTACCAGTTGA-3' SEQ ID NO: 9

[0079] The first step of PCR reaction products was subjected to nucleic acid gel electrophoresis, and smaller fragments were recovered. Figure 2 shown.

[0080] The second PCR used the VHH fragment recovered in the first step as a template, and the reaction was shown in Table 4.

[0081] Table 4: Second step PCR reaction

[0082]

[0083] The primer sequences in Table 4 are as follows:

[0084] AlpVHH-F:

[0085] 5'-CTACAAATGCCTATGCATCCGAGGTGCAGSTSGTGGAGTC-3' SEQ ID NO: 10

[0086] AlpVHH-R:

[0087] 5'-CGGATGTCCTTGCTGCCTGAYGAGGASACRGTGMCCWGG-3' SEQ ID NO: 11

[0088] The second step is to perform nucleic acid gel electrophoresis on the PCR reaction products and recover smaller fragments. Figure 3 shown.

[0089] 3.4 Enzyme digestion of phage vector

[0090] The phagemid vector used was: pComb3XSS, 4992 bp, double-digested with SfiI. See Table 5 for details:

[0091] Table 5: Enzyme digestion system

[0092]

[0093] 3.5 Recombinant phagemid construction

[0094] The VHH fragment obtained in 3.3 and the linearized vector recovered by enzyme digestion in 3.4 were used for homologous recombination reaction. The molar ratio of vector to fragment was 1:2. The homologous recombination kit used was ClonExpress Ultra One Step Cloning Kit V2 (Novizan, C116-01). The system is shown in Table 6 below:

[0095] Table 6: Homologous recombination reaction system

[0096]

[0097] 3.3.6 Transformation of recombinant products and harvesting of antibody library

[0098] Transform TG1 competent cells (Lucigen, 60502-1) with the recombinant product purified in the previous step, using the Bio-RadGene Pulser Xcell™ electroporation system and a 0.2 cm electroporation cup, with the following parameters: 2.5 kV, 25 μF, 200 Ω. Immediately after electroporation, add 37°C preheated SOC medium to resuspend the cells. Incubate at 37°C, 250 rpm for 1 hour for activation. Take a small amount of bacterial solution and dilute it 10 times for storage capacity determination. Spread the remaining bacterial solution on LB / Amp 100 / 2% Glucose plate, incubated at 37℃ for 8h. The next day, the storage capacity was 6.8×10 8 At the same time, the bacteria were collected by cell scraping and 1 / 3 volume of 50% 2YT-glycerol was added to form the antibody library.

[0099] 3.7 Phage antibody library rescue

[0100] The harvested antibody library was inoculated into 2YT / Amp 100 / 2% Glucose medium (starting OD 600 =0.25), 37°C, 220 rpm, and culture until the logarithmic growth phase (OD 600 = 0.5), add a final concentration of 10 10 pfu / ml of M13KO7 helper phage, stand at 37℃ for 30min; culture at 37℃, 150rpm, 30min; centrifuge at 3500g for 10min, discard the supernatant, resuspend the bacteria in 2YTAK / IPTG (final IPTG concentration 0.2mM) medium; culture at 30℃, 220rpm for 10h. Centrifuge at 4℃, 8000g for 10min, collect the supernatant, add 1 / 4 volume of PEG8000 / NaCl, mix well, stand at 4℃ for 1~2h; centrifuge at 4℃, 10000g for 20min; add PBS to resuspend the phage precipitate; centrifuge at 4℃, 12000rpm for 5min, collect the supernatant.

[0101] Example 4: Screening of anti-H1N1-NA protein nanobodies

[0102] The solid phase screening method is used to screen specific antibodies. The specific steps are as follows:

[0103] 1) Antigen plating: Coat 8 wells of Maxsorp 96-well plates with 2 μg / ml (diluted with PBS) H1N1-NA protein at 4°C overnight.

[0104] 2) Host bacteria culture: Take host bacteria TG1 and inoculate it into a suitable volume of fresh 2YT medium. Incubate the culture at 37°C, 250 rpm, and shake for 3-4 hours until OD600 = 0.5. Place it at room temperature for later use.

[0105] 3) Remove the liquid from the wells and wash three times with PBS.

[0106] 4) Block the wells and antibody library ((Input)) with MPBS (final concentration 5% skim milk, diluted in PBS) and incubate at room temperature for 1-2 hours.

[0107] 5) Remove the blocking solution and wash the wells three times with PBS.

[0108] 6) Add the antibody library and incubate at room temperature with shaking (low speed) for 2 hours.

[0109] 7) Wash the wells several times with PBST and PBS respectively.

[0110] 8) Elution: Add freshly prepared 100 mM TEA and incubate at room temperature with shaking (low speed) for 20 minutes.

[0111] 9) Neutralization: Pipette vigorously 10 times, add the eluate into a shaking tube containing 1 / 2 volume of 1M Tris-HCl (pH 7.5), and incubate at room temperature for 5 minutes to neutralize TEA.

[0112] 10) Add the mixture from the previous step to 10 times the volume of the host bacteria and incubate at 37°C with shaking at 250 rpm for 1 h.

[0113] 11) Phage titration: Take a small amount of the culture from the previous step and make a 10-fold gradient dilution with 2YT. Take samples of appropriate dilutions for titration on 2YT plates and incubate at 37°C overnight.

[0114] 12) Transfer the remaining culture to a 50 ml centrifuge tube, centrifuge at 2500 g for 10 min, discard the supernatant, add the original volume of 2YTA resuspended bacterial solution, and continue culturing at 37°C, 250 rpm for 2 h.

[0115] 13) Add M13KO7 helper phage to a final concentration of 10 10phage / ml, 37°C, 250rpm, infection for 30min.

[0116] 14) Transfer the culture to a 50 ml centrifuge tube, centrifuge at 2500 g for 10 min, discard the supernatant, add the original volume of 2YTAK / IPTG (IPTG final concentration 0.2 mM) medium, and incubate at 30°C, 280 rpm, overnight.

[0117] 15) The next day, count the number of infectious monoclonal bacteria titrated in 11) and calculate the number of phages bound in this round (Output)

[0118] 16) At the same time, collect Phage from the overnight culture supernatant as the second round of input phage for the next round of screening.

[0119] 17) Calculate the enrichment constant R = Output / Input for each round.

[0120] 18) When the R value increases significantly, it can be determined that the screening is enriched. The experimental results are shown in Table 7. This screening showed obvious enrichment in the second and third rounds.

[0121] Table 7: Enrichment constants of H1N1-NA protein in three rounds of screening

[0122] Enrichment constant First round R1 Second round R2 Round 3 R3 Output / Input <![CDATA[3.54×10 -7 ]]> <![CDATA[2.18×10 -6 ]]> <![CDATA[8.64×10 -4 ]]>

[0123] Example 5: Identification of positive monoclonal anti-H1N1 protein nanoantibodies - PhageELISA

[0124] 1) Select the Output phage from the previous round where significant enrichment occurred to infect TG1 bacteria. This time, significant enrichment occurred in the third round, so the Output phage from the second round was used.

[0125] 2) Use appropriate amount of Outputphage to infect TG1 host bacteria and apply LB / Amp 100 Plate, 37°C, overnight incubation.

[0126] 3) The next day, pick several (e.g. 190) single clones from the LB / Amp100 plate and add 200 μL / well 2YT / Amp 100 / 2% Glucose medium in a 96-well deep-well plate, 250 rpm, 37°C, overnight culture.

[0127] 4) Inoculate 200 mL / well of 2YT / Amp with the overnight culture from the previous day at a ratio of 1:50 100 / 2% Glucose medium in a 96-well deep-well plate, 250 rpm, 37°C, until the bacterial solution OD600 ≈ 0.5 (the bacteria are in the logarithmic growth phase).

[0128] 5) Add to a final concentration of about 10 10 phage / ml of M13K07 helper phage, 37°C, 250rpm, infection for 30min.

[0129] 6) Centrifuge at 3000g, RT for 10 min, invert and discard the supernatant, add 400mL / well 2YT / Amp100 / Kan50, and resuspend the cells.

[0130] 7) Incubate at 37°C, 250 rpm overnight. Coat the 96-well plate for ELISA tomorrow with 2 μg / ml SpyCatcher003 protein, 100 mL / well; coat overnight at 4°C.

[0131] 8) Take out the 96-well ELISA plate coated yesterday, remove the antigen, and wash three times with PBS, 200 mL / well.

[0132] 9) Add MPBS to block the ELISA plate, 200 mL / well, at room temperature for 2 h.

[0133] 10) Take out the 96-well deep-well plate from 3) and centrifuge at 3000 g for 10 min.

[0134] 11) Pipette 60 mL / well of supernatant into a clean 96-well plate and add an equal volume of 5% skim milk-PBS solution and pre-blocked phage-containing culture supernatant at RT for 1-2 hours.

[0135] 12) Discard the blocking solution in the ELISA plate and wash 4 times with PBST, 200 mL / well.

[0136] 13) Add the culture supernatant prepared in step 11, 100 mL / well, and incubate at room temperature with gentle shaking for 1 hour.

[0137] 14) Wash 8 times with PBST, 200 mL / well.

[0138] 15) Add HRP-anti-M13 antibody, 100 mL / well, and incubate with gentle shaking at room temperature for 30 minutes.

[0139] 16) Wash 6 times with PBST and then wash 2 times with PBS, 200 mL / well.

[0140] 17) Add TMB substrate, 50 mL / well, incubate at room temperature (25°C) or 37°C in the dark for 5-10 min, and observe the color development (blue).

[0141] 18) Add an equal volume of stop solution (1M sulfuric acid solution), 100 mL / well, to terminate the reaction. The reaction solution in the well changes from blue to yellow.

[0142] 19) Within 30 minutes after terminating the reaction, measure the absorbance at 450 nm using an ELISA reader.

[0143] 20) Select positive clones (OD450 value ≥ 0.5) for sequencing.

[0144] 21) After comparing the monoclonal sequences, a positive monoclonal clone (Unique) was obtained and named N1E4.

[0145] N1E4 was sequenced, and the nucleotide and amino acid sequences were as follows:

[0146] Nucleotide sequence (SEQ ID NO: 5):

[0147] 5'-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAG

[0148] ACTCTCCTGTGCAGCCTCTGGAAACATCTTCAATATTGATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTTCCACTATTATTAGTGGTGGTAACACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC

[0149] CAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTTATGTTGAGAGGGAACAGGGCGACTTTGGGGGGCTACCATATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA-3'

[0150] Amino acid sequence (SEQ ID NO: 1):

[0151] QVQLVESGGGLVQPGGSLRLSCAASGNIFNIDAMGWYRQAPGKQRELVAA ISTIISGGNT NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC YVEREQGDFGGLPYDY WGQGTQVTVSS

[0152] Among them, CDR1 (SEQ ID NO: 2): GNIFNIDA; CDR2 (SEQ ID NO: 3): ISTIISGGNT ;

[0153] CDR3 (SEQ ID NO: 4): YVEREQGDFGGLPYDY .

[0154] Example 6: Verification of anti-NA protein nanobody binding activity

[0155] To further confirm the binding activity of the positive single clone, the positive clone was expressed in eukaryotic cells and its binding to the H1N1-NA protein was further verified.

[0156] 6.1 Eukaryotic Expression

[0157] The eukaryotic expression is in the form of nanobody (VHH) fused with the Hinge-Fc (hFc) fragment of human IgG1 subtype antibody, and the expression product is VHH-hFc dimer. The specific expression steps are as follows:

[0158] 1) Construction of recombinant vector:

[0159] a The eukaryotic expression vector pINFUSE-hIgG1-Fc2 was double-digested with EcoRV and NcoI;

[0160] b The positive VHH clone fragment was amplified by PCR, and the specific reaction conditions are shown in Table 8:

[0161] Table 8: Reaction conditions for PCR amplification of positive VHH clone fragments

[0162]

[0163] *Primer sequences are as follows:

[0164] VHH-FcF:GCACTTGTCACGAATTCGATA AGGTGCAGCTCGTGGAGTC SEQ ID NO: 6

[0165] VHH-FcR:TGAGTTTTGTCAGATCTAAC TGAGGAGACGGTGACCTGGG SEQ ID NO: 7

[0166] Note: The underline represents the specific region of the antibody.

[0167] The c-positive VHH fragment was purified by a PCR product recovery kit and then ligated to the restriction linearized vector by homologous recombination. The ligation product was transformed into DH5α competent cells, and 500μL SOC was added to resuspend the cells. After activation at 37°C, 220rpm, 100μL of bacterial solution was applied to LB agarose plates containing a final concentration of 50μg / mL Zeocin and incubated at 37°C overnight. The next day, 3 clones were randomly selected and sent to Qingke Company for sequencing, and the correct recombinant clone was selected for culture and plasmid extraction.

[0168] 2) Transfection: The recombinant expression vector was transfected into 293F cells using PEI.

[0169] 3) Culture: Cells were cultured for 5 days.

[0170] 4) Antibody separation: Collect the culture supernatant and use Protein A to separate and purify VHH-hFc.

[0171] 5) Use ultrafiltration tubes to replace and concentrate the antibody solution.

[0172] 6) Determine the concentration, package and store in an ultra-low temperature refrigerator.

[0173] 6.2 Determination of Nanobody Affinity by Enzyme-Linked Immunosorbent Assay (ELISA)

[0174] The affinity of VHH-hFc to H1N1-NA protein was determined by ELISA. The specific method is as follows:

[0175] 1) Coating: Dilute H1N1-NA protein to 2 μg / mL using PBS, 100 μL / well, and allow to bind overnight at 4°C.

[0176] 2) Washing: Wash 3 times with PBS, 200 μL / well.

[0177] 3) Blocking: 5% MPBS, 200 μL / well, incubate at room temperature for 2 h.

[0178] 4) Primary antibody incubation: 100 μL / well of serially diluted VHH-hFc antibody, incubate at room temperature for 1 hour.

[0179] 5) Washing: Wash 6 times with PBST, 200 μL / well.

[0180] 6) Secondary antibody incubation: Use horseradish peroxidase (HRP)-labeled anti-human IgG-Fc antibody, 1:5000 dilution, 100 μL / well, and bind at room temperature for 30 minutes.

[0181] 7) Washing: Wash with PBST for 6 times and PBS for 3 times, 200 μL / well.

[0182] 8) Color development: Add TMB substrate, 50 μL / well, and react at room temperature for about 5 minutes.

[0183] 9) Stop: Add 1M dilute sulfuric acid, 50 μL / well.

[0184] 10) Read OD value: Use a microplate reader to measure the OD450 signal value.

[0185] 11) Data analysis. The results are shown in Table 9:

[0186] Table 9: Affinity of H1N1-NA positive clones (ELISA)

[0187] serial number N1E4 EC50 (μg / mL) 0.018

[0188] From the ELISA results in Table 9, it can be seen that N1E4 has a very high affinity for H1N1-NA.

[0189] 6.3 Determination of Nanobody Affinity by Bio-Layer Interferometry (BIL)

[0190] Using Sartorius The affinity of VHH-hFc was measured using Protein A sensor, with VHH-hFc (2 μg / mL) as ligand and H1N1-NA and H5N1-NA proteins (10 μg / mL) as analytes. The results are shown in Table 10 and Figure 4 As shown:

[0191] Table 10: Affinity of N1E4 (VHH-Fc) for N1-NA (BLI)

[0192] protein H1N1-NA H5N1-NA Affinity (KD, M) <![CDATA[3.547×10 -11 ]]> -

[0193] The results of the BLI experiment show that N1E4 has a very high affinity for H1N1-NA (pM level) and does not recognize H5N1-NA, that is, it has specific recognition for H1N1-NA.

[0194] Example 7: In vivo preventive and protective effects of anti-NA protein nanobodies

[0195] In order to evaluate the in vivo protective effect of nanoantibodies, 6-8 week old C57BL / 6J mice were selected and randomly divided into 2 groups, 5 mice in each group, namely the control group and the antibody prevention group. Among them, the prevention group was intraperitoneally injected with N1E4-Fc nanoantibody (diluted with PBS) 200 µg / 20g / mouse, while the control group was injected with an equal volume of sterile PBS. 24 h later, the mice were nasally infected with A / PuertoRico / 8 / 34 (H1N1) with an infection dose of 106 EID50 / 30 µL. The mice were weighed every day, and the weight changes and survival rate of the mice were recorded. The weight changes of the mice are shown in Figure 5 As shown, the survival rate is Figure 6 As shown, until 14 days. When the weight loss of mice was ≥25%, they were euthanized. The results of the in vivo prevention and protection experiment in mice showed that the N1E4 nanoantibody can effectively neutralize the virus and resist the infection and invasion of mice by a lethal dose of PR8 H1N1 virus.

[0196] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A nanobody against influenza A virus H1N1 subtype NA protein, characterized in that: The nanobody comprises or consists of the amino acid sequence shown in SEQ ID NO:

1.

2. A nanobody against influenza A virus H1N1 subtype NA protein according to claim 1, characterized in that: The complementary determining regions of the nanobody include CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

4.

3. A DNA molecule encoding the nanobody against the NA protein of influenza A virus H1N1 subtype as described in claim 1 or 2.

4. The DNA molecule according to claim 3, characterized in that The sequence of the DNA molecule is shown in SEQ ID NO:

5.

5. A recombinant vector, characterized in that: The recombinant vector comprises the DNA molecule according to claim 4.

6. A host cell, characterized in that The host cell comprises the DNA molecule according to claim 4 or the recombinant vector according to claim 5.

7. Use of the nanobody according to claim 1 in the preparation of a detection kit for detecting influenza A virus H1N1 subtype NA protein or its content in a sample.

8. Use of the nanobody according to claim 1 in the preparation of a drug for preventing and treating influenza A virus infection.

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