A nano antibody against H7 subtype avian influenza virus and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a nano antibody against H7 subtype avian influenza virus and its application. The nano antibody against H7 subtype avian influenza virus is selected from nano antibody Q74 or nano antibody L125b; the amino acid sequences of the three CDR regions in the nano antibody L125b are shown in SEQ ID NOs: 6-8 respectively; the amino acid sequences of the three CDR regions in the nano antibody Q74 are shown in SEQ ID NOs: 10-12 respectively. The nano antibody has good stability and biological activity, can be applied to the diagnosis and detection of H7 subtype avian influenza virus, and has excellent detection sensitivity and accuracy.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nano antibody against H7 subtype avian influenza virus and an application thereof. Background Art

[0002] Influenza virus is a single-stranded, negative-sense, enveloped, segmented RNA virus belonging to the genus Influenza virus of the family Orthomyxoviridae. Its genome contains 8-stranded negative-sense RNA. The nucleoprotein and matrix protein of influenza virus, namely NP and M1, are classified into four subtypes: A, B, C, and D according to their antigenicity. Avian influenza virus belongs to type A, and its antigenicity is determined by the HA and NA structures on the epitope. These two structures often mutate, resulting in multiple subtypes of avian influenza, among which the most widespread and harmful are subtypes H5, H7, and H9.

[0003] The key to the prevention and control of avian influenza is to make a diagnosis in the early stage of infection. Traditional methods for identifying AIV include cell culture and chicken embryo culture. H7 subtype AIV is isolated and cultured through MDCK subculture, and then a judgment can be made by microscopic observation of the lesions. The development of serology and bioengineering has promoted the development of the field of avian influenza diagnosis. The preliminary diagnostic results of H7 subtype avian influenza virus in most diagnoses are similar to those of other subtypes of avian influenza virus, so it is usually necessary to confirm the avian influenza virus through diagnosis and then verify it through molecular experiments. The mainstream diagnostic methods for avian influenza include pathogen detection, influenza serum detection and molecular biology detection.

[0004] Among all the subtypes of avian influenza virus, the most harmful ones are H5, H7 and H9 subtypes. Among them, there are few studies and antibody screening related to H7 subtype avian influenza. In the current field of diagnostic testing, nano antibodies have shown their considerable development prospects and huge application value. The reason is that nano antibodies have the following advantages:

[0005] 1. Good water solubility: Nanobodies have better solubility in terms of physical and chemical properties because the number of hydrophobic amino acids in the FR2 region accounts for a small proportion of the amino acids in this segment and the hydrophilic molecules are large. If nanobodies are made into drugs, high solubility can improve the utilization rate of the drugs.

[0006] 2. Strong heat resistance: The disulfide bonds in the structure of nanoantibodies greatly enhance the heat resistance of nanoantibodies. After one week at 37°C, the biological activity can be reduced by at least 20%.

[0007] 3. Good refolding properties: Nano antibodies can still regain biological activity after being placed at above 90°C for a long time, while traditional antibodies will undergo irreversible thermal polymerization under such conditions.

[0008] 4. High binding ability with antigens. The convex structure of CDR3 allows it to penetrate deep into the antigen and bind firmly to the antigen. Nanobodies can not only bind to small molecule haptens and peptides, but also to large molecule proteins and viruses. Even when the recognition site is difficult to be recognized by other antibodies, small molecule nanobodies can also recognize specific epitopes.

[0009] 5. Easy to produce: Due to its simple structure, ability to be encoded by a single gene and small molecular weight, nanoantibodies can be successfully expressed in large quantities using yeast, Escherichia coli, etc. through genetic engineering technology.

[0010] 6. Surface effect: The surface effect of nano antibodies makes them have great application value in the field of electrochemical immunosensors. Studies have shown that nano antibodies can be made into a sensor for indirectly determining aflatoxin. When used in the field of feed production, they can also play a role in the detoxification process of AFT, effectively reducing the huge harm caused by AFT.

[0011] 7. Easy to transform and modify: The use of genetic engineering technology to transform VHH is not only mature but also relatively fast, which has promoted the development of nanoantibodies in the therapeutic field.

[0012] 8. Strong penetrability: The penetrating ability of nanoantibodies is much stronger than that of monoclonal antibodies. This advantage makes nanoantibodies of great significance and huge development potential in the field of imaging diagnosis of diseases.

[0013] Therefore, the present invention hopes to propose a nano-antibody with H7 subtype avian influenza virus hemagglutination inhibition activity and virus binding ability, so as to play a role in the diagnosis and detection of H7 subtype avian influenza virus. Summary of the invention

[0014] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a nano antibody against H7 subtype avian influenza virus and its application. The nano antibody has good stability and biological activity, can be used for the diagnosis and detection of H7 subtype avian influenza virus, and has excellent detection sensitivity and accuracy.

[0015] The present invention provides a nanobody against H7 subtype avian influenza virus, selected from nanobody Q74 or nanobody L125b;

[0016] The amino acid sequences of the three CDR regions in the Nanobody L125b are shown in SEQ ID NOs: 6-8, respectively;

[0017] The amino acid sequences of the three CDR regions in the Nanobody Q74 are shown in SEQ ID NOs: 10-12, respectively.

[0018] Preferably, the amino acid sequence of the Nanobody L125b is as shown in SEQ ID NO:5.

[0019] Preferably, the amino acid sequence of the Nanobody Q74 is as shown in SEQ ID NO:9.

[0020] The present invention also provides an isolated nucleic acid molecule encoding the above-mentioned nano antibody against H7 subtype avian influenza virus.

[0021] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0022] The present invention also provides a recombinant expression vector comprising the above nucleic acid molecule.

[0023] The present invention also provides a recombinant bacterium, comprising the above nucleic acid molecule or recombinant expression vector. The recombinant bacterium can be used to express and produce the above anti-H7 subtype avian influenza virus nanoantibody, has the advantages of high yield and low production cost, and has broad application prospects.

[0024] Preferably, the recombinant bacteria is a recombinant yeast.

[0025] The present invention also provides the use of the nano antibody in preparing a diagnosis / detection kit for H7 subtype avian influenza virus.

[0026] The present invention also provides a H7 subtype avian influenza virus diagnosis / detection kit, comprising the above-mentioned nano antibody.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The nanoantibodies against H7 subtype avian influenza virus proposed in the present invention have good stability and biological activity, can be applied to the diagnosis and detection of H7 subtype avian influenza virus, and have excellent detection sensitivity, repeatability and accuracy.

[0029] (2) The present invention adopts a eukaryotic expression system to construct a recombinant bacterium expressing nanoantibodies against H7 subtype avian influenza virus, which effectively reduces the production cost of nanoantibodies and improves accuracy and repeatability. The produced nanoantibody protein has a high purity, and can realize the large-scale production of nanoantibodies against H7 avian influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the recombinant expression vector of anti-H7 avian influenza nanobody Q74.

[0031] Figure 2 Schematic diagram of the recombinant expression vector of anti-H7 avian influenza nanobody L125b.

[0032] Figure 3 It is a PCR verification result diagram of transforming the anti-H7 avian influenza virus nanobody protein plasmids Q74 in pPICZαA and L125bin pPICZαA preserved in Example 4 into the recipient Pichia yeast competent state; wherein, lane 1 is Yeasen2000Marker; lane 2 is the target gene amplification fragment containing plasmid Q74 in pPICZαA; lane 3 is the target gene amplification fragment containing plasmid L125bin pPICZαA; lane 4 is a negative control.

[0033] Figure 4 It is a graph showing the analysis results of protein detection by SDS-PAGE electrophoresis; wherein, lane 1 is Vazyme MP102Maker; lane 2 is the expression result of nano antibody Q74; and lane 3 is the expression result of nano antibody L125b.

[0034] Figure 5 It is a hemagglutination inhibition (HI) diagram for different antigens; among them, the antigen in the first row is H7, the antigen in the second row is H5, the antigen in the third row is H9, the 11th column is the negative control, and the 12th column is the blank control.

[0035] Figure 6 The graph is a hemagglutination inhibition (HI) graph for different antigens. The first row antigen is H7, the second row antigen is H5, the third row antigen is H9, the 11th column is a negative control, and the 12th column is a blank control.

[0036] Figure 7 It is a line graph of the ELISA reaction between the anti-H7 avian influenza nanobody protein Q74 and different coated antigens; the abscissa is the dilution multiple of the expressed anti-H7 avian influenza nanobody protein Q74, and the ordinate is the reading at a wavelength of 650nm after adding the color developing solution.

[0037] Figure 8 It is a line graph of the ELISA reaction between the anti-H7 avian influenza nanobody protein L125b and different coated antigens; the abscissa is the dilution multiple of the expressed anti-H7 avian influenza nanobody protein L125b, and the ordinate is the reading at a wavelength of 650nm after adding the color developing solution. DETAILED DESCRIPTION

[0038] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limitation on the protection scope of the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention.

[0039] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0040] Example 1: Construction of bait vector pGBKT7-H7

[0041] (1) Selecting H7 subtype avian influenza virus sequences

[0042] The sequence of H7 subtype avian influenza virus was searched in the sequences published in the GenBank section of the NCBI website. After sequence alignment of multiple H7 subtype avian influenza virus sequences using DNAman software, the GenBank: MG739458.1 sequence was selected as the target fragment of this experiment and named F-H7.

[0043] (2) The H7 subtype avian influenza virus sequence was connected to the pGBKT7 vector by double restriction digestion experiment

[0044] The target fragment F-H7 and pGBKT7 vector were double-digested with EcoRI and SalI, respectively. The digestion system was: EcoRI 2μL; SalI 2μL; 10×buffer 5μL; F-H7 / vector (pGBKT7) 15μL; sterile distilled water 26μL; double digestion experiment was carried out in a 37℃ water bath. After the digestion reaction lasted for 1.5h, 2.0μL 10×loading buffer was added to terminate the digestion reaction. The products obtained from the digestion experiment were subjected to gel electrophoresis, and the fragment size of the product was determined by the gel imaging system; the target band was intercepted, and after gel recovery, the fragment in the target band was linked to the vector pGBKT7 using T4 ligase in a 16℃ metal ligator for 8 to 14 hours to obtain the pGBKT7 vector containing the target fragment F-H7. Ligation system: T4 Ligase 1μL; 10×T4 buffer 2μL; pGBKT7 after double enzyme digestion 2μL; double enzyme digestion fragment 6μL; sterile distilled water 9μL.

[0045] (3) Transform the pGBKT7 vector containing the target fragment F-H7 into DH5α Escherichia coli competent cells

[0046] Mix 50μL DH5α competent E. coli, 0.5μL pGBKT7 vector containing target fragment F-H7, 1μL 5×KCM and 30μL sterile distilled water; let the mixture stand in a refrigerator at 4℃ for 15min, and then stand in an environment of 20℃~37℃ for 8min; gently add the mixture to 1.0mL of freshly prepared LB liquid culture medium (without antibiotics), blow it gently with a pipette, and culture it in a 37℃ shaker for 1h; centrifuge at low speed for 3min, leaving only 30μL liquid and bottom bacterial sediment; evenly spread the retained bacterial liquid on LB solid culture medium plate (containing kanamycin), and invert and culture it in a 37℃ incubator for 9~15h. The colonies grown are DH5α E. coli containing pGBKT7 vector of target fragment F-H7, and single colonies with good physiological conditions are selected for PCR, electrophoresis and sequencing for verification. After verification, the bait plasmid was successfully constructed and named pGBKT7-H7.

[0047] Example 2: Preparation of bait yeast

[0048] (1) Preparation of bait plasmid using DH5α E. coli containing the pGBKT7 vector containing the target fragment F-H7

[0049] In step (3) of Example 1, select a single colony with good physiological state on the plate of DH5α E. coli containing the pGBKT7 vector of the target fragment F-H7, add it to the freshly prepared LB liquid medium (containing kanamycin), and culture it in a 37°C shaker for 10-15h; use Magen Plasmid Rapid Extraction Kit (Product No.: P1001-03) to extract the plasmid: centrifuge at 13000×g for 1min, and collect 1-5mL of bacteria. Discard the culture medium, gently tap on absorbent paper to absorb the residual liquid, add 250μL Buffer P1 / RNase A mixture, and resuspend the bacteria by high-speed vortex. Add 250μL Buffer P2 to the resuspended liquid, invert and mix 8-10 times, add 350μL Buffer NP3, and immediately invert 8-10 times to completely neutralize the solution. Centrifuge at 13000×g for 2min, place the column in a collection tube, transfer the supernatant to the column, and centrifuge at 13000×g for 30-60s. Discard the filtrate, put the column back into the collection tube, add 500μL Buffer PW1 to the column, and centrifuge at 13000×g for 30-60s. Discard the filtrate, put the column back into the collection tube, add 600μL Buffer PW2 (diluted with anhydrous ethanol) to the column, and centrifuge at 13000×g for 30-60s. Discard the filtrate, put the column back into the collection tube, and centrifuge at 13000×g for 3min to dry the column. Put the column into a sterile 1.5ml centrifuge tube, add 30-100μL Elution Buffer or sterile water to the center of the column membrane, let it stand for 1min, and centrifuge at 13000×g for 1min to elute the DNA. Discard the column and store the plasmid at -20°C. Send the plasmid to the company for sequencing. If the plasmid sequencing is correct, it is the bait vector.

[0050] (2) Preparation of bait strain Y2HGold-H7

[0051] Melt the Carrier DNA in a 4°C environment, heat it in a 100°C water bath for 6 minutes, then place it in an ice-water mixture for 2 minutes, then place it in a 100°C water bath for 6 minutes, then place it in an ice-water mixture for 2 minutes, and finally place it in a 4°C environment for use; add the plasmid pGBKT7-H7 and Carrier DNA to the sterile 1.5mL EP tubes respectively, and shake gently to mix; draw 30μL of yeast competent bacterial solution into the above containing plasmids pGBKT7-H7 and Carrier DNA in the EP tube, and then draw 600μL of PEG / LiAC, gently shake to mix; place the EP tube in a 42℃ water bath for 10min, then take it out and mix it up and down, then put it in a 42℃ water bath for 10min, then take it out and shake it gently; centrifuge at low speed for 2min at room temperature, discard the supernatant, draw 800μL of freshly prepared YPDA liquid culture medium (containing kanamycin) into the EP tube, and place the EP tube in a shaker for 50min; centrifuge at low speed for 2min, discard the supernatant, draw 30μL of freshly prepared YPD liquid culture medium (containing kanamycin) and use an inoculation loop to streak inoculation on a SD / -Trp solid culture medium plate (containing kanamycin), and culture in an incubator for 2 to 4 days; select single colonies with good physiological conditions for PCR, electrophoresis and sequencing for verification. After the verification is correct, the bait strain obtained, that is, the Y2HGold yeast strain introduced with the bait plasmid pGBKT7-H7, is named Y2HGold-H7.

[0052] Example 3: Screening of Nanobody Library

[0053] (1) Mating of bait strain Y2HGold-H7 and nanoantibody library

[0054] Quickly take out a tube of yeast library strain (1.0 mL per tube) and place it on ice to thaw naturally; mix the yeast library strain (1.0 mL per tube) and 1.0 mL of bait strain Y2HGold-H7 (bacterial solution with OD600 of 0.6) in a freshly prepared 50 mL 2×YPDA liquid medium (containing 50 μg / mL kanamycin) in a 2L conical flask and add 1 to 2 mL The library vial was washed with 2×YPDA and added to a 2L conical flask. It was placed in an incubator and cultured at 30°C and 50r / min for about 16h to allow the bait strain Y2HGold-H7 to fully hybridize with the yeast library strain. After 20-22h, a drop of bacterial solution was taken and observed under a phase contrast microscope (40×). If a combination that looks like a clover or Mickey Mouse is found and the number is not less than 5, the next step can be continued. Otherwise, another 4h of culture is required. The bacterial solution was collected and centrifuged at 3000r / min for 10min at room temperature. The supernatant was discarded and the bacteria were collected, and 50mL of The culture flask was rinsed twice with 0.5×YPDA (containing 50 μg / mL kanamycin), and the yeast cells after centrifugation were resuspended with the mixture; the mixture was centrifuged at low speed for 5 min, the supernatant was discarded, 3 mL of 1×YPDA (containing kanamycin) liquid culture medium was pipetted into a centrifuge tube and gently blown; the yeast conjugation culture obtained by the remaining mating was spread on the freshly prepared yeast two-hybrid blue-white plate (150 mm SD / -Trp / -Leu / -ABA-X plate) screening plate culture medium prepared in advance, and the culture was placed in a 30°C incubator in the dark for 3 to 5 days; the plate was taken out on the 3rd day, and the blue single colony was marked. On the 5th day, the blue single colony with a diameter greater than 1.0 mm was picked and marked as a candidate positive clone.

[0055] (2) Screening of candidate positive clones

[0056] The candidate positive clones were streaked on the yeast two-hybrid blue-white plate (150 mm SD / -Trp / -Leu / -ABA-X plate) screening plate medium, and positive control group and negative control group were set up. They were placed in a 30°C incubator for 4 to 8 days to observe their growth. Some relatively weak candidate positive clones were discarded, and the candidate positive clones that still showed blue were kept for the next step of verification.

[0057] (3) PCR identification of candidate positive clones

[0058] Two pairs of primers, T7-F, T7-R and AD-F, AD-R, are used to amplify the target fragment of the positive clones obtained by streaking screening in step (2) by PCR, and then the PCR products are subjected to gel electrophoresis experiment, and finally the size of the amplified fragment is observed to determine whether the candidate positive clone is a false positive. The positive clone is sent for sequencing to obtain the anti-H7 subtype avian influenza virus nanoantibody sequence.

[0059] The nucleotide sequence of T7-R is: 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 1); the nucleotide sequence of T7-F is: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 2);

[0060] The nucleotide sequence of AD-R is: 5′-AGATGGTGCACGATGCACAG-3′ (SEQ ID NO: 3);

[0061] The nucleotide sequence of AD-F is: 5'-TACCACTACAATGGATGATG-3' (SEQ ID NO: 4).

[0062] After screening, nanobody Q74 and nanobody L125b were obtained. The amino acid sequence of nanobody L125b is: QVQLVESGGGSVQAGGSLILSCASS VVANWCMG WFRQAPGKEREGIA AIDIDGTTHYAASVKG RFTITQDKAKSTVFLQMNTLKPEDTAMYFCAA GGSWYCPHLTRYEYNY WGQGTQVTVSS (SEQ ID NO: 5), wherein the underlined portion represents the three complementary determining regions (CDR regions) of the nanobody L125b, and their sequences are marked as SEQ ID NOs: 6-8 in sequence.

[0063] The amino acid sequence of nanobody Q74 is: QVQLVESGGDSVQAGGSLRLSCAAS VSPNWCMG WFRQAPGKEREVIS AIDIDGSTHYAGAVKG RFTISQDKAKNAVYLQMDGLKPEDTAMYYCAA GGSWYCPVLTISEYNY WGQGTQVTVSS (SEQ ID NO: 9), wherein the underlined portion represents the three complementary determining regions (CDR regions) of nanobody Q74, and their sequences are marked as SEQ ID NOs: 10-12 in sequence.

[0064] Example 4: Construction of anti-H7 avian influenza virus nanobody protein plasmid Q74 in pPICZαA and anti-H7 avian influenza virus nanobody protein plasmid L125b in pPICZαA

[0065] (1) By codon optimization, nucleic acid molecules encoding Nanobody Q74 and Nanobody L125b in Example 3 were obtained, and the specific sequence information is as follows:

[0066] The nucleotide sequence of the nucleic acid molecule encoding the anti-H7 subtype avian influenza virus nanobody protein L125b: 5'-ATGGATAACCAGGTGCAACTCGTTGAGAGCGGCGGGGGGTCTGTGCAGGCAGGGGGTTCCTTGATTTTGAGTTGCGCCAGCTCAGTGGTAGCCAACTGGTGTATGGGGTGGTTTCGTCAAGCACCTGGGAAGGAACGTGAAGGGATTGCAGCAATCGACATTGATGGGACCACGCACTACGCAGCAAGTGTCAAGGGCCGCTTTACCATTACCCAGGATAAGGCGAAATCTACGGTTTTCCTGCAGATGAATACCTTAAAACCAGAGGACACAGCTATGTACTTCTGTGCCGCCGGCGGCTCGTGGTACTGTCCACATTTAACACGCTACGAATACAATTATTGGGGCCAAGGGACGCAGGTAACTGTTAGTTCT-3' (SEQ ID NO: 13);

[0067] The nucleotide sequence of the nucleic acid molecule encoding the anti-H7 subtype avian influenza virus nanobody protein Q74: 5'-ATGGATAACCAGGTCCAGCTTGTTGAGTCGGGCGGGGACAGTGTTCAAGCAGGTGGTTCCCTCCGGCTCTCCTGTGCGGCCTCCGTATCACCTAATTGGTGTATGGGCTGGTTTCGCCAAGCCCCAGGGAAGGAGCGGGAAGTCATCAGCGCGATTGACATCGACGGGTCTACACATTATGCTGGCGCCGTCAAGGGGCGGTTTACTATTTCTCAGGATAAGGCCAAAAATGCTGTGTATCTTCAAATGGACGGGCTCAAACCAGAGGACACTGCCATGTACTACTGCGCTGCCGGTGGGAGCTGGTATTGCCCAGTCCTCACCATTTCCGAGTACAACTATTGGGGCCAGGGGACTCAAGTCACGGTCTCTTCA-3' (SEQ ID NO: 14).

[0068] The above nucleic acid molecules were subjected to gene synthesis, and EcoRⅠ and NotⅠ restriction sites were added at both ends of the sequence (synthesized by Shanghai General Company). Then, the obtained target gene fragment and pPICZαA vector were double-digested with EcoRⅠ and NotⅠ, respectively, and connected to obtain two recombinant expression vectors, which were named Q74 in pPICZαA plasmid (the size of the recombinant expression vector was approximately 3946 bp) and L125b in pPICZαA plasmid (the size of the recombinant expression vector was approximately 3946 bp).

[0069] Figure 1-2 Schematic diagram of the recombinant expression vectors of anti-H7 avian influenza nanobody Q74 and nanobody L125b.

[0070] (2) Plasmid extraction, double enzyme digestion to confirm plasmid and sequencing

[0071] The recombinant expression vectors Q74 in pPICZαA and L125b in pPICZαA obtained in step (1) were transformed into the recipient bacteria DH5α respectively, and the bacterial solution was coated on an LB plate containing bleomycin (containing 15 mg / L bleomycin) for recovery and activation. After culturing at 37°C for 16 hours, a single colony was picked and re-transferred to the plate; a portion of the colonies after transfer was picked and transferred to 100 mL of liquid LB culture medium, and the bacteria were shaken on a shaker at 37°C and 200 rpm for 16 hours. Part of the bacterial solution was temporarily divided and stored at 4°C; another portion of the bacterial solution was collected using a 50 mL centrifuge tube, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and plasmid extraction was performed; the positive clone with correct sequencing was expanded and cultured, the plasmid was extracted and stored in a -20°C refrigerator, and the bacterial solution was placed at -80°C for seed preservation using an LB solution containing 15-20% glycerol by volume.

[0072] Example 5: Induced expression of anti-H7 avian influenza virus nanobody protein

[0073] (1) The Q74 in pPICZαA plasmid and the L125b in pPICZαA plasmid extracted in Example 4 were transformed into Pichia pastoris respectively using the Coolaber Pichia pastoris transformation kit (Product No. SK2430).

[0074] 1. Take 0.1-5μg plasmid DNA (linearized plasmid addition amount 5-50μg) and 10μg pre-denatured carrier DNA and add them to the unmelted competent cells, place them in a 30℃ water bath, and mix them by inversion every 15s until the competent cells are completely melted (take them out in time after melting).

[0075] 2. Add 1.4 mL of B2 solution and mix thoroughly by inversion. Incubate in a 30°C water bath for 60 min.

[0076] 3. Centrifuge at 3000 rpm for 3 min, discard the supernatant and keep the bacterial precipitate, add 1 mL of B3 solution to resuspend the bacteria.

[0077] 4. Centrifuge at 3000 rpm for 3 min, discard the supernatant and keep the bacterial precipitate, add 100 μL B3 solution to resuspend the bacteria.

[0078] Spread 100 μL of bacterial solution onto the corresponding plate culture medium and culture at 30°C for 3-5 days until yeast colonies appear on the plate.

[0079] (2) Pick a single colony and perform colony PCR confirmation using aox1 primers (forward primer aox1-f and reverse primer aox1-r). Figure 3 As shown. The cloned colonies of the target bands of about 634 bp and 632 bp in size were re-transferred to the plate and saved; wherein, the AOX1 primer sequence is as follows:

[0080] Forward primer (aox1-f): 5′-TACTATTGCCAGCATTGCTGC-3′ (SEQ ID NO: 15);

[0081] Reverse primer (aox1-r): 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO: 16).

[0082] The PCR reaction system and conditions are as follows:

[0083] Amplification reaction system: PCR Master Mix enzyme 25μL, forward primer (10pmol / μL) 1μL, reverse primer (10pmol / μL) 1μL, gene template 2μL, ddH2O to 50μL.

[0084] Amplification reaction conditions: 98°C for 3 min; 98°C for 15 s, 58°C for 15 s, 72°C for 60 s, 34 cycles; 72°C for 5 min.

[0085] After the PCR reaction was completed, 1% agarose gel electrophoresis was used, and the gel electrophoresis showed target bands of about 634 bp and 632 bp in size.

[0086] (3) Pick a single colony containing the target band, place it in a 250 mL shake flask containing 20 mL BMGY (containing 1% glycerol) medium, seal it with sealing paper, and place it in a shaker at 30°C and 250 rpm to culture until OD600 = 2-6. Centrifuge at 1500-3000g for 5 min at room temperature, collect the bacteria, resuspend the bacteria with 25 mL BMMY (containing 0.5% methanol), and place the resulting bacterial solution in a 250 mL shake flask, seal it with sealing paper, and place it in a shaker at 30°C and 250 rpm to continue growing for 96 h. Add 100% methanol to the culture medium every 24 h to a final concentration of 0.5%. Collect the bacterial solution sample, centrifuge it at 8000g for 20 min, and retain the supernatant of the sample.

[0087] (4) Take a small amount of supernatant and add 4×SDS loading buffer, mix well, boil in boiling water for 10 minutes, take the supernatant and load it on the SDS-PAGE gel hole purchased from GenScript, and add an equal amount of protein Vazyme MP102 Maker, adjust the electrophoresis voltage to 100V and run the gel for 100 minutes. Take the gel block out of the glass plate and gently place it in a staining tank containing Coomassie Brilliant Blue solution on a shaker for 30 minutes; then pour out the Coomassie Brilliant Blue solution in the staining tank, rinse it with clean water, place the gel block in the staining tank on the shaker, decolorize the gel block with clean water for 30 minutes, and clear bands will be visible, indicating that the anti-H7 avian influenza virus nanoantibody protein is successfully expressed (such as Figure 4 shown).

[0088] Example 6: Protein function verification of anti-H7 avian influenza nanobodies

[0089] Anti-H7 avian influenza nanobody has the function of inhibiting hemagglutination, so this embodiment tests the antibody through hemagglutination inhibition (HI) test. The process is as follows: firstly measure the virus titer through hemagglutination (HA) test, prepare four units, and then conduct HI test.

[0090] (1) HA experiment:

[0091] ① Add 25 μL PBS to wells 1-11 of a 96-well plate and 50 μL PBS to well 12;

[0092] ② Add 25 μL of recombinant AIV H7 subtype Re-1 strain HI test antigen (purchased from Harbin Weike Biotechnology Development Company) to the first well, mix well and pipette to the second well, dilute to the 11th well in multiple proportions, discard 25 μL after mixing, and do not add to the 12th well;

[0093] ③ From wells 1 to 11, add 25 μL of diluted PBS to each well;

[0094] ④ Gently shake and mix the 1% red blood cells, add 25 μL to each well of wells 1 to 12; shake, stand at room temperature (24-25°C) for 40 minutes and observe the results; among them, the preparation of 1% chicken red blood cell suspension: extract anticoagulated chicken blood, centrifuge at 700rpm for 5 minutes; wash with PBS. Centrifuge again, absorb the white blood cells on the surface of the precipitated red blood cells, and continue to wash until there are no white blood cells on the surface of the red blood cells, and the PBS washing solution is transparent and colorless. Gently mix at a ratio of PBS: red blood cells = 99:1 to prepare a 1% chicken red blood cell suspension.

[0095] Four-unit configuration: After measuring the HA titer, four-unit antigen (4HAU) is configured by diluting the stock solution 2n-2 times. After configuration, the HA steps are performed to verify the four units. When verifying the configured four units, blood coagulation occurs in the first two wells.

[0096] (1) HI experiment:

[0097] ① Add 25 μL PBS to wells 1-11 of a 96-well plate and 50 μL PBS to well 12;

[0098] ② Add 25 μL of anti-H7 avian influenza virus nanobody protein to the first well, mix well and pipette to the second well, dilute to the tenth well in multiple proportions, mix well and discard 25 μL of PBS, and do not add to the 11th and 12th wells;

[0099] ③ From wells 1 to 11, add 25 μL of diluted AIV H7 subtype Re-1 strain 4 unit antigen suspension to each well and let it stand at room temperature (24-25°C) for at least 30 minutes. Do not add to well 12;

[0100] ④ Gently shake and mix 1% red blood cells, add 25 μL to each well of wells 1 to 12; shake, let stand at room temperature (24-25°C) for 40 minutes, and then observe the results.

[0101] The steps of the H5 and H9 hemagglutination inhibition experiments are the same as those of the H7 subtype Re-1 strain. The H5, H7, and H9 avian influenza hemagglutination inhibition test antigens are Re-8 strain, H7-Re1 strain, and H9 subtype, respectively, and are all avian influenza hemagglutination inhibition test antigens purchased from Harbin Veken Biotechnology Co., Ltd.

[0102] HI results showed that the anti-H7 avian influenza nanobody Q74 had a titer of 9log2 against H7 antigen and had no hemagglutination inhibition against H5 and H9 (e.g. Figure 5 The anti-H7 avian influenza nanoantibody L125b has a titer of 8log2 against H7 antigen and has no hemagglutination inhibition against H5 and H9 (as shown in Figure 6 As shown). Both protein antibodies Q74 and L125b are specific binding anti-H7 avian influenza nanoprotein antibodies.

[0103] Example 7: Application of anti-H7 avian influenza virus nanoantibody protein for specific detection of antigens

[0104] Nanobody Q74 and Nanobody L125b are both specific antibodies against H7 avian influenza. To this end, different antigens are coated and the coated antigens are detected using the screened and expressed Nanobodies. The specific experimental plan is as follows:

[0105] Antigen coating: Take 10μL of H5, H7, and H9 avian influenza hemagglutination inhibition test antigens (Re-8 strain, H7-Re1 strain, and H9 subtype, respectively), dilute 10 times according to the first well antigen (antigen: carbonate coating buffer = 1:9), and start diluting 4 times from the second well to adsorb the antigen on the solid phase carrier polystyrene. That is, after the antigen and coating solution are evenly diluted in the first well, 25μL is drawn to the second well, and diluted once to the seventh well. Only the coating solution is added to the eighth well as a blank control. The final amount of each well is 75μL. After incubation at 37℃ for 2h or 4℃ overnight, the liquid in the plate is discarded;

[0106] Blocking: add 300 μL 3% BSA for 1 h;

[0107] Primary antibody: Anti-H7 avian influenza virus nanobody protein is the primary antibody, diluted with PBS at a ratio of 1:100 and 75 μL of the diluted antibody is added to each well;

[0108] Washing: Wash 3-5 times with PBS containing 0.5% Tween-20, soaking for 5 min each time, wash 5 times;

[0109] Secondary antibody: GenScript mouse anti-his polyclonal antibody was diluted with PBS at a ratio of 1:3000, and 75 μL of the diluted antibody was added to each well and incubated for 1 hour;

[0110] Washing: Wash 3-5 times with PBS containing 0.5% Tween-20, soaking time for 5 minutes each time, wash 5 times;

[0111] Tertiary antibody: Shanghai Bioengineering Company's goat anti-mouse-HRP antibody was diluted with PBS at a ratio of 1:5000, and 75 μL of the diluted antibody was added to each well and incubated for 1 hour;

[0112] Washing: Wash 3-5 times with PBS containing 0.5% Tween-20, soaking time for 5 minutes each time, wash 5 times;

[0113] Color development: Add HRP substrate color development solution for 20 minutes and read the results using an enzyme reader at a wavelength of 650 nm.

[0114] The ELISA results showed that: as the coating antigen (within a certain range) of H7 antigen (Re-1) decreased gradually, the P / N ratio also decreased gradually. Compared with H5 antigen and H9 antigen, the eukaryotic expressed anti-H7 subtype avian influenza virus nanobody Q74 (such as Figure 7 As shown) and L125b (as Figure 8 As shown) have good antigen binding ability to H7 subtype AIV.

[0115] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nanobody against H7 subtype avian influenza virus, characterized in that: Selected from Nanobody Q74 or Nanobody L125b; the amino acid sequences of the three CDR regions in Nanobody L125b are shown in SEQ ID NOs: 6-8, respectively; The amino acid sequences of the three CDR regions in the Nanobody Q74 are shown in SEQ ID NOs: 10-12, respectively.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the Nanobody L125b is shown in SEQ ID NO:

5.

3. The Nanobody according to claim 1, characterized in that The amino acid sequence of the Nanobody Q74 is shown in SEQ ID NO:

9.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the nanobody against H7 subtype avian influenza virus according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 13 or SEQ ID NO:

14.

6. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule according to any one of claims 4-5.

7. A recombinant bacterium, characterized in that: Comprising the nucleic acid molecule according to any one of claims 4-5 or the recombinant expression vector according to claim 6.

8. The recombinant bacterium according to claim 7, characterized in that The recombinant bacteria is a recombinant yeast.

9. Use of the nanobody according to any one of claims 1 to 3 in the preparation of a diagnostic / detection kit for H7 subtype avian influenza virus.

10. A H7 subtype avian influenza virus diagnosis / detection kit, characterized in that: Comprising the Nanobody of any one of claims 1-3.

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