Single-chain antibody for neutralizing avian influenza virus as well as preparation method and application of single-chain antibody

Abstract

The invention provides a single-chain antibody for neutralizing avian influenza virus as well as a preparation method and application thereof, the single-chain antibody has three heavy chain complementary determining regions as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and three light chain complementary determining regions as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The single-chain antibody scFv can be combined on a non-RBD conservative neutralizing epitope of avian influenza virus surface antigen-hemagglutinin, does not depend on ADCC and steric hindrance effects, directly neutralizes H7N9 avian influenza virus in a broad spectrum, and avoids virus drug resistance; the Fc segment of the antibody is not contained, the molecular weight is small, and the penetrating effect is better.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and particularly relates to a single-chain antibody for neutralizing avian influenza virus, as well as a preparation method and use thereof. Background Art

[0002] ScFv (single-chain fragment variable) is formed by connecting the variable region of the heavy chain of an antibody and the variable region of the light chain through a short peptide of 15-20 amino acids, without an Fc fragment. It belongs to a small molecule genetic engineering antibody and has characteristics such as antigen-binding properties, strong penetrability, short in vivo half-life, low immunogenicity, can be expressed in a prokaryotic cell system, and is easy to perform genetic engineering operations.

[0003] The H7N9 avian influenza virus is an influenza A virus that can infect humans through poultry. Human infection with H7N9 avian influenza is an acute respiratory infectious disease that can rapidly develop into severe pneumonia and acute respiratory distress syndrome, with a mortality rate as high as 40%. So far, there is still a lack of effective drugs and vaccines. At present, the emergence of highly pathogenic H7N9 avian influenza virus has posed a serious threat to the poultry farming industry, animal husbandry, and human health, and it is urgent for the health department to take effective control measures.

[0004] Fully human monoclonal antibodies have significant efficacy in preventing and treating avian influenza. They can bind to the receptor-binding site (RBD) or fusion peptide of the surface antigen - hemagglutinin protein of avian influenza virus, inhibit the binding of the virus to the host cell receptor or membrane fusion, thereby neutralizing the avian influenza virus. However, functional epitopes such as RBD are prone to mutation during the virus transmission process, resulting in the easy inactivation of neutralizing antibodies targeting these epitopes. In addition, some antibodies can also bind to antigenic epitopes other than RBD or non-fusion peptides, and neutralize the virus or clear infected cells through the steric hindrance mediated by the Fc segment of the antibody or effects such as ADCC and CDC. If the Fc segment of these antibodies is deleted and transformed into a single-chain antibody scFv, it will lose the function of neutralizing the virus. The molecular weight of the whole antibody is relatively large, with poor permeability, and it is difficult to enrich a sufficient antibody concentration in human or animal organs and tissues. Therefore, the therapeutic effect on severe avian influenza patients is relatively limited. Summary of the Invention

[0005] The single-chain antibody scFv of the present invention can bind to a conserved neutralizing epitope other than RBD of the surface antigen - hemagglutinin of avian influenza virus, directly and broadly neutralize H7N9 avian influenza virus without relying on ADCC and steric hindrance effects, avoiding virus drug resistance; it does not contain the Fc segment of the antibody, has a small molecular weight, and better penetration effect.

[0006] One aspect of the present invention provides an isolated single-chain antibody that neutralizes avian influenza virus. The single-chain antibody has a heavy-chain variable region, a linker peptide, and a light-chain variable region. The heavy-chain variable region has three heavy-chain complementarity-determining regions as shown below:

[0007] The amino acid sequence of the heavy-chain variable region CDR1 is: GFSFSNYG SEQ ID NO.2

[0008] The amino acid sequence of the heavy-chain variable region CDR2 is: ISYDGTNK SEQ ID NO.3

[0009] The amino acid sequence of the heavy-chain variable region CDR3 is: AKGRGPYCSSSICYHGMDV SEQ ID NO.4;

[0010] And has three light-chain complementarity-determining regions as shown below:

[0011] The amino acid sequence of the light-chain variable region CDR1 is: QSVLSGSINMNY SEQ ID NO.6

[0012] The amino acid sequence of the light-chain variable region CDR2 is: WAS

[0013] The amino acid sequence of the light-chain variable region CDR3 is: QQYYSTPLT SEQ ID NO.7.

[0014] Another aspect of the present invention provides an isolated single-chain antibody that neutralizes avian influenza virus, which has a heavy-chain variable region as shown in SEQ ID No: 1 and a light-chain variable region as shown in SEQ ID No: 5.

[0015] Another aspect of the present invention provides an isolated single-chain antibody that neutralizes avian influenza virus, which has a sequence as shown in SEQ ID NO.10.

[0016] Another aspect of the present invention provides a nucleotide sequence that encodes the single-chain antibody that neutralizes avian influenza virus as described above.

[0017] Another aspect of the present invention provides a recombinant vector that contains the nucleotide sequence described above.

[0018] Another aspect of the present invention provides a host cell that contains the foregoing vector or vector group. Preferably, the host cell is prokaryotic or eukaryotic, and more preferably is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0019] Another aspect of the present invention provides a kit that contains the antibody or its antigen-binding fragment as described above.

[0020] Another aspect of the present invention provides the use of the single-chain antibody for neutralizing avian influenza virus in a drug for preventing, treating or alleviating at least one symptom or indication of avian influenza virus infection.

[0021] In the technical solution of the present invention, the drug is an oral or injection preparation.

[0022] Another aspect of the present invention provides a method for preventing, treating or alleviating at least one symptom or indication of avian influenza virus infection, the method comprising administering the antibody or its antigen-binding fragment of any one of the foregoing or the foregoing pharmaceutical composition to a subject.

[0023] In the technical solution of the present invention, the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar damage, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock and death.

[0024] In the technical solution of the present invention, the avian influenza virus is H7N9.

[0025] In the technical solution of the present invention, the pharmaceutical composition or the antibody or its antigen-binding fragment is administered in combination with a second therapeutic agent. The second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, vaccines against avian influenza virus, antibiotics, dietary supplements such as antioxidants and any other palliative therapies for treating avian influenza virus infection, drugs for alleviating the above symptoms or indications.

[0026] In the technical solution of the present invention, the pharmaceutical composition or the antibody or its antigen-binding fragment is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.

[0027] Another aspect of the present invention provides a detection reagent, the detection reagent comprising the antibody as described above.

[0028] Another aspect of the present invention provides the use of the above antibody or its antigen-binding fragment as a detection reagent for use in the following: enzyme-linked immunosorbent assay (ELISA), immunoblot (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immunological PCR.

[0029] In the above immunological detection, the antibody or its antigen-binding fragment can be conjugated alone or through chemical bonds, electrostatic adsorption, or hydrophilic and hydrophobic adsorption, and the conjugate includes conjugates such as horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads, and agarose.

[0030] In the technical solution of the present invention, the detection reagent can be used for non-diagnostic therapeutic purpose detection.

[0031] In the present invention, a method for preparing the above single-chain antibody is provided. The method is to clone the nucleotide sequence encoding the above single-chain antibody of the present invention into a vector plasmid, and then perform protein expression in an expression system, and obtain the single-chain antibody after purification.

[0032] In the present invention, the nucleotide and amino acid sequences of the variable regions of the heavy and light chains of the single-chain antibody are shown as follows.

[0033] Amino acid sequence of the heavy chain variable region:

[0034] QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGLHWVRQAPGKGLDWVAVISYDGTNKYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKGRGPYCSSSICYHGMDVWGQGTTVTVSS SEQ ID NO.1

[0035] Among them, the amino acid sequence of CDR1 of the heavy chain variable region is: GFSFSNYG SEQ ID NO.2

[0036] Amino acid sequence of CDR2 of the heavy chain variable region is: ISYDGTNK SEQ ID NO.3

[0037] Amino acid sequence of CDR3 of the heavy chain variable region is: AKGRGPYCSSSICYHGMDV SEQ ID NO.4

[0038] Amino acid sequence of the light chain variable region: DIVMTQSPDSLAVSLGERATINCKSSQSVLSGSINMNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK SEQ IDNO.5

[0039] Among them, the amino acid sequence of CDR1 of the light chain variable region is: QSVLSGSINMNY SEQ ID NO.6

[0040] The amino acid sequence of the CDR2 in the light chain variable region is: WAS

[0041] The amino acid sequence of the CDR3 in the light chain variable region is: QQYYSTPLT SEQ ID NO.7

[0042] The amino acid sequence of the heavy chain VH of the single-chain antibody as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain VL of the single-chain antibody as shown in SEQ ID NO: 5 are linked by a linker peptide. The linker peptide sequence is as shown in SEQ ID NO.8: GGGGSGGGGSGGGGS SEQ ID NO.8.

[0043] The sequence of the single-chain antibody is as shown in SEQ ID NO.9, MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLSGSINMNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGLHWVRQAPGKGLDWVAVISYDGTNKYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKGRGPYCSSSICYHGMDVWGQGTTVTVSS SEQ ID NO.9.

[0044] Optionally, the gene sequence encoding the single-chain antibody is as shown in SEQ ID NO.10.

[0045] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCAGACAT

[0046] TGTGATGACCCAGAGCCCCGACTCCCTCGCCGTGAGCCTCGGAGAAAGAGCCACCATA

[0047] AACTGCAAGAGCAGCCAGAGCGTGCTGAGCGGCAGCATCAACATGAACTATCTGGCCT

[0048] GGTACCAGCAAAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCATCCACCCGG

[0049] GAAAGCGGAGTGCCCGACAGATTTACCGGATCTGGCAGCGGCACCGACTTCACCCTCA

[0050] CCGTGTCCAGCCTGCAAGCTGAGGACGTGGCCGTGTACTACTGCCAGCAATACTACTCC

[0051] ACCCCCCTGACCTTTGGAGGCGGCACCAAGGTGGAAATCAAGGGTGGCGGTGGCTCGG

[0052] GCGGTGGTGGGTCGGGTGGCGGCGGATCTCAGGTCCAACTCGTCGAGTCAGGCGGCGG

[0053] CGTCGTTCAGCCAGGAAGATCACTCAGACTCAGCTGCGCAGCAAGCGGATTCTCCTTCA

[0054] GCAACTACGGCCTCCACTGGGTCAGACAAGCCCCCGGCAAAGGGCTGGACTGGGTGGC

[0055] TGTGATTAGCTACGATGGGACCAACAAATATTACGCCGATAGCGTGAAGGGCCGGTTCA

[0056] CCATCAGCAGAGACAACAGCAAGAACACACTGCACCTGCAGATGAACTCTCTGAGAGC

[0057] CGAGGACACAGCAGTGTACTACTGCGCCAAGGGGAGAGGACCCTATTGCAGCAGCTCC

[0058] ATCTGCTACCACGGAATGGACGTGTGGGGCCAAGGCACCACCGTGACCGTGAGCTCCCACCATCACCATCACCACCACCACCACCACTGA SEQ ID NO.10

[0059] Among them, the single-chain antibody includes a heavy-chain variable region, a light-chain variable region, and a linker peptide.

[0060] Furthermore, the single-chain antibody further includes a signal peptide, and the signal peptide sequence is as shown in SEQ ID NO.11:

[0061] MHSSALLCCLVLLTGVRA SEQ ID NO.11。

[0062] Beneficial effects

[0063] The single-chain antibody scFv of the present invention was unexpectedly discovered on the basis of the prior research of the inventors of the present invention. The prior patent 201611038444.X provided an anti-H7N9 fully human monoclonal antibody. However, due to its relatively large molecular weight and poor permeability, it is difficult to enrich sufficient antibody concentration in human or animal organs and tissues. Therefore, the therapeutic effect on severe avian influenza patients is relatively limited. Although it is well known that single-chain monoclonal antibodies have the effects of small molecular weight and better penetration effect, usually the loss of the Fc segment means the loss of the function of neutralizing viruses. Therefore, those skilled in the art would not develop the anti-H7N9 fully human monoclonal antibody into a single-chain antibody. However, the inventors of the present invention unexpectedly found that although the single-chain antibody of the present invention does not contain the Fc segment of the antibody, it can directly and broadly neutralize the H7N9 avian influenza virus, can avoid virus drug resistance, and at the same time has the advantages of small molecular weight and better penetration effect. Brief description of the drawings

[0064] Figure 1 SDS-PAGE diagram of the single-chain antibody in Example 1.

[0065] Figure 2 Antibody dose-effect curve in Example 2. Detailed implementation manners

[0066] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention is given, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0067] Preparation and purification of the single-chain antibody in Example 1

[0068] Construct an expression vector

[0069] Clone the target sequence SEQ ID NO.11 into the target vector pcDNA3.1 (provided by Jiangsu Fute Biotechnology Co., Ltd.) using NotI / XbaI to obtain the target plasmid.

[0070] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCAGACAT

[0071] TGTGATGACCCAGAGCCCCGACTCCCTCGCCGTGAGCCTCGGAGAAAGAGCCACCATA

[0072] AACTGCAAGAGCAGCCAGAGCGTGCTGAGCGGCAGCATCAACATGAACTATCTGGCCT

[0073] GGTACCAGCAAAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCATCCACCCGG

[0074] GAAAGCGGAGTGCCCGACAGATTTACCGGATCTGGCAGCGGCACCGACTTCACCCTCA

[0075] CCGTGTCCAGCCTGCAAGCTGAGGACGTGGCCGTGTACTACTGCCAGCAATACTACTCC

[0076] ACCCCCCTGACCTTTGGAGGCGGCACCAAGGTGGAAATCAAGGGTGGCGGTGGCTCGG

[0077] GCGGTGGTGGGTCGGGTGGCGGCGGATCTCAGGTCCAACTCGTCGAGTCAGGCGGCGG

[0078] CGTCGTTCAGCCAGGAAGATCACTCAGACTCAGCTGCGCAGCAAGCGGATTCTCCTTCA

[0079] GCAACTACGGCCTCCACTGGGTCAGACAAGCCCCCGGCAAAGGGCTGGACTGGGTGGC

[0080] TGTGATTAGCTACGATGGGACCAACAAATATTACGCCGATAGCGTGAAGGGCCGGTTCA

[0081] CCATCAGCAGAGACAACAGCAAGAACACACTGCACCTGCAGATGAACTCTCTGAGAGC

[0082] CGAGGACACAGCAGTGTACTACTGCGCCAAGGGGAGAGGACCCTATTGCAGCAGCTCC

[0083] ATCTGCTACCACGGAATGGACGTGTGGGGCCAAGGCACCACCGTGACCGTGAGCTCCCACCATCACCATCACCACCACCACCACCACTGA SEQ ID NO.10

[0084] Single-chain antibody production experiment

[0085] Add 100 μM hypoxanthine and 16 μM thymidine to CellventoTM CHO-210 medium, and add 4 - 8 μM L-glutamine. Resuspend the linear DNA with 1×PBS, adding 50 μL PBS per 5 μg plasmid, and then mix well with a vortex mixer. Take the prepared culture medium, add it to a 6-well plate for preheating, 2 mL per well, and place it in a 37°C incubator for preheating. The passage number of the cells used for transfection is 3 - 15 times. One day before transfection, seed CHO-S cells on a 10 cm culture dish at a cell density of 3.0 - 6.0×10 5 cells / mL and culture for 24 h. After culturing for 24 hours, the cells are in the logarithmic growth phase. Centrifuge to remove the supernatant to fully suspend the cell pellet at the bottom. Add 10 mL of 1×PBS to resuspend the cells, centrifuge at 1000 rpm for 4 min, remove the supernatant, gently slide the centrifuge tube to make the cell pellet at the bottom float. Re-add 10 mL of 1×PBS to resuspend the cells and count them, centrifuge at 1000 rpm for 4 min. According to the cell counting results, add an appropriate amount of 1×PBS to resuspend the cells to a cell density of 4×10 6cells / mL. Take 50 μL of the DNA suspension and add it to 50 μL of the suspension of cells to be transfected. Mix well and add it to an electroporation cuvette with a 5 mm specification, taking care not to have any air bubbles. The electroporation parameters for the CHO-S cell line are: square wave, voltage 160 V, electroporation time 15 ms, one pulse, and the distance between the cuvettes is 5 mm; perform electroporation. After electroporation, quickly add 0.5 mL of the culture medium to the electroporation cuvette using a pipette, pipette up and down 2 - 3 times, aspirate and seed into a pre-warmed 6-well plate. After 24 - 48 hours when the cell growth state has recovered, add G418 to the culture medium. The G418 concentration for CHO-S cells is selected as follows: 1000 - 2000 μg / mL, and the starting concentration is generally 300 - 500 μg / mL. Keep the cells in a selective (antibiotic-containing) culture medium, observe the cell state, and change the medium every 3 - 5 days. Perform resistance screening for 10 days. Add G418 to the culture medium to a final concentration of 1000 μg / mL, add 150 μL per well to a 96-well culture plate, and place it in an incubator to pre-warm; collect and count the surviving cells, dilute them to 20 cells / mL with the culture medium, mix well and add 50 μL per well to the 96-well plate (inoculate 1 cell per well). After culturing for about 10 days, observe the growth of monoclonal cells, and perform ELISA to detect protein expression. Select 1 - 6 monoclonal cells with high expression levels and transfer them to a 6-well plate for culture. After culturing for about 3 days, when the cell density is above 5×10 5 / mL, perform ELISA determination. Transfer those with an expression level of about 0.5 mg / L to a 10 cm culture dish for culture. After culturing in the culture dish for about 3 days, when the cell density is above 1×10 6 / mL, perform ELISA determination, and select the cell line with high expression level for scale-up culture. Centrifuge the cell suspension, discard the supernatant, gently slide the centrifuge tube to make the cell pellet at the bottom float up, and culture with the prepared CellventoTM CHO-210). The cell seeding density is 3 - 5×10 5 / mL, and the rotation speed of the shake flask is 110 - 130 revolutions per minute. Freeze the cells (the freezing medium contains 10% DMSO and is prepared with CellventoTM CHO-210).

[0086] Purification of single-chain antibody

[0087] Centrifuge in a 500 mL conical centrifuge tube at 7500 rpm at 4 °C for 15 min, pour out the supernatant, collect the supernatant, first filter it through a 0.45 μm filter membrane, and then filter it through a 0.22 μm filter membrane. Insert the inlet end of the hose into the bottom of the blue-capped bottle containing the cell supernatant, connect the outlet of the column to the waste liquid cylinder, and make corresponding marks on the blue-capped bottle, hose, column, and waste liquid cylinder. Adjust the peristaltic pump to an appropriate rotation speed and pass through an affinity chromatography Ni column. Elute the single-chain antibody with a 500 mM imidazole solution, and finally change the liquid and concentrate it using an ultrafiltration tube.

[0088] The nucleotide and amino acid sequences of the variable regions of the heavy and light chains of the single-chain antibody are shown as follows.

[0089] Amino acid sequence of the heavy chain variable region:

[0090] QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGLHWVRQAPGKGLDWVAVISYDGTNKYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKGRGPYCSSSICYHGMDVWGQGTTVTVSS SEQ ID NO.1

[0091] Among them,

[0092] Amino acid sequence of CDR1 of the heavy chain variable region: GFSFSNYG SEQ ID NO.2

[0093] Amino acid sequence of CDR2 of the heavy chain variable region: ISYDGTNK SEQ ID NO.3

[0094] Amino acid sequence of CDR3 of the heavy chain variable region: AKGRGPYCSSSICYHGMDV SEQ ID NO.4

[0095] Amino acid sequence of the light chain variable region: DIVMTQSPDSLAVSLGERATINCKSSQSVLSGSINMNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK SEQ IDNO.5

[0096] Among them,

[0097] Amino acid sequence of CDR1 of the light chain variable region: QSVLSGSINMNY SEQ ID NO.6

[0098] Amino acid sequence of CDR2 of the light chain variable region: WAS

[0099] Amino acid sequence of CDR3 of the light chain variable region: QQQYYSTPLT SEQ ID NO.7

[0100] The amino acid sequence of the single-chain antibody heavy chain VH shown as SEQ ID NO: 1 and the amino acid sequence of the single-chain antibody light chain VL shown as SEQ ID NO: 5 are linked by a linker peptide. The linker peptide sequence is shown as SEQ ID NO.8: GGGGSGGGGSGGGGS SEQ ID NO.8.

[0101] The sequence of the single-chain antibody is shown in SEQ ID NO.9: MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLSGSINMNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGLHWVRQAPGKGLDWVAVISYDGTNKYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKGRGPYCSSSICYHGMDVWGQGTTVTVSS SEQ ID NO.9.

[0102] Example 2 Micro-neutralization Experiment

[0103] Using a virus-infected cell model (Madin-Darby canine kidney cells, MDCK), the inhibitory effect of the single-chain antibody on the H7N9 influenza virus (preserved by the Institute of Microbiology, Chinese Academy of Sciences, Beijing) with PR-8 as the backbone was evaluated by a micro-neutralization-ELISA experiment to detect the anti-influenza virus activity of the antibody.

[0104] Ten concentration gradients were set up, successively diluted 10-10 10 times, and three parallel wells were set for each concentration in each group.

[0105] (1) Trypsinize MDCK cells in the logarithmic growth phase, centrifuge and collect them after termination, disperse them evenly to prepare a single-cell suspension. Adjust the cell concentration to 5×10 4 cells / ml with MEM culture medium, inoculate them into a 96-well cell culture dish, and culture them overnight in an incubator at 37°C and 5% CO 2 .

[0106] (2) Dilute the single-chain antibody 10-10 10 times with PBS, mix it with an equal volume of H7N9 influenza virus (100 TCID50) respectively, and incubate at 37°C for 1 h.

[0107] (3) Discard the cell culture supernatant, add the pre-mixed single-chain antibody-virus mixture to the 96-well cell culture dish, incubate at 37°C for 1 h, discard the mixture, and wash twice with PBS.

[0108] (4) Add 100 μL of maintenance medium (add trypsin treated with TPCK at a final concentration of 2 μg / mL to serum-free DMEM with double antibiotics) to each well in the 96-well cell culture plate, and culture it in an incubator at 37°C and 5% CO 2 for 20 h.

[0109] (5) Discard the maintenance solution in the culture dish, and wash the cells once with 100 μL of PBS. Add 50 μL of fixative (acetone: absolute ethanol = 2:3) to each well, and fix the cells at room temperature for 10 min. Discard the fixative, and wash the cells 3 times with 100 mL of PBS to remove the residual acetone.

[0110] (6) Block the cells with 1% BSA for 1 h, and wash the cells once with 100 μL of PBS solution. Dilute the primary antibody (anti-influenza virus nucleoprotein - NP monoclonal antibody) with PBS at a ratio of 1:2000, add 50 μL to each well, and incubate at room temperature for 1 h. Wash the plate 3 times with 100 μL of PBST. Dilute the HRP-conjugated secondary antibody with PBS at a ratio of 1:2000, add 50 μL to each well, and incubate at room temperature for 1 h. Wash the plate 6 times with 100 μL of PBST. Add 50 μL of TMB chromogenic solution to each well. After 10 min of incubation at room temperature in the dark, add 50 μL of 2 M sulfuric acid to each well to terminate the reaction. Read the OD value of each well on a spectrophotometer (450 nm).

[0111] (7) Statistical analysis: Use GraphPad Prism 6.0.1 to analyze the data and draw a dose - effect curve, and calculate the IC50.

[0112] Inhibition rate calculation formula:

[0113] Inhibition rate = [(ODvirus well - ODnegative cell control well) - (ODdrug well - ODnegative cell control well)] ÷ (ODvirus well - ODnegative cell control well) × 100%.

[0114] (8) Results showed that: The single-chain antibody could efficiently neutralize the H7N9 virus, and the IC 50 = 96 ng / mL.

Claims

1. An isolated single-chain antibody that neutralizes avian influenza virus, characterized in that, the single-chain antibody has the following three heavy-chain complementarity-determining regions: The amino acid sequence of heavy-chain variable region CDR1 is: GFSFSNYG SEQ ID NO.2 The amino acid sequence of heavy-chain variable region CDR2 is: ISYDGTNK SEQ ID NO.3 The amino acid sequence of heavy-chain variable region CDR3 is: AKGRGPYCSSSICYHGMDV SEQ ID NO.4; and has the following three light-chain complementarity-determining regions: The amino acid sequence of light-chain variable region CDR1 is: QSVLSGSINMNY SEQ ID NO.6 The amino acid sequence of light-chain variable region CDR2 is: WAS The amino acid sequence of light-chain variable region CDR3 is: QQYYSTPLT SEQ ID NO.

7.

2. An isolated single-chain antibody that neutralizes avian influenza virus, characterized in that, the single-chain antibody has a heavy-chain variable region as shown in SEQ ID No: 1 and a light-chain variable region as shown in SEQ ID No:

5.

3. An isolated single-chain antibody that neutralizes avian influenza virus, characterized in that, the single-chain antibody has the sequence as shown in SEQ ID NO.

9.

4. A nucleotide sequence, characterized in that, it encodes the single-chain antibody that neutralizes avian influenza virus according to any one of claims 1-3.

5. A recombinant vector, characterized in that, it contains the nucleotide sequence according to claim 4.

6. A host cell, characterized in that, it contains the aforementioned vector or vector group; Preferably, the host cell is prokaryotic or eukaryotic; More preferably, it is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or their antigen-binding fragments.

7. Use of the single-chain antibody that neutralizes avian influenza virus according to any one of claims 1-3 in a drug for preventing, treating or alleviating at least one symptom or indication of avian influenza virus infection, wherein the avian influenza virus is H7N9; Preferably, the at least one symptom or indication is selected from the following group: pulmonary inflammation, alveolar damage, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock and death.

8. Use of the single-chain antibody according to any one of claims 1-3 in the preparation of a detection reagent, wherein the detection reagent is a reagent for the following uses: enzyme-linked immunosorbent assay (ELISA), immunoblot (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immunological PCR.

9. A method for preparing the single-chain antibody according to any one of claims 1-3, characterized in that, the preparation method is to clone the nucleotide sequence encoding the single-chain antibody according to any one of claims 1-3 into a vector plasmid, then perform protein expression in an expression system, and obtain the single-chain antibody after purification.

10. A pharmaceutical composition or a detection reagent, characterized in that, The described pharmaceutical composition or a detection reagent comprises the single-chain antibody described in any one of claims 1-3.

Images