A nano antibody against HA1 protein of avian influenza virus and its preparation method and application

Abstract

The present invention discloses a VHH chain of a nano antibody against HA1 protein of avian influenza virus, and the amino acid sequence of the VHH chain is shown in SEQ ID NO: 1. The present invention uses avian influenza virus as a research object, inactivates it and immunizes alpacas, and uses phage display technology to screen and obtain a nano antibody against HA1 protein of avian influenza virus. The present invention provides materials for establishing a broad-spectrum diagnostic method through the preparation of the nano antibody against HA1 protein of avian influenza virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nano antibody against HA1 protein of avian influenza virus and a preparation method and application thereof. Background Art

[0002] There is an antibody that naturally lacks light chains in the peripheral blood of alpacas. This antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, with a molecular weight of only 15Kd, so it is also called Nanobody (Nb). Nanobodies have the advantages of simple structure, good antigen specificity, strong tissue penetration, high affinity, strong stability and easy expression and preparation. The use of phage display technology can construct a nanobody library, and molecular biological methods can be used to screen nanobodies with strong specificity and high affinity from the phage library. Nanobodies have broad application prospects in the diagnosis and treatment of diseases.

[0003] Avian influenza virus belongs to influenza A virus (IAV), and its 8 RNA segments can encode at least 17 proteins. According to the different antigenicity of HA protein and NA protein, influenza A virus is divided into 18 different HA subtypes and 11 different NA subtypes. HA protein is one of the most abundant transmembrane glycoproteins on the surface of influenza virus particles, which forms a trimeric rod-shaped structure with a globular head and a fibrous neck. HA protein usually exists in the form of inactive HA0 precursor protein, which must be cleaved by protease to produce HA1 and HA2 molecules, so that it can play a normal role in the process of viral infection. In addition, HA1 protein has 4 antigenic determinants, is immunogenic, can induce the body to produce neutralizing antibodies, and plays an important role in vaccine development. Alpacas were immunized with HA1 protein, and phage display technology was used to screen broad-spectrum and high-affinity nanoantibodies to provide materials for basic and applied research. At present, there is a lack of efficient and rapid detection technology for IAV. Summary of the invention

[0004] Based on this, the present invention provides a VHH chain of an anti-avian influenza virus HA1 protein nanobody, the amino acid sequence of the VHH chain is shown in SEQ ID NO:1.

[0005] According to another aspect of the present invention, an anti-avian influenza virus HA1 protein nanobody is provided, which is a nanobody against an antigenic epitope of the avian influenza virus HA1 protein and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:1.

[0006] Further, according to the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody or according to the above-mentioned anti-avian influenza virus HA1 protein nanobody, the amino acid sequence is replaced by an amino acid sequence selected from the following:

[0007] a) the amino acid sequence has a degree of identity of at least about 95%, 96%, 97%, 98% or at least 99% to the amino acid sequence of SEQ ID NO: 1;

[0008] b) an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1;

[0009] c) the amino acid sequence differs from the amino acid sequence of SEQ ID NO: 1 by no more than 5, 4, 3, 2 or no more than 1 amino acid;

[0010] and / or

[0011] d) The amino acid sequence has the amino acid sequence shown in SEQ ID NO: 1, including substitution, deletion and / or insertion of 1 to 5 amino acid residues.

[0012] According to another aspect of the present invention, a biomaterial related to the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody, is provided, and the biomaterial is any one of A1) to A12): A1) a nucleic acid molecule encoding the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody;

[0013] A2) an expression cassette containing the nucleic acid molecule of A1);

[0014] A3) a recombinant vector containing the nucleic acid molecule of A1);

[0015] A4) a recombinant vector containing the expression cassette of A2);

[0016] A5) a recombinant microorganism containing the nucleic acid molecule of A1);

[0017] A6) a recombinant microorganism containing the expression cassette of A2);

[0018] A7) a recombinant microorganism containing the recombinant vector in A3);

[0019] A8) a recombinant microorganism containing the recombinant vector of A4);

[0020] A9) a transgenic animal cell line containing the nucleic acid molecule of A1);

[0021] A10) a transgenic animal cell line containing the expression cassette of A2);

[0022] A11) a transgenic animal cell line containing the recombinant vector in A3);

[0023] A12) A transgenic animal cell line containing the recombinant vector of A4).

[0024] Further, according to the above biological material, the nucleotide sequence of the nucleic acid molecule is selected from:

[0025] a) the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2;

[0026] b) a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions;

[0027] c) a nucleotide sequence having a degree of identity of at least about 95%, 96%, 97%, 98% or at least 99% to the nucleotide sequence shown in SEQ ID NO: 2;

[0028] d) a nucleotide sequence encoding an amino acid that is at least about 95%, 96%, 97%, 98% or at least 99% identical to the amino acid set forth in SEQ ID NO: 1;

[0029] e) a nucleotide sequence encoding an amino acid which differs from SEQ ID NO: 1 by no more than 5, 4, 3, 2 or 1 amino acid residues;

[0030] and / or

[0031] f) A nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO: 1, including substitution, deletion and / or insertion of 1 to 5 amino acid residues.

[0032] According to another aspect of the present invention, there is provided a method for producing a VHH chain of an anti-avian influenza virus HA1 protein nanobody or an anti-avian influenza virus HA1 protein nanobody, the method comprising the following steps:

[0033] (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody; and

[0034] (2) Cultivating the transgenic cell to obtain the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody.

[0035] Furthermore, the avian influenza virus is an influenza A virus.

[0036] Furthermore, the avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

[0037] According to another aspect of the present invention, a method for screening VHH chains of anti-avian influenza virus HA1 protein nanoantibodies or anti-avian influenza virus HA1 protein nanoantibodies is provided, the method comprising the following steps:

[0038] (1) emulsifying a certain amount of the purified avian influenza virus with an equal volume of adjuvant, immunizing alpacas 4 to 6 times, then collecting peripheral blood from the alpacas, isolating peripheral blood lymphocytes, and constructing a phage display library against avian influenza virus; and

[0039] (2) Using phage display technology, after 3 to 5 rounds of screening, the VHH chain of the anti-avian influenza virus HA1 protein nanoantibody or the anti-avian influenza virus HA1 protein nanoantibody is screened and obtained.

[0040] Furthermore, the avian influenza virus is avian influenza virus type A and / or avian influenza virus type B avian influenza virus.

[0041] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody; and (b) a pharmaceutically acceptable carrier.

[0042] According to another aspect of the present invention, a recombinant protein is provided, which comprises (a) the sequence of the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the sequence of the above-mentioned anti-avian influenza virus HA1 protein nanobody; and (b) a tag sequence that assists expression and / or purification.

[0043] Furthermore, the tag sequence includes an Fc tag.

[0044] According to another aspect of the present invention, the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the use of the above-mentioned anti-avian influenza virus HA1 protein nanobody, is provided for preparing (a) a medicament, reagent, detection plate, test paper or kit for detecting anti-avian influenza virus HA1 protein molecules; or (b) for preparing a drug for preventing and / or treating avian influenza virus infection; or (c) for distinguishing influenza A virus, influenza B virus and / or influenza C virus;

[0045] Furthermore, the avian influenza virus is an influenza A virus.

[0046] Furthermore, the avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

[0047] Further, the prevention and / or treatment is manifested as increased body weight and / or increased survival rate.

[0048] Beneficial effects of the present invention:

[0049] The present invention uses avian influenza virus as the research object, inactivates it and immunizes alpacas, and uses phage display technology to screen and obtain a broad-spectrum avian influenza virus HA1 protein nano antibody. The present invention provides materials for establishing a broad-spectrum diagnostic method through the preparation of the anti-avian influenza virus HA1 protein nano antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0051] Figure 1 Schematic diagram of nanobody purification results.

[0052] Figure 2 Schematic diagram of the Nb-17 affinity test results.

[0053] Figure 3 Schematic diagram of the indirect immunofluorescence test results of nanoantibodies.

[0054] Figure 4 This is a schematic diagram of the weight changes of mice in the prevention group.

[0055] Figure 5 This is a schematic diagram of the changes in the survival curve of mice in the prevention group.

[0056] Figure 6 This is a schematic diagram of the weight changes of mice in the treatment group.

[0057] Figure 7 This is a schematic diagram of the changes in the survival curve of mice in the treatment group. DETAILED DESCRIPTION

[0058] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0059] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0060] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0061] It should be noted that any process not specifically described below can be implemented or understood by those skilled in the art with reference to the prior art. Reagents and instruments used without manufacturer indicated are considered to be conventional products that can be purchased commercially.

[0062] Unless otherwise specified, the practice of the present invention will adopt the conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. Unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0063] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0064] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0065] As described in the background technology section, the prior art lacks efficient and rapid detection technology for IAV. In order to solve the above problems, the present invention provides a VHH chain of an anti-avian influenza virus HA1 protein nanobody, the amino acid sequence of which is shown in SEQ ID NO:1.

[0066] According to another aspect of the present invention, an anti-avian influenza virus HA1 protein nanobody is provided, which is a nanobody against an antigenic epitope of the avian influenza virus HA1 protein and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:1.

[0067] In a preferred embodiment, according to the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein Nanobody or according to the above-mentioned anti-avian influenza virus HA1 protein Nanobody, the amino acid sequence is replaced by an amino acid sequence selected from the following:

[0068] a) the amino acid sequence has a degree of identity of at least about 95%, 96%, 97%, 98% or at least 99% to the amino acid sequence of SEQ ID NO: 1;

[0069] b) an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1;

[0070] c) the amino acid sequence differs from the amino acid sequence of SEQ ID NO: 1 by no more than 5, 4, 3, 2 or no more than 1 amino acid;

[0071] and / or

[0072] d) The amino acid sequence has the amino acid sequence shown in SEQ ID NO: 1, including substitution, deletion and / or insertion of 1 to 5 amino acid residues.

[0073] In the present invention, "about" refers to a value within the range of ±5% of a particular value. For example, "about 95%" includes ±5% of 95%, or from 90.25 to 99.75.

[0074] According to another aspect of the present invention, a biomaterial related to the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody, is provided, and the biomaterial is any one of A1) to A12): A1) a nucleic acid molecule encoding the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody;

[0075] A2) an expression cassette containing the nucleic acid molecule of A1);

[0076] A3) a recombinant vector containing the nucleic acid molecule of A1);

[0077] A4) a recombinant vector containing the expression cassette of A2);

[0078] A5) a recombinant microorganism containing the nucleic acid molecule of A1);

[0079] A6) a recombinant microorganism containing the expression cassette of A2);

[0080] A7) a recombinant microorganism containing the recombinant vector in A3);

[0081] A8) a recombinant microorganism containing the recombinant vector of A4);

[0082] A9) a transgenic animal cell line containing the nucleic acid molecule of A1);

[0083] A10) a transgenic animal cell line containing the expression cassette of A2);

[0084] A11) a transgenic animal cell line containing the recombinant vector in A3);

[0085] A12) A transgenic animal cell line containing the recombinant vector of A4).

[0086] In a preferred embodiment, according to the above biological material, the nucleotide sequence of the nucleic acid molecule is selected from:

[0087] a) the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2;

[0088] b) a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions;

[0089] c) a nucleotide sequence having a degree of identity of at least about 95%, 96%, 97%, 98% or at least 99% to the nucleotide sequence shown in SEQ ID NO: 2;

[0090] d) a nucleotide sequence encoding an amino acid that is at least about 95%, 96%, 97%, 98% or at least 99% identical to the amino acid set forth in SEQ ID NO: 1;

[0091] e) a nucleotide sequence encoding an amino acid which differs from SEQ ID NO: 1 by no more than 5, 4, 3, 2 or 1 amino acid residues;

[0092] and / or

[0093] f) A nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO: 1, including substitution, deletion and / or insertion of 1 to 5 amino acid residues.

[0094] In the present invention, "about" refers to a value within the range of ±5% of a particular value. For example, "about 95%" includes ±5% of 95%, or from 90.25 to 99.75.

[0095] According to another aspect of the present invention, there is provided a method for producing a VHH chain of an anti-avian influenza virus HA1 protein nanobody or an anti-avian influenza virus HA1 protein nanobody, the method comprising the following steps:

[0096] (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody; and

[0097] (2) Cultivating the transgenic cell to obtain the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody.

[0098] In a preferred embodiment, the avian influenza virus is influenza A virus.

[0099] In a preferred embodiment, the avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

[0100] According to another aspect of the present invention, a method for screening VHH chains of anti-avian influenza virus HA1 protein nanoantibodies or anti-avian influenza virus HA1 protein nanoantibodies is provided, the method comprising the following steps:

[0101] (1) emulsifying a certain amount of the purified avian influenza virus with an equal volume of adjuvant, immunizing alpacas 4 to 6 times, then collecting peripheral blood from the alpacas, isolating peripheral blood lymphocytes, and constructing a phage display library against avian influenza virus; and

[0102] (2) Using phage display technology, after 3 to 5 rounds of screening, the VHH chain of the anti-avian influenza virus HA1 protein nanoantibody or the anti-avian influenza virus HA1 protein nanoantibody is screened and obtained.

[0103] In the present invention, when the number of times, rounds, or other values ​​or parameters are expressed as a range, a preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "4 to 6" is disclosed, the described range should be interpreted as including the range "4 to 6", "4 to 5", "5 to 6", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0104] In a preferred embodiment, the avian influenza virus is avian influenza virus type A and / or avian influenza virus type B avian influenza virus.

[0105] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the above-mentioned anti-avian influenza virus HA1 protein nanobody; and (b) a pharmaceutically acceptable carrier.

[0106] In the present invention, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., a substance that does not cause undesirable biological effects or interact in a deleterious manner with any of its components when administered to an individual.

[0107] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0108] According to another aspect of the present invention, a recombinant protein is provided, which comprises (a) the sequence of the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the sequence of the above-mentioned anti-avian influenza virus HA1 protein nanobody; and (b) a tag sequence that assists expression and / or purification.

[0109] In a preferred embodiment, the tag sequence comprises an Fc tag.

[0110] According to another aspect of the present invention, the VHH chain of the above-mentioned anti-avian influenza virus HA1 protein nanobody, or the use of the above-mentioned anti-avian influenza virus HA1 protein nanobody, is provided for preparing (a) a medicament, reagent, detection plate, test paper or kit for detecting anti-avian influenza virus HA1 protein molecules; or (b) for preparing a drug for preventing and / or treating avian influenza virus infection; or (c) for distinguishing influenza A virus, influenza B virus and / or influenza C virus;

[0111] In a preferred embodiment, the avian influenza virus is influenza A virus.

[0112] In a preferred embodiment, the avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

[0113] In a preferred embodiment, the prevention and / or treatment is manifested as increased body weight and / or increased survival rate.

[0114] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0115] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0116] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0117] Example

[0118] Example 1 Construction of anti-avian influenza virus HA protein nanobody phage display library

[0119] 1. Experimental methods

[0120] 1.1. Immunization of alpacas

[0121] After 50 μg of purified goose astrovirus P2 protein was mixed and emulsified with the same volume of Freund's adjuvant, the alpaca was immunized by multiple subcutaneous injections in the neck. For the first immunization, Freund's complete adjuvant was used; thereafter, Freund's incomplete adjuvant was used for emulsification every two weeks for a total of 5 immunizations. Blood was collected on the 7th day after the last immunization, and the antibody titer in the alpaca serum after 5 immunizations was detected by indirect ELISA (P2 protein as coating antigen, 100 ng / well). When the antibody titer against P2 protein in the serum was not less than 1:64000, the phage display antibody library was constructed.

[0122] 1.2. Construction and panning of VHH phage antibody library

[0123] 1.2.1. Isolation of peripheral blood lymphocytes

[0124] Human peripheral blood lymphocyte separation solution was used to separate alpaca peripheral blood lymphocytes (PBMCs). The specific operation was carried out according to the instructions of the kit. The separated PBMCs were used to extract total cellular RNA.

[0125] 1.2.2 Amplification of VHH gene fragments

[0126] The total RNA of PBMC was extracted using an RNA extraction kit (OMEGA, catalog number R6834-01), and reverse transcription was performed to obtain cDNA. The VHH gene was amplified using nested PCR to finally obtain a VHH fragment with a molecular weight of ∼400 bp. The target gene was recovered by gel excision.

[0127] 1.2.3. Construction of VHH phage display vector

[0128] The VHH gel recovery product and the linearized phagemid vector were connected by homologous recombination. The specific operation was carried out according to the instruction manual (Novozymes, catalog number C113-01).

[0129] 1.2.4. Preparation of SS320 electroporated competent cells

[0130] SS320 Escherichia coli (pre-infected with M13KO7 Helper Phage) was cultured to OD 600nm 0.4, cool rapidly, centrifuge at 4°C, 6000g for 5-10 min, and use pre-cooled ddH 2 After the last centrifugation, resuspend the SS320 competent cells in 10% glycerol water (sterile), 200 μl / tube, and store at -80°C for later use.

[0131] 1.2.5. Transformation of ligation products and harvesting of phage antibody library

[0132] Take out 1 tube of SS320 E. coli competent cells from -80℃ ultra-low temperature storage box, put them in ice bath for 5min to melt, gently add the ligation product to SS320 competent cells, mix well and add to the electroporation cup, use Eppendorf electroporator, set the parameters to 2.5kV, 25μF, 200Ω, 2mm, and perform electroporation transformation. Immediately resuspend the transformed competent cells and transfer them to 20ml of 37℃ preheated SOC medium, 37℃, 120r / min, shake and culture for 1h, take 100μl of the original solution (for backup), and transfer the rest of the culture to 500ml of 2×YT / Amp-Kan medium, and culture at 37℃ for 14-16h. After the culture, the supernatant was collected by centrifugation (10000g / min, 10min, 4°C), 1 / 4 volume of pre-cooled PEG / NaCl solution was added to the supernatant, mixed thoroughly, and allowed to stand at 4°C for 30-60min; finally, the mixture was centrifuged, the precipitate was suspended with 1ml of PBT, the concentration was determined, and the mixture was stored in aliquots at -80°C for later use.

[0133] 1.3. Screening of anti-HA1 protein nanoantibodies

[0134] 1.3.1. Anti-HA1 protein recombinant phage screening

[0135] ELISA plates were coated with HA1 protein, and a sterile PBST solution containing 2% BSA was used as a no-antigen control. Prepared phage solution was added to the ELISA plate, and the phage sample was discarded after incubation at room temperature for 2 hours. 100 μl of freshly prepared 0.1M HCl was added to each well, and the plate was gently shaken at room temperature for 10 minutes. The eluate was quickly neutralized with 1M Tris-HCl (pH 8.0). The eluate was added to 1ml of NEB5aF' bacteria in the logarithmic phase of growth, and the M13 helper phage was added after culturing at 37℃200r / min for 1h. The culture was continued at 37℃200r / min for 1h. Finally, the bacterial solution was transferred to 40ml 2×YT / Amp-Kan medium and cultured for 14-16h. Repeat the operation in 1.2.5 to collect the phage library; use the operation method in 1.1.1 to perform the second and third rounds of screening on the obtained phage library of the first round, and determine the phage enrichment by titration method in each round.

[0136] 1.3.2 Nanoantibody Screening

[0137] 90 single colonies were randomly picked from the enrichment plate, inoculated into a 96-well deep-well plate, with one single colony in each well, and added to 2×YT / Amp-Kan medium for overnight culture; the bacterial solution was centrifuged, and the phages in the supernatant were harvested. The nanoantibodies corresponding to the HA1 protein were screened using the Phage ELISA test method, and the suspected positive bacterial solution selected for identification was sequenced to obtain the nanoantibody sequence.

[0138] 1.3.3 Cloning and expression of nanobodies

[0139] The antibody gene was cloned into the pcDNA3.1-Fc (human) eukaryotic expression vector, the plasmid was extracted and transfected into 293F suspension cells, the supernatant was collected after 5 days, the target antibody was purified using Protein A, and the SDS-PAGE test was performed to analyze the results.

[0140] 1.4. Nanobody affinity detection

[0141] The antibody affinity was detected by SPR test. The CM5 chip was placed in the Biacore 3000 chip slot, and the program was run to activate the chip. The optimal pH value of the sodium acetate solution for protein-chip binding was determined. The A / Puerto Rico / 8 / 34(H1N1)HA1 protein was coupled to the CM5 chip and then blocked. The antibody was diluted to 100nM according to the pre-binding ratio, and diluted 2-fold, with a total of 9 gradients. Take out the microplate, and place the antibody, PBS, and 10mM glycine (pH 2.5) in the microplate according to the set position. Set up and run the program according to the Biacore 3000 operation manual to determine the affinity between the antibody and the HA1 protein.

[0142] 1.5. Nanobody specificity detection

[0143] The specificity of the obtained NP-Nb1 was analyzed by indirect immunofluorescence experiment. The specific steps are: MDCK cells were transfected with HA1-pCAGGS plasmids of different subtypes of avian influenza virus (H1N1, H3N2, H5N1, H5N6, H7N9), and 24 hours after transfection, the cells were washed twice with PBS; 4% paraformaldehyde was added to fix the cells for 15 minutes; PBS was washed three times, and then 2% BSA solution prepared by PBS was added, and the cells were blocked at 37°C for 30 minutes; after being fully washed with PBS buffer, 0.1% Triton X-100 was added to permeabilize the membrane for 15 minutes, and PBS buffer was fully washed; NP-Nb1 nanoantibody diluted with 1% BSA solution was added, incubated for 1 hour, washed, and then anti-Fc-FITC secondary antibody was added, incubated for 30 minutes, washed, DAPI was added, and the cells were detected and photographed with a laser confocal microscope.

[0144] 2. Experimental results

[0145] 2.1. Nanobody screening results

[0146] Using the Phage ELISA test, a nanobody nucleotide sequence (SEQ ID NO: 1) was successfully obtained and named Nb-17.

[0147] 2.2. Nanobody cloning and expression test results

[0148] Through sequencing, it was found that the nanobody gene was successfully cloned into the pcDNA 3.1-Fc eukaryotic expression vector. The constructed nanobody plasmid was transfected into 293F suspension cells, and the cell supernatant was collected after 5 days of culture, and the antibody was purified. The test results showed that the nanobody (such as Figure 1 shown).

[0149] 2.3. Nanobody affinity test results

[0150] The results of affinity test showed that Nb-17 had high affinity activity of 43.2nM (as measured by SPR test). Figure 2 shown).

[0151] 2.4. Results of indirect immunofluorescence test of nanobodies

[0152] An indirect immunofluorescence (IFA) test was performed 24 hours after transfection, and a blank cell control was set up. The test results showed that the screened Nb-17 nanoantibody could bind to the HA1 protein of different subtypes of avian influenza virus (such as Figure 3 This antibody has a broad spectrum.

[0153] Example 2 Preventive and therapeutic study of Nb-17 nanobody on influenza virus infection in C57 mice

[0154] 1. Experimental methods

[0155] 1.1. Test on the prevention of influenza virus infection in C57 mice by Nb-17 nanoantibody

[0156] In order to evaluate the protective effect of Nb-17 nanoantibody on preventing influenza virus infection in mice, 18 C57BL / 6J mice aged 6-8 weeks were selected and randomly divided into 3 groups, 6 mice in each group, namely negative control group (PBS), positive control group (PBS / PR8) and Nb-17 prevention group (200μg / 20g / mouse Nb17 / PR8). 24h after intraperitoneal injection of Nb-17 nanoantibody (200μg / 20g / mouse), the mice were nasally infected with A / Puerto Rico / 8 / 34 (H1N1) at an infection dose of 10 6 EID 50 / 30μL. Weigh the mice every day and record the weight changes and survival rate until day 14. Euthanize the mice when the weight loss is ≥25%.

[0157] 1.2. Therapeutic study of Nb-17 nanoantibody on influenza virus-infected C57 mice

[0158] To evaluate the protective effect of Nb-17 nanoantibody on the treatment of influenza virus infection in mice, 18 C57BL / 6J mice aged 6-8 weeks were selected and randomly divided into 3 groups, 6 mice in each group, namely negative control group (PBS), positive control group (PBS / PR8) and Nb-17 nanoantibody treatment group (500μg / 20g / mouse Nb-17 / PR8). Mice were nasally infected with A / Puerto Rico / 8 / 34(H1N1) at an infection dose of 10 6 EID 50 / 30μL, and Nb-17 nanoantibody (500μg / 20g / mouse) was injected intraperitoneally 24h after infection. The mice were weighed every day, and the weight changes and survival rates of the mice were recorded until day 14.

[0159] When mice lost ≥25% of their body weight,

[0160] 2. Experimental results

[0161] 2.1. Results of the Nb-17 nanoantibody prevention test on influenza virus infection in C57 mice

[0162] The day before C57 mice were infected with the virus, mice were injected with Nb-17 nanoantibodies by intraperitoneal injection. The next day, mice were infected with A / Puerto Rico / 8 / 34(H1N1), which played a preventive role. The weight of mice in the Nb-17 prevention group gradually increased (e.g. Figure 4 The mice in the control group lost more than 25% of their body weight one week after infection, and all of them died (as shown in Figure 5 shown).

[0163] 2.2. Results of the therapeutic experiment of Nb-17 nanoantibody on influenza virus-infected C57 mice

[0164] After C57 mice were infected with A / Puerto Rico / 8 / 34(H1N1), Nb-17 exerted a protective effect by intraperitoneal injection. The body weight of mice in the Nb-17 treatment group first decreased and then gradually recovered (e.g. Figure 6 The mice in the control group lost more than 25% of their body weight one week after infection, and all of them died (as shown in Figure 7 shown).

[0165] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A VHH chain of an anti-avian influenza virus HA1 protein nanobody, characterized in that: The amino acid sequence of the VHH chain is shown in SEQ ID NO:

1.

2. A nanobody against HA1 protein of avian influenza virus, characterized in that: It is a nano antibody targeting the antigenic epitope of HA1 protein of avian influenza virus and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:

1.

3. A biomaterial related to the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1, or the anti-avian influenza virus HA1 protein nanobody according to claim 2, characterized in that: The biological material is any one of A1) to A12): A1) a nucleic acid molecule encoding the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1, or the anti-avian influenza virus HA1 protein nanobody according to claim 2; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) a recombinant microorganism containing the recombinant vector described in A4); A9) a transgenic animal cell line containing the nucleic acid molecule described in A1); A10) a transgenic animal cell line containing the expression cassette described in A2); A11) a transgenic animal cell line containing the recombinant vector described in A3); A12) A transgenic animal cell line containing the recombinant vector described in A4).

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

2.

5. A method for producing a VHH chain of an anti-avian influenza virus HA1 protein nanobody or an anti-avian influenza virus HA1 protein nanobody, characterized in that: The method comprises the following steps: (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1 or the anti-avian influenza virus HA1 protein nanobody according to claim 2 into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody; and (2) Cultivating the transgenic cells to obtain the VHH chain of the anti-avian influenza virus HA1 protein nanobody or the anti-avian influenza virus HA1 protein nanobody.

6. The method according to claim 5, characterized in that The avian influenza virus is type A influenza virus.

7. The method according to claim 5, characterized in that The avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (a) the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1, or the anti-avian influenza virus HA1 protein nanobody according to claim 2; and (b) a pharmaceutically acceptable carrier.

9. A recombinant protein, characterized in that The recombinant protein comprises (a) the sequence of the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1, or the sequence of the anti-avian influenza virus HA1 protein nanobody according to claim 2; and (b) a tag sequence that assists expression and / or purification.

10. The recombinant protein according to claim 9, characterized in that The tag sequence includes an Fc tag.

11. The use of the VHH chain of the anti-avian influenza virus HA1 protein nanobody according to claim 1 or the anti-avian influenza virus HA1 protein nanobody according to claim 2, characterized in that: Used to prepare (a) a medicament, reagent, test plate, test paper or kit for detecting anti-avian influenza virus HA1 protein molecules; or (b) a drug for preventing and / or treating avian influenza virus infection.

12. The use according to claim 11, characterized in that The avian influenza virus is type A influenza virus.

13. The use according to claim 11, characterized in that The avian influenza virus is an H1N1 subtype avian influenza virus, an H3N2 subtype avian influenza virus, an H5N1 subtype avian influenza virus, an H5N6 subtype avian influenza virus and / or an H7N9 subtype avian influenza virus.

14. The use according to claim 11, characterized in that The prevention and / or treatment is manifested as increased body weight and / or increased survival rate.

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