Nano antibody for resisting goose astrovirus P2 protein as well as preparation method and application of nano antibody

Abstract

The invention discloses a VHH chain of an anti-goose astrovirus P2 protein nano antibody. The amino acid sequence of the VHH chain is as shown in SEQ ID NO: 1. Goose astrovirus is used as a research object, the goose astrovirus is inactivated to immunize alpaca, and a bacteriophage display technology is used for screening to obtain a broad-spectrum nano antibody for resisting goose astrovirus P2 protein. Through the preparation of the nano antibody for resisting the goose astrovirus P2 protein, a material is provided for establishing a broad-spectrum diagnosis method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nano antibody against goose astrovirus P2 protein 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] Goose Astrovirus (GAstV) is one of the important viral infectious pathogens that endanger the goose farming industry. When goslings under 3 weeks old are infected with GAstV, the main clinical symptoms include joint swelling, diarrhea, developmental delay, etc.; autopsy shows that the heart and liver are covered with a layer of lime-like white substance, the kidneys are enlarged, and urate deposits are deposited in the ureters; in severe cases, urate deposits are found in the muscles and joints, and the mortality rate is over 50%. The viral capsid protein ORF2 encoded by the GAstV ORF2 gene is the main immunogenic protein. The ORF2 protein contains two proteins, P1 and P2. The P2 protein can stimulate the body to produce an immune response and induce the body to produce protective antibodies. At present, there is a lack of efficient and rapid detection technology for GAstV. Summary of the invention

[0004] Based on this, the present invention provides a VHH chain of an anti-goose astrovirus P2 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-goose astrovirus P2 protein nanobody is provided, which is a nanobody against the goose astrovirus P2 protein antigenic epitope and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:1.

[0006] Furthermore, 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 set forth 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 VHH chain of the above-mentioned anti-goose astrovirus P2 protein nanobody, or a biomaterial related to the above-mentioned anti-goose astrovirus P2 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-goose astrovirus P2 protein nanobody, or the above-mentioned anti-goose astrovirus P2 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] Furthermore, 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-goose astrovirus P2 protein nanobody or an anti-goose astrovirus P2 protein nanobody, the method comprising the following steps:

[0033] (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody; and

[0034] (2) Cultivating the transgenic cells to obtain the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody.

[0035] Furthermore, the goose astrovirus is goose astrovirus type 1 and / or goose astrovirus type 2 goose astrovirus.

[0036] According to another aspect of the present invention, a method for screening VHH chains of anti-goose astrovirus P2 protein nanoantibodies or anti-goose astrovirus P2 protein nanoantibodies is provided, the method comprising the following steps:

[0037] (1) emulsifying a certain amount of the purified goose astrovirus 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 goose astrovirus; and

[0038] (2) Using phage display technology, after 3 to 5 rounds of screening, the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody is screened and obtained.

[0039] Furthermore, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant.

[0040] Furthermore, the immunization is performed by multiple subcutaneous injections in the neck.

[0041] Furthermore, the immunization process includes first immunizing with Freund's complete adjuvant and then immunizing with Freund's incomplete adjuvant.

[0042] Furthermore, the incomplete Freund's adjuvant is used once every two weeks.

[0043] Furthermore, the immunization is performed multiple times, for example, 5 times.

[0044] Furthermore, the time for collecting peripheral blood of the alpaca is 6 to 8 days after the last immunization, for example, the 7th day.

[0045] Furthermore, the time point for constructing the anti-goose astrovirus phage display library is when the antibody titer in the alpaca serum after immunization is detected by indirect ELISA, and the anti-P2 protein antibody titer in the alpaca serum is not less than 1:64000.

[0046] Furthermore, the goose astrovirus is goose astrovirus type 1 and / or goose astrovirus type 2 goose astrovirus.

[0047] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the VHH chain of the above-mentioned anti-goose Astrovirus P2 protein Nanobody, or the above-mentioned anti-goose Astrovirus P2 protein Nanobody; and (b) a pharmaceutically acceptable carrier.

[0048] 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-goose astrovirus P2 protein nanobody, or the sequence of the above-mentioned anti-goose astrovirus P2 protein nanobody; and (b) a tag sequence that assists expression and / or purification.

[0049] Furthermore, the tag sequence includes a 6His tag and / or an HA tag.

[0050] According to another aspect of the present invention, there is provided a use of the VHH chain of the above-mentioned anti-goose astrovirus P2 protein nanobody, or the above-mentioned anti-goose astrovirus P2 protein nanobody, for preparing (a) a medicament, reagent, detection plate, test paper or kit for detecting anti-goose astrovirus P2 protein molecules; or (b) for preparing a drug for preventing and / or treating goose astrovirus infection; or (c) for distinguishing goose astrovirus, Newcastle disease virus and avian influenza virus;

[0051] Furthermore, the goose astrovirus is goose astrovirus type 1 and / or goose astrovirus type 2 goose astrovirus.

[0052] Furthermore, the test paper is colloidal gold test paper.

[0053] Furthermore, in the detection line of the colloidal gold test paper, the dilution gradient of the anti-goose astrovirus P2 protein nanobody and phosphate buffer is 1:0.5 to 1:4, for example, 1:about 1.

[0054] Furthermore, the color development time of the colloidal gold test paper is 1 to 15 minutes, further 5 to 15 minutes, for example, about 10 minutes.

[0055] Furthermore, the minimum virus detection limit of the colloidal gold test paper is 10TCID 50 / ml.

[0056] Beneficial effects of the present invention:

[0057] The present invention uses goose astrovirus as the research object, inactivates it and immunizes alpacas, and uses phage display technology to screen and obtain a broad-spectrum anti-goose astrovirus P2 protein nanoantibody. The present invention provides materials for establishing a broad-spectrum diagnostic method through the preparation of the anti-goose astrovirus P2 protein nanoantibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] 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.

[0059] Figure 1 Schematic diagram of the first round of PCR amplification results.

[0060] Figure 2 This is a schematic diagram of the second round of PCR amplification results.

[0061] Figure 3 Schematic diagram of antibody sequence diversity comparison results.

[0062] Figure 4 Schematic diagram of antibody purification results.

[0063] Figure 5 Schematic diagram of Nb-1 affinity test results.

[0064] Figure 6 Schematic diagram of the results of the indirect immunofluorescence test of Nb-1 nanoantibody.

[0065] Figure 7 Schematic diagram of the results of determining the optimal concentration of the detection line.

[0066] Figure 8 This is a schematic diagram of the results of colloidal gold test strips detecting different pathogens.

[0067] Fig. 9 This is a schematic diagram of the results of determining the optimal color development time of the colloidal gold test strip.

[0068] Fig.10 This is a schematic diagram of the sensitivity test results of the colloidal gold test strip.

[0069] Fig.11 This is a schematic diagram of the repeatability test results of colloidal gold test strips. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] As described in the background technology section, the prior art lacks efficient and rapid detection technology for GAstV. In order to solve the above problems, the present invention provides a VHH chain of an anti-goose astrovirus P2 protein nanobody, the amino acid sequence of which is shown in SEQ ID NO:1.

[0078] The present invention immunizes alpacas with the main immunogenic protein P2 protein, uses phage display technology to screen nano antibodies with good specificity and high affinity, establishes a colloidal gold detection method, and provides a simple, fast and accurate detection product for clinical pathogen detection.

[0079] According to another aspect of the present invention, an anti-goose astrovirus P2 protein nanobody is provided, which is a nanobody against the goose astrovirus P2 protein antigenic epitope and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:1.

[0080] In a preferred embodiment, the amino acid sequence is replaced by an amino acid sequence selected from the group consisting of:

[0081] 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;

[0082] b) an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 1;

[0083] 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;

[0084] and / or

[0085] 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.

[0086] 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.

[0087] According to another aspect of the present invention, a VHH chain of the above-mentioned anti-goose astrovirus P2 protein nanobody, or a biomaterial related to the above-mentioned anti-goose astrovirus P2 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-goose astrovirus P2 protein nanobody, or the above-mentioned anti-goose astrovirus P2 protein nanobody;

[0088] A2) an expression cassette containing the nucleic acid molecule of A1);

[0089] A3) a recombinant vector containing the nucleic acid molecule of A1);

[0090] A4) a recombinant vector containing the expression cassette of A2);

[0091] A5) a recombinant microorganism containing the nucleic acid molecule of A1);

[0092] A6) a recombinant microorganism containing the expression cassette of A2);

[0093] A7) a recombinant microorganism containing the recombinant vector in A3);

[0094] A8) a recombinant microorganism containing the recombinant vector of A4);

[0095] A9) a transgenic animal cell line containing the nucleic acid molecule of A1);

[0096] A10) a transgenic animal cell line containing the expression cassette of A2);

[0097] A11) a transgenic animal cell line containing the recombinant vector in A3);

[0098] A12) A transgenic animal cell line containing the recombinant vector of A4).

[0099] In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is selected from:

[0100] a) the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2;

[0101] b) a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions;

[0102] 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;

[0103] 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;

[0104] 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;

[0105] and / or

[0106] 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.

[0107] 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.

[0108] According to another aspect of the present invention, there is provided a method for producing a VHH chain of an anti-goose astrovirus P2 protein nanobody or an anti-goose astrovirus P2 protein nanobody, the method comprising the following steps:

[0109] (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody; and

[0110] (2) Cultivating the transgenic cells to obtain the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody.

[0111] In a preferred embodiment, the goose Astrovirus is goose Astrovirus type 1 and / or goose Astrovirus type 2 goose Astrovirus.

[0112] According to another aspect of the present invention, a method for screening VHH chains of anti-goose astrovirus P2 protein nanoantibodies or anti-goose astrovirus P2 protein nanoantibodies is provided, the method comprising the following steps:

[0113] (1) emulsifying a certain amount of the purified goose astrovirus 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 goose astrovirus; and

[0114] (2) Using phage display technology, after 3 to 5 rounds of screening, the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody is screened and obtained.

[0115] In the present invention, when the number of times, rounds, days, dilution gradients, time, or other values ​​or parameters are expressed as ranges, preferred ranges, or 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.

[0116] In a preferred embodiment, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant.

[0117] In a preferred embodiment, the immunization is performed by multiple subcutaneous injections in the neck.

[0118] In a preferred embodiment, the immunization process includes first immunizing with Freund's complete adjuvant and then immunizing with Freund's incomplete adjuvant.

[0119] In a preferred embodiment, the incomplete Freund's adjuvant is used once every two weeks.

[0120] In a preferred embodiment, the immunization is performed multiple times, for example, 5 times.

[0121] In a preferred embodiment, the time for collecting peripheral blood of the alpaca is 6 to 8 days after the last immunization, for example, the 7th day.

[0122] In a preferred embodiment, the time point for constructing the anti-goose astrovirus phage display library is when the antibody titer in the alpaca serum after immunization is detected by indirect ELISA, and the anti-P2 protein antibody titer in the alpaca serum is not less than 1:64000.

[0123] In a preferred embodiment, the goose Astrovirus is goose Astrovirus type 1 and / or goose Astrovirus type 2 goose Astrovirus.

[0124] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the VHH chain of the above-mentioned anti-goose Astrovirus P2 protein Nanobody, or the above-mentioned anti-goose Astrovirus P2 protein Nanobody; and (b) a pharmaceutically acceptable carrier.

[0125] 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.

[0126] 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).

[0127] 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-goose astrovirus P2 protein nanobody, or the sequence of the above-mentioned anti-goose astrovirus P2 protein nanobody; and (b) a tag sequence that assists expression and / or purification.

[0128] In a preferred embodiment, the tag sequence includes a 6His tag and / or an HA tag.

[0129] According to another aspect of the present invention, there is provided a use of the VHH chain of the above-mentioned anti-goose astrovirus P2 protein nanobody, or the above-mentioned anti-goose astrovirus P2 protein nanobody, for preparing (a) a medicament, reagent, detection plate, test paper or kit for detecting anti-goose astrovirus P2 protein molecules; or (b) for preparing a drug for preventing and / or treating goose astrovirus infection; or (c) for distinguishing goose astrovirus, Newcastle disease virus and avian influenza virus;

[0130] In a preferred embodiment, the goose Astrovirus is goose Astrovirus type 1 and / or goose Astrovirus type 2 goose Astrovirus.

[0131] In a preferred embodiment, the test paper is colloidal gold test paper.

[0132] In a preferred embodiment, in the detection line of the colloidal gold test paper, the dilution gradient of the anti-goose astrovirus P2 protein nanobody and phosphate buffer is 1:0.5 to 1:4, for example, 1:about 1.

[0133] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0134] In a preferred embodiment, the color development time of the colloidal gold test paper is 1 to 15 minutes, further 5 to 15 minutes, for example, about 10 minutes.

[0135] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0136] In a preferred embodiment, the minimum virus detection limit of the colloidal gold test paper is 10TCID 50 / ml.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] Example

[0141] Example 1 Construction of anti-goose astrovirus P2 protein nanobody phage display library

[0142] 1. Experimental methods

[0143] 1.1. Immunization of alpacas

[0144] 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.

[0145] 1.2. Construction and panning of VHH phage antibody library

[0146] 1.2.1. Isolation of peripheral blood lymphocytes

[0147] 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.

[0148] 1.2.2 Amplification of VHH gene fragments

[0149] 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 ultimately obtain a VHH fragment with a molecular weight of ~400 bp. The target gene was then recovered by gel excision.

[0150] 1.2.3. Construction of VHH phage display vector

[0151] 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).

[0152] 1.2.4. Preparation of SS320 electroporated competent cells

[0153] SS320 Escherichia coli (pre-infected with M13KO7 Helper Phage) was cultured to OD 600nm 0.4, rapidly cooled, centrifuged at 4°C, 6000g for 5-10 min, washed repeatedly with pre-cooled ddH2O (sterile) 3-5 times, resuspended SS320 competent cells with 10% glycerol water (sterile) after the last centrifugation, 200 μl / tube, and stored at -80°C for later use.

[0154] 1.2.5. Transformation of ligation products and harvesting of phage antibody library

[0155] 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.

[0156] 1.2.6 Determination of phage display library capacity and diversity

[0157] The recombinant bacteria after electroporation (the above 100 μl stock solution) were diluted 10-fold in a gradient manner, and 10 -3 -10 -5 10 μl of the dilution solution was applied to LB / Amp-Kan plates and cultured at 37°C for 12 h. The transformation efficiency was calculated and the final storage capacity was 1×10 9 CFU (colony-forming units) phage library. 24 monoclonal bacteria were randomly selected and identified by PCR using VHH-F and VHH-R primers. The target fragment size was about 400 bp. The positive clones were sequenced to determine the diversity of the nanobody.

[0158] 1.3. Screening of anti-P2 protein specific nanoantibodies

[0159] 1.3.1. Selection of anti-P2 protein specific recombinant phage

[0160] The purified P2 protein was coated on the ELISA plate, and the uncoated protein was used as the negative control group, and the plate was incubated at 4°C overnight. The next day, the coating solution was discarded, and the plate was blocked with a sterile PBST solution containing 2% BSA at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed 3-5 times with a sterile PBST solution. The prepared phage solution was added to the ELISA plate and incubated at room temperature for 2 hours. The phage solution was discarded, and the plate was washed 3-5 times with a sterile PBST solution. The plate was patted dry for the last time, and 100 μl of 0.1M HCl was added to each well. The plate was gently shaken at room temperature for 10 minutes. The eluate was neutralized with 1M Tris-HCl (pH 8.0) and added to 1ml of NEB5aF' bacteria in the logarithmic phase. The plate was cultured at 37°C and 200 r / min for 1 hour. M13 helper phage was added, and the plate was cultured at 37°C and 200 r / min for 1 hour. Finally, the liquid was transferred to 40ml of 2×YT / Amp-Kan medium. Repeat the above phage collection process to collect the first round of phage library, and continue to use the 1.3.1 process for the second and third rounds of screening. In each round, the phage enrichment is determined by titration.

[0161] 1.3.2. Screening of Nanobodies

[0162] 90 single colonies were randomly picked from the plate with obvious enrichment, 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; phages in the supernatant were harvested by centrifugation, and the nano-antibodies corresponding to the P2 protein were screened by ELISA, and the selected suspected positive bacterial liquid was identified by Phage ELISA test, and the nano-antibody sequence was obtained by sequencing. The specific operation is as follows:

[0163] (1) Coating: P2 protein was coated on ELISA plate at 100 ng / well at 4°C overnight;

[0164] (2) Blocking: The ELISA plate was turned upside down to remove the supernatant, and 200 μl of sterile PBST solution containing 2% BSA was added to each well, including the negative control group, and blocked at 37°C for 1 h;

[0165] (3) Collect the supernatant: centrifuge the 96-well plate at 4000 rpm / min and 4°C for 10 min;

[0166] (3) After incubation for 1.5 h, discard the supernatant in the ELISA plate, wash three times with PBST and spin dry, add 40 μl of phage (i.e., the supernatant after centrifugation of the 96-deep-well plate), and gently shake at room temperature for 2 h;

[0167] (4) Washing: discard the phages in the ELISA plate, wash with PBST three times and then spin dry, wash with PBST 4-6 times, add 100 μl of HRP-M13 antibody, and incubate at 37°C for 1 h;

[0168] (5) Color development: discard the HRP-M13 antibody in the ELISA plate, wash it three times with PBST and spin dry, add 100 μl of TMB color development solution, leave it at room temperature for 10-15 min, and observe the color change;

[0169] (6) Termination: Add 100 μl of sulfuric acid stop solution to each well to terminate the reaction;

[0170] (7) Reading: Measure the OD of each well at a wavelength of 450 nm using an ELISA reader. 450 Select 10-15 positive clones with higher absorbance values ​​for sequencing verification.

[0171] 1.3.3 Cloning and expression of nanobodies

[0172] The screened nanobody 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 purification medium, and the SDS-PAGE test was verified and the results were analyzed.

[0173] 2. Test results

[0174] 2.1. First round of PCR amplification

[0175] The extracted RNA was transcribed using oligo dT + random primers and reverse primers for the first round of amplification. 2 μl was used as a template to amplify the target gene. The target band of about 500 bp was recovered by gel excision (such as Figure 1 shown).

[0176] 2.2. Second round of PCR amplification

[0177] The product recovered from the first round of PCR gel was used for the second round of PCR amplification, and finally the target gene with a size of about 450 bp was obtained (such as Figure 2 shown).

[0178] 2.3 Phage library construction results

[0179] The phage display library was successfully established with a capacity of 1×10 9 CFU / ml.

[0180] 2.4. Positive clone colony PCR and antibody sequence diversity comparison

[0181] The results of agarose gel electrophoresis of colony PCR showed that all 24 randomly selected monoclones were positive clones, with a positive rate of 100%, and the positive clone rate of the antibody library met the requirements. The 24 randomly selected monoclones were sequenced and transcribed and translated into protein sequences using GENtle software. The comparison showed that all 24 sequences were independent sequences with a diversity of 100% (such as Figure 3 The diversity of the antibody library meets the requirements.

[0182] 2.5. Nanoantibody Screening Results

[0183] Using the Phage ELISA test, a nanobody nucleotide sequence (SEQ ID NO: 1) was successfully obtained and named Nb-1.

[0184] 2.6. Nanobody cloning and expression test results

[0185] 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 4 shown).

[0186] Example 2 Characterization of Anti-Goose Astrovirus P2 Protein Nanobodies

[0187] 1. Experimental methods

[0188] 1.1. Nanobody affinity detection

[0189] Use SPR test to detect antibody affinity. Place the CM5 chip in the Biacore 3000 chip slot, run the program to activate the chip, determine the optimal pH value of sodium acetate solution for protein-chip binding, and block the P2 protein after coupling it to the CM5 chip. Dilute the antibody to 100nM according to the pre-binding ratio, dilute it 2-fold, and dilute it in 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 P2 protein.

[0190] 1.2. Indirect immunofluorescence test of Nb-1 nanobody

[0191] LMH cells with good growth status were plated into 12-well cell plates containing slides. When the cell density grew to 30%, the cell plates were taken out from the 37°C incubator, the culture medium was discarded, and the plates were gently washed 2-3 times with 1× PBS. Then, diluted GAstV (200 TCID 50), and set a negative control. After culturing in a 37°C cell culture incubator for 24 hours, the IFA test was performed. The specific operations were as follows: remove the cell plate, wash the cells twice with PBS; add 4% paraformaldehyde to fix the cells for 15 minutes; wash the cells three times with PBS, add 2% BSA solution prepared by PBS, and block at 37°C for 30 minutes; after fully washing with PBS buffer, add 0.1% Triton X-100 to permeabilize the membrane for 15 minutes, and fully wash with PBS buffer; add NP-Nb1 nanoantibody diluted with 1% BSA solution, incubate for 1 hour, wash, then add anti-Fc-FITC secondary antibody, incubate for 30 minutes, wash, add DAPI, detect with a laser confocal microscope and take pictures.

[0192] 2. Experimental results

[0193] 2.1. Results of the affinity test of nanobodies

[0194] The results of affinity test showed that Nb-1 had high affinity activity of 84fM (e.g. Figure 5 shown).

[0195] 2.2 Results of indirect immunofluorescence test of Nb-1 nanobody

[0196] An indirect immunofluorescence (IFA) test was performed 24 h after infection, and a blank cell control was set up. The test results showed that the screened Nb-1 bound to GAstV and had no reaction with the control group cells (such as Figure 6 shown).

[0197] Implementation case 3: Establishment of colloidal gold detection method

[0198] 1. Experimental methods

[0199] 1.1. Preparation of gold-labeled antibodies

[0200] Take 1mL of colloidal gold solution (stored in a refrigerator at 4 degrees) and place it in a beaker, and add 15μL of 0.2M K2CO3 solution to adjust the pH value of the gold solution. Stir for 15s, add Nb-1 nanoantibody, continue stirring for 15min, add 100μL of 10% BSA blocking solution, with a final BSA concentration of 1%, and stir at room temperature for 10min.

[0201] 1.2. Processing of gold label pad

[0202] Cut the glass fiber membrane to an appropriate size, use a pipette to absorb the gold label pad treatment solution and evenly apply it on the gold label pad, soak it at room temperature for 5 minutes, dry it in a constant temperature oven at 37 degrees, and store it at 4 degrees for later use.

[0203] 1.3. Assembly of colloidal gold test strips

[0204] Take a gold-labeled pad, cut a 4mm wide opening horizontally with scissors, use a suction gun to suck 5 microliters of gold-labeled antibody and drop it on the opening of the gold-labeled pad, repeat labeling multiple times, and put it in a 37-degree oven to dry. Store it in a dry and sealed state.

[0205] Take a NC membrane, draw Nb-1 nanoantibody and PB solution evenly on the NC membrane as the detection line (T line); HRP-labeled Fc secondary antibody and PB mixed solution (1:4) are evenly drawn on the NC membrane as the quality control line (C line). The prepared NC membrane is placed in a 37-degree constant temperature incubator for drying.

[0206] After the gold label pad and NC membrane are dried, take the PVC plate, paste the NC membrane on the PVC plate, and then place the gold label pad of the same size on one end of the colloidal gold, covering 3mm on the NC membrane. At the same time, take the absorbent paper of the same size, place it on the other end of the NC membrane, and also cover about 3mm of the NC membrane. Finally, cover most of the gold label pad with the sample plate, and finally use a strip cutter to cut out 3.98mm wide colloidal gold test strips. Pack in sealed bags and store in a dry and dark place.

[0207] 1.4. Colloidal gold test strip detection and judgment standards

[0208] Mix 20 μL of the sample to be tested and 20 μL of the sample diluent, then drop them into the sample well of the test strip. Let it stand for 5-15 minutes and observe the results. The judgment criteria are:

[0209] If both the C line and the T line are colored, it means that the sample is positive; and observe when the color is brightest.

[0210] If the C line shows color and the T line does not show color, it means that the sample is negative;

[0211] If the C line does not show color, regardless of whether the T line shows color, it means that the test strip has expired and needs to be retested.

[0212] 1.5. Determination of the best color development time

[0213] Take a colloidal gold test strip, place it flat on the table, put a timer next to it, add 20 μl of virus solution (mixed with an equal volume of sample diluent) into the sample well, start timing, and observe the color development of the colloidal gold test strip at the 5th and 10th minutes respectively.

[0214] 1.6. Colloidal gold test strip sensitivity test

[0215] Using the above experimental conditions, test strips were prepared to detect GAstV. The specific experimental operation was as follows: 10 4 TCID 50 The sensitivity of the method was determined by 10-fold serial dilution of GAstV / ml.

[0216] 1.7. Repeatability test of colloidal gold test strips

[0217] Three batches of colloidal gold test strips were prepared and used to test virus samples from the same batch to determine the repeatability of the test strips.

[0218] 2. Experimental results

[0219] 2.1. Determination of the optimal concentration of the detection line (T line)

[0220] The quality control line (C line) was drawn by mixing Fc secondary antibody and PB in a ratio of 1:4 (volume ratio). The detection line (T line) was drawn by diluting Nb-1 nanoantibody (1 mg / ml) and PB in a ratio of 1:1, 1:2 and 1:4, and observing the color of the T line. When the dilution gradient of the T line antibody and PB was 1:1, the color was the best and the color was the darkest (such as Figure 7 shown).

[0221] 2.2. Detection of goose astrovirus using colloidal gold test strips

[0222] In order to verify whether the colloidal gold test strip can specifically detect geese, three pathogens, goose astrovirus (GAstV), Newcastle disease virus (NDV) and avian influenza virus (AIV), were tested respectively. The study showed that the colloidal gold test strip had no binding reaction with NDV and AIV (such as Figure 8 shown).

[0223] 2.3. Determination of color development time

[0224] Take a colloidal gold test strip, place it flat on the table, put a timer next to it, add 20 μl of virus solution (mixed with an equal volume of sample diluent) into the sample well, start timing, and observe the color development of the colloidal gold test strip at the 5th and 10th minutes respectively.

[0225] The test results show that the target band can be seen in the colloidal gold test strip at the 5th and 10th minutes, but the color at the 10th minute is darker than that at the 5th minute. The color development time is determined to be 10 minutes (such as Fig. 9 shown).

[0226] 2.4. Colloidal gold test strip sensitivity test

[0227] GAstV(10 4 TCID 50 / ml), 10 times dilution, a total of 4 dilutions, colloidal gold test strip detection, the test results show that the minimum virus detection of the colloidal gold test strip is 10TCID 50 / ml(such as Fig.10 shown).

[0228] 2.5. Repeatability test of colloidal gold test strips

[0229] In order to verify the repeatability of colloidal gold test strips, three batches of colloidal gold test strips prepared at different times were used to detect GAstV. The results showed that the three batches of colloidal gold test strips could detect GAstV with good repeatability (e.g. Fig.11 shown).

[0230] 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-goose astrovirus P2 protein nanobody, characterized in that: The amino acid sequence of the VHH chain is shown in SEQ ID NO:

1.

2. An anti-goose astrovirus P2 protein nanobody, characterized in that: It is a nanobody targeting the antigenic epitope of goose astrovirus P2 protein and has a VHH chain with an amino acid sequence as shown in SEQ ID NO:

1.

3. The VHH chain of the anti-goose astrovirus P2 protein nanobody according to claim 1 or the anti-goose astrovirus P2 protein nanobody according to claim 2, characterized in that: The amino acid sequence is replaced by an amino acid sequence selected from the following: a) the amino acid sequence has a degree of identity of at least about 95%, 96%, 97%, 98% or at least 99% with the amino acid sequence of SEQ ID NO: 1; 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; c) the amino acid sequence differs from the amino acid sequence shown in SEQ ID NO: 1 by no more than 5, 4, 3, 2 or no more than 1 amino acid; and / or 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.

4. A biomaterial related to the VHH chain of the anti-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3, 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-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3; 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).

5. The biomaterial according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is selected from: a) the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2; b) a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions; 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; 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; 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; and / or 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.

6. A method for producing a VHH chain of an anti-goose astrovirus P2 protein nanobody or an anti-goose astrovirus P2 protein nanobody, characterized in that: The method comprises the following steps: (1) introducing a nucleic acid molecule encoding the VHH chain of the anti-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3 into a recipient cell to obtain a transgenic cell expressing the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody; and (2) culturing the transgenic cells to obtain the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody; Preferably, the goose Astrovirus is goose Astrovirus type 1 and / or goose Astrovirus type 2 goose Astrovirus.

7. A method for screening VHH chains of anti-goose astrovirus P2 protein nanoantibodies or anti-goose astrovirus P2 protein nanoantibodies, characterized in that: The method comprises the following steps: (1) emulsifying a certain amount of the purified goose astrovirus with an equal volume of adjuvant, immunizing alpacas 4 to 6 times, then collecting peripheral blood from the alpacas, separating peripheral blood lymphocytes, and constructing a phage display library against goose astrovirus; and (2) using phage display technology, after 3 to 5 rounds of screening, to screen and obtain the VHH chain of the anti-goose astrovirus P2 protein nanobody or the anti-goose astrovirus P2 protein nanobody; Preferably, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; Preferably, the immunization is performed by multiple subcutaneous injections in the neck; Preferably, the immunization process comprises first immunizing with Freund's complete adjuvant and then immunizing with Freund's incomplete adjuvant; Preferably, the frequency of use of the Freund's incomplete adjuvant is once every two weeks; Preferably, the immunization is performed multiple times, for example, 5 times; Preferably, the time for collecting peripheral blood of alpacas is 6 to 8 days after the last immunization, for example, the 7th day; Preferably, the time point for constructing the anti-goose astrovirus phage display library is when the antibody titer in the alpaca serum after immunization is detected by indirect ELISA, and the antibody titer against P2 protein in the alpaca serum is not less than 1:64000; More preferably, the goose Astrovirus is goose Astrovirus type 1 and / or goose Astrovirus type 2 goose Astrovirus.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (a) the VHH chain of the anti-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3; 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-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the sequence of the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3; and (b) a tag sequence that assists expression and / or purification; Preferably, the tag sequence includes a 6His tag and / or an HA tag.

10. Use of the VHH chain of the anti-goose astrovirus P2 protein nanobody according to claim 1 or 3, or the anti-goose astrovirus P2 protein nanobody according to claim 2 or 3, characterized in that: For use in preparing (a) a medicament, reagent, test plate, test paper or kit for detecting anti-goose astrovirus P2 protein molecules; or (b) for preparing a drug for preventing and / or treating goose astrovirus infection; or (c) for distinguishing goose astrovirus, Newcastle disease virus and avian influenza virus; Preferably, the goose astrovirus is goose astrovirus type 1 and / or goose astrovirus type 2 goose astrovirus; Preferably, the test paper is colloidal gold test paper; More preferably, in the detection line of the colloidal gold test paper, the dilution gradient of the anti-goose astrovirus P2 protein nanobody and phosphate buffer is 1:0.5 to 1:4, for example, 1: about 1; More preferably, the color development time of the colloidal gold test paper is 1 to 15 minutes, preferably 5 to 15 minutes, for example about 10 minutes; More preferably, the minimum virus detection limit of the colloidal gold test paper is 10TCID 50 / ml.

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