A group of shark nanoantibodies targeting shrimp infectious hypodermal and hematopoietic necrosis viruses and preparation methods thereof

Abstract

The present invention discloses a group of shark nano antibodies targeting penaeid shrimp infectious subcutaneous and hematopoietic necrosis virus and a preparation method thereof. The amino acid sequence of the nano antibody is selected from any one of SEQ ID NO.8~14 amino acid sequences, which can be used for the detection of cp, nsp1, and nsp2 antigen proteins of infectious subcutaneous and hematopoietic necrosis virus. The preparation or screening method of the nano antibody comprises the following steps: A: expression and purification of cp, nsp1, and nsp2 antigen proteins; B: construction of shark natural library and / or shark immune library; C: auxiliary phage rescue and purification to obtain phage library; D: panning of anti-cp, anti-nsp1, and anti-nsp2 antigen protein phage library; E: phage-ELISA identification and sequencing, sequence alignment of cp, nsp1, and nsp2 nano antibodies with different CDR3 region sequences. The nano antibody provided by the present invention can specifically identify infectious subcutaneous and hematopoietic necrosis virus, can detect whether aquaculture animals, aquaculture water bodies, and aquaculture feed are contaminated by viruses, etc., and realize rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a group of shark nano antibodies targeting shrimp infectious subcutaneous and hematopoietic necrosis viruses and a preparation method thereof. Background Art

[0002] Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is the smallest virus among the major viruses that affect the world's shrimp farming industry. IHHNV has a wide range of hosts and can infect white shrimp, Penaeus stylirostris, Penaeus monodon, etc. Due to its strong infectivity, shrimp infected with IHHNV will die and grow slowly. Among them, the mortality rate of Penaeus stylirostris infected by IHHNV can reach more than 90%, and white shrimp will cause dwarf deformity syndrome. Although it will not cause death, the deformity rate is high, and the growth is slow and uneven, which can cause up to 50% economic losses, bringing great harm to the shrimp farming industry. IHHNV is mainly transmitted horizontally, mainly through infected shrimp, food chain or cannibalism, and polluted water bodies. Among them, eating diseased shrimp is the most contagious, and infected shrimp can also infect offspring through vertical transmission. With the expansion of my country's shrimp farming industry, the demand for customs disease detection is increasing. The development of early, rapid, efficient and accurate methods for detecting IHHNV disease for non-diagnostic and therapeutic purposes is of great significance for the prevention of diseases in shrimp farming. Studies have shown that the IHHNV gene contains two open reading frames encoding nonstructural proteins nsp1 (nonstructural protein 1), nsp2 (nonstructural protein 2) and one open reading frame encoding viral capsid protein cp (capsid protein).

[0003] At present, there are many detection methods for IHHNV, and the traditional IHHNV detection methods include histopathology, polymerase chain reaction (PCR), real-time quantitative polymerase chain reaction (RT-PCR), monoclonal antibody assay, biosensor and enzyme-linked immunosorbent assay (ELISA). These molecular detection technologies have high sensitivity and reliability, but they require long reaction time, complex instruments and professional operation, and the price is too high, so they cannot be used for continuous monitoring and rapid detection in shrimp farming.

[0004] In recent years, immunological detection based on antigen-antibody binding is a fast, efficient and accurate detection method. Currently, most antibodies used in immunological detection are traditional monoclonal antibodies or polyclonal antibodies. Traditional antibodies have a large molecular weight and are limited in application. Nanobodies are the smallest antibody units known to have antigen recognition activity. The CDR3 region of nanobodies enables them to recognize hidden areas of antigens. They have advantages that traditional antibodies do not have, such as small size, strong penetration, easy modification, and strong stability. Therefore, it is necessary to focus on finding high-affinity nanobodies against nsp1, nsp2 and cp and conduct related research.

[0005] Colloidal gold immunochromatographic test strips are one of the most widely used methods for rapid pathogen detection. The reaction principle of this method is the interaction between antigen and antibody, and it combines immunochromatography and colloidal gold labeling. Compared with traditional immunological methods, this method has the advantages of simple operation, fast speed, no equipment and experience required, etc. Therefore, it is necessary to study how to combine nanoantibodies with colloidal gold technology, and further develop fast, convenient and highly sensitive colloidal gold test strips for pathogen detection, so as to solve the effective prevention and efficient monitoring of diseases in shrimp farming production. Summary of the invention

[0006] The purpose of the present invention is to find high-affinity nanobodies against nsp1, nsp2 and cp to solve the technical problem:

[0007] The first aspect of the present invention provides a nanobody, the amino acid sequence of the nanobody is SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14.

[0008] Furthermore, the nanoantibodies are shark-derived; among them, the anti-cp nanoantibody is shark-derived; the anti-nsp1 nanoantibody is shark-derived; and the anti-nsp2 nanoantibody is shark-derived.

[0009] The second aspect of the present invention provides an application of a nanobody in the detection of infectious subcutaneous and hematopoietic necrosis virus for non-diagnostic purposes. The amino acid sequence of the nanobody is SEQ ID NO.8 or SEQ ID NO.11 for detecting the cp antigen protein of infectious subcutaneous and hematopoietic necrosis virus; or the amino acid sequence of the nanobody is SEQ ID NO.9 or SEQID NO.12 for detecting the nsp1 antigen protein of infectious subcutaneous and hematopoietic necrosis virus; or the amino acid sequence of the nanobody is SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.14 for detecting the nsp2 antigen protein of infectious subcutaneous and hematopoietic necrosis virus.

[0010] The third aspect of the present invention provides an infectious subcutaneous and hematopoietic necrosis virus detection product comprising nano antibodies.

[0011] Furthermore, the infectious subcutaneous and hematopoietic necrosis virus detection product is a reagent, a test strip or a test kit; the test strip includes an immunochromatographic test strip prepared using any one of the labeling technologies of colloidal gold labeling, colloidal carbon labeling, fluorescent microsphere labeling, and nanoparticle labeling.

[0012] Furthermore, the infectious subcutaneous and hematopoietic necrosis virus detection product includes a first antibody combination, a second antibody combination or a third antibody combination; the first antibody combination is used to specifically identify the cp antigen protein, and the first antibody combination is: rabbit polyclonal antibody as the first gold-labeled antibody, the first T-line antibody with an amino acid sequence of SEQ ID NO.11, and goat anti-rabbit antibody as the first C-line antibody; the second antibody combination is used to specifically identify the nsp1 antigen protein, and the second antibody combination is: rabbit polyclonal antibody as the second gold-labeled antibody, the second T-line antibody with an amino acid sequence of SEQ ID NO.12, and goat anti-rabbit antibody as the second C-line antibody; the third antibody combination is used to specifically identify the nsp2 antigen protein, and the third antibody combination is: rabbit polyclonal antibody as the third gold-labeled antibody, the third T-line antibody with an amino acid sequence of SEQ ID NO.13, and goat anti-rabbit antibody as the third C-line antibody.

[0013] The fourth aspect of the present invention provides a method for preparing a nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus, comprising the following steps:

[0014] Step A: expression of cp antigen protein, nsp1 antigen protein or nsp2 antigen protein, comprising: constructing cp recombinant plasmid, nsp1 recombinant plasmid or nsp2 recombinant plasmid; inducing expression of the constructed cp recombinant plasmid, nsp1 recombinant plasmid or nsp2 recombinant plasmid through prokaryotic organisms and purifying to obtain cp antigen protein, nsp1 antigen protein or nsp2 antigen protein;

[0015] Step B: Construction of shark natural library and / or shark immune library;

[0016] Step C: performing auxiliary phage rescue and purification on the constructed shark natural library and / or shark immune library to obtain a phage library and detect its titer;

[0017] Step D: performing two, three, four or five rounds of panning on the phage library to obtain a final eluate;

[0018] Step E: Use the final eluate to prepare the supernatant for phage-ELISA identification and sequencing, and screen the cp nanobody, nsp1 nanobody or nsp2 nanobody with different CDR3 region sequences based on the sequencing results.

[0019] The present invention utilizes phage display technology (phage-ELISA) to screen anti-CP, anti-NSP1 and anti-NSP2 nanoantibodies from natural phage libraries and / or immune phage libraries derived from sharks, and adopts a prokaryotic expression system for production, thereby finding high-affinity anti-CP, anti-NSP1 and anti-NSP2 nanoantibodies and reducing production and research and development costs.

[0020] In addition, the detection product of the present invention utilizes the principle of interaction between antigen and antibody, combines immunochromatography and colloidal gold labeling and other technologies, and compared with traditional immunological methods, this method has the advantages of simple operation, fast speed, no need for equipment and experience, etc. And by combining nano-antibodies with colloidal gold technology, a fast, convenient and highly sensitive colloidal gold test strip is further developed, which can detect whether aquatic animals such as shrimps and aquaculture waters are infected with infectious subcutaneous and hematopoietic necrosis viruses, whether aquaculture feeds are contaminated by infectious subcutaneous and hematopoietic necrosis viruses, etc., to achieve rapid detection of infectious subcutaneous and hematopoietic necrosis viruses, which is conducive to better realizing the effective prevention and efficient monitoring of epidemic diseases in shrimp aquaculture production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed for protection.

[0022] Figure 1 The agarose gel electrophoresis diagram of the cp recombinant plasmid digested by enzymes in Example 1 (M is a marker; 1 is the double-enzyme digested target gene cp gene, the blue arrow points to the target gene band; 2 is the vector);

[0023] Figure 2The agarose gel electrophoresis diagram of the nsp1 recombinant plasmid digested by enzymes in Example 1 of the present invention (M is a marker; 1 is the double-enzyme digested target gene nsp1 gene, the blue arrow points to the target gene band; 2 is the vector);

[0024] Figure 3 The figure is an agarose gel electrophoresis diagram of the nsp2 recombinant plasmid digested by enzymes in Example 1 of the present invention (M is a marker; 1 is the double-enzyme digested target gene nsp2 gene, the blue arrow indicates the target gene band; 2 is the vector);

[0025] Figure 4 This is a diagram of the prokaryotic expression and purification of the cp protein in Example 1 (M displacement marker; 1 is the first culture solution of cp; 2 is the first induction solution of cp; 3 is the third resuspension of cp; 4 is the flow-through FT of cp; 5 is the protein washed with 20 mM imidazole of cp; 6 is the protein washed with 40 mM imidazole of cp; 7 is the protein washed with 60 mM imidazole of cp; 8 is the protein washed with 80 mM imidazole of cp; 9 is the protein of cp; 10 is the protein washed with 1 M imidazole of cp; the blue arrow indicates the protein of cp, with a size of 35 kDa);

[0026] Figure 5 This is a diagram of the prokaryotic expression and purification of the nsp2 protein in Example 1 (M is a marker; 1 is the first culture solution of nsp2; 2 is the first induction solution of nsp2; 3 is the supernatant of nsp2 after ultrasound; 4 is the third resuspension of nsp2; 5 is the flow-through FT of nsp2; 6 is the protein washed with 20 mM imidazole of nsp2; 7 is the protein washed with 40 mM imidazole of nsp2; 8 is the nsp2 protein; 9 is the protein washed with 1 M imidazole of nsp2; the blue arrow indicates the nsp2 protein, which is 45 kDa in size);

[0027] Figure 6 This is a diagram of the prokaryotic expression and purification of the nsp1 protein in Example 1 (M is a marker; 1 is the second resuspension of nsp1; 2 is the first supernatant of nsp1; 3 is the flow-through FT1 of nsp1; 4 is the washing of impurities with 40 mM imidazole of nsp1 before enzyme cleavage; 5 is the elution of the target protein with 500 mM imidazole of nsp1 before enzyme cleavage; the blue arrow points to the nsp1 protein, which is 110 kDa in size);

[0028] Figure 7 It is the reverse screening diagram of nsp1 protease cleavage in Example 1 (M is marker; 1 is the nsp1 protein solution before cleavage after dialysis; 2 is the nsp1 cleavage reaction product; 3 is the nsp1 flow-through liquid FT2; 4 is the nsp1 20 mM imidazole washing protein after cleavage; 5 is the nsp1 40 mM imidazole washing protein after cleavage; 6 is the nsp1 500 mM imidazole elution residual protein after cleavage; the blue arrow indicates);

[0029] Figure 8 The BCA protein concentration determination curve of cp, nsp1, and nsp2 antigen proteins in Example 1 (the horizontal axis is the standard BSA concentration, in mg / mL; the vertical axis is OD 562 nm absorbance value);

[0030] Fig. 9 Figure 2 is a graph of serum ELISA test results in Example 2 (A is the test result of serum titer after immunization with antigen cp; B is the test result of serum titer after immunization with antigen nsp1; C is the test result of serum titer after immunization with antigen nsp2);

[0031] Fig.10 This is the agarose gel electrophoresis diagram of the total RNA from the spleen of the non-immunized shark in Example 2 (M is a marker; 1-12 are the total RNA from the spleen of the non-immunized shark; three complete bands can be observed: 28S, 18S and 5S);

[0032] Fig.11 This is the agarose gel electrophoresis diagram of the total RNA from blood lymphocytes of unimmunized sharks in Example 2 (M is a marker; 1-12 are the total RNA from blood lymphocytes of unimmunized sharks; three complete bands can be observed: 28S, 18S and 5S);

[0033] Fig.12 The agarose gel electrophoresis diagram of the spleen total RNA and blood lymphocyte total RNA of the immunized shark in Example 2 (M is a marker; 1-3 are the spleen total RNA of the immunized shark; 4-6 are the blood lymphocyte total RNA of the immunized shark);

[0034] Fig.13 This is the agarose gel electrophoresis image of the first round PCR amplification product of the non-immunized shark vNAR gene in Example 2 (M is a marker; 1-24 are the amplified bands of the non-immunized shark vNAR target gene);

[0035] Fig.14 This is the agarose gel electrophoresis image of the first round PCR amplification product of the immune shark vNAR gene in Example 2 (M is a marker; 1-3 are the amplified bands of the immune shark vNAR target gene);

[0036] Fig.15 This is the agarose gel electrophoresis image of the fourth recovered product of the non-immunized shark in Example 2 (M is a marker; 1-10 are the bands of the third recovered product after double enzyme digestion of the non-immunized shark);

[0037] Fig.16 This is the agarose gel electrophoresis image of the fourth recovered product of the immune shark in Example 2 (M is a marker; 1-12 are the third recovered product bands after double enzyme digestion of the immune shark);

[0038] Fig.17 This is the original storage capacity determination diagram of the shark natural reservoir in Example 2;

[0039] Fig.18 This is a diagram showing the original library capacity determination of the shark immune library in Example 2;

[0040] Fig.19 Example 2 Agarose gel electrophoresis of the second round PCR amplification products of non-immunized sharks (M is a marker; 1-48 are bands of the second round PCR amplification products of non-immunized sharks);

[0041] Fig. 20 This is the agarose gel electrophoresis image of the second round PCR amplification product of the immune shark in Example 2 (M is a marker; 1-48 are the bands of the second round PCR amplification product of the immune shark);

[0042] Fig.21 This is the sequencing result of the bacterial library of non-immunized sharks in Example 2;

[0043] Fig. 22 This is a diagram showing sequencing results of the immune shark bacterial library in Example 2;

[0044] Fig.23 This is a graph showing the number of positive clones screened by phage-ELISA in Example 3;

[0045] Fig.24 This is a diagram showing the alignment of the Nanobody sequences in Example 4;

[0046] Fig.25 This is a diagram showing the expression and purification results of the shark natural library vNAR nanobody in Example 4 (the blue arrow indicates the purified nanobody protein);

[0047] Fig.26 This is a diagram showing the expression and purification results of vNAR nanobody from the shark immune library in Example 4 (the blue arrow indicates the purified nanobody protein);

[0048] Fig. 27 The shark vNAR nanobody of Example 4 (A is the SDS-PAGE electrophoresis result of the vNAR nanobody; B is the Western blot identification result of the vNAR nanobody; M is a marker; 1-7 are SEQ ID NO. 8-14 vNAR nanobodies respectively);

[0049] Fig.28 This is the ELISA affinity verification diagram of vNAR nanobody in Example 4 (the vertical axis is OD 450nm The absorbance value at the horizontal axis is the concentration of vNAR nanobody);

[0050] Fig.29 is the ultraviolet absorption spectrum of the colloidal gold solution of Example 5;

[0051] Fig.30 The structure diagram of the test strip of Example 5 (wherein 1 is the sample pad; 2 is the gold label pad; 3 is the NC membrane; 4 is the water absorption pad; 5 is the T line antibody; 6 is the C line antibody);

[0052] Fig.31 This is a graph showing the antibody cross-pairing test results for test example 1 (A is the antibody cross-pairing test result for the cp antigen; B is the antibody cross-pairing test result for the nsp1 antigen; C is the antibody cross-pairing test result for the nsp2 antigen; the combination of test strips No. 1 and 2 is nanoantibody as the gold-labeled antibody, rabbit polyclonal antibody as the T-line antibody, and anti-his tag antibody as the C-line antibody; the combination of test strips No. 3 and 4 is rabbit polyclonal antibody as the gold-labeled antibody, nanoantibody as the T-line antibody, and goat anti-rabbit as the C-line antibody);

[0053] Fig.32 This is a graph of the specific test results of the colloidal gold test strip of test example 2 (A is the specific test result of the colloidal gold test strip of cp antigen; B is the specific test result of the colloidal gold test strip of nsp1 antigen; C is the specific test result of the colloidal gold test strip of nsp2 antigen; 1-6 are cp, snp1, snp2, MBP, SWP1 and VP28 protein detection respectively);

[0054] Fig.33 This is a graph of the sensitivity test results of the colloidal gold test strip of test example 3 (A is the sensitivity test result of the colloidal gold test strip of the cp antigen; B is the sensitivity test result of the colloidal gold test strip of the nsp1 antigen; C is the sensitivity test result of the colloidal gold test strip of the nsp2 antigen; 1-6 are 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25μg / mL, 6.25 μg / mL and 3.125 μg / mL, respectively);

[0055] Fig.34 This is a graph showing the test results of the colloidal gold test strips for the hepatopancreas tissue sample of shrimp in Test Example 4 (AJ are the numbers of the 10 shrimps; 1, 2, and 3 are the colloidal gold test strips for cp antigen, nsp1 antigen, and nsp2 antigen, respectively). DETAILED DESCRIPTION

[0056] The detailed features and advantages of the present invention are described in detail in the specific implementation modes below, and the contents are sufficient to enable any person skilled in the art to understand the technical contents of the present invention and implement them accordingly. Moreover, according to the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant objects and advantages of the present invention.

[0057] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0058] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0059] (1) Source of sample materials

[0060] Striped bamboo shark: available at the seafood market in Yazhou District, Sanya City, Hainan Province.

[0061] Shark natural library: purchased from Shenzhen Kangti Life Science Technology Co., Ltd.

[0062] Upstream and downstream primer synthesis and sequencing: carried out by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0063] The pSmart-I vector was directly provided by Bioengineering.

[0064] (2) Sources of reagents and consumables

[0065] Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0066] name Manufacturer Part Number Agar powder Solebao 9002-18-0 Ampicillin Shanghai Bioengineering AB0064 Tryptone Oxoid LP004 Yeast powder Oxoid LP0021B Ni-NTA Cytiva 17531802 5× protein loading buffer (with DTT) Solebao P1040 Prestained protein marker Thermo Fisher LC5615 Non-prestained protein markers Thermo Fisher 26610 SDS-PAGE gel fast stain absci Ab.001.10 Sodium chloride Shanghai Bioengineering A501218-0001 20×PBS Solebao P1032 Imidazole Shanghai Bioengineering A506644-0100 PD-10 Desalting Column Cytiva 17085101 Ultrafiltration tube Merck Millipore UFC901096 BCA Kit Blue Sky P0012S Isopropylthio-β-D-galactoside Solebao I8070 SUMO Protease Blue Sky P2312M Omni-Easy™ One-Step PAGE Gel Rapid Preparation Reagent Yamei PG213 / PG214 10× Tris-Glycine SDS-PAGE electrophoresis buffer Shanghai Bioengineering C520001-0001 Coral Salt Sea Blue CZ0001 MS-222 WEST GENE XB-TB100 Bovine serum albumin (BSA) Solebao A8020-50g Heparin Sodium Solebao H8060 M13KO7 Helper phage NEB N3015s Dog shark peripheral blood lymphocyte isolation kit Haoyang Biology LTS1086F One-component TMB colorimetric solution Solebao PR1200 PEG8000 Bioengineering A100159-0500 Freund's complete adjuvant Sigma-Aldrich F5881 Freund's incomplete adjuvant Sigma-Aldrich F5506 Goat anti-rabbit IgG (HRP) Blue Sky A0208 Trizol Invitrogen 15596026 cDNA Reverse Transcription Kit TAKARA 6215A Q5 High-Fidelity DNA Polymerase NEB M0491S Agarose Gel DNA Recovery Kit TIANGEN DP219-03 SacI-HF NEB R3156V SpeI-HF NEB R3133V General DNA product purification kit TIANGEN DP204-02 T4 DNA ligase NEB M0202V NaAc Blue Sky ST342 PCR Taq enzyme Novozymes P222-01 Immunotube Baroque 12-1275 Immunotube cap Baroque 12-1299 M13KO7 Helper phage NEB N3015s PEG8000 Shanghai Bioengineering A100159-0500 Trypsin Shanghai Bioengineering A003702-0100 10% AEBSF Shanghai Bioengineering C510022 TMB single component colorimetric solution Solebao PR1200 M13Bacteriophage Antibody (HRP), Mouse Mab Sino Biological 11973-MM05T-H BL21 (DE3) competent cells Tiangen CB105-02 Skim milk powder Blue Sky P0216-300g Horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) Blue Sky A0216 His-tag antibody (mouse monoclonal antibody) Blue Sky AH367 HRP-labeled mouse anti-HIS monoclonal antibody Bioengineering D410002-0100 Sartorius CN140 Hangzhou Fenghang FH-NC-8003 Plastic card shell Hangzhou Fenghang FH-SLK-002Y Glass Fiber Hangzhou Fenghang FH-8975 Absorbent pad Hangzhou Fenghang FH-AN3 Base Plate Hangzhou Fenghang FH-DB-8025 Tris Shanghai Bioengineering A100193-0005 sucrose Shanghai Bioengineering A610498-0500 Tween20 Shanghai Bioengineering A600560-0500 Chloroauric acid Shanghai Test 10010711 Trisodium citrate dihydrate Shanghai Bioengineering A610035-0500 Anhydrous potassium carbonate Maclean P816304 Bovine serum albumin (BSA) Solebao A8010 pET-22b(+) vector Bioengineering B540181 Trans-T1 competent cells Bioengineering B528412 Plasmid Extraction Kit Tiangen DP103 BCA Kit Blue Sky P0010 Dialysis Bags Solebao YA1046

[0067] (3) Source of instruments and equipment

[0068] Table 2 Sources and models of instruments and equipment required for the experiment

[0069] Instrument Name Instrument Manufacturer Instrument Model Snow ice machine Xinzhi XB-50 Ultrasonic cell disruptor Xinzhi JY88-IIN Constant temperature metal bath Youning GA15 Decolorization shaker Chilin Bell TS-200 Rotating disk mixer Chilin Bell QB328 Multifunctional ELISA reader Bio Tek Synergy Cytation 5 Protein purification system Bio-Rad NGC Centrifuge Beckman X-30R Ultrapure water preparation system Merk Millipore Milli-Q Direct Gel imaging system Analytik Jena ChemoStudio Plus 515 Plate washer Bio Tek 50TS Vertical Pressure Steam Sterilizer Yamato SQ510 Electronic balance Ohaus PX223ZH Ultra-low temperature refrigerator Haier DW-86L486HC Protein electrophoresis system Bio-Rad Mini-Protean Medical Refrigerator Freezer Haier HYCD-282 Clean bench Haier HCB-1300V Liquid nitrogen tank Haier YDS-35-80 Constant temperature shaking incubator (shaker) smart city ZWY-211B Electric constant temperature incubator (referred to as incubator) smart city ZXDP-B2050 PCR instrument Thermo Fisher Mini Amp Film marking machine Hangzhou Fenghang HGS510 Programmable cutting machine Hangzhou Fenghang HGS201 Wide cutting machine Hangzhou Fenghang HG220S Flat shell pressing machine Hangzhou Fenghang HGS803 Film marking machine Hangzhou Fenghang HGS510

[0070] (4) Solutions and preparation methods required for the experiment

[0071] Table 3 Solutions and methods required for the experiment

[0072] Reagent name Preparation method LB liquid medium 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, autoclave at 121°C for 20 min LB solid medium 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, 1.5% (w / v) agar powder, autoclaved at 121°C for 20 min 10×SDS-PAGE electrophoresis buffer 250 mM Tris-HCl, 2.5 M glycine, 1% (w / v) SDS TBS 10 mM Tris-HCl, pH 8.0, 150 mM NaCl TBST 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween20 Blocking solution 5% skim milk powder dissolved in TBST Electroporation Buffer 25 mM Tris-HCl, 192 mM glycine, 20% methanol, freshly prepared and pre-cooled before use PBST Dilute 20× PBS buffer 20 times to 1× PBS and add 0.1% Tween20 Primary Antibody The primary antibody used in the Western blot experiment was a mouse monoclonal antibody, diluted 1:3000 in blocking solution. Secondary Antibodies The secondary antibody used in the Western blot experiment was goat anti-mouse antibody, diluted 1:5000 in blocking solution. 2×YT liquid medium <![CDATA[0.5 g of NaCl, 1.6 g of Tryptone, 1 g of Yeast Extract, are dissolved in an appropriate amount of double deionized water (ddH2O, Double Distilled Water). After complete dissolution, the volume is made up to 100 mL, autoclaved at 121 °C for 20 min, and stored at 4 °C for later use.]]> PEG / 2.5MNaCl (5×) <![CDATA[Weigh 100 g of PEG-8000 and 75 g of NaCl (2.5 M) and dissolve them in 400 mL of ddH2O at room temperature. Make up the volume to 500 mL, autoclave, stir to cool down, and store at 4°C.]]> 2×YT solid medium (Amp) <![CDATA[Dissolve 0.5 g of NaCl, 1.6 g of Tryptone, 1 g of Yeast Extract, and 1.5 g of Agarose B in an appropriate amount of ddH2O. After complete dissolution, make up the volume to 100 mL, autoclave at 121 °C for 20 min. When the temperature of the medium drops to about 50 °C, add Amp (100 μg / mL), pour the plates. After the medium solidifies, invert and store at 4 °C.]]> 2×YT solid medium (Tet) <![CDATA[Dissolve 0.5 g of NaCl, 1.6 g of Tryptone, 1 g of Yeast Extract, and 1.5 g of Agarose B in an appropriate amount of ddH2O. After complete dissolution, make up the volume to 100 mL, autoclave at 121°C for 20 min. When the temperature of the medium drops to about 50°C, add Tet (10 μg / mL), pour the plates. After the medium solidifies, invert and store at 4°C.]]> Ampicillin (stock solution) Accurately weigh 2.5 g of ampicillin and place it in a 50 mL centrifuge tube. Add 40 mL of sterile water, mix thoroughly to dissolve and make up to 50 mL. Filter with a 0.22 μm filter membrane for sterilization, divide into portions (1 mL / tube), and store at -20°C. Kanamycin (stock solution) Accurately weigh 2.5 g of kanamycin and place it in a 50 mL centrifuge tube. Add 40 mL of sterile water, mix thoroughly to dissolve and make up to 50 mL. Filter with a 0.22 μm filter membrane for sterilization, divide into portions (1 mL / tube), and store at -20°C. Tetracycline (stock solution) Accurately weigh 2.5 g of tetracycline and place it in a 50 mL centrifuge tube. Add 40 mL of anhydrous ethanol and mix thoroughly to dissolve the mixture. Make up to 50 mL of the solution and filter it with a 0.22 μm filter membrane for sterilization. After aliquoting (1 mL / tube), store it at -20°C. CBS Add 3.03 g Na2CO3 and 6.0 g NaHCO3 into 1 L sterile water, dissolve thoroughly, and store at 4°C. 0.25 mg / mL Trypsin Accurately weigh 12.5 mg of Trypsin into a 50 mL centrifuge tube, add sterile water, mix thoroughly and dissolve to make up to 50 mL. <![CDATA[0.2 MK2CO3 aqueous solution]]> Accurately weigh 0.2764 g of anhydrous potassium carbonate powder and add purified water to make up to 10 g. 10% BSA solution Accurately weigh 1 g of bovine serum albumin powder and add purified water to make up to 10 g. Gold complex solution Accurately weigh 0.1 g bovine serum albumin powder and 0.2 g sucrose, pipette 250 μL pH8.0 Tris-HCL, 10 μL Tween20, and then add purified water to 10 g. Gold diluent Accurately weigh 0.2 g bovine serum albumin powder and 0.5 g sucrose, pipette 250 μL pH8.0 Tris-HCL, 10 μL Tween20, and then add purified water to 10 g. Gold standard pad treatment liquid Weigh 5 g of BSA powder and 5 g of sucrose, pipette 10 μL of Tween20, and then add purified water to make up to 100 g. Sample Pad Treatment Solution Weigh 1 g of BSA powder and 1 g of sucrose, pipette 10 μL of Tween20, and then add purified water to make up to 100 g.

[0073] Example

[0074] The expression and purification of cp, nsp1 and nsp2 antigen proteins include the following steps.

[0075] The construction of S1, cp, nsp1, and nsp2 recombinant plasmids, the specific steps are as follows:

[0076] (1) Based on the gene sequences of cp (GenBank: EF633688.1:2521-3510, SEQ ID NO.1), nsp1 (GenBank: EF633688.1:579-2579, SEQ ID NO.2), and nsp2 (GenBank: EF633688.1:523-1614, SEQ ID NO.3) published on NCBI, cp, nsp1, and nsp2 genes were synthesized directly by the direct synthesis method after codon optimization by Shanghai Biotechnology Co., Ltd.

[0077] (2) The target genes cp and nsp2 were respectively subjected to enzyme ligation reaction with the pET-22b (+) vector, and nsp1 was subjected to enzyme ligation reaction with the pSmart-I vector. The enzyme ligation reaction system was: 4 μL of the target gene and 1 μL of the corresponding vector. After gently pipetting to mix, the mixture was reacted at 25°C for 10 min to obtain the first ligation products of cp, nsp1, and nsp2. They were stored at 4°C for later use.

[0078] (3) The pET-22b (+) vector connected with the target genes cp and nsp2 was double-digested with XhoI and NdeI enzymes. The digestion system was: 10× NEBuffer 2 μL, XhoI enzyme 0.5 μL, NdeI enzyme 0.5 μL, pET-22b (+) vector 1 μL, add ddH2O to make the volume 20 μL; digestion conditions were 37℃ 2 h, 80℃ 20 min. After the digestion reaction, the digestion products of the pET-22b (+) vector were detected by 1.5% agarose gel electrophoresis. The electrophoresis results of cp are shown in Figure 1 The electrophoresis results of nsp2 are shown in Figure 2 ; The pSmart-I vector connected to the target gene nsp1 was double-digested with XbaI and XhoI enzymes. The digestion system was: 10× NEBuffer 2 μL, XbaI enzyme 0.5 μL, XhoI enzyme 0.5 μL, pSmart-I vector 1 μL, and ddH2O was added to make the volume 20 μL; the digestion conditions were 37℃ 2 h, 80℃ 20 min. After the digestion reaction, the digestion products of the pSmart-I vector were detected by 1.5% agarose gel electrophoresis. The electrophoresis results of nsp1 are shown in Figure 3 .

[0079] (4) Thaw the TOP 10 competent cells on ice, and add the first ligation products of cp, nsp1, and nsp2 to 50 μL of TOP 10 competent cells, mix gently, and quickly place on ice for 30 min, heat shock in a 42°C metal bath for 30 s, and immediately place on ice for 2 min. Under a sterile environment, add the transformed bacterial solution to 250 μL of non-resistant LB liquid culture medium, shake and culture at 37°C, 220 rpm, and shake for 1 h to obtain the first transformation bacterial solution of cp, nsp1, and nsp2. Subsequently, take 200 μL of the first transformation bacterial solution of cp, nsp1, and nsp2 and spread them on ampicillin (Amp, Amipicilline) resistant LB solid culture medium, and spread the first transformation bacterial solution of nsp1 on kanamycin (Kana, Kanamycin) resistant LB solid culture medium, and incubate them inverted in an incubator at 37°C overnight, which are the first plates of cp, nsp1, and nsp2.

[0080] (5) Pick the monoclonal plaques from the first plate of cp, nsp1, and nsp2 for expansion culture, place on a shaker, and culture at 200 rpm and 37°C for about 6 hours. After extracting the plasmid using a plasmid extraction kit, take an appropriate amount of plasmid and send it to Bioengineering for the first sequencing. The first sequencing results are compared and analyzed, and the similarity is 100%. Therefore, the recombinant protein plasmid vector is successfully constructed, namely the cp plasmid, nsp1 plasmid, and nsp2 plasmid. Mark them and store them in a -20°C refrigerator.

[0081] The specific steps for expression and purification of S2, cp, and nsp2 antigen proteins are as follows:

[0082] (1) According to the first sequencing results, select the cp plasmid and nsp2 plasmid with correct sequence alignment, and transform the cp plasmid and nsp2 plasmid into Rosetta (DE3) competent cells respectively. The transformation method is the same as S1 (5). After the plating is completed, invert and culture in an incubator at 37°C overnight to form the second plate of cp and nsp2. Pick a single colony from the second plate of cp and nsp2 and culture it in 200 μL of Amp-resistant LB liquid culture medium overnight to form the first monoclonal bacterial solution of cp and nsp2. After overnight culture at 37°C and 220 rpm, expand 200 μL of the first monoclonal bacterial solution of cp and nsp2 to 20 mL at a ratio of 1:100 to form the first expansion culture solution of cp and nsp2. Inoculate the first expansion culture solution of cp and nsp2 into 2 L of Amp-resistant LB liquid culture medium and culture it in a shaker at 37°C and 220 rpm for about 3.5 h. When the OD of the first expansion culture solution reaches 0.04, the OD of the first expansion culture solution reaches 0.04. 600 nm=0.5, add IPTG (isopropyl β-D-Thiogalactoside) with a final concentration of 1 mM and induce at 37°C for 4 h to obtain the bacterial solution after the first induction of cp and nsp2. The bacterial liquid after the first induction of cp and nsp2 was transferred to large centrifuge bottles respectively, and centrifuged at 4°C and 4200 rpm for 20 min. The supernatant was discarded and the bacterial pellet was retained as the first pellet of cp and nsp2. Then 20 mL of 1×PBS (phosphate buffered saline, obtained by diluting 20×PBS) was added to resuspend the first pellet of cp and nsp2 to obtain the first resuspension of cp and nsp2; an ultrasonic cell disruptor was used with 30% power, ultrasonication for 2 s, intermittent 4 s, and a total ultrasonication time of 40 min to obtain the second resuspension of cp and nsp2. The second resuspension of cp and nsp2 was divided into 2 mL centrifuge tubes and centrifuged at 4200 rpm and 4°C for 20 min to obtain the second pellet of cp, the second pellet of nsp2, the supernatant after ultrasonication of cp and the supernatant after ultrasonication of nsp2, respectively. The second pellet of cp and the second pellet of nsp2 were resuspended in 20 mL of PBS containing 2M urea to obtain the third resuspension of cp and nsp2.

[0083] (2) The third resuspension of cp and nsp2 was subjected to ultrasonic cell disruption at 30% power, ultrasonicated for 2 s, with an interval of 4 s, and a total ultrasonic time of 5 min to obtain the fourth resuspension of cp and nsp2. The fourth resuspension of cp and nsp2 was divided into 2 mL centrifuge tubes and centrifuged at 4200 rpm and 4°C for 20 min. The supernatant was discarded to obtain the third precipitation of cp and nsp2, which were the cp inclusion bodies and nsp2 inclusion bodies. The cp inclusion bodies and nsp2 inclusion bodies were resuspended in 20 mL of PBS containing 0.3% sodium dodecylamine (SKL) to obtain the fifth resuspension of cp and nsp2. The ultrasonication was performed again under the same ultrasonic conditions as the third resuspension of cp and nsp2 to obtain the sixth resuspension of cp and nsp2, which were the inclusion body solutions of cp and nsp2. The inclusion body solutions of cp and nsp2 were renatured using a PD-10 desalting column to obtain the renatured inclusion body solutions of cp and nsp2.

[0084] (3) Take 5 mL of Ni-NTA (nickel NTA affinity chromatography medium, referred to as nickel column), equilibrate the nickel column with 20 mL of ultrapure water and 1×PBS in turn, inject the renatured cp and nsp2 inclusion body solutions into the nickel column using a 10 mL syringe and a 0.22 μm syringe filter membrane, and the outflow is the cp and nsp2 flow-through FT; wash the nickel column with 20 mL of 20 mM imidazole, 10 mL of 40 mM imidazole, 10 mL of 60 mM imidazole and 10 mL of 80 mM imidazole to remove impurities for cp, and elute the target protein with 20 mL of 200 mM imidazole, which is cp protein; wash the nickel column with 20 mL of 20 mM imidazole and 10 mL of 40 mM imidazole to remove impurities for nsp2, and elute the target protein with 20 mL of 200 mM imidazole, which is nsp2 protein; then use 10 mL of 1 Wash the remaining proteins on the column with 1 M imidazole, namely cp, nsp2; finally, wash the column with 20 mL ddH2O, and preserve the nickel column with 20% ethanol in a 4°C refrigerator.

[0085] (4) Take 40 μL of each of the above cp first culture solution, cp first induced culture solution, cp third resuspension solution, cp flow-through FT, cp 20 mM imidazole washed protein, cp 40 mM imidazole washed protein, cp 60 mM imidazole washed protein, cp 80 mM imidazole washed protein, cp protein and cp 1 M imidazole washed protein samples and mix them with 10 μL of 5× loading buffer (i.e., mixed sample), heat denatured in a metal bath at 99°C for 10 min, centrifuged at 13,000 rpm for 10 min, and prepared the first PAGE gel (polyacrylamide gel, Polyacrylamide GelElectrophoresis) using a 12.5% ​​Omni-Easy™ One-Step PAGE Gel Rapid Preparation Kit. The electrophoresis conditions were 200 V and 35 min. After the electrophoresis was completed, the PAGE gel was stained with SDS-PAGE rapid stain and decolorized with tap water. It is clearly observed that there is a clear band of cp protein at around 35 kDa. The protein is successfully expressed and has good purity. Subsequent experiments can be continued. For specific cp expression and purification diagrams, see Figure 4 .

[0086] The first culture solution of nsp2, the first induction solution of nsp2, the supernatant after nsp2 ultrasound, the third resuspension of nsp2, the flow-through FT of nsp2, the protein washed with 20 mM imidazole, the protein washed with 40 mM imidazole, the protein of nsp2 and the protein washed with 1 M imidazole were mixed with 10 μL of 5× loading buffer, and gel was prepared, electrophoresed, stained and destained. The mixing method and SDS-PAGE electrophoresis method, staining and decolorization method were the same as step S2 (4). It was clearly observed that the nsp2 protein had a clear band of about 45 kDa. The protein was successfully expressed and had good purity. The subsequent experiments can be continued. The specific nsp2 protein expression and purification diagram is shown in the figure. Figure 5 .

[0087] S3. Expression and purification of nsp1 antigen protein. The specific steps are as follows:

[0088] (1) According to the first sequencing results, select the nsp1 plasmid with the correct sequence alignment and transform the nsp1 plasmid into BL21 (DE3) competent cells. The transformation method is the same as S1 (5). After the plating is completed, invert and culture it in an incubator at 37°C overnight to form the nsp1 second plate. Pick a single colony from the nsp1 second plate and culture it in 200 μL of Kana-resistant LB liquid culture medium overnight to form the nsp1 first monoclonal bacterial solution. After overnight culture at 37°C and 220 rpm, expand 200 μL of the nsp1 first monoclonal bacterial solution to 20 mL at a ratio of 1:100 to form the nsp1 first expansion bacterial solution. Inoculate the nsp1 first expansion bacterial solution into 2 L of Kana-resistant LB liquid culture medium and culture it in a shaker at 37°C and 220 rpm for about 3.5 h. When the OD of the first expansion bacterial solution reaches 0.04, the nsp1 first expansion bacterial solution will be inoculated into 2 L of Kana-resistant LB liquid culture medium and cultured in a shaker at 37°C and 220 rpm for about 3.5 h. 600 nm =0.5, add IPTG (isopropyl β-D-Thiogalactoside) with a final concentration of 0.2 mM, induce at 37℃ for 4 h, which is the first induced bacterial solution of nsp1. Then transfer the first induced bacterial solution of nsp1 to large centrifuge bottles, centrifuge at 4℃, 4200 rpm for 20 min, discard the supernatant, keep the bacterial precipitate, which is the first precipitate of nsp1, and then add 20 mL of 1×PBS containing 8 M urea and 0.1% TritonX-100 to resuspend the first precipitate of nsp1, which is the first resuspension of nsp1; use an ultrasonic cell disruptor, 30% power, ultrasonic for 2 s, 4 s interval, total ultrasonic time 40 min, which is the second resuspension of nsp1, and divide the second resuspension of nsp1 into 2 mL centrifuge tubes, centrifuge at 4200 rpm, 4℃ for 20 min, and collect the supernatant, which is the first supernatant of nsp1.

[0089] (2) Take 5 mL of Ni-NTA (nickel NTA affinity chromatography medium, referred to as nickel column), equilibrate the nickel column with 20 mL of ultrapure water and 1×PBS (containing 8 M urea) in turn, inject the first supernatant of nsp1 into the nickel column using a 10 mL syringe and a 0.22 μm syringe filter membrane, incubate for 30 min, and the outflow is the nsp1 flow-through FT1; use 20 mL of 40 mM imidazole to wash the nickel column to remove impurities, which is nsp1 40 mM imidazole impurity wash before enzyme cleavage; use 20 mL of 500 mM imidazole to elute the target protein, which is nsp1 500 mM imidazole elution before enzyme cleavage; then use 10 mL of 1 M imidazole to wash off the remaining protein on the column, which is nsp1 1 M imidazole impurity wash before enzyme cleavage; finally, wash the column with 20 mL of ddH2O, and store the nickel column in 20% ethanol in a 4°C refrigerator.

[0090] (3) Place the target protein eluted with 500 mM imidazole before nsp1 cleavage in a dialysis bag, and place the dialysis bag in a dialysis solution. The dialysis solution is PBS solution (containing 300 mM NaCl and 10% glycerol). Dialyze at 4°C for 24 h. Replace the dialysis solution every 6 h to obtain the nsp1 protein solution before cleavage after dialysis.

[0091] (4) Add SUMO Protease to the nsp1 protein solution after dialysis and before digestion and perform digestion at 4°C overnight. The total digestion system is 5 mL. The specific digestion system is as follows: 10× Reaction Buffer 500 μL, dialyzed nsp1 protein solution 1.6 mL, Sumo Protease 12.5 μL, ddH2O 2.8 mL. The nsp1 digestion reaction product was obtained and purified by nickel column affinity chromatography again. The purification steps were the same as S3 (2). Since both SUMO Protease and the cleaved SUMO tag carried His tags, the nsp1 elution buffer 3 digestion reaction product did not have a His tag. Therefore, the nsp1 digestion reaction product was subjected to nickel column affinity chromatography according to step S3 (2) to obtain nsp1 flow-through FT2, nsp1 20 mM imidazole washed protein after digestion, nsp1 40 mM imidazole washed protein after digestion, and nsp1 500 mM imidazole washed residual protein after digestion. The reaction was then concentrated appropriately according to step S3 (2). The concentration step was the same as S2 (5) to obtain the nsp1 target protein.

[0092] (5) Take the above nsp1 second resuspension, nsp1 first supernatant, nsp1 flow-through FT1, nsp1 40 mM imidazole to wash impurities before enzyme cleavage, nsp1 500 mM imidazole to elute the target protein before enzyme cleavage, and 10 μL of 5× loading buffer for mixing, gel preparation, electrophoresis, staining, and decolorization. The mixing method and SDS-PAGE electrophoresis method, staining and decolorization method are the same as step S2 (4). It is clearly observed that the nsp1 target protein has a clear band of about 110 kDa before enzyme cleavage, which shows that the protein is successfully expressed and has a good purity. Subsequent experiments can be carried out. The specific nsp1 protein expression and purification diagram is shown in Figure 6 .

[0093] The nsp1 protein solution before dialysis and digestion, the nsp1 digestion reaction product, the nsp1 flow-through FT2, the nsp1 20 mM imidazole solution after digestion to wash the impurities, the nsp1 40 mM imidazole solution after digestion to wash the impurities, the nsp1 500 mM imidazole solution after digestion to wash the remaining protein, and the nsp1 protein were mixed with 10 μL of 5× loading buffer, gel preparation, electrophoresis, staining, and decolorization. The mixing method and SDS-PAGE electrophoresis method, staining, and decolorization method were the same as step S2 (4). It was clearly observed that after the nsp1 target protein was digested, there was a clear band at about 80 kDa, indicating that the protein was successfully expressed and had good purity, and subsequent experiments could be carried out. The digestion back screening diagram is shown in Figure 7 .

[0094] Concentration and concentration determination of S4, cp, nsp2 and nsp1 antigen proteins

[0095] The above cp, nsp2, and nsp1 proteins were passed through a PD-10 desalting column to remove salts such as imidazole, and concentrated using a 10 kDa concentrator to obtain cp antigen protein, nsp2 antigen protein, and nsp1 antigen protein. The concentrations of cp antigen protein, nsp2 antigen protein, and nsp1 antigen protein were determined using a BCA kit. The protein concentrations of the concentrated solutions were determined using a BCA kit. The cp antigen protein (diluted 10 times), nsp2 antigen protein (diluted 10 times), and nsp1 antigen protein (diluted 10 times) were measured at OD 562 nm The absorbance values ​​were 0.167, 0.176 and 0.159, respectively. Combined with the fitting equation y=0.2139x+0.1459 in the BCA protein concentration determination standard curve, the concentrations of cp antigen protein, nsp2 antigen protein and nsp1 antigen protein were measured to be approximately 1.0 mg / mL, 1.4 mg / mL and 0.6 mg / mL, respectively. After being packaged at 50 μg / tube, they were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. For specific BCA protein concentration determination standard curves for cp antigen protein, nsp2 antigen protein and nsp1 antigen protein, see Figure 8 .

[0096] Example 2

[0097] The construction of the natural phage library and immune library of the striped bamboo shark includes the following steps:

[0098] S1. The specific steps of feeding, immunization and detection of serum titer of striped bamboo shark are as follows:

[0099] (1) Rearing and immunization of striped bamboo sharks

[0100] Rearing and immunization of striped bamboo sharks: After the healthy striped bamboo sharks (hereinafter referred to as sharks) were bought back, they were temporarily kept in the laboratory breeding area for one week. The salinity of artificial seawater was controlled between 1.020-1.025, the seawater temperature was about 25℃, and the environment was kept away from light as much as possible. Shrimp was fed once a week, and the water was changed every three days to observe its growth status. 12 healthy sharks were selected for the construction of natural libraries, and 3 healthy sharks were used for immunization antigens to construct immune libraries. The 3 selected sharks were marked and numbered, and immunized with nsp1, nsp2 and cp antigen proteins respectively. Before the first immunization of sharks, blood was drawn and serum was separated, and stored at -80℃ for standby use. In the first immunization, 100 μg of nsp1, nsp2 and cp antigen protein solutions were mixed with an equal volume of complete Freund's adjuvant (CAF) and fully emulsified, and the emulsified antigen proteins were injected into sharks at multiple points; for subsequent immunization, 100 μg of antigen solution was mixed with an equal volume of incomplete Freund's adjuvant (IAF) and fully emulsified and injected at multiple points. On average, the sharks were vaccinated once every 12 days, for a total of 6 times. After the last vaccination, blood was drawn and serum was separated, and then the sharks were disposed of harmlessly. The shark vaccination periodic table is shown in Table 4:

[0101] Table 4 Shark immunity periodic table

[0102] Immunization time Immunization Shark-1 Immunization Dosage Shark-2 Immunization Dosage Shark-3 Immunity Dosage 0 day Multiple injections 100 μg cp in CAF 100 μg nsp1 in CAF 100 μg nsp2 in CAF 12 day Multiple injections 100 μg cp in IAF 100 μg nsp1 in IAF 100 μg nsp2 in IAF 24 day Multiple injections 100 μg cp in IAF 100 μg nsp1 in IAF 100 μg nsp2 in IAF 36 days Multiple injections 100 μg cp in IAF 100 μg nsp1 in IAF 100 μg nsp2 in IAF 48 days Multiple injections 100 μg cp in IAF 100 μg nsp1 in IAF 100 μg nsp2 in IAF 60 days Multiple injections 100 μg cp in IAF 100 μg nsp1 in IAF 100 μg nsp2 in IAF

[0103] (2) Double antibody sandwich ELISA to detect the titer of shark serum after immunization: the shark serum extracted above was diluted in series, 100 μL was added to each well of a 96-well plate, and coated overnight at 4°C; the coating solution was discarded, 200 μL PBS was added to each well for washing, and the plates were washed three times, each time for 5 min; 100 μL 5% skim milk powder (blocking solution) was added to each well, and the plates were blocked at room temperature for 1 h; the blocking solution was discarded, 200 μL PBS was added to each well for washing, and the plates were washed three times, each time for 5 min; the three antigens (cp, nsp1, and nsp2 antigen proteins) were diluted to 1 μg / mL with PBS, 100 μL was added to each well, and the plates were incubated at room temperature for 2 h; the antigens were discarded, 200 μL PBST was added to each well for washing, and the plates were washed three times, each time for 5 min; 1:100,000 diluted rabbit polyclonal antibody was added, 100 μL was added to each well, and the plates were incubated at room temperature for 1 h; the primary antibody was discarded, 200 μL PBST was added to each well for washing, and the plates were washed eight times, each time for 5 min min; add 1:250 diluted HRP-goat anti-rabbit monoclonal antibody, 100 μL per well, incubate at room temperature for 1 h; discard the secondary antibody, add 200 μL PBST to each well for washing, wash 8 times, 5 min each time; add 100 μL TMB colorimetric solution to each well, shake and mix, incubate in the dark for 5 min; remove the plate, add 100 μL 1M HCl to each well to stop color development, and detect OD 450 value.

[0104] Immunization results such as Fig. 9 As shown, the effects of multiple immunizations with nsp1 and nsp2 antigens were significant, while the immunization with cp antigen was not so significant, but the antibody titer was nearly doubled compared to before immunization. In general, immunization with the three antigens was effective.

[0105] S2. Sampling of blood and spleen of striped bamboo shark and separation of blood lymphocytes. The specific steps are as follows:

[0106] (1) Blood and spleen sampling of 12 non-immunized and 3 immunized bamboo sharks: Prepare anesthetic seawater with MS-222 fish anesthetic at a final concentration of 120 mg / L. Place the shark in the anesthetic seawater for about 15 minutes. The shark will be motionless and unresponsive. Remove the shark and wrap its gills with moist gauze to keep it moist. Then start blood collection. Use a 1 mL syringe to pierce the caudal vein at 45 degrees. The blood vessel is close to the body surface and the needle can touch the blood vessel just after it is immersed in the subcutaneous tissue. Then remove the syringe needle and immediately inject the blood into the sodium heparin blood collection tube. Slowly turn it upside down to mix thoroughly. About 3-4 mL of blood can be collected from each shark for subsequent blood lymphocyte separation.

[0107] After blood collection, the shark was dissected with a scalpel, the spleen was removed, the blood was rinsed with sterile 1×PBS, and then the spleen was cut into pieces and divided into cryopreservation tubes, quickly frozen in liquid nitrogen, and then immediately stored in a -80°C refrigerator.

[0108] (2) Separation of blood lymphocytes from bamboo sharks: Take a 15 mL sterile centrifuge tube, add 5 mL of separation solution (provided by the dog shark peripheral blood lymphocyte isolation kit), and then slowly add 4 mL of fresh shark anticoagulated blood (carefully add it above the separation solution interface); place the centrifuge tube in a centrifuge, centrifuge at 20°C, 800 × g for 30 min; after centrifugation, carefully remove the top plasma layer, and then carefully transfer the ring-shaped milky white lymphocyte layer in the centrifuge tube to a new 15 mL sterile centrifuge tube; add 10 ml of washing solution (provided by the kit) to the top of the centrifuge tube to mix the cells; place the centrifuge tube in a centrifuge, centrifuge at 20°C, 250 × g for 10 min, and discard the supernatant; use a pipette to aspirate 5 ml of washing solution to resuspend the obtained cells; repeat the centrifugation at 20°C, 250 × g for 10 min. Discard the supernatant and finally obtain about 200 μL of shark blood lymphocytes, which are quickly frozen in liquid nitrogen and then immediately stored in a -80°C refrigerator.

[0109] S3. Extraction and reverse transcription of RNA from spleen and blood lymphocytes of bamboo shark. The specific steps are as follows:

[0110] (1) RNA extraction from spleen and blood lymphocytes: Add 300 μL Trizol to an enzyme-free EP tube, carefully remove the tissue (spleen and blood lymphocytes obtained in step S2 of Example 2) from the cryopreservation tube and place it in Trizol, homogenize it on an ice plate rack until there are no large pieces of tissue, add 700 μL Trizol, and let it stand for 5 min; centrifuge: 4°C, 12,000 × g, centrifuge for 10 min; extraction: add 200 ul chloroform to a new EP tube, draw the supernatant from the previous step and add it to the tube, be careful not to take the middle layer, vortex and oscillate, let it stand for 2 min; centrifuge: 4°C, 12,000 × g, centrifuge for 15 min; precipitation: add 500 μL pre-cooled isopropanol to a new EP tube, draw the supernatant from the previous step and add it to the tube, be careful not to take the middle layer, mix it upside down, and let it precipitate at 4°C for 10 min; centrifuge: 4°C, 12,000 × g, centrifuge for 10 min; washing: discard the supernatant, remove the residual liquid, add 1 mL of pre-cooled 75% ethanol, shake the bottom of the tube, and suspend the precipitate; centrifugation: 4°C, 7500 ×g, centrifuge for 10 min, discard the supernatant, empty for 1 min, remove the residual liquid, and dry; dissolve RNA: add 30 ul DEPC water to the precipitate to dissolve, and obtain spleen total RNA and blood lymphocyte total RNA respectively; detection: after extraction, dilute the samples (spleen total RNA and blood lymphocyte total RNA) by 10 times, and use NanoDrop 1000 ultra-micro UV spectrophotometer and agarose gel electrophoresis to detect the concentration and purity of each RNA sample.

[0111] The agarose gel electrophoresis results of total RNA from spleen and total RNA from blood lymphocytes of unimmunized sharks are shown in Fig.10 , Fig.11 As shown; the agarose gel electrophoresis results of the total RNA of the spleen and blood lymphocytes of the immunized shark are shown Fig.12 As shown, Fig.12 Lanes 1-3 are total RNA bands from the spleen of immunized sharks, and lanes 4-6 are total RNA bands from the blood lymphocytes of immunized sharks. Three complete bands can be observed: 28S, 18S, and 5S, proving that the total RNA from the spleen and blood lymphocytes of the non-immunized and immunized sharks mentioned above is well extracted, the RNA is relatively complete, and the degree of degradation is low, and it can be used for subsequent experiments.

[0112] (2) Reverse transcription was performed using the non-immune and immunized shark spleen total RNA and blood lymphocyte total RNA extracted as the template RNA. The reverse transcription system was as follows (all reagents used were provided in the cDNA reverse transcription kit): 5× PrimeScriptIV cDNA Synthesis Mix 4 μL, Random 6 mers (50 μM) 1 μL, template RNA 5 μg, RNaseFree dH2O to make up to 20 μL; the reverse transcription program was as follows: 30℃ 10 min, 42℃ 20 min, 70℃ 15 min, and stored at 4℃. 5 μL of each of the 12 unimmunized shark spleen cDNA samples and 12 unimmunized shark blood lymphocyte cDNA samples were taken into new 1.5 mL EP tubes and flicked to mix them for subsequent construction of shark natural libraries; 5 μL of each of the 3 immune shark spleen cDNA samples and 3 immune shark blood lymphocyte cDNA samples were taken into new 1.5 mL EP tubes and flicked to mix them for subsequent construction of shark immune libraries.

[0113] S4. Amplification of vNAR fragment, restriction digestion and ligation of pComb3XSS vector. The specific steps are as follows:

[0114] (1) The first round of PCR reaction was carried out using the 12 cDNA samples obtained above as templates (using the Q5 High-Fidelity DNA Polymerase Kit). The first round of PCR reaction system was: Q5 High GC Enhancer 10 μL, Q5 Reaction Buffer 10 μL, Q5 High-Fidelity DNA Polymerase 0.5 μL, cDNA template 1 μL, vNAR-F (10 μM) 2.5 μL, vNAR-R (10 μM) 2.5 μL, dNTPs 1 μL, and ddH2O was added to make the volume to 50 μL; vNAR-F (SEQ ID NO.4): 5'-CGTGGCCCAGGCGGCCATGGCCSMACGGSTTGAACAAACACC-3'; vNAR-R (SEQ ID NO.5): 5'-GCTGGCCGGCCTGGCCWTTCAGTCASARKGGTSCC-3'. The first round of PCR amplification program was: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, full extension at 72°C for 2 min, and storage at 4°C. The denaturation to extension steps totaled 30 cycles. After the first round of PCR reaction, the first amplification product was obtained, which was detected by 1.5% agarose gel electrophoresis. A band of about 350 bp was cut out using an ultraviolet gel excision instrument and recovered using an agarose DNA recovery kit to obtain the vNAR gene, which was the first recovery product. The specific electrophoresis results of the first amplification product of the non-immunized shark vNAR gene are shown in Fig.13 The electrophoresis results of the first amplified product of the immune shark vNAR gene are shown in Fig.14 .

[0115] (2) The non-immunized shark vNAR gene and the immune shark vNAR gene obtained above were used to perform restriction digestion with the pComb3XSS vector, respectively. The restriction digestion system was: 2 μL of 10× NEBuffer, 0.5 μL of SacI enzyme, 0.5 μL of SpeI enzyme, 6 μL of vNAR gene or 1 μL of pComb3XSS vector, and ddH2O was added to make the volume 20 μL; the restriction digestion conditions were 37℃ for 2 h and 80℃ for 20 min. After the restriction digestion reaction, the restriction digestion product of the vNAR gene was recovered using a PCR product recovery kit to obtain a second recovery product; the restriction digestion product of the pComb3XSS vector was detected by 1.5% agarose gel electrophoresis, and a band of about 341 bp was cut out and recovered using a gel recovery kit to obtain a third recovery product. The specific electrophoresis results of the third recovery product of the non-immunized shark are shown in Fig.15 The electrophoresis results of the third recovered product of immune shark are shown in Fig.16 .

[0116] (3) The second recovery product and the third recovery product obtained above were connected. The connection system was: T4 DNA Ligase Buffer (10×) 2 μL, T4 DNA Ligase 1 μL, the second recovery product 20 ng, the third recovery product 64 ng, add ddH2O to make up to 20 μL; the connection condition was 16°C overnight connection to obtain the second connection product. The second connection product was purified by ethanol precipitation: add 1 / 10 volume of 3 M NaAc, then add 2.5 times the volume of anhydrous ethanol, and mix well. Freeze at -20℃ for 1 hour to fully precipitate the DNA; centrifuge at 4℃, 12000 × g for 5 min, and discard the supernatant; add about 1 ml of 70% ethanol, gently tap the bottom of the tube to make the precipitate float slightly to wash and remove salts; centrifuge at 4℃, 12000 × g for 5 min, discard the supernatant and then carefully aspirate the supernatant with a pipette; dry until no obvious residual liquid is visible, dissolve the DNA precipitate with an appropriate amount of water to a concentration of about 100 ng / μL, and obtain the second ligation product after purification from non-immune shark and the second ligation product after purification from immune shark.

[0117] Preparation of S5 and SS320 electroporation competent cells. The specific steps are as follows:

[0118] (1) Pick a single SS320 colony from the streak track of a tetracycline-resistant (Tet) 2×YT plate and place it in 4 mL of 2×YT liquid culture medium. Add Tet at a ratio of 1:1000 and shake overnight at 37°C and 220 rpm to obtain two tubes of seed solution.

[0119] (2) Inoculate the two tubes of seed solution into 400 mL 2×YT liquid medium at a ratio of 1:100, add Tet at the same ratio as above, and shake at 37°C, 220 rpm until OD 600 =0.5, and the first SS320 culture solution was obtained.

[0120] (3) Divide the first culture medium of SS320 into two 400 mL sterile centrifuge bottles, balance them strictly, and pre-cool them on ice for 30 min.

[0121] (4) Centrifuge at 4000 × g for 10 min at 4°C, discard the supernatant, and invert onto absorbent paper for 2 min to fully remove the residual liquid and retain the first bacterial pellet of SS320.

[0122] (5) Add 400 mL of pre-cooled sterile water to each centrifuge bottle and resuspend the SS320 first bacterial pellet by pipetting.

[0123] (6) Centrifuge at 4000 × g for 15 min at 4°C, discard the supernatant, and invert onto absorbent paper for 2 min to obtain the second bacterial precipitate of SS320.

[0124] (7) Add 200 mL of pre-cooled 10% glycerol to each centrifuge bottle and resuspend the SS320 second bacterial pellet by pipetting.

[0125] (8) Centrifuge at 4000 × g for 15 min at 4°C, discard the supernatant, and invert onto absorbent paper for 2 min to obtain the SS320 third bacterial cell precipitate.

[0126] (9) Add 10 mL of pre-cooled 10% glycerol to each centrifuge bottle, pipette and resuspend the second bacterial pellet of SS320, and transfer the bacterial suspension in the two centrifuge bottles into two sterile 15 mL centrifuge tubes.

[0127] (10) Centrifuge at 4000 × g for 15 min at 4°C. Discard the supernatant and invert the tube onto absorbent paper for 2 min. Use a pipette to remove any remaining liquid to obtain the fourth bacterial cell precipitate of SS320.

[0128] (11) Add 1.2 mL of pre-cooled 10% glycerol to each centrifuge tube, pipette and resuspend the SS320 fourth bacterial cell pellet, combine the bacterial suspensions in the two tubes into one tube, and dispense into EP tubes, 100 μL per tube, snap-freeze in liquid nitrogen, and store at -80°C to obtain SS320 E. coli competent cells.

[0129] S6. Construction of bacterial library. The specific steps are as follows:

[0130] (1) Electroporation: Take out 1 tube of SS320 E. coli competent cells from the -80℃ ultra-low temperature freezer, place on ice for 5 minutes to thaw, add 100 ng of the purified second ligation product using a pre-cooled pipette tip, blow gently to evenly mix, transfer to a pre-cooled 1 mm electroporation cup, tap the cup to allow the mixture to flow to the bottom of the cup. Set 1.8 kV, 25 μF, 200Ω, 1 mm spacing, and perform electroporation. Immediately after electroporation, add 900 μL of 37℃ pre-warmed SOC medium to obtain electroporation solution. Quickly remove the electroporation solution from the electroporation cup into a 1.5 ml sterile centrifuge tube, and shake and recover at 37℃, 200 rpm for 1 hour to obtain the recovered bacterial solution. The revived bacterial solution was diluted 10-fold using 2×YT liquid medium, for a total of 6 dilutions (i.e., 100 μL of the revived bacterial solution was diluted to 1000 μL, and then 100 μL was diluted to 1000 μL), and so on for a total of 10 dilutions. 1 -10 6Take 100 μL from each tube of the gradient dilution and spread it evenly on 2×YT(Amp) solid culture medium. Incubate at 37℃ overnight to make the electrotransfer plate. Count the number of colonies on the gradient dilution electrotransfer plate and calculate the connection efficiency using the following formula:

[0131] ,

[0132] Note: E is the ligation efficiency (unit: pfu / 100 ng), D is the dilution factor, and N is the number of monoclones on the plate with the corresponding dilution factor.

[0133] The results of electroporation of non-immunized sharks are as follows Fig.17 As shown, 10 -6 There is one single clone growing on the plate, and the ligation efficiency is calculated to be about 1×10 7 cfu / 100 ng, the library capacity is good.

[0134] The results of electroporation of immune sharks are as follows Fig.18 As shown, 10 -4 There are 13 single clones growing on the plate, and the ligation efficiency is calculated to be about 1.3×10 6 cfu / 100 ng, the library capacity is good.

[0135] (2) Positive clone verification: 48 monoclonal bacteria on the plate were randomly selected and inoculated into a centrifuge tube containing 1 ml of 2×YT(Amp) liquid culture medium. The culture was incubated at 37°C overnight to obtain the second monoclonal bacterial solution. The second monoclonal bacterial solution was used as a template for PCR reaction, which was the second round of PCR reaction. The second round of PCR reaction system was: 2× Rapid Taq Master Mix 10 μL, second monoclonal bacterial solution 1 μL, vNAR-F 1 μL, pComb3XSS-R 1 μL, and ddH2O was added to make the volume up to 20 μL. Among them, pComb3XSS-R (SEQ ID NO.6): 5'-GCCCCCTTATTAGCGTTTGCCATC-3'; the second round of PCR reaction program is: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 5 s, full extension at 72℃ for 5min, and storage at 4℃, wherein the denaturation to extension steps total 28 cycles. After the second round of PCR reaction, the second amplification product was obtained, which was detected by 1% agarose gel electrophoresis, and the positive cloning rate of the purified second ligation product was calculated by the following formula:

[0136] ,

[0137] Note: p is the positive clone rate, Np is the number of single colony PCR positives, and Nt is the total number of PCR reactions. When the positive clone rate is greater than 95%, subsequent experiments will be carried out.

[0138] Positive clone results of non-immunized sharks are as follows Fig.19 As shown, the effective insertion rate of the library reached 100%.

[0139] Positive clone results of immunized sharks are as follows Fig. 20 As shown, the effective insertion rate of the library reached 100%.

[0140] (3) Large-scale construction of bacterial library: 16 electroporation transformation reactions were randomly selected according to the S6 (1) method. All the revived bacterial liquid was concentrated into a 15 mL sterile centrifuge tube and mixed. 100 μL of the revived bacterial liquid was taken to calculate the ligation efficiency and positive clone rate according to the S6 (1) and (2) methods, respectively. The remaining revived bacterial liquid was evenly spread on 6 130 mm square culture medium plates containing 2% agarose with 100 μg / mL (Amp) and 2% glucose, and cultured upright at 37°C overnight.

[0141] Take the square plates that were cultured overnight, add 2 mL of 2×YT liquid culture medium on the surface of each culture plate, gently scrape off the colonies on the 6 square plates and collect the bacterial liquid into the same 50 mL centrifuge tube, add glycerol with a final concentration of 20%, and this is the bacterial library. The library capacity is calculated according to the following formula

[0142] ,

[0143] Note: C is the library capacity, E is the ligation efficiency, and p is the positive clone rate.

[0144] Take 20 μL of the bacterial solution collected on the square plate above and add it to 980 μL of 2×YT liquid medium and measure the OD using a spectrophotometer. 600 Calculate the total OD of the bacterial library according to the following formula: 600 ; Divide the bacterial library solution into 10 tubes, 1 mL / tube, and keep the rest in 50 mL centrifuge tubes and store at -80℃.

[0145] ,

[0146] Note: OD600 is the total bacterial OD 600 , M OD600 The measured OD 600

[0147] The positive clones from the second round of PCR amplification reaction were used for sequencing, and the nanobody fragments in the sequencing results were translated into amino acid sequences and then compared to detect the sequence diversity in the bacterial library.

[0148] The sequencing results of the bacterial library of non-immunized sharks are as follows Fig.21 As shown, 48 single colonies were expanded and cultured and sent for sequencing for comparative analysis, and it was found that they were all gene sequences encoding nanoantibodies, and the library had good diversity and no repetitive sequences.

[0149] The sequencing results of the immune shark bacterial library are as follows Fig. 22 As shown, 48 single colonies were expanded and cultured and sent for sequencing for comparative analysis, and it was found that they were all gene sequences encoding nanoantibodies, and the library had good diversity and no repetitive sequences.

[0150] S7. Construction of phage library. The specific steps are as follows:

[0151] (1) Phage amplification:

[0152] Take out the bacterial library from the -80℃ freezer, thaw on ice, calculate the corresponding bacterial liquid volume according to the following formula, and transfer it to 100 ml of 2×YT liquid culture medium containing 10 μg / mL Tet and 100 μg / mL Amp so that the initial OD600 is 0.1:

[0153] ,

[0154] Note: V is the volume of the transfer bacterial solution (in μL), OD600 is the total OD of the constructed bacterial library 600 .

[0155] Incubate in a constant temperature shaker at 37°C and 250 rpm until the bacterial solution reaches OD 600 =0.5-0.55.

[0156] Calculate and add helper phage M13KO7 according to the following formula so that the number of bacteria: number of phages = 1:20:

[0157] ,

[0158] Note: V is the volume of helper phage added (unit: mL), T_(helper-phage) is the titer of helper phage used, OD 600 is the OD of bacterial solution 600 value.

[0159] The culture was continued in a constant temperature shaker at 37°C and 220 rpm for 30 min.

[0160] Kana with a final concentration of 50 μg / mL and IPTG with a final concentration of 0.2 mM were added respectively, and cultured overnight in a constant temperature shaker at 30°C and 250 rpm to obtain the first induced bacterial solution.

[0161] (2) Preparation of phage library:

[0162] Transfer the bacterial solution after the first induction to a new 50 mL centrifuge tube, centrifuge at 4000 rpm, 4℃ for 20 min, and retain the supernatant, which is the first supernatant. Transfer the first supernatant to a new 50 mL centrifuge tube, add 1 / 4 volume of 4℃ pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly, place on ice for 30 min, centrifuge at 4000 rpm, 4℃ for 20 min, discard the supernatant, and invert on paper for 2 min to obtain the first precipitate. Add 1 ml PBS to resuspend the first precipitate and transfer to a new 1.5 ml centrifuge tube, centrifuge at 12000 rpm, 4℃ for 20 min to obtain the second supernatant. Transfer the second supernatant to a new 1.5 ml centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix well, place on ice for 10 min, centrifuge at 12000 rpm, 4°C for 10 min, discard the supernatant to obtain the second precipitate, add 1 ml PBS to resuspend the second precipitate, centrifuge at 12000 rpm, 4°C for 2 min to obtain the third supernatant, transfer the third supernatant to a new 1.5 ml centrifuge tube, which is the phage library, package at 100 μL / tube, and store at -80°C for a long time.

[0163] (3) Phage library titer detection:

[0164] The SS320 strain stored in a -80℃ refrigerator was streaked on a 2×YT (Tet) solid medium and cultured overnight at 37℃. A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium containing 10 μg / mL Tet and cultured overnight at 37℃. 500 μL of the overnight culture was transferred to 5 mL of 2×YT liquid medium containing 10 μg / ml Tet to obtain the second SS320 culture solution. The culture was carried out at 37℃, 250 rpm for about 45 min-60 min until the OD 600 =0.5-0.55.

[0165] Take 10 μL of the prepared phage library and dilute it 10 times in a 1.5 ml centrifuge tube. -12 , oscillate and mix, add 90 μL of the second SS320 culture solution to each dilution centrifuge tube, mix and incubate at 37°C for 30 min. Take 5 μL from each dilution centrifuge tube and drop it into 2×YT (Amp) solid culture medium, culture at 37°C overnight to obtain the third plate. Count the number of single colonies on the third plate at the dilution that can clearly distinguish single colonies, and calculate the number of phage particles per milliliter of phage solution, that is, the phage library titer, according to the following formula:

[0166] ,

[0167] Note: T is the phage titer (unit: pfu / mL), D is the dilution multiple, and N is the number of single colonies at the corresponding dilution multiple.

[0168] Helper phages were added to the bacterial library for library rescue. After obtaining the phage library, the titer of the phage library was determined. -10 A single colony grew on the gradient, and the final titer was calculated to be 4×10 12 pfu / mL shark natural phage library; shark immune phage library was finally -10 Two single colonies grew on the gradient, and the final titer was calculated to be 8×10 12 pfu / mL of shark immune phage library.

[0169] Example 3

[0170] Using the cp, nsp1, and nsp2 antigen proteins obtained in Example 1, the screening of cp, nsp1, and nsp2 nanoantibodies was performed using striped bamboo sharks, comprising the following steps:

[0171] S1. Screening of anti-cp, nsp1, nsp2 phage libraries. The specific steps are as follows:

[0172] (1) First round of screening: 50 μg of cp, nsp1 and nsp2 antigen proteins were coated in immunotubes with 2 mL of CBS coating solution at 4°C overnight. The CBS coating solution was discarded the next day, and the immunotubes were rinsed three times with 1×PBS for 5 min each time. Then 2 mL of 3% BSA was added as blocking solution, and the blocking was carried out by rotating at room temperature for 2 h. The 3% BSA blocking solution was discarded, and the immunotubes were rinsed three times with 2 mL of 1×PBS for 5 min each time. After discarding the rinsing solution, 2 mL of 1×PBS was added, and then 100 μL of the natural phage library / immune phage library of striped bamboo shark was added. The mixture was incubated by rotating at room temperature for 1 h. The liquid in the immunotubes was discarded, and 2 mL of PBST solution was added and rotated for 20 times. The liquid in the immunotubes was discarded. 1 mL of 0.25 mg / mL trypsin solution was added and the phages were eluted by slowly rotating at room temperature for 30 min. Then, 10 µL of 10% AEBSF protease inhibitor was added to terminate the elution, and the trypsin eluate in the immunotube was transferred to a new 1.5 mL tube, which was the first round of eluate for cp, nsp1, and nsp2.

[0173] (2) First round of eluate titer detection: the method is the same as S7 (3) in Example 2.

[0174] (3) First round of eluate expansion: Culture 3 tubes of 5 mL SS320 bacterial solution to the logarithmic phase in advance, and make the OD 600 nm About 0.5-0.55 for standby use. Take 500 μL of the first round of elution of cp, nsp1, and nsp2 (the remaining elution is stored at 4°C) and add them to the newly cultured 5 mL SS320 bacterial solution. Put it in a shaker at 37°C and 250 rpm for 30 min. Spread all 5.5 mL of the cultured bacterial solution evenly on three 130×130 mm 2×YT square solid culture media (containing 2% glucose, 2% agarose, and 100μg / mL Amp resistance), 3 plates each for cp, nsp1, and nsp2, a total of 9 plates, and culture them at 37°C overnight, which is the fourth plate of cp, nsp1, and nsp2. Add 2 mL of Tet-resistant 2×YT liquid culture medium to the surface of the fourth plate, scrape all the colonies on the plate with a cell scraper and collect the mixed bacterial solution into a 15 mL centrifuge tube, which is the first mixed bacterial solution of cp, nsp1, and nsp2, 6 mL per tube, and measure the OD of the bacterial solution at the same time. 600nm Then add glycerol with a final concentration of 20% to the first mixed bacterial solution of cp, nsp1, and nsp2, and store it in aliquots at -80°C, which is the bacterial library of the first round of eluate of cp, nsp1, and nsp2. 600 nm Substitute the value into the following formula: V (μL) = 10 / OD 600 ×1000, calculate the volume of the first bacterial culture of cp, nsp1, and nsp2, and transfer it to 100 mL of Amp and Tet-resistant 2×YT liquid medium, which is the second bacterial culture of cp, nsp1, and nsp2. Shake at 37°C and 220 rpm until the logarithmic phase and measure the OD 600 nm = about 0.5, and then the helper phage rescue is performed. The volume of helper phage added refers to the following formula (T_(helper-phage) is the titer of the helper phage used):

[0175] .

[0176] After being placed in a 37°C shaker for further culturing for 30 min, kanamycin with a final concentration of 50 μg / mL and IPTG with a final concentration of 0.2 mM were added thereto under a sterile environment, and then placed in a shaker for culturing at 30°C overnight to obtain the third induced bacterial solution of cp, nsp1, and nsp2.

[0177] (4) Purification of the first-round phage sub-library: The method is the same as S7 (2) in Example 2. The bacterial liquid after the third induction of cp, nsp1, and nsp2 is purified to obtain the first-round phage sub-library of cp, nsp1, and nsp2, which serves as the input phage library for the second round of screening.

[0178] (5) Second round of panning: The method is the same as step S1 (1) of Example 3, except that the phage library added is the phage sub-library obtained in the first round of screening, and the second round eluate of cp, nsp1, and nsp2 is screened. The titer detection, amplification and purification steps of the second round eluate are also the same as step S1 (2, 3) of Example 3. Finally, the second round phage sub-library of cp, nsp1, and nsp2 is obtained after purification, which is used as the input phage library for the third round of screening.

[0179] (6) The third round of panning: The method is the same as step S1 (1) of Example 3. The phage library added is the second round phage sub-library of cp, nsp1, and nsp2, and finally the third round eluate of cp, nsp1, and nsp2 is obtained.

[0180] From the three rounds of screening data of cp, nsp1, and nsp2, it can be seen that this method can effectively enrich nanoantibodies that specifically bind to cp, nsp1, and nsp2. Then, phage-ELISA (phage display technology) is used to further test their affinity.

[0181] Table 5 Results of three rounds of panning of anti-cp nanobodies in natural phage library

[0182] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[8×10 12 ]]> <![CDATA[3.2×10 6 ]]> <![CDATA[4×10 -7 ]]> - Round 2 <![CDATA[1.2×10 12 ]]> <![CDATA[4×10 7 ]]> <![CDATA[3.3×10 -5 ]]> 82.5 Round 3 <![CDATA[1.6×10 13 ]]> <![CDATA[2.4×10 8 ]]> <![CDATA[1.5×10 -5 ]]> 0.5

[0183] Recovery rate = elution amount ÷ input amount,

[0184] Enrichment = recovery rate of the next round ÷ recovery rate of the previous round.

[0185] Table 6 Results of three rounds of panning of anti-nsp1 nanobodies in natural phage library

[0186] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[8×10 12 ]]> <![CDATA[2.4×10 6 ]]> <![CDATA[3×10 -7 ]]> - Round 2 <![CDATA[4×10 12 ]]> <![CDATA[4×10 7 ]]> <![CDATA[1×10 -5 ]]> 33 Round 3 <![CDATA[6.4×10 12 ]]> <![CDATA[8×10 7 ]]> <![CDATA[1.25×10 -4 ]]> 12.5

[0187] Table 7 Results of three rounds of panning of anti-nsp2 nanobodies in natural phage library

[0188] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[8×10 12 ]]> <![CDATA[1.6×10 6 ]]> <![CDATA[2×10 -7 ]]> - Round 2 <![CDATA[4×10 12 ]]> <![CDATA[1.2×10 8 ]]> <![CDATA[3×10 -5 ]]> 150 Round 3 <![CDATA[4.8×10 12 ]]> <![CDATA[4×10 8 ]]> <![CDATA[8.3×10 -5 ]]> 2.8

[0189] Table 8 Results of three rounds of panning of anti-cp nanobodies in the immune phage library

[0190] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[4×10 12 ]]> <![CDATA[4×10 5 ]]> <![CDATA[1×10 -7 ]]> - Round 2 <![CDATA[8×10 12 ]]> <![CDATA[4×10 7 ]]> <![CDATA[5×10 -6 ]]> 50 Round 3 <![CDATA[1.2×10 12 ]]> <![CDATA[4×10 6 ]]> <![CDATA[3.3×10 -6 ]]> 0.7

[0191] Table 9 Results of three rounds of panning of anti-nsp1 nanobodies in the immune phage library

[0192] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[4×10 12 ]]> <![CDATA[1.2×10 6 ]]> <![CDATA[3×10 -7 ]]> - Round 2 <![CDATA[4×10 11 ]]> <![CDATA[8×10 6 ]]> <![CDATA[2×10 -5 ]]> 67 Round 3 <![CDATA[2.4×10 12 ]]> <![CDATA[1.2×10 7 ]]> <![CDATA[5×10 -5 ]]> 2.5

[0193] Table 10 Results of three rounds of panning of anti-nsp2 nanobodies in immune phage library

[0194] Selection rounds Input amount (pfu) Elution volume (pfu) Recovery rate Enrichment Round 1 <![CDATA[4×10 12 ]]> <![CDATA[1.6×10 5 ]]> <![CDATA[4×10 -8 ]]> - Round 2 <![CDATA[8×10 12 ]]> <![CDATA[2.4×10 7 ]]> <![CDATA[3×10 -6 ]]> 75 Round 3 <![CDATA[4×10 12 ]]> <![CDATA[4×10 7 ]]> <![CDATA[1×10 -5 ]]> 3.3

[0195] S2. Identification and analysis using phage-ELISA. The specific steps are as follows:

[0196] (1) Preparation of phage supernatant

[0197] Prepare 5 mL culture of SS320 E. coli to OD 600 nm The value is about 0.5 and is reserved. Take 10 μL of cp, nsp1, and nsp2 three rounds of eluate and dilute them in 10-fold concentration gradient (10 μL + 90 μL sterile water in each tube) to 10 -12 , a total of 12 tubes, mix well for later use. Add 90 μL of SS320 logarithmic phase bacterial solution to each tube, pipette to mix well, and incubate at 37°C for 30 min to make the third expansion bacterial solution for cp, nsp1, and nsp2. Take 5 μL from each tube of the third expansion bacterial solution of cp, nsp1, and nsp2 and apply it to the Amp-resistant 2×YT solid culture plate, and evenly apply the bacterial solution, invert and culture at 37°C overnight to make the fifth plate for cp, nsp1, and nsp2. Take a sterile 96-well cell culture plate, add 200 μL of Amp- and Tet-resistant 2×YT liquid culture medium to each well, then use a sterile pipette tip to randomly pick 96 monoclonal clones from the fifth plate and add them to each well, and place them in a 37°C incubator for overnight incubation to make the first cell culture plate for cp, nsp1, and nsp2.

[0198] Take 2 μL of the bacterial solution in each well of the first cell culture plate of cp, nsp1, and nsp2 (the remaining bacterial solution is stored at 4°C) and transfer it to a new 96-well cell culture plate, place it in a 37°C incubator and culture it for 3 hours to form the second cell culture plate of cp, nsp1, and nsp2. Then calculate the volume of helper phage added to each well according to the following formula (T_(helper-phage) is the titer of the helper phage used):

[0199] .

[0200] After being placed in a 37°C incubator for 30 minutes, Kana with a final concentration of 50 μg / mL and IPTG with a final concentration of 0.2 mM were added under a sterile environment, and then placed in a shaker for overnight culture at 30°C to obtain the fourth induced bacterial solution of cp, nsp1, and nsp2. Subsequently, the second cell culture plate of cp, nsp1, and nsp2 was centrifuged at 5000 rpm at 4°C for 10 minutes to obtain the first phage supernatant of cp, nsp1, and nsp2.

[0201] (2) Phage-ELISA identification

[0202] 96-well ELISA plates were coated with cp, nsp1, and nsp2 antigen proteins, respectively. The coating solution was CBS (antigen concentration was 1 ng / μL, and the coating volume was 100 μL / well). At the same time, two 96-well ELISA plates with BSA coating solution (BSA concentration was 1 ng / μL, and the coating volume was 100 μL / well) were set as control groups. They were all placed in a 4°C low-temperature incubator for overnight coating. The next day, the coating solution was discarded, and each well was rinsed three times with 200 μL PBS, each for 10 min. 200 μL of 3% BSA was added as blocking solution and blocked at room temperature for 1 hour. The blocking solution was discarded, and 200 μL PBS was added to each well for washing, and the plates were washed three times, each for 10 min.

[0203] Add 120 µL 3% BSA to each well of the 96-well ELISA plate, then add 80 µL of the first phage supernatant of cp, nsp1, and nsp2 as the primary antibody, incubate at room temperature for 2 h, discard the primary antibody, rinse each well three times with 200 μL PBST, and let stand for 10 min each time. Then add M13 mouse anti-phage monoclonal antibody (HRP labeled) as the secondary antibody, diluted 1:40000 in 3% BSA, add 100 µL to each well, incubate at room temperature for 1 h, discard the secondary antibody, add 200 µL PBST to each well for washing, wash three times at room temperature, and let stand for 10 min each time. Then color development: use TMB single-component color development solution to color each well, add 100 μL to each well, incubate for 5 min, and use 100 μL HCl (1 M) to stop color development. Read OD with an ELISA reader 450 nm Numeric value.

[0204] When processing the color development results, compare the OD values ​​of the cp, nsp1, and nsp2 antigen protein incubation wells with their corresponding BSA-coated control wells. 450 nm The ratios of the values ​​are sorted from large to small. For specific numbers of positive clones screened by phage-ELISA, see Fig.23Finally, 3 positive clones were obtained from cp in the natural library, 2 positive clones were obtained from nsp1, and 5 positive clones were obtained from nsp2; 7 positive clones were obtained from cp in the immune library, 5 positive clones were obtained from nsp1, and 6 positive clones were obtained from nsp2. A total of 28 monoclonal bacterial liquids were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The primer P1 (SEQ ID NO.7) required for sequencing was: 5'-CCAGGCTTTACACTTTATGC-3'. The sequencing result was the result of the second sequencing.

[0205] Example 4

[0206] The expression and identification of the nanobodies against cp, nsp1, and nsp2 antigen proteins obtained in Example 3 comprises the following steps:

[0207] S1. Construction of prokaryotic expression vector. The specific steps are as follows:

[0208] According to the second sequencing results, sequence comparison analysis was performed, and a total of 7 vNAR antibody amino acid sequences with different CDR3 regions were screened. Among them, 3 vNAR sequences were screened from the shark natural library: cp-N-E1 (SEQ ID NO.8), nsp1-N-F9 (SEQ ID NO.9) and nsp2-N-B5 (SEQ ID NO.10). 4 vNAR sequences were screened from the shark immune library: cp-I-C5 (SEQ ID NO.11), nsp1-I-F3 (SEQ ID NO.12), nsp2-I-B12 (SEQ ID NO.13) and nsp2-I-H5 (SEQ ID NO.14). The sequence comparison results are shown in Fig.24 As shown. Seven vNAR antibody sequences were codon optimized, fully synthesized, and recombinant plasmids were constructed by Bioengineering, and then cloned into the pET-28a prokaryotic expression vector to obtain the recombinant plasmids.

[0209] S2. Purify the expression of the recombinant plasmid. The specific steps are as follows:

[0210] Take 1 µL of the recombinant plasmid and add it to 50 µL of BL21 (DE3) competent cells, and place it on ice for 30 min. Then heat shock at 42°C for 90 s, and immediately place it on ice for 5 min. Add 900 µL of LB medium without resistance in a sterile environment, shake and culture at 37°C and 220 rpm for 45 min, which is the second transformation bacterial solution of cp, nsp1, and nsp2. Then take 100 µL of the second transformation bacterial solution of cp, nsp1, and nsp2 and spread it on the Kana-resistant LB solid medium, invert and culture overnight at 37°C, which is the second plate. The next day, pick a single colony from the sixth plate to 5 mL of Kana-resistant LB liquid medium, shake and culture at 37°C and 220 rpm until the logarithmic phase, add glycerol and bacterial solution to the cryovial, and store it at -80°C, which is the BL21 (DE3) glycerol bacteria of vNAR nanoantibody.

[0211] The expression, purification, desalting and concentration steps of vNAR nanobody were the same as those in Example 1 to obtain vNAR nanobody. The prokaryotic expression and purification results of vNAR nanobody (SEQ ID NO. 8-10) screened from shark natural library are shown in Fig.25 As shown, the prokaryotic expression and purification results of vNAR nanobodies (SEQ ID NO.11-14) screened from shark natural library are shown in Fig.26 As shown. The BCA kit was used to determine the concentration of cp, nsp1, and nsp2 nano antibody proteins, and the molecular weights of the nano antibody proteins of SEQ ID NO.8-14 were 14.9 kDa, 15.4 kDa, 14.8 kDa, 15.4 kDa, 15.4 kDa, 15.2 kDa, and 14.6 kDa, respectively; the concentrations of the nano antibody proteins of SEQ ID NO.14-20 were: 1.0 mg / mL, 1.3 mg / mL; 1.6 mg / mL, 0.4 mg / mL, 1.3 mg / mL, 0.7 mg / mL, and 0.1 mg / mL, respectively. After aliquoting, they were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0212] S3. Western blot identification of vNAR nanobody protein. The specific steps are as follows:

[0213] (1) SDS-PAGE electrophoresis: Use the Omni-Easy™ One-Step PAGE Gel Rapid Preparation Kit to prepare 15% PAGE gel. Take 40 μL of vNAR nanobody protein and add 10 μL of 5× loading buffer. Incubate in a 99°C metal bath for 10 min and centrifuge at 13,000 rpm for 10 min. The sample volume is 10 μL and the parameters are set to 200 V for 40 min.

[0214] (2) Electrotransfer: After electrophoresis, remove the PAGE gel and soak it in ddH2O. Immerse the PVDF membrane of the same size as the gel in methanol for activation. Arrange the sponge, filter paper, protein gel and PVDF membrane in order, remove bubbles with a roller, clamp and place in the transfer apparatus. Pour in the pre-cooled transfer solution, cover the transfer apparatus and bury it in ice, set the parameters to 200 mM, 90 min.

[0215] (3) Washing: After transfer, remove the PVDF membrane and wash it 6 times with TBST, 3 times on the front side and 3 times on the back side, each time for 5 min.

[0216] (4) Blocking: Add 5% skimmed milk powder solution as blocking solution and block at room temperature for 2 h.

[0217] (5) Incubation with primary antibody: discard the blocking solution, add 1:3000 diluted mouse anti-His as primary antibody, and incubate at room temperature for 1 h.

[0218] (6) Washing: Wash 6 times with TBST, 3 times on the front side and 3 times on the back side, each time for 5 min.

[0219] (7) Incubation with secondary antibody: Add 1:5000 diluted goat anti-mouse antibody (HRP) as secondary antibody and incubate at room temperature for 1 h.

[0220] (8) Washing: Wash 6 times with TBST, 3 times on the front side and 3 times on the back side, each time for 5 min.

[0221] (9) Add 2 mL of ECL colorimetric solution to the membrane and incubate in the dark for 2 min before illuminating the membrane. The protein specificity is good and there are no extra bands. The specific Western blot identification of shark vNAR nanobody protein after purification is shown in Fig. 27 The band of vNAR nanobody protein is single and free of other bands, with good specificity.

[0222] S4. ELISA verifies the binding ability of vNAR nanobody to cp, nsp1, and nsp2 antigen proteins. The specific steps are as follows:

[0223] (1) Antigen coating: Dilute the cp, nsp1, and nsp2 antigen proteins to 1 μg / mL with CBS coating solution, add 100 μL to each well, and coat overnight at 4°C.

[0224] (2) Washing the plate: Pour out the liquid in the plate and pat dry on absorbent paper (be careful to avoid leaving fingerprints under the ELISA plate when patting dry to affect subsequent readings). Wash each well with 200 μL PBS three times, 5 min each time.

[0225] (3) Blocking: Add 200 μL of 1% BSA solution to each well for 1 h at room temperature.

[0226] (4) Washing: Pour off the blocking solution and wash each well three times with 200 μL PBS, each time for 5 min.

[0227] (5) Primary antibody: Different concentration gradients were set for each vNAR nanoantibody and positive control in 3 parallel groups. The negative control was BSA. 100 μL was added to each well and incubated for 1 h.

[0228] (6) Washing: Discard the liquid in the plate and add 200 μL PBST to each well for washing. Wash three times, 5 min each time.

[0229] (7) Secondary antibody: Add 100 μL vNAR nanoantibody to each well and add diluted enzyme-labeled antibody (mouse anti-his-HRP) to the corresponding well. The secondary antibody for the positive control is goat anti-rabbit-HRP. Incubate at room temperature for 1 h.

[0230] (8) Washing the plate: Discard the liquid in the plate and wash with 200 μL PBST for 8 times, 5 min each time.

[0231] (9) Color development: Add 100 μL of TMB color development solution to each well, shake to mix, and incubate in the dark for 5 min.

[0232] (10) Termination: After color development is completed, add 100 μL 1M HCl to each well to terminate the color development.

[0233] (11) Reading: OD on the microplate reader 450 nm Read the ELISA plate value.

[0234] See the specific vNAR nanobody ELISA affinity verification diagram for Fig.28 As can be seen from the figure, for each antigen protein, the affinity of the nanoantibodies screened from the immune library is stronger than that of the nanoantibodies screened from the natural library.

[0235] Example 5

[0236] The vNAR nanobody obtained in Example 4 was preliminarily applied to the colloidal gold chromatography test strip. The specific steps are as follows:

[0237] (1) Preparation of colloidal gold (20,000th of 30 nm colloidal gold)

[0238] Prepare a clean 250 mL conical flask, wash it repeatedly with pure water 5-6 times, and dry it for later use. Put in a B-type stirring rotor, then put it on an electronic balance to peel it, add 189 g of pure water to the conical flask, and then use a pipette to add 2 g of 2% chloroauric acid (HAuCl4) solution, transfer the conical flask to a heated stirrer to heat it, and when the temperature of the conical flask is heated to about 95°C, turn off the heating function, turn on the stirring function, and quickly add 9 mL of 1% trisodium citrate solution. The solution in the conical flask will slowly change from yellow to dark gray, and finally turn purple-red and react for another 5 minutes. After cooling to room temperature, put it in a 4°C refrigerator for later use. Take 1 mL of the fired colloidal gold solution. The fired colloidal gold solution is wine red, free of impurities or floating matter, and bright in color. Use a UV spectrophotometer to measure its absorption wavelength between 400-600 nm. If Fig.29 As shown, the particle size of colloidal gold is linearly related to its maximum absorption wavelength. The diameter of the colloidal gold particles can be calculated according to the linear equation y=0.4271x+514.56 (x is the particle size, y is the maximum absorption wavelength). The maximum absorption wavelength of colloidal gold is around 520 nm, so the particle size of colloidal gold can be calculated to be 12.7 nm.

[0239] (2) Labeling colloidal gold

[0240] Take 1 mL of colloidal gold solution in a 1.5 mL EP tube, add 10 μL of 0.2 M potassium carbonate (K2CO3) solution, and shake gently to adjust the pH value of colloidal gold. Then add 20 μg of vNAR nanoantibody protein solution, mix and incubate at room temperature for 5 min to label the antibody. Add 30 μL of 10% BSA solution to the antibody-labeled colloidal gold solution, mix and invert at room temperature for 5 min to block the colloidal gold. Place the blocked colloidal gold solution in a centrifuge and centrifuge at 8000 rpm for 6 min. Carefully pipette the supernatant of the colloidal gold solution into a new 1.5 mL centrifuge tube and continue centrifuging at 10000 rpm for 10 min, and discard the supernatant. Collect the precipitates obtained by centrifuging the blocked colloidal gold solution twice and add 100 μL of gold reconstitution solution to dissolve it, and finally obtain 100 μL of colloidal gold concentrate labeled with vNAR antibody.

[0241] (3) Gold laying and drying

[0242] Cut the glass fiber into 0.5 cm wide strips, soak them in the prepared gold label pad treatment solution for 40 min, and dry them at 42°C. After drying, dilute the colloidal gold concentrate labeled with vNAR antibody with 300 μL gold diluent, add it dropwise to the glass fiber, and dry it at 42°C.

[0243] (4) Assembly and identification of test strips

[0244] Use a film drawing machine to draw two different antibodies on the NC membrane at 1 μL / cm as the test line (T line) and quality control line (C line) of the test strip, and dry at 42°C. Cut the polyester into 20 mm wide strips, soak it in the sample pad treatment solution prepared in advance for 40 min, and then dry it at 42°C. Assemble the sample pad, gold label pad, NC membrane, and absorbent paper in order on the PVC base plate, overlap each two pads by about 0.2 cm, and then cut it into 4 mm wide test strips with a strip cutter. For the specific test strip structure diagram, see Fig.30 Then, different concentration gradients of cp antigen protein, nsp1 antigen protein and antigen protein were added to the sample pad for detection.

[0245] Comparative Example 1

[0246] The difference between this comparative example and Example 4 is that the antigen protein to be detected is different.

[0247] Among them, the antigenic proteins used in Comparative Example 1 are MBP (tag protein), SWP1 (shrimp hepatocellular carcinoma virus protein and VP28 (white spot syndrome virus virulence protein), all of which are proteins that have been expressed, purified and stored in the laboratory, and the antigenic proteins used in Example 4 are cp antigenic protein, nsp1 antigenic protein or nsp2 antigenic protein.

[0248] To further illustrate the advantages of the present invention, the antigen proteins used in Comparative Example 1 and Example 4 were subjected to the following test examples 1-6.

[0249] Test Example 1 Nanobody Cross-matching Test

[0250] It is concluded from step S6 in Example 3 that the affinity of the nanobodies screened from the shark immune library is better than that of the nanobodies screened from the shark natural library. Therefore, the three nanobodies cp-I-C5, nsp1-I-F3 and nsp2-I-B12 were used to detect the cp, nsp1, nsp2 antigen proteins in Example 1 and the colloidal gold test strip combination of Example 4, respectively. The combination of C line, T line and gold-labeled antibody in the colloidal gold test strip of the specific vNAR nanobody is shown in Table 11.

[0251] Table 11 Combinations of C-line, T-line and gold-labeled antibodies in colloidal gold test strips of three antigens

[0252] Serial number Gold labeled antibody T-line antibody C-line antibodies A cp-I-C5 cp rabbit polyclonal antibody Anti-his tag antibody A cp rabbit polyclonal antibody cp-I-C5 Goat anti-rabbit B nsp1-I-F3 NSP1 rabbit polyclonal antibody Anti-his tag antibody B NSP1 rabbit polyclonal antibody nsp1-I-F3 Goat anti-rabbit C nsp2-I-B12 nsp2 rabbit polyclonal antibody Anti-his tag antibody C nsp2 rabbit polyclonal antibody nsp2-I-B12 Goat anti-rabbit

[0253] The cp, nsp1, and nsp2 antigen proteins prepared in Example 1 at a concentration of 0.1 mg / mL were added dropwise to the colloidal gold test strips assembled in Comparative Example 1 and Example 4, respectively, and the color development results were observed after 5 minutes.

[0254] Test case 1 results are as follows Fig.31 As shown in the figure, the combination of test strips 1 and 2 is nano antibody as gold label antibody, rabbit polyclonal antibody as T line antibody, and anti-his tag antibody as C line antibody; the combination of test strips 3 and 4 is rabbit polyclonal antibody as gold label antibody, nano antibody as T line antibody, and goat anti-rabbit as C line antibody. When anti-his tag antibody is used as C line antibody, C line is not visible, and the appearance of T line is not as good as the other combination. Therefore, we choose rabbit polyclonal antibody as gold label antibody, nano antibody as T line antibody, and goat anti-rabbit as C line antibody. This antibody cross-pairing combination is the best combination and is used for subsequent specificity and sensitivity tests.

[0255] Test Example 2 Colloidal gold test strip specificity detection

[0256] Select the colloidal gold test strip combination with good color development in test example 1, that is, the gold-labeled antibody is a rabbit polyclonal antibody, the T-line antibody is a nano-antibody (wherein, A is the T-line antibody cp-I-C5 for detecting cp antigen protein; B is the T-line antibody nsp1-I-F3 for detecting snp1 antigen protein; C is the T-line antibody nsp2-I-B12 for detecting snp2 antigen protein), and the C-line antibody is a goat anti-rabbit antibody to prepare colloidal gold test strips for detecting three antigen proteins. Add 6 antigen protein samples (cp, snp1, snp2, MBP, SWP1 and VP28) with a concentration of 0.1 mg / mL to the test strips No. 1-6 in A, B, and C respectively, and observe the line development after about 5 minutes.

[0257] Test case 2 results are as follows Fig.32 As shown, in A, except for the test strip with cp antigen protein added, which has an obvious T line, the other test strips have no T line; in B, except for the test strip with nsp1 antigen protein added, which has an obvious T line, the other test strips have no T line; in C, except for the test strip with nsp2 antigen protein added, which has an obvious T line, the other test strips have no T line; this shows that the test strips for the three antigen proteins (cp, nsp1 and nsp2) have good specificity.

[0258] Test Example 3: Colloidal gold test strip sensitivity test

[0259] Select the colloidal gold test strip combination with good color development in Test Example 1, that is, the gold-labeled antibody is a rabbit polyclonal antibody, the T-line antibody is a nano antibody (wherein, A is the T-line antibody cp-I-C5 for detecting cp antigen protein; B is the T-line antibody nsp1-I-F3 for detecting snp1 antigen protein; C is the T-line antibody nsp2-I-B12 for detecting snp2 antigen protein), and the C-line antibody is a goat anti-rabbit antibody to prepare colloidal gold test strips for detecting three antigen proteins. 100 μL of antigen protein samples with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25μg / mL, 6.25 μg / mL and 3.125 μg / mL were added to the test strips No. 1-6 in the three groups A, B, and C respectively. The specific antigen concentrations in the colloidal gold test strips are shown in Table 10. The line development is observed after about 5-10 minutes.

[0260] Test case 3 results are as follows Fig.33 As shown, the color of the T line becomes lighter with the decrease of the concentration of cp, nsp1 or nsp2 antigen protein. Among them, the test strip sensitivity of cp antigen is 6.25 μg / mL, the test strip sensitivity of nsp1 antigen is 12.5 μg / mL, and the test strip sensitivity of nsp2 antigen is 3.125 μg / mL.

[0261] Test Example 4

[0262] The hepatopancreatic tissue grinding fluid of 10 shrimps (obtained from Putian, Fujian, numbered AJ) was taken and the RT-PCR method was used to detect whether the 10 shrimps were infected with IHHNV. At the same time, the hepatopancreatic tissue grinding fluid of 10 shrimps was diluted and added to the colloidal gold test strip, and the experimental results of the two methods were compared.

[0263] The comparison results of RT-PCR method and colloidal gold test strip are shown in Table 12. The detection results of colloidal gold test strip are consistent with the detection results of RT-PCR method. Among the 10 samples (numbered AJ), three samples C, I, and J are IHHNV positive samples, and the remaining 7 samples are negative samples. Fig.34 As shown, 1-3 are test strips for detecting three antigens, cp, nsp1 and nsp2, in positive samples. It can be seen that the T lines are all visible to varying degrees. This result shows that the colloidal gold immunochromatographic test strip based on nanoantibodies has good accuracy.

[0264] Table 12 Comparison of test results of colloidal gold test strips and RT-PCR

[0265] serial number RT-PCR Colloidal gold test strips A - - B - - C + + D - - E - - F - - G - - H - - I + + J + +

[0266] Test Example 5

[0267] Referring to Test Example 2, 100 μL of pathogenic protein with a concentration of 0.1 mg / mL was tested on a shrimp farming water sample enriched with the gold-labeled antibody being a rabbit polyclonal antibody, the T-line antibody being a nanoantibody (wherein, A: the T-line antibody is cp-I-C5 for detecting cp antigen protein; B: the T-line antibody is nsp1-I-F3 for detecting snp1 antigen protein; C: the T-line antibody is nsp2-I-B12 for detecting snp2 antigen protein), and the C-line antibody is a goat anti-rabbit antibody. The colloidal gold test strip prepared for detection was obtained. Fig.32 The specific test results of three basically identical antigen colloidal gold test strips indicate that the shrimp farming water sample contains IHHNV virus.

[0268] Test Example 6

[0269] Referring to Test Example 2, 100 μL of 0.1 mg / mL pathogenic protein solution enriched with shrimp aquaculture feed was tested, and the colloidal gold test strip prepared by using a rabbit polyclonal antibody as the gold-labeled antibody, a nano antibody as the T-line antibody (wherein, A: the T-line antibody is cp-I-C5 for detecting cp antigen protein; B: the T-line antibody is nsp1-I-F3 for detecting snp1 antigen protein; C: the T-line antibody is nsp2-I-B12 for detecting snp2 antigen protein), and a goat anti-rabbit antibody as the C-line antibody was tested, and the obtained result was the same as Fig.32 The specific test results of the three basically identical antigen colloidal gold test strips indicate that the shrimp farming feed solution contains the IHHNV virus.

[0270] It can be concluded that the vNAR nanoantibody provided by the present invention has good binding with colloidal gold, and a total of three antibody combinations are produced, among which the first antibody combination is used to specifically identify the cp antigen protein, including: rabbit polyclonal antibody as the first gold-labeled antibody, the first T-line antibody with an amino acid sequence of SEQ ID NO.11, and goat anti-rabbit antibody as the first C-line antibody; the second antibody combination is used to specifically identify the nsp1 antigen protein, including: rabbit polyclonal antibody as the second gold-labeled antibody, the second T-line antibody with an amino acid sequence of SEQ ID NO.12, and goat anti-rabbit antibody as the second C-line antibody; the third antibody combination is used to specifically identify the nsp2 antigen protein, including: rabbit polyclonal antibody as the third gold-labeled antibody, the third T-line antibody with an amino acid sequence of SEQ ID NO.13, and goat anti-rabbit antibody as the third C-line antibody. The test strip prepared by the combination of the above three antibodies specifically recognizes the cp, nsp1 and nsp2 proteins of the IHHNV virus. It can quickly detect whether aquatic animals such as shrimp are infected with the IHHNV virus, whether the aquaculture water contains the IHHNV virus, or whether the aquaculture feed and fresh bait are contaminated by the IHHNV virus within 10 minutes, thereby solving the effective prevention and efficient monitoring of epidemics in shrimp farming production, laying the foundation for the rapid detection of the IHHNV virus, and providing a reference model for the early detection of pathogens in other farmed animals. It has wide applicability.

[0271] The terms and expressions used herein are for descriptive purposes only and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean to exclude any equivalent features of the illustrations and descriptions (or parts thereof), and it should be recognized that various modifications that may exist should also be included in the scope of the claims. Other modifications, changes and substitutions may also exist. Accordingly, the claims should be deemed to cover all such equivalents.

[0272] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions may be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. An anti-cp nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus, characterized in that: The amino acid sequence of the anti-cp nanobody is SEQ ID NO.8 or SEQ ID NO.

11.

2. The anti-cp nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus according to claim 1, characterized in that: The anti-cp nanobody is shark-derived.

3. An anti-nsp1 nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus, characterized in that: The amino acid sequence of the anti-nsp1 nanobody is SEQ ID NO.9 or SEQ ID NO.

12.

4. The anti-nsp1 nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus according to claim 3, characterized in that: The anti-nsp1 nanobody is shark-derived.

5. An anti-nsp2 nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus, characterized in that: The amino acid sequence of the anti-nsp2 nanobody is SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.

14.

6. The anti-nsp2 nanobody for detecting infectious subcutaneous and hematopoietic necrosis virus according to claim 5, characterized in that The anti-nsp2 nanobody is shark-derived.

7. A product for detecting infectious subcutaneous and hematopoietic necrosis viruses comprising nanoantibodies, characterized in that: The nanobodies include anti-cp nanobodies for detecting infectious subcutaneous and hematopoietic necrosis virus, anti-nsp1 nanobodies for detecting infectious subcutaneous and hematopoietic necrosis virus, or anti-nsp2 nanobodies for detecting infectious subcutaneous and hematopoietic necrosis virus; The amino acid sequence of the anti-cp nanobody is SEQ ID NO.8 or SEQ ID NO.11; The amino acid sequence of the anti-nsp1 nanobody is SEQ ID NO.9 or SEQ ID NO.12; The amino acid sequence of the anti-nsp2 nanobody is SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.

14.

8. The infectious hypodermal and hematopoietic necrosis virus detection product according to claim 7, characterized in that: The infectious subcutaneous and hematopoietic necrosis virus detection product is a reagent, a test strip or a test kit; the test strip includes an immunochromatographic test strip prepared using any one of the labeling technologies of colloidal gold labeling, colloidal carbon labeling, fluorescent microsphere labeling, and nanoparticle labeling.

9. The infectious hypodermal and hematopoietic necrosis virus detection product according to claim 7, characterized in that: The infectious subcutaneous and hematopoietic necrosis virus detection product includes a first antibody combination, a second antibody combination or a third antibody combination; The first antibody combination is used to specifically identify the cp antigen protein, and the first antibody combination is: rabbit polyclonal antibody as the first gold-labeled antibody, the first T-line antibody with the amino acid sequence of SEQ ID NO.11, and goat anti-rabbit antibody as the first C-line antibody; The second antibody combination is used to specifically identify the nsp1 antigen protein, and the second antibody combination is: rabbit polyclonal antibody as the second gold-labeled antibody, the second T-line antibody with the amino acid sequence of SEQ ID NO.12, and goat anti-rabbit antibody as the second C-line antibody; The third antibody combination is used to specifically identify the nsp2 antigen protein, and the third antibody combination is: rabbit polyclonal antibody as the third gold-labeled antibody, the third T-line antibody with an amino acid sequence of SEQ ID NO.13, and goat anti-rabbit antibody as the third C-line antibody.

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