Set of shark nanobodies targeting infectious hypodermal and haematopoietic necrosis virus of shrimp, and preparation method therefor

Abstract

Provided is a set of shark nanobodies targeting an infectious hypodermal and haematopoietic necrosis virus of shrimp. The amino acid sequence of each nanobody in the set is selected from any one of SEQ ID NOs. 8-14. The nanobodies are useful for detecting cp, nsp1, and nsp2 antigens of the infectious hypodermal and haematopoietic necrosis virus. A preparation or screening method for the nanobodies comprises the following steps: step A: expression and purification of cp, nsp1, and nsp2 antigens; step B: construction of a shark naive library and / or a shark immune library; step C: helper phage rescue and purification to obtain a phage library; step D: panning of anti-cp, anti-nsp1, and anti-nsp2 antigen phage libraries; and step E: identification by phage-ELISA and sequencing, and sequence alignment to obtain cp, nsp1, and nsp2 nanobodies having different CDR3 region sequences.

Description

A group of shark nanobodies targeting infectious hypodermal and hematopoietic tissue necrosis viruses in shrimp and their preparation method

[0001] This application claims priority to Chinese Patent Application No. CN202510188063.2, filed on February 20, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of virus detection technology, specifically to a set of shark nanobodies targeting infectious hypodermal and hematopoietic tissue necrosis viruses in shrimp and their preparation methods. Background Technology

[0003] Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is the smallest of the major viruses affecting the global shrimp farming industry. IHHNV has a wide host range, infecting species such as Litopenaeus vannamei, Litopenaeus chinensis, and Penaeus monodon. Due to its high infectivity, infected shrimp exhibit mortality and slow growth. In Litopenaeus chinensis, the mortality rate can reach over 90%, while in Litopenaeus vannamei, it causes dwarfism syndrome. Although it does not lead to death, the high rate of deformities and slow, uneven growth can result in economic losses of up to 50%, posing a significant threat to the shrimp farming industry. IHHNV is primarily transmitted horizontally, mainly through infected shrimp, the food chain, cannibalism, and contaminated water. The highest infectivity is found in shrimp that consume infected shrimp. Infected shrimp can also infect their offspring vertically. With the expansion of my country's shrimp farming industry, the demand for customs disease testing is constantly increasing. Developing early, rapid, efficient, and accurate methods for detecting IHHNV disease for non-diagnostic and non-treatment purposes is of great significance for disease prevention in shrimp farming. Existing research indicates that the IHHNV gene contains three open reading frames: two encoding nonstructural proteins, nsp1 and nsp2, and one encoding a viral capsid protein, cp.

[0004] Currently, there are many detection methods for IHHNV in various studies. Traditional IHHNV detection methods include histopathology, polymerase chain reaction (PCR), real-time quantitative polymerase chain reaction (RT-PCR), monoclonal antibody assay, biosensors, and enzyme-linked immunosorbent assay (ELISA). These molecular detection technologies have high sensitivity and reliability, but due to the long reaction time, complex instruments, and professional operation required, as well as their high cost, they cannot be used for continuous monitoring and rapid detection in shrimp farming.

[0005] In recent years, antigen-antibody binding-based immunological detection has become a rapid, efficient, and accurate method. Currently, most antibodies used in immunological detection are traditional monoclonal or polyclonal antibodies. Traditional antibodies have large molecular weights, which limits their application. Nanobodies, on the other hand, are the smallest known antibody units with antigen recognition activity. The CDR3 region of nanobodies allows them to recognize hidden regions of antigens, offering advantages that traditional antibodies lack, such as small size, strong penetrability, ease of modification, and high stability. Therefore, it is necessary to focus on finding high-affinity anti-nsp1, nsp2, and cp nanobodies and conducting related research.

[0006] Colloidal gold immunochromatographic test strips are one of the most widely used rapid pathogen detection methods. This method relies on the interaction between antigen and antibody, combining immunochromatography with colloidal gold labeling. Compared to traditional immunological methods, this method offers advantages such as ease of operation, speed, and the absence of required equipment or experience. Therefore, it is necessary to investigate how to combine nanobodies with colloidal gold technology to further develop rapid, convenient, and highly sensitive colloidal gold test strips for pathogen detection, thereby addressing the effective prevention and efficient monitoring of diseases in shrimp farming. Summary of the Invention

[0007] The purpose of this invention is to find nanobodies with high affinity against Nsp1, Nsp2, and CP, and to solve this technical problem:

[0008] The first aspect of the present invention provides a nanobody, wherein 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.

[0009] Furthermore, the nanobody is shark-derived.

[0010] A second aspect of this invention provides the application of nanobodies in the detection of infectious subcutaneous and hematopoietic necrosis virus (ISNV) for non-diagnostic purposes. The nanobodies have amino acid sequences of SEQ ID NO. 8 or SEQ ID NO. 11 for detecting the CP antigen protein of INV; or amino acid sequences of SEQ ID NO. 9 or SEQ ID NO. 12 for detecting the NSP1 antigen protein of INV; or amino acid sequences of SEQ ID NO. 10, SEQ ID NO. 13, or SEQ ID NO. 14 for detecting the NSP2 antigen protein of INV.

[0011] The fourth aspect of this invention provides a detection product for infectious subcutaneous and hematopoietic tissue necrosis viruses, including nanobodies.

[0012] Furthermore, the products for detecting infectious subcutaneous and hematopoietic necrosis virus are reagents, test strips, or kits; test strips include immunochromatographic test strips prepared using any one of the labeling technologies of colloidal gold labeling, colloidal carbon labeling, fluorescent microsphere labeling, and nanoparticle labeling.

[0013] Furthermore, the infectious subcutaneous and hematopoietic necrosis virus (IVZV) detection product includes a first antibody combination, a second antibody combination, or a third antibody combination. The first antibody combination is used to specifically recognize the CP antigen protein, and consists of: a rabbit polyclonal antibody as the first gold-labeled antibody, a first T-line antibody with the amino acid sequence SEQ ID NO.11, and a goat anti-rabbit antibody as the first C-line antibody. The second antibody combination is used to specifically recognize the NSP1 antigen protein, and consists of: a rabbit polyclonal antibody as the second gold-labeled antibody, a second T-line antibody with the amino acid sequence SEQ ID NO.12, and a goat anti-rabbit antibody as the second C-line antibody. The third antibody combination is used to specifically recognize the NSP2 antigen protein, and consists of: a rabbit polyclonal antibody as the third gold-labeled antibody, a third T-line antibody with the amino acid sequence SEQ ID NO.13, and a goat anti-rabbit antibody as the third C-line antibody.

[0014] A fourth aspect of this invention provides a method for preparing nanobodies for detecting infectious subcutaneous and hematopoietic necrosis viruses, comprising the following steps:

[0015] Step A: Expression of cp antigen protein, nsp1 antigen protein, or nsp2 antigen protein, including: constructing cp recombinant plasmid, nsp1 recombinant plasmid, or nsp2 recombinant plasmid; expressing and purifying the constructed cp recombinant plasmid, nsp1 recombinant plasmid, or nsp2 recombinant plasmid through prokaryotes to obtain cp antigen protein, nsp1 antigen protein, or nsp2 antigen protein;

[0016] Step B: Construction of shark natural libraries and / or shark immune libraries;

[0017] Step C: Perform helper phage rescue and purification on the constructed shark natural library and / or shark immune library to obtain phage libraries and test their titers;

[0018] Step D: Perform two, three, four, or five rounds of panning on the phage library to obtain the final eluent;

[0019] Step E: Use the final elution buffer to prepare the supernatant for phage-ELISA identification and sequencing. Based on the sequencing results, screen for cp nanobodies, nsp1 nanobodies, or nsp2 nanobodies with different CDR3 region sequences.

[0020] This invention utilizes phage display technology (phage-ELISA) to screen anti-cp, anti-nsp1, and anti-nsp2 nanobodies from natural phage libraries and / or immune phage libraries derived from sharks. These nanobodies are then produced using a prokaryotic expression system, thereby identifying high-affinity anti-cp, anti-nsp1, and anti-nsp2 nanobodies and reducing production and R&D costs.

[0021] Furthermore, the detection product of this invention utilizes the principle of antigen-antibody interaction, combining immunochromatography and colloidal gold labeling technologies. Compared with traditional immunological methods, this method has advantages such as simple operation, high speed, and no need for equipment or experience. Moreover, by combining nanobodies with colloidal gold technology, a rapid, convenient, and highly sensitive colloidal gold test strip has been developed. This strip can detect whether aquatic animals such as shrimp, aquaculture water, are infected with infectious hypodermal and hematopoietic necrosis virus (IDV), and whether aquaculture feed is contaminated with IV. This enables rapid detection of IV, facilitating more effective prevention and efficient monitoring of diseases in shrimp farming. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 is an agarose gel electrophoresis image of the cp recombinant plasmid digested with enzymes in Example 1 (M is the marker; 1 is the double-digested target gene cp gene, and the blue arrow points to the target gene band; 2 is the vector).

[0024] Figure 2 is an agarose gel electrophoresis image of the nsp1 recombinant plasmid digested by enzymes in Example 1 of the present invention (M is marker; 1 is the nsp1 gene, the target gene band is indicated by the blue arrow; 2 is vector).

[0025] Figure 3 is an agarose gel electrophoresis image of the nsp2 recombinant plasmid digested by enzymes in Example 1 of the present invention (M is marker; 1 is the nsp2 gene, the target gene band is indicated by the blue arrow; 2 is vector).

[0026] Figure 4 shows the prokaryotic expression and purification of cp protein in Example 1 (M shift marker; 1 is the first expansion culture of cp; 2 is the first induction culture of cp; 3 is the third resuspension of cp; 4 is the flow-through filtrate of cp (FT); 5 is cp 20mM imidazole washed protein; 6 is cp 40mM imidazole washed protein; 7 is cp 60mM imidazole washed protein; 8 is cp 80mM imidazole washed protein; 9 is cp protein; 10 is cp 1M imidazole washed protein; the blue arrow points to the cp protein, which is 35kDa).

[0027] Figure 5 shows the prokaryotic expression and purification of NSP2 protein in Example 1 (M is marker; 1 is the first NSP2 expansion culture; 2 is the NSP2 culture after the first induction; 3 is the supernatant of NSP2 after sonication; 4 is the third NSP2 resuspension; 5 is the NSP2 flow-through solution (FT); 6 is NSP2 20mM imidazole washed protein; 7 is NSP2 40mM imidazole washed protein; 8 is NSP2 protein; 9 is NSP2 1M imidazole washed protein; the blue arrow points to the NSP2 protein, which has a size of 45kDa).

[0028] Figure 6 shows the prokaryotic expression and purification of nsp1 protein in Example 1 (M is marker; 1 is the second suspension of nsp1; 2 is the first supernatant of nsp1; 3 is the flow-through buffer FT1 of nsp1; 4 is the nsp1 elution with 40mM imidazole before enzyme digestion; 5 is the nsp1 elution with 500mM imidazole before enzyme digestion; the blue arrow points to the nsp1 protein, which is 110kDa).

[0029] Figure 7 shows the reverse screening diagram of NSP1 protease digestion in Example 1 (M is the marker; 1 is the NSP1 protein solution before digestion after dialysis; 2 is the NSP1 digestion reactant; 3 is the NSP1 flow-through buffer FT2; 4 is the NSP1 20mM imidazole washed protein after digestion; 5 is the NSP1 40mM imidazole washed protein after digestion; 6 is the remaining protein eluted by NSP1 500mM imidazole after digestion; the blue arrow indicates the protein).

[0030] Figure 8 shows the BCA protein concentration curves of the cp, nsp1, and nsp2 antigen proteins in Example 1 (the horizontal axis represents the BSA concentration of the standard, in mg / mL; the vertical axis represents OD). 562nm (Absorbance value at the specified location);

[0031] Figure 9 shows the serum ELISA test results in Example 2 (A is the serum titer test result after antigen cp immunization; B is the serum titer test result after antigen nsp1 immunization; C is the serum titer test result after antigen nsp2 immunization).

[0032] Figure 10 is an agarose gel electrophoresis image of total RNA from the spleen of unimmunized sharks in Example 2 (M is the marker; 1-12 are total RNA from the spleen of unimmunized sharks; three complete bands can be observed: 28S, 18S and 5S).

[0033] Figure 11 is an agarose gel electrophoresis image of total RNA from unimmunized shark blood lymphocytes in Example 2 (M is the marker; 1-12 are total RNA from unimmunized shark blood lymphocytes; three complete bands can be observed: 28S, 18S and 5S).

[0034] Figure 12 shows the agarose gel electrophoresis images of total RNA from the spleen and total RNA from blood lymphocytes of immunized sharks in Example 2 (M is the marker; 1-3 are total RNA from the spleen of immunized sharks; 4-6 are total RNA from blood lymphocytes of immunized sharks).

[0035] Figure 13 is an agarose gel electrophoresis image of the first round of PCR amplification products of the vNAR gene in unimmunized sharks in Example 2 (M is the marker; 1-24 are the amplification bands of the vNAR target gene in unimmunized sharks);

[0036] Figure 14 is an agarose gel electrophoresis image of the first round of PCR amplification products of the vNAR gene in immunized sharks in Example 2 (M is the marker; 1-3 are the amplification bands of the vNAR target gene in immunized sharks).

[0037] Figure 15 is an agarose gel electrophoresis image of the fourth recovered product from the unimmunized shark in Example 2 (M is the marker; 1-10 are the bands of the third recovered product after double enzyme digestion from the unimmunized shark);

[0038] Figure 16 is an agarose gel electrophoresis image of the fourth recovered product from the immunized shark in Example 2 (M is the marker; 1-12 are the bands of the third recovered product after double enzyme digestion of the immunized shark);

[0039] Figure 17 shows the original reservoir capacity measurement of shark natural pool in Example 2;

[0040] Figure 18 shows the initial capacity measurement of the shark immune library in Example 2;

[0041] Figure 19. Agarose gel electrophoresis of the second round PCR amplification products of unimmunized sharks in Example 2 (M is the marker; 1-48 are the bands of the second round PCR amplification products of unimmunized sharks);

[0042] Figure 20 is an agarose gel electrophoresis image of the second round PCR amplification products of the immunized shark in Example 2 (M is the marker; 1-48 are the bands of the second round PCR amplification products of the immunized shark);

[0043] Figure 21 shows the sequencing results of the unimmunized shark bacterial library in Example 2;

[0044] Figure 22 shows the sequencing results of the immune shark bacterial library in Example 2;

[0045] Figure 23 shows the number of positive clones screened by phage-ELISA in Example 3;

[0046] Figure 24 shows the sequence alignment results of the nanobody in Example 4.

[0047] Figure 25 shows the results of expression and purification of shark natural library vNAR nanobodies in Example 4 (blue arrows indicate the purified nanobodies protein);

[0048] Figure 26 shows the results of expression and purification of vNAR nanobodies from the shark immune library in Example 4 (blue arrows indicate the purified nanobodies protein);

[0049] Figure 27 shows 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 the marker; 1-7 are SEQ ID NO.11-14 vNAR nanobody respectively);

[0050] Figure 28 shows the affinity verification graph of vNAR nanobody ELISA in Example 4 (vertical axis is OD). 450nm The absorbance value at the x-axis represents the concentration of vNAR nanobody.

[0051] Figure 29 shows the kinetic detection results of vNAR nanobodies in Example 4 (where A is the kinetic detection result of cp-I-C5, B is the kinetic detection result of nsp1-I-F3, and C is the kinetic detection result of nsp2-I-B12; the horizontal axis is time in seconds, and the vertical axis is the degree of binding in nm).

[0052] Figure 30 shows the ultraviolet absorption spectrum of the colloidal gold solution in Example 5;

[0053] Figure 31 is a structural diagram of the test strip in Example 5 (where 1 is the sample pad; 2 is the gold label pad; 3 is the NC membrane; 4 is the absorbent pad; 5 is the T-line antibody; and 6 is the C-line antibody).

[0054] Figure 32 shows the antibody cross-pairing detection results for Test Example 1 (A is the antibody cross-pairing detection result for CP antigen; B is the antibody cross-pairing detection result for NSP1 antigen; C is the antibody cross-pairing detection result for NSP2 antigen; the combination of test strips 1 and 2 is nanobody as gold-labeled antibody, rabbit polyclonal antibody as T-line antibody, and anti-His-tagged antibody as C-line antibody; the combination of test strips 3 and 4 is rabbit polyclonal antibody as gold-labeled antibody, nanobody as T-line antibody, and goat anti-rabbit as C-line antibody).

[0055] Figure 33 shows the specificity test results of the colloidal gold test strip in Test Example 2 (A is the specificity test result of the colloidal gold test strip for cp antigen; B is the specificity test result of the colloidal gold test strip for nsp1 antigen; C is the specificity test result of the colloidal gold test strip for nsp2 antigen; 1-6 are the detection results of cp, snp1, snp2, MBP, SWP1 and VP28 proteins, respectively).

[0056] Figure 34 shows the sensitivity test results of the colloidal gold test strip in Test Example 3 (A is the sensitivity test result of the colloidal gold test strip for cp antigen; B is the sensitivity test result of the colloidal gold test strip for nsp1 antigen; C is the sensitivity test result of the colloidal gold test strip for nsp2 antigen; 1-6 are 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25 μg / mL, 6.25 μg / mL and 3.125 μg / mL, respectively).

[0057] Figure 35 shows the detection results of colloidal gold test strips on shrimp hepatopancreas tissue samples from Test Example 4 (AJ represents the numbers of 10 shrimp; 1, 2, and 3 are colloidal gold test strips for CP antigen, NSP1 antigen, and NSP2 antigen, respectively). Detailed Implementation

[0058] The following detailed description of the features and advantages of the present invention is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Furthermore, based on the specification, claims and drawings disclosed herein, those skilled in the art can easily understand the related objects and advantages of the present invention.

[0059] It should be noted that in this specification, similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0060] To make the objectives, technical solutions, 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0061] (1) Source of sample materials

[0062] Striped bamboo shark: Purchase intention: Seafood market in Yazhou District, Sanya City, Hainan Province.

[0063] Shark Natural Library: Purchased from Shenzhen Kangti Life Technology Co., Ltd.

[0064] Upstream and downstream primer synthesis and sequencing: carried out by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0065] The pSmart-I vector was provided directly by Sangon Biotech.

[0066] (2) Sources of reagents and consumables

[0067] Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment

[0068] (3) Source of instruments and equipment

[0069] Table 2. Sources and Models of Instruments and Equipment Required for the Experiment

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

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

[0072] Example 1

[0073] The expression and purification of cp, nsp1, and nsp2 antigen proteins include the following steps.

[0074] The construction of S1, cp, nsp1, and nsp2 recombinant plasmids is detailed below:

[0075] (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, the cp, nsp1 and nsp2 genes were directly synthesized by Sangon Biotech Shanghai Co., Ltd. after codon optimization using the direct synthesis method.

[0076] (2) The target genes cp and nsp2 were ligated with pET-22b(+) vector respectively, and nsp1 was ligated with pSmart-I vector. The ligation reaction system was: 4 μL of target gene and 1 μL of corresponding vector. After mixing by pipetting, the mixture was reacted at 25℃ for 10 min to obtain the first ligation products of cp, nsp1 and nsp2. The products were stored at 4℃ for later use.

[0077] (3) The pET-22b(+) vector ligated with the target genes cp and nsp2 was double-digested with XhoI and NdeI enzymes for verification. The digestion system was: 10×NEBuffer 2μL, XhoI enzyme 0.5μL, NdeI enzyme 0.5μL, pET-22b(+) vector 1μL, and ddH2O was added to make up to 20μL. The digestion conditions were 37℃ for 2h and 80℃ for 20min. After the enzyme 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, and the electrophoresis results of nsp2 are shown in Figure 2. The pSmart-I vector ligated with the target gene nsp1 was verified by double digestion 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 up to 20 μL. The digestion conditions were 37℃ for 2 h and 80℃ for 20 min. After the enzyme 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.

[0078] (4) Thaw 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 respectively. After gently mixing, immediately place on ice for 30 min, heat shock in a 42℃ metal bath for 30 s, and immediately ice bath for 2 min. Under aseptic conditions, add the transformed bacterial solution to 250 μL of antibiotic-free LB liquid medium and shake at 37℃ and 220 rpm for 1 h to obtain the first transformed bacterial solutions of cp, nsp1, and nsp2. Subsequently, take 200 μL of the first transformed bacterial solutions of cp, nsp1, and nsp2 and spread them on ampicillin (Amp) resistant LB solid medium, and spread the first transformed bacterial solution of nsp1 on kanamycin (Kana) resistant LB solid medium. Incubate overnight at 37℃ upside down to obtain the first plates of cp, nsp1, and nsp2 respectively.

[0079] (5) Select single-clone plaques from the first plates of cp, nsp1, and nsp2 for expansion culture. Place them on a shaker and incubate at 200 rpm and 37℃ for about 6 hours. After extracting the plasmids using a plasmid mini-prep kit, send an appropriate amount of plasmids to Sangon Biotech for the first sequencing. Compare and analyze the first sequencing results. The similarity is 100%. Therefore, the recombinant protein plasmid vector has been successfully constructed, namely cp plasmid, nsp1 plasmid, and nsp2 plasmid. Label them and store them in a -20℃ freezer.

[0080] The expression and purification of S2, cp, and nsp2 antigen proteins are detailed in the following steps:

[0081] (1) Based on 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 plating, incubate overnight at 37°C in an incubator to obtain the second plate of cp and nsp2. Pick a single colony from the second plate of cp and nsp2 and culture it overnight in 200 μL of Amp-resistant LB liquid medium to obtain the first monoclonal bacterial culture of cp and nsp2. After overnight culture at 37°C and 220 rpm, expand the 200 μL of the first monoclonal bacterial culture of cp and nsp2 to 20 mL at a ratio of 1:100 to obtain the first expanded bacterial culture of cp and nsp2. Inoculate the first expanded bacterial culture of cp and nsp2 into 2L of Amp-resistant LB liquid medium and incubate it in a shaker at 37°C and 220 rpm for about 3.5 h. Wait for the first expanded bacterial culture to OD 600 nmWhen the concentration of CP and NSP2 is 0.5, add IPTG (isopropylβ-D-Thiogalactoside) to a final concentration of 1 mM and induce at 37°C for 4 h. This is the bacterial culture after the first induction of CP and NSP2. After the first induction of CP and NSP2, the bacterial cultures were transferred to large centrifuge flasks and centrifuged at 4200 rpm for 20 min at 4°C. The supernatant was discarded, and the bacterial precipitate was retained as the first CP and NSP2 precipitate. 20 mL of 1×PBS (phosphate buffered saline, obtained by dilution with 20×PBS) was added to resuspend the first CP and NSP2 precipitate as the first resuspension. The second CP and NSP2 resuspension was obtained by sonicating the cells at 30% power for 2 seconds with a 4-second interval for a total sonication time of 40 min using an ultrasonic cell disruptor. The second CP and NSP2 resuspension was aliquoted into 2 mL centrifuge tubes and centrifuged at 4200 rpm for 20 min at 4°C to obtain the second CP precipitate, the second NSP2 precipitate, the supernatant after sonication of CP, and the supernatant after sonication of NSP2. The second CP precipitate and the second NSP2 precipitate were resuspended in 20 mL of PBS containing 2M urea as the third CP and NSP2 resuspension.

[0082] (2) The third suspension of CP and NSP2 was sonicated using an ultrasonic cell disruptor at 30% power for 2 seconds followed by a 4-second interval, for a total sonication time of 5 minutes, to obtain the fourth suspension of CP and NSP2. This fourth suspension was then aliquoted into 2 mL centrifuge tubes and centrifuged at 4200 rpm and 4°C for 20 minutes. The supernatant was discarded, yielding the third precipitate of CP and NSP2, which are CP inclusion bodies and NSP2 inclusion bodies. The CP and NSP2 inclusion bodies were resuspended in 20 mL of PBS containing 0.3% sodium dodecyl sulfate (SKL) to obtain the fifth suspension of CP and NSP2. This fifth suspension was then sonicated again under the same conditions as the third suspension to obtain the sixth suspension of CP and NSP2, which is the CP and NSP2 inclusion body solution. The CP and NSP2 inclusion body solution was renatured using a PD-10 desalting column to obtain the renatured CP and NSP2 inclusion body solution.

[0083] (3) Take 5 mL of Ni-NTA (nickel NTA affinity chromatography medium, abbreviated as nickel column), and equilibrate the nickel column with 20 mL of ultrapure water and 1×PBS. 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. The outflow is the cp and nsp2 flow-through solution (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. Elute the target protein with 20 mL of 200 mM imidazole to obtain the cp protein. Wash the nickel column with 20 mL of 20 mM imidazole and 10 mL of 40 mM imidazole to remove impurities. Elute the target protein with 20 mL of 200 mM imidazole to obtain the nsp2 protein. Then, use 10 mL of PBS to equilibrate the column. The remaining protein on the column was washed away with 1M imidazole, i.e., cp, nsp 21M imidazole to wash away the contaminating protein; finally, the column was washed with 20mL ddH2O and stored in 20% ethanol at 4°C.

[0084] (4) Take 40 μL each of the above-mentioned cp first expansion culture, cp first induction culture, cp third resuspension, 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, mix with 10 μL of 5× loading buffer (this is the mixed sample), heat in a 99℃ metal bath for denaturation for 10 min, centrifuge at 13000 rpm for 10 min, and rinse with 12.5% ​​Omni-Easy TM The first PAGE gel (polyacrylamide gel electrophoresis) was prepared using a one-step rapid PAGE gel preparation kit. Electrophoresis conditions were 200V for 35 min. After electrophoresis, the PAGE gel was stained with SDS-PAGE rapid staining solution and destained with tap water. A clear band of cp protein at approximately 35 kDa was observed, indicating successful protein expression and good purity. Subsequent experiments can proceed. See Figure 4 for a detailed cp expression and purification diagram.

[0085] Take the above-mentioned first expansion culture of NSP2, the first induction culture of NSP2, the supernatant of NSP2 after sonication, the third suspension of NSP2, the flow-through solution of NSP2 (FT), NSP2 20mM imidazole washed protein, NSP2 40mM imidazole washed protein, NSP2 protein and NSP2 1M imidazole washed protein samples and mix them with 10μL of 5× loading buffer. Prepare gel, electrophoresis, staining and destaining. The mixing method and the SDS-PAGE electrophoresis method, staining and destaining method are the same as step S2(4). It is obvious that there is a clear band of NSP2 protein at about 45kDa. The protein expression is successful and the purity is good. Subsequent experiments can be continued. See Figure 5 for the specific NSP2 protein expression and purification diagram.

[0086] The specific steps for the expression and purification of S3 and nsp1 antigen proteins are as follows:

[0087] (1) Based on the first sequencing results, select the nsp1 plasmid with correct sequence alignment and transform it into BL21(DE3) competent cells using the same transformation method as S1(5). After plating, incubate the cells overnight at 37°C in an incubator to obtain the second nsp1 plate. Pick a single colony from the second nsp1 plate and incubate it overnight in 200 μL of Kana-resistant LB liquid medium to obtain the first nsp1 monoclonal culture. Incubate the first nsp1 monoclonal culture overnight at 37°C and 220 rpm. Then, expand the 200 μL of the first nsp1 monoclonal culture to 20 mL at a ratio of 1:100 to obtain the first nsp1 expanded culture. Inoculate the first nsp1 expanded culture into 2 L of Kana-resistant LB liquid medium and incubate it in a shaker at 37°C and 220 rpm for about 3.5 h. Wait for the first expanded culture to OD 600nm When the concentration of Nsp1 was 0.5, IPTG (isopropyl β-D-Thiogalactoside) was added to a final concentration of 0.2 mM, and the mixture was induced at 37°C for 4 h to obtain the first induction culture of Nsp1. The first induction culture of Nsp1 was then transferred to a large centrifuge bottle and centrifuged at 4200 rpm for 20 min at 4°C. The supernatant was discarded, and the bacterial pellet was retained as the first precipitate of Nsp1. The first precipitate of Nsp1 was then resuspended in 20 mL of 1×PBS containing 8 M urea and 0.1% Triton X-100 to obtain the first resuspension of Nsp1. The cell disruptor was used to sonicate the cells at 30% power for 2 s, followed by a 4 s interval, for a total sonication time of 40 min to obtain the second resuspension of Nsp1. The second resuspension of Nsp1 was aliquoted into 2 mL centrifuge tubes and centrifuged at 4200 rpm for 20 min at 4°C. The supernatant was collected as the first supernatant of Nsp1.

[0088] (2) Take 5 mL of Ni-NTA (NiNTA affinity chromatography medium, abbreviated as nickel column), and equilibrate the nickel column with 20 mL of ultrapure water and 1×PBS (containing 8M urea). Inject the first supernatant of nsp1 into the nickel column using a 10 mL syringe and a 0.22 μm syringe filter membrane and incubate for 30 min. The outflow is the nsp1 flow-through solution FT1. Wash the nickel column with 20 mL of 40 mM imidazole to remove impurities. This is the nsp1 40 mM imidazole wash for impurities before enzyme digestion. Elute the target protein with 20 mL of 500 mM imidazole. This is the nsp1 500 mM imidazole wash for the target protein before enzyme digestion. Then wash the remaining protein on the column with 10 mL of 1 M imidazole. This is the nsp1 1 M imidazole wash for impurities before enzyme digestion. Finally, wash the column with 20 mL of ddH2O and store the nickel column in 20% ethanol at 4°C.

[0089] (3) Place the target protein eluted with 500mM imidazole before enzyme digestion into a dialysis bag, and put the dialysis bag into the dialysis solution. The dialysis solution is PBS solution (containing 300mM NaCl and 10% glycerol). Dialyze at 4℃ for 24h, and change the dialysis solution every 6h to obtain the dialysis solution of the undigested nsp1 protein.

[0090] (4) Add SUMO Protease to the nsp1 protein solution after dialysis and before enzyme digestion and digest overnight at 4℃. The total digestion system is 5mL. The specific digestion system is as follows: 10×Reaction Buffer 500μL, dialysis nsp1 protein solution 1.6mL, SUMO Protease 12.5μL, ddH2O 2.8mL. The nsp1 digestion product was obtained and purified again by nickel column affinity chromatography, following the same purification steps as in S3(2). Since both SUMO Protease and the excised SUMO tag have His tags, while the nsp1 Elution buffer 3 digestion product does not have a His tag, the nsp1 digestion product was purified by nickel column affinity chromatography according to step S3(2). The resulting nsp1 flow-through buffer FT2, nsp1 20mM imidazole-washed mixed protein, nsp1 40mM imidazole-washed mixed protein, and nsp1 500mM imidazole-washed remaining protein were obtained. These were then concentrated according to step S3(2), following the same concentration steps as in S2(5), to obtain the nsp1 target protein.

[0091] (5) Take the above-mentioned second suspension of nsp1, first supernatant of nsp1, nsp1 flow-through buffer FT1, nsp1 40mM imidazole to wash off impurities before enzyme digestion, nsp1 500mM imidazole to wash off target protein before enzyme digestion, and mix with 10μL of 5× loading buffer, prepare gel, electrophoresis, staining, and destaining. The mixing method and SDS-PAGE electrophoresis method, staining and destaining method are the same as step S2 (4). It is obvious that the nsp1 target protein has a clear band at about 110kDa before enzyme digestion, which shows that the protein expression is successful and the purity is good. Subsequent experiments can be carried out. See Figure 6 for the specific nsp1 protein expression and purification diagram.

[0092] Take the above-mentioned nsp1 protein solution before enzyme digestion after dialysis, nsp1 enzyme digestion reaction product, nsp1 flow-through buffer FT2, nsp1 20mM imidazole washed impurities after enzyme digestion, nsp1 40mM imidazole washed impurities after enzyme digestion, nsp1 500mM imidazole washed remaining protein after enzyme digestion, and 10μL of 5× loading buffer for mixing, gel preparation, electrophoresis, staining, and destaining. The mixing method and SDS-PAGE electrophoresis method, staining and destaining methods are the same as step S2(4). It is obvious that the target nsp1 protein has a clear band at about 80kDa after digestion, which shows that the protein expression is successful and the purity is good. Subsequent experiments can be carried out. The enzyme digestion screening diagram is shown in Figure 7.

[0093] S4, cp, nsp2 and nsp1 antigen protein concentration and determination

[0094] The CP, NSP2, and NSP1 proteins were passed through a PD-10 desalting column to remove imidazole and other salts, and then concentrated using a 10 kDa concentrator to obtain the CP, NSP2, and NSP1 antigen proteins. The concentrations of the CP, NSP2, and NSP1 antigen proteins were determined using a BCA kit. The protein concentrations of the concentrated solutions were also determined using a BCA kit. The CP antigen protein (10-fold dilution), NSP2 antigen protein (10-fold dilution), and NSP1 antigen protein (10-fold dilution) were analyzed at OD... 562nm The absorbance values ​​were 0.167, 0.176, and 0.159, respectively. Based on the fitted equation y = 0.2139x + 0.1459 in the BCA protein concentration standard curve, the concentrations of CP antigen protein, NSP2 antigen protein, and NSP1 antigen protein were approximately 1.0 mg / mL, 1.4 mg / mL, and 0.6 mg / mL, respectively. After aliquoting into 50 μg tubes, the tubes were flash-frozen in liquid nitrogen and stored at -80°C. The specific BCA protein concentration standard curves for CP antigen protein, NSP2 antigen protein, and NSP1 antigen protein are shown in Figure 8.

[0095] Example 2

[0096] The construction of natural phage and immune libraries from striped bamboo sharks includes the following steps:

[0097] S1. The specific steps for raising, immunizing, and detecting serum titers after immunization of striped bamboo sharks are as follows:

[0098] (1) Feeding and immunization of striped bamboo sharks

[0099] Rearing and Immunization of Striped Bamboo Sharks: After purchasing healthy striped bamboo sharks (hereinafter referred to as sharks), they were temporarily kept in a laboratory breeding area for one week. The salinity of the artificial seawater was controlled between 1.020 and 1.025, the seawater temperature was around 25℃, and the environment was kept as dark as possible. They were fed shrimp once a week, and the water was changed every three days to observe their growth. Twelve healthy sharks were selected for the construction of a natural reservoir, and three healthy sharks were used for immunization antigens to construct an immunization reservoir. The three selected sharks were marked and numbered, and immunized with NSP1, NSP2, and CP antigen proteins, respectively. Before the first immunization, blood was drawn from the sharks and serum was separated and stored at -80℃ for later use. For 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 thoroughly emulsified. The emulsified antigen proteins were injected into the sharks at multiple sites. For subsequent immunizations, 100 μg of antigen solution was mixed with an equal volume of incomplete Freund's adjuvant (IAF), thoroughly emulsified, and injected at multiple sites. On average, sharks are immunized every 12 days, for a total of 6 immunizations. After the last immunization, blood is drawn and serum is separated, and then the sharks are disposed of in a harmless manner. The shark immunization cycle is shown in Table 4:

[0100] Table 4 Shark Immune Cycle Table

[0101] (2) Double-antibody sandwich ELISA detection of post-immunization shark serum titer: The extracted shark serum was serially diluted, and 100 μL was added to each well of a 96-well plate and incubated overnight at 4°C. The coating solution was discarded, and each well was washed with 200 μL of PBS three times for 5 min each time. 100 μL of 5% skim milk powder (blocking solution) was added to each well and blocked at room temperature for 1 h. The blocking solution was discarded, and each well was washed with 200 μL of PBS three times for 5 min each time. The three antigens (cp, nsp1, and nsp2 antigen proteins) were diluted to 1 μg / mL with PBS, and 100 μL was added to each well and incubated at room temperature for 2 h. The antigens were discarded, and each well was washed with 200 μL of PBST three times for 5 min each time. Rabbit polyclonal antibody diluted 1:100000 was added to each well, and incubated at room temperature for 1 h. The primary antibody was discarded, and 200 μL of PBST was added to each well. Wash with PBST 8 times, 5 min each time; add 100 μL of HRP-goat anti-rabbit monoclonal antibody diluted 1:250 per well and incubate at room temperature for 1 h; discard the secondary antibody, add 200 μL of PBST to each well and wash 8 times, 5 min each time; add 100 μL of TMB chromogenic buffer to each well, vortex to mix, and incubate in the dark for 5 min; remove the plate, add 100 μL of 1M HCl to each well to stop the color development, and measure the OD. 450 value.

[0102] The immunization results are shown in Figure 9. Repeated immunizations with NSP1 and NSP2 antigens showed significant effects, while immunization with CP antigen was less effective, although the antibody titer was nearly doubled compared to before immunization. In summary, immunization of sharks with each of the three antigens was effective.

[0103] S2. Sampling of blood and spleen from striped bamboo sharks and separation of blood lymphocytes: The specific steps are as follows:

[0104] (1) Blood and spleen samples were taken from 12 non-immunized and 3 immunized striped bamboo sharks: Seawater for anesthesia was prepared using MS-222 fish anesthetic at a final concentration of 120 mg / L. After the sharks were placed in the seawater for about 15 minutes, they became still and unresponsive. At this time, the sharks were removed and their gills were wrapped with moist gauze to keep them moist. Blood collection was then carried out. A 1 mL syringe was inserted into the tail vein at a 45° angle. The blood vessel was close to the body surface, and the needle could be felt as soon as it was inserted subcutaneously. The syringe needle was then removed, and the blood was injected into a heparin sodium blood collection tube. The tube was slowly inverted to mix thoroughly. Approximately 3-4 mL of blood was collected from each shark for subsequent separation of hemolymphocytes.

[0105] After blood collection, the shark was dissected with a scalpel, the spleen was removed, and the blood was rinsed with sterile 1×PBS. The spleen was then cut into pieces, aliquoted into cryovials, flash-frozen in liquid nitrogen, and immediately stored in a -80°C freezer.

[0106] (2) Isolation of striped bamboo shark blood lymphocytes: Take a 15mL sterile centrifuge tube, add 5mL of separation buffer (provided with the dog shark peripheral blood lymphocyte isolation kit), and then slowly add 4mL of fresh anticoagulated shark blood (carefully add it above the separation buffer interface); place the centrifuge tube in a centrifuge, centrifuge at 20℃, 800×g for 30min; after centrifugation, carefully aspirate the uppermost plasma layer, and then carefully aspirate the ring-shaped milky white lymphocyte layer from the centrifuge tube and transfer it to a new 15mL sterile centrifuge tube; add 10ml of washing buffer (provided with the kit) to the centrifuge tube and mix the cells; place the centrifuge tube in a centrifuge, centrifuge at 20℃, 250×g for 10min, and discard the supernatant; resuspend the obtained cells with 5ml of washing buffer using a pipette; repeat the centrifugation, centrifuge at 20℃, 250×g for 10min. Discard the supernatant, and finally obtain about 200μL of shark blood lymphocytes, flash freeze in liquid nitrogen, and then immediately store them in a -80℃ freezer.

[0107] S3. RNA extraction and reverse transcription from spleen and blood lymphocytes of striped bamboo sharks, with specific steps as follows:

[0108] (1) RNA extraction from spleen and hemolymphocytes: Add 300 μL of Trizol to an enzyme-free EP tube, carefully remove the tissue (spleen and hemolymphocytes obtained in step S2 of Example 2) from the cryopreservation tube and place it in Trizol. Homogenize on an ice plate until no large pieces of tissue remain, then add 700 μL of Trizol. Trizol, let stand for 5 min; Centrifuge: 4℃, 12000×g, for 10 min; Extraction: Add 200 μL of chloroform to a new EP tube, add the supernatant from the previous step to the tube, being careful not to access the middle layer, vortex, let stand for 2 min; Centrifuge: 4℃, 12000×g, for 15 min; Precipitation: Add 500 μL of pre-chilled isopropanol to a new EP tube, add the supernatant from the previous step to the tube, being careful not to access the middle layer, invert and mix well, incubate at 4℃ for 10 min; Centrifuge: 4℃, 12000×g, for 10 min; Wash: Discard the supernatant, aspirate the residual liquid, add 1 mL of pre-chilled 75% ethanol, vortex the bottom of the tube to suspend the precipitate; Centrifuge: 4℃, 7500×g, for 10 min, discard the supernatant, centrifuge empty for 1 min, aspirate the residual liquid, and air dry; Dissolve RNA: Add 30 μL to the precipitate. DEPC was dissolved in water to obtain total RNA from spleen and total RNA from blood lymphocytes. Detection: After extraction, the samples (total RNA from spleen and total RNA from blood lymphocytes) were diluted 10-fold, and the concentration and purity of each RNA sample were detected using a NanoDrop 1000 ultra-micro UV spectrophotometer and agarose gel electrophoresis.

[0109] Figures 10 and 11 show the agarose gel electrophoresis results of total RNA from unimmunized shark spleen and total RNA from hemolymphocytes, respectively. Figure 12 shows the agarose gel electrophoresis results of total RNA from immunized shark spleen and total RNA from hemolymphocytes, with lanes 1-3 representing the total RNA bands from immunized shark spleen and lanes 4-6 representing the total RNA bands from immunized shark hemolymphocytes. Three complete bands (28S, 18S, and 5S) were observed, demonstrating that the total RNA extracted from both unimmunized and immunized shark spleen and hemolymphocytes was of good quality, with relatively intact RNA and low degradation, suitable for subsequent experiments.

[0110] (2) Using the extracted total RNA from unimmunized and immunized shark spleens and total RNA from blood lymphocytes as template RNA, reverse transcription was performed. The reverse transcription system was as follows (all reagents were provided with the cDNA reverse transcription kit): 4 μL of 5×PrimeScript IV cDNA Synthesis Mix, 1 μL of Random 6mers (50 μM), 5 μg of template RNA, and RNase-free dH2O to a final volume of 20 μL. The reverse transcription program was: 30℃ for 10 min, 42℃ for 20 min, 70℃ for 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 placed in a new 1.5 mL EP tube, gently mixed, and used for subsequent construction of a shark natural library. 5 μL of each of the 3 immunized shark spleen cDNA samples and 3 immunized shark blood lymphocyte cDNA samples were placed in a new 1.5 mL EP tube, gently mixed, and used for subsequent construction of a shark immune library.

[0111] The specific steps for S4 and vNAR fragment amplification, pComb3XSS vector digestion and ligation are as follows:

[0112] (1) The first round of PCR reaction was performed 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 as follows: 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 bring the volume to 50 μL; where vNAR-F (SEQ ID NO.4): 5'-CGTGGCCCAGGCGGCCATGGCCSMACGGSTTGAACAAACACC-3'; vNAR-R (SEQ ID NO.5): 5'-GCTGGCCGGCCTGGCCWTTCAGTCASARKGGTSCC-3'. The first round of PCR amplification was as follows: pre-denaturation at 98℃ for 30 seconds, denaturation at 98℃ for 10 seconds, annealing at 58℃ for 30 seconds, extension at 72℃ for 30 seconds, final extension at 72℃ for 2 minutes, and storage at 4℃. The denaturation to extension steps were repeated 30 times. After the first round of PCR, the first amplification product was obtained and detected by 1.5% agarose gel electrophoresis. A band of approximately 350 bp was excised using a UV excimer and recovered using an agarose DNA recovery kit to obtain the vNAR gene, which was the first recovered product. The electrophoresis results of the first amplification product of the unimmunized shark vNAR gene are shown in Figure 13, and the electrophoresis results of the first amplification product of the immunized shark vNAR gene are shown in Figure 14.

[0113] (2) The vNAR gene from non-immunized sharks and vNAR gene from immunized sharks obtained above were digested with pComb3XSS vector. The digestion system was as follows: 2 μL 10×NEBuffer, 0.5 μL SacI enzyme, 0.5 μL SpeI enzyme, 6 μL vNAR gene or 1 μL pComb3XSS vector, and ddH2O was added to bring the volume to 20 μL. The digestion conditions were 37℃ for 2 h and 80℃ for 20 min. After the digestion reaction, the digestion product of vNAR gene was recovered using a PCR product recovery kit to obtain the second recovered product. The digestion product of pComb3XSS vector was detected by 1.5% agarose gel electrophoresis. A band of about 341 bp was excised and recovered using a gel recovery kit to obtain the third recovered product. The electrophoresis results of the third recovered product from non-immunized sharks are shown in Figure 15, and the electrophoresis results of the third recovered product from immunized sharks are shown in Figure 16.

[0114] (3) The second and third recovered products obtained above were used for ligation. The ligation system was as follows: 2 μL of T4 DNA Ligase Buffer (10×), 1 μL of T4 DNA Ligase, 20 ng of the second recovered product, 64 ng of the third recovered product, and ddH2O was added to bring the volume to 20 μL. The ligation conditions were 16℃ overnight to obtain the second ligation product. The second ligation product was purified by ethanol precipitation: 1 / 10 volume of 3M NaAc was added, followed by 2.5 volumes of anhydrous ethanol, and the mixture was stirred well. Freeze at -20℃ for 1 hour to fully precipitate DNA; centrifuge at 12000×g for 5 minutes at 4℃, 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 12000×g for 5 minutes at 4℃, discard the supernatant and then carefully aspirate the supernatant with a pipette; air dry until no obvious residual liquid is visible, dissolve the DNA precipitate in an appropriate amount of water to a concentration of about 100 ng / μL, thus obtaining the second ligation product purified from unimmunized shark and the second ligation product purified from immunized shark.

[0115] The specific steps for preparing S5 and SS320 electrocompetent cells are as follows:

[0116] (1) Pick a single colony of SS320 on the streak trajectory of a 2×YT plate containing tetracycline-resistant (Tet) and put it into 4 mL of 2×YT liquid medium. Add Tet at a ratio of 1:1000 and shake overnight at 37℃ and 220 rpm to obtain two seed culture tubes.

[0117] (2) Inoculate the two tubes of seed culture into 400 mL of 2×YT liquid culture medium at a ratio of 1:100, add Tet at the same ratio as above, incubate at 37℃ and 220 rpm, and shake until OD. 600 =0.5, thus obtaining the first expansion culture of SS320.

[0118] (3) Dispense the first culture of SS320 into two 400mL sterile centrifuge bottles, balance them carefully, and pre-cool them on ice for 30min.

[0119] (4) Centrifuge at 4℃, 4000×g for 10min, discard the supernatant, invert onto absorbent paper for 2min to fully remove residual liquid, and retain the first SS320 cell precipitate.

[0120] (5) Add 400 mL of pre-cooled sterile water to each centrifuge bottle and resuspend the first bacterial precipitate of SS320 by pipetting.

[0121] (6) Centrifuge at 4℃, 4000×g for 15min, discard the supernatant, invert onto absorbent paper for 2min to obtain the second SS320 bacterial precipitate.

[0122] (7) Add 200 mL of pre-cooled 10% glycerol to each centrifuge bottle and resuspend the second SS320 bacterial pellet by pipetting.

[0123] (8) Centrifuge at 4℃, 4000×g for 15min, discard the supernatant, invert onto absorbent paper for 2min to obtain the third bacterial cell precipitate of SS320.

[0124] (9) Add 10 mL of pre-cooled 10% glycerol to each centrifuge bottle, resuspend the second bacterial precipitate of SS320 by pipetting, and transfer the bacterial solution from the two centrifuge bottles into two sterile 15 mL centrifuge tubes.

[0125] (10) Centrifuge at 4℃, 4000×g for 15min, discard the supernatant, invert onto absorbent paper for 2min, and use a pipette to remove the residual liquid to obtain the fourth cell precipitate of SS320.

[0126] (11) Add 1.2 mL of pre-cooled 10% glycerol to each centrifuge tube, resuspend the SS320 fourth bacterial cell precipitate by pipetting, combine the bacterial solutions from the two tubes into one tube, dispense 100 μL into EP tubes, freeze in liquid nitrogen, and store at -80℃ to obtain SS320 Escherichia coli competent cells.

[0127] S6. Construction of the bacterial library. The specific steps are as follows:

[0128] (1) Electroporation: Take one tube of SS320 Escherichia coli competent cells from the -80℃ ultra-low temperature freezer, place it on ice for 5 min to thaw, add 100 ng of purified second ligation product using a pre-cooled pipette tip, gently mix, and transfer to a pre-cooled 1 mm electroporation cuvette. Gently tap the cuvette to allow the mixture to flow to the bottom. Set 1.8 kV, 25 μF, 200 Ω, and 1 mm spacing for electroporation. Immediately after electroporation, add 900 μL of pre-warmed 37℃ SOC medium to obtain the electroporated bacterial solution. Quickly remove the electroporated bacterial solution from the cuvette into a 1.5 ml sterile centrifuge tube and incubate at 37℃ and 200 rpm for 1 h with shaking to obtain the revived bacterial solution. The revived bacterial culture was serially diluted 10-fold using 2×YT liquid medium, for a total of 6 dilutions (i.e., 100 μL of the revived bacterial culture was diluted to 1000 μL, then another 100 μL was diluted to 1000 μL, and so on, for a total of 10 dilutions). 1 -10 6 Take 100 μL from each serially diluted plate and spread it evenly onto 2×YT(Amp) solid medium. Incubate overnight at 37°C to obtain an electroporation plate. Count the number of colonies on the serially diluted electroporation plates and calculate the ligation efficiency using the following formula: E(pfu / 100ng)=N×D×10

[0129] Note: E is the ligation efficiency (unit: pfu / 100ng), D is the dilution factor, and N is the number of single clones on the plate at the corresponding dilution factor.

[0130] The electroconversion results of non-immunized sharks are shown in Figure 17. -6 The plate contains one monoclonal cell, and the calculated connection efficiency is approximately 1 × 10⁻⁶. 7 The cfu / 100ng storage capacity is good.

[0131] The electroporation results of the immune shark are shown in Figure 18. -4 The plate contained 13 monoclonal antibodies, and the calculated ligation efficiency was approximately 1.3 × 10⁻⁶. 6 The cfu / 100ng storage capacity is good.

[0132] (2) Positive clone verification: 48 single clones were randomly selected from the plate and inoculated into centrifuge tubes containing 1 ml of 2×YT(Amp) liquid medium. The culture was incubated overnight at 37°C to obtain the second single clone culture. The second single clone culture was used as a template for PCR reaction, which was the second round of PCR reaction. The second round of PCR reaction system was: 10 μL of 2×Rapid Taq Master Mix, 1 μL of the second single clone culture, 1 μL of vNAR-F, 1 μL of pComb3XSS-R, and ddH2O was added to make up to 20 μL. Among them, pComb3XSS-R (SEQ ID NO.6): 5'-GCCCCCTTATTAGCGTTTGCCATC-3'; The second round of PCR reaction program was: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 5 s, full extension at 72°C for 5 min, and storage at 4°C. The denaturation to extension steps were performed in a total of 28 cycles. After the second round of PCR, the second amplification product was obtained and detected by 1% agarose gel electrophoresis. The positive clone rate of the purified second ligation product was calculated using the following formula:

[0133] Note: p represents the positive cloning rate, Np represents the number of single colonies that tested positive by PCR, and Nt represents the total number of PCR reactions. Subsequent experiments are performed when the positive cloning rate is greater than 95%.

[0134] The positive clone results of non-immunized sharks are shown in Figure 19, with an effective insertion rate of 100% in the library.

[0135] The positive clone results of the immunized shark are shown in Figure 20, with an effective insertion rate of 100% in the library.

[0136] (3) Large-scale construction of bacterial libraries: 16 electroporation transformation reactions were randomly selected according to the S6(1) method. All the recovered bacterial solutions were concentrated into a 15mL sterile centrifuge tube and mixed. 100μL of the recovered bacterial solution was taken and the ligation efficiency and positive clone rate were calculated according to the S6(1) and (2) methods respectively. The remaining recovered bacterial solutions were evenly spread on 6 130mm square culture medium plates containing 100μg / mL (Amp) and 2% glucose in 2% agarose and incubated overnight at 37℃.

[0137] Take a square plate that has been incubated overnight, add 2 mL of 2×YT liquid medium to the surface of each plate, gently scrape off colonies from 6 square plates, and collect the bacterial culture into the same 50 mL centrifuge tube. Add glycerol to a final concentration of 20% to obtain the bacterial library. The library size is calculated using the following formula: C = E × P × 20

[0138] Note: C is the library size, E is the linking efficiency, and p is the positive clone rate.

[0139] Take 20 μL of the bacterial culture collected from the square plate above and add it to 980 μL of 2×YT liquid culture medium. Measure the OD using a spectrophotometer. 600 The total OD of the recorded bacterial library was calculated using the following formula. 600 The bacterial library culture was aliquoted into 10 tubes, 1 mL per tube, with the remainder stored in 50 mL centrifuge tubes at -80°C. OD600 =M OD600 ×50

[0140] Note: T OD600 Total bacterial OD 600 M OD600 For the measured OD 600

[0141] Positive clones from the second round of PCR amplification were sequenced, and the nanobody fragments in the sequencing results were translated into amino acid sequences and then sequenced for alignment to detect sequence diversity in the bacterial library.

[0142] The sequencing results of the non-immunized shark bacterial library are shown in Figure 21. After expanding the culture of 48 single colonies and sending them for sequencing, comparative analysis revealed that they were all gene sequences encoding nanobodies, and the library had good diversity and no repetitive sequences.

[0143] The sequencing results of the immune shark bacterial library are shown in Figure 22. After expanding the culture of 48 single colonies and sending them for sequencing, comparative analysis revealed that they were all gene sequences encoding nanobodies, and the library had good diversity and no repetitive sequences.

[0144] S7. Construction of phage libraries: The specific steps are as follows:

[0145] (1) Phage amplification:

[0146] Remove the bacterial library from the -80°C freezer, thaw it on ice, calculate the corresponding bacterial culture volume using the formula below, and transfer it to 100 ml of 2×YT liquid medium containing 10 μg / mL Tet and 100 μg / mL Amp, so that the initial OD600 is 0.1.

[0147] Note: V is the volume of the transferred bacterial culture (in μL), and OD600 is the total OD of the constructed bacterial library. 600 .

[0148] Incubate at 37°C and 250 rpm in a constant temperature shaker until the bacterial culture reaches OD50. 600 =0.5-0.55.

[0149] Calculate and add helper phage M13KO7 according to the following formula to make the ratio of bacteria to phage count = 1:20:

[0150] Note: V is the volume of helper phage added (in mL), T_(helper-phage) is the titer of the helper phage used, and OD_ 600 OD of bacterial culture 600 value.

[0151] Continue culturing in a constant temperature shaker at 37°C and 220 rpm for 30 min.

[0152] Add 50 μg / mL Kana and 0.2 mM IPTG to the culture solution, respectively, and incubate overnight at 30°C and 250 rpm in a constant temperature shaker to obtain the first induction bacterial culture.

[0153] (2) Preparation of phage libraries:

[0154] After the first induction, transfer the bacterial culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm and 4°C for 20 min. Retain the supernatant, which is the first supernatant. Transfer the first supernatant to a new 50 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl (4°C), mix thoroughly, incubate on ice for 30 min, centrifuge at 4000 rpm and 4°C for 20 min, discard the supernatant, and invert the tube on paper for 2 min to obtain the first precipitate. Resuspend the first precipitate in 1 mL PBS, transfer to a new 1.5 mL centrifuge tube, and centrifuge at 12000 rpm and 4°C 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-chilled 20% PEG / 2.5M NaCl solution, mix well, place on ice for 10 min, centrifuge at 12000 rpm and 4°C for 10 min, discard the supernatant to obtain the second precipitate, add 1 ml of PBS to resuspend the second precipitate, centrifuge at 12000 rpm and 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, aliquot 100 μL / tube, and store at -80°C for long-term storage.

[0155] (3) Phage library titer detection:

[0156] The SS320 strain, stored at -80℃, was streaked onto 2×YT (Tet) solid medium for single colony formation and incubated overnight at 37℃. A single colony was picked from the streak plate and transferred to 5 mL of 2×YT medium containing 10 μg / mL Tet, and incubated 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 expansion culture of SS320. The culture was incubated at 37℃ and 250 rpm for approximately 45-60 minutes until OD was reached. 600 =0.5-0.55.

[0157] Take 10 μL of the prepared phage library and perform sequential 10-fold serial dilutions in a 1.5 ml centrifuge tube, for a total of 12 dilutions to a final volume of 10. -12 After vortexing and mixing, add 90 μL of SS320 second expansion culture to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min. Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT(Amp) solid medium, incubate overnight at 37°C to obtain the third plate. Count the number of single colonies at dilutions that are clearly distinguishable on the third plate, and calculate the number of phage particles per milliliter of phage solution, i.e., the phage library titer, using the following formula: T(pfu / mL)=N×D×400

[0158] Note: T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0159] Helper phages were added to the bacterial library for library rescue. After obtaining the phage library, the phage library titer was determined, and finally, the titer was 10. -10 A single colony grew on the gradient, and the final titer was calculated to be 4 × 10⁻⁶. 12 A shark native phage library at pfu / mL; a shark immune phage library was eventually obtained at 10 pfu / mL. -10 Two single colonies grew on the gradient, and the final titer was calculated to be 8 × 10⁻⁶. 12 Shark immune phage library at pfu / mL.

[0160] Example 3

[0161] Using the CP, NSP1, and NSP2 antigen proteins obtained in Example 1, screening for CP, NSP1, and NSP2 nanobodies using striped bamboo sharks included the following steps:

[0162] The screening steps for S1, anti-cp, nsp1, and nsp2 phage libraries are as follows:

[0163] (1) First round of screening: 50 μg of cp, nsp1 and nsp2 antigen proteins were coated in 2 mL of CBS coating buffer and incubated overnight at 4°C. The next day, the CBS coating buffer was discarded, and the immunotubes were washed three times with 1×PBS for 5 min each time. Then, 2 mL of 3% BSA was added as blocking buffer, and the tubes were incubated at room temperature for 2 h by rotation. The 3% BSA blocking buffer was discarded, and the immunotubes were washed three times with 2 mL of 1×PBS for 5 min each time. After discarding the washing buffer, 2 mL of 1×PBS was added, followed by 100 μL of the native phage library / immunophage library of the striped bamboo shark. The tubes were incubated at room temperature by rotation for 1 h. The liquid in the immunotubes was discarded, and 2 mL of PBST solution was added for 20 rotations, each time slowly rotating for 5 min. The liquid in the immunotubes was discarded. 1 mL of 0.25 mg / mL trypsin solution was added, and the phages were eluted by slow rotation at room temperature for 30 min. Then, add 10 μL of 10% AEBSF protease inhibitor to stop elution, and transfer the trypsin eluent in the immunoassay tube to a new 1.5 mL tube, which is the first round of elution for cp, nsp1, and nsp2.

[0164] (2) First round of eluent potency test: The method is the same as S7(3) in Example 2.

[0165] (3) First round of elution buffer expansion: Incubate 3 tubes of 5mL SS320 bacterial suspension to the logarithmic phase in advance, so that the OD... 600 nmPrepare approximately 0.5-0.55 μL of the first-round elution buffer for cp, nsp1, and nsp2 (store the remaining elution buffer at 4°C) and add it to 5 mL of freshly cultured SS320 bacterial suspension. Incubate on a shaker at 37°C and 250 rpm for 30 min. Spread the remaining 5.5 mL of bacterial suspension evenly onto three 130×130 mm 2×YT square solid medium plates (containing 2% glucose, 2% agarose, and 100 μg / mL Amp resistance), three plates each for cp, nsp1, and nsp2, for a total of nine plates. Incubate overnight at 37°C, resulting in the fourth plate for cp, nsp1, and nsp2. Add 2 mL of Tet-resistant 2×YT liquid medium to the surface of the fourth plate. Use a cell scraper to scrape off all colonies from the plate and collect the mixed bacterial solution into 15 mL centrifuge tubes. This is the first mixed bacterial solution of CP, NSP1, and NSP2, with 6 mL in each tube. At the same time, measure the OD of the bacterial solution. 600nm The results were recorded, and then glycerol to a final concentration of 20% was added to the first mixed bacterial culture of CP, NSP1, and NSP2. The culture was then aliquoted and stored at -80°C, thus forming the first-round elution bacterial library of CP, NSP1, and NSP2. The OD values ​​of the first-round elution bacterial library of CP, NSP1, and NSP2 were recorded. 600nm Substitute the value into the following formula: V(μL)=10 / OD 600 Calculate the volume of the first bacterial culture (cp, nsp1, nsp2) by multiplying by 1000, and transfer it to 100 mL of Amp, Tet-resistant 2×YT liquid medium. This constitutes the second expansion culture of cp, nsp1, and nsp2. After shaking at 37℃ and 220 rpm until the logarithmic growth phase, measure the OD. 600nm = Approximately 0.5, followed by helper phage rescue. The volume of helper phage added is based on the following formula (T_(helper-phage) is the titer of the helper phage used):

[0166] After being placed back into a shaker at 37°C and cultured for another 30 minutes, kanamycin at a final concentration of 50 μg / mL and IPTG at a final concentration of 0.2 mM were added under aseptic conditions. The mixture was then placed back into a shaker at 30°C and cultured overnight to obtain the bacterial culture after the third induction of cp, nsp1, and nsp2.

[0167] (4) Purification of the first round of phage sub-libraries: The method is the same as S7(2) in Example 2. After purification of the bacterial culture after the third induction of cp, nsp1, and nsp2, the first round of phage sub-libraries of cp, nsp1, and nsp2 are obtained and used as the input phage library for the second round of screening.

[0168] (5) Second round of screening: The method is the same as step S1(1) in Example 3, except that the added phage library is the phage sub-library obtained in the first round of screening, and the second round of elution buffers of cp, nsp1, and nsp2 are obtained. The titer detection, amplification, and purification steps of the second round of elution buffers are also the same as steps S1(2, 3) in Example 3. Finally, the second round of phage sub-libraries of cp, nsp1, and nsp2 are obtained after purification and used as the input phage library for the third round of screening.

[0169] (6) Third round of screening: The method is the same as step S1(1) in Example 3. The added phage library is the second round phage sub-library of cp, nsp1, and nsp2, and finally the third round elution buffer of cp, nsp1, and nsp2 is obtained.

[0170] Based on the three rounds of screening data (cp, nsp1, nsp2), this method can effectively enrich nanobodies that specifically bind to cp, nsp1, and nsp2. Then, phage-ELISA (phage display technology) is used for further detection of their affinity.

[0171] Table 5. Results of three rounds of screening for anti-CP nanobodies from the natural phage library.

[0172] Recovery rate = Elution volume ÷ Input volume

[0173] Enrichment degree = Recovery rate of the next round ÷ Recovery rate of the previous round.

[0174] Table 6. Results of three rounds of screening for anti-nsp1 nanobodies from the natural phage library.

[0175] Table 7 Results of three rounds of screening for anti-Nsp2 nanobodies from the natural phage library.

[0176] Table 8 Results of three rounds of screening for anti-CP nanobodies in the phage library.

[0177] Table 9. Results of three rounds of screening for anti-nsp1 nanobodies in the immunophage library.

[0178] Table 10 Results of three rounds of screening for anti-Nsp2 nanobodies in the immunophage library.

[0179] S2. Identification and analysis using phage-ELISA, the specific steps are as follows:

[0180] (1) Preparation of phage supernatant

[0181] Prepare 5 mL of SS320 Escherichia coli and culture until OD. 600nm The value is approximately 0.5, keep it for later use. Take 10 μL of the three eluents (cp, nsp1, nsp2) and perform serial dilutions of 10-fold (10 μL + 90 μL sterile water per tube) to a final concentration of 10. -12 Twelve tubes were prepared and mixed thoroughly. 90 μL of SS320 logarithmic-phase bacterial culture was added to each tube, mixed thoroughly by pipetting, and incubated at 37°C for 30 min to obtain the third expansion culture for CP, NSP1, and NSP2. 5 μL of each of the third expansion culture tubes was then spread onto an Amp-resistant 2×YT solid culture plate, ensuring even distribution. The plates were incubated upside down at 37°C overnight to obtain the fifth plate for CP, NSP1, and NSP2. A sterile 96-well cell culture plate was prepared, and 200 μL of Amp- and Tet-resistant 2×YT liquid medium was added to each well. Then, 96 single colonies were randomly picked from the fifth plate using a sterile pipette tip and added to each well. The plates were incubated at 37°C overnight to obtain the first cell culture plate for CP, NSP1, and NSP2.

[0182] Take 2 μL of bacterial culture from each well of the first cell culture plate (cp, nsp1, and nsp2, with the remaining culture stored at 4℃) and transfer it to a new 96-well cell culture plate. Incubate at 37℃ for 3 hours to create the second cell culture plate (cp, nsp1, and nsp2). Then calculate the volume of helper phage added to each well using the following formula (T_(helper-phage) is the titer of the helper phage used):

[0183] After incubating at 37°C for 30 minutes, Kana (to a final concentration of 50 μg / mL) and IPTG (to a final concentration of 0.2 mM) were added under aseptic conditions. The mixture was then incubated overnight at 30°C on a shaker to obtain the fourth induction bacterial culture for CP, NSP1, and NSP2. Subsequently, the second cell culture plates containing CP, NSP1, and NSP2 were centrifuged at 5000 rpm at 4°C for 10 minutes to obtain the first phage supernatant for CP, NSP1, and NSP2.

[0184] (2) Phage-ELISA identification

[0185] 96-well microplates were coated with CP, NSP1, and NSP2 antigen proteins using CBS (1 ng / μL antigen concentration, 100 μL coating volume / well). Two control groups were prepared using BSA (1 ng / μL BSA concentration, 100 μL coating volume / well). All plates were incubated overnight at 4°C. The next day, the coating solution was discarded, and each well was washed three times with 200 μL PBS for 10 min each time. 200 μL of 3% BSA was added as blocking solution, and the plates were blocked at room temperature for 1 h. The blocking solution was then discarded, and each well was washed three times with 200 μL PBS for 10 min each time.

[0186] Add 120 μL of 3% BSA to each well of a 96-well microplate, followed by 80 μL of cp, NSP1, and NSP2 phage supernatant as primary antibody. Incubate at room temperature for 2 hours, discard the primary antibody, and wash each well three times with 200 μL PBST, incubating for 10 minutes each time. Then add 100 μL of M13 mouse anti-phage monoclonal antibody (HRP-labeled) as secondary antibody, diluted 1:40000 in 3% BSA, and incubate at room temperature for 1 hour. Discard the secondary antibody, and wash each well three times with 200 μL PBST, incubating for 10 minutes each time. Next, develop the color using TMB single-component chromogenic buffer (100 μL per well). Incubate for 5 minutes, then stop the development with 100 μL HCl (1M). Read the OD values ​​using a microplate reader. 450nm Numerical value.

[0187] During colorimetric result processing, the OD values ​​of the cp, nsp1, and nsp2 antigen protein incubation wells and their corresponding BSA-coated control wells were compared. 450nm The values ​​were sorted from largest to smallest. See Figure 23 for a specific chart showing the number of positive clones screened by phage-ELISA. Ultimately, 3 positive clones were obtained from the natural library (cp), 2 from nsp1, and 5 from nsp2; 7 positive clones were obtained from the immune library (cp), 5 from nsp1, and 6 from nsp2, totaling 28 single-clone bacterial cultures. These were sent to Sangon Biotech Shanghai Co., Ltd. for sequencing. The sequencing primer P1 (SEQ ID NO. 7) was 5'-CCAGGCTTTACACTTTATGC-3', and the sequencing results were from the second sequencing run.

[0188] Example 4

[0189] The expression and identification of the nanobodies against cp, nsp1, and nsp2 antigen proteins obtained in Example 3 included the following steps:

[0190] S1. Construction of prokaryotic expression vector, the specific steps are as follows:

[0191] Sequence alignment analysis based on the second sequencing results identified a total of 7 vNAR antibody amino acid sequences with different CDR3 regions. Three vNAR sequences were obtained 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). Four vNAR sequences were obtained 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 alignment results are shown in Figure 24. Codon optimization and full-gene synthesis were performed on the 7 vNAR antibody sequences using bioengineering methods. Recombinant plasmids were constructed and cloned into the pET-28a prokaryotic expression vector to obtain the recombinant plasmids.

[0192] S2. Purify the expression of the recombinant plasmid. The specific steps are as follows:

[0193] Add 1 μL of recombinant plasmid to 50 μL of BL21(DE3) competent cells and incubate on ice for 30 min. Then, heat shock at 42℃ for 90 s, and immediately place on ice for 5 min. Add 900 μL of antibiotic-free LB medium under sterile conditions and incubate on a shaker at 37℃ and 220 rpm for 45 min to obtain the second transformation culture (cp, nsp1, nsp2). Then, spread 100 μL of the second transformation culture (cp, nsp1, nsp2) onto Kana-resistant LB solid medium and incubate overnight at 37℃ (inverted) to obtain the second plate. The next day, pick a single colony from the sixth plate and transfer it to 5 mL of Kana-resistant LB liquid medium. Incubate at 37℃ and 220 rpm until the logarithmic growth phase. Add glycerol and the bacterial culture to cryovials and store at -80℃ to obtain the BL21(DE3) glycerol-containing vNAR nanobody.

[0194] The expression, purification, desalting, and concentration steps of the vNAR nanobodies were the same as in Example 1, resulting in the vNAR nanobodies. Figure 25 shows the prokaryotic expression and purification results of the vNAR nanobodies (SEQ ID NO. 8-10) screened from the shark natural library, and Figure 26 shows the prokaryotic expression and purification results of the vNAR nanobodies (SEQ ID NO. 11-14) screened from the shark natural library. The concentrations of cp, nsp1, and nsp2 nanobody proteins were determined using a BCA kit. The molecular weights of the nanobody proteins in 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 nanobody proteins in 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, the proteins were flash-frozen in liquid nitrogen and stored at -80°C.

[0195] Western blot identification of S3 and vNAR nanobody proteins is performed as follows:

[0196] (1) SDS-PAGE electrophoresis: using Omni-Easy TM Prepare a 15% PAGE gel using the one-step PAGE gel rapid preparation kit. Add 40 μL of vNAR nanobody protein to 10 μL of 5× loading buffer, incubate in a 99℃ metal bath for 10 min, centrifuge at 13000 rpm for 10 min, and load 10 μL of sample. Set the parameters to 200V and incubate for 40 min.

[0197] (2) Electrophoresis: After electrophoresis, remove the PAGE gel and immerse it in ddH2O. Immerse a 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 air bubbles with a roller, clamp them tightly, and place them in the transfer apparatus. Pour in the pre-cooled transfer buffer, cover the transfer apparatus, and bury it in ice. Set the parameters to 200mM for 90min.

[0198] (3) Washing: After the transfer, take out the PVDF membrane and then wash it with TBST 6 times, 3 times on the front side and 3 times on the back side, 5 minutes each time.

[0199] (4) Sealing: Add 5% skim milk powder solution as sealing solution and seal at room temperature for 2 hours.

[0200] (5) Incubation of primary antibody: Discard the blocking solution, add mouse anti-His diluted 1:3000 as primary antibody, and incubate at room temperature for 1 h.

[0201] (6) Washing: TBST wash 6 times, 3 times on the front and 3 times on the back, 5 minutes each time.

[0202] (7) Incubation of secondary antibody: Add 1:5000 diluted goat anti-mouse antibody (HRP) as secondary antibody and incubate at room temperature for 1 h.

[0203] (8) Washing: TBST wash 6 times, front side 3 times, back side 3 times, 5 minutes each time.

[0204] (9) After adding 2 mL of ECL chromogenic solution to the membrane and incubating it in the dark for 2 min, the membrane was photographed. The protein had good specificity and no extra bands. The specific Western blot identification of the shark vNAR nanobody protein after purification is shown in Figure 27. The vNAR nanobody protein band was single and without extra bands, indicating good specificity.

[0205] S4. ELISA is used to verify the binding ability of vNAR nanobodies to CP, NSP1, and NSP2 antigen proteins. Specific steps are as follows:

[0206] (1) Antigen coating: Dilute the cp, nsp1, and nsp2 antigen proteins to 1 μg / mL with CBS coating buffer, 100 μL per well, and coat overnight at 4°C.

[0207] (2) Washing the plate: Pour out the liquid in the plate and pat it dry on absorbent paper (when patting dry, be careful to avoid leaving fingerprints on the bottom of the microplate that may affect subsequent readings). Wash each well with 200 μL PBS 3 times, 5 min each time.

[0208] (3) Sealing: 200 μL of 1% BSA solution per well was used to seal the well at room temperature for 1 h.

[0209] (4) Washing: Discard the blocking solution and wash each well with 200 μL PBS 3 times, 5 min each time.

[0210] (5) Primary antibody: Each vNAR nanobody and positive control were set with different concentration gradients and 3 parallel groups. The negative control was BSA. 100 μL was added to each well and incubated for 1 h.

[0211] (6) Washing: Discard the liquid in the plate, add 200 μL of PBST to each well and wash 3 times, 5 min each time.

[0212] (7) Secondary antibody: Add 100 μL of vNAR nanobody 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.

[0213] (8) Washing: Discard the liquid in the plate and wash with 200 μL PBST, washing 8 times, 5 min each time.

[0214] (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.

[0215] (10) Termination: After the color development is complete, add 100 μL of 1M HCl to each well to terminate the color development.

[0216] (11) Reading: On the OD of the microplate reader 450nm Read the values ​​from the microplate.

[0217] The specific vNAR nanobody ELISA affinity verification diagram is shown in Figure 28. As can be seen from the figure, for each antigen protein, the nanobodies screened from the immune library have stronger affinity than the nanobodies screened from the natural library.

[0218] S5, BLI Experiment

[0219] The kinetics of binding ability of three BLI-based vNAR nanobodies (cp-I-C5, nsp1-I-F3, and nsp2-I-B12) to their corresponding antigens were determined, and the specific steps are as follows:

[0220] (1) Mix the three antigen proteins cp, nsp1 and nsp2 with biotin NHS-PEG. 12 - Mix biotin at a molar ratio of 1:1 and incubate at room temperature for 30 min; remove residual biotin using a PD MiniTrap G-25 desalting column, determine the concentration of biotinylated antigen using a BCA kit, open Octet software, and set up the antigen-antibody kinetic detection process as shown in Table 11.

[0221] Table 11 BLI Experimental Procedure

[0222] (2) Add a total volume of 200 μL of solution to the black 96-well plate in sequence.

[0223] (3) The experimental results were analyzed using Octet BLI Analysis v12.2.2.4 software, and the dissociation constant (KD value) was calculated.

[0224] The BLI results for the nanobody are shown in Figure 29. A is the kinetic detection result of cp-I-C5, B is the kinetic detection result of nsp1-I-F3, and C is the kinetic detection result of nsp2-I-B12.

[0225] The binding rate constant (kon), dissociation rate constant (kdis), and affinity constant (KD) values ​​of the three nanobodies cp-I-C5, nsp1-I-F3, and nsp2-I-B12 are shown in Table 12.

[0226] Table 12 Kon, Kdis, and K of three types of vNAR nanobodies D value

[0227] Example 5

[0228] The vNAR nanobody obtained in Example 4 was initially applied to colloidal gold chromatography test strips. The specific steps are as follows:

[0229] (1) Preparation of colloidal gold (0.02% 30nm colloidal gold)

[0230] Prepare a clean 250mL Erlenmeyer flask, rinse it repeatedly with purified water 5-6 times, and dry it for later use. Place a Type B stirring rotor inside, then place it on an electronic balance to remove the tare. Add 189g of purified water to the Erlenmeyer flask, and then add 2g of 2% chloroauric acid (HAuCl4) solution using a pipette. Transfer the Erlenmeyer flask to a heated stirrer and heat it. When the temperature of the Erlenmeyer flask reaches about 95℃, turn off the heating function, turn on the stirring function, and quickly add 9mL of 1% trisodium citrate solution. The solution in the Erlenmeyer flask will slowly change from yellow to dark gray, and finally turn purplish-red. Let it react for another 5 minutes, cool to room temperature, and then store it in a 4℃ refrigerator for later use. Take 1mL of the prepared colloidal gold solution. The prepared colloidal gold solution is wine-red, free of impurities or floating matter, and has a bright color. Use a UV spectrophotometer to measure its absorption wavelength between 400-600nm. As shown in Figure 30, the particle size of colloidal gold is linearly correlated with its maximum absorption wavelength. According to the linear equation y = 0.4271x + 514.56 (where x is the particle size and y is the maximum absorption wavelength), the diameter of the colloidal gold particles can be calculated. 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.

[0231] (2) Labeled colloidal gold

[0232] Take 1 mL of colloidal gold solution into a 1.5 mL EP tube, add 10 μL of 0.2 M potassium carbonate (K2CO3) solution, and gently shake to adjust the pH of the colloidal gold. Then add 20 μg of vNAR nanobody protein solution, invert and mix at room temperature for 5 min to label the antibody. Add 30 μL of 10% BSA solution to the antibody-labeled colloidal gold solution, invert and mix at room temperature for 5 min to block the colloidal gold. Centrifuge the blocked colloidal gold solution at 8000 rpm for 6 min, carefully aspirate the supernatant of the colloidal gold solution into a new 1.5 mL centrifuge tube, and continue centrifuging at 10000 rpm for 10 min, discarding the supernatant. Collect the precipitates obtained from the two centrifugations of the blocked colloidal gold solution together, and add 100 μL of gold reconstitution solution to reconstitute, finally obtaining 100 μL of colloidal gold concentrate labeled with vNAR antibody.

[0233] (3) Drying the gold leaf

[0234] Cut the glass fiber into strips 0.5 cm wide, immerse them in the pre-prepared gold-labeled pad treatment solution for 40 minutes, then remove and dry at 42°C. After drying, dilute the colloidal gold concentrate labeled with the vNAR antibody in 300 μL of gold diluent and drop it onto the glass fiber, then dry at 42°C again.

[0235] (4) Assembly and identification of test strips

[0236] Two different antibodies were applied at 1 μL / cm onto an NC membrane using a membrane scribing machine to form the test line (T line) and control line (C line) of the test strip, respectively, and then dried at 42°C. Polyester was cut into 20mm wide strips, immersed in pre-prepared sample pad treatment solution for 40 minutes, and then dried at 42°C. The sample pad, gold label pad, NC membrane, and absorbent paper were assembled onto a PVC base plate in sequence, with an overlap of approximately 0.2cm between each pair of pads. The strips were then cut into 4mm wide test strips using a strip cutter. A diagram of the specific test strip structure is shown in Figure 31. Subsequently, different concentration gradients of CP antigen protein, NSP1 antigen protein, and other antigen proteins were added to the sample pad for detection.

[0237] Comparative Example 1

[0238] The difference between this comparative example and Example 4 is that the antigen protein to be tested is different.

[0239] In Comparative Example 1, the antigen proteins used were MBP (tag protein), SWP1 (shrimp hepatocellular carcinoma virus isolated protein) and VP28 (white spot syndrome virus virulence protein), all of which were proteins that had been expressed, purified and preserved in the laboratory. In Example 4, the antigen proteins used were cp antigen protein, nsp1 antigen protein or nsp2 antigen protein.

[0240] To further illustrate the advantages of the present invention, the antigen proteins used in Comparative Example 1 and Example 4 were tested using the following Test Examples 1-6.

[0241] Test Example 1: Nanobody Cross-Pairing Test

[0242] The conclusion drawn from step S6 of Example 3 is that the nanobodies screened from the shark immune library all exhibited better affinity than those 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, and nsp2 antigen proteins from Example 1 and the colloidal gold test strip combination from Example 4. The specific combinations of the C-line, T-line, and gold-labeled antibody in the colloidal gold test strip for the vNAR nanobodies are shown in Table 13.

[0243] Table 13. Combinations of C-line, T-line, and gold-labeled antibody in colloidal gold test strips for three antigens.

[0244] The cp, nsp1, and nsp2 antigen proteins, prepared in Example 1 at a concentration of 0.1 mg / mL, were added 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.

[0245] The results of test example 1 are shown in Figure 32. For test strips 1 and 2, the combination was: nanobody as the gold-labeled antibody, rabbit polyclonal antibody as the T-line antibody, and anti-His-tagged antibody as the C-line antibody. For test strips 3 and 4, the combination was: rabbit polyclonal antibody as the gold-labeled antibody, nanobody as the T-line antibody, and goat anti-rabbit as the C-line antibody. When the anti-His-tagged antibody was used as the C-line antibody, the C-line did not appear, and the T-line appearance was also worse than with the other combination. Therefore, we selected the optimal antibody cross-pairing combination of rabbit polyclonal antibody as the gold-labeled antibody, nanobody as the T-line antibody, and goat anti-rabbit as the C-line antibody, and used it for subsequent specificity and sensitivity tests.

[0246] Test Example 2: Specificity Detection of Colloidal Gold Test Strips

[0247] The colloidal gold test strip combination with good color development in Test Example 1 was selected, namely, a rabbit polyclonal antibody as the gold-labeled antibody, a nanobody as the T-line antibody (where 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 a goat anti-rabbit antibody as the C-line antibody, to prepare colloidal gold test strips for detecting the three antigen proteins. Samples of the six antigen proteins (cp, snp1, snp2, MBP, SWP1, and VP28) at a concentration of 0.1 mg / mL were added to test strips 1-6 of A, B, and C respectively, and the color development was observed after approximately 5 minutes.

[0248] The results of test example 2 are shown in Figure 33. In A, except for the test strip with cp antigen protein, which showed a clear T line, the other test strips did not show a T line; in B, except for the test strip with nsp1 antigen protein, which showed a clear T line, the other test strips did not show a T line; in C, except for the test strip with nsp2 antigen protein, which showed a clear T line, the other test strips did not show a T line. This indicates that the test strips for the three antigen proteins (cp, nsp1, and nsp2) have good specificity.

[0249] Test Example 3: Sensitivity Detection of Colloidal Gold Test Strips

[0250] The colloidal gold test strip combination with good color development in Test Example 1 was selected, namely, the gold-labeled antibody is rabbit polyclonal antibody, the T-line antibody is nanobody (where A is T-line antibody cp-I-C5 for detecting cp antigen protein; B is T-line antibody nsp1-I-F3 for detecting snp1 antigen protein; C is T-line antibody nsp2-I-B12 for detecting snp2 antigen protein), and the C-line antibody is goat anti-rabbit antibody, to prepare colloidal gold test strips for detecting the three antigen proteins. 100 μL of antigen protein samples with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.25 μg / mL, 6.25 μg / mL, and 3.125 μg / mL, respectively, were added to test strips 1-6 in groups A, B, and C. The specific antigen concentrations in the colloidal gold test strips are shown in Table 10. The color development was observed after approximately 5-10 minutes.

[0251] The results of test example 3 are shown in Figure 34. The color of the T line becomes lighter as the concentration of cp, nsp1, or nsp2 antigen proteins decreases. The sensitivity of the test strip for cp antigen is 6.25 μg / mL, the sensitivity of the test strip for nsp1 antigen is 12.5 μg / mL, and the sensitivity of the test strip for nsp2 antigen is 3.125 μg / mL.

[0252] Test Example 4

[0253] Hepatopancreatic tissue homogenate from 10 shrimp (taken from Putian, Fujian, numbered AJ) was used to detect whether the 10 shrimp were infected with IHHNV using RT-PCR. At the same time, the hepatopancreatic tissue homogenate from the 10 shrimp was diluted and added to colloidal gold test strips. The experimental results of the two methods were compared.

[0254] The results of the comparison between RT-PCR and colloidal gold test strip detection are shown in Table 14. The detection results of the colloidal gold test strip are consistent with those of the RT-PCR method. In the 10 samples (numbered AJ), samples C, I, and J were IHHNV positive, and the remaining 7 were negative. As shown in Figure 35, 1-3 are test strips used to detect the three antigens cp, nsp1, and nsp2 in the positive samples, respectively. It can be seen that the T lines are visible to varying degrees in all samples. This result indicates that the colloidal gold immunochromatographic test strip based on nanobodies has good accuracy.

[0255] Table 14 Comparison of detection results between colloidal gold test strips and RT-PCR

[0256] Test Example 5

[0257] Referring to Test Example 2, 100 μL of pathogen protein at a concentration of 0.1 mg / mL was tested from an enriched shrimp farming water sample. The test was performed using a colloidal gold test strip prepared with a rabbit polyclonal antibody as the gold label, a nanobody as the T-line antibody (where A: T-line antibody is cp-I-C5 for detecting cp antigen protein; B: T-line antibody is nsp1-I-F3 for detecting snp1 antigen protein; C: T-line antibody is nsp2-I-B12 for detecting snp2 antigen protein), and a goat anti-rabbit antibody as the C-line antibody. The specificity test results of the three antigen colloidal gold test strips were essentially the same as those in Figure 33, indicating that the shrimp farming water sample contained IHHNV virus.

[0258] Test Example 6

[0259] Referring to Test Example 2, 100 μL of pathogenic protein at a concentration of 0.1 mg / mL was tested using a colloidal gold test strip prepared with rabbit polyclonal antibody as the gold label, nanobody as the T-line antibody (where A: T-line antibody is cp-I-C5 for detecting cp antigen protein; B: T-line antibody is nsp1-I-F3 for detecting snp1 antigen protein; C: T-line antibody is nsp2-I-B12 for detecting snp2 antigen protein), and goat anti-rabbit antibody as the C-line antibody. The results were essentially the same as those in Figure 33, indicating that the shrimp farming feed solution contained IHHNV virus.

[0260] Therefore, it can be concluded that the vNAR nanobody provided by this invention binds well to colloidal gold, producing a total of three antibody combinations. The first antibody combination is used to specifically recognize the CP antigen protein, including: a rabbit polyclonal antibody as the first gold-labeled antibody, a first T-line antibody with the amino acid sequence SEQ ID NO.17, and a goat anti-rabbit antibody as the first C-line antibody; the second antibody combination is used to specifically recognize the NSP1 antigen protein, including: a rabbit polyclonal antibody as the second gold-labeled antibody, a second T-line antibody with the amino acid sequence SEQ ID NO.18, and a goat anti-rabbit antibody as the second C-line antibody; the third antibody combination is used to specifically recognize the NSP2 antigen protein, including: a rabbit polyclonal antibody as the third gold-labeled antibody, a third T-line antibody with the amino acid sequence SEQ ID NO.19, and a goat anti-rabbit antibody as the third C-line antibody. The test strips prepared using the above three antibody combinations specifically recognize the CP, NSP1, and NSP2 proteins of the IHHNV virus. Within 10 minutes, they can rapidly detect whether shrimp and other farmed aquatic animals are infected with the IHHNV virus, whether the aquaculture water contains the IHHNV virus, or whether farmed feed and live bait are contaminated with paraIHHNV virus. This solves the problem of effective prevention and efficient monitoring of diseases in shrimp farming, lays the foundation for rapid detection of the IHHNV virus, and can provide a reference model for the early detection of pathogens in other farmed animals, demonstrating broad applicability.

[0261] The terminology and expressions used herein are for descriptive purposes only, and the invention should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalents (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0262] Similarly, it should be noted that although the present invention has been described with reference to the specific embodiments described above, those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, any changes or modifications to the above embodiments within the scope of the essential spirit of the present invention will fall within the scope of the claims of the present invention.

Claims

1. A nanobody for detecting infectious subcutaneous and hematopoietic necrosis viruses, characterized in that, 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.

2. The nanobody according to claim 1, characterized in that, The nanobody is shark-derived.

3. A detection product for infectious subcutaneous and hematopoietic necrosis viruses comprising the nanobody described in claim 1.

4. The infectious subcutaneous and hematopoietic tissue necrosis virus detection product according to claim 3, characterized in that, The infectious subcutaneous and hematopoietic necrosis virus detection product is a reagent, test strip, or 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.

5. The infectious subcutaneous and hematopoietic tissue necrosis virus detection product according to claim 3, 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 recognize the cp antigen protein. The first antibody combination consists of: a rabbit polyclonal antibody as the first gold-labeled antibody, a first T-line antibody with the amino acid sequence SEQ ID NO.11, and a goat anti-rabbit antibody as the first C-line antibody. The second antibody combination is used to specifically recognize the nsp1 antigen protein. The second antibody combination consists of: a rabbit polyclonal antibody as the second gold-labeled antibody, a second T-line antibody with the amino acid sequence SEQ ID NO.12, and a goat anti-rabbit antibody as the second C-line antibody. The third antibody combination is used to specifically recognize the nsp2 antigen protein. The third antibody combination consists of: a rabbit polyclonal antibody as the third gold standard antibody, a third T-line antibody with the amino acid sequence SEQ ID NO.13, and a goat anti-rabbit antibody as the third C-line antibody.

6. A method for preparing nanobodies for detecting infectious subcutaneous and hematopoietic necrosis viruses, characterized in that, Includes the following steps: Step A: Expression of cp antigen protein, nsp1 antigen protein, or nsp2 antigen protein, including: 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 prokaryotes and purifying it to obtain the cp antigen protein, nsp1 antigen protein, or nsp2 antigen protein; Step B: Construction of shark natural libraries and / or shark immune libraries; Step C: Perform phage rescue and purification on the constructed shark natural library and / or shark immune library to obtain a phage library and test its titer; Step D: Perform two, three, four, or five rounds of panning on the phage library to obtain the final eluent; Step E: Use the final elution buffer to prepare the supernatant for phage-ELISA identification and sequencing. Based on the sequencing results, screen for cp nanobodies, nsp1 nanobodies, or nsp2 nanobodies with different CDR3 region sequences.

Images