Targeted giant salamander iridovirus nano antibody as well as preparation method and application thereof

Abstract

The invention provides a targeted giant salamander iridovirus nano antibody as well as a preparation method and application thereof. The invention discloses a giant salamander iridovirus targeting nano antibody, and aims to provide a giant salamander iridovirus targeting nano antibody which is low in expression cost, low in difficulty and stable in source, can be effectively combined with giant salamander iridovirus, and specifically realizes immunoassay of the giant salamander iridovirus. The amino acid sequences of the nano antibody are shown as SEQ ID NO.1-6, and the nano antibody belongs to the technical field of biological science.

Description

Technical Field

[0001] The invention belongs to the field of biological science technology, and specifically relates to the preparation and application of nano antibodies targeting giant salamander iris virus. Background Art

[0002] The giant salamander, commonly known as the baby fish, is listed as critically endangered (CR) in the IUCN Red List of Threatened Species and is also a Class II protected wild animal in my country. The giant salamander is known as the "living ginseng in the water" because of its tender meat and rich nutrition. At the same time, with the breakthrough of my country's giant salamander breeding technology, large-scale artificial breeding of giant salamanders has been widely promoted throughout the country. The giant salamander iridovirus is the only giant salamander viral disease discovered so far. The spread of this virus has caused large-scale killings in the large-scale breeding of giant salamanders, seriously challenging the healthy development of giant salamander breeding. The giant salamander iridovirus is transmitted through multiple channels: contaminated water, physical contact and ingestion of infected tissues. The detection of giant salamander iridovirus is of great significance, mainly including protecting endangered species, controlling the spread of diseases, ensuring the safety of aquaculture, maintaining the balance of the ecological environment and promoting scientific research. In the aquaculture industry, regular testing can ensure the health and profitability of the farms.

[0003] The methods currently used to detect giant salamander iridovirus include PCR, cell culture, microscopic examination of infected tissues, and immunological methods. PCR technology has high specificity and sensitivity and can quickly and accurately detect target DNA sequences, but it requires professional equipment and technology and may have false positive results. Although the culture method is simple and intuitive, it takes a long time and requires a high number of cells, and it is easy to miss low-concentration samples. Microscopic examination is limited in its diagnostic accuracy when detecting iridovirus due to its lack of sensitivity and specificity and complex operation.

[0004] Immunological methods have high specificity, but may have cross-reactions or low sensitivity due to the influence of antibody quality and pairing. Compared with traditional monoclonal antibodies, recombinant antibodies have lower production costs and better uniformity. Nanobodies, as a cutting-edge recombinant antibody, have the advantages of small molecular weight, low production cost, and simple purification. They are the smallest antigen binding units known so far. However, there are no reports on anti-giant salamander iridovirus nanoantibodies. The anti-giant salamander iridovirus nanoantibodies prepared for the first time by the present invention using phage display technology and prokaryotic expression technology are very necessary and reliable for rapid immune detection of giant salamander iridovirus. . Summary of the invention

[0005] In view of the above-mentioned shortcomings, the first object of the present invention is to provide a nano-antibody targeting giant salamander iridovirus which has low expression cost, low difficulty, stable source, can effectively bind to giant salamander iridovirus, and can specifically realize immune analysis of giant salamander iridovirus.

[0006] The second object of the present invention is to provide a method for preparing nano antibodies targeting giant salamander iridovirus.

[0007] Another object of the present invention is to provide the use of the above-mentioned nanoantibody targeting giant salamander iridescent virus as a giant salamander iridescent virus immunodetection reagent or detection kit.

[0008] To this end, the first technical solution provided by the present invention is as follows:

[0009] A nanobody targeting giant salamander iris virus, wherein the amino acid sequence of the nanobody is shown in SEQ ID NO.1-6.

[0010] The second technical solution provided by the present invention is a method for preparing the above-mentioned nanoantibody targeting giant salamander iris virus, comprising the following steps:

[0011] (1) immunizing alpacas with inactivated giant salamander iris virus, collecting peripheral blood lymphocytes of alpacas for mRNA extraction after immunization, synthesizing the first chain of cDNA using the mRNA as a template, and performing PCR amplification using the obtained cDNA as a template to obtain a DNA fragment encoding a nanobody;

[0012] (2) Connecting the DNA fragment encoding the nanobody to the pComb3xss vector, transforming it into competent cells, constructing an initial nanobody library, and expanding the culture to obtain a nanobody library.

[0013] (3) using the giant salamander iris virus as a coating antigen, and performing enrichment and panning screening on the nanoantibody library to obtain nanoantibody phagemids against the giant salamander iris virus;

[0014] (4) The phagemid is transformed into a host expression bacterium to induce the expression of the nanoantibody, and the nanoantibody sequence targeting the giant salamander iris virus is purified.

[0015] Furthermore, for the nanoantibody targeting giant salamander iris virus, the mRNA extraction in step (1) is performed using a TRIzol kit.

[0016] Furthermore, for the aforementioned nanoantibodies targeting giant salamander iris virus, the primer sequences used in the PCR amplification in step (1) are as follows:

[0017] The nucleotide sequence of primer 1 is shown in SEQ ID NO.7;

[0018] The nucleotide sequence of primer 2 is shown in SEQ ID NO.8;

[0019] The nucleotide sequence of primer 3 is shown in SEQ ID NO.9;

[0020] The nucleotide sequence of primer 4 is shown in SEQ ID NO.10;

[0021] The nucleotide sequence of primer 5 is shown in SEQ ID NO.11.

[0022] Furthermore, for the nanoantibody targeting giant salamander iris virus, the expansion culture described in step (2) is to add the recovered bacterial solution to the culture medium, shake at 37°C for 2 hours, add helper phage to expand the initial library, shake for 2 hours after adding helper phage, add kanamycin, then shake overnight, and collect phage by centrifugation on the next day to complete the construction of the immune library;

[0023] Furthermore, for the above-mentioned nanoantibodies targeting giant salamander iridescent virus, the nanoantibody library described in step (3) is enriched and screened through three rounds of panning, monoclonal colonies are picked and inoculated into super broth culture medium for culture, and then added to the ELISA plate pre-coated with giant salamander iridescent virus. After shaking and washing at room temperature, anti-M13-horseradish peroxidase-labeled antibody is added, and shaking and washing at room temperature again, 3,3',5,5'-tetramethylbenzidine is added for color development, and the monoclonal colonies corresponding to the positive clone wells with absorbance values ​​greater than 1.5 are picked for sequencing.

[0024] Furthermore, the above-mentioned nanoantibodies targeting giant salamander iris virus, the step (4) of transforming the phagemid into the host expression bacteria is to use the Omega plasmid small-scale extraction kit to extract the plasmid of the positive monoclonal colony, and then mix the plasmid with TOP10F' competent cells, place it in an ice bath and then heat shock it in 42°C hot water, then ice bath it again, add super broth medium, shake it at 37°C for one hour, and complete the transformation; inoculate the introduced TOP10F' into the culture medium, shake and culture it until the absorbance OD 600 0.8-1.0, add isopropyl thiogalactoside to 1 mM and induce overnight.

[0025] Furthermore, the above-mentioned nanoantibody targeting giant salamander iris virus, the purification of the nanoantibody described in step (4) is to add BeyoLytic TM The bacteria were lysed by a bacterial active protein extraction reagent, and the supernatant was collected by centrifugation; the Ni-NTA matrix was added to the supernatant collected above, and then added to the purification column after shaking at room temperature; then a phosphate equilibration buffer containing imidazole was added to wash the purification column and elute the impurity protein; then a phosphate elution buffer containing imidazole was added to elute the target protein adsorbed on the nickel column; the nano-antibody was added to a dialysis bag and dialyzed in a phosphate buffer.

[0026] Another technical solution of the present invention is the use of the above-mentioned nano-antibody targeting giant salamander iridovirus in the preparation of a detection reagent or a detection kit for detecting giant salamander iridovirus.

[0027] The last technical solution of the present invention is a detection reagent for detecting giant salamander iridovirus, including the nano antibody targeting giant salamander iridovirus described in the first technical solution.

[0028] Compared with the prior art, the technical solution provided by the present invention has the following advantages:

[0029] 1. The nanoantibody targeting giant salamander iridovirus provided by the present invention is obtained based on a prokaryotic expression method, and has the advantages of low expression cost, low difficulty, and stable source;

[0030] 2. The nanoantibodies targeting giant salamander iridescent virus provided by the present invention can effectively bind to giant salamander iridescent virus and can specifically realize immune analysis of giant salamander iridescent virus; they are suitable for immune detection of giant salamander iridescent virus and will have great application potential in giant salamander iridescent virus screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the agarose gel electrophoresis of the heavy chain variable region gene of the alpaca IgG2 / 3 antibody;

[0032] Figure 2 This is the result graph of 96 phage monoclonal phage-ELISA;

[0033] Figure 3 This is the expression diagram of the nanobody protein analyzed by SDS-PAGE electrophoresis;

[0034] Figure 4 This is the standard curve of the giant salamander iris virus ELISA detection based on nanoantibodies. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific implementation methods, but those skilled in the art should understand that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the technical solution of the present invention, and these modifications and replacements all fall within the scope of protection of the present invention.

[0036] Example 1 Immune library construction

[0037] 1. Alpaca Immunity

[0038] After being cultured in LB medium for 18 hours, the giant salamander iris virus was washed and inactivated antigen of the giant salamander iris virus was prepared by high-temperature sterilization. According to the immune effect of the triple quadruple vaccine, the alpaca with the strongest immune response and the healthiest effect was selected, and the negative serum of the giant salamander iris virus before immunization was taken for use. After the immunization was initiated, five immunizations were performed on the 0th day, the 14th day, the 28th day, the 42nd day and the 56th day respectively. The serum titers were tested seven days after the three immunizations, the four immunizations and the five immunizations, and the peripheral blood was collected for peripheral blood lymphocyte extraction.

[0039] 2. Extraction of Peripheral Blood Lymphocytes

[0040] Slowly add 15 mL of peripheral blood into 15 mL of cell separation solution at a 45-degree tilt, centrifuge at 400 g for 30 min at room temperature, then transfer the lymphocytes to a new centrifuge tube, add 10 mL of PBS buffer, centrifuge again at 400 g for 20 min, slowly aspirate the supernatant, add PBS to resuspend, and count the number of cells.

[0041] 3. cDNA Acquisition

[0042] According to TRIzol TM Total RNA was extracted according to the instructions. After extraction, OD was measured. 260 nm and OD 280 The concentration and purity of the extracted RNA were calculated based on the absorbance value at 0.1 nm. The obtained RNA could be stored in a -80°C refrigerator and used for subsequent reverse transcription to obtain cDNA.

[0043] Using SuperScript TM IV First-Strand Synthesis System reverse transcribes all RNA obtained, and the process is as follows:

[0044] (1) RNA denaturation, the reaction system is as shown in Table 1:

[0045] Table 1

[0046]

[0047] Incubate at 65°C for 5 min and keep on ice for at least 1 min.

[0048] (2) Reverse transcription reaction system, the reaction system is as shown in Table 2:

[0049] Table 2

[0050]

[0051]

[0052] (3) The reverse transcription reaction is as follows:

[0053] The RNA denaturation system of step (1) and the reverse transcription reaction system of step (2) were mixed, incubated at 23° C. for 10 minutes, then at 55° C. for 10 minutes, and finally at 80° C. for 10 minutes to terminate the reaction.

[0054] (4) Removal of RNA:

[0055] Add 1 μL of E. coli RNase H to the above system and incubate at 37°C for 20 minutes.

[0056] (5) Storage of cDNA:

[0057] The cDNA obtained after the above reaction was stored at -20°C for subsequent PCR amplification.

[0058] 4. PCR amplification of the gene sequence of nanoantibodies targeting giant salamander iridovirus

[0059] The one-step method was used to amplify the nanobody gene sequence. The primers were synthesized by Shanghai Bioengineering. The primer sequences are shown in Table 3:

[0060] Table 3

[0061]

[0062] The nanobody gene sequence was amplified by PCR using cDNA as a template, and the amplification reaction system is shown in Table 4:

[0063] Table 4

[0064]

[0065]

[0066] The PCR program is shown in Table 5:

[0067] Table 5

[0068]

[0069] 10 μL of PCR product was identified by agarose gel electrophoresis. Figure 1 ; At the same time, the remaining PCR products are recovered.

[0070] 5. Connection of Nanoantibody Gene Targeting Giant Salamander Iridescent Virus with pComb3xss Vector

[0071] (1) Enzyme digestion of pComb3xss vector, the enzyme digestion system is as follows:

[0072]

[0073] The target fragments were recovered by gel excision and recovery.

[0074] (2) Enzyme cleavage of the nanoantibody gene targeting the giant salamander iris virus, the enzyme cleavage system is as follows:

[0075]

[0076]

[0077] The target gene is recovered after enzyme digestion.

[0078] (3) Connection of the nanoantibody gene targeting giant salamander iris virus and the pComb3xss vector, the connection system is as follows:

[0079]

[0080] The above system was incubated at 16°C overnight, and then incubated at 65°C for ten minutes to terminate the ligation reaction.

[0081] The ligation product was purified and placed in a -20°C refrigerator for subsequent experiments.

[0082] 6. Electroporation of Ligation Products

[0083] Add 3 μL of the above ligation product to 25 μL of competent cells ER2738, and transform the mixture by electroporation under the conditions of 1.8 KV, 200 Ω, and 25 μF; after the transformation is completed, shake and recover for 1 hour to complete the construction of the initial phage library.

[0084] 7. Phage library amplification and expression

[0085] The revived bacterial liquid was added to 200 mL of culture medium. After shaking at 37°C for 2 hours, helper phage was added to expand the initial library. After adding the helper phage, it was shaken for 2 hours. Kanamycin was added and then shaken overnight. The phage was collected by centrifugation the next day to complete the construction of the immune library.

[0086] Example 2 Selection of Nanobodies

[0087] 1. First round of selection

[0088] (1) 10 9 PFU / mL iridovirus was coated onto the ELISA plate and shaken at room temperature for 1 hour;

[0089] (2) Wash three times with PBST and block with 3% skim milk powder for 1 hour;

[0090] (3) Add 100 μL of the phage library constructed in Example 1 and shake at room temperature for 1 hour;

[0091] (4) Wash 10 times with PBST;

[0092] (5) Elution of bound phage

[0093] 100 μl of glycine-HCl (the glycine-HCl is 0.1 M glycine-HCl (pH 2.2)) was added to the wells, incubated at room temperature for 15 minutes, and then immediately neutralized with 1 M Tris-HCl;

[0094] (6) Take 2 μL of eluted phage and add it to 198 μL of ER2738 (OD 0.4-0.6), let it infect for 0.5 h, then take 100 μL and spread it on carbenicillin medium for the first round of panning output titer test;

[0095] (7) Add the remaining phage to 2 mL of ER2738, culture overnight, and collect by centrifugation.

[0096] 2. Second round of selection

[0097] (1) 5x10 8 PFU / mL iridovirus was coated onto the ELISA plate and shaken at room temperature for 1 hour;

[0098] (2) Wash three times with PBST and block with 3% skim milk powder for 1 hour;

[0099] (3) Add 100 μL of the phage library after the first round of panning and shake at room temperature for 1 hour;

[0100] (4) Wash ten times with PBST;

[0101] (5) Elution of bound phage

[0102] 100 μl of glycine-HCl (the glycine-HCl is 0.1 M glycine-HCl (pH 2.2)) was added to the wells, incubated at room temperature for 15 minutes, and then immediately neutralized with 1 M Tris-HCl;

[0103] (6) Take 2 μL of eluted phage and add it to 198 μL of ER2738 (OD 0.4-0.6), let it infect for 0.5 h, then take 100 μL and spread it on carbenicillin medium for the second round of panning phage output titer test;

[0104] (7) Add the remaining phage to 2 mL of ER2738, culture overnight, and collect by centrifugation.

[0105] 3. The third round of selection

[0106] (1) 10 8 PFU / mL iridovirus was coated onto the ELISA plate and shaken at room temperature for 1 hour;

[0107] (2) Wash three times with PBST and block with 3% skim milk powder for 1 hour;

[0108] (3) Add the phage library after R2 panning and shake at room temperature for 1 hour;

[0109] (4) Wash ten times with PBST;

[0110] (5) Elute the bound phages. Add 100 μl of glycine-HCl (0.1 M glycine-HCl (pH 2.2)) to the wells, incubate at room temperature for 15 minutes, and then immediately neutralize with 1 M Tris-HCl;

[0111] (6) 2 μL of eluted phage was added to 198 μL of ER2738 (OD 0.4-0.6), inoculated for 0.5 h, and then 100 μL was plated on carbenicillin medium for R3 phage output titer test;

[0112] (7) Add the remaining phage to 2 mL of ER2738, culture overnight, and collect by centrifugation.

[0113] 4. Screening of Nanobody-Positive Strains

[0114] (1) Thirty-two monoclonal colonies were selected from the output titer test plates after three rounds of screening, inoculated into super broth medium, and cultured overnight with shaking.

[0115] (2) Add the monoclonal colony cultured in step (1) to the pre-coated 8 PFU / mL giant salamander iris virus ELISA plate, shake at room temperature for 1 hour, wash three times with PBST, add 100 μL / well of 100 ng / mL anti-M13-horseradish peroxidase-labeled antibody, shake at room temperature for 1 hour, wash five times with PBST, and add 100 μL / well of 3,3',5,5'-tetramethylbenzidine for color development.

[0116] (3) After the color development is completed (refer to the results Figure 2 ), the monoclonal colonies corresponding to the positive clone wells with absorbance values ​​greater than 1.5 were selected for sequencing, and their gene sequences are shown in SEQ ID NO.1-6.

[0117] Example 3 Prokaryotic expression and purification of anti-giant salamander iris virus nanoantibodies

[0118] 1. Prokaryotic expression of nanobodies

[0119] (1) Use the Omega plasmid miniprep kit to extract the plasmid of the positive monoclonal colony, then mix the plasmid with TOP10F' competent cells, put them in an ice bath for 30 minutes, heat shock them in 42°C hot water for 90 seconds, then put them in an ice bath for 5 minutes, add 1 ml of super broth medium, shake at 37°C for one hour to complete the transformation;

[0120] (2) Inoculate the introduced TOP10F' into the culture medium and culture with shaking until the absorbance reaches OD 600 0.8-1.0, add isopropyl thiogalactoside to 1 mM and induce overnight.

[0121] 2. Purification of Nanobodies

[0122] (1) Collect the overnight culture solution into a centrifuge bottle and centrifuge at 5000g for 20 min;

[0123] (2) Join BeyoLytic TM The bacteria were lysed with bacterial active protein extraction reagent and centrifuged at 16000g for 10 min at 4°C;

[0124] (3) collecting the supernatant for subsequent purification;

[0125] (4) Add Ni-NTA matrix to the supernatant collected above, shake at room temperature for 1 hour, and add to the purification column;

[0126] (6) Add phosphate equilibration buffer containing 2 mM imidazole to wash the purification column and elute the impurities;

[0127] (7) adding phosphate elution buffer containing 50 mM imidazole to elute the target protein adsorbed on the nickel column;

[0128] (8) Take a small amount of the purified nanoantibody and perform SDS-PAGE electrophoresis detection. Figure 3 ;

[0129] (9) The remaining collected nanobodies were added to a dialysis bag and dialyzed in phosphate buffer for 24 hours.

[0130] Application example: Application of nanoantibodies in the immunodetection of giant salamander iris virus

[0131] Direct capture immunoassay of giant salamander iridovirus

[0132] (1) Dilute 1x10 with 0.05 M carbonate buffer 8 The giant salamander iris virus was added to a 96-well ELISA plate at 100 μL / well and coated at 4°C overnight;

[0133] (2) The next day, wash the coated 96-well plate three times with phosphate-Tween buffer;

[0134] (3) Add 300 μL of 3% skim milk powder to each well for blocking. The blocking procedure is incubation with shaking at 37°C for 1 hour.

[0135] (4) After incubation, wash three times with phosphate-Tween buffer

[0136] (5) 100 μL of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0 μg / mL nanoantibodies were added to each well, mixed, and incubated at 37°C for 1 h;

[0137] (5) After the reaction, the mixture was washed three times with phosphate-Tween buffer;

[0138] (6) Add 100 μL of horseradish peroxidase-labeled anti-HA secondary antibody diluted 10,000 times to each well (100 μL / well) and incubate at 37°C for 1 hour;

[0139] (7) After incubation, wash five times with phosphate-Tween buffer;

[0140] (8) Add 100 μL of 3,3',5,5'-tetramethylbenzidine colorimetric solution to each well and react for 15 min in the dark;

[0141] (9) After color development, add 50 μL of 2 M H2SO4 to each well to terminate the reaction.

[0142] The standard curve of the ELISA test for the giant salamander iris virus is shown in Figure 4 ,pass Figure 4 It can be seen that the nanoantibody targeting giant salamander iridovirus provided in the present application can effectively bind to giant salamander iridovirus, thereby specifically realizing immune analysis of giant salamander iridovirus.

Claims

1. A nanobody targeting giant salamander iris virus, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO.1-6.

2. The method for preparing the nanoantibody targeting giant salamander iridescent virus according to claim 1, characterized in that: The steps include: (1) immunizing alpacas with inactivated giant salamander iris virus, collecting peripheral blood lymphocytes of alpacas for mRNA extraction after immunization, synthesizing the first chain of cDNA using the mRNA as a template, and performing PCR amplification using the obtained cDNA as a template to obtain a DNA fragment encoding a nanobody; (2) connecting the DNA fragment encoding the nanobody to the pComb3xss vector, transforming into competent cells, and constructing an initial nanobody library; and expanding and culturing the initial library to obtain a nanobody library; (3) using the giant salamander iris virus as a coating antigen, and performing enrichment and panning screening on the nanoantibody library to obtain nanoantibody phagemids against the giant salamander iris virus; (4) The phagemid is transformed into a host expression bacterium to induce the expression of the nanoantibody, and the nanoantibody sequence targeting the giant salamander iris virus is purified.

3. The nanoantibody targeting giant salamander iridescent virus according to claim 1, characterized in that: The mRNA extraction described in step (1) is performed using a TRIzol kit.

4. The nanoantibody targeting giant salamander iridescent virus according to claim 1, characterized in that: The primer sequences used in the PCR amplification in step (1) are as follows: The nucleotide sequence of primer 1 is shown in SEQ ID NO.7; The nucleotide sequence of primer 2 is shown in SEQ ID NO.8; The nucleotide sequence of primer 3 is shown in SEQ ID NO.9; The nucleotide sequence of primer 4 is shown in SEQ ID NO.10; The nucleotide sequence of primer 5 is shown in SEQ ID NO.

11.

5. The nanobody targeting giant salamander iridescent virus according to claim 1, characterized in that: The initial library described in step (3) is expanded by adding the recovered bacterial solution to the culture medium, shaking at 37°C for 2 hours, adding helper phage to expand the initial library, shaking for 2 hours after adding the helper phage, adding kanamycin, then shaking overnight, and collecting phages by centrifugation the next day to complete the construction of the immune library.

6. The nanobody targeting giant salamander iridescent virus according to claim 1, characterized in that: The nanoantibody library described in step (3) is enriched and screened by three rounds of panning. Monoclonal colonies are picked and inoculated into super broth culture medium for culture, and then added to the ELISA plate pre-coated with giant salamander iridescent virus. After shaking and washing at room temperature, anti-M13-horseradish peroxidase-labeled antibody is added, and shaking and washing at room temperature again, 3,3',5,5'-tetramethylbenzidine is added for color development, and the monoclonal colonies corresponding to the positive clone wells with absorbance values ​​greater than 1.5 are picked for sequencing.

7. The nanobody targeting giant salamander iridescent virus according to claim 1, characterized in that: The step (4) of transforming the phagemid into the host expression bacteria is to use the Omega plasmid small-scale extraction kit to extract the plasmid of the positive monoclonal colony, then mix the plasmid with TOP10F' competent cells, place them in an ice bath and then heat shock them in 42°C hot water, then place them in an ice bath again, add super broth medium, shake at 37°C for one hour to complete the transformation; inoculate the introduced TOP10F' into the culture medium, shake and culture until the absorbance OD 600 0.8-1.0, add isopropyl thiogalactoside to 1 mM and induce overnight.

8. The nanobody targeting giant salamander iridescent virus according to claim 1, characterized in that: Purification of the Nanobody in Step (4) Add BeyoLytic to the overnight cultured bacterial solution TM The bacteria were lysed by a bacterial active protein extraction reagent, and the supernatant was collected by centrifugation; the Ni-NTA matrix was added to the supernatant collected above, and after shaking at room temperature, it was added to a purification column; then a phosphate equilibration buffer containing imidazole was added to wash the purification column and elute the impurity protein; then a phosphate elution buffer containing imidazole was added to elute the target protein adsorbed on the nickel column; the nano-antibody was added to a dialysis bag and dialyzed in a phosphate buffer.

9. Use of the nanoantibody targeting giant salamander iridescent virus according to claim 1 in preparing a detection reagent or a detection kit for detecting giant salamander iridescent virus.

10. A detection reagent for detecting giant salamander iris virus, characterized in that: It includes the nano antibody targeting giant salamander iris virus as described in claim 1.

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