A Nanobody Targeting Carp Herpesvirus Type II and Its Application

Abstract

This invention provides a nanobody targeting carp herpesvirus type II and its application. The amino acid sequence of the nanobody is shown in SEQ ID NO. 1-6. The method includes immunizing alpacas with inactivated carp herpesvirus type II, collecting peripheral blood from the immunized alpacas and separating lymphocytes, and extracting total RNA; synthesizing cDNA by reverse transcription, and amplifying the gene fragment encoding the nanobody; ligating it into the pComb3xss vector, and then transforming it into competent cells to construct a nanobody phage display library; enriching and panning the inactivated virus as a coating antigen to obtain specifically binding phage particles; transforming the selected positive clones into host bacteria for induced expression; and obtaining the anti-carp herpesvirus type II nanobody after separation and purification. This invention aims to provide a carp herpesvirus type II nanobody that is low in expression cost and difficulty, has a stable source, can effectively bind to carp herpesvirus type II, and specifically achieves immunoassay of carp herpesvirus type II.

Description

Technical Field

[0001] This invention belongs to the field of bioscience and technology, specifically relating to a nanobody targeting carp herpesvirus type II and its application. Background Technology

[0002] Hybridized silver carp is an extremely important freshwater aquaculture species in my country. Due to its tender flesh, rich nutrition, and rapid growth, it is widely known as "soft gold of the water" and is a major economic pillar for many fish farmers. However, with the promotion of high-density intensive aquaculture, the frequent occurrence of viral diseases seriously threatens the healthy development of this industry. Among them, carp herpesvirus type II is the culprit causing herpesvirus-induced hematopoietic organ necrosis in crucian carp. This virus has extremely high infectivity and mortality, with rapid onset and high mortality, and can cause latent infection, becoming the number one bottleneck restricting the sustainable development of crucian carp aquaculture in my country. Carp herpesvirus type II has multiple transmission routes, including contaminated water, contact with diseased fish, and vertical transmission. Therefore, establishing a rapid, sensitive, and low-cost detection method for carp herpesvirus type II is of great practical significance for early disease warning, interrupting transmission routes, ensuring the safety of aquaculture, and maintaining the ecological balance of aquatic areas.

[0003] Currently, the main methods for detecting carp herpesvirus type II include polymerase chain reaction (PCR), cell isolation and culture, histopathological observation, and conventional immunological methods. While PCR technology boasts high specificity and sensitivity, accurately detecting viral DNA, it relies on expensive thermal cycling equipment, complex nucleic acid extraction procedures, and specialized operators. Furthermore, it is susceptible to aerosol contamination leading to false positives, hindering its widespread adoption in grassroots aquaculture farms. Although cell culture is the gold standard for virus detection, carp herpesvirus type II exhibits high selectivity for cell lines, making isolation difficult and requiring long culture periods. It is also prone to bacterial contamination, failing to meet the needs of rapid diagnosis during acute outbreaks. Histopathological examination and electron microscopy are limited by equipment costs and cumbersome procedures, and often miss cases in the early stages of infection due to subtle lesions, resulting in low diagnostic efficiency.

[0004] Immunological methods, which detect viral antigen proteins, are intuitive and rapid. However, current detection techniques based on traditional polyclonal or monoclonal antibodies still have significant limitations, such as high production costs, long preparation cycles, and unstable antibody properties. Traditional antibodies have large molecular weights, are easily inactivated by temperature and pH, require cold chain storage, and are difficult to adapt to the harsh environmental conditions of aquaculture sites. Furthermore, large-molecule traditional antibodies struggle to recognize hidden epitopes on the viral surface, potentially leading to insufficient sensitivity. In contrast, nanobodies, as a novel type of recombinant antibody, are the smallest known antigen-binding units. They possess significant advantages such as small molecular weight, extremely stable physicochemical properties, good water solubility, and easy, low-cost, and efficient expression in prokaryotic systems. Currently, there are no reports on nanobodies targeting carp herpesvirus type II, either domestically or internationally. This invention, using phage display and prokaryotic expression technologies, has for the first time prepared high-affinity, high-stability anti-carp herpesvirus type II nanobodies, which is essential and reliable for achieving low-cost, specific, and rapid on-site immunoassay for carp herpesvirus type II. Summary of the Invention

[0005] To address the aforementioned shortcomings, the first objective of this invention is to provide a nanobody targeting cyprinid herpesvirus type II, which has low expression cost, low difficulty, stable source, can effectively bind to cyprinid herpesvirus type II, and can specifically achieve immunoassay of cyprinid herpesvirus type II.

[0006] The second objective of this invention is to provide a method for preparing a nanobody targeting carp herpesvirus type II.

[0007] Another objective of this invention is to provide the application of the above-mentioned cyprinid-targeting cyprinid virus type II nanobody in cyprinid virus type II immunoassay reagents or assay kits.

[0008] Therefore, the first technical solution provided by this invention is as follows: I. A nanobody targeting carp herpesvirus type II The amino acid sequences of the nanobody are shown in SEQ ID NO.1-6.

[0009] II. Preparation method of nanobody targeting carp herpesvirus type II The method includes the following steps: (1) Alpaca were immunized with inactivated carp herpesvirus type II. After immunization, peripheral blood lymphocytes of alpaca were collected for mRNA extraction. The mRNA was used as a template to synthesize the first strand of cDNA. The obtained first strand of cDNA was used as a template for PCR amplification to obtain the DNA fragment encoding nanobody. (2) The DNA fragment encoding the nanobody was ligated to the pComb3xss vector and then transformed into competent cells to construct an initial nanobody library; the initial nanobody library was then expanded and cultured to obtain a nanobody library. (3) Using inactivated carp herpesvirus type II as the coating antigen, the nanobody library was enriched and screened to obtain anti-carp herpesvirus type II nanobody phage particles. (4) Transform the anti-carp herpesvirus type II nanobody phage particles into the host expression bacteria, induce the expression of nanobody, and purify to obtain the anti-carp herpesvirus type II nanobody.

[0010] The mRNA extraction in step (1) was performed using the LeukoLOCK kit.

[0011] The sequences of primers 1-4 used in the PCR amplification described 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.

[0012] In step (2), the construction of the initial nanobody library specifically involves: The DNA fragment encoding the nanobody was ligated to the pComb3xss vector to obtain the ligation product. The ligation product was added to competent cells ER2738 and subjected to electroporation transformation under the conditions of 1.8KV, 200Ω, and 25µF. After shaking and recovery for 1 hour, the recovered strain was obtained as the initial nanobody library, thus completing the construction of the initial phage library.

[0013] The expansion culture of the nanobody library is as follows: the revived strain is added to the culture medium, shaken at 37°C for 2 hours, helper phage is added, and after standing for half an hour, it is shaken again for 2 hours to expand the initial nanobody library. Kanamycin is added, and then shaken overnight. The next day, the phage is collected by centrifugation to complete the construction of the immune library and obtain the nanobody library.

[0014] The specific steps (3) are as follows: Inactivated carp herpesvirus type II (CHVII) was used as a coating antigen and cultured on an ELISA plate to obtain a CHVII-pre-coated ELISA plate. The nanobody library was subjected to three rounds of panning, and different monoclonal colonies were selected and inoculated into super broth medium for culture. The culture medium was then added to the CHVII-pre-coated ELISA plate, and after washing with shaking at room temperature, horseradish peroxidase-labeled anti-HA antibody was added. After washing with shaking at room temperature again, 3,3',5,5'-tetramethylbenzidine was added for color development. Positive monoclonal colonies with absorbance values ​​greater than 1 were selected and sequenced to determine the gene sequence of the positive monoclonal colonies, thus obtaining anti-CHVII nanobody phage particles.

[0015] In step (4), the conversion of anti-carp herpesvirus type II nanobody phage particles into host expression bacteria specifically involves: Nanoparticle phages (i.e., plasmids from positive monoclonal colonies) were extracted using the Omega plasmid mini-extraction kit. 1 μL of plasmid was then mixed with 1 mL of TOP10F' competent cells to form a bacterial suspension. The suspension was then subjected to an ice bath followed by a 42°C hot water shock treatment, and then again to an ice bath before being added to super broth medium. After shaking at 37°C for one hour, the suspension was plated onto a carbenicillin-resistant medium and cultured overnight to obtain fully introduced TOP10F' single bacteria. A portion of the introduced TOP10F' single bacteria was picked from the medium and inoculated into culture medium for shaking culture to obtain a TOP10F' single bacterial suspension. Finally, 1 mL of the TOP10F' single bacterial suspension was transferred to 100 mL of medium for expansion culture until the absorbance OD reached a certain level. 600 The concentration was 0.6-0.8, and isopropyl thiogalactoside was added to a final concentration of 1 mM. The mixture was induced overnight to obtain a bacterial culture containing the host expression bacteria.

[0016] The purification in step (4) specifically involves: TieChui™ super lysis medium was added to the overnight culture containing host expression bacteria to lyse the bacteria. The mixture was then subjected to ultrasonic disruption and centrifugation, and the supernatant was collected. Ni-NTA matrix was added to the collected supernatant, and after shaking at room temperature, the mixture was added to a purification column. Imidazole-containing phosphate equilibration buffer was added to wash the purification column and elute any contaminating proteins. Imidazole-containing phosphate elution buffer was then added to elute the proteins adsorbed on the nickel column (the purification column) to obtain the target protein solution. The target protein solution was added to a dialysis bag, which was then placed in phosphate buffer for dialysis purification to obtain the target carp herpesvirus type II nanobody.

[0017] III. Application of Nanobodies Targeting Carp Herpesvirus Type II Application in the preparation of detection reagents and kits for detecting carp herpesvirus type II.

[0018] IV. A diagnostic reagent for detecting carp herpesvirus type II. This includes the aforementioned nanobodies targeting cheetah herpesvirus type II or nanobodies prepared using the methods described above.

[0019] This invention provides a method for preparing a cyprinid herpesvirus type II nanobody and its application for immunoassay of cyprinid herpesvirus type II. The aim is to provide a cyprinid herpesvirus type II nanobody that is low in expression cost, easy to produce, has a stable source, can effectively bind to cyprinid herpesvirus type II, and specifically achieves immunoassay of cyprinid herpesvirus type II. The amino acid sequence of the nanobody is shown in SEQ ID NO.1-6 and belongs to the field of bioscience and technology.

[0020] Compared with the prior art, the technical solution provided by the present invention has the following advantages: 1. The target carp herpesvirus type II nanobody provided by the present invention is obtained based on the prokaryotic expression method, which has the advantages of low expression cost, low difficulty and stable source.

[0021] 2. The Targeted Carp Herpesvirus Type II Nanobody provided by this invention can effectively bind to carp herpesvirus type II, specifically achieving immunoassay of carp herpesvirus type II; it is suitable for the immunoassay of carp herpesvirus type II and will have great application potential in the detection of carp herpesvirus type II. Attached Figure Description

[0022] Figure 1 This is an agarose gel electrophoresis image of the first-round PCR amplification product of the alpaca heavy chain antibody nanobody gene fragment; Figure 2 This is an agarose gel electrophoresis image of the second-round PCR amplification product of the alpaca heavy chain antibody nanobody gene fragment; Figure 3 This is a graph showing the results of 144 bacteriophage monoclonal phage-ELISA. Figure 4 This is an SDS-PAGE electrophoresis analysis of nanobody protein expression. Figure 5 This is a graph showing the affinity analysis results between the nanobody targeting carp herpesvirus type II and carp herpesvirus type II. Figure 6 This is a standard curve for ELISA detection of carp herpesvirus type II based on a double nanobody sandwich. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that modifications or substitutions to the details and form of the technical solution of the present invention without departing from the technical solution of the present invention shall all fall within the protection scope of the present invention.

[0024] Example 1: Construction of an immune library 1. Alpaca Immunization After being cultured in LB medium for 18 h, carp herpesvirus type II was washed and then sterilized at high temperature to prepare inactivated carp herpesvirus type II antigen. Based on the immunization effect of the trivalent and quadrivalent vaccine, healthy alpacas with the strongest immune response were selected, and carp herpesvirus type II negative serum was collected for use. After immunization was initiated, five immunizations were performed on days 0, 14, 28, 42, and 56. Serum titers and peripheral blood were collected seven days after the third, fourth, and fifth immunizations for peripheral blood lymphocyte extraction.

[0025] 2. Extraction of peripheral blood lymphocytes 15 mL of peripheral blood was slowly added to 15 mL of cell separation medium at a 45-degree angle. The mixture was centrifuged at 400 g for 30 min at room temperature. The lymphocytes were then transferred to a new centrifuge tube, 10 mL of PBS buffer was added, and the mixture was centrifuged again at 400 g for 20 min. The supernatant was slowly aspirated, and the cells were resuspended in PBS. The cell count was then calculated.

[0026] 3. cDNA Acquisition Total RNA was extracted according to the LeukoLOCKl™ instructions. After extraction, the absorbance values ​​at OD260 nm and OD280 nm were measured to calculate the concentration and purity of the extracted RNA. The obtained RNA can be stored at -80°C and used for subsequent reverse transcription to obtain cDNA.

[0027] All obtained RNA was reverse transcribed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit, as follows: (1) RNA denaturation, the reaction system is shown in Table 1: Table 1 After incubating at 65°C for 5 minutes, place on ice to cool rapidly for at least 1 minute.

[0028] (2) Reverse transcription reaction system, the reaction system is shown in Table 2: Table 2 (3) The reverse transcription reaction is as follows: Slowly mix the RNA denaturation system from step (1) and the reverse transcription reaction system from step (2), incubate at 50°C for 60 minutes, then incubate at 95°C for 5 minutes to terminate the reaction, and finally cool on ice.

[0029] (4) Preservation of cDNA: The cDNA obtained after the above reaction was completed was stored at -20℃ for subsequent PCR amplification.

[0030] 4. PCR amplification of the gene sequence of the cyprinid herpesvirus type II nanobody Nested PCR was used to amplify the nanobody gene sequence. Primers were synthesized by PlatinumBio Biotechnology (Shanghai) Co., Ltd., and the primer sequences are shown in Table 3. Table 3 (1) First round of PCR: The first round of PCR was performed using the obtained cDNA as a template for amplification. The amplification reaction system is shown in Table 4. Table 4 The first round of PCR procedures are shown in Table 5: Table 5 The products of the first round of PCR amplification were identified by 1% agarose gel electrophoresis. (See attached image for details.) Figure 1 The target fragments were recovered using a rubber-cutting and recycling method.

[0031] (2) Second round of PCR: The second round of PCR was performed using the gel-recovered products from the first round of PCR as templates. The amplification reaction system is shown in Table 6. Table 6 The second round of PCR procedures are the same as the first round of PCR procedures, as detailed in Table 5.

[0032] The 10 μL PCR product was identified by agarose gel electrophoresis. See the results below. Figure 2 Meanwhile, the remaining PCR products were recovered using a PCR product purification kit.

[0033] 5. Ligation of the cyprinid herpesvirus type II nanobody gene with the pComb3xss vector (1) Enzyme digestion of pComb3xss vector, the enzyme digestion system is shown in Table 7: Table 7 The target fragment is recovered using a gel-cutting and recycling method.

[0034] (2) Enzyme digestion of the cyprinid herpesvirus type II nanobody gene. The enzyme digestion system is shown in Table 8: Table 8 The target gene was recovered after enzyme digestion using a PCR product purification kit.

[0035] (3) Ligation of the cyprinid herpesvirus type II nanobody gene to the pComb3xss vector, the ligation system is shown in Table 9: Table 9 The above system was incubated overnight at 16°C, followed by incubation at 65°C for ten minutes to terminate the linkage reaction, and finally cooled on ice.

[0036] The purified product after ligation was placed in a -20°C freezer for subsequent experiments.

[0037] 6. Electroconversion of the bonding products Add 3 μL of the above ligation product to 25 μL of competent ER2738 cells, and transform the mixture by electroporation at 1.8 KV, 200 Ω, and 25 µF. After transformation, shake and recover for 1 hour to complete the construction of the initial phage library.

[0038] 7. Phage library amplification and expression After the bacterial culture was revived, it was added to 200 mL of medium containing carbenicillin and tetracycline. After shaking at 37°C for 2 hours, helper phages were added to expand the initial library. After adding the helper phages, the culture was allowed to stand for half an hour, followed by shaking for 2 hours. Kanamycin was added, followed by shaking overnight. The phages were collected by centrifugation the next day to complete the construction of the immunological library.

[0039] Example 2: Selection of Nanobodies 1. First round of selection (1) Coating 5 x 10⁹ PFU / mL of carp herpesvirus type II onto an ELISA plate, sealing it with a sealing film, and incubating it overnight at 4°C. (2) Wash three times with PBST, add 3% skim milk powder and seal for 1 hour; (3) Add 100 μL of the phage library constructed in Example 1 and shake at room temperature for 1 hour; (4) Wash with PBST 10 times; (5) Eluting bound phages Add 100 μL of glycine-hydrochloric acid (0.1 M glycine-hydrochloric acid pH 2.2) to the well, incubate at room temperature for 15 minutes, and then immediately neutralize with 1 M Tris-HCl; (6) Take 2 μL of eluted phage and add it to 198 μL of ER2738 (OD 0.4-0.6), let it stand for 0.5 h to infect, and then take 100 μL and spread it on carbenicillin medium for the first round of panning and output titer test; (7) Add the remaining phage to 2 mL of ER2738, incubate overnight, and collect by centrifugation.

[0040] 2. Second round of selection (1) Coating 2.5 x 10⁹ PFU / mL of carp herpesvirus type II onto an ELISA plate, sealing it with a sealing film, and incubating it overnight at 4°C. (2) Wash three times with PBST, add 3% skim milk powder and seal for 1 hour; (3) Add 100 μL of the phage library after the first round of screening and shake at room temperature for 1 hour; (4) Wash ten times with PBST; (5) Elution of bound phages. Add 100 μL of glycine-hydrochloric acid (0.1 M glycine-hydrochloric acid, pH 2.2) to the wells, incubate at room temperature for 15 minutes, and then immediately neutralize with 1 M Tris-HCl; (6) Take 2 μL of eluted phage and add it to 198 μL of ER2738 (OD 0.4-0.6), let it stand for 0.5 h, then take 100 μL and spread it on carbenicillin medium to perform the second round of phage output titer test. (7) Add the remaining phage to 2 mL of ER2738, incubate overnight, and collect by centrifugation.

[0041] 3. Third round of selection (1) Coating 1.25 x 10⁹ PFU / mL of carp herpesvirus type II onto an ELISA plate, sealing it with a sealing film, and incubating it overnight at 4°C. (2) Wash three times with PBST, add 3% skim milk powder and seal for 1 hour; (3) Add the phage library after R2 panning and shake at room temperature for 1 hour; (4) Wash ten times with PBST; (5) Elution of bound phages. Add 100 μL of glycine-hydrochloric acid (0.1 M glycine-hydrochloric acid, pH 2.2) to the well, incubate at room temperature for 15 minutes, and then immediately neutralize with 1 M Tris-hydrochloric acid; (6) 2 μL of eluted phage was added to 198 μL of ER2738 (OD 0.4-0.6), and allowed to stand for 0.5 h. Then, 100 μL was spread on carbenicillin medium and the R3 phage output titer was tested. (7) Add the remaining phage to 2 mL of ER2738, incubate overnight, and collect by centrifugation.

[0042] 4. Screening of nanobody-positive strains (1) Select 48 single colonies from the titer test plates output after three rounds of screening, inoculate them onto super broth medium, and incubate overnight with shaking; (2) Add the monoclonal colonies cultured in step (1) to the ELISA plate pre-coated with 109 PFU / mL carp herpesvirus type II, shake at room temperature for 1 hour, wash three times with PBST, add 100 μL of horseradish peroxidase-labeled anti-HA secondary antibody diluted 600 times with PBS buffer to each well, shake at room temperature for 1 hour, wash five times with PBST, add 100 μL of the prepared TMB colorimetric mixture to each well, and shake at room temperature in the dark for 15 minutes to develop the color. (3) Remove the ELISA plate after color development, add 50 μL of 2 mol / L H2SO4 solution to each well to stop the reaction, and read the absorbance value at 450 nm (see results). Figure 3 Single colonies corresponding to positive clones with absorbance values ​​greater than 1 were selected and sequenced, and their gene sequences are shown in SEQ ID NO.1-6.

[0043] Example 3: Prokaryotic expression and purification of anti-carp herpesvirus type II nanobody 1. Prokaryotic expression of nanobodies (1) The plasmids of positive monoclonal colonies were extracted using the Omega plasmid mini extraction kit. The plasmids were then mixed with TOP10F' competent cells, placed on ice for 30 minutes, then subjected to a 42°C hot water shock for 90 seconds, followed by an ice bath for 5 minutes. 1 ml of super broth medium was added, and the cells were shaken at 37°C for one hour. The cells were then spread on carbenicillin-resistant medium and cultured overnight to complete the transformation. (2) Pick a single TOP10F' bacteria that has been introduced into the culture medium and inoculate it into the culture medium with shaking. Then take 1 mL of the bacterial culture and expand it into 100 mL of culture medium until the absorbance OD reaches the specified value. 600Add isopropyl thiogalactoside to a final concentration of 1 mM (0.6-0.8), and induce overnight.

[0044] 2. Purification of nanobodies (1) Collect the bacterial culture that has been cultured overnight into a centrifuge bottle and centrifuge at 7000 rpm for 5 min at 4℃; (2) Collect the precipitate, add 13.5 mL TBS buffer and 1.5 mL TieChui™ super lysate to lyse the bacteria, shake and incubate for 5 min, sonicate for 5 min, shake and incubate for 10 min, centrifuge at 16000 g for 10 min at 4℃. (3) Collect the supernatant for subsequent purification; (4) Add the Ni-NTA matrix to the collected supernatant, shake at room temperature for 1 hour, and then add it to the purification column; (6) Add phosphate buffer containing 2 mM imidazole, wash the purification column, and elute impurities; (7) Add phosphate elution buffer containing 50 mM imidazole to elute the target protein adsorbed on the nickel column; (8) A small amount of the collected purified nanobody was subjected to SDS-PAGE electrophoresis for detection. The results are shown in the attached document. Figure 4 ; (9) Add the remaining collected nanobodies to the dialysis bag and dialyze in phosphate buffer for 24 hours.

[0045] Example 4: Performance evaluation of anti-carp herpesvirus type II nanobody The affinity analysis of the anti-carp herpesvirus type II nanobody to carp herpesvirus type II is as follows: (1) Add 100 μL of CBS buffer to each well of a 96-well microplate and dilute to a concentration of 5 x 10⁻⁶. 8 PFU / mL carp herpesvirus type II standard solution was coated by sealing the plate with a sealing film and incubating overnight at 4°C. (2) The next day, take out the coated microplate, wash it three times with PBST, add 270 μL of 3% skim milk powder solution to each well, cover it with sealing film and shake at room temperature for 1 h to seal it. (3) Wash the sealed ELISA plate according to step (2), add 100 μL of nanobody diluted with PBS buffer to concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 μg / mL to each well, seal the plate and shake at room temperature for 1 h; (4) Wash the microplate according to step (2), add 100 μL of horseradish peroxidase-labeled anti-HA secondary antibody diluted 600 times with PBS buffer to each well, cover with sealing film and shake at room temperature for 1 h. (5) Take out the microplate, wash it 5 times with PBST, add 100 μL of the prepared TMB colorimetric mixture to each well, and shake it at room temperature in the dark for 15 min to develop the color. (6) Remove the enzyme-labeled plate after color development, add 50 μL of 2 mol / L H2SO4 solution to each well to stop the reaction, and read the absorbance value at 450 nm.

[0046] The binding affinity assay results of the cyprinid nanobody targeting cyprinid herpesvirus type II to cyprinid herpesvirus type II are as follows: Figure 5 As shown; Figure 5 The results showed that the cyprinid nanobody targeting cyprinid herpesvirus type II provided in this application could effectively bind to cyprinid herpesvirus type II. Among them, L1 and L4 showed the strongest binding ability, while L2 showed slightly weaker binding ability. In contrast, L3 showed relatively weak binding ability.

[0047] Example 5: Application of Nanobodies in Immunoassay of Carp Herpesvirus Type II The following is a sandwich immunoassay using two nanobody-based nanoparticles for carp herpesvirus type II: (1) Add 100 μL of nanobody diluted with CBS buffer to each well of a 96-well microplate to a concentration of 5 μg / mL. After sealing the microplate with a sealing film, incubate it overnight at 4°C for coating. (2) The next day, take out the coated microplate, wash it 3 times with PBST, add 270 μL of 3% bovine serum albumin solution to each well, cover it with sealing film and shake at room temperature for 1 h to seal it. (3) Wash the sealed microplate according to step (2), and add 100 μL of PBS buffer to each well to dilute to a concentration of 1x10. 8 5x10 7 2.5x10 7 1.25x10 7 6.25x10 6 3.125x10 6 1.5625x10 6 0 PFU / mL carp herpesvirus type II standard solution, sealed with film and shaken at room temperature for 1 h; (4) Wash the microplate according to step (2), add 100 μL of horseradish peroxidase-labeled nanobody nanobody with a concentration of 2 μg / mL to each well, cover with sealing film and shake at room temperature for 1 h; (5) Take out the microplate, wash it 5 times with PBST, add 100 μL of the prepared TMB colorimetric mixture to each well, and shake it at room temperature in the dark for 15 min to develop the color. (6) Remove the enzyme-labeled plate after color development, add 50 μL of 2 mol / L H2SO4 solution to each well to stop the reaction, and read the absorbance value at 450 nm.

[0048] The standard curve for the ELISA detection of carp herpesvirus type II based on a dual-nanobody sandwich is shown below. Figure 5 As shown, Figure 5 The results showed that the various combinations of the nanobodies targeting carp herpesvirus type II provided in this application could be successfully sandwiched together, enabling highly sensitive and specific detection of carp herpesvirus type II, thereby achieving specific immunoassay for carp herpesvirus type II.

[0049] The sequence involved in this invention is specifically as follows: SEQ ID NO.1: Name: Amino acid sequence 1 of nanobody targeting carp herpesvirus type II QVQLVESGGGSVQAGGSLKLSCLASGFMFSIYEMGWYRQAPGKERELVSSMSDVGRTSYRDSVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCAASFGTLGDAATHWGQGTQVTISG.

[0050] SEQ ID NO.2: Name: Amino acid sequence 2 of nanobody targeting carp herpesvirus type II EVQLAESGGGSVQAGGSLELSCTTSDRFIFPTCGMGWYRQAPGKERELVSSISSDGTISYSDSVKGRFTISRDKAKNTLYLQMNSLKREDTAVYYCAACRLHDPRLYTYWGQGTQVTVSG.

[0051] SEQ ID NO.3: Name: Amino acid sequence 3 of nanobody targeting carp herpesvirus type II QVQLVESGGGSVQAGGSLRLSCAASGSTRGSYSMAWFRQAPGKEREGVATLATNGYTIYADSAKGRFTVSRDNANLANTLYLQMNSLKPEDTAMYYC~AADCGYRMFTTPRASECKYWGQGTQVTVSG.

[0052] SEQ ID NO.4: Name: Amino acid sequence 4 of nanobody targeting carp herpesvirus type II QVQLVESGGGSVQAGGSLSVSCVASGPWYDRRCMGWFRQAPGKEREGVATIDSDGTTSYADSVKGRFTISQDNAKNILYLQMNSLKPEDTAMYYCAATGRLLCNDSTLRASGYWGQGTQVTVSS.

[0053] SEQ ID NO.5: Name: Amino acid sequence 5 of nanobody targeting carp herpesvirus type II QLQLVESGGGSVSAGGSLRLSCAVSGYAYSTYCMGWFRQAPGKEREGVAAVDSDGRTSYADSVEGRFTISQSKNKQILYLQMNSLEPEDTAMYYCAADARLWVGWTGSRWAAQPFAY~WGQGTQVTVSS.

[0054] SEQ ID NO.6: Name: Amino acid sequence 6 of nanobody targeting carp herpesvirus type II QVQLVESGGGVVQAGGSLRLSCAASELDLSTYHVAWFRQAPGKEREFVSLLNKNGVTTSYADSVKGRFTIWRDNAKNTFYLEMNSLEPEDTAVYDCAVDVLFSRLYKFQYAYRGQGTQVTVSS.

[0055] SEQ ID NO.7: Name: Primer 1 Sequence type: DNA (other DNA) Source: synthetic construct 5'-GTCCTGGCTGCTCTTCTACAAG-3'.

[0056] SEQ ID NO.8: Name: Primer 2 Sequence type: DNA (other DNA) Source: synthetic construct 5'-GGTACGTGCTGTTGAACTGTTCC-3'.

[0057] SEQ ID NO.9: Name: Primer 3 Sequence type: DNA (other DNA) Source: synthetic construct 5'-TTAGGCCCAGGCGGCCSAGRTRCARYTGGYGGARTCTGGRGGAGG-3'.

[0058] SEQ ID NO.10: Name: Primer 4 Sequence type: DNA (other DNA) Source: synthetic construct 5'-TTAGGCCGGCCTGGCCRGAGAYGGTGACCTGGGT-3'.

Claims

1. A nanobody targeting carp herpesvirus type II, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.1-6.

2. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 1, characterized in that, The method includes the following steps: (1) Alpaca were immunized with inactivated carp herpesvirus type II. After immunization, peripheral blood lymphocytes of alpaca were collected for mRNA extraction. The mRNA was used as a template to synthesize the first strand of cDNA. The obtained first strand of cDNA was used as a template for PCR amplification to obtain the DNA fragment encoding nanobody. (2) The DNA fragment encoding the nanobody was ligated to the pComb3xss vector and then transformed into competent cells to construct an initial nanobody library; the initial nanobody library was then expanded and cultured to obtain a nanobody library. (3) Using inactivated carp herpesvirus type II as the coating antigen, the nanobody library was enriched and screened to obtain anti-carp herpesvirus type II nanobody phage particles. (4) Transform the anti-carp herpesvirus type II nanobody phage particles into the host expression bacteria, induce the expression of nanobody, and purify to obtain the anti-carp herpesvirus type II nanobody.

3. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 2, characterized in that, The mRNA extraction in step (1) was performed using the LeukoLOCK kit.

4. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 1, characterized in that, The sequences of primers 1-4 used in the PCR amplification described 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.

5. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 2, characterized in that, In step (2), the initial library for constructing the nanobody is: The DNA fragment encoding the nanobody was ligated to the pComb3xss vector to obtain the ligation product. The ligation product was added to competent cells ER2738, followed by electroporation transformation. After shaking and thawing for 1 hour, the thawed strain was obtained as the initial nanobody library. The nanobody library was expanded by adding the revived strain to the culture medium, shaking at 37°C for 2 hours, adding helper phage, letting it stand for half an hour, shaking for another 2 hours, adding kanamycin, shaking overnight, and centrifuging the next day to collect the phage to obtain the nanobody library.

6. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 2, characterized in that, Step (3) is as follows: Inactivated carp herpesvirus type II (CHVII) was used as a coating antigen and cultured on an ELISA plate to obtain a CHVII-pre-coated ELISA plate. The nanobody library was subjected to three rounds of panning, and different monoclonal colonies were selected and inoculated into super broth medium for culture. The culture medium was then added to the CHVII-pre-coated ELISA plate, and after washing with shaking at room temperature, horseradish peroxidase-labeled anti-HA antibody was added. After washing with shaking at room temperature again, 3,3',5,5'-tetramethylbenzidine was added for color development. Positive monoclonal colonies with absorbance values ​​greater than 1 were selected from the positive clone wells, and the positive monoclonal colonies were sequenced and screened to obtain anti-CHVII nanobody phage particles.

7. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 2, characterized in that, In step (4), the conversion of anti-carp herpesvirus type II nanobody phage particles into the host expression bacteria is: Plasmids were extracted from the nanobody phages using the Omega plasmid mini-extraction kit. 1 μL of plasmid was then mixed with 1 mL of TOP10F' competent cells to form a bacterial suspension. The suspension was subjected to an ice bath followed by a 42°C hot water shock treatment, then another ice bath before being added to super broth medium. After shaking at 37°C for one hour, the suspension was plated onto a carbenicillin-resistant medium and cultured overnight to obtain a fully introduced TOP10F' single bacterium. A portion of the introduced TOP10F' single bacterium was picked from the medium and inoculated into a culture medium for shaking culture to obtain a TOP10F' single bacterium suspension. 1 mL of this suspension was then transferred to 100 mL of medium for expansion culture until the absorbance OD reached a certain level. 600 The concentration was 0.6-0.8, and isopropyl thiogalactoside was added to a final concentration of 1 mM. The mixture was induced overnight to obtain a bacterial culture containing the host expression bacteria.

8. The method for preparing the targeted carp herpesvirus type II nanobody according to claim 7, characterized in that, The purification in step (4) is: After lysing the bacteria containing the host expression bacteria with Hammer Super Splitting Fluid, the bacteria were sequentially sonicated and centrifuged, and the supernatant was collected. Ni-NTA matrix was added to the collected supernatant, and after shaking at room temperature, it was added to a purification column. Phosphate equilibration buffer containing imidazole was added to wash the purification column to elute impurities. Phosphate elution buffer containing imidazole was added to elute the proteins adsorbed on the purification column to obtain the target protein solution. The target protein solution was added to a dialysis bag, and the dialysis bag was placed in phosphate buffer for dialysis purification to obtain the target carp herpesvirus type II nanobody.

9. The application of the cyprinid nanobody targeting cyprinid herpesvirus type II as described in claim 1 or the cyprinid nanobody targeting cyprinid herpesvirus type II prepared by any of the preparation methods described in claims 2-8, characterized in that, Application in the preparation of detection reagents and kits for detecting carp herpesvirus type II.

10. A detection reagent for detecting carp herpesvirus type II, characterized in that, This includes the cyprinid nanobody targeting cyprinid herpesvirus type II as described in claim 1 or the cyprinid nanobody targeting cyprinid herpesvirus type II prepared by any of the preparation methods described in claims 2-8.

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