Nanoantibodies targeting giant salamander iridescent virus and their preparation method and application

Abstract

The present invention provides a nano-antibody targeting giant salamander iridescent virus and its preparation method and application; the invention aims to provide a nano-antibody targeting giant salamander iridescent virus with low expression cost, low difficulty, stable source, and the ability to effectively bind to giant salamander iridescent virus and specifically realize immune analysis of giant salamander iridescent virus; the amino acid sequence of the nano-antibody is shown in SEQ ID NO.1-6 and belongs to the field of biological science and technology.

Description

Technical Field

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

[0002] The giant salamander, commonly known as the giant salamander, is listed as Critically Endangered (CR) on the IUCN Red List of Threatened Species and is also a Class II protected wild animal in my country. Due to its tender meat and rich nutritional value, the giant salamander is known as the "living ginseng of water." With breakthroughs in giant salamander aquaculture technology in my country, large-scale captive breeding of giant salamanders has now been widely promoted nationwide. The giant salamander iridovirus is the only known viral disease of giant salamanders. Its spread has caused widespread devastation in large-scale aquaculture, posing a serious challenge to the healthy development of giant salamander aquaculture. The giant salamander iridovirus is transmitted through multiple channels: contaminated water, physical contact, and ingestion of infected tissue. Detection of giant salamander iridovirus is crucial for protecting endangered species, controlling disease transmission, ensuring aquaculture safety, maintaining ecological balance, and promoting scientific research. Regular testing in the aquaculture industry ensures the health and profitability of aquaculture farms.

[0003] Current methods for detecting giant salamander iridescent viruses include PCR, cell culture, microscopic examination of infected tissues, and immunological methods. PCR technology is highly specific and sensitive, enabling rapid and accurate detection of target DNA sequences. However, it requires specialized equipment and techniques and can produce false-positive results. Culture methods, while simple and intuitive, are time-consuming and require a high cell count, making it prone to missing low-concentration samples. Microscopic examination, however, is limited in its sensitivity and specificity, as well as its complexity, limiting its diagnostic accuracy when detecting iridescent viruses.

[0004] Immunological methods have high specificity, but may exhibit cross-reactivity or low sensitivity due to the effects of antibody quality and pairing. Compared to traditional monoclonal antibodies, recombinant antibodies offer lower production costs and greater homogeneity. Nanobodies, a cutting-edge recombinant antibody, offer advantages such as small molecular weight, low production costs, and simplified purification. They are the smallest known antigen-binding units. However, there are currently no reports of anti-giant salamander iridescent virus nanobodies. This invention, using phage display and prokaryotic expression techniques, prepares the first anti-giant salamander iridescent virus nanobodies, which are essential and reliable for rapid immune detection of giant salamander iridescent virus. Summary of the Invention

[0005] In response to the above-mentioned shortcomings, the first object of the present invention is to provide a nanoantibody targeting giant salamander iridescent virus that has low expression cost, low difficulty, stable source, can effectively bind to giant salamander iridescent virus, and can specifically realize immune analysis of giant salamander iridescent virus.

[0006] The second object of the present invention is to provide a method for preparing nanoantibodies targeting giant salamander iridescent virus.

[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 iridescent 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 aforementioned nanoantibody targeting giant salamander iridescent virus, comprising the following steps:

[0011] (1) immunizing alpacas with inactivated giant salamander iridescent virus, collecting peripheral blood lymphocytes from 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 iridescent virus as a coating antigen, and performing enrichment and panning screening on the nanobody library to obtain nanobody phagemids against the giant salamander iridescent 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 iridescent virus is purified.

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

[0016] Furthermore, for the nanoantibody targeting giant salamander iridescent 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 iridescent virus, the expansion culture in step (2) is to add the recovered bacterial solution to the culture medium, shake it at 37°C for 2 hours, add helper phage to expand the initial library, shake it for 2 hours after adding helper phage, add kanamycin, then shake it overnight, and collect the phage by centrifugation the next day to complete the construction of the immune library;

[0023] Furthermore, for the nanoantibody targeting giant salamander iridescent virus, the nanoantibody library described in step (3) is enriched and screened by three rounds of panning, wherein monoclonal colonies are picked and inoculated into super broth culture medium for culture, and then added to an enzyme-labeled plate pre-coated with giant salamander iridescent virus. After washing with shaking at room temperature, anti-M13-horseradish peroxidase-labeled antibody is added, and the plate is washed with shaking 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 an absorbance value greater than 1.5 are picked for sequencing.

[0024] Furthermore, the nanoantibody targeting giant salamander iridescent 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 a 42 ° C hot water heat shock after ice bath, and then ice bath again, add super broth medium, shake at 37 ° C for one hour to complete the transformation; the introduced TOP10F' is inoculated into the culture medium and shaken to the absorbance OD 600 0.8-1.0, add isopropylthiogalactoside to 1 mM and induce overnight.

[0025] Furthermore, the nanoantibody targeting giant salamander iridescent virus is purified in step (4). BeyoLytic TM The bacteria were lysed with a bacterial active protein extraction reagent, and the supernatant was collected by centrifugation; the Ni-NTA matrix was added to the collected supernatant, shaken at room temperature, and then added to a purification column; a phosphate equilibration buffer containing imidazole was then added to wash the purification column and elute the impurities; a phosphate elution buffer containing imidazole was then added to elute the target protein adsorbed on the nickel column; the nanoantibody 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 aforementioned nanoantibody targeting giant salamander iridescent virus in the preparation of a detection reagent or a detection kit for detecting giant salamander iridescent virus.

[0027] The last technical solution of the present invention is a detection reagent for detecting giant salamander iridescent virus, including the nanoantibody targeting giant salamander iridescent virus 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 iridescent virus 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;

[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; it is 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 diagram of the heavy chain variable region gene of the alpaca IgG2 / 3 antibody;

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

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

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

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

[0036] Example 1 Immune library construction

[0037] 1. Alpaca Immunity

[0038] After the giant salamander iridescent virus was cultured in LB medium for 18 hours and washed, it was sterilized at high temperature to prepare the inactivated antigen of giant salamander iridescent virus. According to the immune effect of the triple quadruple vaccine, the alpaca with the strongest immune response effect and the healthiest was selected, and the negative serum of giant salamander iridescent 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 titer was tested seven days after the triple immunization, the quadruple immunization and the pentaimmunization, and the peripheral blood was collected for peripheral blood lymphocyte extraction.

[0039] 2. Peripheral Blood Lymphocyte Extraction

[0040] Slowly add 15 mL of peripheral blood to 15 mL of cell separation solution at a 45-degree angle, 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, resuspend in PBS, and count the cells.

[0041] 3. cDNA Acquisition

[0042] Follow TRIzol TM The total RNA was extracted according to the instructions. After the extraction was completed, the OD 260 nm and OD 280 The concentration and purity of the extracted RNA were calculated based on the absorbance value of 100 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 All RNA obtained was reverse transcribed using the IV First-Strand Synthesis System. 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 minutes and keep on ice for at least 1 minute.

[0048] (2) Reverse transcription reaction system, the reaction system is 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 incubated at 55°C for 10 minutes, and finally incubated at 80°C for 10 minutes to terminate the reaction.

[0054] (4) RNA removal:

[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 Nanobody Gene Sequences Targeting Giant Salamander Iridescent Virus

[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. Ligation of the Nanobody Gene Targeting Giant Salamander Iridescent Virus with the 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 giant salamander iridescent virus, the enzyme cleavage system is as follows:

[0075]

[0076]

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

[0078] (3) The connection between the nanoantibody gene targeting giant salamander iridescent virus and the pComb3xss vector 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 transformation, shake and recover for 1 hour to complete the construction of the initial phage library.

[0084] 7. Phage Library Amplification and Expression

[0085] The recovered 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 helper phage, the culture was shaken for 2 hours. Kanamycin was added and the culture was shaken overnight. The phage was collected by centrifugation the next day to complete the construction of the immune library.

[0086] Example 2 Panning 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 (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) 2 μL of eluted phage was added to 198 μL of ER2738 (OD 0.4-0.6) and allowed to infect for 0.5 h. 100 μL was then plated 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 (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) 2 μL of eluted phage was added to 198 μL of ER2738 (OD 0.4-0.6) and allowed to infect for 0.5 h. 100 μL was then plated on carbenicillin medium for a second round of panning to test the phage output titer.

[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 phage. 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) and allowed to infect for 0.5 h. 100 μL was then plated on carbenicillin medium for R3 phage output titer testing.

[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 titer test plates after three rounds of screening, inoculated into super broth medium, and cultured with shaking overnight.

[0115] (2) Add the monoclonal colony cultured in step (1) to the pre-coated 8 The plate was placed on an enzyme-labeled plate containing PFU / mL giant salamander iridescent virus, shaken at room temperature for 1 hour, washed three times with PBST, and then 100 μL / well of 100 ng / mL anti-M13-horseradish peroxidase-labeled antibody was added. The plate was shaken at room temperature for 1 hour, washed five times with PBST, and then 100 μL / well of 3,3',5,5'-tetramethylbenzidine was added 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 picked for sequencing, and their gene sequences were shown in SEQ ID NO.1-6.

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

[0118] 1. Prokaryotic expression of nanobodies

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

[0120] (2) Inoculate the introduced TOP10F' into the culture medium and shake the culture until the absorbance OD 600 0.8-1.0, add isopropylthiogalactoside 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 minutes;

[0123] (2) Join BeyoLytic TM Bacteria were lysed with bacterial active protein extraction reagent and centrifuged at 16,000 g for 10 min at 4°C;

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

[0125] (4) Ni-NTA matrix was added to the collected supernatant, shaken at room temperature for 1 hour, and added 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) Add phosphate elution buffer containing 50 mM imidazole to elute the target protein adsorbed on the nickel column;

[0128] (8) Take a small amount of collected purified nanoantibodies 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: Nanoantibodies in the immunodetection of giant salamander iridescent virus

[0131] Direct capture immunoassay of giant salamander iridescent virus

[0132] (1) Dilute 1x10 with 0.05M carbonate buffer 8 The giant salamander iridovirus 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 to incubate at 37°C with shaking for 1 hour.

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

[0136] (5) Add 100 μL of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0 μg / mL nanoantibodies to each well, mix well, and incubate at 37°C for 1 hour;

[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 giant salamander iridescent virus ELISA test is shown in Figure 4 ,pass Figure 4 It can be seen that the nanoantibodies targeting giant salamander iridescent virus provided in the present application can effectively bind to giant salamander iridescent virus, thereby specifically realizing immune analysis of giant salamander iridescent virus.

Claims

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

2. 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.

3. A detection reagent for detecting giant salamander iridescent virus, characterized in that: It includes the nanobody targeting giant salamander iridescent virus as described in claim 1.

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