Targeting monopterus albus rhabdovirus nano antibody as well as preparation method and application thereof
By preparing nanobodies targeting the loach rhabdovirus, constructing a nanobodies library, and purifying it, the problems of complexity and high cost of existing detection methods were solved, and rapid and accurate detection of the loach rhabdovirus was achieved.
Patent Information
- Application Number
- CN202511578697.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-01-09
AI Technical Summary
Current technologies lack rapid and accurate methods for detecting loach rhabdovirus. Traditional monoclonal antibodies suffer from cross-reactivity and high costs, and existing methods are complex to operate or require specialized equipment.
Nanobodies targeting the loach rhabdovirus were prepared by immunizing alpacas with inactivated virus, extracting mRNA, constructing a nanobody library, enriching and purifying the nanobodies that specifically bind to the loach rhabdovirus, and applying them to sandwich ELISA detection.
This study provides a low-cost and easy-to-use nanobody detection method that can bind to loach rhabdovirus with high sensitivity and specificity, and is suitable for the immunoassay and detection of loach rhabdovirus.
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Figure CN121293329A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological science, and particularly relates to a nanobody targeting a snakehead rhabdovirus, a preparation method and application thereof. BACKGROUND
[0002] The snakehead (Monopterus albus) is a subtropical freshwater fish widely distributed in East and Southeast Asia. The snakehead is loved by consumers in Asian countries such as China, Japan and South Korea due to its delicious taste and rich nutritional value. The snakehead is one of the important economic aquaculture species in China. However, intensive farming makes farmed snakeheads vulnerable to various diseases, including bacterial infection, parasitic infection and viral infection, which seriously restricts the normal operation of aquaculture and the healthy development of snakehead farming. Among them, the Chinese snakehead rhabdovirus (CrERV) can cause high morbidity in juvenile and adult snakeheads. After infection with the virus, the snakehead will show abnormal movement, gasping on the water surface, loss of balance, anorexia, head swelling and skin bleeding. The snakehead hemorrhagic disease caused by CrERV can cause more than 80% mortality, seriously affecting the production and economic benefits of eel farming.
[0003] Currently, methods for detecting snakehead rhabdovirus include but are not limited to cell culture, pathological analysis, microscopic examination of infected tissues and PCR method. The culture method is simple and intuitive, but it takes a long time and requires a large number of cells, which is easy to miss low-concentration samples. The use of electron microscopy technology for pathogen examination is limited by its lack of sensitivity and specificity, complex operation, and diagnostic accuracy. 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. At present, there is still a need for faster and more accurate detection methods for the rapid detection of snakehead rhabdovirus.
[0004] Immunoassay based on antigen-antibody reaction has the advantages of simple operation, low cost, high throughput and high safety, and has been widely used in the detection of aquatic virus. Antibody is the main factor that determines the sensitivity and accuracy of immunoassay. However, due to the quality and pairing of antibodies, there may be cross-reactions or low sensitivity. Compared with traditional monoclonal antibodies, recombinant antibodies have lower production cost and better uniformity. Nanobody, as a frontier of recombinant antibodies, has the advantages of small molecular weight, low production cost and simple purification, and is the smallest known antigen-binding unit.
[0005] Currently, there is no related report on anti-snakehead rhabdovirus nanobody. SUMMARY
[0006] In view of the above problems, the first object of the present application is to provide a target Rhabdovirus of Misgurnus anguillicaudus nanobody with low expression cost, low difficulty, stable source, capable of effectively combining with Rhabdovirus of Misgurnus anguillicaudus, and capable of specifically realizing immune analysis of Rhabdovirus of Misgurnus anguillicaudus.
[0007] The second object of the present application is to provide a preparation method of the target Rhabdovirus of Misgurnus anguillicaudus nanobody.
[0008] The present application also has an object to provide the application of the above-mentioned target Rhabdovirus of Misgurnus anguillicaudus nanobody as a Rhabdovirus of Misgurnus anguillicaudus immune detection reagent or detection kit.
[0009] To this end, the technical scheme provided by the present application is as follows: One, a target Rhabdovirus of Misgurnus anguillicaudus nanobody, the amino acid sequence of the nanobody is shown in SEQ ID NO. 1-4.
[0010] Two, a preparation method of the target Rhabdovirus of Misgurnus anguillicaudus nanobody, the method comprises the following steps: (1) Inactivated Rhabdovirus of Misgurnus anguillicaudus is used to immunize a llama, and peripheral blood lymphocytes of the llama after immunization are collected for mRNA extraction, cDNA is synthesized by taking the mRNA as a template, and the obtained cDNA is taken as a template for PCR amplification to obtain a DNA fragment encoding a nanobody; (2) The DNA fragment encoding the nanobody is connected with a pComb3xss plasmid vector, and then transformed into a competent cell in the form of Escherichia coli to construct a nanobody initial library (Nanobody library), and then the nanobody initial library is expanded and cultured to obtain a nanobody library; The "library" refers to a solution of host cells (such as Escherichia coli) containing DNA sequences of nanobodies (i.e. the connection product of the fragment encoding the nanobody and the carrier), which is obtained by heat shock transformation treatment to send the recombinant plasmid into the Escherichia coli to make it replicate a large amount of genes.
[0011] (3) The Rhabdovirus of Misgurnus anguillicaudus is used as a coating antigen to enrich and screen the nanobody library to obtain a nanobody phagemid against the Rhabdovirus of Misgurnus anguillicaudus; (4) The nanobody phagemid against the Rhabdovirus of Misgurnus anguillicaudus is transformed into a host expression bacterium to induce and express the nanobody, and the final target Rhabdovirus of Misgurnus anguillicaudus nanobody sequence is obtained by purification.
[0012] In the step (1), the mRNA extraction adopts a TRIzol kit.
[0013] In the step (1), the PCR amplification adopts primers 1-6: The nucleotide sequence of primer 1 is shown in SEQ ID NO. 5; The nucleotide sequence of primer 2 is shown as SEQ ID NO. 6; The nucleotide sequence of primer 3 is shown as SEQ ID NO. 7; The nucleotide sequence of primer 4 is shown as SEQ ID NO. 8; The nucleotide sequence of primer 5 is shown as SEQ ID NO. 9; The nucleotide sequence of primer 6 is shown as SEQ ID NO. 10.
[0014] The PCR amplification in the step (1) is specifically as follows: The first round of PCR amplification is performed according to the following process using primer 1-2 with cDNA as a template: Process 1: 2 min at 98 ℃; Process 2: 10 s at 98 ℃; Process 3: 15 s at 55 ℃; Process 4: 15 s at 72 ℃; Repeat processes 2-4 for 30 cycles; Process 5: 5 min at 72 ℃; Process 6: cooling at 4 ℃.
[0015] The PCR product obtained in the first round is recovered by gel cutting, and then used as a template to perform the second round of amplification according to the following process using primer 3-6: Process 1: 2 min at 98 ℃; Process 2: 10 s at 98 ℃; Process 3: 15 s at 55 ℃; Process 4: 15 s at 72 ℃; Repeat processes 2-4 for 25 cycles; Process 5: 5 min at 72 ℃; Process 6: cooling at 4 ℃.
[0016] The PCR product obtained in the second round is column-recovered to complete the PCR amplification.
[0017] The step (2) is transformed into a competent cell to construct a nanobody initial library, which is specifically as follows: The solution of the DNA fragment encoding the nanobody connected with the pComb3xss plasmid vector is added to the TOP10F' competent cell in the form of Escherichia coli to obtain a recombinant plasmid, and then the recombinant plasmid is transfected into the competent Escherichia coli ER2738 by electroporation; The step (2) of expanding culture of the initial nanobody library is specifically: the recombinant plasmid is added to the super broth medium, and after 2 hours of shaking at 37℃ and 250 rpm, 1 mL of 1*10 13 CFU / mL of helper phage is added to expand the initial library, and after 30 minutes of standing at 37℃ after adding the helper phage, the phage is used to infiltrate E. coli ER2738 containing VHH antibody genes, followed by 2 hours of shaking at 250 rpm, then 200 μL of 70 mg / mL kanamycin is added, followed by overnight shaking at 37℃, and the next day, the phage is collected by centrifugation, and the construction of the nanobody library (immune library) is completed.
[0018] In the step (2), the amount of helper phage added is 400 μL, and the amount of kanamycin added is 200 μL.
[0019] The helper phage is specifically: M13KO7 helper phage purchased from New England Biolabs.
[0020] The nanobody library in the step (3) is subjected to enrichment and screening by panning: First, the nanobody library is subjected to three rounds of panning, and then different single clone colonies are picked and inoculated into 96 deep well plates (each well containing 1 mL of super broth medium) for overnight culture, and the next day, they are added to different wells of an enzyme-labeled plate pre-coated with snake rhabdovirus, different wells are added with different single clone colonies, after washing by shaking at room temperature, anti-M13-horseradish peroxidase labeled antibody is added, and again washed by shaking at room temperature, 3,3',5,5'-tetramethylbenzidine is added for color development, and the single clone colonies corresponding to the wells with an absorbance value OD 450 greater than 1 are selected as positive clone wells, and the single clone colonies corresponding to the positive clone wells are used as nanobody phagemids against snake rhabdovirus.
[0021] In the super broth medium culture, 1 L of ddH2O contains 10 g of MOPS, 30 g of peptone, and 20 g of yeast extract.
[0022] The anti-M13-horseradish peroxidase labeled antibody is diluted 1:2000 with PBS, and 100 μL is added to each well.
[0023] The coating amount of the snake rhabdovirus pre-coated on the enzyme-labeled plate is: 7*10 7 PFU / mL.
[0024] The step (4) of transforming the phagemid into a host expression bacterium is specifically: The plasmid in the positive monoclonal colony is extracted by using an Omega plasmid small extraction kit, then 2 muL of the plasmid is mixed with 50 muL of TOP10F' competent cells in the form of E. coli as host expression bacteria to perform introduction transformation to form a bacterial solution, the bacterial solution is placed in ice water for a period of time, then is subjected to heat shock treatment at 42 DEG C hot water for a period of time, then is placed in ice water again for a period of time, then super broth medium is added, and the solution is shaken at 37 DEG C for one hour, and the heat shock transformation is completed; The TOP10F' competent cell bacterial solution after introduction transformation is inoculated into super broth medium, and is shaken and cultured until the absorbance OD 600 is 0.8-1.0, isopropylthiogalactoside is added to 1 mM for induction, and is subjected to overnight treatment.
[0025] In the step (4), the purification of the nanobody is to add a hammerhead lysis solution to the bacterial solution cultured overnight to lyse the bacteria, and the supernatant is collected after centrifugation; The Ni-NTA matrix is added to the collected supernatant, is shaken at room temperature, and then is added to a purification column to form a nickel column; An imidazole-containing phosphate equilibrium buffer is added to wash and elute the impurities from the purification column; An imidazole-containing phosphate elution buffer is added again to elute the proteins adsorbed on the purification column to obtain a target protein solution; Finally, the target protein solution is added to a dialysis bag, the dialysis bag is placed in a phosphate buffer solution for dialysis treatment to obtain the purified target grass carp rhabdovirus nanobody.
[0026] The purification column specifically adopts a His-tag protein purification kit, and the P2226 type of Biyun Tian Company is specifically adopted.
[0027] Three, an application of the target grass carp rhabdovirus nanobody, in the preparation of a detection product for detecting the grass carp rhabdovirus, the detection product is a detection reagent or a detection kit Four, a detection reagent or a detection kit related to the detection of the grass carp rhabdovirus, comprising a target grass carp rhabdovirus nanobody.
[0028] Five, a sandwich ELISA method for detecting the grass carp rhabdovirus, characterized in that the target grass carp rhabdovirus nanobody is used in the method.
[0029] The present application obtains four nanobodies which can effectively bind to the grass carp rhabdovirus and can specifically realize the immunological analysis of the grass carp rhabdovirus through nanobody phage library construction and phage screening technology; and a sandwich ELISA method based on the sandwich method is constructed for detecting the grass carp rhabdovirus.
[0030] The present application aims to provide a kind of nanobody targeted to Rhabdovirus of Misgurnus anguilus with low expression cost, low difficulty, stable source, compared with traditional monoclonal antibody, it can be mass-produced, and has wide application prospect in the field of Rhabdovirus of Misgurnus anguilus detection.
[0031] Compared with the prior art, the technical scheme provided by the present application has the following advantages: 1、The nanobody targeted to Rhabdovirus of Misgurnus anguilus provided by the present application is obtained based on prokaryotic expression method, and has the advantages of low expression cost, low difficulty and stable source; 2、The nanobody targeted to Rhabdovirus of Misgurnus anguilus provided by the present application can effectively bind to Rhabdovirus of Misgurnus anguilus, and can specifically realize immune analysis of Rhabdovirus of Misgurnus anguilus; it is suitable for immune detection of Rhabdovirus of Misgurnus anguilus, and will play a great application potential in Rhabdovirus of Misgurnus anguilus screening. BRIEF DESCRIPTION OF DRAWINGS
[0032] Figure 1 is the serum titer chart of alpaca immunization Figure 2 is the IgG2 / 3 antibody heavy chain variable region gene agarose gel electrophoresis chart; Figure 3 is the VHH gene agarose gel electrophoresis chart; Figure 4 is the 96 strain phage monoclonal phage-ELISA result chart; Figure 5 is the expression chart of nanobody protein analyzed by SDS-PAGE electrophoresis; Figure 6 is the standard curve chart of Rhabdovirus of Misgurnus anguilus direct ELISA detection based on nanobody; Figure 7 is the standard curve chart of Rhabdovirus of Misgurnus anguilus sandwich ELISA detection based on nanobody. DETAILED DESCRIPTION
[0033] The present application will be further described below in combination with specific embodiments, but those skilled in the art should understand that modifications or replacements of details and forms of the technical scheme of the present application without deviating from the technical scheme of the present application are all within the scope of protection of the present application.
[0034] The embodiments of the present application are as follows: Example 1 I. Construction of immune library 1. Alpaca immunization The grass carp rhabdovirus cultured in LB medium for 18h was washed and prepared into inactivated antigen by high temperature sterilization. According to the immune effect of triple and quadruple vaccine, the strongest immune response and healthiest alpaca were selected, and the negative serum before grass carp rhabdovirus immunization was prepared for use. After the start of immunization, the serum titer was detected seven times after three, four, five, six and seven immunizations, and the peripheral blood was collected for peripheral blood lymphocyte extraction. The serum titer results are shown in Figure 1 .
[0035] 2. Peripheral blood lymphocyte extraction 15 mL of peripheral blood was slowly added to 15 mL of cell separation solution at an angle of 45 degrees, and centrifuged at 400 g for 30 min at room temperature. Then the lymphocytes were transferred to a new centrifuge tube, 10 mL of PBS buffer was added, and centrifuged again at 400 g for 20 min. The supernatant was slowly aspirated, resuspended with PBS, and the cell number was calculated.
[0036] 3. cDNA acquisition According to the instructions of TRIzol™, total RNA was extracted, and after extraction, the OD 260 nm and OD 280 nm absorbance values were measured, and the RNA concentration and purity obtained were calculated. The obtained RNA can be stored in a -80℃ refrigerator and used for subsequent reverse transcription to obtain cDNA.
[0037] All the obtained RNA was reverse transcribed using SuperScript™ IV First-Strand Synthesis System, and the process was as follows: (1) RNA denaturation, reaction system as shown in Table 1: Table 1 65℃ incubate for 5 minutes, and place on ice for at least 1 minute.
[0038] (2) Reverse transcription reaction system, reaction system as shown in Table 2: Table 2 (3) Reverse transcription reaction: Mix step (1) RNA denaturation system and step (2) reverse transcription reaction system, incubate at 23℃ for 10 minutes, then incubate at 55℃ for 10 minutes, and finally incubate at 80℃ for 10 minutes to terminate the reaction.
[0039] (4) Removal of RNA: 1 μL of RNase H was added to the above systemE. coli RNase H, 37°C for 20 minutes.
[0040] (5) Preservation of cDNA: The cDNA obtained from the above reaction was stored at -20°C for subsequent PCR amplification.
[0041] 4. Nested PCR amplification of target Rhabdovirus nanobody gene sequence Nested PCR was used to amplify the nanobody gene sequence. The primers were synthesized by Shanghai Biotech, and the primer sequences are shown in Table 3: Table 3 The nanobody gene sequence was amplified in the first round of PCR using cDNA as the template. The amplification reaction system is shown in Table 4: Table 4 The PCR program is shown in Table 5: Table 5 10 μL of PCR product was identified by agarose gel electrophoresis, and the results are shown in Figure 2 ; at the same time, the remaining PCR product was recovered by gel cutting.
[0042] The PCR product obtained in the first round was used as a template for amplification. The amplification reaction system is shown in Table 6: Table 6 The PCR program is shown in Table 7: Table 7 10 μL of PCR product was identified by agarose gel electrophoresis, and the results are shown in Figure 3 ; at the same time, the remaining PCR product was recovered by column, and was used in the next step of enzyme digestion.
[0043] 5. Ligation of target Rhabdovirus nanobody gene and pComb3xss vector (1) Enzymatic digestion of pComb3xss vector, enzyme digestion system as follows: The target fragment was recovered by gel cutting method, which was used as the target Rhabdovirus nanobody gene.
[0044] (2) Enzymatic digestion of target Rhabdovirus nanobody gene, enzyme digestion system as follows: The target gene after enzyme digestion is recovered.
[0045] (3) Targeting the connection of the Rhabdovirus Nanobody gene of the rice field eel and the pComb3xss vector, and the linking system is as follows: The above system is incubated at 16°C overnight, and then incubated at 65°C for ten minutes to terminate the ligation reaction.
[0046] The linked product is purified and placed in a -20°C refrigerator for subsequent experiments.
[0047] 6. Electroporation of the ligation product 3 μL of the ligation product is added to 25 μL of competent cells ER2738, and the mixture is electroporated at 1.8 KV, 200Ω, and 25 µF; after completion of the transformation, the mixture is shaken and recovered for 1 hour to complete the construction of the initial phage library.
[0048] 7. Amplification and expression of the phage library After the recovery is completed, the bacterial solution is added to 200 mL of culture medium, and after shaking at 37°C for 2 hours, helper phage is added to expand the initial library. After the addition of the helper phage, the mixture is allowed to stand at 37°C for 30 minutes, so that the phage infects the E. coli ER2738 containing the VHH antibody gene, and then the mixture is shaken at 250 rpm for 2 hours. Then, 200 μL of 70 mg / mL kanamycin is added, and then the mixture is shaken overnight at 37°C. The next day, the phage is collected by centrifugation, and the construction of the nanobody library (immune library) is completed. II. Nanobody panning 1. First round of panning (1) 7 x 10 7 PFU / mL of Rhabdovirus is coated on two wells of an enzyme-labeled plate, and the plate is placed in a refrigerator at 4°C overnight; (2) The next day, the plate is washed with PBST three times, and 3% skimmed milk powder is added for blocking for 1 hour; (3) 100 μL of the phage library constructed in Example 1 is added to each well, and the plate is shaken at room temperature for 1 hour; (4) The plate is washed with PBST for 10 times; (5) Elution of the bound phage 100 μl of glycine-hydrochloric acid (the glycine-hydrochloric acid is 0.1 M glycine-hydrochloric acid (pH 2.2), and the plate is incubated at room temperature for 15 minutes, and then 50 μl of 1 M Tris-HCI is immediately added for neutralization; (6) Take 2 μL of eluted phage and add to 198 μL of ER2738 (OD 0.4-0.6), allow to infect for 0.5 h, then take 100 μL and plate on carbenicillin media for output titer test of the first round of selection; (7) Add the remaining phage to 2 mL of ER2738 and shake at room temperature for 1 h; (8) Add 12 μL of CA and shake for 1 h; (9) Add 50 μL of helper phage and shake for 2 h; (10) Add 20 μL of KANA and shake overnight.
[0049] 2. Second round of selection (1) Coat 3.5 x 10 7 PFU / mL of rhabdovirus onto two wells of a microtiter plate and place in the refrigerator at 4°C overnight; (2) The next day, centrifuge the overnight culture at 10,000 g at 4°C for 15 min and transfer the supernatant to a new centrifuge tube; (3) Add 5 mL of 5x PEG / NaCl to the supernatant and incubate on ice for 2 h; (4) Centrifuge the culture at 12,000 rpm for 15 min, discard the supernatant, resuspend in 2 mL of 1% BSA in PBS, filter through a membrane, and store. (This is the phage library after the first round of selection); (5) Wash three times with PBST and block with 3% non-fat dry milk for 1 h; (6) Add 100 μL of the phage library after the first round of selection to each well and shake at room temperature for 1 h; (7) Wash 10 times with PBST; (8) Elute the bound phage Add 100 μl of glycine-hydrochloric acid (the glycine-hydrochloric acid is 0.1 M glycine-hydrochloric acid (pH 2.2) to the wells, incubate at room temperature for 15 minutes, and then immediately add 50 μl of 1 M Tris-HCI for neutralization; (6) Take 2 μL of eluted phage and add to 198 μL of ER2738 (OD 0.4-0.6), allow to infect for 0.5 h, then take 100 μL and plate on carbenicillin media for output titer test of the first round of selection; (7) Add the remaining phage to 2 mL of ER2738 and shake at room temperature for 1 h; (8) Add 12 μL of CA and shake for 1 h; (9) Add 50 μL of helper phage, shake culture for 2 h; (10) Add 20 μL of KANA, shake culture overnight.
[0050] 3. Third round of panning (1) 1.75 x 10 7 PFU / mL of rhabdovirus was coated onto two wells of the ELISA plate and placed in the refrigerator at 4°C overnight; (2) The next day, centrifuge the culture at 10,000 g at 4°C for 15 min, and transfer the supernatant to a new centrifuge tube; (3) Add 5 mL of 5x PEG / NaCl to the supernatant, and incubate on ice for 2 h; (4) Centrifuge the culture at 12,000 rpm for 15 min, discard the supernatant, and resuspend with 2 mL of 1% BSA in PBS, filter membrane, and store. (This is the phage library after the second round of panning); (5) Wash three times with PBST, and add 3% skim milk powder for blocking for 1 h; (6) Add 100 μL of the phage library after the second round of panning to each well, and shake at room temperature for 1 h; (7) Wash 10 times with PBST; (8) Elute the bound phage Add 100 μl of glycine-hydrochloric acid (the glycine-hydrochloric acid is 0.1 M glycine-hydrochloric acid (pH 2.2), incubate at room temperature for 15 min, and then immediately add 50 μl of 1 M Tris-HCI for neutralization; (6) Take 2 μL of the eluted phage and add to 198 μL of ER2738 (OD 0.4-0.6), stand for infection for 0.5 h, and then take 100 μL and spread on a carbenicillin culture medium for first round of panning output titer test; (7) Add the remaining phage to 2 mL of ER2738, shake at room temperature for 1 h; (8) Add 12 μL of CA, shake culture for 1 h; (9) Add 50 μL of helper phage, shake culture for 2 h; (10) Add 20 μL of KANA, shake culture overnight and centrifuge according to the previous process.
[0051] 4. Screening of nanobody positive strains (1) Respectively from the three-wheel screening output titer test plate each other to select 32 single clone colonies, inoculated into super broth medium, shaking culture 4-5 hours after adding 10 μL auxiliary phage overnight.
[0052] The super broth medium contains (1L ddH2O contains 10g MOPS, 30g peptone, 20g yeast extract) (2) Pre-coated 7x10 6 PFU / mL of snake rhabdovirus enzyme plate, 3% skim milk powder blocking, room temperature shaking 1 hour, PBST washing three times, after the step (1) in the culture of single clone colonies into the enzyme plate, room temperature shaking 1 hour, PBST washing three times, adding 100 ng / mL of anti-M13-horseradish peroxidase labeled antibody 100 μL / well, room temperature shaking 1 hour, PBST washing five times, adding 3,3',5,5'-tetramethylbenzidine 100 μL / well for color development.
[0053] (3) After color development (results refer to Figure 4 ), pick the wells with absorbance value greater than 1 as positive clone wells corresponding to the single clone colonies for sequencing, and the gene sequence is shown as SEQ ID NO. 1-4.
[0054] II. Prokaryotic expression and purification of anti-snake rhabdovirus nanobody 1. Prokaryotic expression of nanobody (1) Use Omega plasmid small amount extraction kit to extract plasmid of positive single clone colonies, then mix 2 μL plasmid with TOP10F' competent cells, ice bath for 30 minutes, then place in 42℃ hot water for 90 seconds, then ice bath for 5 minutes, add 1 milliliter super broth medium, 37℃ shaking for one hour, complete transformation.
[0055] (2) Inoculate the TOP10F' introduced into the culture medium, shake culture to absorbance OD 600 0.8-1.0, add isopropylthiogalactoside to 1 mM, change the shaking condition to 30℃ and induce overnight.
[0056] 2. Purification of nanobody (1) Collect the overnight culture into a centrifuge bottle, centrifuge at 5000 rpm at 4℃ for 10 min; (2) Add 1 mL iron hammer lysis solution and 9 mL 1 mM TBS to lyse the bacteria, and break them under ultrasonic crusher for 10 minutes; centrifuge at 16000 g at 4℃ for 10 min; (3) Collect the supernatant for subsequent purification; (4) Add Ni-NTA matrix to the collected supernatant above, shake at room temperature for 30 minutes, and add to the purification column, repeat loading 4 times to form a nickel column; (6) Add 20 mL of 2 mM imidazole-containing phosphate buffer for balancing, wash the purification column, and elute the impurities; (7) Add 50 mM imidazole-containing phosphate elution buffer to elute the target protein adsorbed on the nickel column; (8) Take a small amount of the purified target protein collected for SDS-PAGE electrophoresis detection, and the results are shown in Figure 5 ; (9) Add the remaining collected target protein to a dialysis bag and dialyze in phosphate buffer for 48 hours.
[0057] III. Example application of nanobodies in the immunodetection of Rhabdovirus of Monopterus albus Direct capture immunoassay of Rhabdovirus of Monopterus albus (1) Dilute 1x10 7 of Rhabdovirus of Monopterus albus with phosphate buffer, add 100 μL / well to a 96-well enzyme-labeled plate, and coat overnight at 4°C; (2) The next day, wash the coated 96-well plate three times with phosphate-tween buffer; (3) Add 300 μL of 3% skimmed milk powder to each well for blocking, and the blocking procedure is to shake and incubate at room temperature for 1 hour; (4) After incubation, wash three times with phosphate-tween buffer; (5) Add 100 μL of 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 μg / mL nanobodies to each well, mix well, and shake and incubate at room temperature for 1 hour; (5) After the reaction is completed, wash three times with phosphate-tween buffer; (6) Add 100 μL of diluted 10,000-fold horseradish peroxidase-labeled anti-HA secondary antibody (100 μL / well) to each well, shake and incubate at room temperature for 1 hour; (7) After incubation, wash five times with phosphate-tween buffer; (8) Add 100 μL of 3,3',5,5'-tetramethylbenzidine color developing solution to each well, and react in the dark for 15 min; (9) After color development is completed, add 50 μL of 2 M H2SO4 to each well to terminate the reaction.
[0058] The standard curve of the Rhabdovirus of Monopterus albus ELISA detection is shown in Figure 6 , and the Figure 6It can be seen that the four strains of nanobodies targeting the Rhabdovirus of Misgurnus anguilus provided by the application can effectively bind to the Rhabdovirus of Misgurnus anguilus, thereby specifically achieving immune analysis of the Rhabdovirus of Misgurnus anguilus.
[0059] Sandwich capture immune analysis of the Rhabdovirus of Misgurnus anguilus (1) 5 ug / mL of the Rhabdovirus of Misgurnus anguilus polyclonal antibody diluted with carbonate buffer was coated in advance on an enzyme-labeled plate, and the coating was performed at 4°C overnight; (2) The next day, the 96-well plate after coating was washed three times with phosphate-tween buffer; (3) 300 μL of 3% skimmed milk powder was added to each well for blocking, and the blocking procedure was 37 °C shaking incubation for 1 hour; (4) After incubation, washing was performed three times with phosphate-tween buffer (5) 1x10 7 of the Rhabdovirus of Misgurnus anguilus was diluted, and 100 μL / well was added to the 96-well enzyme-labeled plate, and the coating was performed at 4°C overnight; (6) 2.8x10 7 , 1.4x10 7 , 7x10 6 , 3.5x10 6 , 1.75x10 6 , 8.75x10 5 , 4.38x10 5 , 0 PFU / mL of the Rhabdovirus of Misgurnus anguilus was added to each well, mixed, and incubated at 37 °C for 1 hour; (7) After the reaction was completed, washing was performed three times with phosphate-tween buffer; (8) 100 μL of phosphate-diluted 5 ug / mL nanobodies was added to each well, and incubation was performed at 37 °C for 1 hour; (9) After incubation, washing was performed three times with phosphate-tween buffer; (10) 100 μL of ten-thousand-fold diluted horseradish peroxidase-labeled anti-HA secondary antibody (100 μL / well) was added to each well, and incubation was performed at room temperature for 1 hour; (11) After incubation, washing was performed five times with phosphate-tween buffer; (12) 100 μL of 3,3', 5,5'-tetramethylbenzidine color developing solution was added to each well, and the reaction was performed in the dark for 15 min; (13) After color development was completed, 50 μL of 2 M H2SO4 was added to each well to terminate the reaction.
[0060] The standard curve of the Rhabdovirus of Misgurnus anguilus ELISA detection is shown in Figure 7 , and the detection results are shown in Figure 7It can be seen that the two strains of the targeting grass carp rhabdovirus nanobody provided in the application can effectively bind to the grass carp rhabdovirus, thereby specifically achieving immune analysis on the grass carp rhabdovirus.
[0061] The sequences involved in the application are as follows: SEQ ID No. 1: Name: Targeting grass carp rhabdovirus nanobody amino acid sequence 1 QAAQLQLVESGGGSVQAGGSLRLSCAASGFTYCLSAMSWYRQAPGKEREFVSRIDSDGNTSYADSVKGRFTISQDNGKNTLHLQMNSLAPEDTAMYYCKRDTRPACGGKSYWGRGTQVTVSSG SEQ ID No. 2: Name: Targeting grass carp rhabdovirus nanobody amino acid sequence 2 QAAQLQLVESGGGSVETGGSLTLSCTASGYNDSNFAMAFFRQAPGNEREGVAVRNMWQNTWYGPSVKGRVYASRDTVKNILTLTMNNLRPEDTAMYYCAAARPRLYCTGPDCLAPAFYANWGQGTQVTVSSG SEQ ID No. 3: Name: Targeting grass carp rhabdovirus nanobody amino acid sequence 3 QAAQLQLVESGQGSVEAGGSLTLSCVASGYSDSQFSMAWFRQAPGKEREAVVANNMWNNTWYGDSVKGRIRVYRDNAGNILPLQLNNLQPEDAAMYYCASAQPRTYCARSDCFNQAFYKNWGQGIQVTVSSA SEQ ID No. 4: Name: Targeting grass carp rhabdovirus nanobody amino acid sequence 4 QAAQVQLVESGGGSVQSGGSLRLSCAVSGYDVSRSCMGWFRRAPGKEREGVAGIKTDGTRSYADSVKGRFTVSRDNAKNTLYLEMNSLKPEDTAMYYCAAAPYCRYWNGAALLRSSGFEYWGQGTQVTVSSG SEQ ID No. 5: Name: PCR amplification primer 1 DNA type: other DNA ORGANISM: Artificial Sequence / synthetic construct GTC CTG GCT GCT CTT CTA CAA GG SEQ ID No. 6: NAME: PCR amplification primer 1 DNA TYPE: other DNA ORGANISM: Artificial Sequence / synthetic construct GGT ACG TGC TGT TGA ACT GTT CC SEQ ID No. 7: NAME: PCR amplification primer 1 DNA TYPE: other DNA ORGANISM: Artificial Sequence / synthetic construct CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC SEQ ID No. 8: NAME: PCR amplification primer 1 DNA TYPE: other DNA ORGANISM: Artificial Sequence / synthetic construct CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG SEQ ID No. 9: NAME: PCR amplification primer 1 DNA TYPE: other DNA ORGANISM: Artificial Sequence / synthetic construct CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG SEQ ID No. 10: NAME: PCR amplification primer 1 DNA TYPE: other DNA Organism source: Artificial Sequence / synthetic construct CATGCCATGACTCGCGGCCGGCCTGGCCCTTGCATACTTCATTCGTTCCTG.
Claims
1. A targetable snakehead rhabdovirus nanobody, characterized in that, The amino acid sequence of the nanobody is shown as SEQ ID NO. 1-4.
2. The method for preparing the targetable Rhabdovirus of Monopterus albus nanobody of claim 1, characterized in that, The method comprises the following steps: (1) immunizing the alpaca with inactivated Rhabdovirus of Monopterus albus, collecting peripheral blood lymphocytes of the alpaca after immunization to extract mRNA, synthesizing cDNA by taking the mRNA as a template, and performing PCR amplification by taking the obtained cDNA as a template to obtain a DNA fragment encoding a nanobody; (2) connecting the DNA fragment encoding the nanobody with a pComb3xss vector, then transforming into competent cells to construct a nanobody initial library, and then expanding and culturing the nanobody initial library to obtain a nanobody library; (3) taking the Rhabdovirus of Monopterus albus as a coated antigen, and performing enrichment and screening on the nanobody library to obtain a nanobody phagemid against the Rhabdovirus of Monopterus albus; (4) transforming the nanobody phagemid against the Rhabdovirus of Monopterus albus into a host expression bacterium to express the nanobody, and purifying to obtain a nanobody sequence targeting the Rhabdovirus of Monopterus albus.
3. The method for preparing the targeted loach rhabdovirus nanobody according to claim 2, characterized in that, In the step (1), the PCR amplification is performed by using primers 1-6: The nucleotide sequence of the primer 1 is shown as SEQ ID NO. 5; The nucleotide sequence of the primer 2 is shown as SEQ ID NO. 6; The nucleotide sequence of the primer 3 is shown as SEQ ID NO. 7; The nucleotide sequence of the primer 4 is shown as SEQ ID NO. 8; The nucleotide sequence of the primer 5 is shown as SEQ ID NO. 9; The nucleotide sequence of the primer 6 is shown as SEQ ID NO.
10.
4. The method for preparing the targeted loach rhabdovirus nanobody according to claim 2, characterized in that, In the step (2), the nanobody initial library is constructed by transforming into competent cells, specifically as follows: The solution after connecting the DNA fragment encoding the nanobody with the pComb3xss vector is added into TOP10F' competent cells to obtain a recombinant plasmid, and the recombinant plasmid is transfected into competent E. coli ER2738 by electroporation; In the step (2), the nanobody initial library is expanded and cultured, specifically as follows: the recombinant plasmid is added into super broth medium, after being shaken at 37°C and 250 rpm for 2 hours, auxiliary phages are added and then the mixture is placed at 37°C for 30 minutes, so that the phages are used to infiltrate E. coli ER2738 containing VHH antibody genes, then the mixture is shaken at 250 rpm for 2 hours, 200 μL of 70 mg / mL kanamycin is added, then the mixture is shaken at 37°C overnight, the next day the phages are collected by centrifugation, and the construction of the nanobody library is completed.
5. The method for preparing the targeted loach rhabdovirus nanobody according to claim 1, characterized in that, In the step (3), the nanobody library is subjected to enrichment and screening, specifically as follows: Firstly, the nanobody library was treated by three rounds of panning, then different monoclonal colonies were picked and inoculated into 96 deep well plates (containing 1 mL super broth medium per well) for overnight culture, and then added to the enzyme-labeled plate coated with the rhabdovirus of the grass carp every other day. After washing at room temperature, anti-M13-horseradish peroxidase labeled antibody was added, and then washed again at room temperature. 3,3',5,5'-tetramethylbenzidine was added for color development, and the monoclonal colonies corresponding to the positive clone wells with an absorbance value OD 450 The wells with an absorbance value greater than 1 were used as positive clone wells, and the monoclonal colonies corresponding to the positive clone wells were used as nanobody phagemids against the rhabdovirus of the grass carp.
6. The method for preparing the targeted loach rhabdovirus nanobody according to claim 1, characterized in that, In the step (4), the phagemid is transformed into a host expression bacterium, specifically as follows: The plasmid in the positive monoclonal colony is extracted by using an Omega plasmid small amount extraction kit, then 2 μL of the plasmid is mixed with 50 μL of TOP10F' competent cells as the host expression bacterium to form a bacterial solution, the bacterial solution is placed in ice water for a period of time, then is heated in hot water at 42°C for a period of time, then is placed in ice water again for a period of time, then the super broth medium is added, and the mixture is shaken at 37°C for one hour, and the heat shock transformation is completed. The transformed TOP10F' competent cell broth was inoculated into super broth medium, and shaken to an optical density OD 600 of 0.8-1.0, and induced by adding isopropylthio-galactoside to 1 mM, and treated overnight.
7. The method for preparing the targeted loach rhabdovirus nanobody according to claim 1, characterized in that, In the step (4), the purification of the nanobody is to add iron hammer lysis solution to the bacteria liquid cultured overnight to lyse the bacteria, and collect the supernatant after centrifugation; The Ni-NTA matrix is added to the supernatant, and after oscillation at room temperature, it is added to the purification column; The imidazole-containing phosphate buffer is added to wash the purification column and elute; The imidazole-containing phosphate elution buffer is added again to elute the proteins adsorbed on the purification column to obtain the target protein solution; Finally, the target protein solution is added to the dialysis bag, and the dialysis bag is placed in the phosphate buffer for dialysis treatment to obtain the purified target grass carp rhabdovirus nanobody.
8. Use of the targetable snakehead rhabdovirus nanobody according to any one of claims 1 to 7, characterized in that: The application in the preparation of a detection product for detecting grass carp rhabdovirus.
9. A detection reagent or a detection kit related to the Rhabdovirus of Monopterus albus, characterized in that, The target grass carp rhabdovirus nanobody according to any one of claims 1-7.
10. A sandwich ELISA method for detecting the Monopterus albus rhabdovirus, characterized by, The method uses the target grass carp rhabdovirus nanobody according to any one of claims 1-7.