Nano antibody for detecting vibrio parahaemolyticus as well as preparation method and application of nano antibody

Abstract

The invention discloses a nano antibody for detecting vibrio parahaemolyticus as well as a preparation method and application thereof, and aims to provide a nano antibody for detecting vibrio parahaemolyticus, which is high in detection accuracy, strong in specificity and simple in obtaining method. The amino acid sequence of the nano antibody is as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5. Belongs to the technical field of biological detection.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a nanobody for detecting Vibrio parahaemolyticus; this invention also relates to the nanobody, its preparation method, and its application. Background Technology

[0002] Foodborne illnesses caused by Vibrio parahaemolyticus (Vp) infection pose a significant challenge to seafood safety and human health. As a halophilic Gram-negative bacterium widely found in nearshore seawater, seabed sediments, and shellfish, Vibrio parahaemolyticus possesses extremely strong environmental adaptability and reproductive capacity. It is the leading cause of microbial food poisoning in coastal areas and even some inland regions of my country, and one of the major pathogens causing bacterial enteritis globally. This bacterium can infiltrate and spread throughout the entire "ocean-to-table" food chain, from aquaculture sources, fishing, cold chain transportation, all the way to home kitchens or catering terminals, posing a serious threat to the food processing industry and public health safety. In the aquaculture sector, Vibrio parahaemolyticus severely hinders the large-scale development of shrimp and marine fish farming. It is a major pathogen causing devastating diseases such as "early death syndrome" in shrimp. Improper control of the aquaculture water environment easily leads to infection in aquatic animals. Infected shrimp often exhibit red body, hepatopancreatic necrosis, decreased vitality, and mass mortality, resulting in huge economic losses. For humans, consuming undercooked seafood or coming into contact with cross-contaminated food can easily lead to infection. Patients experience a rapid onset of illness with a short incubation period. Typical clinical manifestations include severe abdominal pain, paroxysmal colic around the navel, and watery or meat-wash-like diarrhea, often accompanied by nausea, vomiting, and fever. Severe cases can even be life-threatening due to dehydration and shock. Therefore, efficient monitoring and control of Vibrio parahaemolyticus is of great demand for ensuring food safety, maintaining public health order, and promoting the sustainable development of aquaculture.

[0003] Currently, screening methods for Vibrio parahaemolyticus mainly encompass molecular biology (such as PCR), microbial isolation and culture, immunoassay, and mass spectrometry. While PCR-based molecular detection boasts excellent specificity and sensitivity, rapidly identifying target DNA, it is highly dependent on sophisticated instruments and skilled personnel, and the risk of false positives is significant. Although bacterial culture has long been considered the "gold standard" for pathogen identification due to its intuitiveness and ease of use, this method is time-consuming and requires a high threshold for bacterial abundance, easily leading to missed detections of low-abundance samples. Mass spectrometry, while enabling high-precision identification through molecular fingerprinting, is limited by high hardware investment and cumbersome procedures. Furthermore, while immunological detection possesses a certain degree of specificity, it is often constrained by antibody titer and concordance, facing challenges such as cross-reactivity and unsatisfactory detection limits. Compared to traditional monoclonal antibodies, recombinant antibodies offer significant advantages in cost control and batch-to-batch uniformity. Therefore, constructing anti-Vibrio parahaemolyticus nanoantibodies based on phage display and prokaryotic expression systems has become an inevitable choice and feasible path to innovate existing detection technologies and make up for the shortcomings of traditional methods. Summary of the Invention

[0004] The purpose of this invention is to provide a nanobody for detecting Vibrio parahaemolyticus that has high accuracy, strong specificity, and a simple acquisition method.

[0005] The present invention also provides a method for preparing nanobodies that specifically recognize Vibrio parahaemolyticus (strain type: ATCC 17802).

[0006] Therefore, the first technical solution provided by the present invention is as follows:

[0007] A nanobody for detecting Vibrio parahaemolyticus, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5.

[0008] Furthermore, the aforementioned nanobody for detecting Vibrio parahaemolyticus uses Vibrio parahaemolyticus strain ATCC 17802.

[0009] The second technical solution provided by this invention is the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus, which includes the following steps in sequence:

[0010] 1) Collect blood samples from immunized camels, isolate and collect camel peripheral blood lymphocytes, extract total RNA of anti-Vibrio parahaemolyticus nanoantibodies from camel peripheral blood lymphocytes, and synthesize cDNA; use cDNA as a template to amplify the heavy chain variable region gene, ligate the amplified camel antibody heavy chain variable region gene with a phage vector, and transform it into competent Escherichia coli to construct an immunized camel nanoantibody phage library;

[0011] 2) Using Vibrio parahaemolyticus as the coating antigen, nanobody phages that can specifically recognize Vibrio parahaemolyticus were obtained through three or more rounds of panning using the pH elution method, and the genes of the nanobody were compared by sequencing analysis.

[0012] 3) The phage vector containing the anti-Vibrio parahaemolyticus nanobody gene was extracted and reintroduced into the host expression bacteria for nanobody expression and purification.

[0013] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus, the total RNA extraction is performed according to the instructions of the TRIzol™ reagent.

[0014] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus, the cDNA is synthesized by reverse transcription using the extracted RNA as a template and the SuperScript™ IV first-strand synthesis system to synthesize the cDNA encoding the antibody gene.

[0015] Furthermore, the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus employs a two-step amplification method. The first step uses upstream primer Call001 and downstream primer Call002, and the second step uses upstream primer 2nd for and downstream primer 2nd reverse to amplify the heavy chain variable region gene.

[0016] The upstream primer Call001 nucleotide is shown in Seq ID NO: 6;

[0017] The downstream primer Call002 nucleotide is shown in Seq ID NO: 7;

[0018] The upstream primer 2nd for nucleotides is shown in Seq ID NO: 8;

[0019] The downstream primer 2nd reverse nucleotide is shown in Seq ID NO: 9.

[0020] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus, the heavy chain variable region gene is ligated to the phage vector by using T4 ligase to ligate the enzyme-digested nanobodies gene fragment and the enzyme-digested vector pComb3xss.

[0021] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Vibrio parahaemolyticus, the phage vector containing the anti-Vibrio parahaemolyticus nanobodies gene is extracted by using the EZNA® Plasmid DNA Mini Kit to extract plasmids of positive phages, and the plasmids are introduced into Escherichia coli TOP10F' by heat shock.

[0022] The nanobody for detecting Vibrio parahaemolyticus provided by this invention is used in the preparation of reagents or kits for detecting Vibrio parahaemolyticus.

[0023] Compared with existing technologies, this invention has screened out nanoantibodies that specifically recognize Vibrio parahaemolyticus, with high detection accuracy and simple acquisition method. It is suitable for immunoassay detection of Vibrio parahaemolyticus in food and is expected to develop high-performance rapid immunoassay products for Vibrio parahaemolyticus detection applications. Attached Figure Description

[0024] Figure 1 These are the results of phage-ELISA for 480 phage monoclonal strains;

[0025] Figure 2 The expression of anti-Vibrio parahaemolyticus nanobodies was analyzed by SDS-PAGE.

[0026] Figure 3 This is a standard curve for a sandwich ELISA of Vibrio parahaemolyticus based on nanobodies. Detailed Implementation

[0027] 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.

[0028] The raw materials used in the following schemes are sourced from the following sources:

[0029] Vibrio parahaemolyticus was purchased from Shandong Huankai Biotechnology Co., Ltd., strain ATCC 17802;

[0030] Both Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma-Aldrich.

[0031] Human peripheral blood lymphocyte separation fluid was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LTS1077;

[0032] The helper phage M13KO7 was purchased from New England Biolabs (Beverly, MA);

[0033] Example 1: Total RNA extraction and cDNA synthesis of anti-Vibrio parahaemolyticus nanobodies

[0034] 1. Extraction of peripheral blood lymphocytes from camels:

[0035] (1) Vibrio parahaemolyticus cultured in LB medium for 18 h was washed and then inactivated with 1% formaldehyde for 24 h to prepare Vibrio parahaemolyticus inactivated antigen. The Vibrio parahaemolyticus inactivated antigen was mixed with Freund's immune adjuvant (CAS: BIO-000001) in equal volume and then injected subcutaneously into camels. The immunization was carried out 3-4 times in a cycle of 14 days. Blood samples were collected from the immunized camels after the third immunization.

[0036] (2) Add 15 mL of human peripheral blood lymphocyte separation solution to a 50 mL centrifuge tube, tilt the centrifuge tube slightly at 45° and slowly add 15 mL of collected camel blood. Centrifuge the prepared cell separation solution containing camel blood at 400 g speed for 30 min at room temperature using a centrifuge with a horizontal rotor.

[0037] (3) Use a pipette to aspirate the cloud-like lymphocytes in the middle of the cell separation fluid containing camel blood that has separated into layers after centrifugation in step (2) into a new 50 mL centrifuge tube.

[0038] (4) Add 10 mL of room temperature PBS buffer to each tube and centrifuge at 400 g for 20 min at room temperature using a horizontal centrifuge.

[0039] (5) Remove the supernatant after centrifugation in step (4), resuspend the precipitate in each tube with 1 mL PBS buffer, mix gently to obtain cell resuspension, and count the number of cells using a hemocytometer.

[0040] 2. Extraction of total RNA

[0041] Total RNA was extracted according to the TRIzol™ reagent instructions, following these steps:

[0042] (1) Transfer the cell resuspension from step 1) to a 1.5 mL centrifuge tube and centrifuge at 4 ℃ and 8000 g for 2 min.

[0043] (2) Remove the supernatant after centrifugation in step (1). Be careful to remove it slowly to prevent vigorous movement from damaging the cell precipitate at the bottom of the centrifuge tube.

[0044] (3) Add 1 mL of TRIzol™ reagent according to the instructions (per 10 mL of TRIzol™ reagent). 7 Add 1 mL of TRIzol™ reagent to each cell.

[0045] (4) Mix thoroughly to lyse the cells until no obvious cell precipitate is visible to the naked eye in the lysate;

[0046] (5) Let the pyrolysis solution from step (4) stand at room temperature for 5 min;

[0047] (6) Add 0.2 mL of chloroform to the lysis solution after standing in step (5), mix thoroughly on a vortex mixer, and let stand at room temperature for 2-3 min;

[0048] (7) Add the lysate after step (6) to a centrifuge tube and place it in a centrifuge and centrifuge at 12000 g at 4℃ for 15 min;

[0049] (8) Carefully remove the centrifuge tube after centrifugation in step (7) from the centrifuge. You can see that the lysate in the centrifuge tube is clearly divided into three layers. Be careful not to shake it violently.

[0050] (9) Carefully aspirate the upper layer of lysate from the centrifuge tube in step (8) and transfer it to another new centrifuge tube, being careful not to aspirate the white flocculent material in the middle layer of the lysate;

[0051] (10) Add 0.5 mL of isopropanol to the upper lysate collected in step (9), gently invert and mix thoroughly, and let stand at room temperature for 10 min.

[0052] (11) Place the centrifuge tubes processed in step (10) into a centrifuge and centrifuge at 12000 g at 4 ℃ for 10 min;

[0053] (12) Carefully discard the supernatant after centrifugation in step (11), and then add 75% (v / v) ethanol to the centrifuge tube in an equal volume to the TRIzol™ reagent;

[0054] (13) Place the centrifuge tubes processed in step (12) into a centrifuge and centrifuge at 7500 g at 4 ℃ for 5 min;

[0055] (14) Carefully discard the supernatant after centrifugation in step (13), invert the centrifuge tube and dry it at room temperature for 5 min;

[0056] (15) After confirming that the centrifuge tubes processed in step (14) are dry, add 20 μL of deionized water to dissolve the precipitate and obtain total RNA;

[0057] (16) OD of total RNA products was measured using a UV spectrophotometer. 260 nm and OD 280 The absorbance value was measured in nm to calculate the concentration and purity of the extracted RNA, which was then stored in an ultra-low temperature freezer at -80 ℃ for later use.

[0058] 3. cDNA Synthesis

[0059] Using the extracted RNA as a template, cDNA encoding the antibody gene was synthesized by reverse transcription using the SuperScript™ IV first-strand synthesis system.

[0060] The process is as follows:

[0061] (1) Add the reagents shown in Table 1 to the PCR tube:

[0062] Table 1

[0063]

[0064] Place the reaction in a PCR instrument, incubate at 65 ℃ for 5 min, and then cool on ice;

[0065] (2) Add reverse transcription reaction solution to the above reaction system. The system is shown in Table 2:

[0066] Table 2

[0067]

[0068] The mixed reaction solution was placed in the PCR instrument, and the conditions were set as shown in Table 3:

[0069] Table 3

[0070]

[0071] (3) Add 1 µL of Escherichia coli RNase H and incubate at 37 °C for 20 min.

[0072] (4) Store the reverse transcription product cDNA library at -80 ℃ for later use.

[0073] Example 2: Amplification of anti-Vibrio parahaemolyticus nanobody gene

[0074] 1. Amplification of the IgG2 / 3 antibody heavy chain variable region (nanobody) gene

[0075] Primers for amplifying the nanobody gene were synthesized by PlatinumBio Biotechnology (Shanghai) Co., Ltd., and the primer sequences are shown in Table 4:

[0076] Table 4

[0077] Note: Underlined bases are enzyme cleavage sites.

[0078] (1) Using cDNA as a template, Call001 as the upstream primer and Call002 as the downstream primer, a first PCR was performed. Then, 2nd for and 2nd reverse were used as the upstream and downstream primers, respectively, for a second PCR to amplify the heavy chain variable region gene.

[0079] For the first round of PCR, add the reagents shown in Table 5-1 to the PCR tube:

[0080] Table 5-1

[0081]

[0082] For the second round of PCR, add the reagents shown in Table 5-2 to the PCR tubes:

[0083] Table 5-2

[0084]

[0085] Set the following conditions in the PCR instrument, and the system is shown in Table 6:

[0086] Table 6

[0087]

[0088] 10 μL of PCR product was detected and verified by 1% agarose gel electrophoresis. The remaining PCR product was used to recover the target gene using the EZNA® Cycle Pure Kit. After purification, the nanobody gene fragment was obtained, and the concentration and purity of the recovered DNA were measured. The fragment was stored at -20 ℃ for later use.

[0089] (2) Agarose gel electrophoresis

[0090] Weigh 0.3 g of agarose and dissolve it in 30 mL of 1×TAE buffer to prepare a 1% agarose gel.

[0091] Microwave the agarose until it is completely dissolved and becomes a colorless, transparent gel solution.

[0092] Once the gel solution has cooled to approximately 50 °C, add 2 μL of nucleic acid dye and mix thoroughly.

[0093] Steps The prepared gel solution is poured into a gel casting tank and cooled and solidified at room temperature;

[0094] Remove the comb and add 10× loading buffer to the PCR product from step (1) into the wells of the fully solidified agarose gel.

[0095] Place the gel in the electrophoresis tank and add 1×TAE buffer, then turn on the electrophoresis apparatus and run it at 100 V for 25 min;

[0096] Remove the gel and observe the results under a UV lamp.

[0097] Example 3: Construction of a phage nanobody library against Vibrio parahaemolyticus

[0098] 1. Enzymatic digestion reaction of vector pComb3Xss and nanobody gene fragment

[0099] Since the nanobody gene fragment obtained in Example 2 has two SfiI restriction sites introduced at both ends of the target gene, it can be directly digested and recombined with the pComb3Xss vector and ligated with T4 ligase.

[0100] The process is briefly described as follows:

[0101] (1) Enzyme digestion of vector pComb3xss

[0102] Add the reagents shown in Table 7 to the PCR tube:

[0103] Table 7

[0104]

[0105] Place in a PCR instrument and incubate overnight at 50 °C.

[0106] Add 22.5 μL of 0.5M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating SfiI enzyme.

[0107] The enzyme digestion effect was detected by 0.7% agarose gel electrophoresis of the enzyme digestion products.

[0108] Recover the enzyme digestion products.

[0109] (2) Enzyme digestion of nanobody gene fragments

[0110] Add the reagents shown in Table 8 to the PCR tube:

[0111] Table 8

[0112]

[0113] Place in a PCR instrument and incubate overnight at 50 °C.

[0114] Add 22.5 μL of 0.5M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating SfiI enzyme.

[0115] The EZNA® Cycle Pure Kit recovers enzyme digestion products.

[0116] 2. Ligation and purification of the vector pComb3Xss and the nanobody gene fragment

[0117] Add the reagents shown in Table 9 to the PCR tube:

[0118] Table 9

[0119]

[0120] Steps Place the PCR tubes containing the reaction solution in a PCR instrument and incubate overnight at 16°C.

[0121] The next day, the steps The reaction solution was incubated in the PCR instrument at 65 °C for 10 min to thermally inactivate the T4 DNA ligase and terminate the ligation reaction, yielding the ligation products of the vector pComb3Xss and the nanobody gene fragment.

[0122] Steps The completed reaction vector pComb3Xss and nanobody gene fragment ligation product were transferred to a 1.5 mL centrifuge tube, 20 μL of 3M sodium acetate (pH=5.2) was added, and then 660 μL of anhydrous ethanol was added.

[0123] Steps Centrifuge tubes were placed at -20 ℃ overnight.

[0124] The next day, the steps Centrifuge tubes containing the vector pComb3Xss and nanobody gene fragment ligation products were placed in a centrifuge and centrifuged at 4 ℃ and 12000 rpm for 30 min.

[0125] End of steps After centrifugation, a white DNA precipitate can be seen in the centrifuge tube. Discard the supernatant and add 75% ethanol to the centrifuge tube to resuspend the precipitate.

[0126] Steps The processed centrifuge tubes were placed in a centrifuge and centrifuged at 4 ℃ and 12000 rpm for 15 min.

[0127] Discard steps After centrifugation, place the centrifuge tube upside down on absorbent paper and let it stand for several minutes to allow any residual ethanol in the centrifuge tube to evaporate.

[0128] Add 30 μL of sterile water to reconstitute the precipitate, and store the purified vector pComb3Xss and nanobody gene fragment ligation product at -20 ℃ for later use.

[0129] 3. Electroporation of the ligation product of vector pComb3Xss and nanobody gene fragment

[0130] (1) Remove the Escherichia coli ER2738 electrotransformation competent cells from the -80 ℃ freezer and thaw them on ice;

[0131] (2) Using 25 μL of electrocompetent cells as one reaction, add 3 μL of the purified ligation product obtained in step 2 and incubate on ice for 10 min;

[0132] (3) The E. coli ER2738 competent cells that have been added with the ligation product in step (2) are quickly transferred to a pre-cooled electroporation cup and electroporated at 1.8 KV, 200 Ω, and 25 µF.

[0133] (4) Add 975 μL of preheated SOC medium at 37 °C within 10 seconds, resuspend the transformed cells and transfer them to a 50 mL centrifuge tube;

[0134] (5) Repeat steps (3) and (4) above 9 times, and transform all 30 μL of vector pComb3Xss and nanobody gene fragment ligation products from step 2 into Escherichia coli ER2738 competent cells.

[0135] (6) Place the competent cells transformed in step (5) in a shaker and revive them at 37 °C and 250 rpm for 1 hour;

[0136] (7) Take 2 µL of the culture solution from step (6) and dilute it 10 times. Then spread it on LB plates containing 50 µg / mL carbenicillin. Place the LB plates in a constant temperature incubator and incubate them upside down at 37 °C overnight.

[0137] (8) The next day, calculate the number of colonies on the plate after culturing in step (7), and calculate the library capacity of the original antibody library in combination with the dilution ratio;

[0138] (9) Randomly select 20 transformants from the plate after culturing in step (7), and culture them overnight in LB medium containing 50 µg / mL carbenicillin. Perform DNA sequencing on the cultured bacteria of the 20 transformants to analyze the diversity of inserted sequences.

[0139] (10) Add the remaining culture medium from step (6) to 200 mL of SB medium, add 200 µL of carbenicillin (50 mg / mL) and 200 µL of tetracycline (20 mg / mL), continue culturing for 2 hours, and then prepare the phage library.

[0140] 4. Construction of nanobody phage display library

[0141] (1) Add 1 mL of 1×10 to the bacterial culture after step 3 (10). 11 The helper phage M13KO7 (cfu / mL) was incubated at 37 °C for 30 min to infect Escherichia coli ER2738 containing nanobody genes.

[0142] (2) Place the bacterial culture after incubation in step (1) on a shaker at 37 ℃ and 250 rpm for 2 h;

[0143] (3) Add 200 µL of kanamycin (70 mg / mL) to the bacterial culture in step (2), and incubate overnight at 37 °C and 250 rpm on a shaker;

[0144] (4) The next day, transfer the bacterial culture from step (3) into a centrifuge bottle and centrifuge at 4 ℃ and 10000 rpm for 30 min;

[0145] (5) Collect the supernatant after centrifugation in step (4), add 50 mL of 5×PEG / NaCl solution, and let stand on ice for 2 h to precipitate the helper phage containing the nanobody gene;

[0146] (6) Centrifuge the supernatant after standing in step (5) at 4 ℃ and 10000 rpm for 30 min. A white phage precipitate will be visible. After removing the supernatant from the centrifuge tube, add 100 mL of PBS to wash and dissolve the white phage precipitate. Add 25 mL of 5×PEG / NaCl solution to reprecipitate. Continue to stand on ice for 2 h.

[0147] (7) Centrifuge the solution after standing in step (6) at 4℃ and 10000 rpm for 30 min. Reconstitute the white phage precipitate after centrifugation with 10 mL of sterile PBS containing 0.1% BSA. Sterilize the precipitate by passing it through a 0.22 µm filter and collect it back into a sterile centrifuge tube. Take 2 µL of the precipitate for titer determination. The result is the volume of the nanobody phage library. Store the remaining phage solution in a -80 ℃ refrigerator for later use.

[0148] Example 4: Screening of anti-Vibrio parahaemolyticus nanobodies

[0149] (1) Vibrio parahaemolyticus was diluted to 10⁻¹⁰ with CBS coating buffer according to different rounds. 9 10 8 10 7 Add 100 μL of cfu / mL solution to each well in a 96-well plate and coat the plate overnight at 4 °C.

[0150] (2) The next day, discard the coating solution, wash three times with PBST, add 3% skim milk powder at 300 μL / well, and incubate at 37 °C for 1 h to block;

[0151] (3) Discard the blocking solution, wash three times with PBST, add 100 μL / well to the phage solution (for phage display nanobody library) prepared in step (7) of Example 3, and incubate at 37 °C for 1 h;

[0152] (4) Aspirate the phage solution from the well after step (3) and vigorously blow it with PBST 10-20 times;

[0153] (5) Add 100 μL / well of 0.1 M glycine-hydrochloric acid (pH = 2.2) to the wells after step (4) treatment, and incubate at 37 °C for 15-30 min to wash away the phages bound to the well plate;

[0154] (6) Add 50 μL / well 1M Tris-HCl (pH=8.8) to the wells after step (5) and incubate at 37 °C for 15-30 min to neutralize the eluted phage solution;

[0155] (7) Aspirate all of the phage solution from step (6) into a centrifuge tube, take 20 μL to determine the titer, and add the remaining phage solution to 2 mL of freshly cultured Escherichia coli ER2738 bacterial suspension (OD200). 600 In a solution containing 0.6-0.8 g / mL, transfer to a 37 ℃ incubator and let stand for 30 min.

[0156] (8) Add 18 mL of LB solution containing 8 μl of carbenicillin (concentration of 20 mg / mL) to the bacterial culture infected by bacteriophage in step (7), and place it in a shaker at 37 ℃ for 1 h;

[0157] (9) Add 12 μL of carbenicillin (final concentration of carbenicillin 20 µg / mL) to the bacterial culture after step (8) and place it in a shaker at 37 ℃ for 1 h.

[0158] (10) Add 50 μL of helper phage (1×10⁻⁶) to the bacterial culture treated in step (9). 11 (cfu / mL), and incubated in a shaker at 37 ℃ for 2 h;

[0159] (11) Add 20 μl of kanamycin (final concentration of kanamycin 70 µg / mL) to the bacterial culture after step (10) and incubate overnight in a shaker at 37 ℃;

[0160] (12) The next day, centrifuge 10,000 g of overnight bacterial culture at 4℃ for 15 min, take the supernatant into a new centrifuge tube, add 5 mL of 5x PEG / NaCl solution, incubate on ice for 2 h, centrifuge 12,000 g at 4℃ for 15 min, reconstitute the phage pellet with 2 mL of sterile PBS containing 0.1% BSA, and then put it into the next round of panning.

[0161] According to the method for preparing the nano-antibody phage library described in steps (4), (5), (6), and (7) of Example 3, Table 10 shows the conditions for three rounds of phage panning.

[0162] Table 10

[0163]

[0164] Example 5: Screening of strongly positive nanoantibody strains

[0165] (1) Prepare a 96-well deep well plate and add 1 mL of SB medium containing 50 μg / mL carbenicillin to each well;

[0166] (2) From the phage titer determination plates after the first, second, and third rounds of screening, 32 single-clone colonies were randomly selected. Each selected single-clone colony was inoculated into a single well of a 96-well plate and then placed in a shaker at 37 ℃ and 250 rpm for several hours (until OD). 600 =0.8);

[0167] (3) Add 10 μL of 1×10⁻⁶ mol / L of the culture solution after step (2) to each well. 11CFU / mL helper phage (the helper phage is a phage without nanobody gene plasmid), incubated at 37 ℃ for 15 min to allow the phage to complete infection;

[0168] (4) Place the deep well plate after step (3) in a shaker at 37 ℃ and 250 rpm overnight for shaking culture;

[0169] (5) The next day, remove the deep well plate from the incubator and let the culture medium stand for later use;

[0170] (6) Dilute Vibrio parahaemolyticus to 10 with CBS buffer. 8 Add 100 μL of cfu / mL to each well of the microplate and incubate overnight at 4°C. Meanwhile, prepare another microplate with CBS coating buffer as a negative control.

[0171] (7) The next day, the coating solution was aspirated from the two microplates prepared in step (6), washed three times with PBST, and 3% skim milk powder was prepared. 270 μL / well was added to the two 96-well microplates and incubated at 37 °C for 1 h.

[0172] (8) Aspirate the blocking solution from the two microplates in step (7), wash three times with PBST, and then add 100 μL / well of the phage culture supernatant from the deep well plate in step (5) after standing. Incubate at 37 °C for 1 h.

[0173] (9) Aspirate the reaction solution from the two microplates in step (8), wash three times with PBST, and then add 100 μL / well of anti-M13-HRP enzyme-labeled antibody (diluted at a volume ratio of 1:2000) and incubate at 37°C for 1 h.

[0174] (10) Aspirate the reaction solution from the two microplates in step (9), wash five times with PBST, add 100 μL / well TMB colorimetric solution, and react at 37 °C for 15 min.

[0175] (11) Add 50 μL / well 2 M H2SO4 to the two ELISA plates after color development in step (10) to stop the reaction, and read the OD in an ELISA reader. 450 The absorbance values ​​are shown in the attached diagram. Figure 1 Initial screening of positive clones.

[0176] Example 6: Prokaryotic expression and purification of anti-Vibrio parahaemolyticus nanobodies

[0177] 1. Prokaryotic expression of nanobodies

[0178] (1) The plasmids of positive phages were extracted using the EZNA® Plasmid DNA Mini Kit and introduced into E. coli TOP10F' by heat shock method.

[0179] The specific steps are as follows: Take out the frozen E. coli TOP10F' competent cells from -80 ℃ and immediately place them on ice for 5 min. Then add 1 μL of the positivist plasmid to each tube of competent cells and continue to place them on ice for 10 min. Subsequently, place the competent cells in a 42 ℃ water bath for 90 s and immediately place them back on ice to cool for several minutes. Add 1 mL of SB medium to each tube of competent cells and culture at 37 ℃ and 250 rpm for 1 h. Finally, take 100 μL of the culture medium from each tube and spread it on a carbenicillin-resistant plate and incubate overnight at 37 ℃.

[0180] (2) Pick a single positive clone from the plate after culture in step (1) and inoculate it into 5 mL of culture medium containing 50 μg / mL SB. Incubate overnight at 37 ℃ and 250 rpm.

[0181] (3) The next day, the bacterial culture from step (2) was transferred to 200 mL of SB culture medium containing 50 μg / mL at a volume fraction of 1%, and cultured at 37°C and 250 rpm for several hours until OD. 600 = 0.8;

[0182] (4) Add IPTG to the bacterial culture after step (3) to make the final concentration 0.5-1 mM, and place it in a shaker at 30 ℃ and 250 rpm overnight;

[0183] (5) Collect the bacterial culture from step (4) overnight into a centrifuge bottle and centrifuge at 4 ℃, 8000 g for 6 min;

[0184] (6) Remove the supernatant after centrifugation in step (5), weigh the bacterial cells, add 10 mL of bacterial lysis buffer, repeatedly blow and aspirate to resuspend the bacterial cells, rotate and invert at room temperature for 15 min to fully lyse the bacteria;

[0185] (7) Centrifuge the bacterial lysate from step (6) at 4 °C and 13000 g for 10 min;

[0186] (8) Collect the supernatant from step (7), which is a soluble protein, for subsequent purification.

[0187] 2. Purification of nanobodies

[0188] (1) Take 1 mL of Ni-NTA matrix from the nickel column and transfer it to a centrifuge tube. Repeat centrifugation 3 times and replace the preservation solution with PBS. Add it to the soluble protein in step 1 (8) and mix by rotating and inverting at room temperature for 1 h.

[0189] (2) Fix the empty purification column vertically, add the reaction solution from step (1) into the empty purification column, collect the sample flow-through, and leave the nickel column on the purification column;

[0190] (3) Add 5 column volumes of equilibration buffer (the equilibration buffer is PBS containing 2 mM imidazole) to the nickel column of step (2) to elute impurities;

[0191] (4) Add 5 column volumes of elution buffer (PBS containing 200 mM imidazole) to the nickel column from step (3), collect the elution buffer in separate tubes until the elution buffer reaches the A column. 280 The signal value at that time is close to 0;

[0192] (5) Collect the eluent obtained in step (4), which contains purified nanobodies, and verify it by SDS-PAGE electrophoresis. See [reference needed]. Figure 2 In the figure: M is a protein standard; 1 is the nanobody expressed by SEQ ID NO.1; 2 is the nanobody expressed by SEQ ID NO.2; 3 is the nanobody expressed by SEQ ID NO.3; 4 is the nanobody expressed by SEQ ID NO.4; and 5 is the nanobody expressed by SEQ ID NO.5.

[0193] in:

[0194] The amino acid sequence of SEQ ID NO.1 is as follows:

[0195] QVQLVESGGGLVQPGGSLRLSCAASGFTFS~NYAMY~WVRQAPGKGLEWIS~TIDSRGGKTLYADSVRG~RFTISRDNANSTLYLQLNSLKIEDTAMYYC~GKGPGTAALKILPHVA~PGQGTLVTVS.

[0196] The amino acid sequence of SEQ ID NO.2 is as follows:

[0197] QVQLVESGGGSVQSGGSLRLSCAASGDTYR~RTCMA~WFRQAPGKEREGVA~ELDSSGSPSYADSVKG~RFTISQDSTKNTLYLQMNSLMPEDTAMYYC~AADACPLYRRTNRYAF~WGQGTQVTVS;

[0198] The amino acid sequence of SEQ ID NO.3 is as follows:

[0199] QVQLVESGGGSVPAGGSLRLSCVASGSTFT~PYCMG~WFRQAPGKEREGVA~SIVTFRGTTSYADSVQG~RFTISQDNAKNTLYLQMNSLNPEDTAMYYC~AAGYSRSSCANFPI~WGQGTQVTVS;

[0200] The amino acid sequence of SEQ ID NO.4 is as follows:

[0201] EVQLVESGGGAVQSGGSLRLSCVASGYTQR~RCRMA~WFRQVPGKEREGVA~SISSGGSPRYTDSVKG~RFTISQDNAQNTLYLQMNSLKPEDTAMYFC~AAFRAAGYCSPMTSVTALGQHQYND~WGQGTQVTVS;

[0202] The amino acid sequence of SEQ ID NO.5 is as follows:

[0203] QVQLVESGGGSVQSGGSLRLSCAASGTTYT~SYCMG~WFRQAPGKEREGVA~VIATFGNTAYADSVKG~RFTISQDNAKTTLYLEMNSLRPEDTAMYYC~AADWRSYCWRTGAGALSVRGFGQ~WGQGTQVTVS;

[0204] (6) Collect and combine the purified nanobody solution obtained in step (4) and put it into a dialysis bag. Dialyze it in PBS at 4 °C for three days, changing the solution twice a day to replace the nanobody dissolution medium.

[0205] Application Example 7: Application of Nanobodies

[0206] 1. Establishment of the standard curve for double-antibody sandwich ELISA

[0207] (1) Dilute the purified rabbit polyclonal antibody with CBS at 2 μg / ml, add 100 µL / well to a 96-well microplate, and coat overnight at 4 ℃;

[0208] (2) The next day, the coating buffer solution in the well plate was aspirated and the plate was washed three times with PBST;

[0209] (3) Add 270 μL / well of 3% blocking solution, incubate at 37 °C for 1 h, then remove the blocking solution and wash the plate three times with PBST;

[0210] (4) Vibrio parahaemolyticus from 10 8 After serially diluting the cfu / mL concentrations, add 100 µL / well to the microplate treated in step (3) and incubate at 37 °C for 1 h.

[0211] (5) Aspirate the liquid from the wells of the ELISA plate after step (4) and wash the plate three times with PBST;

[0212] (6) The nanobodies prepared in Example 6 (the nanobodies expressed by SEQ ID NO.1; SEQ ID NO.2; SEQ ID NO.3; SEQ ID NO.4; and SEQ ID NO.5) were diluted to 1 μg / mL, and then added to the enzyme-labeled plate after step (5) at 100 µL / well for reaction and incubated at 37 °C for 1 h.

[0213] (7) Aspirate the liquid from the wells of the ELISA plate in step (6) and wash the plate three times with PBST;

[0214] (8) Dilute the HRP-labeled Anti-HA mouse monoclonal antibody at a volume ratio of 1:10000 and add 100 μL / well to the well of the enzyme-labeled plate treated in step (7) for reaction, and incubate at 37 °C for 1 h;

[0215] (9) Aspirate the liquid from the wells of the ELISA plate in step (8) and wash the plate five times with PBST;

[0216] (10) Add 100 μL / well of TMB substrate solution to the microplate after step (9) and react at 37 °C in the dark for 10 min;

[0217] (11) Add 50 μL / well 2 M H2SO4 to the chromogenic solution in the microplate of step (10) to terminate the reaction;

[0218] (12) Place the ELISA plate in the ELISA reader and read the OD. 450 ;

[0219] (13) Plot a standard curve with Vibrio parahaemolyticus concentration (log) on ​​the x-axis and absorbance on the y-axis. (Refer to the standard curve.) Figure 3 .

[0220] pass Figure 3The ELISA detection standard curve shows that the anti-Vibrio parahaemolyticus nanobody provided in this application can effectively bind to Vibrio parahaemolyticus, and within a certain concentration range, the absorbance shows a good linear relationship with the bacterial concentration, thus specifically achieving the detection of anti-Vibrio parahaemolyticus.

Claims

1. A nanobody for detecting Vibrio parahaemolyticus, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.

5.

2. The nanobody for detecting Vibrio parahaemolyticus according to claim 1, characterized in that, The strain of Vibrio parahaemolyticus mentioned is ATCC 17802.

3. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 1, characterized in that, The steps are as follows: 1) Collect blood samples from immunized camels, isolate and collect peripheral blood lymphocytes from camels, extract total RNA from anti-Vibrio parahaemolyticus nanoantibodies from camel peripheral blood lymphocytes, and synthesize cDNA. Using cDNA as a template, the heavy chain variable region gene was amplified. The amplified camel antibody heavy chain variable region gene was ligated with a phage vector and transformed into competent Escherichia coli to construct an immune camel nanoantibody phage library. 2) Using Vibrio parahaemolyticus as the coating antigen, nanobody phages that can specifically recognize Vibrio parahaemolyticus were obtained through three or more rounds of panning using the pH elution method, and the genes of the nanobody were compared by sequencing analysis. 3) The phage vector containing the anti-Vibrio parahaemolyticus nanobody gene was extracted and reintroduced into the host expression bacteria for nanobody expression and purification.

4. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 3, characterized in that, The total RNA was extracted according to the instructions for the TRIzol™ reagent.

5. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 3, characterized in that, The cDNA synthesis involves using the extracted RNA as a template and reverse transcription via the SuperScript™ IV first-strand synthesis system to synthesize cDNA encoding the antibody gene.

6. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 3, characterized in that, A two-step amplification method was used. The first step used upstream primer Call001 and downstream primer Call002, and the second step used upstream primer 2ndfor and downstream primer 2ndreverse to amplify the heavy chain variable region gene. The upstream primer Call001 nucleotide is shown in Seq ID NO: 6; The downstream primer Call002 nucleotide is shown in Seq ID NO: 7; The upstream primer 2nd for nucleotides is shown in Seq ID NO: 8; The downstream primer 2nd reverse nucleotide is shown in Seq ID NO:

9.

7. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 3, characterized in that, The heavy chain variable region gene is ligated to the phage vector by using T4 ligase to ligate the enzyme-digested nanobody gene fragment to the enzyme-digested vector pComb3xss.

8. The method for preparing nanobodies for detecting Vibrio parahaemolyticus according to claim 3, characterized in that, The phage vector containing the anti-Vibrio parahaemolyticus nanobody gene was extracted using the EZNA® Plasmid DNA MiniKit to extract plasmids from positive phages, and then introduced into E. coli TOP10F' by heat shock.

9. The application of the nanobody for detecting Vibrio parahaemolyticus as described in claim 1 in the preparation of reagents or kits for detecting Vibrio parahaemolyticus.

Images