Bivalent anti-Vibrio parahaemolyticus nanobody, preparation method, self-sandwich ELISA kit, and detection method.

Abstract

This invention discloses a bivalent anti-Vibrio parahaemolyticus nanobody, its preparation method, a self-sandwich ELISA kit, and a detection method. The aim is to provide a bivalent anti-Vibrio parahaemolyticus nanobody, kit, and detection method with high accuracy, specificity, and sensitivity for detecting Vibrio parahaemolyticus. The amino acid sequence of the bivalent anti-Vibrio parahaemolyticus nanobody is shown in SEQ ID NO.7. This invention belongs to the field of biodetection technology.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a self-sandwich ELISA method for detecting bivalent anti-Vibrio parahaemolyticus nanobodies; this invention also relates to a method for preparing the bivalent anti-Vibrio parahaemolyticus nanobodies. Background Technology

[0002] Vibrio parahaemolyticus (V. parahaemolyticus) is a significant foodborne pathogen and the leading cause of seafood-related bacterial gastroenteritis worldwide. This bacterium is widely distributed in marine and estuarine environments, naturally colonizing aquatic animals such as shrimp, oysters, and crabs. Human infection typically results from consuming contaminated raw or undercooked seafood, or from cross-contamination during food preparation. While infection usually presents as acute diarrhea, abdominal pain, and vomiting, it can develop into life-threatening complications in susceptible individuals. In recent years, driven by rising global sea surface temperatures and the expansion of international seafood trade, the incidence of Vibrio parahaemolyticus infection has surged globally, posing an increasingly serious threat to public health and imposing a significant economic burden.

[0003] Establishing rapid, sensitive, and reliable detection methods for Vibrio parahaemolyticus is crucial for ensuring food supply, safeguarding public health, and reducing economic costs. Among numerous detection strategies, immunoassay methods, particularly enzyme-linked immunosorbent assay (ELISA), have attracted significant attention due to their unique advantages such as high throughput, low operating costs, wide applicability, and high safety. However, traditional immunoassay methods still suffer from limitations such as insufficient sensitivity when detecting trace pathogens.

[0004] The performance of immunoassays fundamentally depends on antibody quality. In recent years, anti-Vibrio parahaemolyticus nanobodies derived from the variable region of camelid heavy chain antibodies have emerged as highly promising immunorecognition elements. Compared to traditional IgG, nanobodies not only possess advantages such as small molecular weight, high physicochemical stability, and ease of genetic engineering, but their extended CDR3 rings can also effectively recognize occult or deeply embedded antigenic epitopes, thereby significantly improving binding affinity and specificity. However, their small size also presents a challenge: a large portion of the active site of nanobodies is exposed on the surface, amplifying the influence of spatial orientation. When used as capture antibodies, nanobodies are typically passively adsorbed onto physical surfaces, which often leads to steric hindrance at the antigen-binding sites, significantly reducing capture efficiency.

[0005] To overcome this limitation, researchers explored various nanobody immobilization strategies. First, the streptavidin-biotin bridging system utilizes streptavidin-coated microplates to capture biotinylated nanobodies, ensuring proper spatial orientation. However, this method relies on additional biotinylation and bridging steps, which not only complicates the detection process but may also impair nanobody activity. Furthermore, the need for pre-coated plates further increases the overall detection cost.

[0006] Another strategy involves the use of biological scaffolds. However, the introduction of biological agents (such as inactivated bacteria or bacteriophages) inevitably raises potential biosafety concerns and poses significant challenges to the reproducibility and standardization of detection.

[0007] Furthermore, multimerization has proven to be an effective strategy for improving binding affinity by significantly expanding the effective spatial coverage of the binding domain. However, fusion- or multimer-based constructs are inherently more complex in terms of protein design, recombinant expression, purification, and quality control, and may also involve additional compromises in analytical performance.

[0008] Overall, while these complex strategies partially mitigate the steric hindrance and reduced capture capacity associated with passive nanobody immobilization, they generally come at the cost of analytical simplicity, introducing cumbersome procedures, increased costs, and potential loss of nanobody activity. In stark contrast, methods for integrating bivalent nanobodies into sandwich ELISA modalities for Vibrio parahaemolyticus detection remain largely unexplored. Specifically, whether bivalent nanobodies can fundamentally improve analytical performance under passive immobilization conditions without requiring complex pretreatment or bridging strategies has not yet been systematically investigated.

[0009] Therefore, there is an urgent need to develop a simple and robust anti-Vibrio parahaemolyticus nanobody capture strategy that does not require bridging, so as to maintain high capture efficiency under conventional passive immobilization, thereby taking into account both ease of operation and wide applicability. Summary of the Invention

[0010] The purpose of this invention is to provide a bivalent anti-Vibrio parahaemolyticus nanobody for detecting Vibrio parahaemolyticus. This bivalent anti-Vibrio parahaemolyticus nanobody has good antigen binding ability and stability. By using biotin-labeled bivalent anti-Vibrio parahaemolyticus nanobody, the accuracy, specificity and sensitivity of Vibrio parahaemolyticus detection can be significantly improved.

[0011] Another objective of this invention is to provide a method for preparing the above-mentioned bivalent anti-Vibrio parahaemolyticus nanobody for detecting Vibrio parahaemolyticus. The method involves steps such as PCR amplification, overlapping PCR splicing, vector ligation, induced expression, and purification to finally obtain a bivalent anti-Vibrio parahaemolyticus nanobody with high detection capability.

[0012] Another objective of this invention is to provide a kit containing the above-mentioned biotin-labeled bivalent anti-Vibrio parahaemolyticus nanoantibody and its detection method, which has high detection accuracy, strong specificity, and high sensitivity.

[0013] Therefore, the first technical solution provided by this invention is as follows:

[0014] A bivalent anti-Vibrio parahaemolyticus nanobody, the amino acid sequence of which is shown in SEQ ID NO.7.

[0015] Furthermore, the preparation method of the above-mentioned bivalent anti-Vibrio parahaemolyticus nanobody includes the following steps in sequence:

[0016] (1) The gene sequence of monovalent anti-Vibrio parahaemolyticus nanobody V1 was amplified by PCR using upstream primer F1 and downstream primer R1 to obtain hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1; the gene sequence of monovalent anti-Vibrio parahaemolyticus nanobody V2 was amplified by PCR using upstream primer F2 and downstream primer R2 to obtain hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2;

[0017] The amino acid sequence of the monovalent anti-Vibrio parahaemolyticus nanobody V1 is shown in SEQ ID NO.1;

[0018] The amino acid sequence of the monovalent anti-Vibrio parahaemolyticus nanobody V2 is as shown in SEQ ID NO.2;

[0019] The upstream primer F1 nucleotide is shown in SEQ ID NO.3;

[0020] The downstream primer R1 nucleotide is shown in SEQ ID NO.4;

[0021] The upstream primer F2 nucleotide is shown in SEQ ID NO. 5;

[0022] The downstream primer R2 nucleotide is shown in SEQ ID NO. 6;

[0023] (2) The hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1 and hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2 prepared in step (1) were amplified by overlap PCR and spliced ​​to obtain bivalent anti-Vibrio parahaemolyticus nanobody gene fragments V1-V2; the bivalent V1-V2 gene fragments obtained by overlap PCR and the vector were recovered by double enzyme digestion, and then the digested V1-V2 fragments and the vector were ligated with ligase to obtain recombinant plasmids containing bivalent anti-Vibrio parahaemolyticus nanobody V1-V2 genes; the constructed recombinant plasmids were transformed into expression strains, and after induction of expression, the bivalent anti-Vibrio parahaemolyticus nanobody was purified to obtain bivalent anti-Vibrio parahaemolyticus nanobody.

[0024] Preferably, the carrier is the pET-22b carrier.

[0025] Preferably, the ligase is a T4 ligase.

[0026] Preferably, the biotin is EZ-Link™ Sulfo-NHS-LC-LC-Biotin powder.

[0027] The second technical solution provided by the present invention is a self-sandwich ELISA kit based on a bivalent anti-Vibrio parahaemolyticus nanobody, comprising a capture antibody and a detection antibody. The capture antibody is the bivalent anti-Vibrio parahaemolyticus nanobody described in the first technical solution; the detection antibody is a biotinylated bivalent anti-Vibrio parahaemolyticus nanobody.

[0028] Furthermore, in the aforementioned self-sandwich ELISA kit based on bivalent anti-Vibrio parahaemolyticus nanobodies, the biotin is EZ-Link™ Sulfo-NHS-LC-LC-Biotin powder.

[0029] Another technical solution of the present invention provides a method for detecting Vibrio parahaemolyticus. The method involves immobilizing the bivalent anti-Vibrio parahaemolyticus nanobody described in the first technical solution onto an ELISA plate as a capture antibody. After blocking with skim milk powder, serially diluted Vibrio parahaemolyticus is used as the antigen. Then, the biotinylated bivalent anti-Vibrio parahaemolyticus nanobody is used as the detection antibody. After amplification of the streptavidin-polymerized horseradish peroxidase signal, the plate is developed using TMB, and finally, the OD value is read using an ELISA reader. 450 The value was calculated based on the standard curve to determine the content of Vibrio parahaemolyticus in the sample.

[0030] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0031] 1. The bivalent anti-Vibrio parahaemolyticus nanobody prepared in this invention significantly improves capture efficiency. In traditional monovalent nanobody detection, due to the exposure of their active sites, passive adsorption on physical surfaces easily leads to steric hindrance, resulting in low capture efficiency. However, the bivalent anti-Vibrio parahaemolyticus nanobody of this invention can bind better to Vibrio parahaemolyticus, greatly improving detection accuracy.

[0032] 2. The bivalent anti-Vibrio parahaemolyticus nanobody detection method of the present invention is simple to operate, requires no complex pretreatment or bridging strategies, reduces the difficulty and cost of operation, and ensures the sensitivity and specificity of detection.

[0033] 3. The self-sandwich ELISA kit of the present invention can accurately and sensitively detect Vibrio parahaemolyticus.

[0034] 4. The detection method of the present invention utilizes biotin-labeled bivalent anti-Vibrio parahaemolyticus nanobody and streptavidin-polymerized horseradish peroxidase signal amplification technology to detect lower concentrations of Vibrio parahaemolyticus. Attached Figure Description

[0035] Figure 1 These are electrophoresis images of the genes for hinged monovalent anti-Vibrio parahaemolyticus nanobodies V-1 and V-2 obtained after PCR amplification, and the gene for the PCR-amplified bivalent anti-Vibrio parahaemolyticus nanobodies.

[0036] Figure 2 The expression of nanobodies V1, V2 and bivalent anti-Vibrio parahaemolyticus nanobodies V1-V2 was analyzed by SDS-PAGE.

[0037] Figure 3 This is a sandwich ELISA standard curve based on bivalent anti-Vibrio parahaemolyticus nanobodies for the detection of Vibrio parahaemolyticus. Detailed Implementation

[0038] 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.

[0039] The raw materials used in the following schemes are sourced from the following sources:

[0040] Vibrio parahaemolyticus was purchased from Shandong Huankai Biotechnology Co., Ltd., strain ATCC 17802;

[0041] Streptavidin-polymerized horseradish peroxidase was purchased from Fitzgerald Industries International, Inc. (Acton, Massachusetts, USA) (lot number C21071404).

[0042] Example 1: Amplification of the monovalent anti-Vibrio parahaemolyticus gene

[0043] (1) Amplification of the gene of the variable region of the heavy chain of monovalent anti-Vibrio parahaemolyticus nanobody

[0044] (1) Based on the gene sequence of the variable region of the heavy chain of the monovalent anti-Vibrio parahaemolyticus nanobody V1 (SEQ ID NO.1) and the gene sequence of the monovalent anti-Vibrio parahaemolyticus nanobody V2 (SEQ ID NO.2), specific primers were designed. The primer sequences were synthesized by Platinum Biotech (Shanghai) Co., Ltd., and their sequences are shown in Table 1. The underline in the F1 sequence indicates the EcoRI restriction site, and the underline in the R2 sequence indicates the HindIII restriction site.

[0045] Table 1

[0046] Primer name Sequence (5'-3') F1 (SEQ ID NO.3) <![CDATA[C GAATTC TCACAAAGTGCAGGTGCAGCTGGTGGAGTC]]> R1 (SEQ ID NO.4) CAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCTGGGTCCCC F2 (SEQ ID NO.5) TTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTGCAGCTCGTGGAGTC R2 (SEQ ID NO.6) <![CDATA[C AAGCTT CTGGCCGGCCTG]]>

[0047] (2) First, the gene sequence of the variable region of the heavy chain of the monovalent anti-Vibrio parahaemolyticus nanobody V1 was amplified by PCR using upstream primer F1 and downstream primer R1 to obtain the hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1. The reaction system is shown in Table 2-1.

[0048] Table 2-1

[0049] reagents Dosage TaKaRa PrimeSTAR Max Ver.2 Premix (2x) 25 μL 10 μM F1 1 μL 10 μM R1 1 μL Monovalent anti-Vibrio parahaemolyticus nanobody V1 250 ng Sterilized water Add to 50 μL

[0050] The monovalent anti-Vibrio parahaemolyticus nanobody V1 is the monovalent anti-Vibrio parahaemolyticus nanobody expressed by SEQ ID NO.1 code disclosed in the applicant's earlier application ZL202512023506.5, application publication number CN121736095A.

[0051] The amino acid sequence shown in SEQ ID NO.1 is as follows:

[0052] QVQLVESGGGLVQPGGSLRLSCAASGFTFS~NYAMY~WVRQAPGKGLEWIS~TIDSRGGKTLYADSVRG~RFTISRDNANSTLYLQLNSLKIEDTAMYYC~GKGPGTAALKILPHVA~PGQGTLVTVS

[0053] Next, the gene sequence of the variable region of the heavy chain of the monovalent anti-Vibrio parahaemolyticus nanobody V2 was amplified by PCR using upstream primer F2 and downstream primer R2 to obtain the hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2. The reaction system is shown in Table 2-2.

[0054] Table 2-2

[0055] reagents Dosage TaKaRa PrimeSTAR Max Ver.2 Premix (2x) 25 μL 10 μM F2 1 μL 10 μM R2 1 μL Monovalent anti-Vibrio parahaemolyticus nanobody V2 250ng Sterilized water Add to 50 μL

[0056] The monovalent anti-Vibrio parahaemolyticus nanobody V2 is the monovalent anti-Vibrio parahaemolyticus nanobody expressed by SEQ ID NO.2 code disclosed in the applicant's earlier application ZL202512023506.5, application publication number CN121736095A.

[0057] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0058] QVQLVESGGGSVQSGGSLRLSCAASGDTYR~RTCMA~WFRQAPGKEREGVA~ELDSSGSPSYADSVKG~RFTISQDSTKNTLYLQMNSLMPEDTAMYYC~AADACPLYRRTNRYAF~WGQGTQVTVS

[0059] Set the following conditions in the PCR instrument, as shown in Table 3:

[0060] Table 3

[0061]

[0062] The PCR product was verified by 1% agarose gel electrophoresis, and the results showed a band at approximately 400 bp. Figure 1 Where M stands for maker; V-1 represents hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1; V-2 represents hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2. PCR products were recovered using the EZNA® Gel Extraction Kit, and the concentration and purity of the recovered DNA were measured and stored at -20 ℃ for later use.

[0063] (2) Agarose gel electrophoresis

[0064] Weigh 0.3 g of agarose and dissolve it in 30 mL of 1×TAE buffer to prepare a 1% agarose gel.

[0065] Microwave until the agarose is completely dissolved, forming a colorless and transparent gel solution;

[0066] Once the gel solution has cooled to approximately 50 °C, add 2 μL of nucleic acid dye and mix thoroughly.

[0067] Steps The prepared gel solution is poured into a gel casting tank and cooled and solidified at room temperature;

[0068] Remove the comb and add 6× loading buffer to the PCR product from step (1) into the wells of the fully solidified agarose gel.

[0069] Place the gel in the electrophoresis tank and add 1×TAE buffer, then turn on the electrophoresis apparatus and run it at 150 V for 10 min;

[0070] Remove the gel and observe the results under a UV lamp.

[0071] Example 2: Construction of bivalent anti-Vibrio parahaemolyticus nanobodies by overlap PCR

[0072] In this experiment, bivalent anti-Vibrio parahaemolyticus nanoantibodies V1-V2 were constructed in the form of V1-Linker-V2. The V1 and V2 genes obtained after the above PCR were linked together using overlap PCR. The reaction system is shown in Table 2-1.

[0073] Table 4

[0074] reagents Dosage TaKaRa PrimeSTAR Max Ver.2 Premix (2x) 25 μL 10 μM F1 1 μL 10 μM R2 1 μL Hinge-linked anti-Vibrio parahaemolyticus nanobody V-1 prepared in Example 1 150 ng Hinge-linked anti-Vibrio parahaemolyticus nanobody V-2 prepared in Example 1 150 ng Sterilized water Add to 50 μL

[0075] Set the conditions in the PCR instrument as shown in Table 3.

[0076] After the reaction was completed, the product was detected by 1% agarose gel electrophoresis, and the results showed a band at approximately 750 bp. Figure 1 (M stands for maker; the bivalent anti-Vibrio parahaemolyticus nanobodies amplified by PCR are V1-V2.) The PCR products were recovered using the EZNA® Gel Extraction Kit, and the concentration and purity of the recovered DNA were measured and stored at -20 ℃ for later use.

[0077] Example 3: Construction of recombinant plasmid of pET-22b(+)-bivalent anti-Vibrio parahaemolyticus nanobody

[0078] 1. Enzyme digestion reaction of vector pET-22b(+) and bivalent anti-Vibrio parahaemolyticus nanobody gene fragment

[0079] Since the bivalent anti-Vibrio parahaemolyticus nanobody gene fragment obtained in Example 2 has EcoRI and HindIII restriction enzyme sites introduced at both ends of the target gene, it can be directly digested and recombined with the pET-22b(+) vector and ligated with T4 ligase.

[0080] The process is briefly described as follows:

[0081] (1) Enzyme digestion of vector pET-22b(+)

[0082] Add the reagents shown in Table 5 to the PCR tube:

[0083] Table 5

[0084] reagents Dosage 10× Cut Smart Buffer 5 μL pET-22b(+) 1 μg EcoRI enzyme 1 μL HindIII enzyme 1 μL Sterilized water Add to 50 μL

[0085] Place in a PCR instrument and incubate overnight at 37 °C.

[0086] Add 22.5 μL of 0.5M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating EcoRI and HindIII enzymes.

[0087] The enzyme digestion effect was detected by 0.7% agarose gel electrophoresis of the enzyme digestion products.

[0088] Recover the enzyme digestion products.

[0089] (2) Enzyme digestion of the bivalent anti-Vibrio parahaemolyticus nanobody gene fragment

[0090] Add the reagents shown in Table 6 to the PCR tube:

[0091] Table 6

[0092] reagents Dosage 10× Cut Smart Buffer 5 μL Bivalent anti-Vibrio parahaemolyticus nanobody gene fragment 1 μg EcoRI enzyme 1 μL HindIII enzyme 1 μL Sterilized water Add to 50 μL

[0093] Place in a PCR instrument and incubate overnight at 37 °C.

[0094] Add 22.5 μL of 0.5M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating EcoRI and HindIII enzymes.

[0095] The EZNA® Cycle Pure Kit recovers enzyme digestion products.

[0096] 1. Ligation and expression of vector pET-22b(+) and bivalent anti-Vibrio parahaemolyticus nanobody gene fragment

[0097] (1) Ligation of vector pET-22b(+) and bivalent anti-Vibrio parahaemolyticus nanobody gene fragment

[0098] Add the reagents shown in Table 7 to the PCR tube:

[0099] Table 7

[0100] reagents Dosage 10 × T4 Ligase Buffer 2 μL pET-22b(+) vector digested with enzymes 120 ng Enzyme-digested bivalent anti-Vibrio parahaemolyticus nanobody gene fragment 60 ng T4 DNA ligase 2 μL Sterilized water Add to 20 μL

[0101] Steps Place the PCR tubes containing the reaction solution in a PCR instrument and incubate overnight at 16°C.

[0102] The next day, the steps The reaction solution was incubated in a PCR instrument at 65 °C for 10 min to thermally inactivate the T4 DNA ligase, thus terminating the ligation reaction and obtaining the ligation product of the vector pET-22b(+) and the bivalent anti-Vibrio parahaemolyticus nanobody gene fragment. It was then stored at -20 °C for later use.

[0103] (2) Expression of recombinant plasmids

[0104] Take 10 μL of the ligation product and introduce it into E. coli DH5α competent cells using a heat shock method.

[0105] The specific steps are as follows: Take 100 μL of frozen *E. coli* DH5α competent cells from -80 °C and immediately place them on ice for 5 min. Then, add 10 μL of ligation product to each tube of competent cells and continue to place them on ice for 10 min. Next, place the competent cells in a 42 °C water bath for 90 s and immediately return them to ice to cool for several minutes. Add 1 mL of SB medium to each tube of competent cells and incubate at 37 °C and 250 rpm for 1 h with shaking. Finally, take 100 μL of the culture medium from each tube and spread it onto a carbenicillin-resistant plate. Incubate overnight at 37 °C.

[0106] In the steps Twenty transformants were randomly selected from the cultured plates and cultured overnight in LB medium containing 50 µg / mL ampicillin. The DNA of the cultured bacteria of these 20 transformants was sequenced, and the successfully transformed transformants were selected.

[0107] Steps The plasmids of the successfully transformed transformants were introduced into Escherichia coli BL21 via heat shock.

[0108] The specific steps are as follows: Remove 30 μL of frozen *E. coli* BL21 cells from -80 °C and immediately place them on ice for 5 min. Add 1 μL of plasmid to each cell tube and continue placing on ice for 10 min. Then, incubate the cells in a 42 °C water bath for 90 s and immediately return them to ice to cool for several minutes. Add 1 mL of SB medium to each cell tube and incubate at 37 °C and 250 rpm for 1 h with shaking. Finally, transfer 100 μL of the culture medium from each tube to a carbenicillin-resistant plate and incubate overnight at 37 °C.

[0109] Picking steps Positive single colonies on the culture plate were inoculated into 5 mL test tubes containing 50 μg / mL SB culture medium and incubated overnight at 37°C and 250 rpm.

[0110] The following day, the inoculum was prepared according to a 1% volume fraction. The overnight culture was transferred to a 100 mL test tube containing 50 μg / mL SB medium and incubated at 37°C and 250 rpm for several hours until OD reached. 600 = 0.8.

[0111] ⑥ To the step After culturing, IPTG was added to the bacterial culture to a final concentration of 0.5 mM, and the culture was placed in a shaker at 16°C and 250 rpm overnight.

[0112] ⑦ Collect the bacterial culture from step ⑥ that has been cultured overnight into a centrifuge bottle and centrifuge at 4 ℃ and 8000 g for 6 min.

[0113] ⑧ Remove the supernatant from step ⑦ after centrifugation, weigh the bacterial cells, add 10 mL of bacterial lysis buffer, repeatedly pipette and aspirate to resuspend the bacterial cells, and rotate and invert at room temperature for 30 min to fully lyse the bacteria;

[0114] ⑨ Centrifuge the bacterial lysate from step ⑧ at 4°C and 16000 g for 10 min.

[0115] ⑩ Collect the supernatant from step ⑨, which contains soluble protein, for subsequent purification.

[0116] (3) Purification of bivalent anti-Vibrio parahaemolyticus nanobodies

[0117] Take 1 mL of the Ni-NTA matrix from the nickel column, transfer it to a centrifuge tube, repeat the centrifugation 3 times, replace the preservation solution with PBS, add it to the soluble protein prepared in step (2), and mix by rotating and inverting at room temperature for 1 h.

[0118] Fix the empty purification column vertically and add the following steps into the empty purification column. The reaction solution was collected, and the sample flow-through was left on the purification column, leaving the nickel column.

[0119] To Steps Add 5 column volumes of equilibration buffer (PBS containing 2 mM imidazole) to the nickel column to elute impurities.

[0120] To Steps Add 5 column volumes of elution buffer (PBS containing 200 mM imidazole) to the nickel column, collect the eluent in separate tubes until the eluent reaches column A. 280The signal value at that time is close to 0.

[0121] Steps The collected eluent contained purified bivalent anti-Vibrio parahaemolyticus nanobodies, as verified by SDS-PAGE electrophoresis. (See attached document). Figure 2 In the figure: M is the protein standard; V1 is the monovalent anti-Vibrio parahaemolyticus nanobody V1 (amino acid sequence as shown in SEQ ID NO.1); V2 is the monovalent anti-Vibrio parahaemolyticus nanobody V2 (amino acid sequence as shown in SEQ ID NO.2); V1-V2 are the bivalent anti-Vibrio parahaemolyticus nanobodies V1-V2.

[0122] The amino acid sequences of the bivalent anti-Vibrio parahaemolyticus nanobodies V1-V2 are shown in SEQ ID NO.7:

[0123] The amino acid sequence shown in SEQ ID NO.7 is as follows:

[0124] QVQLVESGGGLVQPGGSLRLSCAASGFTFS~NYAMY~WVRQAPGKGLEWIS~TIDSRGGKTLYADSVRG~RFTISRDNANSTLYLQLNSLKIEDTAMYYC~GKGPGTAALKILPHVA~PGQGTQVTVS~SGGGGSG GGGSGGGGS~QVQLVESGGGSVQSGGSLRLSCAASGDTYR~RTCMA~WFRQAPGKEREGVA~ELDSSGSPSYADSVKG~RFTISQDSTKNTLYLQMNSLMPEDTAMYYC~AADACPLYRRTNRYAF~WGQGTQVTVS

[0125] ⑥ Step The purified bivalent anti-Vibrio parahaemolyticus nanobody V1-V2 solutions were collected and placed in dialysis bags. Dialysis was performed with PBS at 4 °C for two days, with the solution changed twice a day. The dissolution medium for bivalent anti-Vibrio parahaemolyticus nanobody V1-V2 was changed. The concentration and purity of the bivalent anti-Vibrio parahaemolyticus nanobody were measured after dialysis. The solutions were stored at -20 °C for later use.

[0126] Example 4: Biotin-labeled bivalent anti-Vibrio parahaemolyticus nanobody

[0127] 1. Prepare Thermo Scientific EZ-Link TM Sulfo-NHS-LC-LC-Biotin powder, 1 mg powder is dissolved in 100 μl of ultrapure water.

[0128] 2. 2 mg / mL bivalent anti-Vibrio parahaemolyticus nanobody (coded as shown in SEQ ID NO.7) and EZ-Link TM Sulfo-NHS-LC-LC-Biotin solution was mixed at a biotin to antibody molar ratio of 1:10 and slowly shaken at room temperature for 1 h in the dark.

[0129] 3. Place the mixed solution obtained in step 2 into a dialysis bag and dialyze with PBS at 4 ℃ for two days, changing the solution twice a day. Replace the dissolution medium of the biotinylated divalent anti-Vibrio parahaemolyticus nanobody. Measure the concentration and purity of the biotinylated divalent anti-Vibrio parahaemolyticus nanobody after dialysis and store at -20 ℃ for later use.

[0130] Example 5: Establishment of a standard curve for the detection of Vibrio parahaemolyticus by a self-sandwich ELISA using bivalent anti-Vibrio parahaemolyticus nanobodies.

[0131] (1) The bivalent anti-Vibrio parahaemolyticus nanobody (the bivalent anti-Vibrio parahaemolyticus nanobody expressed by SEQ ID NO.7) prepared in Example 3 was diluted with CBS at 2 μg / ml and added to a 96-well microplate at 100 µL / well. The plate was then coated overnight at 4 °C.

[0132] (2) The next day, the coating buffer in the 96-well microplate was aspirated and the plate was washed three times with PBST.

[0133] (3) Add 270 μL / well of 3% skim milk powder, incubate at 37 °C for 1 h to block, then remove the blocking solution and wash the plate three times with PBST;

[0134] (4) Vibrio parahaemolyticus from 10 8 A series of serial dilutions were performed starting with cfu / mL to obtain different concentrations. 100 µL / well was added to the microplate treated in step (3) and incubated at 37 °C for 1 h.

[0135] (5) Aspirate the liquid from the wells of the ELISA plate after step (4) and wash the plate three times with PBST;

[0136] (6) Dilute the biotinylated bivalent anti-Vibrio parahaemolyticus nanobody prepared in Example 4 to 1 μg / mL, and then add 100 µL / well to the enzyme-labeled plate after step (5) for reaction and incubate at 37 °C for 1 h;

[0137] (7) Aspirate the liquid from the wells of the ELISA plate in step (6) and wash the plate three times with PBST;

[0138] (8) The streptavidin-polymerized horseradish peroxidase was diluted with PBS containing 3% BSA at a volume ratio of 1:40000 and 100 μL / well was added to the microplate treated in step (7) and incubated at 37 °C for 1 h.

[0139] (9) Aspirate the liquid from the wells of the ELISA plate in step (8) and wash the plate five times with PBST;

[0140] (10) Add 100 μL / well TMB substrate solution to the microplate after step (9) and react at 37 °C in the dark for 10 min;

[0141] (11) Add 50 μL / well 2 M H2SO4 to the chromogenic solution in the microplate of step (10) to terminate the reaction;

[0142] (12) Place the ELISA plate in the ELISA reader and read the OD. 450 ;

[0143] (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 .

[0144] pass Figure 3 As can be seen from the ELISA detection standard curve, the bivalent 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, thereby specifically achieving the detection of Vibrio parahaemolyticus.

Claims

1. A bivalent anti-Vibrio parahaemolyticus nanobody, characterized in that, The amino acid sequence of the bivalent anti-Vibrio parahaemolyticus nanobody is shown in SEQ ID NO.

7.

2. The method for preparing the bivalent anti-Vibrio parahaemolyticus nanobody according to claim 1, characterized in that, The steps are as follows: (1) The gene sequence of monovalent anti-Vibrio parahaemolyticus nanobody V1 was amplified by PCR using upstream primer F1 and downstream primer R1 to obtain hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1; the gene sequence of monovalent anti-Vibrio parahaemolyticus nanobody V2 was amplified by PCR using upstream primer F2 and downstream primer R2 to obtain hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2; The amino acid sequence of the monovalent anti-Vibrio parahaemolyticus nanobody V1 is shown in SEQ ID NO.1; The amino acid sequence of the monovalent anti-Vibrio parahaemolyticus nanobody V2 is as shown in SEQ ID NO.2; The upstream primer F1 nucleotide is shown in SEQ ID NO.3; The downstream primer R1 nucleotide is shown in SEQ ID NO.4; The upstream primer F2 nucleotide is shown in SEQ ID NO. 5; The downstream primer R2 nucleotide is shown in SEQ ID NO. 6; (2) The hinged monovalent anti-Vibrio parahaemolyticus nanobody V-1 and hinged monovalent anti-Vibrio parahaemolyticus nanobody V-2 prepared in step (1) were amplified by overlap PCR and spliced ​​to obtain bivalent anti-Vibrio parahaemolyticus nanobody gene fragments V1-V2; the bivalent V1-V2 gene fragments obtained by overlap PCR and the vector were recovered by double enzyme digestion, and then the digested V1-V2 fragments and the vector were ligated with ligase to obtain recombinant plasmids containing bivalent anti-Vibrio parahaemolyticus nanobody V1-V2 genes; the constructed recombinant plasmids were transformed into expression strains, and after induction of expression, the bivalent anti-Vibrio parahaemolyticus nanobody was purified to obtain bivalent anti-Vibrio parahaemolyticus nanobody.

3. The method for preparing bivalent anti-Vibrio parahaemolyticus nanoantibodies according to claim 2, characterized in that, The carrier is pET-22b.

4. The method for preparing bivalent anti-Vibrio parahaemolyticus nanoantibodies according to claim 3, characterized in that, The ligase mentioned is T4 ligase.

5. The method for preparing bivalent anti-Vibrio parahaemolyticus nanoantibodies according to claim 1, characterized in that, The biotin mentioned is EZ-Link™ Sulfo-NHS-LC-LC-Biotin powder.

6. A self-sandwich ELISA kit based on a bivalent anti-Vibrio parahaemolyticus nanobody, characterized in that, It includes a capture antibody and a detection antibody, wherein the capture antibody is the bivalent anti-Vibrio parahaemolyticus nanobody of claim 1; and the detection antibody is the biotinylated bivalent anti-Vibrio parahaemolyticus nanobody of claim 1.

7. The self-sandwich ELISA kit based on bivalent anti-Vibrio parahaemolyticus nanobodies according to claim 1, characterized in that, The biotin mentioned is EZ-Link™ Sulfo-NHS-LC-LC-Biotin powder.

8. A method for detecting Vibrio parahaemolyticus, characterized in that, A bivalent anti-Vibrio parahaemolyticus nanobody was immobilized onto an ELISA plate as a capture antibody. After blocking with skim milk powder, serially diluted Vibrio parahaemolyticus was used as the antigen. The biotinylated bivalent anti-Vibrio parahaemolyticus nanobody was then used as the detection antibody. After amplification of the streptavidin-poly(Hydroxyl peroxidase) signal, TMB was used for color development, and finally, the OD value was read using an ELISA reader. 450 The value was calculated based on the standard curve to determine the content of Vibrio parahaemolyticus in the sample.

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