A nanobody specifically recognizing Staphylococcus aureus, its preparation method and application

Abstract

The present invention discloses a nanobody specifically recognizing Staphylococcus aureus, and a preparation method and application thereof. The present invention adopts ribosome display technology, screens out a pair of nanobody pairs with good pairing effect through constructing a nanobody display library, three rounds of high-throughput screening and ELISA verification, and combines immunochromatography technology for in vivo detection of actual pathogenic bacteria. The nanobodies disclosed by the present invention show high affinity and high specificity when binding to Staphylococcus aureus, can achieve a detection limit, and have advantages such as good stability and low cost compared with monoclonal antibodies, can be mass-produced, and have broad application prospects in the field of Staphylococcus aureus detection and analysis.

Description

Technical Field

[0001] The present invention relates to a nanobody specifically recognizing Staphylococcus aureus, a preparation method thereof, and an application thereof, belonging to the field of nanobodies. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is a Gram-positive coccus. Its pathogenicity is related to a variety of virulence factors it secretes, including adhesins, invasins, and immune evasion proteins. For example, staphylococcal protein A (SpA) expressed on the surface of Staphylococcus aureus can bind to host IgG (immunoglobulin G), interfering with the host immune system, and coagulation factor A (ClfA) can bind to fibrinogen, promoting bacterial adhesion and immune escape. Currently, monoclonal antibodies against Staphylococcus aureus mainly target its surface proteins, such as SpA and ClfA, etc. However, due to the non-specific binding characteristics of staphylococcal protein A and the disadvantages of traditional antibodies, such as long preparation cycle, poor stability, and high cost, it limits their application in rapid detection.

[0003] Currently, the detection methods of Staphylococcus aureus mainly include bacterial culture, polymerase chain reaction (PCR), and immunoassay. Although bacterial culture is the gold standard method, it is time-consuming and requires professional laboratory equipment and personnel, greatly limiting the screening efficiency of Staphylococcus aureus. Although the PCR technique has high sensitivity, it is cumbersome to operate and also requires professional equipment and personnel, and is not suitable for on-site rapid detection. For example, the patent "Primer set for Staphylococcus aureus and kit for detecting or identifying Staphylococcus aureus" (application number: 202411109634.0) discloses a method for detecting Staphylococcus aureus by dual fluorescence quantitative PCR. Although this method attempts to improve the specificity and sensitivity of detection, the inherent problems of the PCR technique itself, such as complex operation and long detection time, still cannot be avoided, limiting its application in on-site rapid detection. Traditional immunoassays, such as ELISA, although having good specificity, have complex operation steps and long detection time. Traditional strip detection methods are simple and fast, but have limited sensitivity and high detection limits, making it difficult to meet the needs of on-site rapid screening. Some emerging detection methods, such as the patent "SERS detection kit and detection method for Staphylococcus aureus" (application number: 202410970199.4) disclose a method for detecting Staphylococcus aureus based on surface-enhanced Raman spectroscopy (SERS). Although it can achieve extremely high detection sensitivity and accuracy, its high instrument and consumable costs limit its popularization in practical applications. Therefore, there is an urgent need for a more rapid, sensitive, and cost-controllable means for detecting Staphylococcus aureus. Summary of the Invention

[0004] Objective of the Invention: The technical problem to be solved by the present invention is to provide a nanobody with high sensitivity and strong specificity that can specifically recognize Staphylococcus aureus, as well as its preparation method and application, which are applied to the rapid and in-situ detection of Staphylococcus aureus.

[0005] Technical Solution: To solve the above technical problem, the present invention provides a nanobody (VHH antibody), and the amino acid sequence of the nanobody is shown as any one of SEQ ID NO: 1-6.

[0006] Among them, in the amino acid sequences shown as SEQ ID NO: 1 (Nb26), SEQ ID NO: 2 (Nb33), SEQ ID NO: 3 (Nb42), SEQ ID NO: 4 (Nb57), SEQ ID NO: 5 (Nb63), and SEQ ID NO: 6 (Nb84), through pairing experiments, it is found that the Nb26 and Nb42 nanobodies have the best pairing effect and can form a paired antibody for use in a sandwich lateral flow chromatography strip.

[0007] The present invention also provides a gene encoding the nanobody.

[0008] Among them, the nucleotide sequence of the gene is shown as any one of SEQ ID NO: 7-12.

[0009] Among them, the sequence shown as SEQ ID NO: 7 is the nucleotide sequence of the Nb26 nanobody, the sequence shown as SEQ ID NO: 8 is the nucleotide sequence of the Nb33 nanobody, the sequence shown as SEQ ID NO: 9 is the nucleotide sequence of the Nb42 nanobody, the sequence shown as SEQ ID NO: 10 is the nucleotide sequence of the Nb57 nanobody, the sequence shown as SEQ ID NO: 11 is the nucleotide sequence of the Nb63 nanobody, and the sequence shown as SEQ ID NO: 12 is the nucleotide sequence of the Nb84 nanobody.

[0010] The present invention also provides a preparation method of the nanobody, including the following steps:

[0011] (1) Extract alpaca lymphocytes, splice and recombine specific gene fragments into a phagemid and transfer it into Escherichia coli to construct a nanobody phage library;

[0012] (2) Use heat-inactivated Staphylococcus aureus as the coating protein to screen for nanobodies against Staphylococcus aureus.

[0013] Among them, the construction method of the nanobody phage display library includes the following steps:

[0014] (1) Amplify the fragment of the gene encoding the nanobody from alpaca lymphocytes, and introduce Sfi I restriction sites at both ends of the nanobody;

[0015] (2) Digest and recombine with the phagemid pComb3XSS, and ligate with T4 ligase to construct a phage display library.

[0016] Specifically, the present invention also provides a method for preparing the above-mentioned nanobody specifically recognizing Staphylococcus aureus, and the method includes the following steps:

[0017] 1: In vitro transcription and translation: Clone the nanobody phage library into a ribosome display vector. Then, using an in vitro transcription and translation system, transcribe the ribosome display vector into mRNA and translate it into a nanobody-ribosome-mRNA complex to form a ribosome display library;

[0018] 2: Affinity screening: Coat inactivated Staphylococcus aureus (ATCC 25923) on a solid-phase carrier (in this experiment, an enzyme-linked immunosorbent assay (ELISA) plate was used). Then, add the ribosome display library obtained by in vitro transcription and translation into the system containing immobilized Staphylococcus aureus, and incubate at an appropriate temperature and time. During this period, the nanobodies displayed on the ribosomes will interact with Staphylococcus aureus. The nanobody-ribosome-mRNA complex that specifically binds to the target will be captured, while the unbound complex will remain in the solution. Remove the unbound nanobody-ribosome-mRNA complex to reduce background noise and improve the specificity of screening. Finally, use a specific elution method to elute the nanobody-ribosome-mRNA complex bound to Staphylococcus aureus from the solid-phase carrier. (In the present invention, acidic elution is used: elute with a buffer solution of low pH, 0.1 M glycine-HCl (pH 2.2). After elution, it is necessary to immediately neutralize with a neutral buffer solution to protect the integrity of mRNA).

[0019] 3: Recover and amplify the VHH gene: Extract mRNA from the ribosome-mRNA complex bound to Staphylococcus aureus. Using the extracted mRNA as a template for reverse transcription PCR (RT-PCR), amplify the VHH gene fragment using primers matching the ribosome display vector. Finally, perform multiple rounds of screening to obtain a specific nanobody with high affinity, and extract mRNA from the eluate for subsequent RT-PCR amplification.

[0020] 4: Clone, express, and purify: Clone the amplified VHH gene fragment into a suitable expression vector, transform an expression system such as Escherichia coli for expression, and purify using affinity chromatography to obtain a soluble nanobody.

[0021] 5: Identification of binding activity and specificity: The binding activity of the purified nanobody to Staphylococcus aureus was identified using ELISA. Meanwhile, cross-reaction experiments were performed using other bacteria (such as Escherichia coli, Salmonella, Vibrio parahaemolyticus, etc.) to verify the specificity of the nanobody.

[0022] The present invention also provides the use of the nanobody or the gene in the preparation of a Staphylococcus aureus nanobody AIE fluorescence probe.

[0023] The present invention also provides a Staphylococcus aureus nanobody AIE fluorescence probe, comprising the nanobody or the gene.

[0024] The present invention also provides the use of the nanobody or the gene in the preparation of a detection reagent or kit for specifically recognizing Staphylococcus aureus.

[0025] The present invention also provides a detection reagent or test strip, comprising the nanobody or the gene.

[0026] The test strip proposed by the present invention can be applied to biological samples such as plasma and urine, food samples such as milk and meat products, and environmental samples such as wastewater and aerosol. When Staphylococcus aureus is present, the test line (T line) and the quality control line (C line) on the test strip show red fluorescence. When Staphylococcus aureus is absent, the quality control line shows red fluorescence while the test line does not develop color.

[0027] The present invention also provides the use of the nanobody or the gene in constructing an immunoassay platform for Staphylococcus aureus.

[0028] The present invention also provides a method for detecting Staphylococcus aureus in biological samples based on a POCT test strip, comprising the following steps:

[0029] (1) Paste the NC membrane, absorbent pad, conjugate pad, and sample pad on the PVC bottom plate and assemble according to the specifications of the test strip; draw the Super monoclonal antibody on the NC membrane as the quality control line (C line), and draw the anti-Staphylococcus aureus nanobody Nb26 on the NC membrane as the test line (T line), and dry for 1 - 2 h.

[0030] (2) Cut into test strips according to the specifications of the test strip using a test strip cutting machine. Drop the AIE fluorescent microspheres labeled with the anti-Staphylococcus aureus nanobody Nb42 onto the conjugate pad and dry at 37 °C for 1 - 2 h; use a pipette to drop it onto the sample pad of the prepared test strip. After chromatography for 5 - 10 min, observe the luminescence of the T and C lines of the test strip under an ultraviolet lamp to evaluate the sensitivity of the method for detecting Staphylococcus aureus in biological samples.

[0031] Nanobody (VHH) is the variable region of a heavy-chain antibody isolated from camelids, with a molecular weight of approximately 15 kDa, only 1 / 10 of that of traditional antibodies. Compared with traditional antibodies, nanobodies have many advantages: higher affinity and specificity, capable of recognizing hidden epitopes that are difficult for traditional antibodies to recognize (such as region A of ClfA); excellent stability, remaining active even under high-temperature or extreme pH conditions; being easily prepared and modified in large quantities through genetic engineering techniques; good tissue penetrability, and lower production costs, etc. Combining nanobodies with lateral flow chromatography can develop rapid diagnostic test strips with higher sensitivity, stronger specificity, better stability, and lower cost, overcoming the limitations of traditional methods and enabling rapid, convenient, and in-situ detection of Staphylococcus aureus, having broad application prospects in the fields of food safety, clinical diagnosis, etc.

[0032] The present invention also provides an application of the above-mentioned nanobody pair specifically recognizing Staphylococcus aureus. The ribosome-displayed nanobody pair of the present invention can specifically bind to Staphylococcus aureus. Combining this pair of nanobodies with the lateral flow chromatography method can achieve in-situ and rapid detection of Staphylococcus aureus. The method includes the following steps:

[0033] (1) Place the NC membrane in a vacuum drying oven and dry it for later use; paste the NC membrane, absorbent pad, conjugate pad, and sample pad on the PVC bottom plate. The sample pad is on the conjugate pad, the conjugate pad is on the NC membrane, and the absorbent pad is about 2 - 4 mm on the NC membrane; draw the SuperC monoclonal antibody on the NC membrane as the control line (C line), and draw the anti-Staphylococcus aureus nanobody Nb26 on the NC membrane as the test line (T line), and dry it in a vacuum drying oven for 1 - 2 h;

[0034] (2) Cut into test strips according to the specifications of the test strips. Add fluorescent microspheres labeled with the anti-Staphylococcus aureus nanobody Nb42 to the conjugate pad, dry at 37 °C for 2 h. Add a certain volume of Tween-20 to the sample to be detected, and use a pipette to drop it on the sample pad of the prepared test strip. After chromatography for 10 min, observe the luminescence of the T and C lines of the test strip under 365 nm ultraviolet light irradiation to evaluate the concentration of Staphylococcus aureus in the sample detected by this method.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0036] 1. Compared with traditional monoclonal antibodies, the ribosome-displayed nanobodies of the present invention have the advantages of small size, high stability, simple preparation, low cost, and large-scale production;

[0037] 2. The immunochromatographic assay provided by the present invention features fast detection speed, strong specificity, and high sensitivity, enabling the detection of Staphylococcus aureus in multiple scenarios, instantaneously, and in situ.

[0038] 3. Traditional monoclonal antibodies are affected by the binding of the Fc region to Staphylococcal protein A (SPA), making it difficult to directly detect Staphylococcus aureus. Instead, they can only indirectly detect the toxic substances secreted by the bacteria, making it difficult to accurately evaluate the infection status of patients and the contamination of water sources and food. The present invention can achieve the direct detection of Staphylococcus aureus.

[0039] 4. The cell-free system of the nanobody library of the present invention can overcome the deficiencies such as biosafety and toxic proteins, and has advantages in expressing recombinant proteins or antibodies. Therefore, developing a cell-free system-based nanobody library is of great value for the efficient screening of nanobodies.

[0040] 5. A pair of Staphylococcus aureus nanobody pairs with particularly good pairing effects was screened out this time, effectively improving the detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 To verify the affinity results of 6 phage-displayed nanobodies by Phage-ELISA, the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0042] Figure 2 For the antigen specificity verification results of Listeria monocytogenes ATCC19115, the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0043] Figure 3 For the antigen specificity verification results of Vibrio parahaemolyticus ATCC17802, the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0044] Figure 4 For the antigen specificity verification results of Rotavirus, the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0045] Figure 5 For the antigen specificity verification results of Norovirus, the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0046] Figure 6 For the antigen specificity verification results of Escherichia coli (wild E. coli), the abscissa represents the nanobody, and the ordinate represents the absorbance value at 450 nm.

[0047] Figure 7 For the specific result of antigen verification of Salmonella ATCC13076, the abscissa is the nanobody, and the ordinate is the absorbance value at 450 nm;

[0048] Figure 8 For detecting Staphylococcus aureus at different concentrations with a Staphylococcus aureus test strip;

[0049] Figure 9 For the specific identification of a Staphylococcus aureus test strip;

[0050] Figure 10 For the optimization of the fluorescence probe pH;

[0051] Figure 11 For screening the pair of nanobodies with the best pairing effect by the checkerboard method. Detailed implementation manners

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0053] Main experimental materials: Standard strain of Staphylococcus aureus (ATCC 25923)

[0054] Main reagents: Chicken ovalbumin and bovine ovalbumin were purchased from Sigma Company in the United States. Horseradish peroxidase (HRP)-anti-M13 monoclonal antibody (product number: 11973-MMO5T) was purchased from Beijing Sino Biological Inc. Skim milk powder, 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic solution, and isopropyl-β-D-thiogalactoside (IPTG) were purchased from Shanghai Sangon Biotech Co., Ltd. LB broth and 2×YT medium were purchased from Qingdao Hope Bio-Technology Co., Ltd.

[0055] Main reagent formulations:

[0056] 1. 2×YT liquid medium: Weigh 31 g of 2×YT powder, dissolve it in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min;

[0057] 2. 2×YT solid medium: Weigh 31 g of 2×YT powder and 18 g of agar, dissolve them in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min;

[0058] 3. LB liquid medium: Weigh 25 g of LB medium, dissolve it in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min;

[0059] 4. 20% Polyethylene Glycol (PEG)-NaCl: 50 g PEG-8000, 36 g NaCl, dissolved by heating with ultrapure water, made up to 250 mL, autoclaved for 15 min;

[0060] 5. Eluent: 0.2 M Glycine (Gly), adjusted to pH = 2.2 with hydrochloric acid, autoclaved for 15 min;

[0061] 6. Neutralization buffer: 1 M Tris (Tris(hydroxymethyl)aminomethane), adjusted to pH = 9.1 with hydrochloric acid, autoclaved for 15 min.

[0062] Example 1: Inactivation of Staphylococcus aureus

[0063] (1) Streak the standard strain of Staphylococcus aureus on an LB plate and incubate at 37 °C for 12 - 14 h;

[0064] (2) Pick a single colony on the plate and inoculate it into 5 ml of LB medium, incubate at 37 °C for 12 - 14 h;

[0065] (3) Spread and count on an LB plate, incubate at 37 °C for 12 - 14 h;

[0066] (4) Pipette 1 ml of the counted bacterial solution, centrifuge at 8000 rpm for 2 min, resuspend with PBS, repeat 3 times;

[0067] (5) Preheat the metal bath to 90 °C in advance, and heat at 90 °C for half an hour to obtain inactivated strains.

[0068] Example 2: Amplification of VHH gene and construction of a nanobody ribosome display library

[0069] (1) RNA extraction: Extract total RNA from B lymphocytes of immunized alpacas;

[0070] (2) cDNA synthesis: Reverse transcribe RNA into cDNA using SuperScript IV reverse transcriptase (reaction system: 1 μg RNA, 1 μL reverse transcriptase, 4 μL 5X buffer, 2 μL 10 mM dNTPs, 1 μL RNase inhibitor, make up to 20 μL with water; reaction conditions: 42 °C for 60 minutes, 70 °C for 15 minutes);

[0071] (3)VHH gene amplification: The VHH gene was PCR amplified from cDNA of multiple B lymphocytes using specific primers VHH-F and VHH-R with SfiI restriction sites (reaction system: 1 μL cDNA, 1 μL DNA polymerase, 5 μL 10X buffer, 1 μL 10 mM dNTPs, 1 μL primer VHH-F, 1 μL primer VHH-R, made up to 50 μL with water; reaction conditions: 95°C for 3 minutes; 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C for 7 minutes; primers are VHH-F (SEQ ID NO:13) and VHH-R (SEQ ID NO:14);

[0072] (4)SfiI digestion: The PCR products and the ribosome display vector pRibVec (purchased from Agilent) were digested with SfiI restriction endonuclease. Among them, the reaction system: 5 μL PCR product or vector, 1 μL SfiI, 2 μL 10X buffer, made up to 20 μL with water; reaction conditions: 37°C for 2 hours;

[0073] (5)Use T4 DNA ligase to ligate the digested VHH gene fragment and the vector pRibVec (reaction system: 1 μL vector, 3 μL PCR product, 1 μL T4 DNA ligase, 2 μL 10X buffer, made up to 20 μL with water; reaction conditions: 16°C overnight);

[0074] (6)In vitro transcription: Use the TNT T7 Quick Master Mix in vitro transcription kit to transcribe the ligation product into mRNA to obtain the nanobody ribosome display library (operate according to the kit instructions, the kit is the TNT® T7 PCRDNA Quick System, purchased from Promega (Beijing) Biotechnology Co., Ltd.).

[0075] Example 3 Screening and Identification of Staphylococcus aureus Ribosome Display Nanobodies

[0076] 1. Antigen coating: The purified Staphylococcus aureus in Example 1 was coated onto the enzyme-linked immunosorbent assay (ELISA) plate, and the coating volume was 100 μL / well, and coated overnight at 4°C.

[0077] 2. Blocking: Use 3% BSA to block non-specific binding sites. The blocking volume was 300 μL / well, and incubated at 37°C for 2 hours.

[0078] 3. Binding: Add the mRNA-ribosome-nanobody complex in the nanolibrary constructed in Example 2, and incubate at 37°C for 60 minutes.

[0079] 4. Washing: Wash the unbound complex with PBST. The number of washing times is 5, and the concentration of the washing solution is 0.05%, which can be adjusted according to the screening rounds.

[0080] 5. Elution: Elute the bound mRNA with 200 µL of 0.1 M Gly-HCl (pH 2.2) for 15 minutes. Immediately add 20 µL of 1 M Tris-HCl (pH 9.1) to neutralize the eluate.

[0081] 6. mRNA recovery: Recover the mRNA in the eluate using a commercial kit.

[0082] 7. RT-PCR amplification: Amplify the recovered VHH nanobody gene using reverse transcription PCR. Among them, the reaction system: 800 ng of cDNA + 1 µL of AlpVh-LD (10 uM) + 1 µL of CH2-R (10 uM) + 7 µL of 10×PCR Buffer + 0.1 µL of Hot STAR Taq DNA Polymerase + 0.4 µL of dNTP mix (10 uM) + add ddH 2 O to 50 uL.

[0083] The reaction program is as follows:

[0084] 1×98°C 15 min

[0085] 35×96°C 1 min

[0086] 35×61.5 / 62.8 / 64.7 / 68°C 1 min

[0087] 35×70°C 1 min

[0088] 1×70°C 10 min

[0089] 1×4°C Store at 4°C

[0090] Among them, the vector is pCMV-3Tag (purchased from Agilent); the primers are synthesized by Shanghai Sangon Biotech Co., Ltd. The specific primers are: CH2-R (as shown in SEQ ID NO:15), AlpVh-LD (as shown in SEQ ID NO:16), P4-S (as shown in SEQ ID NO:17), P4-L (as shown in SEQ ID NO:18), P5 (as shown in SEQ ID NO:19), P6-new (as shown in SEQ ID NO:20), AlpVh-FR1-Fnew (as shown in SEQ ID NO:21).

[0091] Among them, the usage of the primers was referred to the instruction manual of TAKARA's RNAiso reagent: PrimeSTAR high-fidelity DNA polymerase was used to obtain the variable region coding gene of the heavy-chain antibody through nested PCR. In the first round of PCR, cDNA was amplified with primers AlpVh-LD and CH2-R respectively. Then, the first-round amplification products were used as templates for the second-round amplification and amplified with the remaining primers respectively. After recovery and quantification using a recovery kit, they were stored at -20°C for later use.

[0092] 8. Multiple rounds of screening: Repeat steps 1-7 for 3 rounds of screening to gradually increase the screening stringency. Finally, the desired mRNA-ribosome-nanobody complex (amino acid sequences are shown in SEQ ID NO: 1-6) was obtained.

[0093] 9. The checkerboard method was used for paired experiments to screen out a pair of highly sensitive and strongly specific paired antibodies, mainly including the following steps:

[0094] (1) Coat the mRNA-ribosome-nanobody complex (amino acid sequences are shown in SEQ ID NO: 1-6) at 50 μg / ml and incubate overnight.

[0095] (2) Wash the plate 3 times with 0.05% PBST, and incubate with 5% skim milk powder at 37°C for 2 h.

[0096] (3) Wash the plate 3 times with 0.05% PBST, and add the heat-inactivated Staphylococcus aureus obtained in Example 1 at 10 8 cfu / ml and incubate at 37°C for 1 h.

[0097] (4) Wash the plate 3 times with 0.05% PBST, recover the bound mRNA, and incubate at 37°C for 45-60 min.

[0098] (5) Wash the plate 6 times with 0.05% PBST, add 100 μL of horseradish peroxidase (HRP)-anti-M13 monoclonal antibody to each well, and incubate at 37°C for 45-60 min.

[0099] (6) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB chromogenic solution, incubate at 37°C for 15 min and conduct identification; the results are as Figure 11 shown, and the effects of Nb26 and Nb42 are the best.

[0100] Example 4: Synthesis of a Staphylococcus aureus nanobody AIE fluorescence probe

[0101] 1) Take 100 μL of AIE fluorescent microsphere solution (1 wt%), add 500 μL of MES PH6.0 buffer for washing once, and centrifuge at 12000 rpm for 15 min to remove the supernatant.

[0102] 2) Resuspend the microspheres with 500 μL of MES pH 6.0 buffer, then add 30 μL of EDC and 90 μL of NHS (both at a concentration of 3 mg / mL, freshly prepared MES pH 6.0 buffer), rotate and shake in the dark for 30 min, centrifuge at 12,000 rpm for 15 min, and discard the supernatant.

[0103] 3) Wash once with 500 μL of MES pH 6.5 buffer, resuspend the microspheres with 500 μL of MES pH 6.5 buffer, add 20 μg of labeled antibody Nb42 (the amount of antibody varies according to the concentration), rotate and shake in the dark for 2 h, centrifuge at 12,000 rpm for 15 min, and discard the supernatant.

[0104] 4) Resuspend with 500 μL of blocking solution (3% casein), rotate and shake in the dark for 1 h, centrifuge at 12,000 rpm for 15 min, and discard the supernatant.

[0105] 5) Wash once with 500 μL of storage solution (storage solution: 0.02 m Tris + 0.1% Tween - 20 + 0.5% BSA + 0.03% Proclin 300), centrifuge and discard the supernatant.

[0106] 6) Resuspend with 100 μL of storage solution and store at 2 - 8°C.

[0107] Furthermore, adjust the pH of the Staphylococcus aureus nanobody AIE fluorescence probe with potassium carbonate, and the addition amount is between 30 - 70 μL to make the pH 6 - 8. The results are as Figure 10 shown. When the pH is 8, the detection effect of the Staphylococcus aureus nanobody AIE fluorescence probe is the best. Among them, Figure 10 the pH values from left to right are 6, 7, and 8 respectively.

[0108] Example 5

[0109] (1) Coating antigen: Coat different target antigens (such as antigens / proteins of Staphylococcus aureus (S. aureus) ATCC25923, Listeria monocytogenes ATCC19115, Vibrio parahaemolyticus ATCC17802, Rotavirus, Norovirus, Escherichia coli, Salmonella ATCC13076, and the specific concentration needs to be optimized according to the actual antigen characteristics) into different wells of a 96 - well enzyme - linked immunosorbent assay (ELISA) plate respectively, and incubate overnight at 4°C. After washing the plate, block the unbound sites of the ELISA plate with 5% skim milk and incubate at 37°C for 2 h.

[0110] (2) Add ribosome display complex: Add the mRNA-ribosome-protein complex obtained by in vitro transcription and translation (where the mRNA encodes a nanobody library containing different sequences) into each well of the enzyme-linked immunosorbent assay (ELISA) plate coated with different antigens. Incubate at 37 °C for 1 hour. Wash 3 times with 0.05% PBST to remove unbound complex.

[0111] (3) Add detection antibody: Add HRP-labeled anti-His tag antibody and incubate at 37 °C for 45 - 60 minutes. Wash 6 times with 0.05% PBST to remove unbound detection antibody.

[0112] (4) Add chromogenic substrate and measure: Add 100 μL of TMB chromogenic solution to each well, incubate at 37 °C for 15 minutes, and measure the absorbance (OD450nm) using an ELISA reader. Plot the reaction results of each nanobody with different antigens as bar graphs and compare with the blank control to obtain the results as Figures 1 - 7 shown.

[0113] Example 6: Rapid identification steps of test strip

[0114] 1) Place the NC membrane in a vacuum drying oven and dry at 37 °C for 60 - 100 min. According to the structure of the chromatographic test strip, paste the NC membrane on the PVC bottom plate. Press the sample pad on one side against the conjugate pad, the conjugate pad against the NC membrane, and press the absorbent pad on the other side against the NC membrane with a pressure of about 2 - 6 mm to assemble the test strip.

[0115] 2) Use a gold-spraying and membrane-stripping instrument to draw a detection line with Staphylococcus aureus-coated antibody Nb26 (pH 8) at a concentration of 1 - 2 mg / mL, and draw a quality control line with SuperC+ antibody at a concentration of 2.5 mg / mL; the distance between the detection lines is 2 - 4 mm, and place it in a vacuum drying oven to dry for 4 - 6 h.

[0116] 3) Use a test strip cutting machine to cut the test strip into test strips of 2 - 8×60 - 100 mm, seal in bags for later use.

[0117] 4) Sensitivity detection: Pipette 100 μL of amplification products with different initial concentrations (1×10 3 ~1×10 8 CFU / mL), add Tween-20 with a final concentration of 0.05%, drop it on the sample pad of the prepared test strip, and add the set blank control, let it stand for 10 min, and observe the luminescence of the detection line and the quality control line under ultraviolet light excitation at 365 nm. The results are as Figure 8 shown, and the luminescence conditions are 10 8 , 10 7 , 10 6, 10 5 , 10 4 , 10 3 CFU / mL decreased, and the detection limit was 10 4 CFU / mL.

[0118] 5) Specificity detection: Pipette 100 μL of the amplification product respectively, and mix it with Staphylococcus aureus solution (ATCC 25923), Salmonella solution, Shigella solution (ATCC 25931), rotavirus, FOB (brand: Sigma-Aldrich, catalog number: H0267), and norovirus at the same concentration (10 6 CFU / mL). Add Tween-20 with a final concentration of 0.05% to it, and drip it onto the sample pad of the prepared test strip. Let it stand for 10 min, and observe the luminescence of the test line and the control line under the excitation of ultraviolet light at 365 nm. The results are as Figure 9 shown. The nanobody specifically recognizes Staphylococcus aureus. Among them, from left to right are Staphylococcus aureus, Salmonella, Shigella, rotavirus, FOB, and norovirus.

Claims

1. A nanobody that specifically recognizes Staphylococcus aureus, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

2. A gene encoding the Nanobody according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

7.

4. Use of the nanobody according to claim 1 in the preparation of a Staphylococcus aureus nanobody AIE fluorescent probe.

5. A Staphylococcus aureus nanobody AIE fluorescent probe, characterized in that: Comprising the Nanobody according to claim 1.

6. Use of the nanobody according to claim 1 in the preparation of a detection reagent or a kit for specifically recognizing Staphylococcus aureus.

7. A detection reagent or test strip, characterized in that: Comprising the Nanobody according to claim 1.

8. The detection reagent or test strip according to claim 7, characterized in that: It also contains a nanobody with an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 6.

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