Nanobody specifically recognizing staphylococcus aureus and application thereof

Abstract

The application discloses a nano antibody specifically recognizing staphylococcus aureus and a preparation method and application thereof. The ribosome display technology is adopted to screen a pair of nano antibody with good pairing effect through construction of a nano antibody display library, three rounds of high-throughput screening and ELISA verification, and the nano antibody is combined with an immunochromatography technology to be used for in-vivo detection of actual pathogenic bacteria. The nano antibody disclosed by the application shows high affinity and high specificity when being combined with the staphylococcus aureus, can realize a detection limit, and has the advantages of good stability and low cost compared with a monoclonal antibody, can be produced on a large scale, and has a wide application prospect in the field of staphylococcus aureus detection and analysis.

Description

Technical Field

[0001] This invention relates to a nanobody that specifically recognizes Staphylococcus aureus and its application, belonging to the field of nanobodies. This invention is a divisional application of application number 2025102876827, filed on March 12, 2025, entitled "A Nanobody that Specifically Recognizes Staphylococcus aureus, Its Preparation Method and Application," filed on March 12, 2025. Background Technology

[0002] Staphylococcus aureus, belonging to the genus Staphylococcus, is a Gram-positive coccus. Its pathogenicity is related to various virulence factors it secretes, including adhesins, invaginins, 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's immune system, and agglutinating factor A (ClfA) can bind to fibrinogen, promoting bacterial adhesion and immune evasion. Currently, monoclonal antibodies against Staphylococcus aureus mainly target its surface proteins, such as SpA and ClfA. However, the non-specific binding characteristics of Staphylococcus aureus protein A, as well as the drawbacks of traditional antibodies such as long preparation cycles, poor stability, and high costs, limit their application in rapid detection.

[0003] Currently, the main methods for detecting Staphylococcus aureus include bacterial culture, polymerase chain reaction (PCR), and immunoassay. Although bacterial culture is the gold standard method, it is time-consuming and requires specialized laboratory equipment and personnel, greatly limiting the screening efficiency of Staphylococcus aureus. While PCR technology has high sensitivity, it is cumbersome to operate and also requires specialized equipment and personnel, making it unsuitable for rapid on-site testing. For example, the patent "Primer Set for Staphylococcus aureus and Reagent Kit for Detection or Identification of Staphylococcus aureus" (application number: 202411109634.0) discloses a method for detecting Staphylococcus aureus using dual-fluorescence quantitative PCR. Although this method attempts to improve the specificity and sensitivity of the detection, the inherent complexity of PCR technology and its long detection time remain unavoidable, limiting its application in rapid on-site testing. Traditional immunoassays, such as ELISA, while having good specificity, have complex procedures and long detection times. Traditional test strip methods, although simple and fast, have limited sensitivity and high detection limits, making them difficult to meet the needs of rapid on-site screening. Some emerging detection methods, such as the patent "A SERS Detection Kit and Detection Method for Staphylococcus aureus" (application number: 202410970199.4), disclose a Staphylococcus aureus detection method 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 widespread application in practice. Therefore, there is an urgent need for a faster, more sensitive, and cost-effective method for the detection of Staphylococcus aureus. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a highly sensitive and specific nanobody that can specifically identify Staphylococcus aureus and its application, which can be used for rapid and in situ detection of Staphylococcus aureus.

[0005] Technical solution: To solve the above technical problems, the present invention provides a nanobody (VHH antibody), wherein the amino acid sequence of the nanobody is shown in any one of SEQ ID NO: 1 to 6.

[0006] Among them, the amino acid sequences shown in 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) show the best pairing effect between Nb26 and Nb42 nanobodies through pairing experiments. They can form paired antibodies for use in sandwich side-flow chromatography test strips.

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

[0008] The nucleotide sequence of the gene is shown in any one of SEQ ID NO: 7~12.

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

[0010] The present invention also provides a method for preparing the nanobody, comprising the following steps:

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

[0012] (2) Inactivated Staphylococcus aureus was used as the coating protein to screen for anti-Staphylococcus aureus nanobodies.

[0013] The method for constructing the nanobody phage display library includes the following steps:

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

[0015] (2) The phage was digested and recombined with pComb3XSS enzyme, and then ligated with T4 ligase to construct a phage display library.

[0016] Specifically, the present invention also provides a method for preparing the above-described nanobody that specifically recognizes Staphylococcus aureus, the method comprising the following steps:

[0017] 1. In vitro transcription and translation: The nanobody phage library was cloned into a ribosome display vector. Then, using an in vitro transcription and translation system, the ribosome display vector was transcribed into mRNA and translated into a nanobody-ribosome-mRNA complex, forming the ribosome display library.

[0018] 2. Affinity Screening: Inactivated Staphylococcus aureus (ATCC 25923) was coated onto a solid support (ELISA plate was used in this experiment). Then, a ribosome display library obtained from in vitro transcription and translation was added to the system containing immobilized Staphylococcus aureus and incubated at appropriate temperature and time. During this period, the nanobodies displayed on the ribosomes interact with Staphylococcus aureus. Target-specific nanobodies-ribosome-mRNA complexes are captured, while unbound complexes remain in solution. Unbound nanobodies-ribosome-mRNA complexes are removed to reduce background noise and improve screening specificity. Finally, a specific elution method is used to elute the nanobodies-ribosome-mRNA complexes bound to Staphylococcus aureus from the solid support. (This invention uses acidic elution: elution with a low-pH buffer, 0.1 M glycine-HCl (pH 2.2). Immediate neutralization with neutral buffer is required after elution to protect the integrity of the mRNA).

[0019] 3. Recovery and Amplification of the VHH Gene: mRNA was extracted from the ribosome-mRNA complex bound to Staphylococcus aureus. Using the mRNA extracted via reverse transcription PCR (RT-PCR) as a template, the VHH gene fragment was amplified using primers matched to the ribosome display vector. Finally, multiple rounds of screening were performed to obtain high-affinity specific nanobodies, from which mRNA was extracted for subsequent RT-PCR amplification.

[0020] 4. Cloning, expression and purification: The amplified VHH gene fragment is cloned into a suitable expression vector, transformed into expression systems such as E. coli for expression, and purified by affinity chromatography to obtain soluble nanobodies.

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

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

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

[0024] The present invention also provides the application of the nanobody or the gene in the preparation of detection reagents or kits that specifically recognize 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 in this 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 aerosols. When Staphylococcus aureus is present, the test line (T line) and control line (C line) on the test strip show red fluorescence. If Staphylococcus aureus is not present, the control line shows red fluorescence while the test line does not show color.

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

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

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

[0030] (2) Cut the test strips into strips according to the specifications of the test strips using a test strip cutter. Add the AIE fluorescent microspheres labeled with the anti-Staphylococcus aureus nanoantibody Nb42 to the conjugate pad and dry at 37 ℃ for 1-2 h. Add the microspheres to the sample pad used to prepare the test strips using a pipette. After chromatography for 5-10 min, observe the luminescence of the T and C lines of the test strips under a UV lamp to evaluate the sensitivity of this method in detecting Staphylococcus aureus in biological samples.

[0031] Nanobodies (VHHs) are variable regions of heavy-chain antibodies isolated from camelids, with a molecular weight of approximately 15 kDa, only 1 / 10 that of traditional antibodies. Compared to traditional antibodies, nanobodies offer numerous advantages: higher affinity and specificity, enabling them to recognize hidden epitopes that are difficult for traditional antibodies to identify (such as the A region of ClfA); excellent stability, maintaining activity even under high temperatures or extreme pH conditions; ease of mass production and modification through genetic engineering; good tissue penetration; and lower production costs. Combining nanobodies with lateral flow chromatography can lead to the development of 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. This has broad application prospects in food safety, clinical diagnostics, and other fields.

[0032] This invention also provides an application of the above-mentioned nanobody pair that specifically recognizes Staphylococcus aureus. The ribosome-displaying nanobody pair of this invention can specifically bind to Staphylococcus aureus. Combining this nanobody pair with a 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; attach the NC membrane, absorbent pad, conjugate pad, and sample pad to a PVC base plate, with the sample pad on the conjugate pad, the conjugate pad on the NC membrane, and the absorbent pad on the NC membrane, pressing them together by about 2-4 mm; streak the SuperC monoclonal antibody on the NC membrane as the control line (C line), and streak the anti-Staphylococcus aureus nanoantibody Nb26 on the NC membrane as the detection line (T line), and dry it in a vacuum drying oven for 1-2 h;

[0034] (2) Cut the test strips into test strips according to the specifications of the test strips using a test strip cutter. Add fluorescent microspheres labeled with anti-Staphylococcus aureus nanoantibody Nb42 to the conjugate pad and dry at 37 ℃ for 2 h. Add a certain volume of Tween-20 to the sample to be tested and drop it onto the sample pad for preparing the test strips using a pipette. After chromatography for 10 min, observe the luminescence of the T and C lines of the test strips 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 significant advantages:

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

[0037] 2. The immunochromatographic method provided by this invention has fast detection speed, high specificity and high sensitivity, and can realize multi-scenario, real-time and in situ detection of Staphylococcus aureus;

[0038] 3. Traditional monoclonal antibodies are affected by the binding of the Fc terminus to Staphylococcus aureus SPA, making it difficult to directly detect Staphylococcus aureus. They can only indirectly detect the toxic substances secreted by the bacteria, making it difficult to accurately assess the infection status of patients and the contamination of water and food. This invention can achieve direct detection of Staphylococcus aureus.

[0039] 4. The cell-free nanobody library system of the present invention can overcome the shortcomings of biosafety and toxic proteins, and has advantages in expressing recombinant proteins or antibodies; therefore, the development of nanobody libraries based on cell-free systems is of great value for the efficient screening of nanobodies.

[0040] 5. This study identified a pair of Staphylococcus aureus nanoantibody pairs with particularly good pairing performance, which effectively improved the detection results. Attached Figure Description

[0041] Figure 1 To verify the affinity of six phages for nanobodies using Phage-ELISA, the x-axis represents the nanobodies and the y-axis represents the absorbance at 450 nm.

[0042] Figure 2 The results of antigen specificity verification for Listeria monocytogenes ATCC19115 are shown. The horizontal axis represents the nanobody, and the vertical axis represents the absorbance at 450 nm.

[0043] Figure 3 The results show the antigen specificity verification of Vibrio parahaemolyticus ATCC17802. The horizontal axis represents the nanobody, and the vertical axis represents the absorbance at 450 nm.

[0044] Figure 4 The results show the antigen-specificity verification of rotavirus, with the horizontal axis representing the nanobody and the vertical axis representing the absorbance at 450 nm.

[0045] Figure 5 The results of antigen-specific validation for Norovirus are shown, with the horizontal axis representing the nanobody and the vertical axis representing the absorbance at 450 nm.

[0046] Figure 6 The results show the antigen specificity verification of Escherichia coli (wild E. coli), with the horizontal axis representing nanobodies and the vertical axis representing the absorbance at 450 nm.

[0047] Figure 7 The results show the antigen specificity of Salmonella ATCC13076, with the horizontal axis representing the nanobody and the vertical axis representing the absorbance at 450 nm.

[0048] Figure 8 To detect different concentrations of Staphylococcus aureus using test strips;

[0049] Figure 9 For the specificity identification of Staphylococcus aureus test strips;

[0050] Figure 10 Optimization of pH for fluorescent probes;

[0051] Figure 11 To screen the pair of nanobodies with the best pairing effect using the chessboard method. Detailed Implementation

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

[0053] Main experimental material: Staphylococcus aureus standard strain (ATCC 25923)

[0054] Main reagents: Chicken ovalbumin and bovine ovalbumin were purchased from Sigma-Aldrich, USA; horseradish peroxidase (HRP)-anti-M13 monoclonal antibody (catalog number: 11973-MMO5T) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; 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 Haibo Biotechnology Co., Ltd.

[0055] Main reagent formula:

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

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

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

[0059] 4. 20% polyethylene glycol (PEG)-NaCl: Dissolve 50g PEG-8000 and 36g NaCl in ultrapure water by heating, bring the volume to 250mL, and autoclave for 15min.

[0060] 5. Eluent: 0.2M glycine (Gly), add hydrochloric acid to adjust pH to 2.2, autoclave for 15 min;

[0061] 6. Neutralization buffer: 1M tris(hydroxymethyl)aminomethane (Tris), add hydrochloric acid to adjust pH to 9.1, autoclave for 15 min.

[0062] Example 1: Inactivation of Staphylococcus aureus

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

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

[0065] (3) Count the samples on LB plates and incubate at 37°C for 12-14 hours;

[0066] (4) Take 1 ml of the bacterial culture after counting, centrifuge at 8000 rpm for 2 min, resuspend in PBS, and repeat 3 times;

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

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

[0069] (1) RNA extraction: Total RNA was extracted from B lymphocytes of immunized alpacas;

[0070] (2) cDNA synthesis: RNA was reverse transcribed 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, water added to 20 μL; reaction conditions: 42℃ for 60 minutes, 70℃ for 15 minutes).

[0071] (3) VHH gene amplification: VHH gene was amplified by PCR using specific primers VHH-F and VHH-R with SfiI restriction sites on cDNA from multiple B lymphocytes (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, water added to 50 μL; reaction conditions: 95℃ for 3 minutes; 95℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 1 minute, 35 cycles; 72℃ for 7 minutes; primers were VHH-F (SEQ ID NO:13) and VHH-R (SEQ ID NO:14).

[0072] (4) SfiI restriction enzyme digestion: The PCR product and ribosome display vector pRibVec (purchased from Agilent) were digested using SfiI restriction endonuclease. The reaction system consisted of 5 μL PCR product or vector, 1 μL SfiI, 2 μL 10X buffer, and water added to a final volume of 20 μL. The reaction conditions were 37℃ 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, add water to 20 μL; reaction conditions: 16℃ overnight).

[0074] (6) In vitro transcription: The ligation product was transcribed into mRNA using the TNT T7 Quick Master Mix in vitro transcription kit to obtain a nanobody ribosome display library (follow the kit instructions; the kit is the TNT® T7 PCRDNA Rapid System, purchased from Prometheus (Beijing) Biotechnology Co., Ltd.).

[0075] Example 3: Screening and Identification of Nanobodies Displayed by Staphylococcus aureus

[0076] 1. Antigen coating: The purified Staphylococcus aureus from Example 1 was coated onto an ELISA plate at a volume of 100 µL / well and incubated overnight at 4°C.

[0077] 2. Blocking: Block non-specific binding sites using 3% BSA. The blocking volume is 300 µL / well, and incubate at 37°C for 2 hours.

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

[0079] 4. Washing: Wash unbound complexes with PBST. Wash 5 times with a washing buffer concentration of 0.05%, which can be adjusted according to the screening round.

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

[0081] 6. mRNA recovery: mRNA in the eluent was recovered using a commercial kit.

[0082] 7. RT-PCR Amplification: The VHH nanobody gene was amplified and recovered using reverse transcription PCR. The reaction system consisted of: 800 ng cDNA + 1 μL AlpVh-LD (10 μM) + 1 μL CH2-R (10 μM) + 7 μL 10× PCR Buffer + 0.1 μL Hot STAR Taq DNA Polymerase + 0.4 μL dNTP mix (10 μM) + ddH2O to a final volume of 50 μL.

[0083] The reaction procedure is as follows:

[0084] 1 × 98°C for 15 min

[0085] 35 × 96°C for 1 min

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

[0087] 35 × 70°C for 1 min

[0088] 1 × 70°C for 10 min

[0089] Store at 4°C (1×4°C)

[0090] The vector was pCMV-3Tag (purchased from Agilent); the primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the specific primers were: 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), and AlpVh-FR1-Fnew (as shown in SEQ ID NO:21).

[0091] The primer usage followed the instructions for the TAKARA RNAiso reagent: Nested PCR was performed using PrimeSTAR high-fidelity DNA polymerase to obtain the variable region encoding the heavy chain antibody. The first round of PCR amplified cDNA using primers AlpVh-LD and CH2-R. The amplified products from the first round were then used as templates for the second round of amplification with the remaining primers. The amplified products were recovered and quantified using a recovery kit and stored at -20°C for later use.

[0092] 8. Multiple rounds of screening: Repeat steps 1-7 for three rounds of screening to gradually increase the rigor of the screening. Finally, obtain the desired mRNA-ribosome-nanobody complex (amino acid sequence as shown in SEQ ID NO: 1~6).

[0093] 9. The checkerboard method is used to perform pairing experiments to screen for a pair of highly sensitive and specific paired antibodies. This mainly includes the following steps:

[0094] (1) Coat with 50ug / ml of mRNA-ribosome-nanobody complex (amino acid sequence as shown in SEQ ID NO: 1~6) 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 hours;

[0096] (3) Wash the plate three times with 0.05% PBST, then add 10 8 The inactivated Staphylococcus aureus obtained in Example 1 with cfu / ml was incubated at 37°C for 1 hour.

[0097] (4) Wash the plate three 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) enzyme-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℃ for 15 min, and then identify the results; the results are as follows: Figure 11 As shown, Nb26 and Nb42 have the best performance.

[0100] Example 4: Synthesis of AIE fluorescent probe for Staphylococcus aureus nanobody

[0101] 1) Take 100µL of AIE fluorescent microsphere solution (1 wt%), add 500µL of MES pH 6.0 buffer to wash once, centrifuge at 12000rpm for 15min and remove the supernatant;

[0102] 2) Add 500µL of MES pH 6.0 buffer to reconstitute the microspheres, then add 30µL of EDC and 90µL of NHS (both at a concentration of 3mg / mL; the MES pH 6.0 buffer should be freshly prepared before use). In the dark, rotate and vortex for 30 min, then centrifuge at 12000rpm for 15 min and discard the supernatant.

[0103] 3) Wash once with 500µL MES pH 6.5 buffer, reconstitute the microspheres with 500µL MES pH 6.5 buffer, add 20µg labeled antibody Nb42 (the amount of antibody varies depending on the concentration), and rotate and vortex in the dark for 2 hours. Centrifuge at 12000rpm for 15 minutes and discard the supernatant.

[0104] 4) Add 500µL of blocking buffer (3% casein) to reconstitute, rotate and shake in the dark for 1 hour, centrifuge at 12000rpm for 15 minutes and discard the supernatant;

[0105] 5) Add 500µL of preservation solution (preservation solution: 0.02mTris + 0.1%Tween-20 + 0.5%BSA + 0.03%Proclin300) and wash once, then centrifuge to remove the supernatant;

[0106] 6) Redissolve in 100µL of preservation solution and store at 2-8℃.

[0107] Furthermore, the pH of the Staphylococcus aureus nanobody AIE fluorescent probe was adjusted using potassium carbonate, with an addition amount between 30 and 70 μL, to achieve a pH of 6 to 8. The results are as follows... Figure 10 As shown, the detection effect of the Staphylococcus aureus nanobody AIE fluorescent probe was best at pH 8. Among them, Figure 10 The pH values ​​from left to right are 6, 7, and 8.

[0108] Example 5

[0109] (1) Coating antigens: Different target antigens (e.g., Staphylococcus aureus ATCC25923, Listeria monocytogenes ATCC19115, Vibrio parahaemolyticus ATCC17802, Rotavirus, Norovirus, Escherichia coli, Salmonella ATCC13076 antigens / proteins; the specific concentration needs to be optimized according to the actual antigen characteristics) were coated onto different wells of a 96-well ELISA plate and incubated overnight at 4°C. After washing the plate, unbound sites were blocked with 5% skim milk and incubated at 37°C for 2 hours.

[0110] (2) Addition of ribosome display complex: The mRNA-ribosome-protein complex obtained by in vitro transcription and translation (where the mRNA encodes a nanobody library containing different sequences) was added to each well of an ELISA plate coated with different antigens. Incubate at 37°C for 1 hour. Wash three 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 (OD450 nm) using a microplate reader. Plot the reaction results of each nanobody with different antigens into bar graphs and compare them with the blank control to obtain the results as follows. Figures 1-7 The results are shown.

[0113] Example 6: Rapid Identification Steps for Test Strips

[0114] 1) Place the NC membrane in a vacuum drying oven and dry at 37 ℃ for 60-100 min. According to the structure of the chromatography test strip, attach the NC membrane to the PVC base plate, press the sample pad on one side against the conjugate pad, press the conjugate pad against the NC membrane, and press the absorbent pad on the other side against the NC membrane. Press the two sides together by about 2-6 mm to assemble the test strip.

[0115] 2) Using a gold sputtering spectrometer, streak 1-2 mg / mL Staphylococcus aureus-coated antibody Nb26 (pH 8) onto the test line and 2.5 mg / mL SuperC+ antibody onto the control line; the spacing between the test lines should be 2-4 mm, and the sample should be dried in a vacuum drying oven for 4-6 hours.

[0116] 3) Use a test strip cutter to cut the test strips into strips of 2-8×60-100 mm, seal them in bags and set aside.

[0117] 4) Sensitivity detection: Take 100 μL of different initial concentrations (1×10⁻⁶) 3 ~1×10 8 The amplification product (CFU / mL) was added to a final concentration of 0.05% Tween-20, and then dropped onto the sample pad of the prepared test strip. A blank control was also added. After standing for 10 min, the luminescence of the detection line and control line was observed under 365 nm UV excitation. The results are as follows: Figure 8 As shown, the light emission pattern is 10 from left to right. 8 10 7 10 6 10 5 10 4 10 3 Decreasing CFU / mL, detection limit is 10. 4 CFU / mL.

[0118] 5) Specificity detection: Take 100 μL of the amplification product and mix it with the same concentration (10) 6 A mixture of Staphylococcus aureus (ATCC 25923), Salmonella, Shigella (ATCC 25931), rotavirus, FOB (brand: Sigma-Aldrich, catalog number: H0267), and norovirus (CFU / mL) was prepared, and Tween-20 was added to a final concentration of 0.05%. This mixture was then dropped onto the sample pad of the prepared test strip, allowed to stand for 10 min, and the luminescence of the test line and control line was observed under 365 nm UV excitation. Results are as follows: Figure 9 As shown, nanobody antigens specifically recognize Staphylococcus aureus. From left to right, they represent 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:

2.

2. A gene encoding the nanobody of claim 1.

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

8.

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

5. A Staphylococcus aureus nanobody AIE fluorescent probe, characterized in that, Includes the nanobody described in claim 1.

6. The use of the nanobody of claim 1 in the preparation of a detection reagent or kit that specifically recognizes Staphylococcus aureus.

7. A detection reagent or test strip, characterized in that, Includes the nanobody described in claim 1.

8. The detection reagent or test strip according to claim 7, characterized in that, It also contains nanobodies with amino acid sequences as shown in any one of SEQ ID NO: 3 to 6.

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