Nanometer antibody capable of specifically recognizing staphylococcus aureus as well as preparation method and application of nanometer antibody

Abstract

The invention discloses a nano antibody capable of specifically recognizing staphylococcus aureus as well as a preparation method and application of the nano antibody. A ribosome display technology is adopted, a nano antibody pair with a good pairing effect is screened out through construction of a nano antibody display library, three rounds of high-throughput screening and ELISA verification, and the nano antibody pair is used for in-vivo detection of actual pathogenic bacteria in combination with an immunochromatography technology. The nano antibody disclosed by the invention shows high affinity, high specificity and achievable detection limit when being combined with staphylococcus aureus, has the advantages of good stability, low cost and the like 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] The invention relates to a nano antibody specifically recognizing Staphylococcus aureus and a preparation method and application thereof, belonging to the field of nano antibodies. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is a Gram-positive coccus. Its pathogenicity is related to the multiple virulence factors it secretes, including adhesins, invasins, and immune escape proteins. For example, Staphylococcal protein A (SpA) expressed on the surface of Staphylococcus aureus can bind to host IgG (immunoglobulin G) to interfere with the host immune system, and clumping factor A (ClfA) can bind to fibrinogen to promote bacterial adhesion and immune escape. At present, monoclonal antibodies against Staphylococcus aureus mainly target its surface proteins, such as SpA and ClfA. However, due to the non-specific binding characteristics of Staphylococcus aureus protein A, as well as the shortcomings of traditional antibodies such as long preparation cycle, poor stability, and high cost, its application in rapid detection is limited.

[0003] At present, 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, which greatly limits the screening efficiency of Staphylococcus aureus. Although PCR technology has high sensitivity, it is cumbersome to operate and also requires professional equipment and personnel, which is not suitable for rapid on-site 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 with dual fluorescence quantitative PCR. Although this method attempts to improve the specificity and sensitivity of detection, the inherent operational complexity and long detection time of PCR technology itself cannot be avoided, which limits its application in rapid on-site detection. Traditional immunoassays, such as ELISA, have good specificity, but their operation steps are complicated and the detection time is long. Although the traditional test strip detection method is simple and fast, it has limited sensitivity and high detection limit, which is 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), discloses a Staphylococcus aureus detection method based on surface enhanced Raman spectroscopy (SERS). Although extremely high detection sensitivity and accuracy can be achieved, its high instrument and consumable costs limit its promotion in practical applications. Therefore, there is an urgent need for a faster, more sensitive and cost-controlled means as a detection method for 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 highly specific nano-antibody that can specifically identify Staphylococcus aureus and 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 problems, the present invention provides a nanobody (VHH antibody), the amino acid sequence of the nanobody is shown in any one of SEQ ID NOs: 1 to 6.

[0006] Among them, in 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), pairing experiments showed that nanoantibodies Nb26 and Nb42 have the best pairing effect and can form paired antibodies for sandwich lateral flow chromatography test strips.

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

[0008] Wherein, the nucleotide sequence of the gene is shown in any one of SEQ ID NOs: 7 to 12.

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

[0010] The present invention also provides a method for preparing the nanobody, comprising the following steps: (1) Extract alpaca lymphocytes, splice and recombinant specific gene fragments into phagemids and transfer them into Escherichia coli to construct a nanoantibody phage library; (2) Inactivated Staphylococcus aureus was used as coating protein to screen for anti-Staphylococcus aureus nanoantibodies.

[0011] Wherein, the method for constructing the nanobody phage display library comprises the following steps: (1) Amplifying a fragment encoding a nanobody gene from alpaca lymphocytes and introducing Sfi I restriction sites at both ends of the nanobody; (2) Recombination with phagemid pComb3XSS and ligation with T4 ligase to construct a phage display library.

[0012] Specifically, the present invention also provides a method for preparing the above-mentioned nanoantibody that specifically recognizes Staphylococcus aureus, the method comprising the following steps: 1: In vitro transcription and translation: The nanobody phage library is cloned into a ribosome display vector. Afterwards, the ribosome display vector is transcribed into mRNA using an in vitro transcription and translation system, and translated into a nanobody-ribosome-mRNA complex to form a ribosome display library; 2: Affinity screening: Inactivated Staphylococcus aureus (ATCC 25923) is coated on a solid phase carrier (ELISA plate is used in this experiment). After that, the ribosome display library obtained by in vitro transcription and translation is added to the system containing immobilized Staphylococcus aureus and incubated at a suitable temperature and time. During this period, the nanoantibodies displayed on the ribosomes will interact with Staphylococcus aureus. The nanoantibody-ribosome-mRNA complex that specifically binds to the target will be captured, while the unbound complex will remain in the solution. Remove the unbound nanoantibody-ribosome-mRNA complex to reduce background noise and improve the specificity of the screening. Finally, a specific elution method is used to elute the nanoantibody-ribosome-mRNA complex bound to Staphylococcus aureus from the solid phase carrier. (The present invention uses acidic elution: elution with a low pH buffer, 0.1 M glycine-HCl (pH 2.2). After elution, it is necessary to neutralize with a neutral buffer immediately to protect the integrity of the mRNA).

[0013] 3: Recover and amplify VHH genes: Extract mRNA from the ribosome-mRNA complex bound to Staphylococcus aureus. Use the mRNA extracted by reverse transcription PCR (RT-PCR) as a template and amplify the VHH gene fragment using primers matching the ribosome display vector. Finally, multiple rounds of screening are performed to obtain high-affinity specific nanobodies, and mRNA is extracted from the eluate for subsequent RT-PCR amplification.

[0014] 4: Cloning, expression and purification: The amplified VHH gene fragment is cloned into a suitable expression vector, transformed into an expression system such as Escherichia coli for expression, and purified by affinity chromatography to obtain soluble nanobodies.

[0015] 5: Binding activity and specificity identification: The binding activity of the purified nanobody with Staphylococcus aureus was identified by ELISA. At the same time, cross-reaction experiments were performed using other bacteria (Escherichia coli, Salmonella, Vibrio parahaemolyticus, etc.) to verify the specificity of the nanobody.

[0016] The present invention also provides the use of the nanobody or the gene in preparing a Staphylococcus aureus nanobody AIE fluorescent probe.

[0017] The present invention also provides a Staphylococcus aureus nanobody AIE fluorescent probe, comprising the nanobody or the gene.

[0018] The present invention also provides the use of the nano antibody or the gene in preparing a detection reagent or a kit for specifically identifying Staphylococcus aureus.

[0019] The present invention also provides a detection reagent or a test strip, comprising the nano antibody or the gene.

[0020] 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 aerosols. When Staphylococcus aureus is present, the detection line (T line) and the quality control line (C line) on the test strip show red fluorescence. If Staphylococcus aureus is not present, the quality control line shows red fluorescence and the detection line does not show color.

[0021] The present invention also provides the use of the nanobody or the gene in constructing an immunoassay platform for Staphylococcus aureus.

[0022] The present invention also provides a method for detecting Staphylococcus aureus in a biological sample based on a POCT test strip, comprising the following steps: (1) Paste the NC membrane, absorbent pad, conjugate pad, and sample pad on the PVC base plate and assemble according to the specifications of the test strip; draw a line of Super monoclonal antibody on the NC membrane as the quality control line (C line) and draw a line of anti-Staphylococcus aureus nanoantibody Nb26 on the NC membrane as the detection line (T line) and dry for 1-2 h.

[0023] (2) According to the specifications of the test strips, the test strips were cut into test strips using a test strip cutting machine, and the AIE fluorescent microspheres labeled with the anti-Staphylococcus aureus nanoantibody Nb42 were added to the conjugate pad and dried at 37°C for 1-2 h. The microspheres were added dropwise to the sample pad of the prepared test strips using a pipette. After chromatography for 5-10 min, the luminescence of the T and C lines of the test strips was observed under ultraviolet light to evaluate the sensitivity of this method in detecting Staphylococcus aureus in biological samples.

[0024] Nanobodies (VHH) are the variable regions of heavy chain antibodies isolated from camelids, with a molecular weight of about 15 kDa, which is only 1 / 10 of traditional antibodies. Compared with traditional antibodies, nanobodies have many advantages: higher affinity and specificity, able to recognize hidden epitopes that are difficult for traditional antibodies to recognize (such as the A region of ClfA); excellent stability, even at high temperatures or extreme pH conditions can still maintain activity; easy to prepare and modify in large quantities through genetic engineering technology; good tissue penetration, and lower production costs. Combining nanobodies with lateral flow chromatography can develop rapid diagnostic test strips with higher sensitivity, stronger specificity, better stability, and lower cost, overcome the limitations of traditional methods, and achieve rapid, convenient, and in-situ detection of Staphylococcus aureus, which has broad application prospects in food safety, clinical diagnosis and other fields.

[0025] The present invention also provides an application of the above-mentioned nanoantibody pair that specifically recognizes Staphylococcus aureus. The ribosome-displayed nanoantibody pair of the present invention can specifically bind to Staphylococcus aureus. Combining the pair of nanoantibodies with a lateral flow chromatography method can achieve in situ and rapid detection of Staphylococcus aureus. The method comprises the following steps: (1) Place the NC membrane in a vacuum drying oven and dry it for later use; stick the NC membrane, water absorbent pad, conjugate pad, and sample pad on the PVC base plate, with the sample pad on the conjugate pad, the conjugate pad on the NC membrane, and the water absorbent pad on the NC membrane by about 2-4 mm; draw a line of SuperC monoclonal antibody on the NC membrane as the quality control line (C line), and draw a line of 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; (2) According to the specifications of the test strips, the test strips were cut into strips using a test strip cutting machine. The fluorescent microspheres labeled with the anti-Staphylococcus aureus nanoantibody Nb42 were added to the conjugate pad and dried at 37 °C for 2 h. A certain volume of Tween-20 was added to the sample to be tested and dripped onto the sample pad of the prepared test strip using a pipette. After chromatography for 10 min, the luminescence of the T and C lines of the test strips was observed under 365 nm ultraviolet light to evaluate the method for detecting the concentration of Staphylococcus aureus in the sample.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Compared with traditional monoclonal antibodies, the ribosome-displayed nanoantibodies of the present invention have the advantages of small size, high stability, simple preparation, low cost, and large-scale production; 2. The immunochromatographic method provided by the present invention has fast detection speed, strong specificity and high sensitivity, and can realize multi-scenario, immediate and in-situ detection of Staphylococcus aureus; 3. Traditional monoclonal antibodies are affected by the binding of the Fc end with Staphylococcus aureus SPA, making it difficult to detect Staphylococcus aureus directly. They can only detect toxic substances secreted by bacteria indirectly, making it difficult to accurately assess the infection status of patients and the contamination status of water sources and food. The present invention can achieve direct detection of Staphylococcus aureus. 4. The cell-free system of the nanoantibody library 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 a nanoantibody library based on a cell-free system is of great value for the efficient screening of nanoantibodies; 5. This time, a pair of Staphylococcus aureus nanoantibodies with particularly good matching effect was screened out, which effectively improved the detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 To verify the affinity results of 6 phage-displayed nanobodies by Phage-ELISA, the horizontal axis is the nanobodies and the vertical axis is the absorbance value at 450 nm; Figure 2 The results of antigen specificity verification of Listeria monocytogenes ATCC19115 are shown in Figure 1. The horizontal axis is the nanoantibody and the vertical axis is the absorbance value at 450 nm. Figure 3 The results of antigen specificity verification of Vibrio parahaemolyticus ATCC17802 are shown in Figure 1. The horizontal axis is the nanoantibody and the vertical axis is the absorbance value at 450 nm. Figure 4 The results of antigen specificity verification of rotavirus are shown in Figure 2. The horizontal axis is the nanoantibody and the vertical axis is the absorbance value at 450 nm. Figure 5 The results of antigen specificity verification of Norovirus are shown in Figure 2. The horizontal axis is the nanoantibody and the vertical axis is the absorbance value at 450 nm. Figure 6 The results of antigen specificity verification of Escherichia coli (wild E. coli), the horizontal axis is the nanoantibody, and the vertical axis is the absorbance value at 450nm; Figure 7 The antigen validation specificity result of Salmonella ATCC13076, the horizontal axis is the nanoantibody, and the vertical axis is the absorbance value at 450nm; Figure 8 The Staphylococcus aureus test strips are used to detect different concentrations of Staphylococcus aureus; Fig. 9 To identify the specificity of the Staphylococcus aureus test strip; Fig.10 Optimization of pH for fluorescent probes; Fig.11 To screen a pair of nanoantibodies with the best pairing effect through the chessboard method. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0029] Main experimental materials: Staphylococcus aureus standard strain (ATCC 25923) Main reagents: Chicken ovalbumin and bovine ovalbumin were purchased from Sigma, USA; horseradish peroxidase (HRP) enzyme-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) colorimetric solution, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; LB broth and 2×YT culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0030] Main reagent formula: 1. 2×YT liquid medium: weigh 31 g of 2×YT powder, dissolve it in 1000 mL of ultrapure water, and sterilize it by high pressure at 121°C for 15 min; 2. 2×YT solid medium: weigh 31 g 2×YT powder and 18 g agar, dissolve in 1000 mL ultrapure water, and sterilize at 121°C for 15 min; 3. LB liquid medium: weigh 25 g LB medium, dissolve in 1000 mL ultrapure water, and sterilize at 121°C for 15 min; 4. 20% polyethylene glycol (PEG)-NaCl: 50 g PEG-8000, 36 g NaCl, dissolve in ultrapure water by heating, dilute to 250 mL, and sterilize by high pressure for 15 min; 5. Eluent: 0.2 M glycine (Gly), add hydrochloric acid to adjust pH = 2.2, and sterilize by high pressure for 15 min; 6. Neutralization buffer: 1M Tris, add hydrochloric acid to adjust pH to 9.1, and sterilize by high pressure for 15 minutes.

[0031] Example 1: Inactivation of Staphylococcus aureus (1) Streak the standard strain of Staphylococcus aureus onto an LB plate and incubate at 37°C for 12-14 h; (2) Pick a single colony from the plate, inoculate it into 5 ml of LB medium, and incubate at 37°C for 12-14 h; (3) Spread and count on LB plates and incubate at 37°C for 12-14 h; (4) Take 1 ml of the counted bacterial solution, centrifuge at 8000 rpm for 2 min, resuspend in PBS, and repeat three times; (5) The metal bath is preheated to 90°C in advance and heated at 90°C for half an hour to inactivate the strain.

[0032] Example 2: VHH gene amplification and construction of nanobody ribosome display library (1) RNA extraction: Extract total RNA from B lymphocytes of immunized alpacas; (2) cDNA synthesis: Use SuperScript IV reverse transcriptase to reverse transcribe RNA into cDNA (reaction system: 1 μg RNA, 1 μL reverse transcriptase, 4 μL 5X buffer, 2 μL 10 mM dNTPs, 1 μL RNase inhibitor, add water to 20 μL; reaction conditions: 42°C for 60 minutes, 70°C for 15 minutes); (3) VHH gene amplification: cDNA from multiple B lymphocytes was amplified by PCR 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, add water 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 are VHH-F (SEQ ID NO:13) and VHH-R (SEQ ID NO:14); (4) SfiI digestion: Use SfiI restriction endonuclease to digest the PCR product and ribosome display vector pRibVec (purchased from Agilent). The reaction system: 5 μL PCR product or vector, 1 μL SfiI, 2 μL 10X buffer, add water to 20 μL; reaction conditions: 37℃ 2 hours; (5) Use T4 DNA ligase to ligate the VHH gene fragment after digestion 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°C overnight); (6) In vitro transcription: Transcribe the ligation product into mRNA using the TNT T7 Quick Master Mix in vitro transcription kit to obtain the nanobody ribosome display library (follow the instructions of the kit, the kit is TNT® T7 PCRDNA Quick System, purchased from Promega (Beijing) Biotechnology Co., Ltd.).

[0033] Example 3 Screening and identification of S. aureus ribosome-displayed nanobodies 1. Antigen coating: The Staphylococcus aureus purified in Example 1 was coated onto an ELISA plate at a coating volume of 100 µL / well at 4°C overnight.

[0034] 2. Blocking: Use 3% BSA to block non-specific binding sites. The blocking volume is 300 µL / well and incubate at 37°C for 2 hours.

[0035] 3. Binding: Add the mRNA-ribosome-nanobody complex in the nanolibrary constructed in Example 2 and incubate at 37° C. for 60 minutes.

[0036] 4. Washing: Use PBST to wash the unbound complexes. Wash 5 times with a washing solution concentration of 0.05%, which can be adjusted according to the screening rounds.

[0037] 5. Elution: Elute the bound mRNA with 200 µL 0.1 M Gly-HCl (pH 2.2) for 15 minutes. Immediately thereafter, add 20 µL 1 M Tris-HCl (pH 9.1) to neutralize the eluate.

[0038] 6. Recover mRNA: Use a commercial kit to recover mRNA from the eluate.

[0039] 7. RT-PCR amplification: Reverse transcription PCR amplification was used to recover the VHH nanoantibody gene. The reaction system: cDNA 800ng + AlpVh-LD (10uM) 1uL + CH2-R (10uM) 1uL + 10×PCR Buffer 7uL + Hot STAR Taq DNA Polymerase 0.1uL + dNTP mix (10uM) 0.4uL + add ddH2O to 50uL.

[0040] The reaction procedure is as follows: 1×98°C 15min 35×96°C 1min 35×61.5 / 62.8 / 64.7 / 68°C 1min 35×70°C 1min 1×70°C 10min 1×4°C Store at 4°C Among them, the vector is pCMV-3Tag (purchased from Agilent); the primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and 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).

[0041] The primer usage was carried out in accordance with the instructions for RNAiso reagent of TAKARA: PrimeSTAR high-fidelity DNA polymerase was used to obtain the variable region encoding gene of the heavy chain antibody by nested PCR. The first round of PCR amplified cDNA with primers AlpVh-LD and CH2-R respectively. Then, the first round of amplification products were used as templates for the second round of amplification, and were amplified with the remaining primers respectively, recovered and quantified by the recovery kit, and stored at -20°C for future use.

[0042] 8. Multiple rounds of screening: Repeat steps 1-7 for 3 rounds of screening, gradually improving the rigor of the screening. Finally, the desired mRNA-ribosome-nanobody complex (amino acid sequence as shown in SEQ ID NO: 1-6) is obtained.

[0043] 9. Use the chessboard method to conduct a pairing experiment to screen out a pair of highly sensitive and highly specific paired antibodies, which mainly includes the following steps: (1) Coating with 50ug / ml of mRNA-ribosome-nanobody complex (amino acid sequence as shown in SEQ ID NO: 1-6) and incubating overnight; (2) Wash the plate three times with 0.05% PBST and incubate with 5% skim milk powder at 37°C for 2 h; (3) Wash the plate 3 times with 0.05% PBST and add 10 8 cfu / ml The inactivated Staphylococcus aureus obtained in Example 1 was incubated at 37°C for 1 h; (4) Wash the plate three times with 0.05% PBST to recover bound mRNA and incubate at 37°C for 45-60 min; (5) Wash the plate six times with 0.05% PBST, add 100 μL horseradish peroxidase (HRP) enzyme-anti-M13 monoclonal antibody to each well, and incubate at 37°C for 45-60 min; (6) Wash the plate 7 times with 0.05% PBST, add 100 μL TMB colorimetric solution, incubate at 37°C for 15 min and identify the results. Fig.11 As shown, Nb26 and Nb42 have the best effects.

[0044] Example 4: Synthesis of AIE fluorescent probe of gold-staphylococcus nanobody 1) Take 100 µL of AIE fluorescent microsphere solution (1 wt%), add 500 µL MES PH6.0 buffer to wash once, centrifuge at 12000 rpm for 15 min and remove the supernatant; 2) Add 500µL MES PH6.0 buffer to re-dissolve the microspheres, then add 30µL EDC and 90µL NHS (both at a concentration of 3 mg / mL, MES PH6.0 buffer prepared immediately before use), rotate and shake in the dark for 30 minutes, centrifuge at 12000rpm for 15 minutes, and remove the supernatant; 3) Add 500µL MES PH 6.5 buffer to wash once, add 500µL MES PH6.5 buffer to re-dissolve the microspheres, add 20µg labeled antibody Nb42 (the amount of antibody varies according to the concentration), rotate and shake in the dark for 2h, centrifuge at 12000rpm for 15min, and remove the supernatant; 4) Add 500 µL of blocking solution (3% casein) to re-dissolve, rotate and shake in the dark for 1 hour, centrifuge at 12000 rpm for 15 minutes, and remove the supernatant; 5) Add 500µL preservation solution (preservation solution: 0.02mTris+0.1%Tween-20+0.5%BSA+ 0.03%Proclin300), wash once, centrifuge and remove the supernatant; 6) Add 100 µL of storage solution to reconstitute and store at 2-8°C.

[0045] Furthermore, potassium carbonate was used to adjust the pH of the AIE fluorescent probe of the aureobacterium nanobody, and the amount added was between 30 and 70 uL, so that the pH was 6 to 8. Fig.10 As shown, when pH is 8, the detection effect of Staphylococcus aureus nanobody AIE fluorescent probe is the best. Fig.10 The pH from left to right is 6, 7, and 8.

[0046] Example 5

[0047] (1) Coating antigen: Different target antigens (e.g., antigens / proteins of Staphylococcus aureus (S. aureus) ATCC25923, Listeria monocytogenes (Listeria monocytogenes) ATCC19115, Vibrio parahaemolyticus (Vibrio parahaemolyticus) ATCC17802, rotavirus, Norovirus, Escherichia coli, and Salmonella (Salmonella) ATCC13076, the specific concentration needs to be optimized according to the actual antigen characteristics) are coated in different wells of a 96-well ELISA plate and incubated at 4°C overnight. After washing the plate, block the unbound sites of the ELISA plate with 5% skim milk and incubate at 37°C for 2 hours.

[0048] (2) Add ribosome display complex: Add the mRNA-ribosome-protein complex obtained by in vitro transcription and translation (where the mRNA encodes a library of nanoantibodies with different sequences) to each well of the ELISA plate coated with different antigens. Incubate at 37°C for 1 hour. Wash three times with 0.05% PBST to remove unbound complexes.

[0049] (3) Add detection antibody: Add HRP-labeled anti-His tag antibody and incubate at 37°C for 45-60 minutes. Wash six times with 0.05% PBST to remove unbound detection antibody.

[0050] (4) Add color substrate and measure: Add 100 μL TMB color development solution to each well, incubate at 37°C for 15 minutes, and measure the absorbance (OD450nm) using an enzyme reader. Plot the reaction results of each nanobody with different antigens into a bar graph and compare them with the blank control to obtain the following results: Figures 1 to 7 Results shown.

[0051] Example 6: Test strip rapid identification steps 1) Place the NC membrane in a vacuum drying oven and dry it at 37 °C for 60-100 min. According to the structure of the chromatography test strip, the NC membrane is pasted on the PVC bottom plate, with the sample pad on one side pressing the binding pad, the binding pad pressing the NC membrane, and the water-absorbing pad on the other side pressing the NC membrane with a pressure of about 2-6 mm to assemble the test strip.

[0052] 2) Use a gold spray film streaking instrument to draw the test line with 1-2 mg / mL Staphylococcus aureus coated antibody Nb26 (pH 8), and draw the quality control line with 2.5 mg / mL SuperC+ antibody; the spacing between the test lines is 2 - 4 mm, and place in a vacuum drying oven to dry for 4- 6 hours.

[0053] 3) Use a test strip cutting machine to cut the test strips into 2-8×60-100 mm test strips and seal them for later use.

[0054] 4) Sensitivity test: Pipette 100 μL of different initial concentrations (1×10 3 ~1×10 8 CFU / mL) amplification product, and add Tween-20 with a final concentration of 0.05%, drip it on the sample pad of the prepared test strip, and drip the set blank control, let it stand for 10 minutes, and observe the luminescence of the test line and quality control line under 365 nm ultraviolet light excitation. The results are as follows Figure 8 As shown, the luminous situation is 10 from left to right 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 CFU / mL decreases, and the detection limit is 10 4 CFU / mL.

[0055] 5) Specificity detection: Take 100 μL of the amplified product and add it to the same concentration (10 6 CFU / mL) of Staphylococcus aureus liquid (ATCC 25923), Salmonella liquid, Shigella liquid (ATCC 25931), rotavirus, FOB (brand: Sigma-Aldrich, catalog number: H0267), and norovirus were mixed, and Tween-20 with a final concentration of 0.05% was added to it, and dropped onto the sample pad of the prepared test strip, and allowed to stand for 10 minutes. The luminescence of the test line and the quality control line was observed under 365 nm ultraviolet light excitation. The results are as follows Fig. 9 As shown, the nanoantibody antigen specifically recognizes Staphylococcus aureus. From left to right, they are Staphylococcus aureus, Salmonella, Shigella, rotavirus, FOB, and Norovirus.

Claims

1. A nanobody, characterized in that The amino acid sequence of the nanobody is shown in any one of SEQ ID NOs: 1 to 6.

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 any one of SEQ ID NOs: 7 to 12.

4. A method for preparing the nanobody according to claim 1, characterized in that: The following steps are involved: (1) Extract alpaca lymphocytes, splice and recombinant specific gene fragments into phagemids and transfer them into Escherichia coli to construct a nanoantibody phage library; (2) Inactivated Staphylococcus aureus was used as coating protein to screen for anti-Staphylococcus aureus nanoantibodies.

5. The preparation method according to claim 4, characterized in that: The method for constructing the nanobody phage display library comprises the following steps: (1) Amplifying a fragment encoding a nanobody gene from alpaca lymphocytes and introducing Sfi I restriction sites at both ends of the nanobody; (2) Recombination with phagemid pComb3XSS and ligation with T4 ligase to construct a phage display library.

6. Use of the nanobody according to claim 1 or the gene according to claim 2 or 3 in the preparation of a Staphylococcus aureus nanobody AIE fluorescent probe.

7. A Staphylococcus aureus nanobody AIE fluorescent probe, characterized in that: Comprising the nanobody according to claim 1 or the gene according to claim 2 or 3.

8. Use of the nanobody according to claim 1 or the gene according to claim 2 or 3 in the preparation of a detection reagent or a kit for specifically recognizing Staphylococcus aureus.

9. A detection reagent or test strip, characterized in that: Comprising the nanobody according to claim 1 or the gene according to claim 2 or 3.

10. Use of the nanobody according to claim 1 or the gene according to claim 2 or 3 in constructing an immunoassay platform for Staphylococcus aureus.

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