Nanobody targeting staphylococcus aureus and use thereof

Abstract

This invention discloses a nanobody targeting Staphylococcus aureus and its applications. The invention utilizes nanobody phage library construction and phage screening technology to obtain a pair of Staphylococcus aureus nanobody pairs with good pairing performance, and constructs a detection kit based on sandwich ELISA for the detection of actual live bacteria in food. The nanobody disclosed in this invention exhibits high affinity and high specificity when binding to Staphylococcus aureus, and compared with monoclonal antibodies, it has advantages such as good stability and low cost, allowing for large-scale production and showing broad application prospects in the field of Staphylococcus aureus detection and analysis. The detection kit disclosed in this invention can achieve 4.27^10 4 The detection limit of cfu / ml.

Description

Technical Field

[0001] This invention relates to a nanobody targeting Staphylococcus aureus and its application, belonging to the field of nanobodies targeting Staphylococcus aureus. This invention is a divisional application of application number 202510287680.8, filed on March 12, 2025, entitled "A Nanobody Targeting Staphylococcus aureus, its Preparation Method and Application," filed on March 12, 2025. Background Technology

[0002] Staphylococcus aureus is a Gram-positive coccus belonging to the genus Staphylococcus. It grows in grape-like clusters, and its cell wall is composed of peptidoglycan and teichoic acid. It expresses various virulence factors, such as staphylococcal protein A (SpA) and agglutination factor A (ClfA), mediating bacterial adhesion, host tissue invasion, and immune evasion. Staphylococcus aureus infection can cause localized purulent infections, pneumonia, pseudomembranous colitis, etc., and in severe cases, can develop into systemic infections such as sepsis and septicemia, even leading to death. Foodborne Staphylococcus aureus infection is currently very common, causing acute gastroenteritis. Existing monoclonal antibodies mainly target surface proteins such as SpA and ClfA, but the non-specific binding of SpA, as well as the high cost and poor stability of traditional antibodies, limit their application in rapid detection.

[0003] Current methods for detecting Staphylococcus aureus mainly include bacterial culture, PCR, and immunoassay. Bacterial culture, as the gold standard, is time-consuming, typically requiring 24-48 hours to obtain results, making it unsuitable for rapid detection. While PCR technology offers high sensitivity, it is complex to operate, requiring specialized equipment and personnel, and is unsuitable for rapid on-site testing or in resource-limited areas. Immunoassay, represented by enzyme-linked immunosorbent assay (ELISA) kits, has become an important method for pathogen detection due to its advantages of low operational difficulty, rapid detection speed, and in-situ detection capabilities. Traditional monoclonal antibodies, composed of heavy and light chains, are the most commonly used detection elements in immunoassays. However, high cost, poor stability, and difficulty in large-scale production limit the application of monoclonal antibodies as immunoassay elements. Furthermore, the presence of the SpA protein on the surface of Staphylococcus aureus binds to the Fc terminus of traditional monoclonal antibodies, severely interfering with detection accuracy. Therefore, there is an urgent need to develop a monoclonal antibody alternative targeting Staphylococcus aureus.

[0004] Nanobodies (VHHs) are unique antibody fragments discovered in camel-like animals, containing only a single heavy chain variable region, which avoids interference from the SpA protein on the surface of Staphylococcus aureus. With a molecular weight of approximately 15 kDa, nanobodies are much smaller than traditional antibodies, exhibiting better tissue penetration and the ability to bind to epitopes that are difficult for traditional antibodies to reach. Currently, nanobodies have shown great potential in various biotechnology fields, including pathogen detection, human diagnosis, and treatment. Therefore, developing a Staphylococcus aureus detection kit based on nanobodies has broad application prospects. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nanobody with high sensitivity and strong specificity targeting Staphylococcus aureus and its application.

[0006] Technical Solution: To solve the above-mentioned technical problems, this invention provides a nanobody targeting Staphylococcus aureus. The nanobody is a VHH antibody with the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. This nanobody exhibits excellent binding performance and specificity to Staphylococcus aureus.

[0007] The VHH antibody fragment has the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0008] Secondly, the present invention also provides a method for preparing the above-described Staphylococcus aureus-targeting nanobody, the method comprising the following steps:

[0009] (1) Staphylococcus aureus (ATCC25923) was cultured overnight, plated and counted, and then sterilized in a 90℃ metal bath for 30 min to obtain inactivated Staphylococcus aureus.

[0010] (2) Unimmunized alpaca lymphocytes were extracted and total RNA was extracted. cDNA was synthesized and the fragment encoding the VHH gene was amplified by PCR. After digestion with a fast digestion enzyme, the fragment was re-ligated with the phage particle pComb3XSS using T4 ligase. The recombinant phage particle was then transformed into E. coli TG1 to construct a nanobody natural phage library. The library size was determined to be approximately 2 × 10⁻⁶. 10 The sequence has low repetition and good diversity;

[0011] (3) Inactivated Staphylococcus aureus was coated onto enzyme-labeled wells, and recombinant phage was added and incubated for a period of time. Non-specifically bound recombinant phage was washed away with PBST, and specifically bound phage was eluted with acid. The eluted phage was amplified, and the titer was determined for the next round of screening or analysis. 3-5 rounds of screening were performed following the "adsorption-washing-elution-amplification" steps. By changing the concentration of PBST, phage incubation time, and other conditions during the screening process, the screening was progressively improved to obtain nanobody phages with stronger affinity and specificity.

[0012] (4) After several rounds of screening, 50 phages were randomly selected for Phage-ELISA identification. Salmonella enteritidis, Escherichia coli, Vibrio parahaemolyticus protein, Listeria monocytogenes protein, SARS-CoV-2 S protein, norovirus protein, and rotavirus protein were used as negative controls, and PBS was used as a blank control. Ten recombinant phage display nanobodies with better performance and specific binding to Staphylococcus aureus were obtained. These 10 nanobody phages were amplified, plasmids were extracted, and sequenced to obtain 6 nanobodies.

[0013] Furthermore, the method for constructing the nanobody phage display library of the present invention mainly 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] Furthermore, the pH elution screening method mainly includes the following steps:

[0017] (1) Wrapped in 10 6 -10 8 Add cfu / mL inactivated Staphylococcus aureus to 400μL wells and coat at 4℃ for 10-14h.

[0018] (2) Wash the plate 5-10 times with 0.05-0.25% PBST, and block with bovine serum albumin (BSA) or ovalbumin (OVA) at 37°C for 1-2 hours;

[0019] (3) Wash the plate 5-10 times with 0.05-0.25% PBST, and add 2×10 10 Recombinant phage, incubated at 37°C for 60-120 min;

[0020] (4) Wash the plate 5-10 times with 0.05-0.25% PBST, incubate with 100-200 μL of 0.1M Gly-HCl (pH=2.2) for 8-10 min, and add 30-45 μL of 1M Tris-HCl (pH=9.1). Measure the titer and pick single phages for phage-ELISA identification.

[0021] Thirdly, this invention employs a checkerboard method for pairing experiments to screen for a pair of highly sensitive and specific paired antibodies, possessing the nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.5, and the amino acid sequences of SEQ ID NO.8 and SEQ ID NO.11, and mainly includes the following steps:

[0022] (1) Coating with 100ul, 50ug / ml nanobody and incubating overnight;

[0023] (2) Wash the plate 3 times with 0.05% PBST, and incubate with 5% skim milk powder at 37°C for 2 hours;

[0024] (3) Wash the plate three times with 0.05% PBST, then add 10 8 Inactivated Staphylococcus aureus at CFU / ml was incubated at 37°C for 1 hour.

[0025] (4) Wash the plate 3 times with 0.05% PBST, add the phage supernatant after panning, and incubate at 37℃ for 45-60 min;

[0026] (5) Wash the plate 6 times with 0.05% PBST, add 100-200 μL of anti-M13 secondary antibody to each well, and incubate at 37℃ for 45-60 min;

[0027] (6) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB colorimetric solution, incubate at 37℃ for 10-15 min and then identify.

[0028] Furthermore, this invention utilizes the successfully paired nanobodies described above as the coating antibody and the labeling antibody, respectively, to develop an ELISA detection kit. The coating antibody is Nb2, and the labeling antibody is Nb5, coupled with HRP enzyme as an enzyme-labeled secondary antibody. The kit mainly includes the following steps:

[0029] (1) Coated plates were obtained by overnight incubation with 100ul Nb2 nanobody at a concentration of 50ug / ml;

[0030] (2) Wash the plate 3 times with 0.05% PBST, and incubate with 5% skim milk powder at 37°C for 2 hours;

[0031] (3) Wash the plate three times with 0.05% PBST, and dilute each solution 10 μL. 8 10 7 106 10 5 10 4 10 3 10 2 A standard curve was plotted using standard strains with cfu / ml, and the test sample was added and incubated at 37°C for 1 hour.

[0032] (4) Wash the plate 5 times with 0.05% PBST, add enzyme-labeled secondary antibody, and incubate at 37°C for 1 hour;

[0033] (5) Wash the plate 6 times with 0.05% PBST, add colorimetric solution, incubate at 37℃ for 20 min, add 2M sulfuric acid as stop solution, and read A at 450nm.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0035] (1) Compared with traditional monoclonal antibodies, the phage-displaying nanoantibody of the present invention has the advantages of small size, high stability, simple preparation, low cost and large-scale production.

[0036] (2) The six nanobody sequences of this invention are reported for the first time both domestically and internationally, and have high innovation.

[0037] (3) This invention screened out a pair of highly sensitive and specific nanobody pairs;

[0038] (4) The ELISA kit constructed in this invention can achieve 4.27 × 10⁻⁶ ppm. 4 The detection limit for CFU / ml is much lower than that of existing immunological methods. Attached Figure Description

[0039] Figure 1 The results of Phage-ELISA validation of the affinity of six phage-displaying nanobodies were shown; the x-axis represents the phage clone number; the y-axis represents the absorbance at 450 nm.

[0040] Figures 2-8 Salmonella ATCC13076 was used respectively. Figure 2 ), wild Escherichia coli ( Figure 3 Vibrio parahaemolyticus ATCC17802 Figure 4 Listeria monocytogenes ATCC19115 Figure 5 ), SARS-CoV-2 S protein ( Figure 6 Norovirus antigen protein Figure 7), Rotavirus antigen protein ( Figure 8 The results of antigen specificity verification are shown on the x-axis as phage clone number and on the y-axis as absorbance at 450 nm.

[0041] Figure 9 The results of the nanobody pairing experiment;

[0042] Figure 10 The standard curve for the ELISA kit developed for this invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0044] Main experimental materials:

[0045] The standard strain of Staphylococcus aureus (ATCC25923), Escherichia coli BL21(DE3), Escherichia coli TG1, helper phage M13K07, and phage particle pComb3XSS were preserved in our laboratory.

[0046] Main reagents:

[0047] Chicken egg albumin and bovine egg albumin were purchased from Sigma-Aldrich, USA. Horseradish peroxidase (HRP)-anti-13 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, isopropyl-β-D-thiogalactoside (IPTG), and HRP enzyme conjugation kit were purchased from Shanghai Sangon Biotech Co., Ltd. LB broth and 2×YT medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0048] Main reagent formula:

[0049] 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.

[0050] 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.

[0051] 3. LB liquid medium: Weigh 25g of LB medium, dissolve it in 1000mL of ultrapure water, and autoclave at 121℃ for 15min.

[0052] 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.

[0053] 5. Eluent: 0.2M glycine (Gly), add hydrochloric acid to adjust pH to 2.2, autoclave for 15 min.

[0054] 6. Neutralization buffer: 1M tris(hydroxymethyl)aminomethane (Tris), add hydrochloric acid to adjust pH to 9.1, autoclave for 15 min.

[0055] Example 1: Inactivation of Staphylococcus aureus

[0056] (1) Streak the standard strain of Staphylococcus aureus on LB plates and incubate at 37°C for 12-14 hours;

[0057] (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.

[0058] (3) Count the samples on LB plates and incubate at 37°C for 12-14 hours;

[0059] (4) Take 1 mL of the bacterial culture after counting, centrifuge at 8000 rpm for 2 min, resuspend in PBS, and repeat 3 times;

[0060] (5) Preheat the metal bath to 90°C, and heat the strain at 90°C for half an hour to obtain an inactivated strain.

[0061] Example 2: Construction of a natural library of phage-displayed nanoantibodies

[0062] (1) Peripheral blood lymphocytes were extracted from multiple alpacas;

[0063] (2) Add 2 mL of Trizol and 1 mL of isopropanol to the cells, invert several times to mix, and let stand at room temperature for 10-20 min.

[0064] (3) Centrifuge at 10000-14000g for 10-20min, discard the supernatant, and obtain the RNA precipitate from the cells;

[0065] (4) Add 3 mL of 75% ethanol, invert several times to mix, let stand at room temperature for 10 min, centrifuge at 10000-14000g for 10-20 min, and discard the supernatant.

[0066] (5) Invert the tube at room temperature for 5-10 minutes to air dry or vacuum dry. Add 50 μL of DEPC-ddH2O to dissolve the RNA. Detect the RNA quality and concentration using gel electrophoresis. Use the RevertAid RT reverse transcription kit (Thermo Fisher Scientific, K1691) to reverse transcribe the extracted RNA to obtain a cDNA template.

[0067] (6) VHH fragment / pComb3XSS digestion: 5 μg VHH fragment or pComb3XSS, 100 μL RNase free H2O, 2 μL Sfi I fast digestion enzyme, 10 μL fast digestion enzyme buffer; reaction conditions: 37℃ for 60 min.

[0068] (7) Cloning of VHH fragment into phage plasmid pComb3XSS: 30g VHH fragment digested with enzymes, 100ng of digested plasmid pComb3XSS, 1μL T4 ligase, 2μL T4 ligase buffer, 10μL RNase-free H2O. Reaction conditions: 16℃ for 12h.

[0069] (8) 100-200 ng pComb3XSS-VHH recombinant phage particles were transformed into E. coil TG1 competent cells, and after culturing at 37℃ for 12 h, all single colonies on the eluted plate were used to form a phage nanobody library.

[0070] (9) Take 50-100 μL of the eluted phage library and inoculate it into 5 mL of 2×YT / ampicillin (Amp) medium (the working concentration of ampicillin is 50 ug / ml). When the medium reaches the logarithmic growth phase, add helper phage M13K07 according to the infection ratio of Escherichia coli: phage = 1 cfu / mL: 20 pfu / mL, and let it stand at 37℃ for 1 h.

[0071] (10) Add the entire 5 mL culture system to 50 mL of 2×YT / Amp / kanamycin (kana) medium (the working concentration of AMP is 50 ug / ml and the working concentration of kana is 100 ug / ml), and incubate at 37℃ for 12 h.

[0072] (11) Centrifuge at 12000-16000g for 10-15min, take the supernatant and add 12mL PEG8000 / NaCl (30%, sterile), and let stand on ice for 4-6h.

[0073] (12) Centrifuge at 12000-16000g for 30-40 min, discard the supernatant, resuspend the precipitate in 1 mL PBS, add 200-300 μL PEG8000 / NaCl (30%, sterile), and let stand on ice for 1-2 h.

[0074] (13) Centrifuge at 12000-16000g for 30-40 min, discard the supernatant, resuspend the precipitate in 200-300μL PBS, and take 10μL of phage to determine the recombinant phage titer, which can reach 2×10⁻⁶. 10 Phages are dispensed for later screening.

[0075] Example 3: Screening of Staphylococcus aureus phage-displayed nanobodies

[0076] (1) In a clean bench, wash the enzyme-labeled plate 5 times with sterile water and sterilize it with ultraviolet light for 60 minutes.

[0077] (2) Dilute the inactivated Staphylococcus aureus with PBS to a final concentration of 10. 8 Add 100-200 μL of diluted Staphylococcus aureus to each well of the microplate and coat at 4°C for 12 h.

[0078] (3) Wash the plate 5 times with PBS, pat dry with sterile paper, add 300 μL of 3% BSA-PBS blocking solution to each well, and block at 37°C for 2 h.

[0079] (4) Wash the plate 5 times with PBS, blot dry with sterile paper, and take the titer of the above-constructed solution as 2×10⁻⁶. 10 The recombinant phage library was mixed with 200 μL PBS and added to an ELISA plate, where it was incubated at 37°C for 120 min.

[0080] (5) Wash the plate 5 times with 0.1% PBST, pat dry with sterile paper, add 100 μL Gly-HCl buffer, incubate at 37°C for 10 min, aspirate the elution product, and quickly add 30-45 μL Tris-HCl buffer.

[0081] (6) Take 10 μL of phage for serial dilution, determine the titer of the eluted phage, calculate the panning recovery rate, and amplify and purify the remaining phage for the next round of screening or analysis; the amplification steps are the same as the phage library amplification steps;

[0082] (7) Steps (1)-(6) constitute the first round of amplification. The selection steps for rounds 2-5 are largely the same, with the amount of phage introduced in each round being 2×10⁻⁶. 10 The PFU / well concentration of Staphylococcus aureus was decreased sequentially to 5 × 10⁻⁶. 6 -1×10 8 The phage was blocked alternately with cfu / mL, 1-3% OVA-PBS and 1-3% BSA-PBS blocking solutions. The binding time between the phage and Staphylococcus aureus was 120-130 min, and the elution buffer concentration was 0.05%-0.25% PBST. The specific panning scheme is shown in Table 1.

[0083] Table 1. Screening process for Staphylococcus aureus phage-displayed nanobodies

[0084]

[0085] Example 4: Screening and Identification of Specific Phage Clones

[0086] After five rounds of screening, 50 phage-displaying nanobody single colonies were selected for amplification and identification by phage-ELISA. The specific operation steps are as follows:

[0087] (1) Select 25 single colony clones and inoculate them into 1 mL of 2×YT / Amp liquid medium. Incubate at 37℃ and 220 rpm for 12-14 h.

[0088] (2) Take 100-150 μL of the above culture and add it to 1 mL of 2×YT / Amp liquid culture medium. Mix well and shake at 220 rpm for 2-3 h until the logarithmic growth phase.

[0089] (3) Add helper phage M13K07 to each tube at a ratio of cell:phage = 1:1, incubate at 37℃ for 15-20 min, and then shake at 220 rpm for 30-45 min.

[0090] (4) Centrifuge at 8000-10000rpm for 2-5min at 4℃, then resuspend in 1-1.5ml of 2×YT / Amp / Kana. Incubate at 37℃ with shaking at 250rpm for 10-14h.

[0091] (5) After the culture is completed, centrifuge at 8000-10000 rpm for 10-12 min, aspirate the supernatant into a sterile centrifuge tube, label it, and store it at 4℃ for ELISA identification.

[0092] (6) Take 100-200 μL of 10 μg solution per well. 8 CFU / mL Staphylococcus aureus, 1 μg / mL bovine serum albumin, and ovalbumin were coated onto an ELISA plate and coated at 4°C for 12 h.

[0093] (7) Discard the coating solution, wash the plate 3 times with 0.05% PBST, add 250-350 μL of 3-5% skim milk to each well for blocking, and incubate at 37°C for 2 hours.

[0094] (8) Wash the plate three times with 0.05% PBST. Add 100 μL of the selected phage supernatant culture medium to each well coated with Staphylococcus aureus, bovine serum albumin and ovalbumin. Incubate at 37°C for 45-60 min.

[0095] (9) Wash the plate 6 times with 0.05% PBST, add 100-200 μL of anti-M13 secondary antibody to each well, and incubate at 37℃ for 45-60 min.

[0096] (10) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB colorimetric solution, incubate at 37℃ for 10-15 min, add 50 μL of 2M H2SO4 to each well, and measure A at 450 nm.

[0097] (11) Of the 50 clones selected, all 50 clones were able to bind to Staphylococcus aureus; among them, clones Nb2, Nb5, Nb7, Nb11, Nb18, Nb32, Nb34, and Nb47 showed good binding ability and specificity to Staphylococcus aureus. These 8 clones were amplified and sequenced using designed primers, and 6 different phage display nanobody amino acid sequences (VHH antibodies) were obtained. Their amino acid sequences are shown in SEQ ID NO.7 (Nb2), SEQ ID NO.8 (Nb5), SEQ ID NO.9 (Nb7), SEQ ID NO.10 (Nb11), SEQ ID NO.11 (Nb18), and SEQ ID NO.12 (Nb32), and their corresponding nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6. Figure 1 The binding ability of six selected recombinant phage clones to Staphylococcus aureus was shown. The horizontal axis represents the phage clone number, and the vertical axis represents the absorbance at 450 nm.

[0098] (12) To further investigate whether the screened phage display proteins have high specificity, different target antigens (Salmonella ATCC13076, wild Escherichia coli, Vibrio parahaemolyticus ATCC17802, Listeria monocytogenes ATCC19115, SARS-CoV-2 S protein, Norovirus antigen protein, and Rotavirus antigen protein) were coated into different wells of a 96-well ELISA plate, with a bacterial coating concentration of 10-1. 8 The concentration of cfu / ml and protein coating concentration were both 1ug / ml, and the plates were incubated overnight at 4°C. After washing, the unbound sites of the ELISA plate were blocked with 5% skim milk and incubated at 37°C for 2 hours. Subsequent verification steps were the same as steps (7)-(10) in this embodiment, and the verification results are as follows. Figure 2-8 As shown.

[0099] Example 5: Highly sensitive and highly specific nanobody pairing

[0100] (1) Six expression plasmids containing Staphylococcus aureus nanobodies (synthesized by Genscript Biotech, with Ndel restriction site at the 5' end, Xhol restriction site at the 3' end, and PET25b expression vector) were transformed into Escherichia coli BL21 (DE3). After being cultured to the logarithmic growth phase, the engineered bacteria were induced to express the nanobodies using isopropyl-β-D-thiogalactoside (IPTG). The expressed NP protein was purified by Ni-NAT column to obtain high-purity protein. The Staphylococcus aureus nanobodies were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA.

[0101] (2) Coating nanobodies with 100ul and 50ug / ml respectively, and incubating overnight;

[0102] (3) Wash the plate 3 times with 0.05% PBST, and incubate with 5% skim milk powder at 37°C for 2 hours;

[0103] (4) Wash the plate three times with 0.05% PBST, then add 10 8 Inactivated Staphylococcus aureus at CFU / ml was incubated at 37°C for 1 hour.

[0104] (5) Wash the plate 3 times with 0.05% PBST, add the selected phage supernatant at a concentration of 1:1, and incubate at 37℃ for 45-60 min;

[0105] (6) Wash the plate 6 times with 0.05% PBST, add 100-200 μL of anti-M13 secondary antibody to each well, and incubate at 37℃ for 45-60 min;

[0106] (7) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB colorimetric solution, and incubate at 37°C for 10-15 min;

[0107] (8) Add 50 μL of 2M H2SO4 to each well and measure A at 450 nm. The pairing results are as follows: Figure 9 As shown.

[0108] Example 6: Specific application of the present invention: Construction of a sandwich ELISA detection kit for Staphylococcus aureus based on paired nanobodies.

[0109] (1) HRP enzyme was conjugated to labeled antibodies using the HRP enzyme conjugation kit (periodate method) from Shanghai Sangon Biotech Co., Ltd. as enzyme-labeled secondary antibodies;

[0110] (2) Coated plates were obtained by overnight incubation with 100 μL of 50 μg / ml Nb2 nanobody;

[0111] (3) Wash the plate 3 times with 0.05% PBST, and incubate with 5% skim milk powder at 37°C for 2 hours;

[0112] (4) Wash the plate three times with 0.05% PBST, and dilute each solution 10⁻⁶ times. 8 10 7 10 6 10 5 10 4 10 3 10 2 A standard curve was plotted using standard strains with cfu / ml, and the test sample was added and incubated at 37°C for 1 hour.

[0113] (5) Wash the plate 5 times with 0.05% PBST, add enzyme-labeled secondary antibody, and incubate at 37°C for 1 hour;

[0114] (6) Wash the plate 6 times with 0.05% PBST, add colorimetric solution, incubate at 37℃ for 20 min, add 2M sulfuric acid as stop solution, and read A at 450nm.

[0115] The standard curve plotted using this kit is as follows: Figure 10 As shown, the equation of the standard curve is .

Claims

1. A nanobody targeting Staphylococcus aureus, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

11.

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:

5.

4. The use of the nanobody of claim 1 in the preparation of a kit for detecting Staphylococcus aureus.

5. A kit for detecting Staphylococcus aureus, characterized in that, The kit contains the nanobody as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit also contains nanobodies with amino acid sequences as shown in SEQ ID NO.

12.

7. The reagent kit according to claim 5, characterized in that, The kit also includes detection reagents for enzyme-linked immunosorbent assay (ELISA).

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