Staphylococcus aureus targeting nano antibody and application thereof
By constructing nano-antibody targeting Staphylococcus aureus and constructing an ELISA detection kit, the existing detection methods are solved, which are time-consuming, complex, cost-effective and poor accuracy, and fast, low-cost and high-specific detection is achieved.
Patent Information
- Application Number
- CN202510544433.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-04
AI Technical Summary
The existing Staphylococcus aureus detection methods have problems such as long time, complex operation, high cost, poor stability and detection accuracy that are interfered with SpA protein, making it difficult to meet the fast, low-cost and high-specific detection needs.
Develop nano-antibodies targeting Staphylococcus aureus, use phage display technology to construct a nano-antibody library, obtain highly sensitive and specific nano-antibody pairs through multiple rounds of screening, and build an ELISA detection kit.
Fast and low-cost large-scale production is achieved, with the detection limit reaching 4.27×104CFU/ml, which significantly improves the sensitivity and specificity of the detection and avoids interference from SpA protein.
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Abstract
Description
Technical Field
[0001] The present invention relates to a nanobody targeting Staphylococcus aureus and its applications, belonging to the field of nanobodies targeting Staphylococcus aureus. The present invention is a divisional application of the invention with the title: A nanobody targeting Staphylococcus aureus, its preparation method and applications, application number: 202510287680.8, and application date: March 12, 2025. Background Art
[0002] Staphylococcus aureus is a Gram-positive coccus belonging to the genus Staphylococcus, arranged in grape-like clusters. Its cell wall is composed of peptidoglycan and teichoic acid, and expresses a variety of virulence factors, such as staphylococcal protein A (SpA) and clumping factor A (ClfA), which mediate bacterial adhesion, host tissue invasion, and immune escape. Staphylococcus aureus infection can cause local suppurative infections, pneumonia, pseudomembranous enteritis, etc. In severe cases, it can develop into systemic infections such as sepsis and septicemia, and even lead 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 problems such as 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 detection methods for Staphylococcus aureus mainly include bacterial culture methods, PCR techniques, and immunoassay methods. As the gold standard, the bacterial culture method takes a long time, usually requiring 24 - 48 hours to obtain results, and it is difficult to meet the needs of rapid detection. Although the PCR technique is highly sensitive, it is complex to operate, requires professional equipment and personnel, and is not suitable for on-site rapid detection and areas with limited resources. Immunoassay methods represented by enzyme-linked immunosorbent assay kits have become important detection means for pathogenic bacteria due to their low operation difficulty, fast detection speed, and ability to achieve in-situ detection. Traditional monoclonal antibodies are composed of heavy chains and light chains and are the most commonly used detection elements in immunoassay methods. However, problems such as high cost, poor stability, and difficulty in large-scale production restrict the application of monoclonal antibodies as immunoassay elements. Moreover, since the SpA protein exists on the surface of Staphylococcus aureus and binds to the Fc end of traditional monoclonal antibodies, seriously interfering with the accuracy of detection, there is an urgent need to develop a monoclonal antibody alternative element targeting Staphylococcus aureus.
[0004] Nanobody (VHH) is a unique antibody fragment discovered in camelids. It contains only one variable heavy chain region and can avoid the interference of SpA protein on the surface of Staphylococcus aureus. The molecular weight of nanobody is about 15 kDa, which is much smaller than that of traditional antibodies, and has better tissue penetration, enabling it to bind to epitopes that are difficult to reach by traditional antibodies. At present, nanobodies have shown great potential in different biotechnological fields such as pathogenic microorganism detection, human diagnosis, and treatment. Therefore, developing a Staphylococcus aureus detection kit based on nanobodies has broad application prospects. Summary of the Invention
[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a nanobody targeting Staphylococcus aureus with high sensitivity and strong specificity and its application.
[0006] Technical Solution: To solve the above technical problem, the present invention provides a nanobody targeting Staphylococcus aureus. The nanobody is a VHH antibody and has 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 has excellent binding performance and specificity to Staphylococcus aureus.
[0007] Among them, the VHH antibody fragment has the nucleotide sequences 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] In the second aspect, the present invention also provides a preparation method of the above-mentioned nanobody targeting Staphylococcus aureus, which includes the following steps: (1) Overnight culture of Staphylococcus aureus (ATCC25923), plate counting, and then placing the bacteria in a 90°C metal bath for high-temperature sterilization for 30 min to obtain inactivated Staphylococcus aureus; (2) Extract unimmunized alpaca lymphocytes, extract total RNA, synthesize cDNA, amplify the fragment encoding the VHH gene by PCR, digest with a fast cutter enzyme, and then reconnect with the phagemid pComb3XSS using T4 ligase. Transfer the recombinant phagemid into Escherichia coli TG1 to construct a nanobody natural phage library, and measure the library capacity to be about 2×10 10 , with a low sequence repetition and good diversity; (3) Coat the inactivated Staphylococcus aureus onto the enzyme-linked immunosorbent assay (ELISA) wells, add the recombinant phage and incubate for a period of time. Wash away the non-specifically bound recombinant phage using PBST, elute the specifically bound phage with acid, amplify the eluted phage, measure the titer, and use it for the next round of panning or analysis. Perform 3 - 5 rounds of screening according to the steps of "adsorption - washing - elution - amplification". By changing conditions such as the concentration of PBST and the phage incubation time during the screening process, gradually progress layer by layer to screen for nanobody phages with stronger affinity and specificity; (4) After several rounds of screening, randomly select 50 phages for Phage-ELISA identification. Use proteins of Salmonella enteritidis, Escherichia coli, Vibrio parahaemolyticus, Listeria monocytogenes, SARS-CoV-2 S protein, norovirus protein, and rotavirus protein as negative controls, and PBS as a blank control; Obtain 10 recombinant phage-displayed nanobodies with better effects and specific binding to Staphylococcus aureus. Amplify, extract plasmids, and sequence these 10 nanobody phages to obtain 6 nanobodies.
[0009] Furthermore, the method for constructing the nanobody phage display library of the present invention mainly includes the following steps: (1) Amplify the fragment encoding the nanobody gene from alpaca lymphocytes and introduce Sfi I restriction enzyme sites at both ends of the nanobody; (2) Digest and recombine with the phagemid pComb3XSS, and use T4 ligase for ligation to construct the phage display library.
[0010] Furthermore, the pH elution method for screening mainly includes the following steps: (1) Coat 10 6 -10 8 cfu / mL of inactivated Staphylococcus aureus into 400 μL ELISA wells and coat at 4 °C for 10 - 14 h; (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 h; (3) Wash the plate 5 - 10 times with 0.05 - 0.25% PBST, add 2 × 10 10 recombinant phages, and incubate at 37 °C for 60 - 120 min; (4) Wash the plate 5 - 10 times with 0.05 - 0.25% PBST, incubate with 100 - 200 μL of 0.1 M Gly-Hcl (pH = 2.2) for 8 - 10 min, and add 30 - 45 μL of 1 M Tris-Hcl (pH = 9.1). Measure the titer and pick monoclonal phages for phage-ELISA identification.
[0011] Thirdly, the present invention uses the checkerboard method to conduct paired experiments and screen out a pair of highly sensitive and strongly specific paired antibodies with nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.5 and amino acid sequences of SEQ ID NO.8 and SEQ ID NO.11, mainly including the following steps: (1) Coat 100 μl of nanobody at 50 μg / ml and incubate overnight; (2) Wash the plate 3 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, add 10 8 cfu / ml of heat-inactivated Staphylococcus aureus and incubate at 37°C for 1 h; (4) Wash the plate 3 times with 0.05% PBST, add the phage supernatant after panning and incubate at 37°C for 45 - 60 min; (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°C for 45 - 60 min; (6) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB chromogenic solution, incubate at 37°C for 10 - 15 min and conduct identification.
[0012] Furthermore, the present invention uses the successfully paired nanobodies as the coating antibody and the labeled antibody respectively to develop an ELISA detection kit. The coating antibody is Nb2, and the labeled antibody is Nb5 conjugated with HRP enzyme as the enzyme-labeled secondary antibody, mainly including the following steps: (1) Coat 100 μl of Nb2 nanobody at 50 μg / ml and incubate overnight to obtain the coated plate; (2) Wash the plate 3 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, dilute 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 cfu / ml of standard strains to draw a standard curve, add the test sample and incubate at 37°C for 1 h; (4) Wash the plate 5 times with 0.05% PBST, add the enzyme-labeled secondary antibody and incubate at 37°C for 1 h; (5) Wash the plate 6 times with 0.05% PBST, add the chromogenic solution, incubate at 37°C for 20 min, add 2 M sulfuric acid as the stop solution and read the A450nm value.
[0013] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: (1) Compared with traditional monoclonal antibodies, the phage-displayed nanobodies of the present invention have the advantages of small size, high stability, simple preparation, low cost, and large-scale production; (2) The six nanobody sequences of the present invention are reported for the first time at home and abroad, with high innovation; (3) A pair of highly sensitive and specific nanobody pairs have been screened out in the present invention; (4) The ELISA kit constructed in the present invention can achieve a detection limit of 4.27×10 4 CFU / ml, which is much lower than the existing immunological methods. Description of the drawings
[0014] Figure 1 Results of verifying the affinity of 6 phage-displayed nanobodies by Phage-ELISA; the abscissa is the phage clone number; the ordinate is the absorbance value at 450 nm; Figures 2 to 8 Respectively, using Salmonella ATCC13076 ( 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 ) to verify the specificity results, the abscissa is the phage clone number; the ordinate is the absorbance value at 450 nm; Figure 9 Results of the nanobody pairing experiment; Figure 10 Standard curve of the ELISA kit developed in the present invention. Detailed implementation manners
[0015] The technical solutions of the present invention will be further described below with reference to the drawings.
[0016] Main experimental materials: The standard strain of Staphylococcus aureus (ATCC25923), Escherichia coli BL21(DE3), Escherichia coli TG1, helper phage M13K07, and phagemid pComb3XSS are stored in our laboratory.
[0017] Main reagents: Chicken ovalbumin and bovine ovalbumin were purchased from Sigma, USA. Horseradish peroxidase (HRP)-anti-β monoclonal antibody (product number: 11973-MMO5T) was purchased from Beijing Sino Biological Inc. Skim milk powder, 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution, isopropyl-β-D-thiogalactoside (IPTG), and HRP enzyme coupling kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. LB broth and 2×YT medium were purchased from Qingdao Hope Bio-Technology Co., Ltd.
[0018] Formulas of main reagents: 1. 2×YT liquid medium: Weigh 31 g of 2×YT powder, dissolve it in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min.
[0019] 2. 2×YT solid medium: Weigh 31 g of 2×YT powder and 18 g of agar, dissolve them in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min.
[0020] 3. LB liquid medium: Weigh 25 g of LB medium, dissolve it in 1000 mL of ultrapure water, and autoclave at 121 °C for 15 min.
[0021] 4. 20% polyethylene glycol (PEG)-NaCl: 50 g of PEG-8000, 36 g of NaCl, dissolve them by heating with ultrapure water, make up the volume to 250 mL, and autoclave for 15 min.
[0022] 5. Elution buffer: 0.2 M glycine (Gly), adjust the pH to 2.2 with hydrochloric acid, and autoclave for 15 min.
[0023] 6. Neutralization buffer: 1 M tris(hydroxymethyl)aminomethane (Tris), adjust the pH to 9.1 with hydrochloric acid, and autoclave for 15 min.
[0024] Example 1. Inactivation of Staphylococcus aureus (1) Streak the standard strain of Staphylococcus aureus on an LB plate and incubate at 37 °C for 12 - 14 h; (2) Pick a single colony on the plate and inoculate it into 5 mL of LB medium, incubate at 37 °C for 12 - 14 h; (3) Spread and count on an LB plate, incubate at 37 °C for 12 - 14 h; (4) Pipette 1 mL of the counted bacterial solution, centrifuge at 8000 rpm for 2 min, resuspend with PBS, and repeat 3 times; (5) Preheat the metal bath to 90 °C in advance, and heat the strain at 90 °C for half an hour to obtain the inactivated strain.
[0025] Example 2: Construction of a phage display nanobody natural library (1) Extract peripheral blood lymphocytes from multiple alpacas; (2) Add 2 mL of Trizol to the cells, add 1 mL of isopropanol, invert several times to mix evenly, and let stand at room temperature for 10 - 20 min; (3) Centrifuge at 10000 - 14000 g for 10 - 20 min, discard the supernatant, and obtain the RNA precipitate of the cells; (4) Add 3 mL of 75% ethanol, invert several times to mix evenly, let stand at room temperature for 10 min, centrifuge at 10000 - 14000 g for 10 - 20 min, and discard the supernatant.
[0026] (5) Invert and dry at room temperature for 5 - 10 min or vacuum dry, add 50 uL of DEPC-ddH2O to dissolve the RNA, detect the RNA quality by gel electrophoresis and measure the concentration. Use the RevertAid RT reverse transcription kit (Thermo Fisher, K1691) to reverse transcribe the extracted RNA to obtain the cDNA template.
[0027] (6) Digestion of VHH fragment / pComb3XSS: 5 μg of VHH fragment or pComb3XSS, 100 μL of RNase free H2O, 2 μL of Sfi I fast digestion enzyme, 10 μL of fast digestion enzyme buffer; reaction conditions: react at 37 °C for 60 min.
[0028] (7) Clone the VHH fragment into the phagemid pComb3XSS: 30 VHH digested fragments, 100 ng of digested plasmid pComb3XSS, 1 μL of T4 ligase, 2 μL of T4 ligase buffer, 10 μL of RNase free H2O. Reaction conditions: react at 16 °C for 12 h.
[0029] (8) Transform 100 - 200 ng of pComb3XSS-VHH recombinant phagemid into E. coil TG1 competent cells. After culturing at 37 °C for 12 h, elute all single colonies on the plate to form a phage nanobody library.
[0030] (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 μg / ml). When the medium reaches the logarithmic growth phase, add the helper phage M13K07 at an infection ratio of Escherichia coli:phage = 1 cfu / mL:20 pfu / mL, and let stand at 37 °C for 1 h.
[0031] (10) Add the above 5 mL culture system to 50 mL of 2×YT / Amp / Kanamycin (kana) medium (the working concentration of AMP is 50 μg / ml, and the working concentration of kana is 100 μg / ml), and culture at 37 °C for 12 h.
[0032] (11) Centrifuge at 12000 - 16000 g for 10 - 15 min, take the supernatant, add 12 mL of PEG8000 / NaCl (30%, sterile), and let it stand on ice for 4 - 6 h.
[0033] (12) Centrifuge at 12000 - 16000 g for 30 - 40 min, discard the supernatant, resuspend the precipitate with 1 mL of PBS, add 200 - 300 μL of PEG8000 / NaCl (30%, sterile), and let it stand on ice for 1 - 2 h.
[0034] (13) Centrifuge at 12000 - 16000 g for 30 - 40 min, discard the supernatant, resuspend the precipitate with 200 - 300 μL of PBS, take 10 μL of the phage to measure the titer of the recombinant phage, which can reach 2×10 10 , aliquot the phage for later screening.
[0035] Example 3: Screening of Staphylococcus aureus phage-displayed nanobodies (1) In a laminar flow hood, wash the ELISA plate 5 times with sterile water and sterilize it with ultraviolet light for 60 min; (2) Dilute the heat-inactivated Staphylococcus aureus with PBS to a final concentration of 10 8 cfu / mL, add the diluted Staphylococcus aureus to the ELISA plate at 100 - 200 μL / well, and coat it at 4 °C for 12 h; (3) Wash the plate 5 times with PBS, pat it dry with sterile paper, add 300 μL of 3% BSA-PBS blocking solution to each well, and block it at 37 °C for 2 h; (4) Wash the plate 5 times with PBS, pat it dry with sterile paper, take the above-constructed recombinant phage library with a titer of 2×10 10 and mix it with 200 μL of PBS, then add it to the ELISA plate and bind it at 37 °C for 120 min; (5) Wash the plate 5 times with 0.1% PBST, pat it dry with sterile paper, add 100 μL of Gly-HCl buffer, incubate it at 37 °C for 10 min, aspirate the elution product, and quickly add 30 - 45 μL of Tris-HCl buffer; (6) Take 10 μL of the phage for serial dilution, measure 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 step is the same as that of the phage library amplification step; (7) Steps (1)-(6) are the amplification process of the first round. The panning steps of the 2nd - 5th rounds are generally the same. The input amount of phage per round is 2×10 10 pfu / well, and the coating concentration of Staphylococcus aureus decreases round by round to 5×10 6 -1×10 8 cfu / mL. 1 - 3% OVA-PBS and 1 - 3% BSA-PBS blocking solutions are used for alternate blocking. The binding time of the input phage to Staphylococcus aureus is 120 - 130 min, and the concentration of the eluent is 0.05% - 0.25% PBST. The specific panning scheme is shown in Table 1.
[0036] Table 1 Screening process of Staphylococcus aureus phage-displayed nanobody
[0037] Example 4. Screening and identification of specific phage clones After five rounds of screening, 50 phage-displayed nanobody single colonies are picked for amplification and phage-ELISA identification. The specific operation steps are as follows: (1) Pick 25 single colony clones and inoculate them into 1 mL of 2×YT / Amp liquid medium, and culture at 37°C and 220 rpm for 12 - 14 h.
[0038] (2) Take 100 - 150 μL of the above culture and add them to 1 mL of 2×YT / Amp liquid medium respectively, mix well and shake culture at 220 rpm for 2 - 3 h until the logarithmic growth phase.
[0039] (3) Add helper phage M13K07 to each tube at a ratio of cell:phage = 1:1. After incubating at 37°C for 15 - 20 min, shake culture at 220 rpm for 30 - 45 min.
[0040] (4) Centrifuge at 4°C and 8000 - 10000 rpm for 2 - 5 min, then add 1 - 1.5 ml of 2×YT / Amp / Kana to resuspend. Shake culture at 37°C and 250 rpm for 10 - 14 h.
[0041] (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 at 4°C for ELISA identification.
[0042] (6) Take 100 - 200 μL of Staphylococcus aureus with a concentration of 10 8 cfu / ml and 1 μg / mL bovine serum albumin and ovalbumin and coat them onto the ELISA plate, and coat at 4°C for 12 h.
[0043] (7) Discard the coating solution, wash the plate 3 times with 0.05% PBST, add 250 - 350 μL of 3 - 5% defatted milk to each well for blocking, and incubate at 37 °C for 2 h.
[0044] (8) Wash the plate 3 times with 0.05% PBST, add 100 μL of the phage supernatant culture solution after panning to each well coated with Staphylococcus aureus, bovine serum albumin, and ovalbumin, and incubate at 37 °C for 45 - 60 min.
[0045] (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 °C for 45 - 60 min.
[0046] (10) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB chromogenic solution, incubate at 37 °C for 10 - 15 min, add 50 μL of 2M H2SO4 to each well, and measure A450nm.
[0047] (11) Among the 50 clones picked, all 50 clones could bind to Staphylococcus aureus; among them, clones Nb2, Nb5, Nb7, Nb11, Nb18, Nb32, Nb34, and Nb47 had good binding ability and specificity to Staphylococcus aureus. These 8 clones were amplified and sequenced using the designed primers, and 6 different phage - displayed nanobody amino acid sequences (VHH antibodies) were obtained by analysis. Their amino acid sequences are shown as 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), SEQ ID NO.12 (Nb32), and the corresponding nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6. Figure 1 The binding ability of 6 selected recombinant phage clones to Staphylococcus aureus was demonstrated. The abscissa is the number of phage clones, and the ordinate is the absorbance value at 450 nm.
[0048] (12) To further explore whether the selected phage-displayed 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, Rotavirus antigen protein) were respectively coated in different wells of a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The coating concentration of bacteria was 10 8 cfu / ml, and the coating concentration of proteins was 1 μg / ml. Incubate overnight at 4°C. After washing the plate, the unbound sites of the ELISA plate were blocked with 5% skim milk and incubated at 37°C for 2 hours. The subsequent verification steps were the same as those in steps (7)-(10) of this example, and the verification results are as Figures 2 - 8 shown.
[0049] Example 5: Highly Sensitive and Highly Specific Nanobody Pairing (1) Six expression plasmids containing Staphylococcus aureus nanobodies (synthesized by GenScript Biotech Corporation, with the restriction enzyme site at the 5' end being NdeI and the restriction enzyme site at the 3' end being XhoI, and the expression vector being PET25b) were transformed into Escherichia coli BL21 (DE3). After culturing to the logarithmic growth phase, isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of the engineered bacteria. The expressed NP protein was purified by Ni-NTA column to obtain high-purity protein, and the Staphylococcus aureus nanobodies were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA; (2) Coat 100 μl of nanobodies at a concentration of 50 μg / ml and incubate overnight; (3) Wash the plate 3 times with 0.05% PBST and incubate with 5% skim milk powder at 37°C for 2 h; (4) Wash the plate 3 times with 0.05% PBST, add 10 8 cfu / ml heat-inactivated Staphylococcus aureus and incubate at 37°C for 1 h; (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°C for 45 - 60 min; (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°C for 45 - 60 min; (7) Wash the plate 7 times with 0.05% PBST, add 100 μL of TMB chromogenic solution and incubate at 37°C for 10 - 15 min; (8) Add 50 μL of 2 M H2SO4 to each well and measure A450nm. The paired results are as Figure 9 shown.
[0050] Example 6. Specific application of the present invention: Construction of a sandwich ELISA detection kit for Staphylococcus aureus based on paired nanobodies (1) Use the HRP enzyme conjugation kit (periodate method) of Shanghai Sangon Biotech Co., Ltd. to conjugate the labeled antibody with HRP enzyme as the enzyme-labeled secondary antibody; (2) Coat 100 μL of 50 μg / ml Nb2 nanobody and incubate overnight to obtain the coated plate; (3) Wash the plate 3 times with 0.05% PBST and incubate with 5% skim milk powder at 37°C for 2 h; (4) Wash the plate 3 times with 0.05% PBST, dilute 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 cfu / ml of the standard strain to draw the standard curve, and add the test sample, incubate at 37°C for 1 h; (5) Wash the plate 5 times with 0.05% PBST, add the enzyme-labeled secondary antibody, and incubate at 37°C for 1 h; (6) Wash the plate 6 times with 0.05% PBST, add the chromogenic solution, incubate at 37°C for 20 min, add 2 M sulfuric acid as the stop solution, and read A450nm.
[0051] The standard curve drawn using this kit is as Figure 10 shown, and the standard curve equation is .
Claims
1. A nanobody targeting Staphylococcus aureus, characterized in that, Its amino acid sequence is as shown in SEQ ID NO:
8.
2. A gene encoding the nanobody according to claim 1.
3. The gene according to claim 2, wherein Its nucleotide sequence is as shown in SEQ ID NO:
2.
4. Use of the nanobody according to 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 according to claim 1.
6. The kit according to claim 5, characterized in that, The kit further contains a nanobody whose amino acid sequence is as shown in any one of SEQ ID NOs. 9 to 12.
7. The kit according to claim 5, characterized in that, The kit further includes detection reagents for enzyme-linked immunosorbent assay.