Staphylococcus aureus targeting nano antibody as well as preparation method and application thereof

Abstract

The invention discloses a targeted staphylococcus aureus nano antibody as well as a preparation method and application thereof. According to the invention, a pair of staphylococcus aureus nano-antibody pairs with a good pairing effect is obtained through nano-antibody phage library construction and phage screening technologies, and a detection kit which is based on sandwich method ELISA and can be used for detecting actual living bacteria in food is constructed. The nano antibody disclosed by the invention shows high affinity and high specificity 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. The detection kit disclosed by the invention can realize the detection limit of 4.27104 cfu / ml.

Description

Technical Field

[0001] The invention relates to a nano antibody targeting Staphylococcus aureus and a preparation method and application thereof, belonging to the field of nano antibodies targeting Staphylococcus aureus. Background Art

[0002] Staphylococcus aureus is a Gram-positive coccus belonging to the genus Staphylococcus. It is arranged in grape-like clusters. Its cell wall is composed of peptidoglycan and teichoic acid, and it 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 infection, pneumonia, pseudomembranous colitis, etc. In severe cases, it can develop into systemic infection such as sepsis and septicemia, and even lead to death. Foodborne Staphylococcus aureus infection is currently very common, leading to acute gastroenteritis. Existing monoclonal antibodies mainly target surface proteins such as SpA and ClfA, but the nonspecific binding of SpA, as well as the high cost and poor stability of traditional antibodies, limit their application in rapid detection.

[0003] The current detection methods of Staphylococcus aureus mainly include bacterial culture, PCR technology and immunoassay. As the gold standard, bacterial culture is time-consuming and usually takes 24-48 hours to obtain results, which is difficult to meet the needs of rapid detection. Although PCR technology has high sensitivity, it is complicated to operate and requires professional equipment and personnel. It is not suitable for rapid on-site detection and areas with limited resources. Immunoassay represented by enzyme-linked immunosorbent assay kits has become an important detection method for pathogens due to its advantages of low operation difficulty, fast detection speed, and in-situ detection. Traditional monoclonal antibodies are composed of heavy chains and light chains and are the most commonly used detection elements for immunoassays. However, problems such as high cost, poor stability, and difficulty in large-scale production restrict the application of monoclonal antibodies as immunoassay elements. In addition, since the presence of SpA protein on the surface of Staphylococcus aureus will bind to the Fc end of traditional monoclonal antibodies, it will seriously interfere with the accuracy of detection. Therefore, it is urgent to develop a monoclonal antibody replacement element targeting Staphylococcus aureus.

[0004] Nanobodies (VHH) are a unique antibody fragment found in camelids, containing only one heavy chain variable region, which can avoid interference from the SpA protein on the surface of Staphylococcus aureus. Nanobodies have a molecular weight of about 15 kDa, which is much smaller than traditional antibodies. They have better tissue penetration and can bind to epitopes that are difficult for traditional antibodies to reach. Currently, nanobodies have shown great potential in different biotechnology fields such as pathogen detection and human diagnosis and treatment. Therefore, the development of Staphylococcus aureus detection kits 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 nano-antibody targeting Staphylococcus aureus with high sensitivity and strong specificity, as well as a preparation method and application thereof.

[0006] Technical solution: To solve the above technical problems, the present invention provides a nanobody targeting Staphylococcus aureus, wherein the nanobody is a VHH antibody having an amino acid sequence as 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. The nanobody has excellent binding performance and specificity with Staphylococcus aureus.

[0007] Wherein, the VHH antibody fragment has a nucleotide sequence as 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 a second aspect, the present invention also provides a method for preparing the above-mentioned nanoantibody targeting Staphylococcus aureus, the method comprising the following steps: (1) Staphylococcus aureus (ATCC25923) was cultured overnight, plated and counted, and then the bacteria were sterilized in a 90°C metal bath for 30 min to obtain inactivated Staphylococcus aureus; (2) Lymphocytes of unimmunized alpacas were extracted and total RNA was extracted. cDNA was synthesized and the fragment encoding the VHH gene was amplified by PCR. After digestion with the fast-cutting enzyme, it was reconnected with the phagemid pComb3XSS using T4 ligase. The recombinant phagemid was transferred into Escherichia coli TG1 to construct a natural phage library of nanobodies. The library capacity was determined to be approximately 2×10 10 , the sequence repetition is low and the diversity is good; (3) The inactivated Staphylococcus aureus is coated on the enzyme-labeled well, and the recombinant phage is put into the well for incubation for a period of time. The non-specifically bound recombinant phage is washed with PBST, the specifically bound phage is acid-eluted, the eluted phage is amplified, and the titer is measured for the next round of selection or analysis. According to the "adsorption-washing-elution-amplification" steps, 3-5 rounds of screening are performed, and the conditions such as the concentration of PBST and the incubation time of phage are changed during the screening process, so as to screen the nano-antibody phage with stronger affinity and specificity; (4) After several rounds of screening, 50 phages were randomly selected for Phage-ELISA identification, using Salmonella enteritidis, Escherichia coli, Vibrio parahaemolyticus protein, Listeria monocytogenes protein, SARS-CoV-2 S protein, Norovirus protein, and Rotavirus protein as negative controls, and PBS as a blank control; 10 recombinant phage-displayed nanoantibodies with better specific binding to Staphylococcus aureus were obtained, and these 10 nanoantibody phages were amplified, plasmid extracted, and sequenced to obtain 6 nanoantibodies.

[0009] Furthermore, the method for constructing a nanobody phage display library of the present invention mainly 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.

[0010] Furthermore, the pH elution method screening mainly comprises the following steps: (1) Coated with 10 6 -10 8 cfu / mL of inactivated Staphylococcus aureus to 400 μL of ELISA wells and coat at 4°C for 10-14h; (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 and add 2×10 10 Recombinant phage, 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 0.1M Gly-Hcl (pH=2.2) for 8-10 min, add 30-45 μL 1M Tris-Hcl (pH=9.1), measure the titer, and select monoclonal phage for phage-ELISA identification.

[0011] In the third aspect, the present invention uses a chessboard method to perform a pairing experiment to screen out a pair of highly sensitive and highly specific paired antibodies having 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, which mainly comprises the following steps: (1) Coat with 100ul, 50ug / ml nanoantibody and incubate 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 of inactivated Staphylococcus aureus, incubated at 37°C for 1 h; (4) Wash the plate three times with 0.05% PBST, add the selected phage supernatant, and incubate at 37°C for 45-60 min; (5) Wash the plate six 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 seven times with 0.05% PBST, add 100 μL TMB colorimetric solution, incubate at 37°C for 10-15 min, and perform identification.

[0012] Furthermore, the present invention uses the above successfully paired nanobodies as coating antibodies and labeled antibodies to develop an ELISA detection kit, wherein the coating antibody is Nb2, the labeled antibody is Nb5 and is coupled with HRP enzyme as an enzyme-labeled secondary antibody, which mainly comprises the following steps: (1) Coating with 100ul 50ug / ml Nb2 nanobody and incubating overnight to obtain a coated plate; (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 three times with 0.05% PBST and 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 sample to be tested, and incubate at 37°C for 1 h; (4) Wash the plate five times with 0.05% PBST, add enzyme-labeled secondary antibody, and incubate at 37°C for 1 h; (5) Wash the plate six times with 0.05% PBST, add the colorimetric solution, incubate at 37°C for 20 min, add 2 M sulfuric acid as the stop solution, and read the value at A450 nm.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Compared with traditional monoclonal antibodies, the phage-displayed nanoantibodies 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 and are highly innovative; (3) The present invention screened out a pair of highly sensitive and specific nanoantibodies; (4) The ELISA kit constructed by the present invention can achieve 4.27×10 4 The detection limit of CFU / ml is much lower than that of existing immunological methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of the affinity of 6 phage-displayed nanobodies verified by Phage-ELISA; the horizontal axis is the phage clone number; the vertical axis is the absorbance value at 450nm; Figure 2~Figure 8 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 ) The results of antigen specificity verification, the horizontal axis is the phage clone number; the vertical axis is the absorbance value at 450nm; Fig. 9 The results of the nanobody pairing experiment; Fig.10 This is the standard curve of the ELISA kit developed by the present invention. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described below in conjunction with the accompanying 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 were preserved by our laboratory.

[0017] Main reagents: Chicken ovalbumin and bovine ovalbumin were purchased from Sigma, USA; horseradish peroxidase (HRP) enzyme-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) colorimetric solution, isopropyl-β-D-thiogalactopyranoside (IPTG), and HRP enzyme coupling kit were purchased from Shanghai Sangon Biotechnology Co., Ltd.; LB broth and 2×YT culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0018] Main reagent formula: 1. 2×YT liquid culture 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.

[0019] 2. 2×YT solid culture 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.

[0020] 3. LB liquid culture medium: Weigh 25 g LB culture medium, dissolve it in 1000 mL ultrapure water, and sterilize it by high pressure at 121°C for 15 min.

[0021] 4. 20% polyethylene glycol (PEG)-NaCl: 50 g PEG-8000, 36 g NaCl, heat and dissolve in ultrapure water, make up to 250 mL, and sterilize under high pressure for 15 min.

[0022] 5. Eluent: 0.2 M glycine (Gly), add hydrochloric acid to adjust pH = 2.2, and sterilize under high pressure for 15 minutes.

[0023] 6. Neutralization buffer: 1M Tris, add hydrochloric acid to adjust pH to 9.1, and sterilize by high pressure for 15 minutes.

[0024] 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., and the strain is heated at 90° C. for half an hour to obtain an inactivated strain.

[0025] Example 2: Construction of a naive library of phage-displayed nanobodies (1) extracting peripheral blood lymphocytes from multiple alpacas; (2) Add 2 mL of Trizol and 1 mL of isopropanol to the cells, invert several times to mix, and leave at room temperature for 10-20 min; (3) Centrifuge at 10,000-14,000 g for 10-20 min, discard the supernatant, and obtain the RNA precipitate from the cells; (4) Add 3 mL of 75% ethanol, invert several times to mix, leave at room temperature for 10 min, centrifuge at 10,000-14,000 g for 10-20 min, and discard the supernatant.

[0026] (5) Invert at room temperature for 5-10 minutes to air dry or vacuum dry, add 50uL DEPC-ddH2O to dissolve RNA, and detect RNA quality and concentration by gel electrophoresis. Use RevertAid RT reverse transcription kit (Thermo Fisher, K1691) to reverse transcribe the extracted RNA to obtain cDNA template.

[0027] (6) VHH fragment / pComb3XSS digestion: 5 μg VHH fragment or pComb3XSS, 100 μL RNase free H2O, 2 μL Sfi I enzyme, 10 μL enzyme buffer; reaction conditions: 37°C for 60 min.

[0028] (7) Cloning of VHH fragment into phagemid pComb3XSS: 30 VHH fragment, 100 ng of digested plasmid pComb3XSS, 1 μL T4 ligase, 2 μL T4 ligase buffer, 10 μL RNase free H2O. Reaction conditions: 16°C for 12 h.

[0029] (8) 100-200 ng of pComb3XSS-VHH recombinant phagemid was transformed into E.coil TG1 competent cells. After culturing at 37°C for 12 h, all single colonies on the eluted plate constituted the phage nanoantibody 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 ug / ml). When the culture medium reaches the logarithmic growth phase, add helper phage M13K07 at an infection ratio of E. coli: phage = 1 cfu / mL: 20 pfu / mL, and incubate at 37°C for 1 h.

[0031] (10) Add all 5 mL of the above culture system into 50 mL of 2×YT / Amp / kanamycin (kana) medium (working concentration of AMP is 50 μg / ml, working concentration of kana is 100 μg / ml) and culture at 37°C for 12 h.

[0032] (11) Centrifuge at 12000-16000g for 10-15 min. Take the supernatant and add 12 mL of PEG8000 / NaCl (30%, sterile) and place on ice for 4-6 h.

[0033] (12) Centrifuge at 12000-16000g for 30-40 min, discard the supernatant, resuspend the pellet in 1 mL of PBS, add 200-300 μL of PEG8000 / NaCl (30%, sterile), and let stand on ice for 1-2 h.

[0034] (13) Centrifuge at 12000-16000g for 30-40min, discard the supernatant, resuspend the precipitate in 200-300μL PBS, and take 10μL of phage to measure the recombinant phage titer, which can reach 2×10 10 , and aliquot the phage for later screening.

[0035] Example 3: Screening of Staphylococcus aureus phage-displayed nanobodies (1) In a clean bench, wash the ELISA plate with sterile water five times and sterilize it with ultraviolet light for 60 minutes; (2) Dilute the inactivated Staphylococcus aureus with PBS to a final concentration of 10 8 cfu / mL, diluted Staphylococcus aureus was added to the ELISA plate at 100-200 μL / well and coated at 4°C for 12 h; (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; (4) Wash the plate 5 times with PBS, pat dry with sterile paper, and take the titer of the above-constructed 10 The recombinant phage library was mixed with 200 μL PBS, added to the ELISA plate, and allowed to bind at 37°C for 120 min; (5) Wash the plate 5 times with 0.1% PBST, pat dry with sterile paper, add 100 μL of Gly-HCl buffer, incubate at 37°C for 10 min, aspirate the eluted product, and quickly add 30-45 μL of Tris-HCl buffer; (6) Take 10 μL of phage for gradient dilution, measure the titer of 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; (7) Steps (1) to (6) are the first round of amplification. The selection steps for rounds 2 to 5 are basically the same. The amount of phage input in each round is 2 × 10 10 pfu / well, and the coating concentration of Staphylococcus aureus decreased to 5×10 6 -1×10 8 cfu / mL, 1-3% OVA-PBS and 1-3% BSA-PBS blocking solutions were used for alternate blocking, the phage binding time with Staphylococcus aureus was 120-130 min, and the concentration of the elution solution was 0.05%-0.25% PBST. The specific panning scheme is shown in Table 1.

[0036] Table 1 Screening process of Staphylococcus aureus phage displayed nanoantibodies

[0037] Example 4: Screening and identification of specific phage clones After five rounds of screening, 50 phage-displaying nanoantibody single colonies were selected for amplification and phage-ELISA identification. The specific steps are as follows: (1) Select 25 single colony clones and inoculate them into 1 mL of 2×YT / Amp liquid culture medium. Culture at 37°C and 220 rpm for 12-14 h.

[0038] (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 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, incubate at 37°C for 15-20 min, and then shake culture at 220 rpm for 30-45 min.

[0040] (4) Centrifuge at 8000-10000 rpm for 2-5 min at 4°C, then add 1-1.5 ml 2×YT / Amp / Kana to resuspend. Incubate at 37°C with shaking at 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 with a number, and store it at 4°C for ELISA identification.

[0042] (6) Take 100-200 μL of 10 8 cfu / ml Staphylococcus aureus, 1 ug / mL bovine serum albumin and ovalbumin were coated on the ELISA plate at 4℃ for 12h.

[0043] (7) Discard the coating solution, wash the plate three times with 0.05% PBST, add 250-350 μL 3-5% skim milk to each well for blocking, and incubate at 37°C for 2 h.

[0044] (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, BSA, and OVA, and incubate at 37°C for 45-60 min.

[0045] (9) Wash the plate six 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 seven times with 0.05% PBST, add 100 μL TMB colorimetric solution, incubate at 37°C for 10-15 min, add 50 μL 2M H2SO4 to each well, and measure A450nm.

[0047] (11) Among the 50 clones picked, all 50 clones were able to bind to Staphylococcus aureus; among them, clones Nb2, Nb5, Nb7, Nb11, Nb18, Nb32, Nb34, and Nb47 had good binding ability and specificity with Staphylococcus aureus. These 8 clones were amplified and sequenced using the designed primers, and 6 phage-displayed nanoantibody amino acid sequences (VHH antibodies) with different sequences were obtained through analysis. 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 the 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 6 selected recombinant phage clones to Staphylococcus aureus is shown. The horizontal axis is the number of the phage clone, and the vertical axis is the absorbance value at 450nm.

[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, and Rotavirus antigen protein) were coated in different wells of a 96-well ELISA plate, and the bacterial coating concentration was 10 8 cfu / ml, protein coating concentration was 1ug / ml, and incubated at 4°C overnight. After washing, 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 steps (7) to (10) of this embodiment. The verification results were as follows: Figure 2-8 shown.

[0049] Example 5: Highly sensitive and highly specific nanoantibody pairing (1) Six expression plasmids containing Staphylococcus aureus nanobodies (synthesized by GenScript Biotechnology Co., Ltd., with Ndel at the 5' end and Xhol at the 3' end, and the expression vector PET25b) were transformed into Escherichia coli BL21 (DE3), cultured to the logarithmic growth phase, and then induced to express the engineered bacteria using isopropyl-β-D-thiogalactoside (IPTG). The expressed NP protein was purified using a Ni-NAT column to obtain a high-purity protein, and the Staphylococcus aureus nanobodies were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA; (2) Coating with 100ul and 50ug / ml of nanoantibodies respectively and incubating overnight; (3) Wash the plate three 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 and add 10 8 cfu / ml of inactivated Staphylococcus aureus, incubated at 37°C for 1 h; (5) Wash the plate three 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 six 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 seven times with 0.05% PBST, add 100 μL TMB colorimetric solution, and incubate at 37°C for 10-15 min; (8) Add 50 μL 2M H2SO4 to each well and measure A450nm. Fig. 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) The labeled antibody was conjugated to HRP enzyme as an enzyme-labeled secondary antibody using the HRP enzyme conjugation kit (periodate method) from Shanghai Shenggong Biotechnology Co., Ltd.; (2) Coating with 100uL 50ug / ml Nb2 nanobody and incubating overnight to obtain a coated plate; (3) Wash the plate three times with 0.05% PBST and incubate with 5% skim milk powder at 37°C for 2 h; (4) Wash the plate three times with 0.05% PBST and 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 sample to be tested, and incubate at 37°C for 1 h; (5) Wash the plate five times with 0.05% PBST, add enzyme-labeled secondary antibody, and incubate at 37°C for 1 h; (6) Wash the plate six times with 0.05% PBST, add the colorimetric solution, incubate at 37°C for 20 min, add 2 M sulfuric acid as a stop solution, and read the value at A450 nm.

[0051] The standard curve drawn using this kit is as follows Fig.10 As shown, the standard curve equation is .

Claims

1. A nanobody targeting Staphylococcus aureus, characterized in that: Its amino acid sequence is shown in any one of SEQ ID NOs. 7 to 12.

2. A gene encoding the nanobody according to claim 1, characterized in that Its nucleotide sequence is shown in any one of SEQ ID NO.1~6.

3. A method for preparing the nanobody according to claim 1, characterized in that: The following steps are included: (1) extracting alpaca lymphocytes, splicing and recombining specific gene fragments into phagemids and transferring them into Escherichia coli to construct a nanobody phage library; (2) Inactivated Staphylococcus aureus was used as the coating protein, and the anti-Staphylococcus aureus nanoantibodies were screened by pH elution method; (3) Phage-ELISA was used to identify the binding ability and specificity with Staphylococcus aureus, and plaque sequencing was performed on the specific target Staphylococcus aureus.

4. The preparation method according to claim 3, characterized in that: The Staphylococcus aureus is produced by a prokaryotic expression system; the pH value of the pH elution method in step (2) is 2-10.

5. The preparation method according to claim 3, characterized in that: The proteins used to identify phage specificity were inactivated Staphylococcus aureus, bovine serum albumin, and ovalbumin.

6. 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.

7. Use of the nanobody according to claim 1 in constructing an immunoassay platform for Staphylococcus aureus.

8. Use of the nanobody according to claim 1 in preparing a kit for detecting Staphylococcus aureus.

9. A kit for detecting Staphylococcus aureus, characterized in that: The kit contains the nanobody according to claim 1.

10. The kit according to claim 9, characterized in that The kit also includes a detection reagent for enzyme-linked immunosorbent assay.

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