A nanobody for detecting salmonella typhimurium

Abstract

The application discloses a nanobody for detecting salmonella typhimurium, and aims to provide a nanobody for detecting salmonella typhimurium, which has high detection accuracy, high specificity and simple obtaining method. The amino acid sequence of the nanobody is shown in SEQ-Styp NO. 1, SEQ-Styp NO. 2, SEQ-Styp NO. 3 and SEQ-Styp NO. 4, and the nanobody belongs to the technical field of biological detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a nanobody for detecting Salmonella typhimurium. BACKGROUND

[0002] Salmonella infection caused by salmonellosis is a common bacterial disease, which poses a serious threat to food production and safety. Food poisoning and diarrhea caused by Salmonella seriously endanger human life and health every year worldwide. Salmonella typhimurium (S. typh) is one of the most common pathogens in the Salmonella genus, which can cause bacterial diarrhea and has the highest incidence rate after Salmonella infection. It is one of the important zoonotic pathogens that cause foodborne diseases worldwide. As one of the foodborne pathogens, Salmonella typhimurium can spread throughout the food chain, from animal feed, primary production to family or catering services, posing a serious threat to the livestock industry and public health safety. Improper feeding management can cause animals such as cattle and sheep to be infected with Salmonella typhimurium, and people can easily get sick through contaminated food or contact with infected animals. Salmonella typhimurium also hinders the large-scale breeding of the sheep industry. After infection, sick sheep often show signs of weakness, anorexia, and elevated body temperature. For lambs, obvious diarrhea symptoms appear, and in severe cases, they can even die. Therefore, the monitoring of Salmonella typhimurium has a great application demand in the food safety, public health, and livestock breeding industries.

[0003] The current common detection methods for Salmonella typhimurium include molecular detection technology (PCR technology), bacterial culture method, immunological method, and mass spectrometry. PCR-based molecular detection technology has excellent specificity and sensitivity, and can accurately detect target DNA sequences in a short time. However, the implementation of PCR technology requires professional equipment and technical personnel, and may produce false positive results affecting the detection effect. The bacterial culture method is simple and the results are intuitive, and it is the "gold standard" method for bacterial detection, but the entire detection process requires a long time and has high requirements for the number of bacteria, and there is a significant omission rate for low-concentration samples. Mass spectrometry can directly detect molecular characteristics and has high accuracy, but the equipment cost is high and the operation process is complex. The specificity of the immunological method is relatively high, but it may be affected by the quality and matching of antibodies, and there may be cross-reactions or low sensitivity. Compared with traditional monoclonal antibodies, recombinant antibodies have lower production costs and higher morphological consistency. Therefore, it is necessary and feasible to develop anti-Salmonella typhimurium nanobodies prepared by phage display technology and prokaryotic expression technology. SUMMARY

[0004] The application aims to provide a nanobody for detecting Salmonella typhimurium, which has high detection accuracy, high specificity and simple obtaining method.

[0005] The application also provides a preparation method of the nanobody for specifically recognizing Salmonella typhimurium (strain type: ATCC 14028).

[0006] To this end, the application provides a first technical solution.

[0007] A nanobody for detecting Salmonella typhimurium, wherein the amino acid sequence of the nanobody is shown in SEQ-Styp NO. 1, SEQ-Styp NO. 2, SEQ-Styp NO. 3 and SEQ-Styp NO. 4.

[0008] Further, the nanobody for detecting Salmonella typhimurium described above has a strain of ATCC 14028.

[0009] The application provides a second technical solution, which is a preparation method of the nanobody for detecting Salmonella typhimurium described above, and sequentially comprises the following steps.

[0010] 1) Collecting blood samples of immunized lama, separating and collecting peripheral blood lymphocytes of the lama, extracting total RNA of the nanobody for anti-Salmonella typhimurium of the peripheral blood lymphocytes of the lama, and synthesizing cDNA; taking the cDNA as a template, amplifying a heavy chain variable region gene, and after the amplified lama antibody heavy chain variable region gene is connected with a phagemid vector, transforming into competent Escherichia coli to construct a nanobody phage library of the immunized lama;

[0011] 2) Taking Salmonella typhimurium as a coating antigen, using pH elution method, and through 3 rounds or more than 3 rounds of panning, obtaining the nanobody phage capable of specifically recognizing Salmonella typhimurium, and through sequencing analysis and comparison of the nanobody gene;

[0012] 3) Extracting the phagemid vector containing the nanobody gene for anti-Salmonella typhimurium and re-introducing into a host expression bacterium to perform expression and purification of the nanobody protein.

[0013] Further, the preparation method of the nanobody for detecting Salmonella typhimurium described above, wherein the extraction of the total RNA is performed according to the instruction of TRIzol TM Reagent.

[0014] Further, the preparation method of the nanobody for detecting Salmonella typhimurium described above, wherein the synthesis of the cDNA is performed by taking the extracted RNA as a template and using SuperScript TMIV The first chain synthesis system reverses transcription to synthesize the cDNA of the antibody coding gene.

[0015] Further, in the preparation method of the nanobody for detecting Salmonella typhimurium, the F1 is used as the upstream primer for amplifying the heavy chain variable region gene, and R1, R2 and R3 are used as the downstream primers for amplifying the heavy chain variable region gene.

[0016] The upstream primer F1 is shown in SEQ ID NO: 5.

[0017] The downstream primer R1 is shown in SEQ ID NO: 6.

[0018] The downstream primer R2 is shown in SEQ ID NO: 7.

[0019] The downstream primer R3 is shown in SEQ ID NO: 8.

[0020] Further, in the preparation method of the nanobody for detecting Salmonella typhimurium, the VHH gene fragment after enzyme digestion is connected with the vector pComb3xss after enzyme digestion by using T4 ligase.

[0021] Further, in the preparation method of the nanobody for detecting Salmonella typhimurium, the extraction of the phagemid vector containing the nanobody gene against Salmonella typhimurium is performed by using Plasmid DNA Mini Kit. The plasmid of the positive phagemid is extracted by using Plasmid DNA Mini Kit, and the plasmid is introduced into E.coli TOP10F' by heat shock method.

[0022] The nanobody for detecting Salmonella typhimurium provided by the application is used for preparing a reagent or a kit for detecting Salmonella typhimurium.

[0023] Compared with the prior art, the nanobody for specifically recognizing Salmonella typhimurium is screened out, the detection accuracy is high, the method is simple, and the nanobody is suitable for immunological analysis and detection of Salmonella typhimurium in food, and high-performance rapid immunological detection products are expected to be developed in the detection of Salmonella typhimurium. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the result of phage-ELISA of 96 phage monoclonal antibodies;

[0025] Figure 2 is the expression of the nanobody protein against Salmonella typhimurium analyzed by SDS-PAGE;

[0026] Figure 3This is a standard curve for a double-antibody sandwich ELISA of Salmonella typhimurium based on nanobodies. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that modifications or substitutions to the details and form of the technical solution of the present invention without departing from the technical solution of the present invention shall all fall within the protection scope of the present invention.

[0028] Example 1: Total RNA extraction and cDNA synthesis of anti-Salmonella typhimurium nanobody

[0029] 1. Extraction of peripheral blood lymphocytes from alpacas:

[0030] (1) After washing the Salmonella typhimurium cultured in LB medium for 15 hours, Salmonella typhimurium inactivated antigen was prepared by high temperature sterilization at 120℃ for 30 minutes. The Salmonella typhimurium inactivated antigen was mixed with an equal volume of immune adjuvant and then injected subcutaneously into the alpaca. The alpaca was immunized 3-4 times in a cycle of 14 days. Blood samples of the immunized alpaca could be collected after the third immunization.

[0031] (2) Add 15 mL of peripheral blood lymphocyte separation solution to a 50 mL centrifuge tube, tilt the centrifuge tube slightly at 45° and slowly add 15 mL of collected alpaca blood. Centrifuge the prepared cell separation solution containing alpaca blood at 400 g speed for 30 min at room temperature using a centrifuge with a horizontal rotor.

[0032] (3) Use a pipette to aspirate the cloudy lymphocytes in the middle of the liquid that appears to be layered after centrifugation in step (2) into a new 50mL centrifuge tube;

[0033] (4) Add 10 mL of room temperature PBS buffer to each tube and centrifuge at 400 g for 20 min at room temperature using a horizontal centrifuge.

[0034] (5) Remove the supernatant after centrifugation in step (4), resuspend the precipitate in each tube with 1 mL of PBS buffer, mix gently to obtain cell resuspension, and count the number of cells using a hemocytometer.

[0035] 2. Extraction of total RNA

[0036] According to TRIzol TM The Reagent instructions describe the steps for total RNA extraction as follows:

[0037] (1) Transfer the cell resuspension from step 1) to a 1.5 mL centrifuge tube and centrifuge at 4°C and 8000 g for 2 min.

[0038] (2) Remove the supernatant after centrifugation in step (1). Be careful to remove it slowly to prevent vigorous action from damaging the cell precipitate at the bottom of the centrifuge tube.

[0039] (3) Add TRIzol according to the instructions. TM Reagent reagent (per 10) 7 Add 1 mL of reagent to each cell;

[0040] (4) Mix thoroughly to lyse the cells until no obvious cell precipitate is visible to the naked eye in the lysate.

[0041] (5) Let the pyrolysis solution treated in step (4) stand at room temperature for 5 minutes;

[0042] (6) Add 0.2 mL of chloroform (1 / 5 volume of TRIzol) to the lysis buffer after standing in step (5). TM Mix thoroughly on a vortex mixer (v / v) and let stand at room temperature for 2-3 minutes.

[0043] (7) Add the lysate after step (6) to a centrifuge tube and place it in a centrifuge and centrifuge at 12000g 4℃ for 15min;

[0044] (8) Carefully remove the centrifuge tube after centrifugation in step (7) from the centrifuge. You can see that the lysate in the centrifuge tube is clearly divided into three layers. Be careful not to shake it violently.

[0045] (9) Carefully aspirate the upper layer of lysate from the centrifuge tube in step (8) and transfer it to another new centrifuge tube. Be careful not to aspirate the white flocculent material in the middle layer of the lysate.

[0046] (10) Add 0.5 mL of isopropanol (1 / 2 volume of TRIzol) to the upper lysate collected in step (9). TM Mix thoroughly by gently inverting the container (v / v), and let stand at room temperature for 10 minutes.

[0047] (11) Place the centrifuge tubes processed in step (10) into a centrifuge and centrifuge at 12000g at 4℃ for 10min;

[0048] (12) Carefully discard the supernatant after centrifugation in step (11), then add TRIzol to the centrifuge tube. TM An equal volume of 75% (v / v) ethanol;

[0049] (13) Place the centrifuge tubes processed in step (12) into a centrifuge and centrifuge at 7500g at 4℃ for 5min;

[0050] (14) Carefully discard the supernatant after centrifugation in step (13), invert the centrifuge tube and dry it at room temperature for 5 minutes;

[0051] (15) After confirming that the centrifuge tubes processed in step (14) are dry, add 20 μL of deionized water to dissolve the precipitate and obtain total RNA;

[0052] (16) Measurement of OD of total RNA products using ultraviolet spectrophotometer 260 nm and OD 280 The absorbance value was measured in nm to calculate the concentration and purity of the extracted RNA, which was then stored in an ultra-low temperature freezer at -80℃ for later use.

[0053] 3. cDNA Synthesis

[0054] Using the extracted RNA as a template, SuperScript was used. TM IV first-strand synthesis system reverse transcription synthesizes cDNA encoding antibody genes.

[0055] The process is as follows:

[0056] (1) Add the reagents shown in Table 1 to the PCR tube:

[0057] Table 1

[0058]

[0059] Place the reaction in a PCR instrument, incubate at 65°C for 5 minutes, and then cool on ice.

[0060] (2) Add reverse transcription reaction solution to the above reaction system, as shown in Table 2:

[0061] Table 2

[0062]

[0063]

[0064] The mixed reaction solution was placed in the PCR instrument, and the conditions were set as shown in Table 3:

[0065] Table 3

[0066]

[0067] (3) Add 1 μL of LE.coli RNase H and incubate at 37°C for 20 min.

[0068] (4) Store the reverse transcription product cDNA library at -80℃ for later use.

[0069] Example 2: Gene amplification of anti-Salmonella typhimurium nanobody

[0070] 1. Amplification of the IgG2 / 3 antibody heavy chain variable region (VHH) gene

[0071] Primers for amplifying the VHH antibody gene were synthesized by Shanghai Bioengineering Co., Ltd., and the primer sequences are shown in Table 4.

[0072] Table 4

[0073]

[0074] Note: Underlined bases are enzyme cleavage sites.

[0075] (1) Using cDNA as a template, F1 as the upstream primer, and R1, R2, and R3 as the downstream primers, the heavy chain variable region gene was amplified.

[0076] Add the reagents shown in Table 5 to the PCR tube:

[0077] Table 5

[0078]

[0079] Set the following conditions in the PCR instrument, and the system is shown in Table 6:

[0080] Table 6

[0081]

[0082] 10 μL of PCR product was analyzed and verified by 1% agarose gel electrophoresis, and the remaining PCR products were processed using... The Cycle Pure Kit recovers the target gene, purifies it to obtain the VHH antibody gene fragment, and measures the concentration and purity of the recovered DNA. It is then stored at -20°C for later use.

[0083] (2) Agarose gel electrophoresis

[0084] ① Weigh 0.25g of agarose and dissolve it in 25mL of TAE buffer to prepare a 1% agarose gel;

[0085] ② Heat in a microwave oven until the agarose is completely dissolved, forming a colorless and transparent gel solution;

[0086] ③After the gel solution cools to about 50°C, add 1 μL of nucleic acid dye and mix thoroughly;

[0087] ④ Pour the gel solution prepared in step ③ into the gel casting tank and allow it to cool and solidify at room temperature;

[0088] ⑤ Remove the comb, add 10× loading buffer to the PCR product from step (1), and add it to the corresponding well of the completely solidified agarose gel;

[0089] ⑥ Place the gel into the electrophoresis tank and add 1×TAE buffer, then turn on the electrophoresis apparatus and run it at 100V for 30 minutes;

[0090] ⑦ Remove the gel and observe the results under a UV lamp.

[0091] Example 3: Construction of a phage nanobody library against Salmonella

[0092] 1. Enzymatic digestion reaction of vector pComb3Xss and nanobody gene fragment

[0093] Since the VHH antibody gene fragment obtained in Example 2 has two SfiI restriction sites introduced at both ends of the target gene, it can be directly digested and recombined with the pComb3Xss vector and ligated with T4 ligase.

[0094] The process is briefly described as follows:

[0095] (1) Enzyme digestion of vector pComb3xss

[0096] ① Add the reagents shown in Table 7 to the PCR tube:

[0097] Table 7

[0098]

[0099] ② Place in a PCR instrument and incubate overnight at 50°C;

[0100] ③ Add 22.5 μL of 0.5 M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating SfiI enzyme;

[0101] ④ The enzyme digestion products were analyzed by 0.7% agarose gel electrophoresis to detect the enzyme digestion effect;

[0102] ⑤ Recover the enzyme digestion products.

[0103] (2) Enzyme digestion of VHH antibody gene fragment

[0104] ① Add the reagents shown in Table 8 to the PCR tube:

[0105] Table 8

[0106]

[0107]

[0108] ② Place in a PCR instrument and incubate overnight at 50°C;

[0109] ③ Add 22.5 μL of 0.5 M EDTA, mix well, and terminate the enzymatic digestion reaction by inactivating SfiI enzyme;

[0110] ④ Cycle Pure Kit recovers enzyme digestion products.

[0111] 2. Ligation and purification of vector pComb3Xss and nanobody gene fragments

[0112] ① Add the reagents shown in Table 9 to the PCR tube:

[0113] Table 9

[0114]

[0115] ② Place the PCR tube containing the reaction solution from step ① into a PCR instrument and react overnight at 16°C;

[0116] ③ The next day, the reaction solution from step ② was incubated in the PCR instrument at 65°C for 10 minutes to allow the T4 DNA ligase to be thermally inactivated, thus terminating the ligation reaction.

[0117] ④ Transfer the ligation product of the vector pComb3Xss and VHH gene fragment from step ③ to a 1.5 mL centrifuge tube, add 20 μL of 3M sodium acetate (pH = 5.2), and then add 660 μL of anhydrous ethanol;

[0118] ⑤ Place the centrifuge tubes from step ④ at -20°C overnight;

[0119] ⑥ The next day, place the centrifuge tube containing the ligation product from step ⑤ into a centrifuge and centrifuge at 4°C and 12,000 rpm for 30 minutes.

[0120] ⑦ After centrifugation in step ⑥, a white DNA precipitate can be seen in the centrifuge tube. Discard the supernatant and add 75% ethanol to the centrifuge tube to resuspend the precipitate.

[0121] ⑧ Place the centrifuge tubes processed in step ⑦ into a centrifuge and centrifuge at 4℃ and 12000rpm for 15min;

[0122] 9. Discard the supernatant after centrifugation in step 8. Invert the centrifuge tube onto absorbent paper and let it stand for several minutes to allow any residual ethanol in the centrifuge tube to evaporate.

[0123] ⑩ Add 30 μL of sterile water to reconstitute the precipitate, and store the sample at -20℃ for later use.

[0124] 3. Electroporation of the ligation product of vector pComb3Xss and VHH gene fragment

[0125] (1) Remove E. coli ER2738 electrotransformed competent cells from the -80℃ freezer and thaw them on ice;

[0126] (2) Using 25 μL of electrocompetent cells as one reaction, add 3 μL of the purified ligation product obtained in step 2 and incubate on ice for 10 min;

[0127] (3) The E.coli ER2738 competent cells with the ligation product added in step (2) were rapidly transferred to a pre-cooled electroporation cup and electroporated under the conditions of 1.8KV, 200Ω, and 25μF.

[0128] (4) Add 975 μL of preheated SOC medium at 37℃ within 10 seconds, resuspend the transformed cells and transfer them to a 50 mL centrifuge tube.

[0129] (5) Repeat steps (3) and (4) above 9 times, and transform all 30 μL of the ligation product from step 2 into E.coli ER2738 competent cells;

[0130] (6) Place the competent cells transformed in step (5) in a shaker and revive them at 37°C and 250 rpm for 1 hour.

[0131] (7) Take 2 μL of the culture solution from step (6) and dilute it 10 times. Then spread it on LB plates containing 50 μg / mL carbenicillin. Place the LB plates in a constant temperature incubator and incubate them upside down at 37°C overnight.

[0132] (8) The next day, calculate the number of colonies on the plate after the culture in step (7), and calculate the library capacity of the original antibody library in combination with the dilution ratio;

[0133] (9) Randomly select 16 transformants from the plate after culturing in step (7), and culture them overnight in LB medium containing 50 μg / mL carbenicillin. Perform DNA sequencing on the cultured bacteria of the 16 transformants to analyze the diversity of inserted sequences.

[0134] (10) Add the remaining culture medium from step (6) to 200 mL of SB medium, add 200 μL of carbenicillin (50 mg / mL) and 200 μL of tetracycline (20 mg / mL), continue culturing for 2 hours, and then prepare the phage library.

[0135] 4. Construction of nanobody phage display library

[0136] (1) Add 1 mL of 1×10⁻⁶ bacteria to the bacterial culture culture after step 3 (10). 13 Helper phages at cfu / mL were incubated at 37°C for 30 min to infect E. coli ER2738 containing the VHH antibody gene.

[0137] (2) Place the bacterial culture after incubation in step (1) on a shaker at 37°C and 250 rpm for 2 hours;

[0138] (3) Add 200 μL of kanamycin (70 mg / mL) to the bacterial culture in step (2), and incubate overnight at 37°C and 250 rpm on a shaker;

[0139] (4) The next day, transfer the bacterial culture from step (3) into a centrifuge bottle and centrifuge at 4°C and 10,000 rpm for 30 min.

[0140] (5) Collect the supernatant after centrifugation in step (4), add 50 mL of 5×PEG / NaCl, and let stand on ice for 2 h to precipitate helper phages containing the VHH antibody gene;

[0141] (6) Centrifuge the supernatant after standing in step (5) at 4℃ and 10000rpm for 30min. A white phage precipitate will be visible. After removing the supernatant from the centrifuge tube, add 100mL of PBS to wash and dissolve the white phage precipitate. Add 25mL of 5×PEG / NaCl to reprecipitate. Continue to stand on ice for 2h.

[0142] (7) Centrifuge the solution after standing in step (6) at 4℃ and 10000rpm for 30min. Reconstitute the white phage precipitate after centrifugation with 10mL of sterile PBS containing 0.1% BSA. Sterilize the precipitate by passing it through a 0.22μm filter and collect it back into a sterile centrifuge tube. Take 2μL of the precipitate for titer determination. The result is the library capacity of the nanobody phage library. Store the remaining phage solution at -80℃ for later use.

[0143] Example 4: Screening of anti-Salmonella typhimurium nanobodies

[0144] (1) Salmonella typhimurium was sequentially diluted to 10⁻¹⁰ using CBS coating buffer according to different round numbers. 9 10 8 10 7 Add 100 μL of cfu / mL to each well of a 96-well plate and coat overnight at 4°C.

[0145] (2) The next day, discard the coating solution, wash four times with PBST, add 3% skim milk powder at 250 μL / well, and incubate at 37°C for 1 h.

[0146] (3) Discard the blocking solution, wash four times with PBST, add 100 μL / well to the phage solution (for phage display nanobody library) prepared in step (7) of Example 3, label it as Input, and incubate at 37°C for 1 h;

[0147] (4) Aspirate the phage solution from the well after step (3) and vigorously blow and aspirate with PBST 10-20 times;

[0148] (5) Add 100 μL / well of 0.1 M glycine-hydrochloric acid (pH = 2.2) to the wells after step (4) treatment, and incubate at 37°C for 10 min to wash away the phages bound to the well plate;

[0149] (6) Add 50 μL / well of 1M Tris-HCl (pH=8.8) to the wells after step (5) to neutralize the eluted phage solution;

[0150] (7) Aspirate all of the phage solution from step (6) into a centrifuge tube, take 2 μL to determine the titer, and add the remaining phage solution to 2 mL of freshly cultured E. coli ER2738 bacterial suspension (OD200). 600 =0.8), transfer to a 37℃ incubator and let stand for 30 minutes;

[0151] (8) Add 6 mL of LB solution containing 50 μg / mL carbenicillin to the bacterial culture infected by bacteriophage in step (7), and place it in a shaker at 37°C for 2 h.

[0152] (9) Add 1 mL of helper phage (1 × 10⁻⁶) to the bacterial culture treated in step (8). 13 Add 91 mL of LB medium and carbenicillin (final concentration 50 μg / mL), and incubate at 37°C in a shaker for 2 h.

[0153] (10) Add kanamycin (final concentration of 70 μg / mL) to the bacterial culture after step (9) and incubate overnight in a shaker at 37°C.

[0154] (11) The next day, following the method for preparing the nanobody phage library described in steps (4), (5), (6), and (7) of Example 3, the phages were precipitated with 5×PEG / NaCl. The phage precipitate was then reconstituted with 2 mL of sterile PBS containing 0.1% BSA. The resulting phages were labeled as Output and then added to the next round of panning. Table 10 shows the conditions for the three rounds of phage panning.

[0155] Table 10

[0156]

[0157] Example 5: Screening of strongly positive nanoantibody strains

[0158] (1) Prepare a 96-well deep well plate and add 1 mL of SB medium containing 50 μg / mL carbenicillin to each well;

[0159] (2) From the Output phage titer assay plates after the first, second, and third rounds of screening, 32 single-clone colonies were randomly selected. Each selected single-clone colony was inoculated into a single well of a 96-well plate and then incubated at 37°C and 250 rpm for several hours (until OD). 600 =0.8);

[0160] (3) Add 10 μL of 1×10⁻⁶ mol / L of the culture solution after step (2) to each well. 13 cfu / mL helper phage (the helper phage is a phage without the VHH gene plasmid), incubate at 37℃ for 15 min to allow the phage to complete infection;

[0161] (4) Place the deep well plate after step (3) in a shaker at 37°C and 250 rpm for 2 hours;

[0162] (5) Add kanamycin to the deep well plate after culturing in step (4) to a final concentration of 70 μg / mL, and incubate overnight at 37°C and 250 rpm in a shaker.

[0163] (6) The next day, remove the deep well plate from the incubator and let the culture medium stand for later use;

[0164] (7) Dilute Salmonella Typhimurium to 10⁻⁶ CBS buffer. 8 Add 100 μL of cfu / mL to each well of the microplate and incubate overnight at 4°C. Meanwhile, prepare another microplate and add CBS coating buffer as a negative control.

[0165] (8) The next day, the coating solution was aspirated from the two microplates prepared in step (7), washed four times with PBST, and 3% skim milk powder was prepared. 250 μL / well was added to the two 96-well microplates and incubated at 37°C for 1 hour.

[0166] (9) Aspirate the blocking solution from the two microplates in step (8), wash with PBST four times, and then add 100 μL / well of the phage culture supernatant from the deep well plate in step (6) after standing. Incubate at 37°C for 1 h.

[0167] (10) Aspirate the reaction solution from the two microplates in step (9), wash four times with PBST, add 100 μL / well of anti-M13-HRP enzyme-labeled antibody (1:2000 dilution), and incubate at 37°C for 1 h.

[0168] (11) Aspirate the reaction solution from the two microplates in step (10), wash five times with PBST, add 100 μL / well TMB colorimetric solution, and react at 37°C for 15 min.

[0169] (12) Add 50 μL / well 2M H2SO4 to the two microplates after color development in step (11) to stop the reaction, and read the OD in a microplate reader. 450 The absorbance values ​​are shown in the attached diagram. Figure 1 Initial screening of positive clones.

[0170] Example 6: Prokaryotic Expression and Purification of Anti-Salmonella Typhimurium Nanobodies

[0171] 1. Prokaryotic expression of nanobody proteins

[0172] (1)Reference Plasmids were extracted from positive phages using the Plasmid DNA Mini Kit and introduced into E. coli TOP10F' using a heat-shock method.

[0173] The specific steps are as follows: Remove the frozen E. coli TOP10F' competent cells from -80℃ and immediately place them on ice for 5 min. Then, add 1 μL of the positive phage plasmid to each tube of competent cells and continue to place them on ice for 10 min. Subsequently, place the competent cells in a 42℃ water bath for 90 s and immediately place them back on ice to cool for several minutes. Add 1 mL of SB medium to each tube of competent cells and culture at 37℃ and 250 rpm for 1 h. Finally, take 100 μL of the culture medium from each tube and spread it onto a CA-resistant plate and incubate overnight at 37℃.

[0174] (2) Pick a single positive clone from the plate after culturing in step (1) and inoculate it into 5 mL of SB culture medium containing 50 μg / mL. Place it in a shaker at 37℃ and 250 rpm overnight.

[0175] (3) The next day, the bacterial culture from step (2) overnight was transferred to 200 mL of SB culture medium containing 50 μg / mL at an inoculation rate of 1%, and incubated at 37°C and 250 rpm for several hours until OD. 600 =0.8;

[0176] (4) Add IPTG to the bacterial culture after step (3) to make the final concentration 0.5-1mM, and place it in a shaker at 30℃ and 250rpm overnight.

[0177] (5) Collect the bacterial culture cultured overnight in step (4) into a centrifuge bottle and centrifuge at 4℃ and 5000g for 20min;

[0178] (6) Remove the supernatant after centrifugation in step (5), weigh the bacterial cells, add 10 mL of bacterial lysis buffer, repeatedly blow and aspirate to resuspend the bacterial cells, rotate and invert at room temperature for 15 min to fully lyse the bacteria.

[0179] (7) Centrifuge the bacterial lysate from step (6) at 4°C and 15000g for 10 min;

[0180] (8) Collect the supernatant from step (7), which is a soluble protein, for subsequent purification.

[0181] 2. Purification of nanobody proteins

[0182] (1) Take 1 mL of Ni-NTA matrix from the nickel column and transfer it to a centrifuge tube. Repeat centrifugation 3 times and replace the preservation solution with PBS. Add it to the soluble protein in step 1 (8) and mix by rotating and inverting at room temperature for 1 h.

[0183] (2) Fix the empty purification column vertically, add the reaction solution from step (1) into the empty purification column, collect the sample flow-through, and leave the nickel column on the purification column.

[0184] (3) Add 5 column volumes of equilibration buffer (PBS containing 10 mM imidazole) to the nickel column from step (2) to elute impurities;

[0185] (4) Add 5 column volumes of elution buffer (PBS containing 250 mM imidazole) to the nickel column from step (3), collect the elution buffer in separate tubes until the elution buffer reaches column A. 280 The signal value at that time is close to 0;

[0186] (5) Collect the eluent obtained in step (4), which contains purified nanobody protein, and verify it by SDS-PAGE electrophoresis. (See [reference]). Figure 2 In the figure: M is the protein standard; 1 is the nanobody protein expressed by SEQ-Styp NO.1; 2 is the nanobody protein expressed by SEQ-Styp NO.2.

[0187] 3 is the nanobody protein expressed by SEQ-Styp NO.3; 4 is the nanobody protein expressed by SEQ-Styp NO.4.

[0188] (6) Collect and combine the purified nanobody solution obtained in step (4) and put it into a dialysis bag. Dialyze it in PBS at 4°C for three days, changing the solution twice a day to replace the nanobody dissolution medium.

[0189] Application example: Application of nanobodies

[0190] 1. Establishment of the standard curve for double-antibody sandwich ELISA

[0191] (1) Dilute the polyclonal antibody serum with CBS at a ratio of 1:1000, add 100 μL / well to a 96-well microplate, and coat overnight at 4°C;

[0192] (2) The next day, the coating buffer solution in the well plate was aspirated and the plate was washed three times with PBST.

[0193] (3) Add 270 μL / well of 3% skim milk powder blocking solution, incubate at 37°C for 1 h, then remove the blocking solution and wash the plate three times with PBST;

[0194] (4) Salmonella typhimurium from 10 7 After serially diluting the cfu / mL concentration, add 100 μL / well to the microplate treated in step (3) and incubate at 37°C for 1 h.

[0195] (5) Aspirate the liquid from the wells of the ELISA plate after step (4) and wash the plate three times with PBST;

[0196] (6) Dilute the nanobody to 40 μg / mL, and then add 100 μL / well to the enzyme-labeled plate after step (5) for reaction and incubate at 37°C for 1 h;

[0197] (7) Aspirate the liquid from the wells of the ELISA plate in step (6) and wash the plate three times with PBST;

[0198] (8) Dilute the HRP enzyme-labeled Anti-HA mouse monoclonal antibody at 1:10000 and add 100 μL / well to the well plate treated in step (7) and incubate at 37°C for 1 h.

[0199] (9) Aspirate the liquid from the wells of the ELISA plate in step (8) and wash the plate five times with PBST;

[0200] (10) Add 100 μL / well TMB chromogenic substrate solution to the microplate after step (9) and react at 37°C in the dark for 15 min.

[0201] (11) Add 50 μL / well 2M H2SO4 to the chromogenic solution in step (10) to terminate the reaction;

[0202] (12) Place the ELISA plate in the ELISA reader and read the OD. 450 ;

[0203] (13) Plot a standard curve with Salmonella typhimurium concentration (log) on ​​the x-axis and absorbance on the y-axis. (Refer to...) Figure 3 .

[0204] pass Figure 3 The ELISA detection standard curve shows that the anti-Salmonella typhimurium nanobody provided in this application can effectively bind to anti-Salmonella typhimurium nanoviruses, and within a certain concentration range, the absorbance shows a good linear relationship with the bacterial concentration, thereby specifically achieving the detection of anti-Salmonella typhimurium nanoviruses.

Claims

1. A nanobody for detecting Salmonella typhimurium, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

3.

2. The application of the nanobody for detecting Salmonella typhimurium as described in claim 1 in the preparation of reagents or kits for detecting Salmonella typhimurium.

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