Nanometer antibody for detecting salmonella typhimurium

Abstract

The invention discloses a nano antibody for detecting salmonella typhimurium, and aims to provide a nano antibody for detecting salmonella typhimurium, which is high in detection accuracy, strong in specificity and simple in obtaining method. The amino acid sequences of the nano antibody are as shown in SEQ (sequence)-Styp NO.1, SEQ (sequence)-Styp NO.2, SEQ (sequence)-Styp NO.3 and SEQ (sequence)-Styp NO.4; belongs to the technical field of biological detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a nano antibody for detecting Salmonella typhimurium. Background Art

[0002] Salmonellosis caused by Salmonella infection is a common bacterial disease that poses a serious threat to food production and safety. Every year, food poisoning and diarrhea caused by Salmonella around the world seriously endanger human life and health. Salmonella typhimurium (S.typh) is one of the most common pathogens in the genus Salmonella. It can cause bacterial diarrhea. The incidence rate after infection ranks first among Salmonella infections. 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 households or catering service institutions, posing a serious threat to the breeding industry and public health safety. Improper feeding and management can cause animals such as cattle and sheep to be infected with Salmonella typhimurium, and people are prone to illness through contaminated food or contact with infected animals. Salmonella typhimurium also hinders the large-scale breeding of sheep. After infection with the bacteria, sick sheep often show weakness, anorexia, and fever. Lambs will have obvious diarrhea symptoms, and even die in severe cases. Therefore, the monitoring of Salmonella typhimurium has great application needs in food safety, public health and animal husbandry.

[0003] Currently, common detection methods for Salmonella typhimurium include molecular detection technology (PCR technology), bacterial culture method, immunological method and mass spectrometry. Molecular detection technology represented by PCR has excellent specificity and sensitivity, and can accurately detect the target DNA sequence in a short time. However, the implementation of PCR technology requires professional equipment and technicians, and may produce false positive results that affect the detection effect. The bacterial culture method is easy to operate and the results are intuitive. It is the "gold standard" method for bacterial detection, but the entire detection process takes a long time and has high requirements for the number of bacteria, and there is a significant omission rate for the inspection of low-concentration samples. Mass spectrometry can directly detect molecular features with high accuracy, but the equipment cost is expensive and the operation process is complicated. The specificity of immunological methods is high, but it may be affected by the quality and pairing of antibodies, and there are problems of cross-reaction 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 nanoantibodies prepared by phage display technology and prokaryotic expression technology. Summary of the invention

[0004] The purpose of the present invention is to provide a nano antibody for detecting Salmonella typhimurium, which has high detection accuracy, strong specificity and a simple acquisition method.

[0005] The present invention also provides a method for preparing a nano antibody that specifically recognizes Salmonella typhimurium (strain type: ATCC 14028).

[0006] To this end, the first technical solution provided by the present invention is as follows:

[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] Furthermore, in the above-mentioned nanobody for detecting Salmonella typhimurium, the strain of Salmonella typhimurium is ATCC 14028.

[0009] The second technical solution provided by the present invention is the preparation method of the above-mentioned nanoantibody for detecting Salmonella typhimurium, which comprises the following steps in sequence:

[0010] 1) collecting blood samples from immune alpacas, separating and collecting peripheral blood lymphocytes from alpacas, extracting total RNA of anti-Salmonella typhimurium nanoantibodies from peripheral blood lymphocytes from alpacas, and synthesizing cDNA; using cDNA as a template, amplifying heavy chain variable region genes, connecting the amplified alpaca antibody heavy chain variable region genes to a phagemid vector, and then transforming them into competent Escherichia coli to construct an immune alpaca nanoantibody phage library;

[0011] 2) Using Salmonella typhimurium as the coating antigen, the pH elution method was used to obtain nanobody phages that can specifically recognize Salmonella typhimurium after 3 or more rounds of panning, and the genes of the nanobody were compared by sequencing analysis;

[0012] 3) Extracting the phagemid vector containing the anti-Salmonella typhimurium nanobody gene and re-introducing it into the host expression bacteria to express and purify the nanobody protein.

[0013] Furthermore, in the above-mentioned method for preparing nanoantibodies for detecting Salmonella typhimurium, the total RNA is extracted according to TRIzol TM Total RNA was extracted according to the instructions of the Reagent.

[0014] Furthermore, in the above-mentioned method for preparing nanoantibodies for detecting Salmonella typhimurium, the synthesis of cDNA is based on the extracted RNA as a template, using SuperScript TMIV first-strand synthesis system reverse transcribes and synthesizes cDNA of antibody encoding gene.

[0015] Furthermore, in the above-mentioned method for preparing a nanobody for detecting Salmonella typhimurium, the amplification of the heavy chain variable region gene F1 is an upstream primer, and R1, R2, and R3 are used as downstream primers to amplify the heavy chain variable region gene;

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

[0017] The nucleotide of downstream primer R1 is shown in SEQ ID NO: 6;

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

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

[0020] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Salmonella typhimurium, the heavy chain variable region gene is connected to the phagemid vector by connecting the VHH gene fragment after enzyme digestion with the vector pComb3xss after enzyme digestion using T4 ligase.

[0021] Furthermore, in the above-mentioned method for preparing a nanobody for detecting Salmonella typhimurium, the phagemid vector containing the anti-Salmonella typhimurium nanobody gene is extracted. Plasmid DNA Mini Kit was used to extract the plasmid of positive phagemid, and the plasmid was introduced into E.coli TOP10F' by heat shock method.

[0022] The nano antibody for detecting Salmonella typhimurium provided by the present invention is used for preparing a reagent or a kit for detecting Salmonella typhimurium.

[0023] Compared with the prior art, the present invention screened out nano-antibodies that specifically recognize Salmonella typhimurium, has high detection accuracy, and is simple to obtain. It is suitable for immunoassay detection of Salmonella typhimurium in food, and is expected to develop high-performance rapid immunoassay products in the application of Salmonella typhimurium detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the result of phage-ELISA of 96 phage monoclonals;

[0025] Figure 2 is SDS-PAGE analysis of the expression of anti-Salmonella typhimurium nanobody protein;

[0026] Figure 3This is the standard curve of the Salmonella typhimurium double antibody sandwich ELISA based on nanoantibodies. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific implementation methods, but those skilled in the art should understand that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the technical solution of the present invention, and these modifications and replacements all fall within the scope of protection of the present invention.

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

[0029] 1. Extraction of alpaca peripheral blood lymphocytes:

[0030] (1) After the Salmonella typhimurium cultured in LB medium for 15 hours is washed, the inactivated Salmonella typhimurium antigen is prepared by high temperature sterilization at 120° C. for 30 minutes, the inactivated Salmonella typhimurium antigen is mixed with an immune adjuvant in equal volumes, and then the alpaca is immunized by subcutaneous injection. The immunization is performed 3-4 times in a cycle of 14 days, and a blood sample of the immunized alpaca can be collected after the third immunization;

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

[0032] (3) Using a pipette, pipette the cloudy lymphocytes in the liquid layer after centrifugation in step (2) into a new 50 mL 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.

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

[0035] 2. Total RNA Extraction

[0036] According to TRIzol TM The total RNA was extracted according to the instructions of the reagent. The steps are as follows:

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

[0038] (2) Aspirate the supernatant after centrifugation in step (1). Be careful to aspirate slowly to avoid damaging the cell pellet at the bottom of the centrifuge tube due to violent movements;

[0039] (3) Add TRIzol according to the instructions TM Reagent (10 7 cells were added with 1 mL of reagent);

[0040] (4) Mix thoroughly to lyse the cells until no obvious cell precipitation is visible in the lysis solution;

[0041] (5) standing the lysate treated with lysis in step (4) at room temperature for 5 minutes;

[0042] (6) Add 0.2 mL of chloroform (1 / 5 volume of TRIzol TM , v / v), mix thoroughly on a vortexer, and let stand at room temperature for 2-3 min;

[0043] (7) adding the lysate treated in step (6) into a centrifuge tube and centrifuging at 12000 g for 15 min at 4°C;

[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 the lysate in the centrifuge tube in step (8) and transfer it to another new centrifuge tube, being careful not to aspirate the white flocculent in the middle layer of the lysate;

[0046] (10) Add 0.5 mL of isopropanol (1 / 2 volume of TRIzol TM , v / v), gently invert upside down to mix thoroughly, and let stand at room temperature for 10 min;

[0047] (11) placing the centrifuge tube treated in step (10) in a centrifuge and centrifuging at 12000 g at 4°C for 10 min;

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

[0049] (13) Place the centrifuge tube treated in step (12) in a centrifuge and centrifuge at 7500 g at 4° C. for 5 min;

[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 tube treated in step (14) is dry, add 20 μL of deionized water to dissolve the precipitate to obtain total RNA;

[0052] (16) UV spectrophotometer to measure the OD of total RNA product 260 nm and OD 280 The concentration and purity of the extracted RNA were calculated based on the absorbance value at nm and stored in a -80°C ultra-low temperature refrigerator for later use.

[0053] 3. cDNA Synthesis

[0054] Using the extracted RNA as a template, SuperScript TM IV first-strand synthesis system reverse transcribes and synthesizes cDNA of antibody encoding gene.

[0055] The process is as follows:

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

[0057] Table 1

[0058]

[0059] The reaction was placed in a PCR instrument, incubated at 65°C for 5 min, and then cooled on ice;

[0060] (2) Continue to add reverse transcription reaction solution to the above reaction system, the system is shown in Table 2:

[0061] Table 2

[0062]

[0063]

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

[0065] Table 3

[0066]

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

[0068] (4) Store the reverse transcription product cDNA library in a -80°C refrigerator for future use.

[0069] Example 2: Anti-Salmonella typhimurium nanobody gene amplification

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

[0071] The primers for amplifying the VHH antibody gene were synthesized by Shanghai Bioengineering Company, and the primer sequences are shown in Table 4:

[0072] Table 4

[0073]

[0074] Note: The underlined bases are restriction enzyme cleavage sites.

[0075] (1) Using cDNA as a template, F1 as an upstream primer, and R1, R2, and R3 as 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] The following conditions were set in the PCR instrument, the system is shown in Table 6:

[0080] Table 6

[0081]

[0082] Take 10 μL of PCR product and verify it by 1% agarose gel electrophoresis, and use the rest of the PCR product to The target gene was recovered by Cycle Pure Kit, and the VHH antibody gene fragment was obtained after purification. The concentration and purity of the recovered DNA were measured and stored at -20°C for future use.

[0083] (2) Agarose gel electrophoresis

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

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

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

[0087] ④ Pour the gel solution processed in step ③ into the gel making tank and cool and solidify at room temperature;

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

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

[0090] ⑦ Remove the gel and place it under ultraviolet light to observe the results.

[0091] Example 3: Construction of anti-Salmonella phage nanobody library

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

[0093] Since the VHH antibody gene fragment obtained in Example 2 has introduced two SfiI restriction sites 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 PCR instrument, 50℃, incubate overnight;

[0100] ③ Add 22.5 μL 0.5 M EDTA and mix well to inactivate the SfiI enzyme and terminate the enzyme digestion reaction;

[0101] ④ Detect the effect of enzyme cleavage by 0.7% agarose gel electrophoresis;

[0102] ⑤Recover the enzyme cleavage products.

[0103] (2) Enzymatic cleavage of VHH antibody gene fragments

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

[0105] Table 8

[0106]

[0107]

[0108] ② Place in PCR instrument, 50℃, incubate overnight;

[0109] ③ Add 22.5 μL 0.5 M EDTA and mix well to inactivate the SfiI enzyme and terminate the enzyme digestion reaction;

[0110] ④ The digestion products were recovered by Cycle Pure Kit.

[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 in step ① into a PCR instrument and react at 16°C overnight;

[0116] ③ The next day, the reaction solution of step ② was continued to be incubated in a PCR instrument at 65°C for 10 min to heat-inactivate T4 DNA ligase and terminate the ligation reaction;

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

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

[0119] ⑥ The next day, place the centrifuge tube containing the ligation product in step ⑤ into a centrifuge and centrifuge at 4°C, 12000 rpm for 30 min;

[0120] ⑦ After the 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] ⑧Put the centrifuge tube treated in step ⑦ into a centrifuge and centrifuge at 4°C, 12000 rpm for 15 min;

[0122] ⑨Discard the supernatant after centrifugation in step ⑧, invert the centrifuge tube on absorbent paper, and let it stand for a few minutes to evaporate the residual ethanol in the centrifuge tube.

[0123] ⑩ Add 30 μL of sterile water to re-dissolve the precipitate and store the sample at -20°C for later use.

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

[0125] (1) Take out the E. coli ER2738 electroporation competent cells from the -80°C refrigerator and thaw on ice;

[0126] (2) Take 25 μL of electroporated competent cells as one reaction, add 3 μL of the purified ligation product from step 2, and incubate on ice for 10 min;

[0127] (3) The E. coli ER2738 competent cells to which the ligation product has been added in step (2) are quickly transferred to a pre-cooled electroporation cuvette and transformed by electroporation at 1.8 KV, 200 Ω, and 25 μF;

[0128] (4) Within 10 seconds, quickly add 975 μL of 37°C preheated SOC medium, 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 shake at 37°C and 250 rpm for 1 hour to recover;

[0131] (7) Take 2 μL of the culture solution of step (6) and perform 10-fold gradient dilution, then spread them on LB plates containing 50 μg / mL carbenicillin, place the LB plates in a constant temperature incubator, and culture them upside down at 37°C overnight;

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

[0133] (9) 16 transformants were randomly selected from the plate cultured in step (7), and cultured overnight in LB medium containing 50 μg / mL carbenicillin. DNA sequencing was performed on the cultured bacteria of the 16 transformants to analyze the diversity of the inserted sequences;

[0134] (10) The remaining culture medium in step (6) was added to 200 mL of SB medium, and 200 μL of carbenicillin (50 mg / mL) and 200 μL of tetracycline (20 mg / mL) were added. The culture was continued for 2 hours, and then the phage library was prepared.

[0135] 4. Construction of Nanobody Phage Display Library

[0136] (1) Add 1 mL of 1×10 13 cfu / mL of helper phage, and incubate at 37°C for 30 min to allow the phage to infect E. coli ER2738 containing the VHH antibody gene;

[0137] (2) placing the bacterial solution incubated in step (1) on a shaker at 37°C and 250 rpm for 2 h;

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

[0139] (4) The next day, the bacterial solution from step (3) was transferred to a centrifuge bottle and centrifuged at 10,000 rpm for 30 min at 4°C;

[0140] (5) Collecting the supernatant after centrifugation in step (4), adding 50 mL of 5×PEG / NaCl, and standing on ice for 2 h to precipitate the helper phage containing the VHH antibody gene;

[0141] (6) The supernatant after standing in step (5) was centrifuged at 4°C, 10,000 rpm for 30 min. A white phage precipitate was visible. The supernatant in the centrifuge tube was aspirated and 100 mL of PBS was added to wash and dissolve the white phage precipitate. 25 mL of 5×PEG / NaCl was added to re-precipitate, and the mixture was allowed to stand on ice for 2 h.

[0142] (7) The solution after standing in step (6) was centrifuged at 4°C, 10000 rpm for 30 min, and the white phage precipitate after centrifugation was re-dissolved with 10 mL of sterile PBS containing 0.1% BSA, and sterilized by 0.22 μm filter membrane and re-collected into a sterile centrifuge tube, and 2 μL was taken for titer determination. The determination result is the library capacity of the nanobody phage library, and the remaining phage solution was placed in a -80°C refrigerator for use.

[0143] Example 4 Screening of Anti-Salmonella Typhimurium Nanobodies

[0144] (1) Use CBS coating buffer to dilute Salmonella typhimurium to 10 9 , 10 8 , 10 7 cfu / mL, 100 μL / well was added to a 96-well plate and coated 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 of the phage solution (the phage-displayed nanoantibody library) prepared in step (7) of Example 3, marked as Input, and incubate at 37°C for 1 h;

[0147] (4) Aspirate the phage solution in the wells treated in step (3) and vigorously pipette and aspirate 10-20 times with PBST;

[0148] (5) Add 100 μL / well of 0.1 M glycine-HCl (pH = 2.2) to the wells treated in step (4) and incubate at 37°C for 10 min to elute the bound phages on the plate;

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

[0150] (7) Pipette all the phage solution from step (6) into a centrifuge tube, take 2 μL of it to measure the titer, and add the remaining phage solution to 2 mL of freshly cultured E. coli ER2738 bacterial solution (OD 600 =0.8), moved to a 37°C incubator and allowed to stand for 30 min;

[0151] (8) Add 6 mL of LB solution containing 50 μg / mL carbenicillin to the bacterial solution infected with phage in step (7), and incubate in a shaker at 37°C for 2 h;

[0152] (9) Add 1 mL of helper phage (1×10 13 cfu / mL), supplemented with 91 mL of LB medium and carbenicillin (final concentration of 50 μg / mL), and cultured in a shaker at 37°C for 2 h;

[0153] (10) adding kanamycin (final concentration of 70 μg / mL) to the bacterial solution treated in step (9), and incubating the mixture in a shaker at 37° C. overnight;

[0154] (11) The next day, according to the method for preparing the nanobody phage library described in (4), (5), (6), and (7) in step 4 of Example 3, phages were precipitated with 5×PEG / NaCl, and the phage precipitates were re-dissolved with 2 mL of sterile PBS containing 0.1% BSA. The obtained phages were marked as Output and put into 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 Nanobody Strong Positive 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) Randomly pick 32 monoclonal colonies from the plates of Output phage titer determination after the first, second, and third rounds of screening, and inoculate each monoclonal colony into a single well of a 96-deep-well plate. Then place the plate in a shaker at 37°C and 250 rpm for several hours (until OD 600 =0.8);

[0160] (3) Add 10 μL of 1×10 13 cfu / mL helper phage (the helper phage is a phage without VHH gene plasmid), and stand at 37°C for 15 min to allow the phage to complete infection;

[0161] (4) placing the deep-well plate after standing in step (3) in a shaking incubator at 37° C. and 250 rpm for 2 h;

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

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

[0164] (7) Dilute Salmonella typhimurium to 10 8 cfu / mL, 100 μL / well was added to the ELISA plate and incubated at 4°C overnight. At the same time, another ELISA plate was prepared and CBS coating buffer was added as a negative control;

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

[0166] (9) Aspirate the blocking solution in the two ELISA plates in step (8), wash four times with PBST, and then add 100 μL / well of the phage culture supernatant in the deep-well plate after standing in step (6) to the two ELISA plates, and incubate the reaction at 37°C for 1 h;

[0167] (10) Aspirate the reaction solution from the two ELISA plates in step (9), wash four times with PBST, add 100 μL / well 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 ELISA plates 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 of 2M H2SO4 to the two color-developed ELISA plates in step (11) to terminate the reaction and place in an ELISA reader to read the OD 450 The absorbance value of Figure 1 , preliminary screening of positive clones.

[0170] Example 6 Prokaryotic expression and purification of anti-Salmonella typhimurium nanobodies

[0171] 1. Prokaryotic expression of nanobody protein

[0172] (1)Reference Plasmid DNA Mini Kit was used to extract the plasmid of positive phagemid, and the plasmid was introduced into E.coli TOP10F' by heat shock method.

[0173] Specifically as follows: take out the frozen E. coli TOP10F' competent cells from -80℃ and immediately place them on ice for 5 minutes, then add 1μL of positive phagemid plasmid to each tube of competent cells and continue to place them on ice for 10 minutes, then place the competent cells in a 42℃ water bath for 90 seconds and immediately put them back on ice to cool for a few minutes, add 1mL of SB culture medium to each tube of competent cells and shake and culture them at 37℃, 250rpm in a shaker for 1 hour, finally take 100μL of the culture solution from each tube and spread it on a CA-resistant plate and culture it in a 37℃ incubator overnight;

[0174] (2) Pick a single colony of the positive clone on the plate after culturing in step (1) and inoculate it into 5 mL of culture medium containing 50 μg / mL SB, and culture it in a shaking incubator at 37°C and 250 rpm overnight;

[0175] (3) The next day, the overnight culture solution of step (2) was transferred to 200 mL of culture medium containing 50 μg / mL SB at a 1% inoculum volume and cultured in a shaker at 37°C and 250 rpm for several hours until the OD 600 =0.8;

[0176] (4) adding IPTG to the bacterial solution cultured in step (3) to a final concentration of 0.5-1 mM, and incubating overnight in a shaking incubator at 30° C. and 250 rpm;

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

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

[0179] (7) centrifuging the bacterial lysate from step (6) at 4°C and 15,000 g for 10 min;

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

[0181] 2. Purification of Nanobody Protein

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

[0183] (2) fixing the empty purification column vertically, adding the reaction solution of step (1) into the empty purification column, collecting the sample flow-through liquid, and leaving the nickel column on the purification column;

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

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

[0186] (5) The eluate obtained in step (4) is collected to contain the purified nanobody protein, and is verified by SDS-PAGE electrophoresis. Figure 2 , in the figure: M is a protein standard; 1 is a nanobody protein expressed by SEQ-Styp NO.1; 2 is a 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) The purified nanobody solution obtained in step (4) was collected and combined and placed in a dialysis bag, and dialyzed against PBS at 4°C for three days, with the solution being replaced twice a day to replace the dissolution medium of the nanobody.

[0189] Application examples: Application of nanobodies

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

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

[0192] (2) The next day, aspirate the coating buffer in the well plate and wash the plate three times with PBST;

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

[0194] (4) Salmonella typhimurium from 10 7 After a series of dilutions were made starting from 100 μL / well, 100 μL / well was added to the ELISA plate treated in step (3) and incubated at 37°C for 1 h.

[0195] (5) aspirating the liquid in the wells of the ELISA plate after treatment in step (4), and washing the plate three times with PBST;

[0196] (6) diluting the nanobody to 40 μg / mL, then adding 100 μL / well to the ELISA plate treated in step (5) for reaction, and incubating at 37°C for 1 h;

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

[0198] (8) After diluting HRP enzyme-labeled Anti-HA mouse monoclonal antibody at 1:10000, 100 μL / well was added to the ELISA plate treated in step (7) for reaction and incubated at 37° C. for 1 h;

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

[0200] (10) Add 100 μL / well of TMB colorimetric substrate solution to the ELISA plate treated in step (9) and react at 37°C in the dark for 15 min;

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

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

[0203] (13) Plot a standard curve with the concentration of Salmonella typhimurium (log) as the horizontal axis and the absorbance as the vertical axis. Figure 3 .

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

Claims

1. A nanobody for detecting Salmonella typhimurium, characterized in that: The amino acid sequences of the nanobody are shown in SEQ-StypNO.1, SEQ-StypNO.2, SEQ-StypNO.3 and SEQ-StypNO.

4.

2. The nanobody for detecting Salmonella typhimurium according to claim 1, characterized in that The strain of the Salmonella typhimurium is ATCC14028.

3. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 1, characterized in that: The steps are as follows: 1) collecting blood samples from immunized alpacas, separating and collecting peripheral blood lymphocytes from alpacas, extracting total RNA of anti-Salmonella typhimurium nanoantibodies from peripheral blood lymphocytes from alpacas, and synthesizing cDNA; Using cDNA as a template, amplify the heavy chain variable region gene, connect the amplified alpaca antibody heavy chain variable region gene with the phagemid vector, and then transform it into competent Escherichia coli to construct an immune alpaca nanoantibody phage library; 2) Using Salmonella typhimurium as the coating antigen, the pH elution method was used to obtain nanobody phages that can specifically recognize Salmonella typhimurium after 3 or more rounds of panning, and the genes of the nanobody were compared by sequencing analysis; 3) Extracting the phagemid vector containing the anti-Salmonella typhimurium nanobody gene and re-introducing it into the host expression bacteria to express and purify the nanobody protein.

4. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 3, characterized in that: The total RNA was extracted according to TRIzol TM Total RNA was extracted according to the instructions of the Reagent.

5. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 3, characterized in that: The cDNA synthesis is based on the extracted RNA as a template, using SuperScript TM IV first-strand synthesis system reverse transcribes and synthesizes cDNA of antibody encoding gene.

6. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 3, characterized in that: The heavy chain variable region gene amplification F1 is used as the upstream primer, and R1, R2, and R3 are used as the downstream primers to amplify the heavy chain variable region gene; The upstream primer F1 nucleotide is shown in SEQ ID NO: 5; The downstream primer R1 nucleotide is shown in SEQ ID NO: 6; The downstream primer R2 nucleotide is shown in SEQ ID NO: 7; The nucleotide sequence of the downstream primer R3 is shown in SEQ ID NO:

8.

7. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 3, characterized in that: The heavy chain variable region gene is connected to the phagemid vector by connecting the VHH gene fragment after enzyme cutting with the vector pComb3xss after enzyme cutting using T4 ligase.

8. The method for preparing a nanobody for detecting Salmonella typhimurium according to claim 3, characterized in that: The phagemid vector containing the anti-Salmonella typhimurium nanobody gene is extracted Plasmid DNA MiniKit was used to extract the plasmid of positive phagemid, and the plasmid was introduced into E. coli TOP10F' by heat shock method.

9. Use of the nanobody for detecting Salmonella typhimurium according to claim 1 for preparing a reagent or a kit for detecting Salmonella typhimurium.

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