Nano antibody for detecting escherichia coli O157: H7 type and preparation method thereof

Abstract

The invention discloses a nano antibody for detecting escherichia coli O157: H7 type and a preparation method of the nano antibody. The invention aims to provide the nano antibody for detecting the escherichia coli O157: H7 type, which is high in detection accuracy and simple in obtaining method. The amino acid sequence of the nano antibody is as shown in SEQ ID NO.1-3; belongs to the technical field of biological medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a nano antibody for detecting Escherichia coli O157:H7. Background Art

[0002] E. coli O157:H7 is a highly pathogenic bacterium belonging to the Enterohemorrhagic E. coli (EHEC) group. This bacterium causes severe food poisoning by producing Shiga toxins, which lead to symptoms including abdominal pain, bloody diarrhea and vomiting. In severe cases, it may cause hemolytic uremic syndrome, which is particularly dangerous for children and the elderly. It is mainly transmitted through contaminated food and water. By timely and accurate detection of E. coli O157:H7, food contamination incidents can be prevented, food safety can be ensured, and public health can be protected. In addition, detection helps to quickly track and control the source of infection, meet the legal and regulatory requirements of the food industry, and maintain the reputation of enterprises and the stability of the market.

[0003] The methods currently used for detecting E. coli O157:H7 include PCR, bacterial culture, immunological methods, and mass spectrometry. PCR technology has high specificity and sensitivity, and can quickly and accurately detect the target DNA sequence, but it requires professional equipment and technology, and there may be false positive results. Although the culture method is simple and intuitive, it takes a long time and has high requirements for the number of bacteria, and it is easy to miss low-concentration samples. Mass spectrometry can directly detect molecular characteristics with high accuracy, but the equipment is expensive and the operation is complicated. Immunological methods have high specificity, but they may have cross-reactions or low sensitivity due to the quality and pairing of antibodies. Compared with traditional monoclonal antibodies, recombinant antibodies have lower production costs and better uniformity. Therefore, it is very necessary and feasible to develop anti-E. coli O157:H7 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 method for constructing a heavy chain antibody variable region gene library by immunizing alpacas, extracting peripheral blood lymphocytes, and combining phage display technology to screen specific anti-Escherichia coli O157:H7 nanoantibodies. The antibody has a high affinity for the O157:H7 nanoantibody, is suitable for the immune detection of Escherichia coli O157:H7 in food, and has high detection accuracy.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned nanoantibody for detecting Escherichia coli O157:H7 type.

[0006] To this end, the present invention first provides a nanobody against Escherichia coli O157:H7, the amino acid sequence of the encoded protein is SEQ ID NO: 1-3;

[0007] The second technical solution of the present invention is to provide a method for preparing the above-mentioned nanoantibody for detecting Escherichia coli O157:H7 type, which comprises the following steps in sequence:

[0008] 1) extracting total RNA from alpaca peripheral blood lymphocytes for anti-Escherichia coli O157:H7 nanoantibodies and performing cDNA synthesis;

[0009] 2) Anti-E. coli O157:H7 nanoantibody gene amplification

[0010] Using cDNA as a template, F1 as an upstream primer, and R1, R2, and R3 as downstream primers, the heavy chain variable region gene is amplified simultaneously; the target gene of the PCR product after amplification is recovered, and the VHH antibody gene fragment is obtained after purification;

[0011] 3) Construction of a phage nanobody library against Escherichia coli O157:H7

[0012] 3.1) Enzyme digestion of the vector pComb3Xss to obtain the digested pComb3xss vector;

[0013] 3.2) Enzymatically digesting the VHH antibody gene fragment to obtain the digested VHH gene fragment;

[0014] 3.3) The pComb3xss vector after restriction digestion and the VHH gene fragment after restriction digestion were connected using T4 DNA ligase to obtain a ligation product;

[0015] 3.4) The ligation product was transformed into E.coli ER2738 competent cells, and cultured to construct an immune alpaca nanobody phage library;

[0016] 4) Screening the genes of anti-Escherichia coli O157:H7 nanoantibodies from the nanoantibody phage library;

[0017] 5) Introducing the plasmid containing the anti-E. coli O157:H7 nanobody gene into the host bacteria to express and purify the nanobody;

[0018] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Escherichia coli O157:H7, the total RNA extraction of the anti-Escherichia coli O157:H7 nanobodies is carried out according to TRIzol TM Total RNA was extracted according to the instructions.

[0019] Furthermore, in the above-mentioned method for preparing nanoantibodies for detecting Escherichia coli O157:H7, the cDNA synthesis 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.

[0020] Further, in the above-mentioned method for preparing nanoantibodies for detecting Escherichia coli O157:H7 type, the upstream primer F1 nucleotide is shown in SEQ ID NO: 4; the downstream primer R1 nucleotide is shown in SEQ ID NO: 5, the downstream primer R2 nucleotide is shown in SEQ ID NO: 6, and the downstream primer R3 nucleotide is shown in SEQ ID NO: 7.

[0021] Furthermore, in the above-mentioned method for preparing nanoantibodies for detecting Escherichia coli O157:H7, the target gene is recovered by Cycle Pure Kit.

[0022] Further, in the above-mentioned method for preparing nanoantibodies for detecting Escherichia coli O157:H7 type, step 3.4) is to add the ligation product to E. coli ER2738 electroporated competent cells, resuscitate and perform gradient dilution, and then spread them on LB plates containing carbenicillin, invert and culture overnight, calculate the number of colonies on the plate, and calculate the library capacity of the original antibody library based on the dilution ratio; randomly pick transformants on the LB plate, culture them overnight in LB medium containing carbenicillin, perform DNA sequencing, add the remaining culture solution to SB medium, add carbenicillin and tetracycline and continue to culture to obtain a bacterial solution containing nanoantibody phage.

[0023] Furthermore, in the above-mentioned method for preparing nanobodies for detecting Escherichia coli O157:H7, the purification is carried out using Ni-NTA matrix purification.

[0024] The last technical solution of the present invention is the use of the above-mentioned nanoantibody for detecting Escherichia coli O157:H7 as a reagent for preparing a detection reagent for Escherichia coli O157:H7.

[0025] Compared with the prior art, the technical solution provided by the present invention immunizes alpacas, extracts peripheral blood lymphocytes, constructs a heavy chain antibody variable region gene library, and combines phage display technology to screen specific anti-Escherichia coli O157:H7 nanoantibodies. The antibodies have high affinity for O157:H7 nanoantibodies and are suitable for immune detection of Escherichia coli O157:H7 in food, and will have great application potential in Escherichia coli O157:H7 screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result graph of phage ELISA of 96 phage strains;

[0027] Figure 2 This is the expression diagram of the nanobody protein analyzed by SDS-PAGE;

[0028] Figure 3 is a binding analysis diagram of the nanobody binding to Escherichia coli O157:H7;

[0029] Figure 4 This is the standard curve of the Escherichia coli O157:H7 double antibody sandwich ELISA based on nanoantibodies. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1: Total RNA extraction and cDNA synthesis of anti-Escherichia coli O157:H7 nanoantibodies

[0032] 1. Extraction of alpaca peripheral blood lymphocytes:

[0033] (1) Mixing the inactivated anti-E. coli O157:H7 antigen with an equal amount of immune adjuvant, immunizing alpacas by subcutaneous injection, and performing 3-4 immunizations at intervals of 14 days. After the third immunization, blood samples of the immunized alpacas are collected; 15 mL of alpaca peripheral blood lymphocyte separation fluid (from Tianjin Haoyang Biology) is first added to the collected blood sample, and 15 mL of blood is slowly added to a 50 mL centrifuge tube at a slight tilt of 45 degrees;

[0034] (2) Place the centrifuge tube in a horizontal centrifuge and centrifuge at 400 g for 30 minutes at room temperature;

[0035] (3) After centrifugation, remove the separation solution and use a pipette to transfer the cloudy lymphocytes in the middle to a new 50 mL centrifuge tube;

[0036] (4) Add 10 mL of room temperature PBS buffer to the tube and centrifuge at 400 g for 20 minutes.

[0037] (5) Remove the supernatant and add 1 mL of room temperature PBS buffer to each tube for resuspending. Mix gently and count the number of cells using a hemocytometer.

[0038] 2. Total RNA Extraction

[0039] According to TRIzol TM The total RNA was extracted according to the instructions of the manufacturer as follows:

[0040] (1) Pour the cell resuspension into a 1.5 mL centrifuge tube and centrifuge at 8000g for 2 minutes at 4°C;

[0041] (2) Discard the supernatant gently, taking care not to disrupt the cell pellet at the bottom of the centrifuge tube;

[0042] (3) Add 1 mL of TRIzol to the above tube. TM (1mL per 10 7 cells);

[0043] (4) Mix the cells thoroughly until no obvious cell precipitation is visible in the lysis solution;

[0044] (5) Leave to stand at room temperature for 5 minutes;

[0045] (6) Add 0.2 mL of chloroform to the tube, mix the solution thoroughly by inverting it, and let it stand at room temperature for 2-3 minutes;

[0046] (7) Place the centrifuge tube in a centrifuge and centrifuge at 12000g at 4°C for 15 minutes;

[0047] (8) Carefully remove the centrifuge tube from the centrifuge. You can see that the lysate is clearly divided into three layers.

[0048] (9) Transfer the upper layer of lysate in the above tube to another new centrifuge tube;

[0049] (10) Add 0.5 mL of isopropanol to the tube, mix thoroughly by inverting, and let stand at room temperature for 10 minutes;

[0050] (11) Place the centrifuge tube in a centrifuge and centrifuge at 12000g at 4°C for 10 minutes;

[0051] (12) Carefully discard the supernatant and add TRIzol TM An equal volume of 75% ethanol;

[0052] (13) Place the centrifuge tube in a centrifuge and centrifuge at 7500 g at 4°C for 5 minutes;

[0053] (14) Carefully discard the supernatant, invert the centrifuge tube, and dry it at room temperature for 5 minutes;

[0054] (15) Add 20 μL of deionized water to the above tube to dissolve the precipitate and obtain total RNA;

[0055] (16) UV spectrophotometer to measure OD 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.

[0056] 3. cDNA Synthesis

[0057] Using the extracted RNA as a template, SuperScript TM IV First-Strand Synthesis System reverse transcribes and synthesizes cDNA of antibody encoding gene. The process is as follows:

[0058] (1) Add the reagents shown in Table 1 to the PCR tube:

[0059] Table 1

[0060]

[0061]

[0062] The reaction was placed in a PCR instrument, incubated at 65°C for 5 minutes, and then cooled on ice;

[0063] (2) Continue to add reverse transcription reaction solution to the above reaction system, the system is shown in Table 2:

[0064] Table 2

[0065]

[0066] Place in a PCR instrument and set the conditions as shown in Table 3.

[0067] Table 3

[0068]

[0069] (3) Add 1 μL E.coli RNase H and incubate at 37°C for 20 min.

[0070] (4) Store the reverse transcription product cDNA library in a -80°C refrigerator for future use.

[0071] Example 2: Anti-Escherichia coli O157:H7 Nanobody Gene Amplification

[0072] 1. Amplification of IgG2 / 3 antibody heavy chain variable region (VHH) gene

[0073] The primers for amplifying the VHH antibody gene were synthesized by Shanghai Bioengineering Company, and the primer sequences are shown in Table 4 below:

[0074] Table 4

[0075]

[0076]

[0077] The underlined bases are restriction enzyme cleavage sites

[0078] (1) Using cDNA as template, F1 as upstream primer, and R1, R2, and R3 as downstream primers, the heavy chain variable region gene was amplified simultaneously.

[0079] (3) Add the reagents shown in Table 5 to the PCR tube:

[0080] Table 5

[0081]

[0082] Set the following conditions in the PCR instrument as shown in Table 6.

[0083] Table 6

[0084]

[0085] 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.

[0086] (2) Agarose gel electrophoresis

[0087] ① Weigh 0.2 g agarose and dissolve it in 20 mL TAE buffer to make 1% agarose gel;

[0088] ② Heat in a microwave until the agarose is completely dissolved and becomes colorless and transparent;

[0089] ③ When the gel cools to about 50°C, add 1 μL of nucleic acid dye and mix thoroughly;

[0090] ④ Pour into the glue making tank and cool and solidify at room temperature;

[0091] ⑤ Remove the comb and add 10 μL 10× loading buffer per 100 μL PCR product to the corresponding wells;

[0092] ⑥Run at 100V for 30 minutes;

[0093] ⑦Observe the results under ultraviolet light.

[0094] Example 3: Construction of phage nanobody library against Escherichia coli O157:H7

[0095] 1. Enzyme digestion reaction of vector pComb3Xss and nanobody gene fragment

[0096] Since the obtained VHH antibody gene fragment has two SfiI restriction sites introduced at both ends, it can be directly digested and recombined with the pComb3Xss vector and ligated with T4 ligase. The process is briefly described as follows:

[0097] (1) Enzyme digestion of vector pComb3xss

[0098] ① Add the reagents listed in Table 7 to the PCR tube.

[0099] Table 7

[0100]

[0101] ② Place in PCR instrument, 50℃, incubate overnight;

[0102] ③ Add 22.5 μL 0.5 M EDTA and mix well to inactivate the SfiI enzyme;

[0103] ④ Detect the effect of enzyme cleavage by 0.7% agarose gel electrophoresis;

[0104] ⑤Recover the enzyme cleavage products.

[0105] (2) Enzymatic cleavage of VHH antibody gene fragments

[0106] ① Add the reagents listed in Table 8 to the PCR tube.

[0107] Table 8

[0108]

[0109] ② Place in PCR instrument, 50℃, incubate overnight;

[0110] ③ Add 22.5 μL 0.5 M EDTA and mix well to inactivate the SfiI enzyme;

[0111] ④ Cycle Pure Kit recovers the enzyme digestion products.

[0112] 2. Ligation and purification of vector pComb3xss and nanobody gene fragments

[0113] ① Add the reagents listed in Table 9 to the PCR tube.

[0114] Table 9

[0115]

[0116] ② Place in PCR instrument and react at 16℃ overnight;

[0117] ③ The next day, incubate at 65°C for 10 minutes to heat-inactivate T4 DNA ligase;

[0118] ④ Transfer the ligation product to a 1.5 mL centrifuge tube, add 20 μL 3M sodium acetate (pH = 5.2), and then add 660 μL anhydrous ethanol;

[0119] ⑤Place the centrifuge tube at -20℃ overnight;

[0120] ⑥ The next day, centrifuge the ligation product at 4°C, 12000 rpm for 30 minutes;

[0121] ⑦ At this time, white DNA precipitate can be seen. Discard the supernatant and resuspend the precipitate in 75% ethanol;

[0122] ⑧ Centrifuge at 12000 rpm for 15 minutes at 4°C;

[0123] ⑨Discard the supernatant, invert the centrifuge tube, and let it stand for a few minutes to evaporate the remaining ethanol.

[0124] ⑩ Add 30 μL of sterile water to re-dissolve the precipitate and store the sample at -20°C for later use.

[0125] 3. Electrotransformation of the pComb3X vector and the ligation product of the single-stranded gene fragment

[0126] (1) Take out the E. coli ER2738 electroporation competent cells from the -80°C refrigerator and thaw on ice;

[0127] (2) Take 25 μL of electroporated competent cells as one reaction, add 3 μL of the purified ligation product and incubate on ice for 10 minutes;

[0128] (3) The mixture was quickly transferred to a pre-cooled electroporation cup and electroporated at 1.8 KV, 200 Ω, and 25 μF.

[0129] (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;

[0130] (5) Repeat the above steps 9 times, and transform a total of 30 μL of the ligation product into E. coli ER2738 competent cells;

[0131] (6) Place the transformed cells in a shaker at 37°C and 250 rpm for 1 hour to recover;

[0132] (7) Take 2 μL of the culture and perform 10-fold gradient dilutions, then spread them 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;

[0133] (8) The next day, the number of colonies on the plate was counted and the capacity of the original antibody library was calculated based on the dilution ratio;

[0134] (9) 16 transformants were randomly selected from the plate, cultured overnight in LB medium containing 50 μg / mL carbenicillin, and DNA sequencing was performed to analyze the diversity of the inserted sequences;

[0135] (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 to obtain a bacterial solution, and then the phage library was prepared.

[0136] 4. Construction of Nanobody Phage Display Library

[0137] (1) Add 1 mL of 1×10 13 cfu / mL of helper phage, and incubate at 37°C for 30 minutes to allow the phage to infect E. coli ER2738 containing the VHH antibody gene;

[0138] (2) placing the bacterial solution from step (1) in a shaker at 37°C and 250 rpm for 2 h;

[0139] (3) Add 200 μL of kanamycin (70 mg / mL kanamycin) and culture overnight in a shaker at 37°C and 250 rpm;

[0140] (4) The next day, the bacterial solution from step (3) was transferred to a centrifuge bottle and centrifuged at 10,000 rpm for 30 minutes at 4°C;

[0141] (5) Collect the supernatant, add 50 mL of 5× PEG / NaCl, and place on ice for 2 h to precipitate the helper phage;

[0142] (6) Centrifuge the cells at 4°C, 10,000 rpm for 30 min. A white phage precipitate is visible. Add 100 mL of PBS to wash the precipitate, add 25 mL of 5× PEG / NaCl to precipitate, and place on ice for 2 h.

[0143] (7) Centrifuge at 10,000 rpm for 30 min at 4°C, reconstitute with 10 mL of sterile PBS containing 0.1% BSA, and sterilize through a 0.22 μm filter. Take 2 μL to measure the titer, which is the storage capacity of the nanobody phage library. The remaining phages are placed in a -80°C refrigerator for future use.

[0144] Example 4 Screening of anti-Escherichia coli O157:H7 nanobodies

[0145] (1) E. coli O157:H7 was diluted to 10% in CBS coating buffer in different rounds. 9 , 10 8 , 10 7 cfu / mL, 100 μL / well was added to a 96-well plate and coated overnight at 4°C;

[0146] (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 hour;

[0147] (3) Discard the blocking solution, wash four times with PBST, add 100 μL / well of phage-displayed nanoantibody library, marked as Input, and incubate at 37°C for 1 hour;

[0148] (4) Aspirate the phages in the wells by vigorously pipetting PBST 10-20 times;

[0149] (5) Add 100 μL / well 0.1 M glycine-HCl (pH = 2.2) to elute the phages and incubate at 37°C for 10 min;

[0150] (6) Add 50 μL / well 1 M Tris-HCl (pH = 8.8) to neutralize the eluted phage;

[0151] (7) Collect the supernatant, take 2 μL to measure the titer, and add 2 mL of freshly cultured E. coli ER2738 culture (OD 600 =0.8), and incubated at 37°C for 30 minutes;

[0152] (8) Add 18 mL of LB solution containing 8 μL of carbenicillin (50 mg / mL) and incubate in a shaker at 37 °C for 1 h;

[0153] (9) Add 12 μL of carbenicillin (50 mg / mL) and incubate in a shaker at 37 °C for 1 hour;

[0154] (10) Add 50 μL of helper phage (1×10 13 cfu / mL), placed in a 37°C shaker for 2 hours; added 20 μL of kanamycin (70 mg / mL), placed in a 37°C shaker for overnight shaking;

[0155] (11) The next day, according to the method for preparing the nanobody phage library in steps 4(4)-(7) of Example 3 above, phages were precipitated with 5×PEG / NaCl, and the phages 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.

[0156] Example 5 Screening of Nanobody Strong Positive Strains

[0157] (1) Randomly pick 32 monoclonal colonies from the plates of Output phage titer determination after the first, second, and third rounds of screening, inoculate them into 1 mL of SB medium containing 50 μg / mL carbenicillin, and place them in a shaking incubator at 37°C and 250 rpm for several hours (OD 600 =0.8);

[0158] (2) Add 10 μL of helper phage (1×10 13 cfu / mL), and incubate at 37°C for 15 minutes to allow the phage to complete infection;

[0159] (3) Incubate at 37°C, 250 rpm in a shaker for 2 h;

[0160] (4) Add 1 μL of 70 mg / mL kanamycin and incubate overnight at 37°C and 250 rpm in a shaker;

[0161] (5) The next day, the culture medium was left to stand for later use;

[0162] (6) Dilute E. coli O157:H7 to 10% using CBS buffer. 8 cfu / mL, 100 μL / well was added to the ELISA plate at 4°C overnight, and only CBS coating buffer was added to the other plate as a negative control;

[0163] (7) The next day, discard the coating solution, wash four times with PBST, prepare 3% skim milk powder, add 250 μL / well to the 96-well plate, and incubate at 37°C for 1 hour;

[0164] (8) Filter out the blocking solution, wash four times with PBST, add 100 μL / well of phage culture supernatant, and incubate at 37°C for 1 hour;

[0165] (9) The above solution was discarded, washed four times with PBST, and 100 μL / well anti-M13-HRP enzyme-labeled antibody (1:2000 dilution) was added, and incubated at 37°C for 1 hour;

[0166] (10) Discard the secondary antibody in the well, wash the plate four times with PBST, add 100 μL / well TMB colorimetric solution, and react at 37°C for 15 min;

[0167] (11) Add 50 μL / well 2M H2SO4 to terminate the reaction and place in a microplate reader to read the OD 450 The absorbance value of Figure 1 .

[0168] Example 6 Prokaryotic expression and purification of anti-Escherichia coli O157:H7 nanoantibody

[0169] 1. Prokaryotic expression of nanobody protein

[0170] (1)Reference Plasmid DNA mini Kit was used to extract the plasmid of positive phagemid, and the plasmid was mixed with E. coli TOP10F' chemically competent cells and rapidly heated in a 42°C water bath for 90 seconds. Subsequently, the mixture was rapidly cooled. Cultured at 37°C in a shaker at 200 rpm for 1 hour, and 100 μL of the culture solution was evenly spread on a medium containing carbenicillin to sort the successfully transformed colonies, and the culture dish was incubated at 37°C overnight;

[0171] (2) Inoculate the positive clone into 5 mL of LB culture medium containing 50 μg / mL and culture overnight in a shaker at 37°C and 250 rpm;

[0172] (2) The next day, the overnight culture was transferred to 200 mL of LB culture medium containing 50 μg / mL according to a 1% inoculum, and cultured in a shaker at 37°C and 250 rpm for several hours until the OD 600 =0.8;

[0173] (3) Add IPTG to a final concentration of 0.5 mM and culture overnight in a shaker at 30°C and 250 rpm;

[0174] (4) Collect the overnight culture solution into a centrifuge bottle and centrifuge at 5000 g for 20 min at 4°C;

[0175] (5) Discard the supernatant, weigh the cells, add 4 mL of bacterial lysis buffer for every 1 g of cells, repeatedly pipette and resuspend the cells, and invert at room temperature for 15 minutes to fully lyse the bacteria;

[0176] (6) Centrifugation at 15,000 g for 10 min at 4°C;

[0177] (7) Collect the supernatant, which is the soluble protein, for subsequent purification.

[0178] 2. Purification of Nanobody Protein

[0179] (1) Take the Ni-NTA matrix and centrifuge it three times to replace the storage solution with PBS. Add the above soluble protein and mix it upside down at room temperature for 1 hour.

[0180] (2) Fixing the purification column vertically, adding the above-mentioned mixture containing Ni-NTA matrix and the protein sample to be purified into the empty purification column, and collecting the sample flow-through;

[0181] (3) Add 5 column volumes of equilibration buffer (PBS containing 10 mM imidazole) to elute the impurities;

[0182] (4) Add 5 column volumes of elution buffer (PBS containing 250 mM imidazole) and collect the eluate until the flow-through is at A. 280 The signal value is close to 0;

[0183] (5) detecting the purified antibody by SDS-PAGE electrophoresis;

[0184] (6) The purified nanoantibody solution was collected and placed in a dialysis bag, and dialyzed in PBS for three days, with the solution changed twice a day to remove excess imidazole and other impurities.

[0185] Analysis: See Figure 2 , the SDS-PAGE results show that after purification by Ni-NTA matrix, a nanobody with a size of about 17 kDa was obtained.

[0186] Experimental Example 1 Affinity of Anti-Escherichia coli O157:H7 Nanobodies

[0187] 1. Direct ELISA analysis of the affinity of nanobodies

[0188] (1) Use CBS to convert 10 8 cfu / mL E. coli O157:H7 type standard solution was added to the ELISA plate at 100 μL / well and coated overnight at 4°C;

[0189] (2) The next day, the coating buffer in the well plate was discarded and the plate was washed four times with PBST;

[0190] (3) Add 3% skim milk powder blocking solution at 270 μL / well and incubate at 37°C for 1 hour;

[0191] (4) Discard the liquid in the wells, wash the plate four times with PBST, add 0-20 μg / mL nanoantibodies at 100 μL / well, mix well, and incubate at 37°C for 1 hour;

[0192] (5) Discard the liquid in the wells, wash the plate four times with PBST, add 1:10000 diluted HRP enzyme-labeled Anti-HA mouse monoclonal antibody (100 μL / well), and incubate at 37°C for 1 hour;

[0193] (6) Discard the liquid in the wells, wash the plate five times with PBST, add 100 μL / well TMB colorimetric substrate solution, and react at 37°C in the dark for 15 min;

[0194] (7) Add 50 μL / well 2M H2SO4 to terminate the reaction;

[0195] (8) Read OD in a microplate reader450 ;

[0196] (9) The affinity of the nanobody was analyzed using the nanobody concentration as the horizontal axis and the absorbance as the vertical axis.

[0197] Analysis: Direct ELISA was performed by coating the target analyte, E. coli O157:H7, directly on the ELISA plate. Figure 3 It can be seen that all three nanobodies have a strong affinity for E. coli O157:H7, and still maintain a high absorbance at 1.25μg / mL. The absorbance of VHH3 decreases when it is less than 0.625μg / mL, while VHH1 and 2 still maintain a high absorbance. This comparison shows that among the three nanobodies, VHH1 and VHH2 have similar affinities with E. coli O157:H7, and are greater than VHH3.

[0198] Application Example 1 Application of Nanobodies

[0199] 1. Establishment of double antibody sandwich ELISA standard curve

[0200] (1) Dilute the monoclonal antibody to 2 μg / mL with CBS, add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4°C;

[0201] (2) The next day, the coating buffer in the well plate was discarded and the plate was washed four times with PBST;

[0202] (3) Add 270 μL / well of 3% skim milk powder blocking solution and incubate at 37°C for 1 hour;

[0203] (4) Add 2 μg / mL nanobody (100 μL) and an equal volume of a series of concentrations of Escherichia coli O157:H7 standard solution, mix well, and incubate at 37 °C for 1 h;

[0204] (5) Discard the liquid in the wells and wash the plate four times with PBST;

[0205] (6) Add 1:10000 diluted HRP enzyme-labeled Anti-HA mouse monoclonal antibody (100 μL / well) and incubate at 37°C for 1 hour;

[0206] (7) Discard the liquid in the wells and wash the plate five times with PBST;

[0207] (8) Add 100 μL / well TMB colorimetric substrate solution and incubate at 37°C in the dark for 15 min;

[0208] (9) Add 50 μL / well 2M H2SO4 to terminate the reaction;

[0209] (10) Read OD in a microplate reader 450 ;

[0210] (11) Draw a standard curve with the concentration (log) of E. coli O157:H7 as the horizontal axis and the absorbance as the vertical axis.

[0211] Analysis: A double antibody sandwich ELISA was constructed based on monoclonal antibody-nanoantibody for the detection of Escherichia coli O157:H7. Figure 4 As shown, at a bacterial concentration of 10 3 -10 9 The logarithm was transformed in the range of cfu / mL and four-parameter fitting was performed. The corresponding fitting curves R of the three nanoantibodies 2 They were 0.9773 (VHH1), 0.9911 (VHH2), and 0.9908 (VHH3) respectively. 450nm The value obtained by multiplying the value of the three blank standard errors is taken as the Y value, and the X value in the four-parameter fitting curve is calculated. Then, the minimum detection limit of the method can be obtained by exponential conversion. Based on this, the minimum detection limits of the sandwich enzyme-linked immunosorbent assay method formed by the three nanoantibodies and monoclonal antibodies can be calculated to be 7×10 4 (VHH1), 8×10 4 (VHH2), 2×10 5 It can be found that when using equal amounts of monoclonal antibodies as capture antibodies and nanobodies as detection antibodies, the performance comparison of the three nanobodies is similar to the aforementioned affinity test results, and the performance of VHH1 and VHH2 is not much different, and both are higher than VHH3.

Claims

1. A nanobody for detecting Escherichia coli O157:H7, characterized in that: The amino acid sequence of the nanobody is shown in any one of SEQ ID NO.1-3.

2. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 1, characterized in that: The steps are as follows: 1) extracting total RNA from alpaca peripheral blood lymphocytes for anti-Escherichia coli O157:H7 nanoantibodies and performing cDNA synthesis; 2) Anti-E. coli O157:H7 nanoantibody gene amplification Using cDNA as a template, F1 as an upstream primer, and R1, R2, and R3 as downstream primers, the heavy chain variable region gene is amplified simultaneously; the target gene of the PCR product after amplification is recovered, and the VHH antibody gene fragment is obtained after purification; 3) Construction of a phage nanobody library against Escherichia coli O157:H7 3.1) Enzyme digestion of the vector pComb3Xss to obtain the digested pComb3xss vector; 3.2) Enzymatically digesting the VHH antibody gene fragment to obtain the digested VHH gene fragment; 3.3) The pComb3xss vector after restriction digestion and the VHH gene fragment after restriction digestion were connected using T4 DNA ligase to obtain a connection product; 3.4) The ligation product was transformed into E.coliER2738 competent cells, and cultured to construct an immune alpaca nanobody phage library; 4) Screening the genes of anti-Escherichia coli O157:H7 nanoantibodies from the nanoantibody phage library; 5) The plasmid containing the anti-Escherichia coli O157:H7 nanobody gene is introduced into the host bacteria to express and purify the nanobody.

3. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: The total RNA extraction of the anti-E. coli O157:H7 nanoantibody was carried out according to TRIzol TM Total RNA was extracted according to the instructions.

4. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: The cDNA synthesis was 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.

5. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: The upstream primer F1 nucleotide is shown in SEQ ID NO: 4; the downstream primer R1 nucleotide is shown in SEQ ID NO: 5; the downstream primer R2 nucleotide is shown in SEQ ID NO: 6; and the downstream primer R3 nucleotide is shown in SEQ ID NO:

7.

6. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: The target gene recovery method CyclePureKit kit.

7. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: Step 3.4) is to add the ligation product to E. coli ER2738 electroporated competent cells, recover and perform gradient dilution, and then spread them on LB plates containing carbenicillin, invert and culture overnight, calculate the number of colonies on the plate, and calculate the library capacity of the original antibody library based on the dilution ratio; randomly pick transformants on the LB plate, culture them overnight in LB medium containing carbenicillin, perform DNA sequencing, add the remaining culture solution to SB medium, add carbenicillin and tetracycline and continue to culture to obtain a bacterial solution containing nano-antibody phage.

8. The method for preparing a nanobody for detecting Escherichia coli O157:H7 according to claim 2, characterized in that: The purification was performed using Ni-NTA matrix purification.

9. Use of the nanobody for detecting Escherichia coli O157:H7 according to claim 1 as a reagent for preparing a detection reagent for Escherichia coli O157:H7.

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