CN115043939A - A kind of Omp16 specific nanobody for detecting Brucella, expression vector and application thereof - Google Patents

Abstract

The invention relates to the technical field of immunodetection, in particular to an Omp16 specific nano antibody for detecting brucella, an expression vector and application thereof, wherein a brucella OMP16 nano antibody nucleic acid sequence is obtained by screening OMP16 recombinant protein through a brucella nano antibody display library established at the early stage, the nano antibody can specifically recognize brucella outer membrane protein OMP16 and can effectively block the combination of brucella positive serum and Omp16, and therefore, the antibody can be used for establishing a detection method for blocking ELISA, colloidal gold and the like to detect brucella.

Description

A kind of Omp16 specific nanobody for detecting Brucella, expression vector and the same application

technical field

The invention relates to the technical field of immunodetection, in particular to an Omp16-specific nanobody for detecting Brucella, an expression vector and an application thereof.

Background technique

Brucellosis ("brucellosis" for short) is a chronic multi-organ damage zoonotic infectious disease caused by Brucella. comorbidity, but also one of the seven most important infectious diseases that are most easily overlooked." Brucellosis is mainly acquired through direct or indirect contact with infected animals or animal products. When humans suffer from brucellosis, it will cause fever, muscle and joint pain, etc., and in severe cases, they will lose their ability to work completely; livestock infected with brucellosis will cause abortion and infertility, causing huge economic losses to the breeding industry, and a serious threat to human food safety and public health. health and safety.

Accurate diagnosis is an extremely important part of brucellosis prevention and control. Brucellosis diagnosis is further divided into serological diagnosis and etiological diagnosis. Compared with the commonly used brucellosis serological diagnosis method, brucellosis etiological diagnosis has higher requirements on laboratory biosafety level and laboratory personnel. At present, the etiological diagnosis methods of brucellosis mainly include bacterial isolation, biochemical identification and typing, nucleic acid detection, etc. Among them, nucleic acid detection includes AMOS-PCR, Bruce-ladder PCR, real-time PCR, MLVA and other methods. The development of the above-mentioned etiological diagnosis methods needs to be completed in a professional brucellosis laboratory, the operators must undergo special training, and a test takes at least 2 hours. Therefore, it is necessary to develop a rapid on-site diagnostic method for the etiology of brucellosis.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the present invention provides an Omp16-specific nanobody for detecting Brucella, an expression vector and an application thereof. The nanobody can specifically recognize the Brucella outer membrane protein OMP16, and can effectively block the cloth The positive serum of L. rubella binds to Omp16. Therefore, using this antibody, detection methods such as blocking ELISA and colloidal gold can be established to detect brucellosis.

To achieve the above object, the technical scheme adopted in the present invention is:

A kind of Omp16 specific nano antibody of detecting Brucella, this specific nano antibody is prepared by following steps:

(1) Inducible expression of Omp16

The pGEX4T-1-Omp16 plasmid stored in the laboratory was transformed into BL21(DE3), the single clone was picked and activated to the logarithmic growth phase (OD600nm to 0.6-0.8), cooled to room temperature, and IPTG with a final concentration of 1mM was added. , transferred to 22°C, cultured at 200r/min for 16h to induce protein expression; centrifuged at 6000r/min for 10min at room temperature, discarded the supernatant, resuspended the cells with 1/20 volume of PBS, and disrupted the cells by ultrasonication for 3s, intermittently for 3s , 12000r/min, centrifuge at 4°C for 10min, separate supernatant and precipitate;

(2) Purification of Omp16 protein

Take the supernatant after sonication, use a 0.45 μM filter to remove impurities, and place it on ice for later use; take 1 mL of GSTresin into the chromatography column, settle naturally with the medium, and add 10 times the volume of PBS to equilibrate the column; add the filtered expression supernatant , adjust the flow rate to 0.5mL/min, and adsorb the target protein; after loading, add 10-20 times the volume of PBS to elute the impurity protein until the effluent absorbance at 280nm is close to 0, and use elution buffer (50mM Tris-Cl , 10mM reduced glutathione) to elute the target protein, control the flow rate to be 0.5mL/min, collect the effluent, detect the protein content of the effluent by SDS-PAGE, and dialyze the protein into PBS solution, measure the protein concentration, and divide the Store in freezer at -80°C;

(3) Panning of Omp16-specific phage

The purified Omp16 protein was diluted with PBS buffer to 200 μg/mL, and coated on a 96-well microtiter plate with 100 μL per well, a total of 3 wells were coated, and a PBS control well was set at the same time, and coated overnight at 4°C; discarded Coating solution, after washing the plate three times with PBS'T, add 200 μL 2.5% nonfat milk powder, block at 37°C for 2h; discard the blocking solution, add 5×10 ¹⁰ PFU/well of phage diluted with 2.5% nonfat milk powder, and incubate at 37°C for 2h; Discard the phage supernatant, wash the plate 10 times with PBS'T, wash the plate 5 times with PBS, add 100 μl of freshly prepared 0.1 M triethylamine to each well, let stand for 10 min at room temperature, and quickly aspirate the eluate with an equal volume of 1 M Tris-HCl. (pH=7.4) to neutralize the eluate; take 400 μL of the panning product to rescue the panned phage according to the library rescue method, and detect the phage titer at the same time; carry out the second and third rounds of the rescued phage according to the same method. The coating concentrations of Omp16 in the second and third rounds of panning were 100 and 50 μg/mL, respectively;

(4) Preparation of Nanobody Crude Extract

Randomly pick 96 phage monoclones from the final round of panning into 100 μL LB/AMP-GLU medium, and cultivate at 200 r/min at 37°C for 8 hours; 10 μL of each clone was transferred to 500 μL TB medium, 37°C Cultivate to logarithmic growth phase, add 10 μL 100mM IPTG, continue to culture for 12-14 h; centrifuge at 12,000 r/min for 2 min, collect the cells, freeze-thaw at -80 °C and 37 °C for three times to lyse the cells, add 500 μL PBS, incubate at 4 °C for 30 min. The bacterial cells were suspended, and the supernatant after centrifugation was the crude extract.

Further, in step (3), the method for library rescue comprises the following steps:

Take 100 μL of the phage display library, inoculate it in 100 mL of 2×YT/AMP-GLU medium (containing 1 μg/mL ampicillin and 2% glucose), and cultivate it to logarithmic growth phase at 37°C, 200 r/min; add 20 MOI doses The helper phage M13K07 was left at 37°C for 30min, centrifuged at 2800g for 10min at room temperature, the supernatant was discarded, and the pellet was resuspended in 200mL 2×YT/AMP-KAN (containing 1μg/mL ampicillin and kanamycin) medium at 37°C , cultured at 200r/min for 14-16h; 3800g, centrifuged at 4°C for 30min, collected the supernatant, added 1/5 volume of PEG/NaCl (200g PEG6000, 146g NaCl dissolved in water, and made up to 1L), mixed well, kept on ice Set for 6-8h; centrifuge at 3800g for 30min at 4°C to discard the supernatant, add 1mL PBS solution to resuspend the pellet, incubate overnight at 4°C on a shaker to fully dissolve the phage particles, centrifuge at 12000g for 15min at 4°C, collect the supernatant, and use logarithmic Growth phase TG1 determination of rescue phage titers.

The invention also discloses the above-mentioned expression vector for detecting the Omp16 specific nanobody of Brucella, which is constructed by the following methods:

The gene sequence of NbA4 was inserted into the pCMV-HRP expression vector through the two restriction sites of Pst I and Not I, the recombinant plasmid was constructed, the correctness of the recombinant plasmid was verified by PCR and sequencing, and the endotoxin-free pCMV-NbA4-HRP was extracted For recombinant plasmids, Lipo8000 (Biyuntian) was used, and the following reaction system was prepared in each well of a 12-well plate: Lipo80001.6 μL, pCMV-NbA4-HRP 1 μg, Opti-mem 50 μL, and the plasmid was transfected into HEK with a fusion degree of 60-70% In -293T cells, the supernatant was collected after 48h to detect the expression and activity of NbA4-HRP.

The expression vector of a kind of Omp16 specific nano-antibody that detects Brucella of the present invention can be used for constructing the detection of bovine serum Brucella antibody based on NbA4-HRP blocking ELISA method, specifically, constructs through the following steps:

(1) Antigen coating: set the concentration of Omp16 to 1, 2, and 4 μg/mL, coat 100 μL/well on a 96-well ELISA plate, and incubate at 4°C overnight;

(2) Blocking: Wash the plate three times with PBS'T solution, add 200 μL of 2.5% nonfat dry milk to each well, and block at 37°C for 1 hour;

(3) Incubation with primary antibody: The collected NbA4-HRP expression supernatant was diluted 2, 4, 6, 8, 16, 32, and 64-fold with blocking solution, added to the ELISA plate, and incubated at 37°C for 1 h;

(4) Color development and termination: Add 100 μL of TMB solution for color development at room temperature for 10 min, 50 μL of 3M H ₂ SO ₄ to terminate the reaction, read the absorbance at 450 nm, analyze the test results, and determine the concentration of antigen and antibody that blocks ELISA.

During detection, Omp16 was diluted to 2 μg/mL, coated on a 96-well microtiter plate, overnight at 4°C; 2.5% nonfat milk powder was added, and blocked at 37°C for 1 h. Serum and nanobodies were mixed at a ratio of 1:20 for serum and 1:4 for HPR-NbA4, and 100 μL per well was added to the microtiter plate, incubated at 37°C for 1 h, and after washing the plate, TMB and 3M H2SO4 were added for color development, and detected at 450 nm. absorbance.

The nanobody of the present invention can specifically recognize Brucella outer membrane protein OMP16, and can effectively block the binding of Brucella positive serum and Omp16. Therefore, by using this antibody, detection methods such as blocking ELISA and colloidal gold can be established. Test for brucellosis.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Fig. 1 Result of purification of prokaryotic expressed Omp16 by GST affinity chromatography;

In the figure: lane 1: uninduced bacterial solution; lane 2: precipitate after IPTG induction; lane 3: supernatant after IPTG induction; lane 4: eluted impurity protein; lane 5-9: eluted target protein.

Figure 2 shows the results of indirect ELISA detection of Omp16 antibody titers in camel serum.

Figure 3 shows the results of three rounds of panning titers for Omp16-specific phages.

Fig. 4 is indirect ELlSA detection Omp16 specific Nanobody crude extract.

Figure 5 is the sequence analysis of the Omp16-specific Nanobody NbA4.

Figure 6 shows the results of the determination of the binding titer of HRP-NbA4 to Omp16.

Figure 7 shows the detection of Omp16 antibody in bovine serum by blocking ELISA based on HRP-NbA4.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Example

1. Expression and purification of Omp16 recombinant protein and determination of antibody titer in camel serum

(1) Inducible expression of Omp16

The pGEX4T-1-Omp16 plasmid stored in the laboratory was transformed into BL21(DE3), the single clone was picked and activated to the logarithmic growth phase (OD600nm to 0.6-0.8), cooled to room temperature, and IPTG with a final concentration of 1mM was added. , transferred to 22 ℃, 200r/min culture for 16h, induced expression of protein. Centrifuge at 6000 r/min at room temperature for 10 min, discard the supernatant, resuspend the cells with 1/20 volume of PBS, disrupt the cells by sonicating for 3 s, intermittently for 3 s, centrifuge at 12,000 r/min, 4 °C for 10 min, separate the supernatant and the precipitate, take Uninduced, supernatant and pellet after induction were identified by SDS-PAGE.

(2) Purification of Omp16 protein

The supernatant after sonication was taken, filtered with a 0.45 μM filter to remove impurities, and placed on ice for later use. 1 mL of GSTresin will be taken into the chromatography column, and the medium will settle naturally, and 10 times the volume of PBS will be added to equilibrate the column. The filtered expression supernatant was added, and the flow rate was adjusted to 0.5 mL/min to adsorb the target protein. After loading, add 10-20 times the volume of PBS to elute impurity proteins until the effluent absorbance at 280nm is close to 0. The target protein was eluted with elution buffer (50 mM Tris-Cl, 10 mM reduced glutathione), the flow rate was controlled at 0.5 mL/min, and the effluent was collected. The protein content of the effluent was detected by SDS-PAGE, the protein was dialyzed into PBS solution, the protein concentration was determined, and the aliquots were frozen and stored in a -80°C refrigerator.

(3) Determination of Omp16 antibody titer in camel serum

ELISA detection of camel serum titer: The prepared Omp16 was coated on an ELISA plate at a concentration of 2 μg/mL, and incubated at 4°C overnight. The plate was washed three times with PBS containing 0.5% Tween-20 (PBS'T) as the wash solution. PBS'T solution containing 2.5% nonfat dry milk was added, the ELISA plate was blocked at 37°C for 1 h, and the plate was washed three times. Diluted with blocking solution (unimmunized camel serum and three-immunized camel serum (dilution multiples of 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ ), added to the ELISA plate at 100 μL/well, Incubate at 37 °C for 1 h. Wash the plate three times, add rabbit anti-camel IgG polyclonal antibody at a dilution ratio of 1:2000, and incubate at 37 °C for 1 h. After washing the plate three times, use horseradish peroxidase-labeled goat anti-rabbit IgG, and incubate at 37 °C for 1 h After washing the plate three times, add TMB for color development at room temperature for 10 min, 3M sulfuric acid to stop the reaction, read the absorbance at a wavelength of 450 nm with a microplate reader, and analyze the camel immune effect.

2. Construction of phage display library of Brucella A19 vaccine strain

2.1 RNA extraction and cDNA preparation

2 × 10 ⁷ lymphocytes from the camel's peripheral blood after A19 immunization were taken, and the total RNA of the peripheral blood lymphocytes was extracted using the MiniBEST Universal RNA Extraction Kit of TaKaRa Company, and the RNA concentration and purity were determined. Use Thermo Fisher's RevertAid First Strand cDNA Synthesis Kit, oligo(dT) ₁₈ primer to obtain cDNA, and the specific reverse transcription system is as follows:

PBMC total RNA 1 μg, oligo(dT) ₁₈ 1 μL, DEPC H ₂ O added to 12 μL, mixed, incubated at 65°C for 5 min, and stored at 4°C. The following reaction components were added to the above system, specifically 5×reaction buffer 4μL, RiblockRNase lnhibitor (20U/μL) 1μL, 10mM dNTP Mix 2μL, ReverAid M-MμLV RT (200U/μL) 1μL. After mixing, reverse transcription for 60 min at 42 °C and 5 min at 70 °C to terminate the reaction. Reverse transcripts were stored at 4°C for short-term storage and at -20 or -80°C for long-term storage.

2.2 Nested PGR amplification of VHH gene

The primer sequences for nested PCR amplification of VHH gene are as follows:

The first round PCR amplification gene size is about 700bp,

Upstream primer CALL0015'-GTCCTGGCTGCTCTTCTACAAGG-3';

Downstream primer CALL0025'-GGTACGTGCTGTTGAACTGTTCC-3';

The second round PCR amplification gene size is about 400bp,

Upstream primer VHH-FOR 5'-CAGGTGCAGCTGCAGGAGTCTGGGGGAGR-3';

Downstream primer VHH-REV 5'-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3'.

DNA Polymerase High Fidelity进行PCR扩增，反应体系如下：cDNA(或第一轮PCR回收产物)2μL，上游引物2μL，下游引物2μL，

HiFi Buffer II 5μL，10mM dNTP Mix 4μL，

HiFi DNAPolymerase 1μL，H₂O 34μL。Use Beijing Full Gold

DNA Polymerase High Fidelity was used for PCR amplification. The reaction system was as follows: 2μL of cDNA (or the first-round PCR recovery product), 2μL of upstream primer, 2μL of downstream primer,

HiFi Buffer II 5μL, 10mM dNTP Mix 4μL,

HiFi DNAPolymerase 1 μL, H ₂ O 34 μL.

The PGR reaction program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 40s, including 18 cycles of the first round of PGR amplification, 28 cycles of the second round of PGR amplification, and the final 72°C The total extension is 10min. Agarose gel electrophoresis was used to identify the amplification, and the gel recovery reagent was used to recover the band of about 400bp, and the concentration of the recovered product was determined.

2.3 Construction of VHH phage display library

(1) Construction of pMECs-VHH recombinant vector

The pMECs empty vector and the recovered VHH gene were double digested with restriction enzymes Pst I and Not I, and the specific reaction system was as follows: 10×Buffer 5 μL; Quick cut Pst I 1 μL; Quick cut Not I 1 μL; pMECs (or VHH) 1ug; H20 up to 50μL. Incubate for 1 h at 37°C. The digested fragments were recovered and the concentration was determined. The vector and the target fragment were ligated and recovered using the following system: 10×T4 DNA ligase buffer 50μL, pMECs digested product 6.9ug, VHH 2.3ug, T4 DNA ligase 5ug, H20 up to 500μL. Connect at 16°C for 8h. The ligation product was recovered and the recovered concentration was determined.

(2) Preparation of TG1 competent cells

Pick a single colony of TG1 into 10 mL of 2×YT medium, 37 ° C, 200 r/min shaking culture to logarithmic growth phase. Transfer to 800mL 2×YT medium at a ratio of 1:100, 37°C, 200r/min shaking culture for about 2h to logarithmic growth phase, put on ice for 30min, and wait for the bacterial solution to cool. The cells were washed twice with pre-cooled sterile ultrapure water and 10% glycerol under the conditions of 2200×g, 4°C, and centrifugation for 10 min. Centrifuge for the last time, discard the supernatant, add 1.6 mL of 10% glycerol to resuspend the cells, and store in 100 μL aliquots per tube at -80°C.

(3) Electrotransformation of recombinant plasmids

Take 100 μL of TG1 competent and 5 μL of recombinant plasmid, mix well, and let stand on ice for 5 min. The mixture was added to a pre-cooled 0.1 mL rotor beaker. Adjust the voltage of the electrotransformer to 1.8KV and shock the cells. Resuspend cells in 2 mL of pre-warmed SOC medium. Take 100 μL of the resuspended cells for gradient dilution, spread them onto an ampicillin LB plate, and culture at 37°C for 12-14 hours to calculate the library capacity. The rest of the cells were directly spread on the ampicillin LB plate, cultured at 37°C for 8-10 h, collected with 50% glycerol, and frozen at -80°C for later use.

(4) Positive rate and diversity identification of the library

Pick 96 single clones of transformants into LB ampicillin medium, 37 ° C, 220r/min shaking culture for 6h, by using primers MP57 (5'-TTATGCTTCCGGCTCGTATG-3') and VHH-REV to carry out bacterial liquid PCR identification of transformants, The transformant positive rate was calculated. The reaction system is as follows: 10 μL of 2×rTaq Mix, 71 μL of MP5, 1 μL of VHH-REV, 1 μL of bacterial solution, and 207 μL of ddH. The reaction program was 94°C for 5min, 94°C for 30sec, 55°C for 30sec, 72°C for 40s, 30 cycles of amplification, 72°C for 10min, the positive band was about 500bp. 10 positive clones were randomly selected and sequenced using MP57 primers to compare the diversity of the library.

3. Panning of Omp16-specific Nanobodies

(1) Rescue of phage library

Take 100 μL of the phage display library, inoculate it in 100 mL of 2×YT/AMP-GLU medium (containing 1 μg/mL ampicillin and 2% glucose), and cultivate to logarithmic growth phase at 37° C. and 200 r/min. Add 20 MOI doses of helper phage M13K07 and let stand at 37°C for 30 min. Centrifuge at 2800 g for 10 min at room temperature, discard the supernatant, resuspend the pellet in 200 mL of 2×YT/AMP-KAN (containing 1 μg/mL ampicillin and kanamycin) medium, and culture at 37°C for 14-16 h at 200 r/min. Centrifuge at 3800g for 30min at 4°C, collect the supernatant, add 1/5 volume of PEG/NaCl (200g PEG6000, 146g NaCl dissolved in water, dilute to 1L), mix well, and let stand on ice for 6-8h. Centrifuge at 3800 g for 30 min at 4°C to discard the supernatant, add 1 mL of PBS solution to resuspend the pellet, and incubate overnight at 4°C on a shaker to fully dissolve the phage particles. The supernatant was collected by centrifugation at 12000g at 4°C for 15min, and the rescue phage titer was determined using TG1 in logarithmic growth phase.

(2) Panning of Omp16-specific phage

The purified Omp16 protein was diluted with PBS buffer to 200 μg/mL, and coated on a 96-well microtiter plate, with 100 μL per well, and 3 wells were coated in total. At the same time, PBS control wells were set and coated overnight at 4°C. The coating solution was discarded, and after washing the plate three times with PBS'T, 200 μL of 2.5% nonfat milk powder was added, and the plate was blocked at 37°C for 2 hours. Discard the blocking solution, add 5×10 ¹⁰ PFU/well of phage diluted with 2.5% nonfat milk powder, and incubate at 37°C for 2h. Discard the phage supernatant, wash the plate 10 times with PBS'T, wash the plate 5 times with PBS, add 100 μl of freshly prepared 0.1 M triethylamine to each well, let stand for 10 min at room temperature, and quickly aspirate the eluate with an equal volume of 1 M Tris-HCl. (pH=7.4) to neutralize the eluate. Take 400 μL of panning products to rescue the panned phage according to the library rescue method, and detect the phage titer at the same time. The rescued phages were subjected to the second and third rounds of panning according to the same method. The coating concentrations of Omp16 in the second and third rounds of panning were 100 and 50 μg/mL, respectively.

(3) Preparation of Nanobody Crude Extract

96 phage monoclones from the final round of panning were randomly selected into 100 μL LB/AMP-GLU medium, and cultured at 37°C at 200 r/min for 8 h. 10 μL of each clone was transferred to 500 μL TB medium, cultured at 37°C to logarithmic growth phase, 10 μL of 100 mM IPTG was added, and the culture was continued for 12-14 h. Centrifuge at 12000 r/min for 2 min, collect the cells, freeze-thaw at -80°C and 37°C three times to lyse the cells, add 500 μL of PBS and incubate at 4°C for 30 min to resuspend the cells. After centrifugation, the supernatant is the crude extract.

(4) ELISA detection of crude phage extracts

The purified Omp16 recombinant protein and the control protein GST were respectively coated in a 96-well microtiter plate at a concentration of 2 μg/mL, and incubated at 4°C overnight. Wash the ELISA plate 4 times with PBS'T, add blocking solution, 200 μl per well, and block at room temperature for 1 h. After washing the plate, 100 μL of crude extract was added for ELISA reaction, and the target antibody was detected using mouse anti-HA-labeled antibody and HRP-labeled goat anti-mouse secondary antibody. Use TMB to develop color at room temperature for 10 min, 3M H ₂ SO ₄ to terminate the reaction, read the absorbance at 450 nm wavelength, and analyze the test results. The positive clones were picked for sequence analysis, and an Omp16-specific nanobody sequence QVQLQESGGGMVQPGSLRLTCVGDGAFFKLVDMSWVRQAPGKRLEWVASINSGGDRTYYADSVKGRFTISRDNAKNTLYLQLNSLKTEDTAMYHCALGMPRAAGWDQVGPGTQVTVSS was obtained (as shown in Figure 5).

(5) Recombinant Nanobody Recognition Omp16 Protein Western Blot Identification

Brucella A19 and S2 were cultured for 36 h respectively, centrifuged at 12000 r/min for 2 min, the precipitate was resuspended in PBS, boiled for inactivation, sonicated, centrifuged at 12000 r/min at 4°C for 10 min, and the supernatant was removed for SDS-PAGE and transfer. membrane. The crude nanobody extract was added as the primary antibody, and mouse anti-HA tag antibody and HRP-goat anti-mouse IgG were used as the secondary antibody. After incubation, the ECL luminescent liquid developed color to verify that the ELlSA-positive nanobody crude extract was bound to Brucella In the case of Omp16.

4. Expression of Horseradish Peroxidase-Conjugated Nanobodies

The gene sequence of NbA4 was inserted into the pCMV-HRP expression vector through the two restriction sites of Pst I and Not I, and the recombinant plasmid was constructed, and the correctness of the recombinant plasmid was verified by PGR and sequencing. The endotoxin-free pCMV-NbA4-HRP recombinant plasmid was extracted. Using Lipo8000 (Biyuntian), the following reaction system was prepared in each well of a 12-well plate: Lipo80001.6 μL, pCMV-NbA4-HRP 1 μg, Opti-mem 50 μL, and the plasmid was transfected into HEK-293T cells with a confluence of 60-70%. , the supernatant was collected after 48 h to detect the expression and activity of NbA4-HRP.

5. Establishment of ELISA method based on NbA4-HRP blocking

5.1 Determination of working concentration of antigen and antibody by checkerboard method

(1) Antigen coating: set the concentration of Omp16 to 1, 2, and 4 μg/mL, coat with 100 μL/well on a 96-well ELISA plate, and incubate at 4°C overnight.

(2) Blocking: Wash the plate three times with PBS'T solution, add 200 μL of 2.5% nonfat milk powder to each well, and block at 37°C for 1 h.

(3) Incubation with primary antibody: The collected NbA4-HRP expression supernatant was diluted 2, 4, 6, 8, 16, 32, and 64-fold with blocking solution, added to the ELISA plate, and incubated at 37°C for 1 h.

(4) Color development and termination. Add 100 μL of TMB solution for color development at room temperature for 10 min, 50 μL of 3M H ₂ SO ₄ to terminate the reaction, read the absorbance at 450 nm, analyze the test results, and determine the concentration of antigen and antibody that blocks ELISA.

5.2 Detection of Brucella antibodies in bovine serum based on HRP-NbA4 blocking ELISA

Omp16 was diluted to 2 μg/mL, coated on a 96-well microtiter plate, overnight at 4°C. 2.5% skimmed milk powder was added, and the cells were blocked at 37°C for 1 h. Serum and nanobodies were mixed in the ratio of serum 1:20 and HPR-NbA41:4, 100 μL per well was added to the microtiter plate, incubated at 37°C for 1 h, after washing the plate, TMB and 3M H2SO4 were added for color development, and the absorbance at 450 nm was detected. .

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

sequence listing

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Claims (6)

1. An Omp16 specific nano antibody for detecting Brucella, which is characterized in that: the specific nano antibody is prepared by the following steps:

(1) induction of expression of Omp16

Transforming the pGEX4T-1-Omp16 plasmid preserved in a laboratory into BL21(DE3), selecting a single clone to activate to a logarithmic growth phase, cooling to room temperature, adding IPTG with the final concentration of 1mM, transferring to 22 ℃, culturing at 200r/min for 16h, and inducing to express proteins; centrifuging at 6000r/min at normal temperature for 10min, discarding supernatant, resuspending thallus with 1/20 volume of PBS, ultrasonically crushing thallus at 3s with 3s of ultrasound, intermittently crushing thallus for 3s, centrifuging at 12000r/min at 4 deg.C for 10min, and separating supernatant and precipitate;

(2) purification of Omp16 protein

Taking the supernatant after ultrasonic treatment, filtering the supernatant by using a 0.45 mu M filter to remove impurities, and placing the supernatant on ice for later use; adding 1mL of GST resin into a chromatographic column, naturally settling with a medium, and adding 10-time volume of PBS to balance the column; adding the filtered expression supernatant, adjusting the flow rate to be 0.5mL/min, and adsorbing the target protein; after the sample loading is finished, adding PBS with 10-20 times volume to elute the impure protein until the absorbance of the effluent is close to 0 at 280nm, eluting the target protein by using an elution buffer solution, controlling the flow rate to be 0.5mL/min, collecting the effluent, detecting the protein content of the effluent by SDS-PAGE, dialyzing the protein into the PBS solution, determining the protein concentration, and subpackaging and freezing to a refrigerator at-80 ℃;

(3) panning of Omp 16-specific phages

Diluting the purified Omp16 protein to 200 mu g/mL by using PBS buffer solution, coating the protein on a 96-well enzyme label plate with each well being 100 mu L, coating 3 wells in total, setting PBS control wells at the same time, and coating overnight at 4 ℃; discarding the coating solution, washing the plate with PBS' T for three times, adding 200 μ L of 2.5% skimmed milk powder, and sealing at 37 deg.C for 2 hr; the blocking solution was discarded and 2.5% skim milk diluted 5X 10 phage was added ¹⁰ PFU/well, incubation at 37 ℃ for 2 h; discarding the phage supernatant, washing the plate 10 times with PBS' T, washing the plate 5 times with PBS, adding 100 μ l of freshly prepared 0.1M triethylamine to each well, standing at room temperature for 10min, sucking out the eluate, and rapidly neutralizing the eluate with an equal volume of Tris-HCI with pH of 7.4 at 1M; taking 400 mu L of elutriation products to rescue the elutriation phages according to a library rescue method, and simultaneously detecting the titer of the phages; carrying out second round and third round of panning on the rescued phage according to the same method, wherein the coating concentrations of Omp16 of the second round and the third round of panning are 100 and 50 mu g/mL respectively;

(4) preparation of crude extract of nano antibody

Randomly picking 96 final panning phage monoclonals into 100 mu L LB/AMP-GLU culture medium, and culturing at 37 ℃ for 8h at 200 r/min; transferring 10 μ L of each clone culture into 500 μ L of TB medium, culturing at 37 deg.C to logarithmic phase, adding 10 μ L of 100mM IPTG, and culturing for 12-14 hr; centrifuging at 12000r/min for 2min, collecting thallus, repeatedly freezing and thawing at-80 deg.C and 37 deg.C for three times to lyse thallus, adding 500 μ L PBS, incubating at 4 deg.C for 30min, and resuspending thallus, centrifuging, and collecting supernatant to obtain crude extract.

2. The Omp16 specific nanobody for detecting Brucella according to claim 1, wherein: in the step (3), the library rescue method comprises the following steps:

taking 100 mu L of phage display library, inoculating the phage display library into 100mL of 2 XYT/AMP-GLU culture medium, and culturing at 37 ℃ and 200r/min until logarithmic phase; adding 20 MOI doses of helper phage M13K07, standing at 37 deg.C for 30min, centrifuging at 2800g room temperature for 10min, discarding supernatant, resuspending the precipitate with 200mL 2 XYT/AMP-KAN culture medium, and culturing at 37 deg.C for 14-16h at 200 r/min; 3800g, centrifuging at 4 deg.C for 30min, collecting supernatant, adding 1/5 volume of PEG/NaCl, mixing, and standing on ice for 6-8 hr; 3800g, centrifuging at 4 deg.C for 30min, discarding supernatant, adding 1mL PBS solution to resuspend the precipitate, shaking and incubating overnight at 4 deg.C to fully dissolve phage particles, centrifuging at 12000g at 4 deg.C for 15min, collecting supernatant, and determining rescue phage titer by using TG1 in logarithmic phase.

3. The expression vector of Omp16 specific nanobody for detecting Brucella according to any of claims 1-2, wherein: the construction method comprises the following steps:

the gene sequence of NbA4 is inserted into a pCMV-HRP expression vector through two restriction enzyme cutting sites of Pst I and Not I, a recombinant plasmid is constructed, the correctness of the recombinant plasmid is verified through PCR and sequencing, the pCMV-NbA4-HRP recombinant plasmid without endotoxin is extracted, and the following reaction system is prepared in each hole by using a Lipo8000 and 12-hole plate: lipo80001.6 μ L, pCMV-NbA4-HRP 1 μ g, Opti-mem 50 μ L, plasmid was transfected into HEK-293T cells with a fusion degree of 60-70%, and after 48h the supernatant was harvested for NbA4-HRP expression and activity.

4. The use of the expression vector of Omp 16-specific nanobody of brucella detection according to any of claims 1-2, characterized in that: can be used for constructing a method for detecting bovine serum brucella antibody based on NbA4-HRP blocking ELISA.

5. The use of claim 4, wherein: is constructed by the following steps:

(1) antigen coating: setting the concentration of Omp16 as 1, 2 and 4 mug/mL, coating the Omp16 on a 96-well enzyme label plate by 100 mug/well, and incubating overnight at 4 ℃;

(2) and (3) sealing: washing the plate with PBS' T solution for three times, adding 200 μ L of 2.5% skimmed milk powder into each well, and sealing at 37 deg.C for 1 h;

(3) incubation of primary antibody: carrying out gradient dilution on the collected NbA4-HRP expression supernatant by 2, 4, 6, 8, 16, 32 and 64 times by using a confining liquid, adding the diluted solution into an enzyme label plate, and incubating for 1h at 37 ℃;

(4) color development and termination: adding 100 μ L TMB solution, developing at room temperature for 10min, 50 μ L3M H ₂ SO ₄ After the reaction is stopped, the absorbance value of 450nm is read, the test result is analyzed, and the antigen antibody using concentration for blocking ELISA is determined.

6. The use of claim 4, wherein: during detection, Omp16 is diluted to 2 mug/mL and coated in a 96-hole enzyme label plate, and the temperature is kept overnight at 4 ℃; adding 2.5% skimmed milk powder, sealing at 37 deg.C for 1 hr; according toMixing serum and nano antibody at the ratio of 1: 20 and HPR-NbA 41: 4, adding 100 μ L of serum into enzyme label plate, incubating at 37 deg.C for 1h, washing plate, adding TMB and 3M H ₂ SO ₄ Developing color, and detecting absorbance at 450 nm.

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