CN116854829A

CN116854829A - Antibody SEB27, fusion protein SEB27-vHRP, preparation method and application

Info

Publication number: CN116854829A
Application number: CN202310916601.6A
Authority: CN
Inventors: 王新; 张瑶; 张鹏飞; 万阳丽; 常冠红; 周婷
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2023-10-10

Abstract

The application provides an antibody SEB27, a fusion protein SEB27-vHRP, a preparation method and application thereof, wherein the fusion protein comprises an IgG antibody signal peptide, a hemagglutinin tag, an antibody SEB27, a connecting peptide, horseradish peroxidase and a histidine tag; the fusion protein can be used for detecting staphylococcus aureus enterotoxin B in food. The fusion protein SEB27-vHRP provided by the application has the advantages that the horseradish peroxidase is not needed to be subjected to secondary antibody incubation, the subsequent chromogenic reaction can be directly performed after the primary antibody incubation is finished, the detection time is shortened, and the application prospect is wide.

Description

Antibody SEB27, fusion protein SEB27-vHRP, preparation method and application

Technical Field

The application belongs to the technical field of food detection, relates to enterotoxin B immunological detection, and in particular relates to an antibody SEB27, a fusion protein SEB27-vHRP, a preparation method and application.

Background

Enterotoxin is staphylococcus aureusStaphylococcus aureus) A potent exotoxin of the gastrointestinal tract synthesized throughout the log phase or from the exponential phase to the stationary phase, has superantigenic activity and is very resistant to heat treatment, low pH and proteolytic enzymes, which enables them to remain active in contaminated foods and to trigger food poisoning. Studies have shown that a lethal dose of 20 ng/kg of enterotoxin B, at lower concentrations, can lead to disability in humans. Although staphylococcus aureus is easily inactivated by cooking with heat, it is known that staphylococcus aureus enterotoxin B #Staphylococcalenteratoxin B, SEB) has heat-resistant properties and does not lose toxic and superantigenic activity after heating, so detection of s.

The Chinese patent with the publication number of CN110498854B discloses an antibody against staphylococcus aureus enterotoxin B, and an ELISA analysis method based on the antibody is provided in the patent, and although SEB can be detected by the method, the method needs secondary antibody incubation, is complex in operation and takes longer detection time.

Disclosure of Invention

Aiming at the defects of the prior art, the application aims to provide an antibody SEB27, a fusion protein SEB27-vHRP, a preparation method and application thereof, and solves the technical problem that the detection time for enterotoxin B immunological detection in the prior art is long.

In order to solve the technical problems, the application adopts the following technical scheme:

a fusion protein SEB27-vHRP comprising an IgG antibody signal peptide, a hemagglutinin tag, an antibody SEB27, a linker peptide, horseradish peroxidase, and a histidine tag.

The application also has the following technical characteristics:

the nucleotide sequence of the fusion protein is shown as sequence ID number in a nucleotide or amino acid sequence table.

The application also provides a preparation method of the fusion protein SEB27-vHRP, which specifically comprises the following steps: the gene fragment of the antibody SEB27 is connected to an expression vector pCMV-N1-HRP, then the recombinant vector is transformed into escherichia coli, single colony is selected for sequencing after culture, clones of a sequencing result are selected for culture, plasmids are extracted, then the plasmids are transfected into mammalian cells, and cell culture liquid containing the fusion protein SEB27-vHRP is collected after culture.

Specifically, the nucleotide sequence of the expression vector pCMV-N1-HRP is shown as sequence ID number in a nucleotide or amino acid sequence table.

The application also provides application of the fusion protein SEB27-vHRP in detecting staphylococcus aureus enterotoxin B in food.

For the application described above, specifically and optionally, the enzyme-linked immunosorbent assay is used to detect staphylococcus aureus enterotoxin B in food.

For the use as described above, in particular and optionally, the detection of staphylococcus aureus enterotoxin B in food products is performed by a protein immunoblotting method.

The application also protects the antibody SEB27, and the nucleotide sequence of the antibody is shown as sequence ID number 1 in a nucleotide or amino acid sequence table.

The application also provides a preparation method of the antibody SEB27, which specifically comprises the following steps:

step one, obtaining camel-derived lymphocyte RNA.

Step two, amplification of VHH gene fragment:

step 2.1, reverse transcription PCR:

and (3) taking the camel-derived lymphocyte RNA obtained in the step one as a template, and performing reverse transcription PCR to synthesize cDNA.

Step 2.2, first round PCR amplification of nested PCR was performed:

the first round of PCR amplification was performed using the cDNA synthesized by reverse transcription in step 2.1 as a template, and primer CALL001 and primer CALL002 were used to identify and recover the PCR product.

The sequence of the primer CALL001 is as follows: 5'-gtcctggctgctcttctacaagg-3'.

The primer CALL002 has the sequence: 5'-ggtacgtgctgttgaactgttcc-3'.

Step 2.3, performing a second round of PCR amplification of nested PCR:

and 2, taking the recovered PCR product in the step 2.2 as a template, and adopting a primer VHH-FOR and a primer VHH-REV to carry out a second round of PCR amplification, and identifying and recovering the PCR product, wherein the PCR product is the VHH gene fragment.

The sequence of the primer VHH-FOR is as follows: 5 '-caggtgcagctgcaggagtctggggagr-3'.

The sequence of the primer VHH-REV is as follows: 5'-ctagtgcggccgctgaggagacggtgacctgggt-3'.

Step three, constructing and identifying a nano antibody library:

performing enzyme digestion reaction on the phage display vector and the VHH gene fragment obtained in the step 2.3, recovering enzyme digestion products, connecting the phage display vector with the enzyme digestion products of the VHH gene fragment, and recovering connection products; transferring the connection product into competent cells by adopting electrotransformation, and obtaining nano antibody library bacterial liquid after screening and culturing; and (3) screening and culturing the nano antibody library bacterial liquid again and extracting phage to obtain the camel-derived single-domain heavy chain antibody library.

Step four, affinity panning and identification of the nanobody:

and (3) carrying out multiple rounds of panning and identification on the camel source single-domain heavy chain antibody library obtained in the step (III) by adopting enterotoxin B to obtain positive clones, and extracting and purifying proteins after the positive clones are subjected to amplification culture to obtain the antibody SEB27.

Compared with the prior art, the application has the following technical effects:

and (I) as the fusion protein SEB27-vHRP provided by the application is provided with horseradish peroxidase, secondary antibody incubation is not needed, and subsequent chromogenic reaction can be directly carried out after the primary antibody incubation is finished, so that the detection time is shortened, and the application prospect is wide.

Compared with the existing monoclonal antibody, the fusion protein SEB27-vHRP provided by the application has stronger specificity for recognizing enterotoxin B and wider application range.

Drawings

FIG. 1 shows the results of indirect ELISA assays for panning positive clones in example 2.

FIG. 2 shows the results of potency detection of the fusion protein SEB27-vHRP in example 4.

FIG. 3 shows the results of sensitivity assay of the fusion protein SEB27-vHRP in example 4.

FIG. 4 shows the results of specific identification of the fusion protein SEB27-vHRP in example 4.

FIG. 5 shows the results of detection by protein immunoblotting.

FIG. 6 is a schematic structural diagram of a fusion protein. In fig. 6: igGK singnal peptide the IgG antibody signal peptide, HA the hemagglutinin tag, SEB27 the antibody SEB27, linker the connecting peptide, vHRP the horseradish peroxidase, 6 XHis the histidine tag.

The following examples illustrate the application in further detail.

Detailed Description

In the application, the following components are added:

VHH gene fragments refer to heavy chain variable region fragments of nanobodies.

MOI is a measure of phage, which is specifically meant to be the multiplicity of infection.

ELISA refers to an ELISA assay.

All media and reagents used in the present application are those known in the art, for example, unless otherwise specified:

the formulations of the LB/Amp-GLU solid medium and the LB/Amp-GLU liquid medium adopt the formulations known in the prior art, and 1L of LB/Amp-GLU liquid medium comprises the following components: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 100 mg of ampicillin and 20 g of glucose; the LB/Amp-GLU solid medium was further supplemented with 15 agar g on the basis of the above formulation.

The 2 XYT/Amp-GLU liquid medium is prepared by adopting a formula known in the prior art, and 1L of the 2 XYT/Amp-GLU liquid medium comprises the following components: tryptone 16g, yeast extract 10g, naCl 40g, ampicillin 100 mg, glucose 20 g.

The formula of the 2 XYT/Amp-Kan liquid culture medium adopts a formula known in the prior art, and 1L of the 2 XYT/Amp-GLU liquid culture medium comprises the following components: tryptone 16g, yeast extract 10g, naCl 40g, ampicillin 100 mg, kanamycin 50 mg.

The phosphate buffer solution adopts a conventional phosphate buffer solution known in the prior art, and the pH value of the phosphate buffer solution is 7.2-7.4.

The lymphocyte separation medium was a Ficoll-Paque PLUS lymphocyte separation medium known in the art and obtained from Merck company of America.

Trizol reagent known in the art is available from Merck, america.

The reverse transcription PCR kit was a HiFi-Script cDNA first strand synthesis kit known in the art, hiFiScript cDNA Synthesis Kit, cat# CW2569, available from (Beijing) Kangji Biotech Co.

The nested PCR reaction adopts a TransTaq DNA Polymerase High Fidelity (HiFi) PCR kit known in the prior art, the product number is AP131-11, and the kit is purchased from Beijing full-scale gold biotechnology Co.

The restriction enzymes used in the digestion reaction are Pst I and Not I known in the prior art, and the digestion reaction Buffer solution adopts Cut Smart Buffer known in the prior art, both of which are purchased from NEB (Beijing) company.

T4 DNA ligase used in the ligation reaction was T4 DNA ligase known in the prior art, and ligase buffer was T4 DNA Ligase Buffer known in the prior art, and T4 DNA ligase and ligase buffer were purchased from NEB (Beijing).

The PCR product recovery kit was a Cycle Pure KitPCR product recovery kit known in the art and available from Omega Bio-Tek under the designation D6492.

The following specific embodiments of the present application are provided, and it should be noted that the present application is not limited to the following specific embodiments, and all equivalent changes made on the basis of the technical scheme of the present application fall within the protection scope of the present application.

Example 1:

the present example shows a staphylococcus aureus enterotoxin B nanobody SEB27, the nucleotide sequence of which is shown below: 5'-gagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggatacaccttcgatacgaacaccatggcctggtttcgccaggctccagggaaggagcgcgagggggtcgcaacttactatactggtgctggtggtcgtgctttatactatgccgacttcgtgaagggccgattcgccatctcccaagacaacgccaagaacacggtgtatctgcaaatggacagcctgaaacctgaggacactgccatgtactactgtgcgtcaacagtcccccgccgggcgatatctggtgcccctaaacccagtgactttgattattggggccaggggacccaggtcaccgtctcctca-3'.

The amino acid sequence of the antibody is as follows: ESGGGSVQAGGSLRLSCAASGYTFDTNTMAWFRQAPGKEREGVATYYTGAGGRALYYADFVKGRFAISQDNAKNTVYLQMDSLKPEDTAMYYCASTVPRRAISGAPKPSDFDYWGQGTQVTVSS.

In this example, the antibody comprises framework regions FR comprising the amino acid sequences of FR1, FR2, FR3 and FR4 and complementarity determining regions CDR comprising the amino acid sequences of CDR1, CRD2 and CDR 3.

Example 2:

the embodiment provides a preparation method of a staphylococcus aureus enterotoxin B nanobody SEB27 in the embodiment 1, which specifically comprises the following steps:

step one, obtaining camel-derived lymphocyte RNA:

step 1.1, immunization of Bactrian camels:

taking enterotoxin B as an immunogen, and immunizing an adult male Alshan Bactrian camel by adopting a subcutaneous multipoint injection mode to perform five rounds of immunization. The primary immunization is carried out by emulsifying Freund's complete adjuvant and an equal volume of immune antigen, and the immune dose is 100 mug/patient. The immunization was boosted once every two weeks afterwards, and injection was performed after emulsification with Freund's incomplete adjuvant and an equal volume of immunogen, at an immunization dose of 50. Mu.g/dose.

Step 1.2, lymphocyte separation:

after seven days of last immunization in step 1.1, 200mL of peripheral blood was collected with a disposable plastic blood bag containing an anticoagulant, and then the blood sample was diluted with an equal volume of phosphate buffer solution. After balancing the lymphocyte separation liquid to room temperature, 15mL was sucked into the lymphocyte separation tube with the porous partition plate, and then the lymphocyte separation tube was centrifuged at room temperature for 30s with a centrifugal force of 1000g so that the lymphocyte separation liquid was located just below the porous partition plate.

After balancing the diluted blood sample to room temperature, adding 30mL of the blood sample into each of the centrifuged lymphocyte separation tubes, adjusting the braking acceleration of the centrifuge to 0, and centrifuging the lymphocyte separation tubes at room temperature for 10min by using a centrifugal force of 1000 g. After centrifugation, the red blood cells are positioned at the bottom of the lymphocyte separation tube, the uppermost layer is blood plasma, and a layer of ring-packed milky white substance between the blood plasma and the white transparent lymphocyte separation liquid is the lymphocyte.

The uppermost plasma was carefully removed with a dropper until the plasma was 5-10 mm from the cell layer, lymphocytes were collected with a dropper into another clean 50mL centrifuge tube, and then at least 10 volumes of ice-bath phosphate buffer solution was added to the centrifuge tube, mixed upside down, and centrifuged at a centrifugal force of 250g for 10min at a temperature of 4 ℃. After centrifugation, the supernatant was discarded, the cells were resuspended in 45mL of ice-bath phosphate buffer, centrifuged at a centrifugal force of 250g at 4℃for 10min, and the above-mentioned resuspension centrifugation was repeated twice to ensure that the lymphocytes were washed thoroughly.

After the final centrifugation, lymphocytes were resuspended in 10mL of ice-bath phosphate buffer, counted with a hemocytometer, and after counting, the lymphocytes were packed into 1.5mL centrifuge tubes, each tube was filled with 1X 10 ⁷ Separating cells, centrifuging at 4deg.C for 10min with centrifugal force of 250g, removing supernatant after centrifuging, and retaining lymphocyte sediment; the lymphocyte precipitate is directly used for RNA extraction or is preserved at-80 ℃ for standby.

Step 1.3, lymphocyte RNA extraction:

adding 1mL of Trizol reagent into lymphocyte sediment, blowing off lymphocyte agglomerates at the bottom of a centrifuge tube by using a liquid transfer device, scattering the lymphocyte agglomerates to obtain lymphocyte lysate, and then adding 1/5 volume of chloroform into the lymphocyte lysate; then the centrifugal tube cover is covered tightly, vigorously shaken for 15s, and after standing for 5min at room temperature, centrifuged for 10-15 min at the temperature of 4 ℃ with the centrifugal force of 12000 g.

After centrifugation, carefully sucking the upper water phase into a new centrifuge tube, adding 1/2 volume of isopropanol, reversing and uniformly mixing, standing at room temperature for 10min, centrifuging at the temperature of 4 ℃ for 10min by using a centrifugal force of 12000g, carefully discarding the supernatant after centrifugation, adding an equal volume of 75% ethanol, swirling and fully washing, flicking the bottom of the tube to suspend cell sediment, centrifuging at the temperature of 4 ℃ for 5min by using a centrifugal force of 7500g, discarding the supernatant after centrifugation, opening a centrifuge tube cover, standing at room temperature for 5-10 min, and obtaining RNA sediment. Adding 30-100 mu L of RNase-free water into the RNA precipitate, and taking a small amount of the RNA to measure the OD after the RNA precipitate is completely dissolved ₂₆₀ Value and OD ₂₆₀ /OD ₂₈₀ The ratio, the concentration and quality of total RNA are determined, and the rest solution is preserved at-80 ℃.

Step two, amplification of VHH gene fragment:

step 2.1, reverse transcription PCR:

the RNA extracted in step 1.3 was used as a template to synthesize cDNA by reverse transcription PCR, and the reaction solution of reverse transcription PCR was prepared according to the instructions of the reverse transcription PCR kit, specifically as shown in Table 1. The reaction conditions of reverse transcription PCR are specifically: incubation was preceded by a 30min incubation at a temperature of 42℃and followed by a 5min incubation at a temperature of 85 ℃. After the completion of reverse transcription PCR, the obtained cDNA was frozen at-20℃for use.

TABLE 1 reverse transcription PCR System

Step 2.2, first round PCR amplification of nested PCR was performed:

the first round of PCR amplification was performed using the cDNA synthesized by reverse transcription in step 2.1 as a template, and the reaction solution for the first round of PCR amplification was prepared according to the instructions of the PCR kit, as shown in Table 2. The reaction conditions for the first round of PCR amplification are specifically: pre-denaturing for 10min at 94 ℃; then carrying out 28 cycles of reaction at 94 ℃ for 30s, reaction at 55 ℃ for 30s and reaction at 72 ℃ for 30 s; finally, the extension is carried out for 10min at a temperature of 72 ℃. After the first round of PCR amplification was completed, the target band around 700bp was recovered after identifying the PCR product by 1.2% agarose gel electrophoresis, and the concentration of the recovered product was measured.

TABLE 2 first round PCR amplification System

In table 2, the sequence of primer CALL001 is: 5'-gtcctggctgctcttctacaagg-3'; the primer CALL002 has the sequence: 5'-ggtacgtgctgttgaactgttcc-3'.

Step 2.3, performing a second round of PCR amplification of nested PCR:

the second round of PCR amplification was performed using the recovered product from step 2.3 as a template, and the reaction solution for the second round of PCR amplification was prepared according to the instructions of the PCR kit, as shown in Table 3. The reaction conditions for the second round of PCR amplification are specifically: pre-denaturing for 10min at 94 ℃; then carrying out 30 cycles of reaction at 94 ℃ for 30s, reaction at 55 ℃ for 30s and reaction at 72 ℃ for 30 s; finally, the extension is carried out for 10min at a temperature of 72 ℃. After the second round of PCR amplification is finished, 1.2% agarose gel electrophoresis is adopted to identify the PCR product, then the target band near 400bp is recovered, and the concentration of the recovered product is measured, wherein the recovered product is the VHH gene fragment.

TABLE 3 second round PCR amplification System

In Table 3, the sequences of the primers VHH-FOR are: 5 '-caggtgcagctgcaggagtctggggagr-3'; the sequence of the primer VHH-REV is as follows: 5'-ctagtgcggccgctgaggagacggtgacctgggt-3'.

Step three, constructing and identifying a nano antibody library:

step 3.1, constructing a recombinant vector:

the phage display vector pMECS and the VHH gene fragment obtained in step 2.3 were subjected to an overnight cleavage reaction, the cleavage system being specifically shown in table 4. And (3) after the enzyme digestion reaction is finished, recovering enzyme digestion products by using a PCR product recovery kit, and measuring the concentration of the recovered products. The recovered products of phage display vector pMECS and VHH gene fragment were then subjected to overnight ligation at a temperature of 16 ℃ with the ligation system shown in table 5. And after the ligation reaction is finished, recovering the ligation product by using a PCR product recovery kit.

Table 4, enzyme digestion System

TABLE 5 connection System

Step 3.2, electrotransformation of ligation product:

adding the connection product obtained in the step 3.1 into 800 mu L of TG1 competent cells, then split charging the cells into electric rotating cups, setting split charging quantity of each electric rotating cup to be 50 mu L, setting parameters of electric rotating instrument equipment to be 1800V, 25 mu F,200 omega and 1 mm, carrying out electric conversion, placing the electric-converted thalli in a shaking table at 37 ℃ for 220rpm expansion culture for 1 h, coating the culture on a flat plate containing LB/Amp-GLU solid culture medium, wherein the coating quantity of each flat plate is 1mL, and placing the flat plate in an incubator at 37 ℃ for inversion culture for 6-8 h after the flat plate is dried. And finally, adding 1mL of LB/Amp-GLU liquid culture medium on each flat plate, scraping off lawn by using cells, adding 1/3 volume of 50% glycerol to obtain nano antibody library bacterial liquid, uniformly mixing the nano antibody library bacterial liquid, sub-packaging, and preserving at-80 ℃ for later use.

Step 3.3, rescue of the initial library:

adding the nanometer antibody library bacterial solution obtained in step 3.2 of 1mL into 2 XYT/Amp-GLU culture medium of 100 mL, culturing in shaking table of 220rpm at 37deg.C to logarithmic phase, and measuring OD _600nm And calculating the bacterial load. Adding 20 MOI helper phage M13K07 into the culture solution, mixing, standing at 37deg.C for 30min, centrifuging at 4000g centrifugal force at room temperature for 30min, discarding supernatant after centrifuging, adding 500mL 2 XYT/Amp-Kan, culturing again for 14 h, centrifuging at 4deg.C for 30min with 4000g centrifugal force, collecting supernatant, adding 1/5 volume of precooled sterile PEG/NaCl solution, mixing, standing on ice for 8h, centrifuging at 4deg.C for 30min with 4000g centrifugal force, collecting precipitate, adding about 500 μL phosphate buffer solution, suspending precipitate, incubating overnight at 4deg.C, centrifuging to obtain supernatant, and storing the camel source single domain heavy chain antibody library at 4deg.C.

Step four, affinity panning and identification of the nanobody:

step 4.1, affinity panning of nanobody:

step 4.1.1, first round panning:

first, enterotoxin B was diluted with phosphate buffer solution to a final concentration of 50. Mu.g/mL and coated overnight at 4 ℃. The next day was washed 5 times with 10mM PBST (phosphate buffer solution containing 0.1% Tween-20 (v/v)), and then, phosphate buffer solution containing 5% skimmed milk was added thereto, followed by blocking at 37℃for 1 hour. Then washed 6 times with PBST, 100. Mu.L of the camelid single domain heavy chain antibody pool obtained in step 3.3 was added to each well (titre about 5X 10) ¹¹ CFU/mL), 37 ℃ for 2 hours.

After the incubation, unbound phage were discarded, washed 10 times with PBST, 100. Mu.L of freshly prepared 0.1M triethylamine solution was added to each well, and after 10min standing at room temperature, the eluate was collected and rapidly neutralized with an equal volume of 1M Tris-HCl (pH 7.4) to obtain eluted phage; titers were determined by taking 10 μl of eluted phage, amplifying the remaining E.coli TG1 strain used to infect 25mL of the E.coli strain grown to log phase, precipitating the amplified phage with PEG/NaCl solution the third day, and determining the phage titer.

Step 4.1.2, continuing panning:

the first round of panning was completed and two rounds of panning were performed. The specific procedure for the second and third panning was essentially the same as that of step 4.1.1, except that the enterotoxin B concentrations were 25. Mu.g/mL and 12.5. Mu.g/mL, respectively.

Step 4.2, identification of positive phage clones:

randomly picking 48 clones from the plate after the third round of panning in step 4.1.2, preparing crude nanobody extract, determining positive clones by indirect ELISA, and selecting OD ₄₅₀ Clones 2.1 times greater than negative control were positive clones.

In this example, 43 positive clones were obtained, designated SEB1, SEB2, SEB3, SEB4, SEB5, SEB6, SEB7, SEB8, SEB9, SEB11, SEB12, SEB13, SEB14, SEB15, SEB17, SEB18, SEB19, SEB20, SEB21, SEB22, SEB23, SEB24, SEB25, SEB27, SEB28, SEB29, SEB30, SEB31, SEB32, SEB33, SEB34, SEB35, SEB37, SEB38, SEB39, SEB40, SEB41, SEB42, SEB43, SEB44, SEB45, SEB46, SEB48, and the P/N values of the positive clones are shown in FIG. 1, wherein the P/N values of the SEB27 clones are the greatest. After the SEB27 clone is subjected to amplification culture, the protein is extracted and purified, so that the antibody SEB27 can be obtained, and the antibody SEB27 can be used as a primary antibody for detecting staphylococcus aureus enterotoxin B in foods.

Example 3:

this example shows a fusion protein SEB27-vHRP comprising an IgG antibody signal peptide, a hemagglutinin tag, the antibody SEB27 of example 1, a linker peptide, horseradish peroxidase and a histidine tag as shown in FIG. 6.

The nucleotide sequence of the fusion protein is as follows: 5'-atggagaccgacaccttactgctgtgggtgctgctgttatgggtgcccggtagcaccggcgactacccatacgatgttccagattacgctctgcaggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggatacaccttcgatacgaacaccatggcctggtttcgccaggctccagggaaggagcgcgagggggtcgcaacttactatactggtgctggtggtcgtgctttatactatgccgacttcgtgaagggccgattcgccatctcccaagacaacgccaagaacacggtgtatctgcaaatggacagcctgaaacctgaggacactgccatgtactactgtgcgtcaacagtcccccgccgggcgatatctggtgcccctaaacccagtgactttgattattggggccaggggacccaggtcaccgtctcctcagcggccgccagtagtagtggcagtggtatgcagctgacccccacattctacgataactcttgtcccaacgtgtccaacatcgttcgtgacatcatcgtgaatgagctgaggagcgaccctaggatcgccgccagcattctgaggctgcacttccacgactgcttcgtgaatggctgcgacgcctccattttactggataacaccaccagctttcgtaccgaaaaggacgctttcggcaacgccaacagcgctcgtggcttcagcgtgatcgatcgtatgaaagccgccgtggaaagcgcttgtcccggcacagtgtcttgtgccgatttattaaccattgccgcccagcagtccgtgacactggccggcggacctagctggagagtgcctctgggtcgtagggattctttacaagcctttttagatttagccaacgccaatttacccgcccctttcttcactttacctcagctcaaggacagcttcagaaacgtgggtttaaataggagcagcgatttagtggctttatccggcggccacacatttggcaagagccagtgtcgtttcattatggatcgtctgtacaatttcagcaacaccggtttacccgaccccacactgaacaccacctatctccagactttaagaggtttatgccctttaaacggcaatctgtccgctttagtggatttcgacttacgtacccccacaatcttcgacaacaaatactacgtgaatttagaggagcagaagggtttaatccagagcgaccaagaactgttttccagccccgatgccaccgacaccatccctctggtgaggagcttcgccaactccacccagaccttcttcaacgcctttgtggaggccatggatcgtatgggcaacattacccctctgaccggcacccaaggtcagattcgtaggaactgtagggtggtgaatagcaacagcgatttacatcatcaccaccatcaccactaa-3'.

The amino acid sequence of the fusion protein is as follows: METDTLLLWVLLLWVPGSTGDYPYDVPDYALQESGGGSVQAGGSLRLSCAASGYTFDTNTMAWFRQAPGKEREGVATYYTGAGGRALYYADFVKGRFAISQDNAKNTVYLQMDSLKPEDTAMYYCASTVPRRAISGAPKPSDFDYWGQGTQVTVSSAAASSSGSGMQLTPTFYDNSCPNVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPDATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRRNCRVVNSNSDLHHHHHHH.

Example 4:

the present example provides a method for preparing the fusion protein SEB27-vHRP of the embodiment 3, which specifically comprises the following steps: the SEB27 clone obtained in example 2 was subjected to amplification culture, followed by extraction of a plasmid containing a gene fragment of the antibody SEB27, digestion of the plasmid, ligation of the plasmid onto the expression vector pCMV-N1-HRP, and transformation of the recombinant vector pCMV-N1-HRP-SEB27 into E.coli DH 5. Alpha. 10 single colonies were selected from the transformation plate for sequencing, clones from the sequencing result were selected for culture, plasmids were extracted with the deindotoxin plasmid extraction kit, and then transfected into HEK293T cells, and after 48 hours of culture, cell culture broth containing the fusion protein SEB27-vHRP was collected. The nucleotide sequence of the expression vector pCMV-N1-HRP is shown as sequence ID number in a nucleotide or amino acid sequence table.

In this example, ELISA was used to identify the titer of the fusion protein SEB27-vHRP, and the result is shown in FIG. 2, wherein the fusion protein has horseradish peroxidase activity, and the titer is 1:1000.

in the embodiment, a sandwich ELISA detection method is adopted to identify the sensitivity of the fusion protein SEB27-vHRP, and the specific process is as follows:

diluting the enterotoxin B rabbit polyclonal antibody to 10 mug/mL by using a phosphate buffer solution, adding 100 mug/hole, and coating at 4 ℃ overnight; the next day after washing 5 times with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)), 200. Mu.L of 5% skimmed milk powder was added and blocked at 37℃for 1 hour. 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL, 78.13ng/mL, 39.06ng/mL, 19.53ng/mL, 9.77ng/mL, 4.88ng/mL, 2.44ng/mL, 1.22ng/mL, 0.61ng/mL and 0.31ng/mL enterotoxin B were then added to each well, and after incubation for 1 hour at 37 ℃, 100. Mu.L of fusion protein SEB27-vHRP was added to each well and incubated for 1 hour at 37 ℃. After the incubation, PBST was washed 5 times, 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate solution was added, developed in a dark place for 15 minutes, and then 2M sulfuric acid solution was added to terminate the reaction, and OD was measured ₄₅₀ And drawing a standard curve.

This practice isIn the example, as shown in FIG. 3, the linear range of the standard curve is 9.77-312.5 ng/mL, the linear equation is y= 0.2318lgx-0.1699, R ² 0.9054, the minimum limit of detection was 9.77ng/mL, indicating that the sensitivity of the fusion protein SEB27-vHRP was higher in detecting enterotoxin B.

In the embodiment, the specificity of the fusion protein SEB27-vHRP is identified by adopting a sandwich ELISA detection method, and the specific process is as follows:

diluting the enterotoxin B rabbit polyclonal antibody to 10 mug/mL by using a phosphate buffer solution, adding 100 mug/hole, and coating at 4 ℃ overnight; the following day was washed 5 times with PBST (phosphate buffer containing 0.05% Tween-20 (v/v)), 200. Mu.L of 5% nonfat dry milk was added, after 1 hour of blocking at 37℃was washed 5 times, then 100. Mu.L of 10. Mu.g/mL enterotoxin A was added, incubated at 37℃for 1 hour, 100. Mu.L of the fusion protein SEB27-vHRP was added, and incubated at 37℃for 1 hour. After the incubation, PBST was washed 5 times, 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate solution was added, developed in a dark place for 15 minutes, and then 2M sulfuric acid solution was added to terminate the reaction, and OD was measured ₄₅₀ 。

In this example, as shown in FIG. 4, the sandwich ELISA established based on SEB27-vHRP did not cross react with enterotoxin A, demonstrating that SEB27-vHRP exhibited better specificity for enterotoxin B.

Example 5:

the application of the fusion protein SEA33-vHRP of the embodiment 3 for detecting the staphylococcus aureus enterotoxin B in the food is provided in the embodiment, and the enzyme-linked immunosorbent assay is adopted for detecting the staphylococcus aureus enterotoxin B in the food, and the specific process of the application is as follows:

diluting the enterotoxin B rabbit polyclonal antibody to 10 mug/mL by using a phosphate buffer solution, adding 100 mug/hole, and coating at 4 ℃ overnight; the following day was washed 5 times with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)), 200. Mu.L of 5% skimmed milk powder was added, after blocking at 37℃for 1 hour, PBST was washed 5 times, then 100. Mu.L of milk containing different concentrations of enterotoxin B was added, after incubation at 37℃for 1 hour, 100. Mu.L of fusion protein SEB27-vHRP was added, and incubation at 37℃for 1 hour. After the incubation was completed, PBST was washed 5 times, 100. Mu.L of 3,3', 5' -tetramethylammonium chloride was addedThe substrate solution of benzidine was developed in the dark for 15min, then 2M sulfuric acid solution was added to terminate the reaction, and OD was measured ₄₅₀ 。

In this example, as shown in Table 6, enterotoxin B standard substances with final concentrations of 50 and 250ng/mL are added into milk, and the detection result is close to the actual addition concentration, which indicates that the immunological detection of the staphylococcus aureus enterotoxin B is carried out by adopting a sandwich ELISA detection method established based on the fusion protein SEB27-vHRP, and the detection method has good accuracy.

TABLE 6 results of enterotoxin B immunological tests

Example 6:

the application of the fusion protein SEA33-vHRP of the embodiment 3 for detecting the staphylococcus aureus enterotoxin B in food is provided in the embodiment, and the staphylococcus aureus enterotoxin B in the food is detected by adopting a protein immunoblotting method, and the specific process of the application is as follows:

after separating different concentrations of staphylococcus aureus enterotoxin B by SDS-PAGE gel electrophoresis, transferring to PVDF membrane, then sealing 2 h by skimmed milk, washing 5 times by TBST, incubating PVDF membrane with fusion protein SEB27-vHRP for 2 h at 37 ℃, washing 5 times by TBST, and developing and photographing.

In this example, as shown in fig. 5, as the concentration of staphylococcus aureus enterotoxin B increases, the band gray value on the PVDF membrane gradually increases, and in the immunoblotting detection, the incubation of the second antibody is not required, thus saving the experimental time and the reagent consumption.

Claims

1. A fusion protein SEB27-vHRP, comprising an IgG antibody signal peptide, a hemagglutinin tag, an antibody SEB27, a linker peptide, horseradish peroxidase, and a histidine tag.

2. The fusion protein SEB27-v hrp of claim 1 wherein the nucleotide sequence of the fusion protein is as follows: 5'-atggagaccgacaccttactgctgtgggtgctgctgttatgggtgcccggtagcaccggcgactacccatacgatgttccagattacgctctgcaggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggatacaccttcgatacgaacaccatggcctggtttcgccaggctccagggaaggagcgcgagggggtcgcaacttactatactggtgctggtggtcgtgctttatactatgccgacttcgtgaagggccgattcgccatctcccaagacaacgccaagaacacggtgtatctgcaaatggacagcctgaaacctgaggacactgccatgtactactgtgcgtcaacagtcccccgccgggcgatatctggtgcccctaaacccagtgactttgattattggggccaggggacccaggtcaccgtctcctcagcggccgccagtagtagtggcagtggtatgcagctgacccccacattctacgataactcttgtcccaacgtgtccaacatcgttcgtgacatcatcgtgaatgagctgaggagcgaccctaggatcgccgccagcattctgaggctgcacttccacgactgcttcgtgaatggctgcgacgcctccattttactggataacaccaccagctttcgtaccgaaaaggacgctttcggcaacgccaacagcgctcgtggcttcagcgtgatcgatcgtatgaaagccgccgtggaaagcgcttgtcccggcacagtgtcttgtgccgatttattaaccattgccgcccagcagtccgtgacactggccggcggacctagctggagagtgcctctgggtcgtagggattctttacaagcctttttagatttagccaacgccaatttacccgcccctttcttcactttacctcagctcaaggacagcttcagaaacgtgggtttaaataggagcagcgatttagtggctttatccggcggccacacatttggcaagagccagtgtcgtttcattatggatcgtctgtacaatttcagcaacaccggtttacccgaccccacactgaacaccacctatctccagactttaagaggtttatgccctttaaacggcaatctgtccgctttagtggatttcgacttacgtacccccacaatcttcgacaacaaatactacgtgaatttagaggagcagaagggtttaatccagagcgaccaagaactgttttccagccccgatgccaccgacaccatccctctggtgaggagcttcgccaactccacccagaccttcttcaacgcctttgtggaggccatggatcgtatgggcaacattacccctctgaccggcacccaaggtcagattcgtaggaactgtagggtggtgaatagcaacagcgatttacatcatcaccaccatcaccactaa-3'.

3. A method for preparing the fusion protein SEB27-v hrp according to claim 1, which comprises the following steps: connecting a gene fragment of an antibody SEB27 to an expression vector pCMV-N1-HRP, then converting the recombinant vector into escherichia coli, selecting single colony for sequencing after culturing, selecting clones of a sequencing result for culturing, extracting plasmids, then transfecting the plasmids into mammalian cells, and collecting a cell culture solution after culturing, wherein the cell culture solution contains a fusion protein SEB27-vHRP;

the nucleotide sequence of the expression vector pCMV-N1-HRP is shown as sequence ID number in a nucleotide or amino acid sequence table.

4. Use of the fusion protein SEB27-v hrp according to claim 1 for detecting staphylococcus aureus enterotoxin B in food.

5. The use according to claim 4, wherein the detection of staphylococcus aureus enterotoxin B in the food product is performed by enzyme-linked immunosorbent assay.

6. The use according to claim 4, wherein the detection of staphylococcus aureus enterotoxin B in the food product is performed by a protein immunoprinting method.

7. An antibody SEB27, which is characterized in that the nucleotide sequence of the antibody is shown as sequence ID number 1 in a nucleotide or amino acid sequence table.

8. A method for preparing the antibody SEB27 according to claim 7, wherein the method specifically comprises the following steps:

step one, obtaining camel-derived lymphocyte RNA;

step two, amplification of VHH gene fragment:

step 2.1, reverse transcription PCR:

taking camel-derived lymphocyte RNA obtained in the first step as a template, and performing reverse transcription PCR to synthesize cDNA;

step 2.2, first round PCR amplification of nested PCR was performed:

using the cDNA synthesized by reverse transcription in the step 2.1 as a template, and adopting a primer CALL001 and a primer CALL002 to carry out a first round of PCR amplification, identifying and recovering PCR products;

the sequence of the primer CALL001 is as follows: 5'-gtcctggctgctcttctacaagg-3';

the primer CALL002 has the sequence: 5'-ggtacgtgctgttgaactgttcc-3';

step 2.3, performing a second round of PCR amplification of nested PCR:

taking the recovered PCR product in the step 2.2 as a template, and adopting a primer VHH-FOR and a primer VHH-REV to carry out a second round of PCR amplification, and identifying and recovering the PCR product, wherein the PCR product is the VHH gene fragment;

the sequence of the primer VHH-FOR is as follows: 5 '-caggtgcagctgcaggagtctggggagr-3';

the sequence of the primer VHH-REV is as follows: 5'-ctagtgcggccgctgaggagacggtgacctgggt-3';

step three, constructing and identifying a nano antibody library:

performing enzyme digestion reaction on the phage display vector and the VHH gene fragment obtained in the step 2.3, recovering enzyme digestion products, connecting the phage display vector with the enzyme digestion products of the VHH gene fragment, and recovering connection products; transferring the connection product into competent cells by adopting electrotransformation, and obtaining nano antibody library bacterial liquid after screening and culturing; screening and culturing the nano antibody library bacterial liquid again and extracting phage to obtain a camel-derived single-domain heavy chain antibody library;

step four, affinity panning and identification of the nanobody: