CN116023486A - Staphylococcus aureus alpha-hemolysin nano antibody, preparation method and application thereof - Google Patents

Abstract

The invention provides a staphylococcus aureus alpha-hemolysin nano antibody, a preparation method and application thereof, wherein the antibody is named as staphylococcus aureus alpha-hemolysin nano antibody HLA17. The preparation method of the antibody comprises the steps of firstly panning a nano antibody capable of specifically binding with target molecule alpha-hemolysin in a camel-derived immune nano antibody library, and then obtaining staphylococcus aureus alpha-hemolysin nano antibody HLA17 by means of phage amplification or genetic engineering recombinant expression. The staphylococcus aureus alpha-hemolysin nano antibody disclosed by the invention can specifically identify alpha-hemolysin, and has wider application range than a conventional monoclonal antibody, and has stronger specificity of identifying alpha-hemolysin.

Description

Staphylococcus aureus alpha-hemolysin nano antibody, preparation method and application thereof

Technical Field

The invention belongs to the technical field of food detection, relates to alpha-hemolysin immunological detection, and in particular relates to a staphylococcus aureus alpha-hemolysin nano-antibody, a preparation method and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus) is a common food-borne pathogenic bacterium that can cause a variety of infections and diseases in humans and animals, including skin infection, pneumonia, endocarditis, toxic shock syndrome, sepsis, and the like. Staphylococcus aureus can express a variety of toxins to disrupt host homeostasis, including enterotoxins, haemolysins, toxic shock syndrome toxin 1, leukocidal agents, etc., wherein haemolysins are the most important causative agents of staphylococcus aureus infection as exotoxins of staphylococcus aureus, can damage erythrocytes, platelets, and destroy lysosomes causing ischemia and necrosis. According to the research, the alpha hemolysin is the one with the strongest pathogenic effect and the most widely studied hemolysin with the most definite and clear mechanism.

Alpha-hemolysin is a small beta-barrel pore-forming toxin, toxic to many mammalian cells, secreted as a water-soluble monomer (33.2 kDa), and assembled on the host cell membrane to form a transmembrane oligomeric heptamer (232.4 kDa) channel that allows molecules with a molecular weight less than 2kDa to pass through, e.g., potassium ions, sodium ions, thereby causing host cell lysis or death. Hla has been shown to be the primary virulence factor used by Staphylococcus aureus to evade the host immune system or antibiotics, and is therefore particularly important for the detection of alpha-hemolysin in tissue samples and food samples.

The ELISA method is a brand new ELISA method proposed in the ELISA assay for IgG quantitative determination in 1971 by Engvall and Perlmann. After antigen and antibody are combined on the surface of a solid phase carrier, adding a marking enzyme (mainly comprising horseradish peroxidase and alkaline phosphatase) to form a still active conjugate, then catalyzing a substrate to generate a color reaction, and enabling the displayed color shade to have a linear relation with the corresponding antigen and antibody, thereby realizing an analysis method for detecting a sample. The method not only has the specificity of antigen-antibody, but also retains the high sensitivity of enzyme-linked amplification reaction, thus being a basic immunization method and being widely applied.

At present, staphylococcus aureus alpha-hemolysin antibodies prepared by research at home and abroad are all polyclonal antibodies or monoclonal antibodies for recognizing surface antigens of the staphylococcus aureus alpha-hemolysin antibodies, and the preparation of the traditional monoclonal antibodies is time-consuming, labor-consuming and low in yield, so that the method is an important problem in immunological detection of alpha-hemolysin.

In 1993, hamers-Casterman et al first found an antibody naturally deleted of the light chain from serum of camelids such as dromedaries, bactreeIma, llama, etc., called a heavy chain antibody, which lacks the heavy chain constant region CH1 except for the light chain portion, and the antigen binding site consisted of only the heavy chain variable region, and this single domain antibody consisting of only the heavy chain variable region was the smallest natural intact antigen binding fragment, named nanobody, with a molecular weight of about 15 kDa. Nanobodies have the same domains as human immunoglobulin chain variable regions: 4 conserved sequence regions (framework regions-FR 1/2/3/4) surround 3 highly denatured antigen-binding regions (complementarity determining regions-CDR 1/2/3). CDR3 is the main site of action for nanobody antigen recognition and specificity, with an average length of 18 amino acids, more than the number of amino acids of CDR3 in mice and humans.

Compared with monoclonal antibodies and polyclonal antibodies, the nano-antibodies have the following advantages:

first, the structure is simple, the molecular weight is small, the size is small (2×2×4 nm), and the tissue can be penetrated rapidly, including blood brain barrier, inflammation site, tumor tissue, etc. Second, the affinity is high, and the antigen epitope which is inaccessible to the conventional antibody is recognized. Thirdly, the solubility is good, the stability is high, the extreme environment is extremely resistant, and the highly stable conformation can be still maintained. Fourth, it is easy to express in a variety of host systems, can be relatively low cost, and is easy to mass produce.

From the above description, nanobodies have great development prospects for improving the sensitivity and specificity of immunological detection of alpha-hemolysin, but no report on nanobodies of staphylococcus aureus alpha-hemolysin is currently seen.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide an alpha-hemolysin nano-antibody of staphylococcus aureus, a preparation method and application thereof, and solves the technical problem that the sensitivity and the specificity of the immunological detection of the alpha-hemolysin in the prior art are to be further improved.

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

a staphylococcus aureus alpha-hemolysin nano-antibody, which is characterized in that the antibody is named as staphylococcus aureus alpha-hemolysin nano-antibody HLA17; the nucleotide sequence of the staphylococcus aureus alpha-hemolysin nano antibody HLA17 is as follows:

5’-GAGTCTGGGGGAGGCTCGGTCCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTCAGTCTCTGGATAT AGCGTCTATAACACCTGCATGGGCTGGTACCGCCAGGGTACAGGCCAGAAGCGCGACGAAGTCGCTATTATTGATAGTGATGGTAGCACATACTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGACGACAATTCTTTGAATCTGGAAATGGACAGCCTGAAACCTGACGACACTGGGGTGTACTACTGTGGGTTAGAACGAGGCCCGTGTAGCTTCCGTGGCGAGTATGACGGTTCTCGCCGCTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA-3’。

the invention also has the following technical characteristics:

the invention also provides a preparation method of the staphylococcus aureus alpha-hemolysin nano antibody, which comprises the steps of firstly panning a nano antibody library of camel source immunity to obtain the nano antibody capable of specifically binding with target molecule alpha-hemolysin, and then obtaining the staphylococcus aureus alpha-hemolysin nano antibody HLA17 by means of phage amplification or genetic engineering recombinant expression.

The method specifically comprises the following steps:

step one, obtaining camel source lymphocyte RNA.

Step two, amplification of VHH gene fragment:

step 2.1, reverse transcription PCR:

and (3) performing reverse transcription PCR (polymerase chain reaction) to synthesize cDNA (complementary deoxyribonucleic acid) by taking the camel lymphocyte RNA obtained in the step one as a template.

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’-CAGGTGCAGCTGCAGGAGTCTGGGGGAGR-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 screening, culturing and extracting phage again to obtain the camel-derived single-domain heavy chain antibody library.

Step four, affinity panning and identification of the nanobody:

and (3) adopting alpha-hemolysin to perform multi-round panning on the camel source single domain heavy chain antibody library obtained in the step (III) to obtain positive clones, and then performing sequencing identification on plasmids extracted from the positive clones.

Step five, preparation of large amounts of HLA17 nanobody:

transferring the HLA17 cloned plasmid obtained in the step four into a host cell for protein expression, and culturing and extracting to obtain staphylococcus aureus alpha-hemolysin nano antibody HLA17; or the phage displayed with the positive nano antibody is infected with host cells, and after culturing and extracting, phage amplification liquid containing staphylococcus aureus alpha-hemolysin nano antibody HLA17 is obtained.

The invention also protects the application of the staphylococcus aureus alpha-hemolysin nano-antibody in the staphylococcus aureus alpha-hemolysin immunological detection.

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

the staphylococcus aureus alpha-hemolysin nano-antibody disclosed by the invention can specifically recognize alpha-hemolysin, and has wider application range than a conventional monoclonal antibody, and has stronger specificity of recognizing the alpha-hemolysin.

The staphylococcus aureus alpha-hemolysin nano antibody has small relative molecular mass and strong stability, can be produced in a large scale by adopting a prokaryotic expression system, has low cost and high yield, and has wide application prospect.

The following examples illustrate the invention in further detail.

Drawings

FIG. 1 is a direct ELISA assay result for panning positive clones; in the figure, the ordinate represents the OD450 value, the abscissa represents the number of clones, the shorter square column on the left side of each number represents the negative control (i.e., phosphate buffer), and the longer square column on the right side represents the bacterial liquid of the positive clone.

FIG. 2 shows the result of SDS-PAGE electrophoresis gel identification of the target protein.

Fig. 3 is a direct ELISA standard curve established with nanobody HLA17.

FIG. 4 shows the results of a nanobody specificity assay.

Detailed Description

In the invention, the following components are added:

phage amplification refers to the preparation of phage particles displaying the alpha-hemolysin nanobody by means of bioamplification of phage displaying the anti-alpha-hemolysin nanobody.

The way of gene engineering recombinant expression refers to that a gene fragment of the nanobody is connected with an expression vector to construct and obtain a recombinant vector, and then the recombinant vector is transformed into a host cell to carry out protein expression to obtain the nanobody.

HLA refers to human leukocyte antigen.

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, reagents and carriers used in the present invention are those known in the art, for example:

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, 100mg of ampicillin and 20g of glucose; LB/Amp-GLU solid medium was further added with 15g of agar based on 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 100mg, and glucose 20g.

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-Kan liquid culture medium comprises the following components: tryptone 16g, yeast extract 10g, naCl 40g, ampicillin 100mg, kanamycin 50mg.

Phage display vector pMECS, a phage display vector known in the art, was used and stored in the laboratory where the inventors were located.

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.

Nested PCR reactions using the prior art known in the

DNA Polymerase High Fidelity (HiFi) PCR kit, accession number AP131-11, was purchased from Beijing full gold Biotechnology Co., ltd.

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

Examples:

the embodiment provides a preparation method of staphylococcus aureus alpha-hemolysin nanobody HLA17 in embodiment 1, which specifically comprises the following steps:

step one, obtaining camel source lymphocyte RNA:

step 1.1, immunization of Bactrian camels:

the alpha-hemolysin is used as immunogen, and five rounds of immunization are carried out by immunizing adult male Alshan Bactrian camel by subcutaneous multipoint injection. 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 RNase-free water into RNA precipitate, taking a small amount of measured RNA OD260 value and OD260/OD280 ratio after the RNA precipitate is completely dissolved, determining the concentration and quality of total RNA, and preserving the rest solution 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 a30 min 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’-CAGGTGCAGCTGCAGGAGTCTGGGGGAGR-3’。

the sequences of the primers VHH-REV are:

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 into electric rotating cups, wherein the split charging amount of each electric rotating cup is 50 mu L, then setting the parameters of electric rotating instrument equipment to be 180V, 25 mu F,200 omega and 1mm, carrying out electric conversion, placing the thalli subjected to electric conversion in a shaking table at 37 ℃ for expansion culture at 220rpm for 1h, coating the culture on a flat plate containing LB/Amp-GLU solid culture medium, wherein the coating amount of each flat plate is 1mL, and placing the flat plate in a culture box 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 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:

1mL of the nanobody library bacterial liquid obtained in the step 3.2 is taken and added into 100mL of 2 XYT/Amp-GLU culture medium, then a fungus shaking tube is placed in a shaking table at 220rpm, and is cultured to logarithmic phase at 37 ℃, OD600nm is measured, and the bacterial load is calculated. Adding 20MOI 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, centrifuging at 4deg.C for 30min, 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, centrifuging at 4deg.C overnight, incubating to obtain supernatant, and storing the supernatant 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, alpha-hemolysin was diluted with phosphate buffer solution to a final concentration of 50. Mu.g/mL and coated overnight at 4 ℃. The next day, after washing 5 times with 10mM PBST (phosphate buffer solution containing 0.1% Tween-20 (v/v)), phosphate buffer solution containing 5% bovine serum albumin or 5% ovalbumin was added, and the mixture was blocked at 37℃for 1 hour. Then washed 6 times with PBST, and 100. Mu.L of the camel-derived single domain heavy chain antibody library obtained in step 3.3 (titer about 5X 10) was added to each well ¹¹ cfu/mL), incubation at 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 alpha-hemolysin concentration was 25. Mu.g/mL and 12.5. Mu.g/mL, respectively.

Step 4.2, identification of positive phage clones:

48 clones were randomly picked from the plate after the third round of panning in step 4.1.2, crude nanobody extract was prepared, positive clones were determined using indirect ELISA, and clones with OD450 greater than 2-fold of negative control were selected as positive clones.

In this example, 45 positive clones were obtained, and P/N values of the positive clones were designated as HLA1, HLA2, HLA3, HLA4, HLA5, HLA6, HLA7, HLA8, HLA10, HLA11, HLA12, HLA13, HLA14, HLA15, HLA16, HLA17, HLA18, HLA19, HLA21, HLA22, HLA23, HLA24, HLA25, HLA26, HLA27, HLA28, HLA29, HLA30, HLA31, HLA32, HLA33, HLA34, HLA36, HLA37, HLA38, HLA39, HLA40, HLA41, HLA42, HLA43, HLA44, HLA45, HLA46, HLA47, and HLA48, respectively, are shown in fig. 1, and P/N values of the positive clones are the largest as can be seen in fig. 1.

In this example, the plasmid extracted from HLA17 clone was sequenced, and the nucleotide sequence obtained by sequencing was:

5’-GAGTCTGGGGGAGGCTCGGTCCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTCAGTCTCTGGATAT AGCGTCTATAACACCTGCATGGGCTGGTACCGCCAGGGTACAGGCCAGAAGCGCGACGAAGTCGCTATTATTGATAGTGATGGTAGCACATACTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGACGACAATTCTTTGAATCTGGAAATGGACAGCCTGAAACCTGACGACACTGGGGTGTACTACTGTGGGTTAGAACGAGGCCCGTGTAGCTTCCGTGGCGAGTATGACGGTTCTCGCCGCTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA-3’。

the amino acid sequence of the codon table is as follows:

5’-ESGGGSVQAGGSLRLSCSVSGYSVYNTCMGWYRQGTGQKRDEVAIIDSDGSTYYADSVKGRFTISQDN DDNSLNLEMDSLKPDDTGVYYCGLERGPCSFRGEYDGSRRYYGMDYWGKGTQVTVSS-3’。

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

Step five, mass preparation of nano antibodies:

as a specific scheme of the embodiment, the preparation is carried out in a form of recombinant expression of genetic engineering, and the specific process is as follows:

the HLA17 cloned plasmid obtained in step 4.2 was extracted and transferred into E.coli Top 10F. A single colony was picked from the transformation plate and inoculated into 5mL of LB liquid medium containing ampicillin, and cultured overnight at 37℃with shaking at 220 r/min. Inoculating the overnight culture into 300mL of LB liquid medium containing ampicillin at an inoculum size (v/v) of 1%, and performing shaking culture at 37 ℃ and 220 r/min; when the cell concentration OD600 of the culture reached 0.5, 1mM IPTG was added to the culture, followed by shaking culture at 30℃and 220r/min for 8-12 hours. The culture is centrifuged at 8000rpm at 4 ℃ for 20min to collect bacterial precipitate, 5mL of precooled phosphate buffer solution is used for resuspension of bacterial, after ultrasonic crushing for 10min, 8000rpm is used for centrifugation for 20min to obtain supernatant, and the supernatant is subjected to affinity chromatography purification to obtain target protein.

In the embodiment, SDS-PAGE electrophoresis gel is adopted to identify the target protein, and the result is shown in figure 2, the band of the target protein is between 15kDA and 25Kda, and the target protein is proved to be staphylococcus aureus alpha-hemolysin nano antibody HLA17.

As an alternative to this example, phage amplification can also be used for preparation as follows:

phage displaying positive nanobodies were added to 20mL of E.coli TG1 inoculated culture and cultured with shaking at 37℃and 220rpm for 12h. Transferring the culture into another centrifuge tube, centrifuging at 10000rpm for 10min at 4deg.C, transferring the supernatant into a new centrifuge tube, adding 1/6 volume of PEG/NaCl solution, standing at 4deg.C for 4 hr, centrifuging at 10000rpm for 10min at 4deg.C, and discarding the supernatant; and adding a small amount of phosphate buffer solution to clean phage, centrifuging at 10000rpm for 10min at 4 ℃, discarding the supernatant, and adding 1mL of phosphate buffer solution to carry out resuspension to obtain phage amplification liquid containing staphylococcus aureus alpha-hemolysin nano antibody HLA17.

Sensitivity and specificity identification of antibodies:

(A) The invention adopts ELISA method to identify the sensitivity of the prepared staphylococcus aureus alpha-hemolysin nano antibody HLA17, and the specific process of the identification is as follows:

diluting the alpha-hemolysin 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 5 washes with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)), 300. Mu.L of 3% skimmed milk powder was added and blocked at 37℃for 1 hour. 10000ng/mL, 5000ng/mL, 1000ng/mL, 750ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 75ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 10ng/mL, 7.5ng/mL, 5ng/mL, 2.5ng/mL and 1ng/mL of alpha-hemolysin are then added to each well, and after incubation for 1 hour at 37 ℃, 100. Mu.L of 10. Mu.g/mL of staphylococcus aureus alpha-hemolysin nanobody HLA17 is added to each well, and incubation is performed for 1 hour at 37 ℃.

After the incubation, 100. Mu.L of horseradish peroxidase-labeled anti-HA antibody was added at 1:10000 dilution, incubated at 37℃for 1 hour, 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate solution was added, developed for 15min in the absence of light, OD450 was measured, and a standard curve was drawn. As shown in FIG. 3, the linear range of the standard curve is 10-1000 ng/mL, the linear equation is y=0.0035x+0.2551, R ² 0.9955, the lowest detection limit is 7.5ng/mL, and shows better sensitivity.

(B) The invention adopts ELISA method to identify the specificity of the prepared staphylococcus aureus alpha-hemolysin nano antibody HLA17, and the specific process of the identification is as follows:

diluting the alpha-hemolysin rabbit polyclonal antibody to 10 mug/mL with phosphate buffer solution (pH 7.4), adding 100 mug/hole, and coating at 4 ℃ overnight; after washing 3 times with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)), 300. Mu.L of 3% skimmed milk powder was added, after blocking at 37℃for 1 hour, washing 3 times, then 100. Mu.L of culture supernatant of HLA gene knocked out strain was added, incubation was performed at 37℃for 1 hour, 100. Mu.L of 10. Mu.g/mL nanobody HLA17 was added, and incubation was performed at 37℃for 1 hour.

After the incubation, 100. Mu.L of horseradish peroxidase-labeled anti-HA antibody was added at 1:10000 dilution, incubated at 37℃for 1 hour, 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate solution was added, developed for 15min in the absence of light, and OD450 was measured. As shown in fig. 4, HLA17 nanobody did not cross-react with culture supernatant of HLA gene knockout strain, indicating that nanobody HLA17 exhibited better specificity.

And (3) verifying the effect of antibody detection:

the invention adds alpha-hemolysin standard with known concentration into milk sample without alpha-hemolysin to analyze the accuracy of antibody detection, which comprises the following steps: diluting the alpha-hemolysin rabbit polyclonal antibody to 10 mug/mL with phosphate buffer solution (pH 7.4), adding 100 mug/hole, and coating at 4 ℃ overnight; after washing 3 times with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)), 300. Mu.L of 3% skimmed milk powder was added, blocking was performed at 37℃for 1 hour, washing 3 times with PBST (phosphate buffer solution containing 0.05% Tween-20 (v/v)) was further performed, 100. Mu.L of milk containing an alpha-hemolysin concentration was then added, incubation was performed at 37℃for 1 hour, and 100. Mu.L of nanobody HLA17 at 10. Mu.g/mL was added, and incubation was performed at 37℃for 1 hour.

After the incubation, 100. Mu.L of horseradish peroxidase-labeled anti-HA antibody was added at 1:10000 dilution, incubated at 37℃for 1 hour, 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate solution was added, developed for 15min in the absence of light, and OD450 was measured. As shown in Table 1, the final concentrations of 50, 100 and 500ng/mL of alpha-hemolysin standard substances are added into milk, and the detection result is close to the actual addition concentration, which indicates that the staphylococcus aureus alpha-hemolysin nano antibody HLA17 provided by the invention is adopted for carrying out the immunological detection of the staphylococcus aureus alpha-hemolysin, and has good accuracy.

TABLE 6 results of immunological detection of alpha-hemolysin

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Claims (3)

1. A staphylococcus aureus alpha-hemolysin nano-antibody, which is characterized in that the antibody is named as staphylococcus aureus alpha-hemolysin nano-antibody HLA17; the nucleotide sequence of the staphylococcus aureus alpha-hemolysin nano antibody HLA17 is as follows:

5’-GAGTCTGGGGGAGGCTCGGTCCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTCAGTCTCTGGATAT AGCGTCTATAACACCTGCATGGGCTGGTACCGCCAGGGTACAGGCCAGAAGCGCGACGAAGTCGCTATTATTGATAGTGATGGTAGCACATACTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGACGACAATTCTTTGAATCTGGAAATGGACAGCCTGAAACCTGACGACACTGGGGTGTACTACTGTGGGTTAGAACGAGGCCCGTGTAGCTTCCGTGGCGAGTATGACGGTTCTCGCCGCTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA-3’。

2. a method for preparing staphylococcus aureus alpha-hemolysin nano-antibody according to claim 1, which comprises the following steps:

step one, obtaining camel source lymphocyte RNA;

step two, amplification of VHH gene fragment:

step 2.1, reverse transcription PCR:

performing reverse transcription PCR (polymerase chain reaction) to synthesize cDNA (complementary deoxyribonucleic acid) by taking camel-derived lymphocyte RNA obtained in the first step as a template;

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’-CAGGTGCAGCTGCAGGAGTCTGGGGGAGR-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; the nano antibody library bacterial liquid is subjected to screening culture and phage extraction again to obtain a camel-derived single-domain heavy chain antibody library;

step four, affinity panning and identification of the nanobody:

adopting alpha-hemolysin to carry out multi-round panning on the camel source single domain heavy chain antibody library obtained in the step three to obtain positive clones, and then carrying out sequencing identification on plasmids extracted from the positive clones;

step five, preparation of large amounts of HLA17 nanobody:

transferring the HLA17 cloned plasmid obtained in the step four into a host cell for protein expression, and culturing and extracting to obtain staphylococcus aureus alpha-hemolysin nano antibody HLA17; or the phage displayed with the positive nano antibody is infected with host cells, and after culturing and extracting, phage amplification liquid containing staphylococcus aureus alpha-hemolysin nano antibody HLA17 is obtained.

3. Use of the staphylococcus aureus alpha-hemolysin nanobody of claim 1 for the immunological detection of staphylococcus aureus alpha-hemolysin.

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