CN117986359A - Nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A and application thereof - Google Patents

Abstract

The invention discloses a nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A and application thereof, which is obtained by screening a phage display natural nanometer antibody library, wherein the nanometer antibody SEA-4-13 comprises protein or polypeptide with an amino acid sequence shown as SEQ ID NO.1, and the nanometer antibody has high affinity and can be combined with staphylococcus aureus enterotoxin A; the nano antibody provided by the invention can be applied to the fields of immunodetection, antigen enrichment, purification, toxin neutralization and the like; the amino acid sequence provided by the invention can be used as a precursor, and can be modified by random or site-directed mutagenesis technology to obtain mutants with better properties in terms of yield, water solubility, affinity, specificity, stability and the like.

Description

Nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A and application thereof

Technical Field

The invention relates to the technical field of nanobodies, in particular to a nanobody SEA-4-13 for resisting staphylococcus aureus enterotoxin A and application thereof.

Background

Enterotoxins (Staphylococcal enterotoxins, SEs) are a series of structurally related, similarly virulent, antigenically different extracellular proteins produced by staphylococcus aureus, and have a molecular weight of between 22.6 and 28.6KDa and consist of 220 to 240 amino acids. Is named because it causes acute gastroenteritis and gastrointestinal toxic reactions in humans and mammals, with staphylococcus aureus enterotoxin a (Staphylococcal enterotoxinA, SEA) being one of the most common enterotoxins, about 80% of staphylococcal food poisoning events being associated with SEA. Meanwhile, SEA has strong tolerance and has toxicity after high heat treatment and pepsin digestion. Thus, conventional cooking and food processing methods have difficulty in completely inactivating SEA, resulting in the occurrence of food poisoning events caused by SEA.

The heavy chain antibody (HEAVY CHAIN anti body, HCAb) with naturally deleted light chain exists in camelid body, and the variable region (Variable domains of HEAVY CHAIN of HEAVY CHAIN anti body, VHH) is a single domain heavy chain antibody, which is also called nanobody due to its small size. Nanobodies are currently known antigen-binding fragments of minimal molecular weight, with a molecular weight of only 15kDa, approximately one tenth of conventional IgG antibodies.

At present, traditional antibodies such as polyclonal antibodies, monoclonal antibodies and the like for resisting SEA exist, and the traditional antibodies have the defects of complex preparation process, high cost and the like; therefore, there is a need to develop a new solution to improve the problems of conventional antibodies.

Disclosure of Invention

The invention aims to provide a nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A and application thereof, and overcomes the defects of complex preparation process and high cost of the existing polyclonal antibody and monoclonal antibody.

In one aspect, the invention provides a nanobody SEA-4-13 for resisting staphylococcus aureus enterotoxin A, wherein the nanobody comprises an amino acid sequence shown as SEQ ID NO. 1.

Optionally, the nanobody comprises at least one of a monomer, a bivalent antibody, and a multivalent antibody.

In a second aspect, the invention provides a gene encoding nanobody SEA-4-13, the nucleotide sequence of which is shown in SEQ ID NO. 9.

In a third aspect, the invention provides the use of the nanobody-encoding gene in the construction of an expression vector.

In a fourth aspect, the present invention provides a drug conjugate comprising the nanobody, wherein the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

In a fifth aspect, the invention provides the use of the nanobody SEA-4-13 in a pharmaceutical composition.

In a sixth aspect, the invention provides the use of the nanobody SEA-4-13 in immunodetection.

In a seventh aspect, the present invention provides recombinant proteins constructed from the nanobody SEA-4-13.

The beneficial effects of the invention include:

(1) The preparation method of the nano antibody provided by the invention has the advantages of simple flow and low cost;

(2) The nano antibody provided by the invention is derived from phage display natural nano antibody library (AlpSDab-P) of Sichuan apak biotechnology limited company, and has the advantages of large library capacity, strong diversity and the like;

(3) The nano antibody provided by the invention can be specifically combined with SEA, and can be applied to the fields of SEA immunodetection, enrichment and purification and the like;

(4) The amino acid sequence of the nano antibody provided by the invention can be used as a precursor, and can be modified by random or site-directed mutagenesis technology to obtain mutants with better properties (yield, water solubility, affinity, specificity, stability and the like).

Drawings

FIG. 1 shows the results of a phase-ELISA for identifying anti-SEA positive cloned phages in example 1;

FIG. 2 is a schematic diagram of the amino acid sequence and domain of SEA-4-13 nanobody prepared in example 2;

FIG. 3 is a SDS-PAGE identification of the anti-SEA nanobody SEA-4-13 prepared in example 2;

FIG. 4 is a western blot identification chart of the anti-SEA nanobody SEA-4-13 prepared in example 2;

FIG. 5 shows the results of a specific assay for the anti-SEA nanobody SEA-4-13.

Detailed Description

The invention will be further illustrated by the following examples in conjunction with the accompanying drawings.

In one aspect, the embodiment of the invention provides a screening method of a nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A.

In a second aspect, an embodiment of the invention provides a nanobody SEA-4-13 against staphylococcus aureus enterotoxin A, wherein the nanobody comprises an amino acid sequence shown as SEQ ID NO. 1.

In some embodiments, nanobody SEA-4-13 comprises four Framework Regions (FR) and three complementarity determining regions (Complementarity-DETERMINING REGION, CDRs); the amino acid sequences of the framework regions (FR 1-FR 4) are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8; the amino acid sequences of the complementarity determining regions (CDR 1-CDR 3) are shown as SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7; the CDR region is mainly responsible for SEA recognition, the FR region structure is relatively stable, and the structure of the nanobody is mainly maintained.

In some embodiments, the nanobody comprises at least one of a monomer, a bivalent antibody, a multivalent antibody.

In some embodiments, the nucleic acid molecule of the nanobody comprises DNA, RNA, cDNA.

In some embodiments, the amino acid sequence can be used as a precursor, and modified by random or site-directed mutagenesis techniques to obtain mutants with better properties in terms of yield, water solubility, affinity, specificity, stability, etc.

In a third aspect, embodiments of the present invention provide a gene encoding nanobody SEA-4-13, the nucleotide sequence of which is shown in SEQ ID NO. 9.

In a fourth aspect, the present embodiment provides an application of the gene encoding the nanobody in constructing an expression vector.

In some embodiments, the nucleic acid sequences are 85-90% similar due to degeneracy of the genetic code.

In a fifth aspect, embodiments of the present invention provide a drug conjugate comprising the nanobody, where the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

In a sixth aspect, embodiments of the present invention provide the use of the nanobody SEA-4-13 in a pharmaceutical composition.

In a seventh aspect, embodiments of the present invention provide recombinant proteins constructed from the nanobody SEA-4-13.

In some embodiments, the recombinant protein is expressed by a host cell, including E.coli, yeast cells, mammalian cells.

In particular, the host cell comprises an expression vector or a nucleic acid sequence encoding a SEA-4-13 nanobody integrated into the host cell genome.

In particular, the host cell is capable of expressing the nanobody and a polypeptide comprising the amino acid sequence of the nanobody.

In an eighth aspect, embodiments of the present invention provide an application of the nanobody SEA-4-13 in immunodetection.

In some embodiments, the application includes enzyme-linked immunosorbent assay, immunochromatography, immuno-chip, SEA immunodetection, enrichment purification.

In particular, the invention can be used for providing the ability of protein or polypeptide containing the amino acid sequence of the nanometer antibody SEA-4-13 to pair and identify SEA in SEA immunodetection, and establishing a detection method for SEA.

In the embodiment of the invention, the culture mediums are all commercially available, and the LB liquid culture medium is from Beijing Soy Bao technology Co., ltd; the self-induction medium was from apaker biotechnology limited, sichuan.

Example 1

The embodiment 1 of the invention provides a panning method of a nano antibody, which comprises the following steps:

S1, diluting SEA antigen to 10 mug/mL by phosphate buffer solution with pH=8.6, adding 100 mug LSEA antigen into a 96-well plate, and incubating for 12 hours at 4 ℃; after incubation was completed, 96-well plates were washed 3 times with PBST solution;

S2, after washing, adding 300 mu L of 3% protein blocking solution into each hole, wherein the protein blocking solution is formed by alternately using 3% BSA and 3% OVA, and incubating for 2 hours at 37 ℃; after incubation was completed, 96-well plates were washed 5 times with PBST solution;

S3, after washing, adding 100 mu L of phage display natural nano antibody library into each hole, and incubating for 1h at 37 ℃; after incubation was completed, 96-well plates were washed 10 times with PBST solution;

s4, after washing, adding 100 mu L of Gly-HCl eluent with pH value of 2.2 into each hole, and culturing for 8min on a horizontal shaking table;

S5, after the culture is completed, adding 10 mu L of neutralization buffer Tris-HCl with pH=9.0 into 100 mu L of eluent in each hole to neutralize to obtain 110 mu L of neutralization solution;

S6, taking 10 mu L of neutralizing solution and measuring phage titer on a flat plate; the remaining 100 μl of neutralization solution was used to infect ER2738 amplified phage and the next round of screening was performed;

The panning conditions for each round were progressively harsher to cause high affinity nanobody to be obtained by panning, four rounds of panning were performed together, and the panning conditions and experimental parameters for each round are shown in table 1.

TABLE 1 panning conditions and experimental parameters for anti-SEA nanobodies

Example 2

The embodiment 2 of the invention provides a self-induction mode expression anti-SEA nanobody SEA-4-31, which comprises the following steps:

D1, converting phage vector pcomb3xss-C4bp alpha-SEA-4-31 into chemically competent cells E.coli Rosetta at 42 ℃ for 90s by a heat shock method to obtain bacterial liquid I;

D2, adding 50 mu L of bacterial liquid and 5 mu L of ampicillin (Amp) into 5mL of LB liquid culture medium, and carrying out shake culture for 12h at 37 ℃ and 220rpm under the final concentration of Amp of 100mg/mL to obtain bacterial liquid II;

After the culture is completed, inoculating the second bacterial liquid into 100mL of self-induction culture medium with an inoculum size of 1% (v/v), adding 100 mu L of Amp, and carrying out shaking culture at 37 ℃ and 250rpm for 3.5 hours until the logarithmic phase (OD ₆₀₀ reaches 0.5-0.7) to obtain a first culture;

D4, placing the first culture at 25 ℃ and shaking culture at 180rpm for 12 hours to induce protein expression to obtain a second culture;

D5, centrifuging the second culture at 12000rpm/min and 4 ℃ for 20min; re-suspending the precipitate obtained by centrifugation through 20mL of PBS buffer solution to obtain a bacterial body weight suspension;

Adding 20mg of lyase into the heavy suspension, and performing enzymolysis for 30min at 25 ℃ to obtain an enzymolysis solution;

D7, adding 15 mu L of PMSF into the enzymolysis liquid, wherein the final concentration of the PMSF is 1mmol/L, crushing for 30min at 4 ℃ by a cell ultrasonic crusher at 300W, and stopping crushing when the solution is observed to become clear and transparent from turbidity during crushing, so as to obtain a crushed product;

d8, centrifuging the crushed product at 10000rpm/min and 4 ℃ for 20min, and collecting supernatant; filtering the supernatant with a 0.22 μm water-based filter membrane, removing impurities, and purifying;

The purification comprises the following substeps:

D81, adding 2mL of Ni-NTA column material into the purification column, washing with distilled water with 5-10 times of column volume after Ni-NTA naturally subsides, removing impurities, and balancing the purification column by using 15mL of balancing buffer solution;

d82, adding the supernatant to the purification column in batches, slowly flowing out, and collecting the flow-through liquid of each time;

D83, equilibrate the purification column with 15mL equilibration buffer (containing 50mM imidazole), rinse the purification column of the hybrid protein;

d84, after washing, eluting the target protein bound in the purification column by using 5mL of elution buffer (containing 300mM imidazole), and collecting target protein liquid to store in a centrifuge tube in 1.5 mL;

D85, transferring the collected target protein solution into a 3kDa dialysis bag, dialyzing by PBS (phosphate buffer solution) with the concentration of 10mmol/L and the pH value of 7.4 at the temperature of 4 ℃, and replacing the dialysate every 8 hours for 1D;

D86, after the dialysis is finished, taking protein liquid, and measuring the protein concentration by using Nano Drop 2000; after the measurement, the sample was packed in a 1.5mL centrifuge tube and stored in a freezer at-20 ℃.

Property detection

1. Positive clone identification of nanobodies

Positive clones were identified by phage-ELISA comprising the steps of:

in example 1, at rounds 3 and 4 of panning, 48 clones were randomly selected for phage amplification on each plate for phage titer determination;

experimental group: diluting the SEA antigen concentration to 0.5 mug/mL by using PBS buffer solution, adding the SEA antigen concentration into a 96-well plate, adding 100 mug of SEA antigen into each well, and coating at 4 ℃ for 12 hours;

Blank group: the BSA concentration was diluted to 10. Mu.g/mL using PBS buffer, 100. Mu.L of each well was added and coated for 12h at 4 ℃; after coating was completed, washing 3 times with PBST solution; the PBST solution is PBS buffer solution, 0.5% Tween-20 is added, and the final concentration of PBS in the PBST solution is 10mmol/L;

after washing, 300. Mu.L of 3% BSA was added to each well and blocked at 37℃for 2 hours; after the sealing is completed, PBST is added for washing 3 times;

100 mu L of phage amplification solution is added to each well of a 96-well plate of an experimental group and a control group, and incubated for 1h at 37 ℃;

After the incubation is completed, 100 mu L of diluted HRP-M13 secondary antibody is added to each well, and the mixture is incubated for 1h at 37 ℃; after the incubation is completed, adding PBST solution for washing for 4 times;

After the washing is finished, 100 mu L of TMB color developing solution is added into each hole, and the color development reaction is carried out for 10min at 37 ℃ in a dark place;

After the color development reaction is finished, 50 mu L of 2mol/L H ₂SO₄ stop solution is added into each hole;

Measuring an absorption value at 450nm by using an enzyme label instrument; selecting experimental phage clones with OD ₄₅₀ being 10 times greater than that of a control as positive clones, sequencing 44 positive clones as shown in figure 1 to obtain 7 different amino acid sequences; selecting the nanometer antibody SEA-4-13 with better performance for gene sequencing and subsequent identification;

The amino acid sequence of the nanometer antibody SEA-4-13 is shown as SEQ ID NO.1, comprises a framework region (FR 1-4) and a complementarity determining region (CDR 1-3), and the amino acid sequence IMGT number and the structural domain schematic diagram are shown as figure 2;

The amino acid sequence of the SEA-4-13 framework region (FR 1-FR 4) of the nano antibody is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8; the amino acid sequences of the complementarity determining regions (CDR 1-CDR 3) are shown as SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7; the CDR region is mainly responsible for SEA recognition, the FR region structure is relatively stable, and the structure of the nano antibody is mainly maintained;

the nucleotide sequence of the nanometer antibody SEA-4-13 is shown as SEQ ID NO. 9.

2. Identification of nanobody SEA-4-13

Identification was performed by polypropylene gel electrophoresis (SDS-PAGE) and immunoblotting (Western blot),

SDS-PAGE results are shown in FIG. 3; lane M is Maker, lane 1 is nanobody obtained in example 2;

western blot results are shown in FIG. 4; lane M is Maker, lane 1 is nanobody obtained in example 2;

The results in FIGS. 3 and 4 show that SEA-4-13 nanobody was successfully expressed and purified, and the nanobody was of higher purity, and the molecular weight of the nanobody was close to that predicted by software, about 16kDa.

3. Specific analysis of the anti-SEA nanobody SEA-4-13

The specificity of the nanobody to enterotoxin SEA, SEB, SEC, SED and SEE was determined by phase-ELISA, comprising the steps of:

Diluting SEA antigen, SEB antigen, SEC antigen, SED antigen and SEE antigen to a concentration of 0.5 mug/mL by using PBS buffer solution, respectively adding 100 mug/well into a 96-well plate, and coating for 12 hours at 4 ℃;

After coating, adding PBST solution to wash for 3 times; the PBST solution is PBS buffer solution, 0.5% Tween-20 is added, and the final concentration of PBS in the PBST solution is 10mmol/L;

after washing, 300. Mu.L of 3% BSA was added to each well and blocked at 37℃for 2 hours; after the sealing is completed, PBST is added for washing 3 times;

after washing, respectively adding SEA-4-13 phage amplification solution into each antigen, and incubating at 37 ℃ for 1h with 100 mu L/hole;

after the incubation is completed, 100 mu L of diluted HRP-M13 secondary antibody is added to each well, and the mixture is incubated for 1h at 37 ℃; after incubation was completed, washed 4 times with PBST;

After the washing is finished, 100 mu L of TMB color developing solution is added into each hole, and the color development reaction is carried out for 10min at 37 ℃ in a dark place;

After the color reaction is completed, 50 mu L of 2mol/L H ₂SO₄ stop solution is added into each hole;

Measuring an absorption value at 450nm by using an enzyme label instrument; the binding results of each enterotoxin to nanobody SEA-4-13 are shown in FIG. 5; the nanometer antibody SEA-4-13 has higher affinity with SEA, has weak combination with SEE, and has no obvious combination with other enterotoxins measured, thus indicating that the nanometer antibody provided by the invention has better specificity.

While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Claims (8)

1. The nanometer antibody SEA-4-13 for resisting staphylococcus aureus enterotoxin A is characterized by comprising an amino acid sequence shown as SEQ ID NO. 1.

2. The nanobody of claim 1, wherein the nanobody comprises at least one of a monomer, a bivalent antibody, a multivalent antibody.

3. The gene for encoding the nanobody SEA-4-13 as claimed in claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 9.

4. Use of the gene according to claim 3 for constructing an expression vector.

5. The drug conjugate of nanobody composition of claim 1, wherein the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

6. Use of the nanobody SEA-4-13 according to any of claims 1-2 in a pharmaceutical composition.

7. Use of the nanobody SEA-4-13 according to any of claims 1-2 in an immunoassay.

8. The recombinant protein constructed by nanobody SEA-4-13 according to any of claims 1-2.