Paired nanoantibodies for detecting Staphylococcus aureus enterotoxin A and their applications

Abstract

The present invention discloses a paired nanobody for detecting Staphylococcus aureus enterotoxin A and its application, wherein the paired nanobody comprises nanobody SEA-4-03 and nanobody SEA-4-05, wherein the amino acid sequence of the nanobody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of the nanobody SEA-4-05 is shown in SEQ ID NO: 2. The paired nanobody of the present invention can avoid SpA interference when detecting SEA, and can be used in the fields of immunoassay, drug development, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to paired nano antibodies for detecting Staphylococcus aureus enterotoxin A and applications thereof. Background Art

[0002] Staphylococcus aureus is one of the most common pathogens that can cause severe staphylococcal food poisoning (SFP). Its pathogenicity mainly depends on staphylococcal enterotoxins (SEs). These SEs are toxic proteins with similar structures. Among them, staphylococcal enterotoxin A (SEA) is the most common because of its strongest toxicity. Staphylococcal enterotoxin A has excellent thermal stability and resistance to proteolytic digestion. Conventional food processing processes are difficult to destroy the toxicity of staphylococcal enterotoxin A. Therefore, the development of sensitive and rapid detection methods for staphylococcal enterotoxin A is crucial for food safety, clinical diagnosis and environmental monitoring.

[0003] Currently, various methods for detecting Staphylococcal enterotoxin in different samples (food, water, and even body fluids) have been reported. These include enzyme-linked immunosorbent assay (ELISA), test strips, electrochemical analysis, and biosensors. In fact, ELISA based on antigen-antibody recognition is currently the most widely used method for rapid screening of Staphylococcal enterotoxin A due to its high throughput, simple operation, and high degree of automation. However, Staphylococcal protein A (SpA) is a surface protein commonly found in the cell wall of Staphylococcus aureus. It can strongly and nonspecifically bind to the crystallizable fragment (Fc) region of immunoglobulins of common mammals (pigs, guinea pigs, mice, dogs, monkeys, etc.), resulting in false positive results in immunoassays based on IgG antibodies to detect Staphylococcal enterotoxin A.

[0004] Single-domain heavy chain antibodies, also known as nanobodies, are a type of variable region derived from the naturally missing light chain in camelids, namely the heavy chain antibody. Its molecular weight is only one-tenth of that of traditional IgG antibodies (about 15kDa). In recent years, nanobodies have gained wide attention in the fields of life science research, drug development, medical diagnosis, food safety, and environmental monitoring, mainly because compared with traditional IgG antibodies, they have the following advantages: small molecular weight, high affinity, strong stability, good solubility, strong tissue penetration, and the ability to recognize hidden antigen epitopes. Nanobodies have advantages in various performance aspects and can provide another attractive immunoreagent option to solve the interference problem of Staphylococcal protein A (SpA) in Staphylococcal enterotoxin A (SEA) immunoassay. Summary of the invention

[0005] The purpose of the present invention is to provide a paired nanobody and application for detecting Staphylococcus aureus enterotoxin A. The paired nanobody obtained by screening can avoid SpA interference when detecting SEA, providing a powerful tool for reducing the harm of SEA.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A paired nanobody for detecting Staphylococcus aureus enterotoxin A, comprising nanobody SEA-4-03 and nanobody SEA-4-05, wherein the amino acid sequence of nanobody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of nanobody SEA-4-05 is shown in SEQ ID NO: 2.

[0008] Furthermore, the nanobody SEA-4-03 includes at least one of a monomer, a bivalent antibody, and a multivalent antibody; the nanobody SEA-4-05 includes at least one of a monomer, a bivalent antibody, and a multivalent antibody.

[0009] A derivative product comprising the paired Nanobodies, wherein the derivative product is selected from any one of the following A, B, C and D:

[0010] A: a nucleic acid molecule encoding the paired nanobody;

[0011] B: A recombinant vector, comprising a nucleic acid molecule encoding the paired nanobody, or the recombinant vector expresses the paired nanobody;

[0012] C: A host cell, in which the paired Nanobody is expressed, or a nucleic acid molecule encoding the paired Nanobody or a recombinant vector as described in B is contained in the host cell;

[0013] D: A pharmaceutical composition comprising the paired nanoantibodies, or at least one of A, B, and C.

[0014] Furthermore, the pharmaceutical composition also contains one or more pharmaceutically acceptable carriers.

[0015] The present invention also provides a recombinant protein constructed by the paired nano-antibodies.

[0016] The present invention also provides the use of the paired nano-antibodies in the preparation of Staphylococcus aureus enterotoxin A inhibitory drugs.

[0017] The present invention also provides the use of the paired nano-antibodies in the preparation of Staphylococcus aureus enterotoxin A immunological detection reagent.

[0018] The present invention also provides the use of the paired nano-antibodies in the preparation of a Staphylococcus aureus enterotoxin A immunoassay kit.

[0019] The present invention also provides a Staphylococcus aureus enterotoxin A immunoassay kit, wherein the kit carries the paired nano-antibodies.

[0020] The present invention also provides a method for preparing a paired nanobody for detecting Staphylococcus aureus enterotoxin A, comprising the following steps:

[0021] Step 1: Screening of Nanobodies:

[0022] The SEA antigen was diluted with phosphate buffer, added to a 96-well plate, incubated at 37°C for 1 h, and then washed with PBST solution;

[0023] After washing, 3% protein blocking solution was added and incubated at 37°C for 2 h, followed by washing with PBST solution;

[0024] After washing, the phage-displayed natural nanobody library was added and incubated at 37°C, followed by washing with PBST solution;

[0025] After washing, Gly-HCl eluent at pH = 2.2 was added, reacted on a horizontal shaker, and then Tris-HCl neutralization buffer at pH = 9.0 was added to obtain a neutralized phage solution;

[0026] The neutralized phage solution was sampled to measure the titer, and the remaining neutralized phage solution was used to infect ER2738 to amplify the phage and perform the next round of screening;

[0027] Step 2: Identification of anti-SEA nanobodies by phage-ELISA:

[0028] In the fourth round of screening, 16 clones were randomly selected for phage amplification on the plate where the eluted phage titer was determined;

[0029] The SEA antigen and BSA were diluted to 0.5 μg / mL using PBS buffer, added to a 96-well plate at 100 μL / well, and coated at 4°C for 12 h;

[0030] Wash three times with PBST solution, add 3% BSA to a 96-well plate, 300 μL / well, and block at 37°C for 2 h;

[0031] Wash three times with PBST solution, add 100 μL of phage amplification solution to each well, and incubate at 37°C for 1 h;

[0032] Wash three times with PBST solution, add 100 μL of diluted HRP-M13 secondary antibody to each well, and incubate at 37°C for 1 h;

[0033] Wash three times with PBST solution, add 100 μL TMB colorimetric solution to each well, react at 37°C in the dark for 10 min, and after the reaction, add 50 μL 2 mol / L H2SO4 stop solution to each well;

[0034] The absorbance value at 450 nm was measured using an ELISA instrument; the phage clones in the experimental group whose OD450 was 10 times greater than that of the control were selected as positive clones, and the positive clones were sequenced to obtain a total of 2 different amino acid sequences, which were named SEA-4-03 or SEA-4-05 respectively; the amino acid sequence of the nanoantibody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of the nanoantibody SEA-4-05 is shown in SEQ ID NO: 2.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The preparation method of the paired nanobodies provided by the present invention has a simple process and low cost;

[0037] 2. The paired nanoantibodies provided by the present invention can sandwich bind SEA and can be applied to the development of double antibody sandwich immunoassays for detecting SEA, antigen enrichment and purification, and neutralizing drug development;

[0038] 3. The amino acid sequence of the paired nanoantibodies provided by the present invention can be used as a precursor and modified by random or site-directed mutagenesis technology to obtain mutants with better properties (yield, water solubility, affinity, specificity, stability, etc.).

[0039] 4. The paired nanoantibodies provided by the present invention can avoid the interference of SpA when constructing an immunoassay method for detecting SEA. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of phage-ELISA identification of anti-SEA positive clone phage in Example 1;

[0041] Figure 2 This is the polyacrylamide gel electrophoresis identification result of the nanobody SEA-4-03;

[0042] Figure 3 This is the immunoblotting identification result of nanobody SEA-4-03;

[0043] Figure 4 The specificity analysis results of nanoantibodies SEA-4-03 and SEA-4-05;

[0044] Figure 5 This is the SEA curve of the nanobody SEA-4-03 phage double antibody sandwich detection;

[0045] Figure 6 This is the SEA curve detected by nanoantibody SEA-4-05 phage double antibody sandwich. DETAILED DESCRIPTION

[0046] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0047] In the first aspect, the present invention provides a paired nanobody for detecting Staphylococcus aureus enterotoxin A, including nanobody SEA-4-03 and nanobody SEA-4-05, wherein the amino acid sequence of the nanobody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of the nanobody SEA-4-05 is shown in SEQ ID NO: 2. In some embodiments, the nanobody SEA-4-03 includes at least one of a monomer, a bivalent antibody, and a multivalent antibody; the nanobody SEA-4-05 includes at least one of a monomer, a bivalent antibody, and a multivalent antibody. The amino acid sequence can be used as a precursor and modified by random or site-directed mutagenesis technology to obtain mutants with better properties in terms of yield, water solubility, affinity, specificity, stability, etc.

[0048] In a second aspect, the present invention provides a recombinant protein constructed by the paired nanobodies (nanobody SEA-4-03 and nanobody SEA-4-05). In some embodiments, the recombinant protein is expressed by a host cell, and the host cell includes Escherichia coli, yeast cells, mammalian cells, etc.

[0049] Specifically, the host cell contains an expression vector or a nucleic acid sequence encoding the SEA-4-03 or SEA-4-05 nanobody is integrated into the host cell genome.

[0050] In a third aspect, the present invention provides the application of the nano-antibodies to SEA-4-03 and SEA-4-05 in the fields of immunoassay and drug development.

[0051] In some embodiments, the applications include enzyme-linked immunosorbent assay, immunochromatography, immunochip, SEA immunoassay, and antigen enrichment and purification.

[0052] Specifically, the ability of the present invention to pair proteins or polypeptides containing the amino acid sequence of nanobody SEA-4-03 or nanobody SEA-4-05 and recognize SEA can be utilized in SEA immunoassay to establish a detection method for SEA.

[0053] In a fourth aspect, the present invention provides a derivative product comprising the paired Nanobodies, wherein the derivative product is selected from any one of the following A, B, C and D:

[0054] A: a nucleic acid molecule encoding the paired nanobody;

[0055] B: A recombinant vector, comprising a nucleic acid molecule encoding the paired nanobody, or the recombinant vector expresses the paired nanobody;

[0056] C: A host cell, in which the paired Nanobody is expressed, or a nucleic acid molecule encoding the paired Nanobody or a recombinant vector as described in B is contained in the host cell;

[0057] D: A pharmaceutical composition, comprising the paired Nanobodies, or at least one of A, B, and C. The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0058] In a fifth aspect, the present invention also provides a method for preparing a paired nanobody for detecting Staphylococcus aureus enterotoxin A, comprising the following steps:

[0059] Step 1: Screening of Nanobodies:

[0060] The SEA antigen was diluted with phosphate buffer, added to a 96-well plate, incubated at 37°C for 1 h, and then washed with PBST solution;

[0061] After washing, 3% protein blocking solution was added and incubated at 37°C for 2 h, followed by washing with PBST solution;

[0062] After washing, the phage-displayed natural nanobody library was added and incubated at 37°C, followed by washing with PBST solution;

[0063] After washing, Gly-HCl eluent at pH = 2.2 was added, reacted on a horizontal shaker, and then Tris-HCl neutralization buffer at pH = 9.0 was added to obtain a neutralized phage solution;

[0064] The neutralized phage solution was sampled to measure the titer, and the remaining neutralized phage solution was used to infect ER2738 to amplify the phage and perform the next round of screening;

[0065] Step 2: Identification of anti-SEA nanobodies by phage-ELISA:

[0066] In the fourth round of screening, 16 clones were randomly selected for phage amplification on the plate where the eluted phage titer was determined;

[0067] The SEA antigen and BSA were diluted to 0.5 μg / mL using PBS buffer, added to a 96-well plate at 100 μL / well, and coated at 4°C for 12 h;

[0068] Wash three times with PBST solution, add 3% BSA to a 96-well plate, 300 μL / well, and block at 37°C for 2 h;

[0069] Wash three times with PBST solution, add 100 μL of phage amplification solution to each well, and incubate at 37°C for 1 h;

[0070] Wash three times with PBST solution, add 100 μL of diluted HRP-M13 secondary antibody to each well, and incubate at 37°C for 1 h;

[0071] Wash three times with PBST solution, add 100 μL TMB colorimetric solution to each well, react at 37°C in the dark for 10 min, and after the reaction, add 50 μL 2 mol / L H2SO4 stop solution to each well;

[0072] The absorbance value at 450 nm was measured using an ELISA instrument; the phage clones in the experimental group whose OD450 was 10 times greater than that of the control were selected as positive clones, and the positive clones were sequenced to obtain a total of 2 different amino acid sequences, which were named SEA-4-03 or SEA-4-05 respectively; the amino acid sequence of the nanoantibody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of the nanoantibody SEA-4-05 is shown in SEQ ID NO: 2.

[0073] Example 1

[0074] The screening of nanobodies includes the following steps:

[0075] S1. Dilute the SEA antigen to 10 μg / mL with phosphate buffer at pH 8.6, add 100 μL of SEA antigen to a 96-well plate, incubate at 4°C for 12 h; after incubation, wash three times with PBST solution;

[0076] S2. After washing, add 300 μL of 3% protein blocking solution (all percentages in this application are by mass) to each well. The protein blocking solution is 3% BSA (bovine serum albumin) and 3% OVA alternately. Incubate at 37°C for 2 hours. After incubation, wash 5 times with PBST solution. 3% BSA means that 100 ml of solution contains 3 grams of BSA; 3% OVA means that 100 ml of solution contains 3 grams of OVA.

[0077] S3. After washing, add 100 μL of phage-displayed natural nanoantibody library (the phage-displayed natural nanoantibody library (AlpSDab-P) of Sichuan Apak Biotechnology Co., Ltd.) to each well, incubate at 37°C for 1 hour; after incubation, wash 10 times with PBST solution;

[0078] S4. After washing, add 100 μL of Gly-HCl elution solution (pH=2.2) to each well and react on a horizontal shaker for 8 min.

[0079] S5. After the reaction is completed, 10 μL of the eluate in each well is added with 10 μL of a neutralization buffer solution (pH = 9.0) Tris-HCl to obtain 110 μL of a neutralization solution;

[0080] S6, take 10 μL of the neutralization solution to measure the phage titer on the plate; the remaining 100 μL of the neutralization solution is used to infect ER2738 to amplify the phage and perform the next round of screening;

[0081] The screening conditions of each round were gradually stringent to enable the screening of high-affinity nanoantibodies. A total of four rounds of screening were performed. The screening conditions and experimental parameters of each round are shown in Table 1.

[0082] Table 1 Screening conditions and parameters of anti-SEA nanobodies

[0083]

[0084] Example 2

[0085] The anti-SEA nanobody was identified by phage-ELISA, comprising the following steps:

[0086] S1. During the fourth round of screening in Example 1, 16 clones were randomly selected on the plate for measuring the titer of the eluted phage for phage amplification;

[0087] S2. Dilute the SEA antigen and BSA to 0.5 μg / mL using PBS buffer, add 100 μL / well to each well of a 96-well plate, and coat at 4°C for 12 h.

[0088] S3, wash three times with PBST (0.5% vol Tween-20) solution, add 3% BSA to the 96-well plate, 300 μL / well, and block at 37°C for 2 h;

[0089] S4, wash three times with PBST (0.5% vol Tween-20) solution, add 100 μL phage amplification solution to each well, and incubate at 37°C for 1 h;

[0090] S5. Wash three times with PBST (0.5% vol Tween-20) solution, add 100 μL diluted HRP-M13 secondary antibody to each well, and incubate at 37°C for 1 h.

[0091] S6. Wash three times with PBST (0.5% vol Tween-20) solution, add 100 μL TMB colorimetric solution to each well, and perform colorimetric reaction at 37°C in the dark for 10 min. After the colorimetric reaction is completed, add 50 μL 2 mol / L H2SO4 stop solution to each well.

[0092] S7. Use an ELISA reader to measure the absorbance at 450nm; select the experimental group phage clones with OD450 greater than 10 times the control as positive clones, a total of 6 strains, such as Figure 1 As shown, 6 positive clones were sequenced and 2 different amino acid sequences were obtained, which were named SEA-4-03 or SEA-4-05 respectively; the amino acid sequence of nanobody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of nanobody SEA-4-05 is shown in SEQ ID NO: 2.

[0093] Example 3

[0094] Identification of Nanobody SEA-4-03:

[0095] The nanobody SEA-4-03 was identified by SDS-PAGE and Western blot.

[0096] The results of polyacrylamide gel electrophoresis were as follows Figure 2 As shown; Lane M is Maker, and Lane 1 is the Nanobody SEA-4-03 obtained in Example 2;

[0097] The results of immunoblotting were as follows Figure 3 As shown; Lane M is Maker, and Lane 1 is the Nanobody SEA-4-03 obtained in Example 2;

[0098] Figure 2 , Figure 3 The results in show that the SEA-4-03 nanobody was successfully expressed and purified, and the nanobody had a high purity.

[0099] Example 4

[0100] Specificity analysis of anti-SEA nanobody SEA-4-03 and nanobody SEA-4-05:

[0101] The specificity of the nanobody to the enterotoxin SEA antigen, SEB antigen, SEC antigen, SED antigen and SEE antigen was determined by phage-ELISA, comprising the following steps:

[0102] S1. Dilute SEA antigen, SEB antigen, SEC antigen, SED antigen, and SEE antigen to a concentration of 0.5 μg / mL using PBS buffer, add 100 μL per well to a 96-well plate, and coat at 4°C for 12 h.

[0103] S2, wash three times with PBST (0.5 vol% Tween-20) solution, add 3% BSA, 300 μL / well, and block at 37°C for 2 h;

[0104] S3, wash three times with PBST solution, add SEA-4-03 or SEA-4-05 phage amplification solution to each antigen, 100 μL / well, and incubate at 37°C for 1 h;

[0105] S4, wash three times with PBST solution, add diluted HRP-M13 secondary antibody, 100 μL / well, incubate at 37°C for 1 h;

[0106] S5, wash three times with PBST solution, add TMB colorimetric solution, 100 μL / well, colorimetric reaction at 37°C in the dark for 10 min, add 50 μL 2 mol / L H2SO4 stop solution to each well;

[0107] S6. Measure the absorbance at 450nm using an ELISA instrument. The results of the binding of each enterotoxin with SEA-4-03 and SEA-4-05 phage amplification solutions are shown in the figure. Figure 4 As shown, Nanobody SEA-4-03 and Nanobody SEA-4-05 have a higher affinity with SEA and bind to SEE, while having no obvious binding to other measured enterotoxins, indicating that the Nanobody provided by the present invention has good specificity.

[0108] Example 5

[0109] The pairing binding ability of nanoantibodies SEA-4-03 and SEA-4-05 to SEA was analyzed by double antibody sandwich phage-ELISA:

[0110] S1. Dilute the expressed nanobody SEA-4-03 protein to a concentration of 0.5 μg / mL using PBS buffer, add 100 μL per well to a 96-well plate, and coat at 4°C for 12 h.

[0111] S2, wash three times with PBST (0.5% vol Tween-20) solution, add 3% BSA, 300 μL / well, and block at 37°C for 2 h;

[0112] S3, wash three times with PBST (0.5% vol Tween-20) solution, add different concentrations of SEA standard (1000, 750, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 ng / mL), 100 μL / well, incubate at 37°C for 2 h;

[0113] S4. Wash the wells three times with PBST (0.5% vol Tween-20) solution and add 100 μL phage amplification solution (1×10 9 pfu / mL), incubated at 37°C for 1 h;

[0114] S5. Wash three times with PBST (0.5% vol Tween-20) solution, add 100 μL diluted HRP-M13 secondary antibody to each well, and incubate at 37°C for 1 h.

[0115] S6. Wash three times with PBST (0.5% vol Tween-20) solution, add 100 μL TMB colorimetric solution to each well, and colorimetric reaction at 37°C in the dark for 10 min. After the colorimetric reaction is completed, add 50 μL 2 mol / L H2SO4 stop solution to each well.

[0116] S7, the absorbance at 450nm is measured by ELISA; the test results are as follows Figure 5 As the concentration of SEA increases, the absorbance gradually increases, reaching a plateau when the SEA concentration is 500 ng / mL, and the absorbance value no longer increases, indicating that the maximum binding capacity of the nanoantibody SEA-4-03 has been reached. Therefore, the absorbance values ​​of SEA concentrations of 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 ng / mL were selected to draw the detection curve. The results are shown in the figure. Figure 6 As shown, within this range, the fitting curve is y=0.0051x (R 2 =0.992), indicating that there is a good linear relationship between SEA concentration and absorbance.

[0117] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A paired nanobody for detecting Staphylococcus aureus enterotoxin A, characterized in that: The antibody is nanobody SEA-4-03 or nanobody SEA-4-05, the amino acid sequence of nanobody SEA-4-03 is shown in SEQ ID NO: 1, and the amino acid sequence of nanobody SEA-4-05 is shown in SEQ ID NO:

2.

2. The paired Nanobody according to claim 1, characterized in that The nanobody SEA-4-03 is a monomer; the nanobody SEA-4-05 is a monomer.

3. A derivative product comprising the paired nanobody according to claim 1, characterized in that: The derivative product is selected from any one of the following A, B, C and D: A: A nucleic acid molecule encoding the paired nanobody according to claim 1; B: A recombinant vector, comprising a nucleic acid molecule encoding the paired nanobody according to claim 1, or the recombinant vector expresses the paired nanobody according to claim 1; C: A host cell expressing the paired Nanobody according to claim 1, or containing a nucleic acid molecule encoding the paired Nanobody according to claim 1 or a recombinant vector according to B; D: A pharmaceutical composition comprising the paired nanoantibody according to claim 1, or at least one of A, B, and C.

4. A recombinant protein constructed by the paired nanoantibody as claimed in claim 1.

5. Use of the paired nanobody as claimed in claim 1 in the preparation of an immunological detection reagent for Staphylococcus aureus enterotoxin A.

6. Use of the paired nanoantibodies as claimed in claim 1 in the preparation of a Staphylococcus aureus enterotoxin A immunoassay kit.

7. A Staphylococcus aureus enterotoxin A immunoassay kit, characterized in that: The kit carries the paired nanobody according to claim 1.

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