CN113087807B - Shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigen and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine and antigen detection, and particularly relates to a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens and a preparation method thereof. The invention comprises the following steps: a. prokaryotic expression and purification of the recombinant protein of the Shiga toxin B subunit; b. preparing a short peptide containing a biotin group; c. the recombinant protein and the biotinylation short peptide are subjected to in vitro enzyme fusion modification; d. preparing a coupling probe of biotinylation Shiga toxin B subunit recombinant protein and streptavidin marked quantum dots. The probe can effectively detect the expression of various tumor cell surface carbohydrate antigens, such as Gb3 and Gb4, in combination with flow cytometry, and can be applied to detection and diagnosis by taking the carbohydrate antigens such as Gb3 and Gb4 as tumor markers. The carbohydrate antigen detection method has the advantages of low preparation cost, strong detection specificity and high sensitivity.

Description

Probe and preparation method based on Shiga toxin B subunit recombinant protein for detecting carbohydrate antigen

technical field

The invention belongs to the technical field of biomedicine/antigen detection, and in particular relates to a Shiga toxin B subunit recombinant protein-based probe and a preparation method for detecting carbohydrate antigens.

Background technique

Carbohydrate antigen is a tumor marker. Changes in glycosylation level and structure are common features of tumor cells, so some carbohydrate antigens can be used as markers for tumor detection, diagnosis and treatment. Carbohydrate antigens are not unique to a specific type of tumor, and different carbohydrate antigens are specific in different malignant tumors, and their application as a combined marker can further improve the accuracy of malignant tumor diagnosis. Common tumor-associated carbohydrate antigens include Gb3, Gb4, Globo-H, GM2, GD2, GD3, Fuc-GM1, LeY, SSEA, forssman, etc.

Glycosphingolipid Gb ₃ is a neutral carbohydrate antigen with a structure of Gal1-4Gal1-4Glc-Cer. Glycosphingolipid Gb ₃ widely exists on the surface of mammalian cell membrane and plays a very important role in cell growth, differentiation and signal transduction. The abnormal expression of glycosphingolipid Gb ₃ can cause a series of pathological reactions in the body. The current research results show that the overexpression of glycosphingolipid Gb ₃ is related to the metastasis of colon cancer, and it is found that almost all cancer cell metastasis is accompanied by changes in cell surface glycosylation.

The current detection of Gb ₃ is mainly by western blotting experiments using commercial anti-Gb ₃ antibodies. Its application is limited due to the long experimental period and the expensive commercialization of antibodies.

Shiga toxin is a virulence protein factor produced by Shigella and some Escherichia coli, which can specifically bind to the Gb ₃ carbohydrate antigen expressed by human host cells. Shiga toxin is a complex composed of A subunit protein and B subunit protein, A subunit protein has virulence activity, B subunit has Gb ₃ carbohydrate antigen binding activity. Shiga toxins include Shiga toxin 1 and Shiga toxin 2. Shiga toxin B subunit proteins include Shiga toxin 1B subunit and Shiga toxin 2B subunit.

The function of the Shiga toxin B subunit is to specifically bind the Gb ₃ carbohydrate antigen. Shiga toxin B subunit does not have the toxic activity of Shiga toxin when it exists alone, but maintains the binding property of Shiga toxin to Gb ₃ carbohydrate antigen, so the recombinant Shiga toxin B subunit protein can be recognized as Gb ₃ The unit develops detection probes for Gb ₃ carbohydrate antigens in tumor cells.

However, there is no relevant report on the effective use of Shiga toxin B subunit to prepare the detection probe of Gb ₃ .

SUMMARY OF THE INVENTION

Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a probe and a preparation method based on Shiga toxin B subunit recombinant protein for detecting carbohydrate antigens,

Technical scheme: In order to realize the above-mentioned purpose, the technical scheme adopted in the present invention is:

The invention is a probe based on the recombinant protein of Shiga toxin B subunit for detecting carbohydrate antigens. First, the recombinant protein of Shiga toxin B subunit is exogenously expressed, and then the recombinant protein of Shiga toxin B subunit is combined with biotinylated short protein. The peptide was subjected to in vitro enzymatic fusion modification to obtain a biotinylated Shiga toxin B subunit recombinant protein. Finally, the biotinylated Shiga toxin B subunit recombinant protein was coupled with streptavidin-labeled quantum dots to form a probe.

Said exogenous expression is included in prokaryotic E. coli BL21 (DE3).

An object of the present invention is to provide a probe for detecting carbohydrate antigens based on Shiga toxin B subunit recombinant protein, the probe is formed by coupling the following two parts:

a. Biotinylated Shiga toxin B subunit recombinant protein,

b. Streptavidin-labeled quantum dots;

Wherein, the biotinylated Shiga toxin B subunit recombinant protein is obtained by in vitro enzymatic fusion modification of the following components:

c. Shiga toxin B subunit recombinant protein,

d. Biotinylated short peptides;

Wherein, the Shiga toxin B subunit recombinant protein is obtained from Shiga toxin B subunit protein by modifying the carboxyl terminus, and the carboxyl terminus modification is: modifying the Myc tag on the carboxyl terminus of the Shiga toxin B subunit protein, Transpeptidase A recognition site, His tag sequence;

The Shiga toxin B subunit protein includes Shiga toxin 1B subunit protein and Shiga toxin 2B subunit protein;

The biotinylated short peptide is a short peptide containing a biotin group.

Further, the amino acid sequence of the Shiga toxin 2B subunit recombinant protein is shown in SEQ ID No.2, and the nucleotide sequence encoding the Shiga toxin 2B subunit recombinant protein is shown in SEQ ID No.3; The amino acid sequence of the Shiga toxin 1B subunit recombinant protein is shown in SEQ ID No.6, and the nucleotide sequence encoding the Shiga toxin 1B subunit recombinant protein is shown in SEQ ID No.7.

Further, the biotinylated short peptide includes a short peptide and at least one biotin molecule, wherein, the short peptide is a polypeptide containing a glycine tag required for the enzymatic fusion, and can also be any short peptide sequence performed on the short peptide sequence. A polypeptide having the same function by changing and increasing or decreasing it, preferably, the amino acid structure composition of one of the short peptides selected in the embodiment of the present invention is shown in SEQ ID No.4. The biotin molecule includes any biotin analog that can bind to streptavidin, such as 2-iminobiotin.

Further, the method for the in vitro enzymatic fusion modification is to use a pH7.5 PBS buffer containing Tris, _NaCl , and CaCl as an enzyme reaction system, and add Shiga toxin B subunit recombinant protein, Biotinylated peptides and transpeptidase A were shaken at 4-25°C for 1-5 hours to obtain the biotinylated Shiga toxin B subunit recombinant protein.

Further, the enzyme reaction system comprises pH6.0-8.0PB buffer of 50mM Tris, 150mM NaCl, 5mM CaCl ₂ ; the concentration of the Shiga toxin B subunit recombinant protein is 10-50 μM, and the biological The concentration of the short peptide is 300-400 μM, and the concentration of the transpeptidase A is 2-5 μM.

Further, the preparation method of the probe is as follows: the biotinylated Shiga toxin B subunit recombinant protein and the conjugated probe of streptavidin-labeled quantum dots are mixed in a pH6.0-8.0 PBS buffer, 4 Protect from light at -25°C, and shake the reaction for 30min-2h.

Further, the concentration of the biotinylated Shiga toxin B subunit recombinant protein is 1 μM; the concentration of the streptavidin-labeled quantum dots is 5-50 nM, and the oscillation speed is 10-200 rpm.

Another object of the present invention is to provide a method for preparing a probe based on Shiga toxin B subunit recombinant protein for detecting carbohydrate antigens, comprising the following steps:

1) Prokaryotic expression and purification of Shiga toxin B subunit recombinant protein;

2) Preparation of short biotin peptides: solid-phase synthesis of short biotin peptides based on Fmoc method;

3) In vitro enzymatic fusion modification of the Shiga toxin B subunit recombinant protein and biotinylated short peptide to obtain a biotinylated Shiga toxin B subunit recombinant protein;

4) The biotinylated Shiga toxin B subunit recombinant protein is coupled with streptavidin-labeled quantum dots to prepare the probe.

Another object of the present invention is to provide an application of a probe based on the recombinant protein of Shiga toxin B subunit for detecting carbohydrate antigens, for the micro-detection of carbohydrate antigens in cells, wherein the carbohydrate antigens include Gb3, Gb4; the micro-detection of carbohydrate antigens in cells refers to the detection of tumor markers of carbohydrate antigens in cells.

Further, the probe was incubated with the cells to be tested at 4-37°C, and after centrifugation with PBS, the fluorescence was detected by flow cytometry, the excitation wavelength was ultraviolet or blue light, and the emission spectrum was 605nm±5nm; The concentration of the probe is 5-50 nM, and the incubation time is 30 min-2 h.

Beneficial effects: Compared with the prior art, the Shiga toxin B subunit recombinant protein-coupled probe and preparation method provided by the present invention have the following advantages: the present invention has successfully constructed a Shiga toxin-based Detection of carbohydrate antigens such as Gb4, Globo-H, GM2, GD2, GD3, Fuc-GM1, LeY, SSEA, forssman, etc., especially the detection method of Gb ₃ . The method has the advantages of low cost, simple method, short experimental period and high detection sensitivity, which lays a foundation for the detection of relevant indicators in the fields of biology, medicine, and food.

Description of drawings

Figure 1 is a schematic diagram of the technical route.

Fig. 2 is the detection chart of key parameters in the purification process of Shiga toxin 2B subunit recombinant protein;

Figure 3 is an SDS-PAGE graph; lane M is the standard protein sample, lane 1 is the purified Shiga toxin 2B subunit recombinant protein Stx2B-His.

Figure 4 is an SDS-PAGE graph; lane M is the standard protein sample, lane 1 is the purified Shiga toxin 2B subunit recombinant protein Stx2B-His, and lane 2 is the purified biotinylated Shiga toxin 2B subunit Recombinant protein Stx2B-biotin.

Figure 5 is a flow cytometry analysis of the ability of the quantum dot-modified Shiga toxin 2B subunit recombinant protein to detect the carbohydrate antigen Gb ₃ on the surface of K562 cells (human myeloid leukemia cells) . In the experiment, PBS was used as the blank control NC, and two concentration gradients of 250 nM and 500 nM were set. A is the flow shift diagram, and B is the histogram of the mean fluorescence intensity (n=3, p<0.05).

Figure 6 is a flow cytometry analysis of the ability of the quantum dot-modified Shiga toxin 2B subunit recombinant protein to detect the carbohydrate antigen Gb ₃ on the surface of Raji cells (human lymphoma cells) . In the experiment, PBS was used as a blank control, and two concentration gradients of 250 nM and 500 nM were set. A is the flow shift diagram, and B is the histogram of the mean fluorescence intensity (n=3, p<0.05).

Figure 7 is a flow cytometry analysis of the ability of the quantum dot-modified Shiga toxin 2B subunit recombinant protein to detect the carbohydrate antigen Gb ₃ on the surface of Caco-2 cells (human cloned colon adenocarcinoma cells) . In the experiment, PBS was used as a blank control, and two concentration gradients of 250 nM and 500 nM were set. A is the flow shift diagram, and B is the histogram of the mean fluorescence intensity (n=3, p<0.05).

Figure 8 is a flow cytometry analysis of the ability of quantum dot-modified Shiga toxin 2B subunit recombinant protein to detect carbohydrate antigen Gb ₃ on the surface of HT-29 cells (human colorectal adenocarcinoma cells) . In the experiment, PBS was used as a blank control, and two concentration gradients of 250 nM and 500 nM were set. A is the flow shift diagram, and B is the histogram of the mean fluorescence intensity (n=3, p<0.05).

Detailed ways

The invention belongs to the technical field of biomedicine and antigen detection, and in particular relates to a probe based on Shiga toxin B subunit recombinant protein for detecting carbohydrate antigens, and a preparation method. As shown in FIG. 1 , the preparation of the probe of the present invention includes steps :

a. Prokaryotic expression and purification of Shiga toxin B subunit recombinant protein;

b. Preparation of short peptides containing biotin groups: solid-phase synthesis of biotin short peptides based on Fmoc method;

c. In vitro enzymatic fusion modification of the above recombinant protein and biotinylated short peptide;

d. Preparation of conjugated probes of biotinylated Shiga toxin B subunit recombinant protein and streptavidin-labeled quantum dots.

Wherein, the method for preparing the above-mentioned Shiga toxin B subunit recombinant protein, the steps are as follows:

1) Design the target gene sequence, add MYC tag, transpeptidase A (Sortase A) recognition site and His tag to its carboxyl terminus.

2) using Escherichia coli to express the target gene fragment;

3) using nickel column purification to obtain the target protein;

4) linking the target protein with a short peptide with a biotin group under the catalysis of transpeptidase A;

5) The target protein with biotinylation is obtained by nickel column purification.

The probe of the present invention, combined with flow cytometry, can effectively detect the expression of various tumor cell surface carbohydrate antigens, especially Gb3, and can be applied to the detection and diagnosis of Gb3 and other carbohydrate antigens as tumor markers. The carbohydrate antigen detection method has the advantages of low preparation cost, strong detection specificity and high sensitivity.

The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood, however, that these examples do not limit the scope of the invention.

Example

The embodiment uses Shiga toxin 2B subunit recombinant protein and Gb3 as examples.

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims. .

Terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 Design of the recombinant protein sequence of Shiga toxin 2B subunit

Find the protein sequence of Shiga toxin 2B subunit (GenBank ID: AAD25446.1, SEQ ID No. 1), and modify its carboxyl terminus. First, a MYC tag (EQKLISEEDLNGAA) and a recognition site LPETGG (L: leucine; P: proline; E: glutamic acid) for cleavage by transpeptidase A were added to the carboxyl terminus of the Shiga toxin 2B subunit protein. ; T: threonine; G: glycine); then add histidine purification tag His, namely HHHHHH (H: histidine), the recombinant protein is named Stx2B-His, and its amino acid sequence is as SEQ ID No.2 shown.

After the protein sequence is designed, the protein sequence is handed over to a commercial company for codon optimization. The optimized gene sequence (SEQ ID No. 3) was inserted into the pET22b(+) plasmid, and the plasmid was transformed into E. coli BL21 (DE3). This strain is a fermentation strain containing the target gene.

Example 2 Expression and purification of Shiga toxin 2B subunit recombinant protein

Take 20 μL of glycerol bacteria containing recombinant protein Stx2B-His to 3 mL of ampicillin-resistant LB medium (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L), 37 ℃, 200rpm overnight culture, the next day according to 4% of the inoculum was transferred to TB fermentation medium (peptone 12g/L, yeast powder 24g/L, glycerol 4g/L, KH ₂ PO ₄ 23.1g/L, K ₂ HPO ₄ 125.4g/L). After reaching an OD ₆₀₀ of 0.6-1.2, the inducer IPTG with a final concentration of 0.4 mM was added for induction. The conditions of protein expression were 20 °C, 200 rpm, and the expression time was 24 h. Exogenous expression can be performed in prokaryotic E. coli BL21 (DE3).

After fermentation, the cells were collected by centrifugation at 9000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in wall-breaking buffer (10 mM Tris, 500 mM NaCl, pH 8.0), and then disrupted with an ultrasonic crusher. The crushing conditions were: ice bath, running for 2 s, stopping for 3 s, and running for 20 min. After the fragmentation was completed, centrifugation at 9000 rpm for 10 min was repeated three times to completely remove cell debris to obtain a clear fragmented supernatant. The broken supernatant was incubated with the nickel column for 2-4 h at 4°C, and then the nickel column was washed with 25mM imidium to remove impurity proteins. The recombinant protein Stx2B-His was washed down with a gradient of wall breaking buffer containing 25mM-500mM imidazole. The purified protein was desalted, lyophilized, and kept at -20°C for later use.

Figure 2 is a diagram showing the detection of key parameters in the process of recombinant protein purification. Figure 3 is the SDS-PAGE image of the purified recombinant protein. The grayscale analysis of Image J software shows that the purity of the purified Stx2B-His is above 95%.

Example 3 Biotinylation modification and purification of Shiga toxin 2B subunit recombinant protein

The enzyme reaction system was 50 mM Tris, 150 mM NaCl, 5 mM CaCl ₂ buffer, pH 7.5 PBS buffer, the concentration of Shiga toxin 2B subunit recombinant protein substrate was 20 μM, the concentration of biotin short peptide was 400 μM, and the final concentration of transpeptidase A was The concentration was 5 μM, wherein the short peptide used for the biotin short peptide was a polypeptide containing a glycine tag required for the enzymatic fusion. The reaction conditions were 16°C, 200rpm, 2h. After the reaction was completed, the resulting protein was purified using a nickel column. Rinse the nickel column with 1x PBS buffer and collect the flow-through. The flow-through protein was desalted, lyophilized, and placed at -20°C for use. The protein was named Stx2B-biotin.

The analysis of the SDS-PAGE results in Figure 4 shows that the molecular weight of Stx2B-biotin is significantly changed compared with that of Stx2B-His, and the purity of the purified Stx2B-biotin is above 95%.

Example 4 Biotinylated Shiga toxin 2B subunit recombinant protein modified by conjugated probe of streptavidin-labeled quantum dots

The quantum dot modification system was 1x PBS solution, pH 7.0; the concentration of Stx2B-biotin was 1 μM, and the concentration of streptavidin-labeled quantum dots was 20 nM. The reaction conditions were room temperature, protected from light, 50 rpm, and protected from light for 2 h. After the reaction is completed, it is directly used in subsequent experiments without purification; the product of this step can also be packaged. Store at 2-8°C.

Example 5 Quantum dot-labeled Shiga toxin 2B subunit recombinant protein is used for the detection of cell surface carbohydrate antigen Gb3

The cells to be tested in the logarithmic growth phase were inoculated into 6-well plates and cultured for 24 hours; after resuspending, the cells in each well were collected in a single flow tube, centrifuged to remove the supernatant, and the cells were washed twice with PBS, and 100 μL of the above-mentioned cells were added to each tube. The prepared quantum dot-modified recombinant type II Shiga toxin 2B subunit constitutes a probe with a concentration of 20 nM; incubate at 4°C for 30 min; remove the supernatant by centrifugation, wash the cells twice with PBS, and centrifuge for 5 min; add 200 μL to each tube Cells were resuspended in PBS and detected on the machine.

The analysis of Figure 5, Figure 6, Figure 7 and Figure 8 shows that for the negative cell group K562 cells, there is no obvious shift and average fluorescence intensity difference; for the positive cell group Raji, Caco-2, HT-29 cells, compared with the PBS control, The displacement and the average fluorescence intensity have obvious changes, and the concentration gradient can be seen, indicating that this embodiment can achieve the above purpose. At the same time, due to the excellent luminescence properties of quantum dots, the background is low, and the emission spectrum is narrow and symmetrical.

The above example shows that the recombinant protein of Shiga toxin 2B subunit can specifically bind to the glycosphingolipid Gb ₃ receptor on the surface of the cell membrane; at the same time, combined with flow cytometry, the cell screening can be completed within 1 hour at the fastest, saving a lot of money time required for the experiment. Similarly, this example is also applicable to Shiga toxin 1B subunit protein which has the same function as Shiga toxin 2B subunit protein to prepare a coupled probe based on Shiga toxin 1B subunit recombinant protein. Similarly, this embodiment is also applicable to other carbohydrate antigens on the cell membrane, such as Gb4 which is similar in structure and function, and other carbohydrate antigens such as Globo-H, GM2, GD2, GD3, Fuc-GM1, LeY, SSEA , Forssman et al.

Example 6 Conjugated probe of streptavidin-labeled quantum dots Modified biotinylated Shiga toxin 1B subunit recombinant protein

The design of the recombinant protein sequence of Shiga toxin 1B subunit, the expression and purification of the recombinant protein of Shiga toxin 1B subunit, and the biotinylation modification and purification of the recombinant protein of Shiga toxin 1B subunit, the same as in Examples 1, 2, and 3, wherein , the amino acid sequence of Shiga toxin 1B subunit protein is shown in SEQ ID No. 5, the amino acid sequence of Shiga toxin 1B subunit recombinant protein is shown in SEQ ID No. 6, and the protein encoding the Shiga toxin 1B subunit recombinant protein is shown in SEQ ID No. 6. The nucleotide sequence is shown in SEQ ID No.7; the short peptide is also shown in SEQ ID No.4.

The quantum dot modification method is the same as that in Example 4, and the obtained probe is used for the detection of cell surface carbohydrate antigens such as Gb3 and Gb4.

The above is only the preferred embodiment of the present invention, it should be pointed out that: for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (7)

1. A shiga toxin B subunit recombinant protein-based probe for detection of carbohydrate antigens, characterized in that: the carbohydrate antigen is Gb3, and the probe is formed by coupling the following two parts:

a. biotinylation Shiga toxin B subunit recombinant protein,

b. labeling quantum dots with streptavidin;

the biotinylation Shiga toxin B subunit recombinant protein is obtained by performing in-vitro enzymatic fusion modification on the following components:

c. a recombinant protein of a subunit B of the shiga toxin,

d. biotinylation of the short peptide;

wherein the Shiga toxin B subunit recombinant protein is obtained by modifying Shiga toxin B subunit protein through a carboxyl terminal, and the carboxyl terminal is modified by: modifying Myc tag, transpeptidase A recognition site and His tag sequence at the carboxyl terminal of the Shiga toxin B subunit protein;

the Shiga toxin B subunit protein is Shiga toxin 1B subunit protein or Shiga toxin 2B subunit protein;

the biotinylation short peptide is a short peptide containing a biotin group, and the amino acid sequence of the short peptide is shown as SEQ ID No. 4;

the method for in vitro enzyme method fusion modification comprises the following steps of₂The PBS buffer solution with the pH value of 7.5 is an enzyme reaction system, Shiga toxin B subunit recombinant protein, biotin short peptide and transpeptidase A are added into the enzyme reaction system, and the biotinylation Shiga toxin B subunit recombinant protein is obtained after oscillation reaction for 1 to 5 hours at the temperature of 4 to 25 ℃;

the amino acid sequence of the Shiga toxin 2B subunit recombinant protein is shown as SEQ ID No.2, and the amino acid sequence of the Shiga toxin 1B subunit recombinant protein is shown as SEQ ID No. 6.

2. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: the biotinylation short peptide comprises a short peptide and at least one biotin molecule, wherein the short peptide is a polypeptide containing a glycine tag, and the biotin molecule is any biotin analogue capable of being combined with streptavidin.

3. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: the enzyme reaction system comprises 50mM Tris, 150mM NaCl and 5mM CaCl₂pH6.0-8.0PBS buffer.

4. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: the concentration of the Shiga toxin B subunit recombinant protein added into the enzyme reaction system is 10-50 mu M, the concentration of the biotin short peptide is 300-400 mu M, and the concentration of the transpeptidase A is 2-5 mu M.

5. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: the preparation method of the probe comprises the steps of mixing the coupling probe of the biotinylation shiga toxin B subunit recombinant protein and the streptavidin marked quantum dot in PBS buffer solution with the pH value of 6.0-8.0, keeping out of the sun at the temperature of 4-25 ℃, and carrying out oscillation reaction for 30min-2 h.

6. The shiga toxin B subunit recombinant protein-based probe for the detection of carbohydrate antigens according to claim 5, wherein: the concentration of the biotinylation Shiga toxin B subunit recombinant protein is 1 mu M; the concentration of the streptavidin marked quantum dots is 5-50nM, and the oscillation speed is 10-200 rpm.

7. The method for preparing a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to any one of claims 1 to 6, wherein: the method comprises the following steps:

1) prokaryotic expression and purification of the recombinant protein of the Shiga toxin B subunit;

2) preparing biotin short peptide;

3) the shiga toxin B subunit recombinant protein is subjected to in vitro enzymatic fusion modification with the biotinylation short peptide to obtain the biotinylation shiga toxin B subunit recombinant protein;

4) and coupling the biotinylation shiga toxin B subunit recombinant protein with streptavidin marked quantum dots to prepare the probe.

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