CN113087807A - Shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigen and preparation method thereof - Google Patents
Shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigen and preparation method thereof Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of biomedicine/antigen detection, and particularly relates to a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens and a preparation method thereof.
Background
Carbohydrate antigen is a tumor marker. Changes in glycosylation levels and structure are common features of tumor cells, and thus certain carbohydrate antigens can be used as markers for the detection, diagnosis and treatment of tumors. The carbohydrate antigen is not specific to a certain tumor, different carbohydrate antigens have specificity in different malignant tumors, and the application of the carbohydrate antigen 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, and the like.
Glycosphingolipid Gb3Is a neutral carbohydrate antigen, and the structure of the neutral carbohydrate antigen is Gal1-4Gal1-4 Glc-Cer. Glycosphingolipid Gb3Widely exists on the surface of the cell membrane of mammals and plays an important role in the aspects of cell growth, differentiation, signal conduction and the like. Glycosphingolipid Gb3The abnormal expression of (A) can cause a series of pathological reactions of the body. The results of the current study show that: glycosphingolipid Gb3The overexpression of (a) is associated with metastasis of colon cancer, and it is now found that almost all cancer cell metastasis is accompanied by changes in cell surface glycosylation.
Current pair Gb3Mainly using commercial anti-Gb3Antibodies were subjected to western blot experiments. The application is limited due to long experimental period, expensive commercial antibody and the like.
Shiga toxin is a virulence protein factor produced by Shigella and some colibacillus, and can be specifically combined with Gb indicated by human host cells3A carbohydrate antigen. Shiga toxin is a complex composed of A subunit protein and B subunit protein, wherein the A subunit protein has virulence activity, and the B subunit protein has Gb3Carbohydrate antigen binding activity. Shiga toxins include two classes, shiga toxin 1 and shiga toxin 2. The Shiga toxin B subunit protein comprises a Shiga toxin 1B subunit and a Shiga toxin 2B subunit.
The function of the Shiga toxin B subunit is to specifically bind Gb3A carbohydrate antigen. Shiga toxin subunit BHas no toxic activity of Shiga toxin when singly existing, but maintains the Shiga toxin to Gb3The binding property of carbohydrate antigen, therefore, the recombinant Shiga toxin B subunit protein can be used as Gb3Recognition unit for developing tumor cells Gb3A detection probe for carbohydrate antigens.
However, no effective method for preparing Gb from Shiga toxin B subunit exists3The detection probe of (1).
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a probe for detecting carbohydrate antigens based on recombinant proteins of Shiga toxin B subunits and a preparation method thereof,
the technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
the invention relates to a probe for detecting carbohydrate antigen based on Shiga toxin B subunit recombinant protein.
The exogenous expression is included in prokaryotic escherichia coli e.coli BL21(DE 3).
One object of the present invention is to provide a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens, which is formed by coupling the following two moieties:
a. biotinylation Shiga toxin B subunit recombinant protein,
b. streptavidin marks quantum dots;
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 proteins comprise Shiga toxin 1B subunit protein and Shiga toxin 2B subunit protein;
the biotinylation short peptide is a short peptide containing a biotin group.
Furthermore, the amino acid sequence of the Shiga toxin 2B subunit recombinant protein is shown as SEQ ID No.2, and the nucleotide sequence for coding the Shiga toxin 2B subunit recombinant protein is shown as SEQ ID No. 3; the amino acid sequence of the Shiga toxin 1B subunit recombinant protein is shown as SEQ ID No.6, and the nucleotide sequence of the Shiga toxin 1B subunit recombinant protein is shown as SEQ ID No. 7.
Further, the biotinylated short peptide comprises 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 a polypeptide with the same function by changing or increasing or decreasing the sequence of the short peptide, and preferably, the amino acid structural composition of one selected short peptide in the embodiment of the invention is shown in SEQ ID No. 4. The biotin molecule includes any biotin analogue which can bind streptavidin, such as 2-iminobiotin (2-iminobiotin).
Further, the method for fusion modification by in vitro enzyme method 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 ℃.
Further, the enzyme reaction system comprises 50mM Tris, 150mM NaCl and 5mM CaCl2pH6.0-8.0PB buffer; the concentration of the recombinant protein of the Shiga toxin B subunit 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.
Further, the preparation method of the probe comprises the steps of mixing the coupling probe of the biotinylated 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.
Further, the concentration of the biotinylated Shiga toxin B subunit recombinant protein is 1 mu M; the concentration of the streptavidin marked quantum dot is 5-50nM, and the oscillation speed is 10-200 rpm.
Another object of the present invention is to provide a method for preparing a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens, comprising the following steps:
1) prokaryotic expression and purification of the recombinant protein of the Shiga toxin B subunit;
2) preparation of biotin short peptide: solid-phase synthesis of biotin short peptides based on Fmoc method;
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 and the streptavidin marked quantum dot to prepare the probe.
Another objective of the present invention is to provide an application of a shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens, which is used for micro-detection of carbohydrate antigens of cells, wherein the carbohydrate antigens comprise Gb3 and Gb 4; the trace detection of the carbohydrate antigen of the cell refers to the detection of a tumor marker of the carbohydrate antigen in the cell.
Further, standing and incubating the probe and the cell to be detected at 4-37 ℃, carrying out flow cytometry to detect fluorescence after centrifugal PBS (phosphate buffer solution) cleaning, wherein the excitation wavelength is ultraviolet or blue light, and the emission spectrum is 605nm +/-5 nm; wherein the concentration of the probe is 5-50nM, and the incubation time is 30min-2 h.
Has the advantages that: compared with the prior art, the Shiga toxin B subunit recombinant protein coupling probe for detecting carbohydrate antigens and the preparation method thereof provided by the invention have the following advantages: the invention successfully constructs the detection of carbohydrate antigens such as Gb4, Globo-H, GM2, GD2, GD3 and Fu based on Shiga toxinc-GM1, LeY, SSEA, forssman, etc., especially Gb3The method of (1). The method has the advantages of low cost, simple and convenient method, short experimental period, high detection sensitivity and the like, and lays a foundation for the detection of relevant indexes in the fields of biology, medicine, food and the like.
Drawings
FIG. 1 is a schematic diagram of a route according to the present technology.
FIG. 2 is a diagram of key parameter detection in the purification process of Shiga toxin 2B subunit recombinant protein;
FIG. 3 is an SDS-PAGE pattern; lane M is the standard protein sample, and lane 1 is the purified Shiga toxin 2B subunit recombinant protein Stx 2B-His.
FIG. 4 is an SDS-PAGE pattern; lane M is a standard protein sample, Lane 1 is purified Shiga toxin 2B subunit recombinant protein Stx2B-His, and Lane 2 is purified biotinylated and modified Shiga toxin 2B subunit recombinant protein Stx 2B-biotin.
FIG. 5 shows the detection of Shiga toxin 2B subunit recombinant protein modified by quantum dot analysis by flow cytometryK562 cells (human) Myeloid leukemia cell)Surface carbohydrate antigen Gb3The ability of the cell to perform. Two concentration gradients of 250nM and 500nM were set in the experiment with PBS as a blank control NC. A is flow displacement graph, B is mean fluorescence intensity histogram (n is 3, p)<0.05)。
FIG. 6 shows the detection of Shiga toxin 2B subunit recombinant protein modified by quantum dot analysis by flow cytometryRaji cell (human) Lymphoma cell)Surface carbohydrate antigen Gb3The ability of the cell to perform. PBS was used as a blank in the experiment and two concentration gradients of 250nM and 500nM were set. A is flow displacement graph, B is mean fluorescence intensity histogram (n is 3, p)<0.05)。
FIG. 7 shows the detection of Shiga toxin 2B subunit recombinant protein modified by quantum dot analysis by flow cytometryCaco-2 cells (human clonal colon adenocarcinoma cells)Surface carbohydrate antigen Gb3The ability of the cell to perform. PBS was used as a blank in the experiment and two concentration gradients of 250nM and 500nM were set. A is flow displacement graph, B is mean fluorescence intensity histogram (n is 3, p)<0.05)。
FIG. 8 flow cytometry analysis of quantaDot-modified Shiga toxin 2B subunit recombinant protein detectionHT-29 cells (human) Colorectal adenocarcinoma cells)Surface carbohydrate antigen Gb3The ability of the cell to perform. PBS was used as a blank in the experiment and two concentration gradients of 250nM and 500nM were set. A is flow displacement graph, B is mean fluorescence intensity histogram (n is 3, p)<0.05)。
Detailed Description
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, wherein as shown in figure 1, the preparation of the probe comprises the following steps:
a. prokaryotic expression and purification of the recombinant protein of the Shiga toxin B subunit;
b. preparation of a short peptide containing a biotin group: solid-phase synthesis of biotin short peptides based on Fmoc method;
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 method for preparing the Shiga toxin B subunit recombinant protein comprises the following steps:
1) designing a target gene sequence, and adding a MYC label, a transpeptidase A (sortase A) recognition site and a His label at the carboxyl terminal of the target gene sequence.
2) Expressing a target gene segment by using escherichia coli;
3) purifying by using a nickel column to obtain target protein;
4) connecting target protein with short peptide with biotin group under catalysis of transpeptidase A;
5) purifying with nickel column to obtain target protein with biotinylation.
The probe can effectively detect the expression of various tumor cell surface carbohydrate antigens, particularly Gb3, by combining with flow cytometry, and can be applied to detection and diagnosis by taking the carbohydrate antigens such as Gb3 and the like as tumor markers. The carbohydrate antigen detection method has the advantages of low preparation cost, strong detection specificity and high sensitivity.
The invention is further described with reference to the following figures and examples. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.
Examples
The example uses shiga toxin 2B subunit recombinant protein and Gb3 as an illustration.
The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
Terms used in the present invention generally have 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 Shiga toxin 2B subunit recombinant protein sequences
The protein sequence of the Shiga toxin 2B subunit (GenBank ID: AAD25446.1, SEQ ID No.1) is searched, and the carboxyl terminal is modified. Firstly, adding MYC label (EQKLISEEDLNGAA) and recognition site LPETGG (L: leucine; P: proline; E: glutamic acid; T: threonine; G: glycine) cut by transpeptidase A at the carboxyl terminal of the sequence of Shiga toxin 2B subunit protein; then, a histidine purification tag His, namely HHHHHHHHHH (H: histidine), was added thereto, and the recombinant protein was named Stx2B-His, and its amino acid sequence was shown in SEQ ID No. 2.
After the protein sequence was designed, the protein sequence was submitted to a commercial company for codon optimization. The optimized gene sequence (SEQ ID No.3) was inserted into pET22b (+) plasmid, and the plasmid was transformed into E.coli BL21(DE 3). The strain is a fermentation strain containing a target gene.
Example 2 expression and purification of Shiga toxin 2B subunit recombinant proteins
mu.L of glycerol strain containing the recombinant protein Stx2B-His was added to 3mL of ampicillin-resistant LB medium (peptone 10g/L,5g/L yeast powder and 10g/L sodium chloride), culturing at 37 deg.C overnight at 200rpm, inoculating TB fermentation medium (peptone 12g/L, yeast powder 24g/L, glycerol 4g/L, KH) in the next day according to 4% inoculum size2PO4 23.1g/L，K2HPO4125.4g/L) until the concentration of the bacteria reaches OD600After 0.6-1.2, the final concentration of 0.4mM inducer IPTG was added for induction, and the conditions for protein expression were 20 ℃, 200rpm, and 24 hours. Exogenous expression can be carried out in prokaryotic E.coli BL21(DE 3).
After the fermentation, the cells were collected by centrifugation at 9000rpm for 5min and the supernatant was discarded. After the mycelia were resuspended in a wall-breaking buffer (10mM Tris, 500mM NaCl, pH8.0), the mycelia were disrupted by an ultrasonication apparatus under the following conditions: and (5) carrying out ice bath, running for 2s, stopping running for 3s, and running for 20 min. After the disruption was completed, centrifugation was carried out at 9000rpm for 10min, which was repeated three times to completely remove cell debris, thereby obtaining a clear cell-disrupted supernatant. The wall-broken supernatant was incubated with a nickel column at 4 ℃ for 2-4 hours, and then the nickel column was washed with a 25mM imidazole solution to remove foreign proteins. The recombinant protein Stx2B-His was washed down with a wall-breaking buffer gradient containing 25mM-500mM imidazole. The purified protein is desalted, freeze-dried and placed at-20 ℃ for later use.
FIG. 2 is a graph of key parameter detection during recombinant protein purification. FIG. 3 is an SDS-PAGE Image of the purified recombinant protein, and 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 proteins
The enzyme reaction system is 50mM Tris, 150mM NaCl, 5mM CaCl2Buffer, pH7.5PBS buffer, Shiga toxin 2B subunit recombinant protein substrate concentration of 20 μ M, biotin short peptide concentration of 400 μ M, transpeptidase A final concentration of 5 μ M, wherein, the short peptide used by the biotin short peptide is a polypeptide containing glycine tag required for the enzymatic fusion. The reaction conditions were 16 ℃, 200rpm, 2 h. After the reaction was completed, the resulting protein was purified using a nickel column. The nickel column was washed with 1x PBS buffer and the flow-through was collected. The protein was desalted and lyophilized, and was placed at-20 ℃ for use, and was designated Stx 2B-biotin.
SDS-PAGE analysis in FIG. 4 shows that Stx2B-biotin has a significant change in molecular weight compared with Stx2B-His, and the purity of purified Stx2B-biotin is above 95%.
Example 4 modification of biotinylated Shiga toxin 2B subunit recombinant protein by coupled probe of streptavidin-labeled quantum dot
The quantum dot modification system is 1x PBS solution, pH7.0; stx2B-biotin concentration was 1. mu.M, and streptavidin-labeled quantum dots concentration was 20 nM. The reaction conditions were room temperature, protected from light, 50rpm, and protected from light for 2 h. After the reaction is finished, the product is directly used for subsequent experiments without purification; the product obtained in this step can be packaged. Storing at 2-8 deg.C.
Example 5 application of Shiga toxin 2B subunit recombinant protein marked by quantum dots to detection of carbohydrate antigen Gb3 on cell surface
Inoculating cells to be detected in logarithmic growth phase in a 6-hole plate, and culturing for 24 hours; after resuspension, collecting cells in each hole in a single flow tube, centrifuging to remove supernatant, washing the cells for 2 times by PBS (phosphate buffer solution), and adding 100 mu L of the prepared quantum dot modified recombinant Shiga toxin type II 2B subunit into each tube to form a probe with the concentration of 20 nM; standing and incubating for 30min at 4 ℃; centrifuging to remove supernatant, washing cells with PBS for 2 times, and centrifuging for 5 min; add 200. mu.L PBS to each tube to resuspend the cells and test on the machine.
The analysis of fig. 5, 6, 7 and 8 shows that, for the negative cell population K562 cells, there are no obvious differences in shift and average fluorescence intensity; for positive cell populations Raji, Caco-2 and HT-29, the shift and average fluorescence intensity of the cells are obviously changed compared with PBS control, and concentration gradient can be seen, which indicates that the embodiment can achieve the purpose. Meanwhile, due to the excellent light-emitting characteristics of the quantum dots, the background is low, and the emission spectrum is narrow and symmetrical.
The above examples illustrate that the recombinant protein of Shiga toxin 2B subunit can specifically bind to the surface glycosphingolipidGb on the cell membrane3Receptor binding; meanwhile, by combining with the flow cytometry, the cell screening can be finished within 1h at the fastest, and the time required by the experiment is greatly saved. In the same way, the embodiment is also suitable for preparing the Shiga toxin 1B subunit protein with the completely same function as the Shiga toxin 2B subunit proteinBecomes a coupling probe based on the recombinant protein of the Shiga toxin 1B subunit. Similarly, this example also applies to other carbohydrate antigens on cell membranes, such as Gb4, which are similar in structure and function, and to other carbohydrate antigens, such as Globo-H, GM2, GD2, GD3, Fuc-GM1, LeY, SSEA, forssman, etc.
Example 6 modification of biotinylated Shiga toxin 1B subunit recombinant protein by streptavidin-labeled Quantum dot coupled Probe
Designing a recombinant protein sequence of a Shiga toxin 1B subunit, expressing and purifying the recombinant protein of the Shiga toxin 1B subunit, and biotinylating, modifying and purifying the recombinant protein of the Shiga toxin 1B subunit, wherein the amino acid sequence of the protein of the Shiga toxin 1B subunit is shown as SEQ ID No.5, the amino acid sequence of the recombinant protein of the Shiga toxin 1B subunit is shown as SEQ ID No.6, and the nucleotide sequence encoding the recombinant protein of the Shiga toxin 1B subunit is shown as SEQ ID No.7 in the same examples 1, 2 and 3; the short peptide is shown as SEQ ID No. 4.
The quantum dot modification method is the same as that in example 4, and the obtained probe is used for detecting carbohydrate antigens such as Gb3 and Gb4 on the cell surface.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Sequence listing
<110> university of south of the Yangtze river
<120> probe for detecting carbohydrate antigen based on Shiga toxin B subunit recombinant protein and preparation method thereof
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<213> Shiga toxin 2B subunit protein ()
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Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp
1 5 10 15
Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp Thr Ser Arg
20 25 30
Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr
35 40 45
Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu
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Val Gln Phe Asn Asn Asp
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Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp
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Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp Thr Ser Arg
20 25 30
Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr
35 40 45
Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu
50 55 60
Val Gln Phe Asn Asn Asp Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
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Asn Gly Ala Ala Leu Pro Glu Thr Gly Gly His His His His His His
85 90 95
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gctgattgtg caaaaggtaa aattgaattt tccaagtaca acgaagacga tacctttacc 60
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agcgcacagc tgaccggtat gaccgttacc attaagagca gcacctgcga aagcggtagc 180
ggctttgccg aagttcagtt taataatgat gaacagaagc tgattagcga agaagatctg 240
aatggtgccg cactgccgga aaccggtggt catcatcatc atcaccat 288
<210> 4
<211> 11
<212> PRT
<213> short peptide ()
<400> 4
Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Lys
1 5 10
<210> 5
<211> 69
<212> PRT
<213> Shiga toxin 1B subunit protein ()
<400> 5
Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr Lys Tyr Asn Asp
1 5 10 15
Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu Leu Phe Thr Asn
20 25 30
Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln Ile Thr Gly Met
35 40 45
Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly Gly Gly Phe Ser
50 55 60
Glu Val Ile Phe Arg
65
<210> 6
<211> 95
<212> PRT
<213> Shiga toxin 1B subunit recombinant protein ()
<400> 6
Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr Lys Tyr Asn Asp
1 5 10 15
Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu Leu Phe Thr Asn
20 25 30
Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln Ile Thr Gly Met
35 40 45
Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly Gly Gly Phe Ser
50 55 60
Glu Val Ile Phe Arg Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
65 70 75 80
Gly Ala Ala Leu Pro Glu Thr Gly Gly His His His His His His
85 90 95
<210> 7
<211> 285
<212> DNA
<213> Shiga toxin 1B subunit recombinant protein ()
<400> 7
acccctgatt gcgtgaccgg caaagtggaa tataccaaat ataatgatga cgataccttc 60
accgtgaaag tgggcgataa agaactgttt accaatcgtt ggaatctgca gagtctgctg 120
ctgagtgccc agattaccgg tatgaccgtg accattaaga ccaatgcatg tcataatggc 180
ggtggcttta gcgaagtgat ttttcgtgaa cagaaactga ttagcgaaga agatctgaat 240
ggtgcagcac tgccggaaac cggtggccat catcatcatc accat 285
Claims (10)
1. A shiga toxin B subunit recombinant protein-based probe for detection of carbohydrate antigens, characterized in that: the probe is formed by coupling the following two parts:
a. biotinylation Shiga toxin B subunit recombinant protein,
b. streptavidin marks quantum dots;
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 comprises a shiga toxin 1B subunit protein and a shiga toxin 2B subunit protein;
the biotinylation short peptide is a short peptide containing a biotin group.
2. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: 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.
3. 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.
4. The shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigens according to claim 1, characterized in that: the method for in vitro enzyme method fusion modification comprises the following steps of2The pH7.5PBS buffer solution is used as 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 ℃.
5. The shiga toxin B subunit recombinant protein-based probe for the detection of carbohydrate antigens according to claim 4, wherein: the enzyme reaction system comprises 50mM Tris, 150mM NaCl and 5mM CaCl2pH6.0-8.0PB buffer; the concentration of the recombinant protein of the Shiga toxin B subunit 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.
6. 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.
7. The shiga toxin B subunit recombinant protein-based probe for the detection of carbohydrate antigens according to claim 6, wherein: the concentration of the biotinylation Shiga toxin B subunit recombinant protein is 1 mu M; the concentration of the streptavidin marked quantum dot is 5-50nM, and the oscillation speed is 10-200 rpm.
8. 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 7, 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 and the streptavidin marked quantum dot to prepare the probe.
9. Use of a shiga toxin B subunit recombinant protein based probe for the detection of carbohydrate antigens according to any one of claims 1 to 7, characterized in that: a trace amount of carbohydrate antigens for cells, the carbohydrate antigens including Gb3, Gb 4; the trace detection of the carbohydrate antigen of the cell refers to the detection of a tumor marker of the carbohydrate antigen in the cell.
10. Use of a shiga toxin B subunit recombinant protein based probe for the detection of carbohydrate antigens according to claim 9, characterized in that: standing and incubating the probe and cells to be detected at 4-37 ℃, washing by centrifugal PBS, and detecting fluorescence by flow cytometry, wherein the excitation wavelength is ultraviolet or blue light, and the emission spectrum is 605nm +/-5 nm; wherein the concentration of the probe is 5-50nM, and the incubation time is 30min-2 h.
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