One-step enzyme chemical detection method for tumor-associated Tn antigen and application of one-step enzyme chemical detection method

Abstract

The invention belongs to the technical field of biomedicine, and relates to a one-step enzyme chemical detection method for tumor-associated Tn antigens and application of the one-step enzyme chemical detection method, C1GALT1 glycosyl transferase is utilized to realize efficient labeling of Tn antigens in lysate, living cells and biological samples through a one-step enzyme chemical method, fluorescence imaging and Tn enrichment can be carried out, and the Tn antigens in the lysate, the living cells and the biological samples can be efficiently labeled through the C1GALT1 glycosyl transferase. And identification of site-specific Tn protein sites can be carried out through mass spectrometry. Compared with the detection effect of the traditional lectin and the previous two-step enzyme chemical method, the marking efficiency of the method is improved by at least one order of magnitude. The one-step enzyme chemical marker for the Tn antigen can realize multi-dimensional and omnibearing high-specificity, high-sensitivity, stable and rapid detection of the Tn antigen, and has great significance in the aspects of functional research of the Tn antigen in an organism, disease diagnosis and treatment, prognosis, targeted intervention and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a method for detecting tumor-associated Tn antigen, in particular to a one-step enzymatic chemical detection method for tumor-associated Tn antigen and its application. Background Art

[0002] The prior art discloses that abnormal glycosylation changes are a known cancer hallmark. Tumor-associated carbohydrate antigens (TACAs) are a class of biopolysaccharide markers highly expressed on the cell surface during tumor development, including Tn antigen, T / TF antigen, Lewis blood group antigen, complex oligosaccharides containing 1,2-linked fucose, and Globo series antigens, etc.

[0003] Research discloses that Tn antigen (GalNAcα1-Ser / Thr / Tyr, CD175) is a truncated expression form of the initiating monosaccharide of Mucin-type O-glycan synthesis. In normal cells, it will be further extended to synthesize other O-glycans, so its expression level is very low. However, its expression level is greatly increased in many tumor cells / tissues, such as colon cancer, breast cancer, lung cancer, ovarian cancer, and pancreatic cancer, etc. Research shows that 95% of colon cancer tissues and 90% of breast cancer tissues highly express Tn antigen. Moreover, multiple studies have proved that the high expression of Tn antigen is closely related to tumor metastasis or poor prognosis. Therefore, targeted therapy of Tn antigen has currently become a new approach for tumor treatment.

[0004] The existing detection techniques for Tn antigen mainly rely on lectins, antibodies, and mass spectrometry techniques. The two most widely used lectins are: Vicia villosa agglutinin / lectin (VVA) and Helix pomatia agglutinin (HPA). However, their recognition specificities are not strong. They not only recognize Tn antigen but also recognize many other sugar chain structures, resulting in the problem of non-specific binding. The commercial antibodies for detecting Tn antigen are similar to most other sugar chain antibodies. Due to the low immunogenicity of sugar chains, the detection sensitivity and specificity of their antibodies are not strong. There are few Tn antigen antibodies on the current market, and most of them can only be used for immunohistochemistry and immunofluorescence labeling, and cannot be used for western blot (WB) detection and immunoprecipitation enrichment. Currently, some mass spectrometry techniques have been developed for site-specific proteomic identification of Tn antigen modification, but the methods based on mass spectrometry detection cannot perform labeling and imaging analysis simultaneously at the levels of lysate and living cells. Therefore, there is still a lack of methods with high specificity, high sensitivity, and comprehensive analysis for the existing Tn antigen detection. The lack of methods limits the in-depth exploration and wide application of Tn antigen.

[0005] Based on the current state of the art, the inventors of the present application intend to provide a method for comprehensively and efficiently specifically labeling, enriching, and detecting Tn antigen, specifically relating to a one-step enzymatic chemical detection method for tumor-associated Tn antigen and its applications. Summary of the Invention

[0006] The object of the present invention is to provide a method for comprehensively and efficiently specifically labeling, enriching, and detecting Tn antigen based on the current state of the art, specifically relating to a one-step enzymatic chemical detection method for tumor-associated Tn antigen and its applications.

[0007] The one-step enzymatic chemical detection method of the present invention can not only simply, rapidly, and efficiently label, image, and enrich Tn antigen at the levels of live cells and lysates, but also maintain high-efficiency specificity in detecting complex patient samples, and can further be applied to site-specific Tn proteomic identification. The inventive method is not limited to the applications described above, and those skilled in the art can clearly understand other technical applications not mentioned herein through the following description.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A one-step enzymatic chemical detection method for tumor-associated Tn antigen, comprising:

[0010] A1) Using protein C1GALT1(34-363) as a glycosyltransferase to catalyze the donor substrate azidosugar for enzymatic chemical labeling of different samples containing Tn antigen;

[0011] A2) Performing a click reaction on the sample processed in step A1) with an alkyne-containing tag compound to obtain a Tn antigen labeled with the alkyne-containing tag compound;

[0012] A3) Alternatively, combining the two-step labeling in steps A1) and A2) into one step, first performing a click chemical reaction on the donor substrate azidosugar and the alkyne-containing tag compound to form UDP-galactose-tag compound, and then directly catalyzing the UDP-galactose-tag compound with the glycosyltransferase C1GALT1(34-363) for one-step enzymatic labeling of Tn antigen.

[0013] The donor substrate azidosugar in A1) can also be a donor substrate alkynyl sugar, and then the compound containing an azide tag is used for the click reaction in the corresponding A2).

[0014] Correspondingly, the UDP-galactose-tag compound in A3) can also be the product of the reaction between an alkynyl sugar and an azide tag compound.

[0015] The tag compounds in the present invention include but are not limited to enrichment tags and fluorescent tags. Other small molecule compounds and macromolecules that can be Tn-labeled by C1GALT1 glycosyltransferase, including protein tags, nucleic acid tags, polypeptide tags, etc., are also protected by this patent.

[0016] The present invention provides a highly stable and active human glycosyltransferase C1GALT1 / T-synthase (UniportID: Q9NS00), and the high stability of its activity requires the coexistence of its chaperone protein C1GALT1C1(29-318) / COSMC(29-318). The specific information of the protein is as follows:

[0017] B1) The full length of C1GALT1 (Uniport ID: Q9NS00) glycosyltransferase contains 363 amino acids: MASKSWLNFLTFLCGSAIGFLLCSQLFSILLGEKVDTQPNVLHNDPHARHSDDNGQNHLEGQMNFNADSSQHKDENTDIAENLYQKVRILCWVMTGPQNLEKKAKHVKATWAQRCNKVLFMSSEENKDFPAVGLKTKEGRDQLYWKTIKAFQYVHEHYLEDADWFLKADDDTYVILDNLRWLLSKYDPEEPIYFGRRFKPYVKQGYMSGGAGYVLSKEALKRFVDAFKTDKCTHSSSIEDLALGRCMEIMNVEAGDSRDTIGKETFHPFVPEHHLIKGYLPRTFWYWNYNYYPPVEGPGCCSDLAVSFHYVDSTTMYELEYLVYHLRPYGYLYRYQPTLPERILKEISQANKNEDTKVKLGNP;

[0018] The complete sequence of the B2)C1GALT1C1 (Uniport ID: Q96EU7) protein contains 318 amino acids: MLSESSSFLKGVMLGSIFCALITMLGHIRIGHGNRMHHHEHHHLQAPNKEDILKISEDERMELSKSFRVYCIILVKPKDVSLWAAVKETWTKHCDKAEFFSSENVKVFESINMDTNDMWLMMRKAYKYAFDKYRDQYNWFFLARPTTFAIIENLKYFLLKKDPSQPFYLGHTIKSGDLEYVGMEGGIVLSVESMKRLNSLLNIPEKCPEQGGMIWKISEDKQLAVCLKYAGVFAENAEDADGKDVFNTKSVGLSIKEAMTYHPNQVVEGCCSDMAVTFNGLTPNQMHVMMYGVYRLRAFGHIFNDALVFLPPNGSDND.

[0019] Proteins obtained by mutating and / or deleting and / or adding the amino acid sequences shown in B1) and / or B2), which have more than 80% identity with the proteins shown in B1) and B2) and have the same function, are all within the scope of protection of the present invention.

[0020] Fusion proteins with the same function obtained by inserting a fusion tag or a signal peptide at the N-terminus and / or C-terminus of B1) and / or B2) are all within the scope of protection of the present invention.

[0021] Proteins that are homologous but from different families and proteins with the same function but different origins as the protein B1) and / or B2) are all within the scope of protection of the present invention.

[0022] Both proteins described in the present invention are truncated expressions: C1GALT1(34 - 363) and C1GALT1C1(29 - 318), and GFP, His tags and a secretion peptide signal are added at the N-terminus. In order to obtain a purified protein B1) with stable activity, C1GALT1(34 - 363) and C1GALT1C1(29 - 318) are co-expressed in mammalian cells HEK293F.

[0023] The expression systems of the proteins C1GALT1(34 - 363) and C1GALT1C1(29 - 318) in the present invention can also be other prokaryotic and eukaryotic expression systems.

[0024] The present invention also provides any one of the following applications of the Tn antigen labeling method:

[0025] Application of the Tn antigen labeling method in the labeling, enrichment and imaging of Tn antigen in cell lysates;

[0026] Application of the Tn antigen labeling method in the labeling, enrichment and imaging of Tn antigen on living cells;

[0027] Application of the Tn antigen labeling method in the labeling, enrichment and imaging of Tn antigen in biological samples;

[0028] The biological samples in C3) include but are not limited to various clinical samples of humans or animals, including blood, tissues, biological fluids, etc., which are all within the scope of this patent protection.

[0029] The present invention provides an enzymatic chemical specific and efficient detection method for tumor-related Tn antigen and its application. The present invention uses C1GALT1 glycosyltransferase for enzymatic chemical labeling of Tn antigen. Due to the recognition specificity of C1GALT1 glycosyltransferase for Tn antigen and the reaction specificity of bioorthogonal reaction (click chemistry), the high specificity of labeling is guaranteed; due to the signal amplification effect of the label group (fluorescent group, biotin group, etc.), the high sensitivity of labeling is guaranteed; in addition, the activity of C1GALT1 glycosyltransferase co-expressed and purified with the chaperone protein C1GALT1C1 is very stable (it can be placed at 4°C for half a year and still have activity), and the repeatability of the labeling effect in a complex system reflects the stability of this method; the labeling of Tn antigen by C1GALT1 glycosyltransferase can achieve one-step enzymatic chemical labeling. Compared with the previous two-step enzymatic chemical labeling method, it is not only simpler and faster in operation, but also more efficient. Compared with the detection effect of the traditional lectin VVA, the labeling efficiency of the present invention is significantly improved by at least one order of magnitude; in addition, the cleavable biotin enrichment tag developed by the present invention can also identify the site-specific Tn protein sites by mass spectrometry. Therefore, the Tn antigen labeling of the present invention can achieve multi-dimensional and all-round detection from cell lysates, living cells, biological samples to the identification of Tn-modified protein sites, which is of great significance for the functional research of Tn antigen in the body and in disease diagnosis, treatment, prognosis and targeted intervention. Description of the Drawings

[0030] Figure 1 SDS-PAGE diagram after the expression and purification of C1GALT1 and C1GALT1C1;

[0031] Figure 2 SDS-PAGE fluorescence imaging diagram for the comparison of the two-step method and the one-step enzymatic labeling method;

[0032] Figure 3 WB diagram for the comparison of the two-step method and the one-step enzymatic labeling method;

[0033] Figure 4Figure for comparing the labeling effects of one-step enzyme labeling method and lectin VVA;

[0034] Figure 5 Imaging of Tn antigen on the surface of living cells under a confocal microscope;

[0035] Figure 6 SDS-PAGE fluorescence imaging of one-step enzyme labeling method for labeling patient serum and tissues. Specific implementation mode

[0036] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0037] In the present invention, unless otherwise specified, the materials, reagents, etc. used are all purchased conventionally, and the experimental methods used are also conventional methods.

[0038] Example 1: Preparation of glycosyltransferase C1GALT1(34-363)

[0039] First, eukaryotic expression vectors of C1GALT1(34-363) and C1GALT1C1(29-318) are cloned and constructed by conventional methods, and a signal peptide sequence, His tag and GFP tag are inserted in sequence at the N-terminus of the target gene.

[0040] Human glycosyltransferase CIGALT1(34-363) and its molecular chaperone CIGALT1C1(29-318) are secreted and expressed using mammalian cells HEK293F. The specific operations are as follows:

[0041] 1. HEK293F suspension cell culture and transfection (taking 200 mL as an example)

[0042] 1) HEK293F cell culture: Under suspension cell culture conditions (5% CO2, 100 rpm / min), when the cell growth state is good (survival rate above 98%) and shows a doubling growth state (the cells proliferate 1-1.2 times every other day), cell seeding is carried out, and the seeding density is 1.0×10 6 cells / mL, with a total of 200 mL of culture medium.

[0043] 2) Transfection: Transfection is carried out 24 h after seeding. When the density of HEK293F cells is detected to be 2.0×10 6cells / mL. Take 200 μg of CIGALT1(34-363) plasmid vector and 200 μg of CIGALT1C1(29-318) plasmid vector, add them to 1 mL of cell culture medium, and mix well. Then add 800 μg of PEI transfection reagent and mix again. The mixed plasmid DNA-PEI transfection reagent medium is placed in an incubator at 37 °C and allowed to stand for 15 min. The mixed medium containing the transfection reagent and plasmid is slowly added dropwise to 200 mL of HEK293F cells to complete the transfection (Day 0).

[0044] 3) Transfection observation and cell monitoring: 24 h after the transfection is completed (Day 1), add 20 mL of cell growth supplement to promote the secretion and expression of the target protein. Monitor the cell viability daily after transfection. When the viability drops to 80% (Day 5-7), harvest the medium.

[0045] 2. Purification of glycosyltransferase C1GALT1 and its molecular chaperone C1GALT1C1

[0046] 1) Take the medium in 1.3), centrifuge at 500 g to remove the cell pellet, and retain the supernatant medium.

[0047] 2) Filter the collected supernatant cell medium through a 0.45 μM filter membrane to remove cell debris.

[0048] 3) Add nickel column packing to the filtered medium and incubate with shaking at 4 °C overnight.

[0049] 4) Load the overnight-bound protein solution and packing mixture onto a column for purification, wash with 20 mM imidazole for 10 column volumes; then elute the target protein with 5 column volumes of 300 mM imidazole.

[0050] 5) Centrifuge and concentrate the eluate to finally obtain the target glycosyltransferase C1GALT1 and C1GALT1C1 protein complex.

[0051] 6) Detect the successful expression of the target protein by SDS-PAGE (as Figure 1 shown).

[0052] The glycosyltransferase C1GALT1 and its co-expressed and purified molecular chaperone C1GALT1C1 protein will serve as the key enzyme for catalysis in subsequent experiments and will be referred to as T-synthase in the following text.

[0053] Example 2: Application of Tn antigen C1GALT1 enzymatic labeling on Jurkat cell lysate

[0054] 1. Preparation of Jurkat cell lysate: Jurkat cells were cultured to the logarithmic growth phase, harvested, centrifuged at 500 g for 5 min, the supernatant was removed, and the lower cell pellet was taken. The cells were lysed with 1% SDS in 50 mM Tris-HCl and then centrifuged at 12,000 g at high speed for 5 min. The upper clear cell lysate was taken for BCA protein quantification.

[0055] 2. First step of the two-step enzyme labeling reaction system: 20 μL of cell lysate (30 μg of protein), 20 μL of labeling buffer, 0.5 μL of 5 mM UDP-6-N3-Gal, 1 μL of 100 mM Mn 2+ 1 μL, 3 μL of T-synthase (200 μg / mL), and finally ddH2O was added to make up the total volume to 50 μL. React at 37 °C for 2 h. The labeling buffer (the same below) is a mixed buffer of 50 mM Tris-HCl pH 7.5, 5% NP-40, and 125 mM NaCl. The control group of the two-step enzyme labeling method is the reaction system without T-synthase.

[0056] 3. Second step of the two-step enzyme labeling reaction system: The reaction solution after 2 h of the first-step labeling reaction was subjected to protein precipitation with methanol-chloroform, redissolved with 46.25 μL of 1% SDS in 50 mM Tris-HCl, 0.5 μL of 5 mM CY3-Alkyne / Biotin-Alkyne was added, 1 μL of 5 mM CuSO4 and 1 μL of 30 mM BTTP were added, and then 1.25 μL of 100 mM L-ascorbic acid was added. React with shaking at room temperature for 30 min.

[0057] 4. One-step enzyme labeling reaction system: 20 μL of cell lysate (30 μg of protein), 20 μL of labeling buffer, 0.5 μL of 5 mM UDP-Gal-Tag (fluorescent CY3 or enriched Biotin tag), 1 μL of 100 mM Mn 2+ 1 μL, 3 μL of T-synthase (200 μg / mL), and finally ddH2O was added to make up the total volume to 50 μL. React at 37 °C for 2 h. The control group of the one-step enzyme labeling method is the reaction system without T-synthase.

[0058] 5. Detection: SDS-PAGE fluorescence photography detection (CY3-labeled samples) or WB detection (Biotin tag-labeled samples) was performed with equal protein loading amounts. For the CY3-labeled sample group, after separating the proteins by SDS-PAGE, fluorescence photography was directly performed at an excitation wavelength of 555 nm (such as Figure 2(as shown). For the sample group labeled with Biotintag, after separating proteins by SDS-PAGE, transfer them to a 0.45 μm PVDF membrane. After blocking the membrane with 5% skim milk at room temperature for 2 h, incubate with streptavidin-conjugated horseradish peroxidase antibody (streptavidin-HRP) diluted 1:8000 at room temperature for 15 min. Wash 3 times with TBST, 10 min each time, and then expose on the machine.

[0059] 6. The results are as Figure 2 and Figure 3 shown: With the same protein loading amount, the one-step enzymatic labeling is significantly better than the two-step enzymatic labeling.

[0060] Example 3: Comparison of the labeling effects of one-step enzymatic labeling method and lectin VVA

[0061] 1. Perform WB detection by loading different protein contents of Jurkat cell lysate and bovine submaxillary mucin (BSM) protein solution respectively. At the same time, perform the one-step enzymatic labeling reaction in Example 2 on the two proteins to obtain labeled protein solutions, and directly perform SDS-PAGE fluorescence photography and.

[0062] 2. For the WB detection group, after separating samples with different protein contents by SDS-PAGE, transfer them to a 0.45 μm PVDF membrane. After blocking with 5% skim milk at room temperature for 1 h, incubate with VVA antibody at 2 μg / mL at room temperature for 30 min. After incubation, wash 3 times with TBST, 10 min each time, and then incubate with streptavidin-conjugated horseradish peroxidase antibody (streptavidin-HRP) diluted 1:8000 at room temperature for 15 min, wash 3 times with TBST, 10 min each time, and then expose on the machine.

[0063] 3. The results are as Figure 4 shown: Compared with the currently commercial traditional lectin VVA labeling method, the one-step enzymatic labeling method is at least 10 times better than the traditional method for labeling cell lysate, and for labeling BSM mucin, the one-step enzymatic labeling method is significantly better than the traditional method by more than one order of magnitude.

[0064] Example 4: Detection of Tn antigen on the surface of living cells by one-step enzymatic labeling method

[0065] 1. Culture Jurkat cells to the logarithmic growth phase, harvest the cells, centrifuge at 500 g for 5 min, remove the supernatant, take the lower cell pellet, and resuspend it with PBS + 5% FBS.

[0066] 2. One-step enzymatic labeling: Sequentially add 100 mM Mn 2+1 μL of 5 mM UDP-Gal-Tag (fluorescent CY3 tag), 0.5 μL, 3 μL of T-synthase (200 μg / mL), and react with shaking at 37°C for 1 h. The control group is the reaction system without T-synthase.

[0067] After the reaction, centrifuge at 500 g for 5 min, remove the supernatant, wash the cells 3 times with PBS + 5% FBS, add DAPI dye to resuspend the cells, then prepare slides and observe under a confocal microscope (as Figure 5 shown).

[0068] 4. The results show that: Fluorescent signals can be seen on the cell membrane surface in the experimental group, while no fluorescent signals are seen in the control group.

[0069] Example 5: Application of one-step enzyme labeling method on complex biological samples

[0070] 1. Take serum samples for BCA protein quantification.

[0071] 2. Take about 1 mg of liver cancer and cervical cancer tumor tissues, grind and lyse them thoroughly with 200 μL of 1% SDS 50 mM Tris-HCl, centrifuge at 12000 g at high speed for 5 min, take the supernatant tissue lysate for BCA protein quantification.

[0072] 3. One-step enzyme labeling reaction system (same as Example 2): 20 μL of serum / tissue lysate with the same protein amount, 20 μL of labeling buffer, 0.5 μL of 5 mM UDP-Gal-Tag (fluorescent CY3 tag), 1 μL of 100 mM Mn 2+ 1 μL, 3 μL of T-synthase, and finally make up to a total volume of 50 μL with ddH2O. React at 37°C for 2 h. The control group of the one-step enzyme labeling method is the reaction system without T-synthase.

[0073] 4. Separate the protein samples after the reaction by SDS-PAGE and take fluorescent photos (as Figure 6 shown).

[0074] 5. The results show that: The one-step enzyme method can specifically and efficiently label complex biological samples such as serum and tissue lysates, and has clinical application value and advantages.

[0075] The description of the above examples is only for understanding the method of the present invention. However, the present invention is not limited to the specific details in the above embodiments. For those skilled in the art, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A one-step enzymatic chemical detection method for tumor-associated Tn antigen, characterized in that, It includes: A1) First, perform a click chemical reaction on UDP-azidosugar and an alkyne-tagged compound to generate a UDP-galactose-tagged compound, and then directly catalyze it using the human glycosyltransferase C1GALT1 (T-synthase) to label the Tn antigen by a one-step enzymatic chemical method; A2) Use the human glycosyltransferase C1GALT1 (T-synthase) to catalyze the donor substrate azidosugar for the first-step enzymatic chemical labeling of the Tn antigen, and then perform a click reaction with the alkyne-containing tagged compound to obtain the Tn antigen labeled with the tagged compound through two steps; A3) In A1) and A2), the donor sugar substrates are UDP-Gal-CY3, UDP-Gal-Biotin, and UDP-6N3-Gal, as well as other small and large molecules with functional tags derived from UDP-Gal by chemical methods.

2. The one-step enzymatic chemical detection method for tumor-related Tn antigen according to claim 1, characterized in that, The highly stable and active human glycosyltransferase C1GALT1 / T-synthase described above is: B1) The amino acid sequence of C1GALT1 (Uniport ID: Q9NS00) is: MASKSWLNFLTFLCGSAIGFLLCSQLFSILLGEKVDTQPNVLHNDPHARHSDDNGQNHLEGQMNFNADSSQHKDENTDIAENLYQKVRILCWVMTGPQNLEKKAKHVKATWAQRCNKVLFMSSEENKDFPAVGLKTKEGRDQLYWKTIKAFQYVHEHYLEDADWFLKADDDTYVILDNLRWLLSKYDPEEPIYFGRRFKPYVKQGYMSGGAGYVLSKEALKRFVDAFKTDKCTHSSSIEDLALGRCMEIMNVEAGDSRDTIGKETFHPFVPEHHLIKGYLPRTFWYWNYNYYPPVEGPGCCSDLAVSFHYVDSTTMYELEYLVYHLRPYGYLYRYQPTLPERILKEISQANKNEDTKVKLGNP; B2) Human protein C1GALT1C1 / COSMC (Uniport ID: Q96EU7) is a chaperone protein of glycosyltransferase C1GALT1 / T-synthase. Its full-length amino acid sequence is: MLSESSSFLKGVMLGSIFCALITMLGHIRIGHGNRMHHHEHHHLQAPNKEDILKISEDERMELSKSFRVYCIILVKPKDVSLWAAVKETWTKHCDKAEFFSSENVKVFESINMDTNDMWLMMRKAYKYAFDKYRDQYNWFFLARPTTFAIIENLKYFLLKKDPSQPFYLGHTIKSGDLEYVGMEGGIVLSVESMKRLNSLLNIPEKCPEQGGMIWKISEDKQLAVCLKYAGVFAENAEDADGKDVFNTKSVGLSIKEAMTYHPNQVVEGCCSDMAVTFNGLTPNQMHVMMYGVYRLRAFGHIFNDALVFLPPNGSDND; B3) A protein with more than 80% identity to the proteins shown in B1) and B2) and having the same function, obtained by mutating and / or deleting and / or adding to the amino acid sequences shown in B1) and / or B2); B4) A fusion protein with the same function obtained by inserting a fusion tag or signal peptide at the N-terminus and / or C-terminus of B1) and / or B2).

3. The one-step enzymatic chemical detection method for tumor-associated Tn antigen according to claim 1, characterized in that, The small and large molecules with functional tags such as azide, CY3, and Biotin, obtained by chemically derivatizing UDP-Gal, are selected from compounds such as polypeptides, nucleic acids, proteins, and lipids.

4. The one-step enzymatic chemical detection method for tumor-related Tn antigen according to claim 1, characterized in that, Among them, C1GALT1 and C1GALT1C1 are co-expressed.

5. The application of the enzyme chemical labeling method according to claim 1, characterized by any one of the following: C1) Application in the labeling, enrichment, and imaging of Tn antigen in cell lysates; C2) Application in the labeling, enrichment, and imaging of Tn antigen on live cells; C3) Application in the labeling, enrichment, and imaging of Tn antigen in biological samples; C4) The biological samples described in C3) include, but are not limited to, various clinical samples of humans or animals, including blood, tissues, and biological fluids; C5) The enzyme chemical labeling method according to claim 1 is applied to a detection kit; C6) The enzyme chemical labeling method according to claim 1 is applied to the preparation of disease diagnosis and treatment, prognosis products, and targeted intervention drugs.

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