SUMO-Luc probe, synthetic method and application

Abstract

The invention belongs to the technical field of biology, and relates to an SUMO-Luc probe, a synthesis method and application. The molecular formula of the SUMO modified capture probe is SUMO-C17H16N4O4S2, the SUMO is human SUMO-1, the nucleotide sequence of the SUMO is as shown in SEQ ID NO: 1, and the amino acid sequence of the SUMO is as shown in SEQ ID NO: 2. According to the present invention, the gene cloning is adopted to obtain the polypeptide coding the human-derived SUMO-1 protein, the SUMO-C17H16N4O4S2 is obtained through the chemical synthesis method, the structure of the SUMO-C17H16N4O4S2 is stable, and the chemical synthesis and the detection application are convenient. The SUMO-C17H16N4O4S2 fluorescent probe has the advantages that the specificity is high, and the sensitivity is relatively high.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a SUMO-Luc probe, a synthesis method and an application thereof, and specifically refers to a SUMO-modified capture probe (SUMO-(S, E)-2-(6-(4-acetylaminobutyric-2-amido)benzo[d]thiazol-2-yl)-4,5-dihydrothiazolidine-4-carboxylic acid) (SUMO-C 17 H 16 N 4 O 4 S 2 ), its synthesis method and application in tumor detection. Background Art

[0002] Breast cancer seriously threatens human life and health, and is the most common cancer in women. It is also one of the most common causes of morbidity and mortality in the world today. Therefore, the prevention and treatment of breast cancer has become a global health issue and a key disease for prevention and treatment by the World Health Organization.

[0003] At present, the pathogenesis of breast cancer is not fully understood, and there are no effective preventive measures. It is well known that the occurrence and development of most cancers, including breast cancer, is a chronic pathological change process. The current diagnostic methods for breast cancer can only diagnose cancer when the tumor has grown to a certain size, which is too late in many cases. The current treatment of breast cancer is still mainly drug therapy, radiotherapy and surgical resection, but there is no effective diagnosis and treatment method so far. Although surgery removes the tumor lesions, it also partially or completely removes the corresponding tissues and organs, which lose their normal functions; while radiotherapy kills tumor cells with radiation, it also damages the body's normal tissue cells, which greatly limits tumor treatment; drug therapy has a weak efficacy and strong toxic side effects, which causes great physical and psychological trauma to patients. Like other cancers, the effective prevention and treatment of breast cancer also needs to clarify and explain the pathogenesis of breast cancer and how to discover and diagnose it early, so early detection of breast cancer is particularly important.

[0004] Small ubiquitin-related modifier (SUMO) modification is an important dynamic reversible protein post-translational modification, which has been discovered for more than 20 years. So far, more than 3,000 SUMO-modified proteins have been discovered and confirmed, and SUMO modification has important regulatory effects on the functions of target proteins, such as gene transcription regulation, protein stability, signal transduction, cell sublocalization, enzyme activity, cell cycle regulation, cell differentiation, etc. At present, four different SUMO protein subtypes have been identified in mammals, namely SUMO-1, SUMO-2, SUMO-3 and SUMO-4. With the participation of E1 activating enzyme, E2 conjugating enzyme and E3 ligase, the C-terminal diGly of SUMO molecules is covalently bound to the lysine side chain ε-NH2 on the target protein through an isopeptide bond, regulating the structure and function of the substrate protein. For example, the lysine residues at positions 254, 266, and 289 of the PTEN (phosphatidylinositol-3,4,5-trisphosphate-3-phosphatase) protein can be modified by SUMO-1 and SUMO-2, which downregulate the PI3K / AKT pathway and inhibit cell proliferation and tumor growth. SUMOylation of p53 occurs at the lysine residue at position 386, and members of the PIAS (protein inhibitor of activated STATs) family can enhance the stability of p53. At the same time, studies have also confirmed that SUMOylation can enhance the transcriptional activity of p53, leading to cell apoptosis. Multiple proteins that interact with p53 can be SUMOylated, and the role of core factors of signaling pathways such as NF-κB and PTEN in tumor development at least partially depends on the activity of SUMO proteins. Therefore, SUMOylation is one of the most promising targets for cancer detection and treatment in the future. By studying relevant SUMO fluorescent probes and understanding the dynamic changes in SUMOylation levels during tumor development, new ideas can be opened up for early diagnosis and treatment of tumors.

[0005] SUMOylation of proteins is a dynamic and reversible process, and the deSUMOylation process is regulated by deSUMOylation enzymes. DeSUMOylation enzymes (DSPs) are composed of a group of SUMO-specific protease families, SENPs (Sentrin / SUMO-specific protease). SENPs can regulate the SUMOylation level and activity of target proteins, and their own expression or activity is also regulated by some regulatory factors. Therefore, deSUMOylation enzymes are considered to be drug treatment targets. Through sequence alignment, it was found that SENPs all have an enzyme active region of about 200 amino acids, belonging to C48 cysteine ​​proteases, which can cut the isopeptide bond between SUMO and target proteins. Based on this characteristic of DSP, a molecular probe targeting cysteine ​​in the active center of DSP is designed, which can specifically recognize and covalently bind to cysteine ​​without cutting the isopeptide bond between SUMO and target proteins, so that deSUMOylation reaction will not occur.

[0006] Since SUMOylation is a dynamic process, it is very difficult to locate and dynamically detect SUMOylation in cells. Currently, there is an urgent need for a detection method to accurately locate and dynamically monitor SUMOylation. In particular, in some tumor cells, the degree of SUMOylation is closely related to the malignancy and prognosis of the tumor. Therefore, the cell localization, quantitative analysis and tracking of dynamic modification changes of SUMOylation will help in the diagnosis and prognosis of malignant tumors. Summary of the invention

[0007] In order to solve the problem in SUMOylation detection, the present invention provides a SUMOylation modification capture probe, which can quickly detect the subcellular localization, modification degree and dynamic modification change process of SUMOylation modified proteins in cells.

[0008] The technical solution of the present invention is:

[0009] A SUMO-Luc probe, whose molecular formula is SUMO-C 17 H 16 N 4 O 4 S 2 , the structural formula is shown in the following formula (I):

[0010]

[0011] The SUMO is human SUMO-1; the nucleotide sequence of the human SUMO-1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0012] The above-mentioned method for synthesizing the SUMO-Luc probe comprises the following steps:

[0013] Step 1. SUMO1(1-97) was subcloned into the pTXB1 vector and expressed in Rosetta 2(DE3)pLacI cells. When OD600 reached 0.8, the culture was cooled to 18°C ​​for 1 hour and then induced with 0.5mM IPTG. The cells were grown at 18°C ​​for 12-16 hours, centrifuged at 4000rpm for 10 minutes, and purified immediately.

[0014] Step 2: Resuspend the cells obtained in step 1 in buffer A (20 mM HEPES, 50 mM sodium acetate, 75 mM NaCl, water, pH 6.5) supplemented with DNase I, and lyse with ultrasound. Clear the suspension by centrifugation and filter through a 0.45 μm filter.

[0015] Step 3. The clarified lysate from step 2 was loaded onto a pre-equilibrated chitin resin (NEB S6651L) and incubated overnight at 7°C with overhead rotation. The resin was washed with 1L high salt buffer (20mM HEPES, 50mM sodium acetate, 500mM NaCl, solvent is water, pH 6.5), and then washed with 100mL buffer A. Elution was started by adding 50mL sodium 2-mercaptoethanesulfonate solution (0.5g MesNa, 100mM buffer A). After incubation at 7°C for 24-36 hours, the eluate was collected and concentrated to less than 5mL. The protein was purified into buffer A by size exclusion chromatography (hilload 26 / 600Superdex 75pg, GE Healthcare), analyzed on a gel, concentrated, and stored at -80°C to obtain SUMO1-mesna.

[0016] Step 4: The SUMO1-mesna obtained in step 3 was reacted with hydrazine monohydrate. After 30 minutes at room temperature, the mixture was concentrated, filtered, separated and purified (hilload 16 / 600 Superdex 75 pg) with buffer B (20 mM MES, 50 mM NaCl, solvent is water, pH 6.7). The SUMO1-mesna protein was verified by LC-MS, and the mixture was dissolved in a mixed solution of TFA and DCM in a volume ratio of 1:1, stirred at room temperature for 1 hour, monitored by TLC, and the organic phase was concentrated in vacuo. The residue was dried overnight under high vacuum to obtain SUMO1-mesna C-terminal azide.

[0017] Step 5: Treat the SUMO1-mesna C-terminal azide solution (concentration 0.5-6.0 mM, solvent is water) in step 4 in nitrous acid at -10°C for 2-10 minutes to obtain SUMO1-NHNH2 acyl azide which is easily hydrolyzed.

[0018] Step 6. Add sodium nitrite (1M, 50μL, solvent is water) and citric acid (200mM, 50μL, solvent is water) to the SUMO1-NHNH2 solution obtained in step 5 at -10°C. After 2 minutes, add link-amino-D-fluorescein (10mM link-amino-D-fluorescein, 1.5M HEPES, pH 8.0, total volume is 200μL) and incubate at 30°C for 3 minutes. Analyze the reaction by LC-MS, dilute to 40mL, and dialyze into buffer C (20mM MES, solvent is water, pH 6.0) overnight. Then, in buffer C, elute with a gradient of 0-300mM NaCl solution and purify by cation exchange chromatography. Mix the protein-containing fractions and dialyze into a low pH buffer (20mM NaOAc, solvent is water, pH 4.5). SUMO1-Luc was obtained by cation exchange chromatography (Resource S) with gradient elution of 0-300 mM NaCl solution in low pH buffer. SUMO1-Luc was concentrated, quickly frozen in liquid nitrogen, stored at -80°C, and verified by LC-MS.

[0019] The use of the SUMOylation capture probe of the present invention for detecting SUMOylation modification includes cellular localization, quantitative analysis and / or dynamic modification change analysis of SUMOylation modification, and these characteristics are used in the preparation of reagents for cancer diagnosis and / or prognosis.

[0020] The beneficial effects of the present invention are as follows: the present invention uses gene cloning to obtain a polypeptide encoding a human SUMO-1 protein, and obtains SUMO-C by a chemical synthesis method. 17 H 16 N 4 O 4 S 2 The structure is stable, convenient for chemical synthesis and detection applications, SUMO-C 17 H 16 N 4 O 4 S 2 Fluorescent probes are highly specific and sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the three-dimensional spatial structure diagram of the human SUMO-1 molecule.

[0022] Figure 2 Mass spectra for SUMO protein identification.

[0023] Figure 3 SUMO-1-C 17 H 16 N4 O 4 S 2 Fluorescent probe identification mass spectra.

[0024] Figure 4 SUMO-1-C 17 H 16 N 4 O 4 S 2 The luminescence intensity of the fluorescent probe molecule was detected in MCF7. DETAILED DESCRIPTION

[0025] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0026] Example 1

[0027] Cloning and expression of human SUMO-1 gene

[0028] Total RNA was extracted from human breast cancer MCF-7 cells, mRNA was purified, mRNA was reverse transcribed, and cDNA library was constructed. Primers were designed and PCR was used to screen the human SUMO1 gene. The 5' end primer was 5'-ATGTCTGACCAGGAGGCAAAACCT-3' (SEQID NO: NM_003352.8), and the 3' end primer was 5'-CTAAACTGTTGAATGACCCCCCGT-3' (SEQ ID NO: NM_003352.8). The obtained positive monoclonal was sequenced for gene nucleotide sequencing. Gene sequencing results showed that the sequence encoding human SUMO-1 consisted of 306 nucleotides, and the sequence from 5' to 3' was (SEQ ID NO: 1):

[0029] ATGTCTGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAA

[0030] GGAAGGTGAATATATTAAACTCAAAGTCATTGGACAGGATAGCAGTGAGAT

[0031] TCACTTCAAAGTGAAAATGACAACACATCTCAAGAAACTCAAAGAATCAT

[0032] ACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGTTTCCTCTTTGAGG

[0033] GTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATGGAGGAA

[0034] GAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGT

[0035] TTAG

[0036] Encodes a human SUMO-1 protein molecule, whose amino acid sequence is (SEQ ID NO: 2; NP_001005781.1):

[0037] Met-Ser-Asp-Gln-Glu-Ala-Lys-Pro-Ser-Thr-Glu-Asp-Leu-Gly-Asp-Lys-Lys-Glu-Gly-Glu-Tyr-Ile-Lys-Leu-Lys-Val-Ile-Gly-Gln-Asp-Ser-Ser-Glu-Ile-His-Phe-Lys-Val -Lys-Met-Thr-Thr-His-Leu-Lys-Lys-Leu-Lys-Glu-Ser-Yyr-Cys-Gln-Arg-Gln-Gly-Val-Pro-Met-Asn-Ser-Leu-Arg-Phe-Leu-Phe-Glu-Gly-Gln-Arg-Ile-Ala-Asp-Asn-His-Th r-Pro-Lys-Glu-Leu-Gly-Met-Glu-Glu-Glu-Asp-Val-Ile-Glu-Val-Tyr-Gln-Glu-Gln-Thr-Gly-Gly-His-Ser-Thr-Val

[0038] (MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESY CQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV)

[0039] The three-dimensional structure of human SUMO-1 molecule is as follows Figure 1 shown.

[0040] Specifically, the cloning of the gene of SUMO-1 molecule includes:

[0041] 1) Total RNA extraction from human MCF7 cells:

[0042] ① Take MCF7 cells cultured in a 35 mm culture dish, add 1 ml RNAiso Plus (Trizol, TAKARA, Japan), place on a decolorization shaker, and lyse for 5 minutes at room temperature.

[0043] ② Transfer the cell lysate to a 1.5 ml centrifuge tube, add 200 μl of chloroform to the tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0044] ③Put the centrifuge tube in ② into a low-temperature refrigerated centrifuge and centrifuge at 12000 rpm for 15 minutes at 4°C.

[0045] ④ Transfer the supernatant to a 1.5 ml RNase-Free centrifuge tube, add 500 μl of isopropanol, gently invert the centrifuge tube to mix the liquid, and let it stand at room temperature for 10 minutes.

[0046] ⑤ Place the centrifuge tube in ④ into a low-temperature refrigerated centrifuge and centrifuge at 12000 rpm for 10 min at 4°C.

[0047] ⑥ Remove the supernatant, add 1 ml of 75% ethanol to the centrifuge tube, and gently blow to precipitate the RNA.

[0048] ⑦ Place the centrifuge tube in ⑥ into a low-temperature refrigerated centrifuge and centrifuge at 5000 rpm for 3 minutes at 4°C.

[0049] ⑧ Carefully remove the supernatant with a pipette and leave it at room temperature for a few minutes to dry the RNA.

[0050] ⑨ Add 30 μl of RNase-Free water to the centrifuge tube in ⑧ to dissolve the RNA.

[0051] 2) Construction of human cDNA library:

[0052] I.cDNA first strand synthesis (mRNA reverse transcription):

[0053] ① Add 1.0 μl of total RNA from human MCF7 cells, 1.0 μl of Oligo(dT)primer and 5.0 μl of RNase-Free ddH 2 O, make the total volume reach 7μl, mix and centrifuge briefly (2000rpm, 30s), incubate at 72℃ for 10 minutes after centrifugation; after incubation, incubate the centrifuge tube at 4℃ for 2 minutes.

[0054] ② Add the following reagents to the above centrifuge tube: 2.0μl 5×M-MLV Buffer, 0.5μl 10mM dNTPMix, 0.5μl 40U / μl RNase Inhibitor and 0.25μl RTase M-MLV. Mix the reagents in the centrifuge tube and centrifuge briefly (2000rpm, 30s). Incubate at 42℃ for 60min, then at 70℃ for 15s. After incubation, place the centrifuge tube on ice to terminate the synthesis and set aside.

[0055] II. Amplification of SUMO1 gene by polymerase chain reaction (PCR):

[0056] ① Mix 1 μl cDNA template, 0.25 μl Taq enzyme, 10 μl 5× PCR buffer, 1 μl 10 mM dNTP, 1.0 μl 5' PCR primer, 1.0 μl 3' PCR primer and 35.75 μl ddH 2 O was mixed in a PCR tube and centrifuged to allow the mixture to accumulate at the bottom of the tube.

[0057] ② Amplify in a PCR instrument according to the following program: 95℃, 5min; 35 cycles: 94℃, 30sec, 56℃, 30sec, 72℃, 1min. After the cycle, incubate at 72℃ for 10min and store in a -80℃ refrigerator after PCR.

[0058] 3) Human SUMO-1 gene cloning and screening:

[0059] After the PCR reaction, the PCR reaction product was electrophoresed using 1% agarose gel. After 30 minutes of constant voltage at 120V, it was placed on a gel imager for observation and photography. The gel containing the target fragment of SUMO-1 was cut and recovered using an agarose gel DNA recovery kit. The recovered target fragment was connected to the pTXB1 vector and transformed into DH5α competent cells. The plate was coated and double screened with ampicillin and blue-white staining. A single colony was picked and the size of the inserted fragment was detected by PCR with M13 primers. Positive colonies were picked, and the plasmid was extracted by shaking the bacteria and sent to Sangon Biotech (Shanghai) Co., Ltd. for nucleotide sequencing and translated into amino acid sequence.

[0060] 4) SUMO protein expression and in vitro purification

[0061] pTXB1-SUMO1 was transformed into E. coli BL21, and positive clones were screened and cultured in shake flasks. When OD 600 reached 0.8, the culture was cooled to 18°C ​​for 1 h and induced with 0.5 mM IPTG. The cells were grown at 18°C ​​for 12-16 h, harvested, sonicated, and purified by affinity purification columns. The proteins were then identified by MS (e.g. Figure 2).

[0062] Example 2: SUMO1-MesNa Synthesis

[0063] Step 1. SUMO1(1-97) was subcloned into the pTXB1 vector and expressed in Rosetta2(DE3)pLacI cells as described above. When OD600 reached 0.8, the culture was cooled to 18°C ​​for 1 h and induced with 0.5 mM IPTG. The cells were grown at 18°C ​​for 12-16 h, harvested, centrifuged at 4000 rpm for 10 min, and purified immediately.

[0064] Step 2: Resuspend the cells in step 1 in buffer A (20 mM HEPES, 50 mM sodium acetate, 75 mM NaCl, water, pH 6.5) supplemented with DNase I and lyse with ultrasound. Clear the suspension by centrifugation and filter through a 0.45 μm filter.

[0065] Step 3. The clarified lysate from step 2 was loaded onto a pre-equilibrated chitin resin (NEB S6651L) and incubated overnight at 7°C with overhead rotation. The resin was washed with 1L of high salt buffer (20mM HEPES, 50mM sodium acetate, 500mM NaCl, solvent is water, pH 6.5), and then washed with 100mL of buffer A. Elution was started by adding 50mL of sodium 2-mercaptoethanesulfonate solution (MesNa, 100mM buffer A). After incubation at 7°C for 24-36 hours, the eluate was collected and concentrated to less than 5mL. The protein was purified into buffer A by size exclusion chromatography (hilload26 / 600Superdex 75pg, GE Healthcare), analyzed on a gel, concentrated, and stored at -80°C.

[0066] Example 3: SUMO-C 17 H 16 N 4 O 4 S 2 Probe synthesis

[0067] Step 1. The above SUMO1-mesna was reacted with hydrazine monohydrate (64% water, 500 μL added to 100 mL eluent). After 30 minutes at room temperature, the mixture was concentrated, filtered, separated and purified (hilload 16 / 600 Superdex 75 pg) with a buffer solution (20 mM MES, 50 mM NaCl, solvent is water, pH 6.7). The SUMO1-mesna protein was verified by LC-MS, and the mixture was dissolved in TFA:DCM=1:1 (volume ratio), stirred at room temperature for 1 h, monitored by TLC, and the organic phase was concentrated in vacuo. The residue was dried overnight under high vacuum to obtain SUMO1-mesna C-terminal azide.

[0068] Step 2: Treat the C-terminal azide solution of the concentration (0.5-6.0 mM) in step 1 in nitrous acid at -10°C for 2-10 minutes to obtain an acyl azide that is easily hydrolyzed.

[0069] Step 3. Add sodium nitrite (1M, 50μL, solvent is water) and citric acid (200mM, 50μL, solvent is water) to the SUMO1-NHNH2 solution obtained in step 2 at -10°C. After 2 minutes, add link-amino-D-fluorescein (10mM in 1.5M HEPES pH 8.0, 200μL) and incubate at 30°C for 3 minutes. Analyze the reaction by LC-MS, dilute to 40mL, and dialyze into buffer (20mM MES, solvent is water pH 6.0) overnight. In the same buffer, elute with a gradient of 0-300mM NaCl and purify by cation exchange chromatography. Mix the protein-containing fractions and dialyze into a low pH buffer (20mM NaOAc, solvent is water pH 4.5). In low pH buffer, 0-300 mM NaCl gradient elution was used and further purified by cation exchange chromatography (Resource S). SUMO1-Luc was concentrated, quickly frozen in liquid nitrogen, stored at -80°C, and verified by LC-MS to obtain the product SUMO1-Luc (such as Figure 3 ).

[0070] Example 4: SUMO-C 17 H 16 N 4 O 4 S 2 Application of probe molecules in MCF7 cells

[0071] MCF7 cells were inoculated into 35 mm cell culture plates, and fusion experiments were performed when the cell confluence reached about 40%. 17 H 16 N 4 O4 S 2 The probe molecules were incubated with MCF7 cells and detected by ELISA after 36 hours. Figure 4 As shown, the luminescence intensity of the SUMO probe incubation group was significantly higher than that of the control group.

[0072] The probe detection method is:

[0073] Since SUMO protein can specifically recognize the lysine residues of substrate proteins, the modified proteins convert SUMO-C to SUMO through the action of enzymes related to the SUMO process. 17 H 16 N 4 O 4 S 2 The probe molecule directly binds to the substrate. Since the luciferase contained in the probe molecule will emit light with luciferin as the substrate, the wavelength is 540-600nm, and the light intensity can reflect the expression of luciferase, that is, the amount of SUMO protein in the cell. The SUMO modification in the cell can be located, quantified, and the dynamic changes can be monitored.

Claims

1. A SUMO-Luc probe, characterized in that The molecular formula of the SUMO-Luc probe is SUMO-C 17 H 16 N4O4S2, the structural formula is shown in the following formula (I): The SUMO is human SUMO-1; the nucleotide sequence of the human SUMO-1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. The method for synthesizing the SUMO-Luc probe according to claim 1, characterized in that: The following steps are involved: Step 1, SUMO1 (1-97) was subcloned into the pTXB1 vector and expressed in Rosetta 2 (DE3) pLacI cells; when OD600 reached 0.8, the culture was cooled to 18 ° C and maintained for 1 hour, and then induced with 0.5 mM IPTG; the cells were grown at 18 ° C for 12-16 hours, collected, centrifuged at 4000 rpm for 10 minutes, and purified immediately; Step 2, resuspend the cells obtained in step 1 in buffer A supplemented with DNase I, and lyse them by ultrasound; remove the suspension by centrifugation and filter through a 0.45 μm filter; The buffer A is specifically: 20mM HEPES, 50mM sodium acetate, 75mM NaCl, the solvent is water, pH 6.5; Step 3: The clarified lysate from step 2 was loaded onto the pre-equilibrated chitin resin and incubated overnight at 7°C with overhead rotation; the resin was washed with 1L high salt buffer and then with 100mL buffer A; elution was started by adding 50mL sodium 2-mercaptoethanesulfonate solution; after incubation at 7°C for 24-36 hours, the eluate was collected and concentrated to less than 5mL; the protein was purified by size exclusion chromatography into buffer A, analyzed on a gel, concentrated, and stored at -80°C to obtain SUMO1-mesna; The high salt buffer is specifically: 20mM HEPES, 50mM sodium acetate, 500mM NaCl, the solvent is water, pH 6.5; the sodium 2-mercaptoethanesulfonate solution is specifically: 0.5g MesNa, 100mM buffer A; Step 4, reacting the SUMO1-mesna obtained in step 3 with hydrazine monohydrate; after 30 minutes at room temperature, concentrating, filtering, separating and purifying with buffer B; the SUMO1-mesna protein was verified by LC-MS, dissolving the mixture in a mixed solution of TFA and DCM in a volume ratio of 1:1, stirring at room temperature for 1 hour, monitoring by TLC, concentrating the organic phase in vacuo, and drying the residue under high vacuum overnight to obtain SUMO1-mesna C-terminal azide; The buffer B is specifically: 20mM MES, 50mM NaCl, the solvent is water, pH 6.7; Step 5, treating the SUMO1-mesna C-terminal azide solution (concentration 0.5-6.0 mM, solvent is water) in step 4 in nitrous acid at -10°C for 2-10 minutes to obtain SUMO1-NHNH2 acyl azide that is easily hydrolyzed; The concentration of the SUMO1-mesna C-terminal azide solution is 0.5-6.0 mM, and the solvent is water; Step 6. Add sodium nitrite (1M, 50μL, solvent is water) and citric acid (200mM, 50μL, solvent is water) to the SUMO1-NHNH2 (2mM, 100μL) solution obtained in step 5 at -10°C; 2 minutes later, add link-amino-D-fluorescein and incubate at 30°C for 3 minutes; analyze the reaction by LC-MS, dilute to 40mL, and dialyze into buffer C overnight; then in buffer C, gradient elute with 0-300mM NaCl solution, and purify by cation exchange chromatography; mix the protein-containing parts and dialyze into a low pH buffer; in a low pH buffer, gradient elute with 0-300mM NaCl solution, and further purify by cation exchange chromatography to obtain SUMO1-Luc; concentrate SUMO1-Luc, quickly freeze in liquid nitrogen, store at -80°C, and verify by LC-MS; The concentration of SUMO1-NHNH2 is 2mM, the solvent is water, and the dosage is 100μL; the concentration of sodium nitrite is 1M, the solvent is water, and the dosage is 50μL; the concentration of citric acid is 200mM, the solvent is water, and the dosage is 50μL; The link-amino-D-luciferin is specifically: 10 mM link-amino-D-luciferin, 1.5 M HEPES, pH 8.0, total volume 200 μL; The buffer C is 20 mM MES, the solvent is water, and the pH is 6.0; the pH buffer is 20 mM NaOAc, the solvent is water, and the pH is 4.

5.

3. Use of the SUMO-Luc probe according to claim 1 in the preparation of a reagent for cancer diagnosis and / or prognosis.

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