CN114507278A

CN114507278A - Usu probe UFM1-Lys-TAMRA and synthetic method thereof

Info

Publication number: CN114507278A
Application number: CN202210101211.9A
Authority: CN
Inventors: 李佳斌; 陈聪; 李芳�; 路家琦; 许国强
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-05-17
Anticipated expiration: 2042-01-27
Also published as: CN114507278B

Abstract

The invention belongs to the technical field of protein synthesis, and particularly relates to a Usher probe UFM1-Lys-TAMRA and a synthesis method thereof, wherein the preparation method comprises the following steps of utilizing Usher activating enzyme to activate the C end of the Usher and then carrying out in-situ hydrazinolysis to prepare Usher hydrazide; converting the utahenine hydrazide into utahenine thioester through in-situ oxidation; directly aminolyzing the eugenol thioester to obtain the eugenol probe UFM 1-Lys-TAMRA. The invention firstly uses the Ushinagin activating enzyme UBA5 catalyzed in-situ activation strategy to prepare the Ushinagin hydrazide UFM1-NHNH₂And further converting the acyl hydrazine into the eugenol thioester UFM1-Mesna based on a hydrazide method, and further obtaining the eugenol probe UFM1-Lys-TAMRA by a direct ammonolysis method. Has the characteristics of high preparation yield, high synthesis purity, simple operation, mass preparation, low synthesis cost and the like.

Description

Usin probe UFM1-Lys-TAMRA and synthetic method thereof

Technical Field

The invention belongs to the technical field of protein synthesis, and particularly relates to a Usher probe UFM1-Lys-TAMRA and a synthesis method thereof.

Background

U. jelisine (UFM1) is a ubiquitin-like modifier that can be covalently linked to a substrate protein through a series of specific enzymatic cascades to form a dynamically reversible post-translational modification, a process known as Ufmylation. The euphorbia humifusa is involved in a plurality of important physiological processes, such as endoplasmic reticulum homeostasis, vesicle transport, transcription control, DNA damage response and the like, and the abnormality of the processes is also related to a plurality of human diseases, such as cancer, diabetes, schizophrenia, ischemic heart disease and the like. The protein jewish essence modification is a dynamic reversible process, and jewish essence combined on a substrate can be specifically removed by the enzyme de-jewish essence (UfSPs). The enzyme de-kosher is a key factor in a protein kosher regulation signal and is also considered as a potential drug target. Therefore, the research on the enzyme activity property of the jewish-melting enzyme and the screening of the specific small-molecule inhibitor of the jewish-melting enzyme are of great significance for understanding the control function and mechanism of the jewish-melting enzyme and the diagnosis and treatment of diseases.

The fluorescent probe based on the activity is an important tool for detecting the activity of the deuterosin-removing enzyme and screening the inhibitor of the deuterosin-removing enzyme in high flux.

Disclosure of Invention

The invention aims to provide a synthesis method of Uygur probe UFM1-Lys-TAMRA, which has the characteristics of high yield, high purity, simple operation and mass preparation.

According to the technical scheme of the invention, the synthesis method of Uighur probe UFM1-Lys-TAMRA comprises the following steps,

s1: activating the C end of the eusin by using eusin activating enzyme (UBA5) and then performing in-situ hydrazinolysis to prepare eusin hydrazide;

s2: converting the utahenine hydrazide into utahenine thioester through in-situ oxidation;

s2: directly aminolyzing the eugenol thioester to obtain the eugenol probe UFM 1-Lys-TAMRA.

The invention designs a novel Ujewish element fluorescent probe UFM1-Lys-TAMRA which can be applied to monitoring the activity of Ujewish element removing enzyme and high-flux screening of Ujewish element removing enzyme inhibitor and the like, and the principle is as follows: deutahenisin can specifically recognize isopeptide bond at the tail end of UFM1-Lys-TAMRA and hydrolyze the isopeptide bond to release a fluorescent group Lys-TAMRA. When excited by polarized light, UFM1-Lys-TAMRA emits primarily polarized light, while free Lys-TAMRA emits primarily depolarized light, and degauscultation enzyme activity can be detected by monitoring the change in fluorescence polarization. Compared with the currently reported Ushin fluorescent probe UFM1-AMC, the fluorescent probe UFM1 has the advantages that the change of fluorescence polarization is detected, the interference of the fluorescence of an inhibitor can not be caused when a small-molecule inhibitor is screened, and the UFM1 is connected with a lysine side chain amino group on a fluorescent group Lys-TAMRA through an isopeptide bond, so that Ushin modification under physiological conditions can be better simulated.

Specifically, the amino acid sequence of U. jelisii is shown as SEQ ID No.2 and can be obtained by expression of recombinant bacteria, the recombinant bacteria are escherichia coli BL21(DE3) which is used for transfecting a target gene (the nucleotide sequence is shown as SEQ ID No. 3), and the steps are as follows:

1a, selecting UFM1 monoclonal colony to 16mL LB culture medium containing ampicillin resistance, culturing at 37 deg.C and 200rpm for 14 h;

1b, adding the bacterial liquid cultured in the step 1a into 1L LB culture medium containing ampicillin resistance, continuously culturing at 37 ℃ and 200rpm, adding 800 mu L of 1.0M IPTG (isopropyl-beta-D-thiogalactoside) to induce the bacterial liquid when the OD 600 light absorption value of the bacterial liquid reaches 0.6-0.8, and continuously culturing at 16 ℃ and 180rpm for 18 hours;

1c, centrifugally collecting the bacterial liquid after the culture in the step 1b, removing the supernatant, fully suspending the obtained thalli by using a lysis buffer (30mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), 150mM NaCl, pH 7.4) (the thalli obtained by each 1L of LB culture medium is suspended in 20mL of the lysis buffer), and lysing the thalli by using an ultrasonic disruptor;

1d, centrifuging the bacterial lysate obtained in 1c by using a high-speed refrigerated centrifuge (12000rpm, 30min, 4 ℃), collecting the supernatant, and adding the supernatant to the supernatant according to a volume ratio of 100: 1, adding trifluoroacetic acid, stirring at room temperature for 20min to precipitate impure protein, centrifuging at high speed (12000rpm, 15min, 4 ℃) and collecting supernatant;

1e, transferring the supernatant collected in step 1d to a dialysis bag intercepted by 3KDa, placing the dialysis bag in a dialysis buffer solution (30mM HEPES, pH 7.4) for dialysis at room temperature for 6 hours, replacing the dialysis buffer solution, and repeating dialysis once;

and 1f, concentrating the protein solution dialyzed by the 1e until the final concentration of the protein is 4mg/mL to obtain the kosher concentrated solution.

Further, the amino acid sequence of the kosher activating enzyme is shown as SEQ ID No.1 and can be obtained by expression of recombinant bacteria, the recombinant bacteria are escherichia coli BL21(DE3) transfected with a target gene (the nucleotide sequence is shown as SEQ ID No. 4), and the steps are as follows:

2a, picking His₆-UBA5 monoclonal colonies were cultured in 16mL LB medium containing ampicillin resistance at 37 ℃ for 14h at 200 rpm;

2b, adding the bacterial liquid cultured in the step 2a into 1L LB culture medium containing ampicillin resistance, continuing to culture at 37 ℃ and 200rpm, adding 800 mu L of 1.0M IPTG to induce the bacterial liquid when the light absorption value of the OD 600 of the bacterial liquid reaches 0.6-0.8, and continuing to culture at 37 ℃ and 180rpm for 18 hours;

2c, centrifugally collecting the bacterial liquid after the culture in the step 2b, discarding the supernatant, fully suspending the obtained bacterial cells with a lysis buffer (30mM HEPES, 150mM NaCl, pH 7.4) (the obtained Escherichia coli is suspended in 20mL of the lysis buffer per 1L of LB medium), and lysing the bacterial cells by using an ultrasonic disruptor;

2d, centrifuging the cell disruption solution obtained in the step 2c by using a high-speed refrigerated centrifuge, collecting a supernatant, adding the supernatant into 3mL of Ni-NTA beads for incubation, eluting by using a high-concentration imidazole buffer (30mM HEPES, 150mM NaCl, 400mM imidazole, pH 7.4), and collecting an eluent;

2e, the eluate was transferred to a 3KDa cut-off dialysis bag and dialyzed in dialysis buffer (30mM HEPES, 150mM NaCl, 1mM DTT, pH 7.4) at 4 ℃ for 12 h;

2f, concentrating the protein solution dialyzed by the 2e until the final protein concentration is 2mg/mL to obtain a UBA5 concentrated solution.

Further, the specific operation of step S1 is: adding magnesium chloride, ATP (adenosine triphosphate), hydrazine and the jewish enzyme into the jewish solution, adding a lysis buffer solution, adjusting the pH to 7.0-8.0, and reacting to obtain the jewish hydrazide.

Further, the hydrazine is selected from one or more of hydrazine hydrochloride, hydrazine hydrate and hydrazine acetate.

Further, the molar ratio of the jewish essence to the jewish essence activating enzyme is 45-60: 1; preferably, the molar ratio is 50: 1.

preferably, 2mL of 1M hydrazine hydrochloride, 200. mu.L of 100mM ATP, 200. mu.L of 1M MgCl are added to 10mL of the kosher protein concentrate (4 mg/mL)₂2mL of 2mg/mL UBA5, and 20mL of lysis buffer.

Specifically, adding magnesium chloride, ATP, hydrazine hydrochloride and UBA5 concentrated solution obtained from 2f into the U. sequin protein concentrated solution obtained from 1f, diluting the volume of the reaction solution to be twice of that of the 1f protein concentrated solution by using a lysis buffer solution, uniformly mixing, adjusting the pH of the solution to 7.5, then placing the solution in a 37 ℃ oven, stirring the solution by using a rotor for 12 hours, after the reaction is finished, purifying the reaction solution by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain the Usequin hydrazide UFM1-NHNH₂。

Further, the specific operation of step S2 is:

dissolving the utahenoside hydrazide in a buffer solution containing guanidine hydrochloride, adding an acidic sodium nitrite solution, and reacting at the temperature of-15 to-5 ℃;

adding mesna (sodium mercaptoethanesulfonate), adjusting the pH value to 4.0-6.0, and reacting to obtain the kosher thioester.

Further, the molar ratio of the utahenzine hydrazide to the sodium nitrite to the mesna is 1: 8-12: 35-50 parts of; preferably, the molar ratio is 1: 10: 40.

preferably, 0.03mmol of sodium nitrite and 20mg of mesna are added into 30mg of the utahensu hydrazide.

Specifically, 1 equivalent of UFM1-NHNH was added₂Dissolving in PBS buffer solution containing 6.0M guanidine hydrochloride with pH of 3.0, adding 10 times equivalent of acidic sodium nitrite solution, and reacting at-10 deg.C for 20 min; then adding 40 times of equivalent of mesna, adjusting the pH value to 5.0, and reacting for 20min at room temperature; after the reaction is finished, purifying by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain Usherbet thioester UFM 1-Mesna;

further, the specific operation of step S3 is: dissolving the isorhodin thioester in a mixed solvent, adding sodium thiophenolate and Lys-TAMRA, and reacting to obtain the isorhodin probe UFM 1-Lys-TAMRA.

Further, the mixed solvent is a mixed solution of an organic solvent and a buffer salt.

Further, the organic solvent is dimethyl sulfoxide, the buffer salt is HEPES, and the molar ratio of the dimethyl sulfoxide to the HEPES is 5-7: 1, preferably, the concentration of dimethyl sulfoxide in the mixed solvent is 6M, and the concentration of HEPES is 1M.

Further, the molar ratio of the isorhodin thioester, sodium thiophenolate and Lys-TAMRA is 1: 160-250: 25-40; preferably, the molar ratio is 1: 200: 30.

preferably, 26.4mg of sodium thiophenolate, 16mg Lys-TAMRA is added per 10mg of the jewish thioester.

Specifically, dissolving the prepared UFM1-Mesna protein dry powder with an organic solvent-buffer salt mixed buffer solution, adding 0.2M sodium thiophenolate, adding 30 equivalents of Lys-TAMRA, dissolving, stirring at 37 ℃ for reaction for 2h, quenching the reaction solution with PBS buffer solution containing 6.0M guanidine hydrochloride with the pH value of 3.0 after the reaction is completed, centrifuging (12000rpm, 15min, 4 ℃), purifying the supernatant by using semi-preparative high performance liquid chromatography, collecting the purified solution (the elution gradient is 30-60% acetonitrile, 30min), and freeze-drying to obtain the Ushernin fluorescent probe M1-Lys-TAMRA.

Compared with the prior art, the technical scheme of the invention has the following advantages: the invention firstly uses the Ushinagin activating enzyme UBA5 catalyzed in-situ activation strategy to prepare the Ushinagin hydrazide UFM1-NHNH₂And further converting the acyl hydrazine into the eugenol thioester UFM1-Mesna based on a hydrazide method, and further obtaining the eugenol probe UFM1-Lys-TAMRA by a direct ammonolysis method. Has the characteristics of high preparation yield, high synthesis purity, simple operation, mass preparation, low synthesis cost and the like.

Drawings

FIG. 1 shows Ustilagin hydrazide UFM1-NHNH₂The high performance liquid chromatogram of (1).

FIG. 2 is Ustilagin hydrazide UFM1-NHNH₂Mass spectrum of (2).

FIG. 3 is a high performance liquid chromatogram of Usin probe UFM 1-Lys-TAMRA.

FIG. 4 is a mass spectrum of Usin probe UFM 1-Lys-TAMRA.

FIG. 5 is a schematic diagram of the synthesis of Utilin probe UFM 1-Lys-TAMRA.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

A single colony of UFM1 was picked into 16mL LB medium containing ampicillin resistance and cultured at 37 ℃ and 200rpm for 14 h. Subsequently, 16mL of the bacterial solution was added to 1L of LB medium (1g of peptone, 1g of NaCl, 0.5g of yeast powder, 5mg of Amp, 100mL of double distilled water) containing ampicillin resistance, and the culture was continued at 37 ℃ and 200rpm, and when the OD 600 absorbance of the bacterial solution reached 0.6 to 0.8, 800. mu.L of 1.0M IPTG was added to make the final concentration of IPTG 0.8mM to induce the bacterial solution, and the culture was continued at 16 ℃ and 180rpm for 18 hours.

Centrifugally collecting the cultured bacterial liquid, removing the supernatant, fully suspending the obtained thalli by using 20mL of lysis buffer (30mM HEPES, 150mM NaCl, pH 7.4), and lysing the thalli by using an ultrasonication instrument; the obtained bacterial lysate was centrifuged (12000rpm, 30min, 4 ℃) by using a high-speed refrigerated centrifuge, and the supernatant was collected, 200. mu.L of trifluoroacetic acid was added to the supernatant, and the mixture was stirred at room temperature for 20min to precipitate impure proteins, and the supernatant was collected after high-speed centrifugation (12000rpm, 15min, 4 ℃).

The collected supernatant was transferred to a 3kDa cut-off dialysis bag and placed in 500mL of dialysis buffer (30mM HEPES, 150mM NaCl, pH 7.4) for dialysis at room temperature for 6h, the dialysis buffer was changed, and the dialysis was repeated once.

And concentrating the dialyzed protein solution until the final protein concentration is 4mg/mL to obtain 10mL of protein concentrate.

Example 2

Picking His₆Single colonies of UBA5 were cultured in 16mL of LB medium containing ampicillin resistance at 37 ℃ and 200rpm for 14 hours. Adding 16mL of the bacterial liquid into 1L of LB culture medium containing ampicillin resistance, continuing the culture at 37 ℃ and 200rpm, and adding 800 μ L of 1.0M IPTG to enable I to be added when the OD 600 light absorption value of the bacterial liquid reaches 0.6-0.8The PTG final concentration is 0.8mM, and the bacterial liquid is induced and cultured for 20 hours at 37 ℃ and 180 rpm;

the cultured bacterial solution was collected by centrifugation, the supernatant was discarded, and the resulting cells were thoroughly resuspended in 20mL of lysis buffer (30mM HEPES, 150mM NaCl, pH 7.4), and lysed using an ultrasonication apparatus.

The obtained bacterial disrupted solution was centrifuged using a high-speed refrigerated centrifuge (12000rpm, 30min, 4 ℃), the supernatant was collected, the supernatant was added to 3mL of Ni-NTA beads for incubation, and eluted using 4mL of a high-concentration imidazole buffer (30mM HEPES, 150mM NaCl, 400mM imidazole, pH 7.4), and the eluate was collected.

The eluate was transferred to a dialysis bag with a 3kDa cut-off and dialyzed at 4 ℃ for 12 hours in 1L of dialysis buffer (30mM HEPES, 150mM NaCl, 1mM DTT, pH 7.4). And concentrating the dialyzed protein solution until the final protein concentration is 2mg/mL to obtain 3mL of protein concentrate.

Example 3

Adding 2mL of 1M magnesium chloride, 200 μ L of 100mM ATP, 2mL of 1M hydrazine hydrochloride (pH 7.4) and 2mL of UBA5 concentrated solution into the obtained 10mL of UFM1 protein concentrated solution, diluting the reaction solution to 20mL by using a lysis buffer solution, uniformly mixing, adjusting the pH of the solution to 7.5, then placing the solution in a 37 ℃ oven, stirring for 12h, after the reaction is finished, purifying the reaction solution by using semi-preparative high performance liquid chromatography (the elution gradient is 30-60% acetonitrile, 30min), collecting the purified solution, and freeze-drying to obtain the UJUJUJUN hydrazide M1-NHNH₂About 30 mg. Obtained hydrazide UFM1-NHNH₂The high performance liquid chromatogram of (A) is shown in FIG. 1, and the mass spectrum is shown in FIG. 2.

Example 4

30mg of UFM1-NHNH₂Dissolving in 3mL PBS buffer solution containing 6.0M guanidine hydrochloride with pH of 3.0, adding 150 μ L0.2M acidic sodium nitrite solution, and reacting at-10 deg.C for 20 min; adding 20mg of mesna, adjusting the pH value to 5.0, and reacting for 20min at room temperature; after the reaction is finished, semi-preparative high performance liquid chromatography is used for purification (the elution gradient is 30-60% acetonitrile, 30min), the purified solution is collected, and the UFM1-Mesna is obtained after freeze drying.

Example 5

10mg of UFM1-Mesna protein dry powder was mixed with 1mL of dimethyl sulfoxide: dissolving 6M guanidine hydrochloride and 1M HEPES (6: 1) mixed solvent, adding 26.4mg of thiophenol sodium, adding 16mg of Lys-TAMRA, stirring at 37 ℃ for reaction for 2h, after the reaction is finished, quenching the reaction liquid by using 3mL of PBS buffer solution containing 6.0M guanidine hydrochloride and having the pH value of 3.0, centrifuging (12000rpm, 15min and 4 ℃), purifying the supernatant by using semi-preparative high performance liquid chromatography, collecting the target component, and freeze-drying to obtain the Ushin probe UFM 1-Lys-TAMRA.

The high performance liquid chromatogram of the obtained Ushinsen probe UFM1-Lys-TAMRA is shown in FIG. 3, the mass spectrum is shown in FIG. 4, and the overall synthesis schematic diagram is shown in FIG. 5.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

<110> Suzhou university

<120> Usin probe UFM1-Lys-TAMRA and synthetic method thereof

<130> 1.17

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 405

<212> PRT

<213> (Artificial Synthesis)

<400> 1

His His His His His His Ala Glu Ser Val Glu Arg Leu Gln Gln Arg

1 5 10 15

Val Gln Glu Leu Glu Arg Glu Leu Ala Gln Glu Arg Ser Leu Gln Val

20 25 30

Pro Arg Ser Gly Asp Gly Gly Gly Gly Arg Val Arg Ile Glu Lys Met

35 40 45

Ser Ser Glu Val Val Asp Ser Asn Pro Tyr Ser Arg Leu Met Ala Leu

50 55 60

Lys Arg Met Gly Ile Val Ser Asp Tyr Glu Lys Ile Arg Thr Phe Ala

65 70 75 80

Val Ala Ile Val Gly Val Gly Gly Val Gly Ser Val Thr Ala Glu Met

85 90 95

Leu Thr Arg Cys Gly Ile Gly Lys Leu Leu Leu Phe Asp Tyr Asp Lys

100 105 110

Val Glu Leu Ala Asn Met Asn Arg Leu Phe Phe Gln Pro His Gln Ala

115 120 125

Gly Leu Ser Lys Val Gln Ala Ala Glu His Thr Leu Arg Asn Ile Asn

130 135 140

Pro Asp Val Leu Phe Glu Val His Asn Tyr Asn Ile Thr Thr Val Glu

145 150 155 160

Asn Phe Gln His Phe Met Asp Arg Ile Ser Asn Gly Gly Leu Glu Glu

165 170 175

Gly Lys Pro Val Asp Leu Val Leu Ser Cys Val Asp Asn Phe Glu Ala

180 185 190

Arg Met Thr Ile Asn Thr Ala Cys Asn Glu Leu Gly Gln Thr Trp Met

195 200 205

Glu Ser Gly Val Ser Glu Asn Ala Val Ser Gly His Ile Gln Leu Ile

210 215 220

Ile Pro Gly Glu Ser Ala Cys Phe Ala Cys Ala Pro Pro Leu Val Val

225 230 235 240

Ala Ala Asn Ile Asp Glu Lys Thr Leu Lys Arg Glu Gly Val Cys Ala

245 250 255

Ala Ser Leu Pro Thr Thr Met Gly Val Val Ala Gly Ile Leu Val Gln

260 265 270

Asn Val Leu Lys Phe Leu Leu Asn Phe Gly Thr Val Ser Phe Tyr Leu

275 280 285

Gly Tyr Asn Ala Met Gln Asp Phe Phe Pro Thr Met Ser Met Lys Pro

290 295 300

Asn Pro Gln Cys Asp Asp Arg Asn Cys Arg Lys Gln Gln Glu Glu Tyr

305 310 315 320

Lys Lys Lys Val Ala Ala Leu Pro Lys Gln Glu Val Ile Gln Glu Glu

325 330 335

Glu Glu Ile Ile His Glu Asp Asn Glu Trp Gly Ile Glu Leu Val Ser

340 345 350

Glu Val Ser Glu Glu Glu Leu Lys Asn Phe Ser Gly Pro Val Pro Asp

355 360 365

Leu Pro Glu Gly Ile Thr Val Ala Tyr Thr Ile Pro Lys Lys Gln Glu

370 375 380

Asp Ser Val Thr Glu Leu Thr Val Glu Asp Ser Gly Glu Ser Leu Glu

385 390 395 400

Asp Leu Met Ala Lys

405

<210> 2

<211> 83

<212> PRT

<213> (Artificial Synthesis)

<400> 2

Met Ser Lys Val Ser Phe Lys Ile Thr Leu Thr Ser Asp Pro Arg Leu

1 5 10 15

Pro Tyr Lys Val Leu Ser Val Pro Glu Ser Thr Pro Phe Thr Ala Val

20 25 30

Leu Lys Phe Ala Ala Glu Glu Phe Lys Val Pro Ala Ala Thr Ser Ala

35 40 45

Ile Ile Thr Asn Asp Gly Ile Gly Ile Asn Pro Ala Gln Thr Ala Gly

50 55 60

Asn Val Phe Leu Lys His Gly Ser Glu Leu Arg Ile Ile Pro Arg Asp

65 70 75 80

Arg Val Gly

<210> 3

<211> 252

<212> DNA

<213> (Artificial Synthesis)

<400> 3

atgagtaaag tatcatttaa gataacacta acgtccgacc cgcgcctgcc gtacaaagtc 60

ctgagcgttc cggaaagcac cccgtttacc gctgtgttaa agttcgccgc ggaagagttc 120

aaagtgccag cggctacctc tgcaattatc accaacgacg gtatcggcat caacccggcg 180

cagactgcgg gtaatgtttt tttgaagcac ggcagcgagc tgcgtattat tccgcgtgat 240

cgtgttggtt aa 252

<210> 4

<211> 1233

<212> DNA

<213> (Artificial Synthesis)

<400> 4

atgcaccacc atcaccacca tgctgaatca gtagaacgcc tgcaacagcg tgttcaagaa 60

cttgagcgcg aattagcgca ggagcgcagc ctgcaagttc cgcgttccgg cgatggtgga 120

ggcggcagag ttcgtatcga gaagatgagc agcgaggttg tggacagcaa tccgtatagc 180

cgtcttatgg cgctgaagcg catgggcatc gtttcggact acgagaagat tcgtaccttt 240

gctgtggcga ttgttggtgt gggtggcgtt ggttccgtga ctgctgaaat gctgacccgt 300

tgtggtatag gcaaactgtt gttgttcgat tacgacaaag tggaactggc aaacatgaat 360

cgtctgttct ttcagccgca tcaggcaggt ctgtcgaaag tgcaagcggc ggagcacacc 420

ctgcgtaaca tcaacccgga tgttttattc gaagttcata attacaacat caccacggta 480

gagaacttcc agcactttat ggatcgcatc agcaatggtg gcttggagga gggcaagcct 540

gttgacctgg ttctatcttg tgttgacaac ttcgaagcac gtatgaccat taacaccgcg 600

tgcaatgagc tgggtcagac ctggatggaa agcggtgtct cagaaaacgc cgtgtctggc 660

catattcagc tgattattcc gggtgaatct gcgtgctttg cctgcgcgcc accgctggtc 720

gtggccgcga acatcgatga aaaaaccctg aagcgtgaag gcgtgtgcgc ggctagcctc 780

ccaacgacga tgggtgtcgt ggctggcatt ctggtccaga atgttcttaa gtttctgttg 840

aatttcggca ccgttagttt ttacctgggt tataacgcaa tgcaggattt tttcccgacc 900

atgagcatga aaccgaatcc gcagtgtgat gatcgcaact gccgtaaaca acaagaggaa 960

tacaaaaaga aggtggcggc gctgccgaaa caggaggtga ttcaagaaga ggaagagatc 1020

atccacgaag ataacgaatg gggtatcgag ctggtatctg aggtcagcga ggaggagttg 1080

aagaacttca gcggtccggt gccggacctg ccggaaggta tcaccgtggc ctatacgatc 1140

ccgaaaaagc aagaggacag cgtaactgaa ttgaccgttg aggacagcgg cgaatccttg 1200

gaggacctga tggctaaaat gaaaaacatg taa 1233

Claims

1. A synthesis method of Usin probe UFM1-Lys-TAMRA is characterized by comprising the following steps,

s1: in-situ hydrazinolysis is carried out after the Usherbet activating enzyme is used for activating the C end of the Usherbet to prepare the Usherbet hydrazide;

2. The method for synthesizing the Ustilagin probe UFM1-Lys-TAMRA of claim 1, wherein the amino acid sequence of the Ustilagin activating enzyme is shown in SEQ ID No. 1.

3. The method for synthesizing Uyghur probe UFM1-Lys-TAMRA of claim 1 or 2, wherein the step S1 comprises the following specific operations: adding magnesium chloride, ATP, hydrazine and the Ustilagin activating enzyme into the Ustilagin solution, adjusting the pH to 7.0-8.0, and reacting to obtain the Ustilagin hydrazide.

4. The method for synthesizing utahensis probe UFM1-Lys-TAMRA as claimed in claim 3, wherein the molar ratio of utahensis to utahensis activating enzyme is 45-60: 1.

5. the method for synthesizing Uygur probe UFM1-Lys-TAMRA of claim 1, wherein the specific operation of step S2 is:

adding mesna, adjusting the pH value to 4.0-6.0, and reacting to obtain the kosher thioester.

6. The method for synthesizing the Ustilagin probe UFM1-Lys-TAMRA of claim 5, wherein the molar ratio of Ustilagin hydrazide, sodium nitrite and mesna is 1: 8-12: 35-50.

7. The method for synthesizing Uygur probe UFM1-Lys-TAMRA of claim 1, wherein the specific operation of step S3 is: dissolving the isorhodin thioester in a mixed solvent, adding sodium thiophenolate and Lys-TAMRA, and reacting to obtain the isorhodin probe UFM 1-Lys-TAMRA.

8. The method for synthesizing Uygur probe UFM1-Lys-TAMRA of claim 7, wherein said mixed solvent is a mixed solution of an organic solvent and a buffer salt.

9. The method of synthesizing the Usher probe UFM1-Lys-TAMRA of claim 7, wherein the molar ratio of the Usher thioester, the sodium thiophenolate, and the Lys-TAMRA is 1: 160-250: 25-40.

10. A kosher probe UFM1-Lys-TAMRA prepared by the synthetic method of any one of claims 1-9.