CN113789335B

CN113789335B - Leech tyrosine sulfotransferase gene and application thereof

Info

Publication number: CN113789335B
Application number: CN202111353784.2A
Authority: CN
Inventors: 周正雄; 汪仁; 曾道平; 高萌; 徐晟�; 李洁; 孙彬; 周佳宇
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-03-15
Anticipated expiration: 2041-11-16
Also published as: CN113789335A

Abstract

The invention discloses a leech tyrosine sulfotransferase gene and application thereof, belonging to the field of biological medicine. The catalytic ability of the product is greatly improved compared with acyl sulfotransferase, human-derived tyrosine sulfotransferase and bovine-derived tyrosine sulfotransferase. Compared with other tyrosine sulfotransferases, the hirudin tyrosine sulfotransferase obtained by the application has stronger substrate preference for recombinant hirudin, is suitable for preparing natural hirudin on a large scale, and provides a new mode for synthesizing the natural hirudin.

Description

Leech tyrosine sulfotransferase gene and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, relates to a leech tyrosine sulfotransferase gene and application thereof, and particularly relates to a method for excavating, expressing and sulfonating a leech tyrosine sulfotransferase gene to modify recombinant hirudin.

Background

According to the statistics of the world health organization, about 1800 million people die of cardiovascular diseases every year, and account for 31 percent of the death people all over the world. In order to suppress and reduce the mortality of cardiovascular diseases, there is a need to actively search for and develop drugs for treating cardiovascular diseases. Natural hirudin (Sulfo-hirudin) is the strongest thrombin inhibitor found so far and is widely used clinically for treating cardiovascular diseases. However, as a small molecule active peptide in leeches, the isolation and purification of natural hirudin is extremely difficult. Therefore, the recombinant hirudin expressed by microbial cells is generally used as a substitute clinically, but the efficacy of the recombinant hirudin is only one tenth of that of natural hirudin. The main reason for this difference is that the tyrosine residue of recombinant hirudin is not modified by sulfonation, whereas the tyrosine residue at position 63 of natural hirudin is not modified by sulfonation⁶³Tyr is modified to tyrosine sulfonate by sulfonation with tyrosine sulfotransferase (TPS). Therefore, the analysis of the recombinant hirudin sulfonation modification process by the leech tyrosine sulfotransferase gene and the natural process are excavatedThe biosynthesis of hirudin is particularly important.

With the development of genome sequencing technology, transcriptome sequencing technology, synthetic biology technology and protein characterization technology, various leech source genomes and transcriptome data are published, but related analysis work is always stopped, and analysis of a hirudin sulfonation modification path and biosynthesis of natural hirudin are seriously hindered.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a leech tyrosine sulfotransferase and a coding gene thereof; the invention also aims to solve the technical problem of providing the application of the leech tyrosine sulfotransferase in catalyzing the sulfonation modification of recombinant hirudin into natural hirudin.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a leech tyrosine sulfotransferase gene has a nucleotide sequence shown as SEQ ID number 2 or SEQ ID number 3.

A recombinant expression vector or a recombinant strain containing the leech tyrosine sulfotransferase gene.

Further, the strain is escherichia coli or pichia pastoris.

A set of amplimers of leech tyrosine sulfotransferase gene comprises the following primer sequences:

F1：5'-ATGGCTGCTTTC ATAATCGAGATTATTG-3'，

R1：5'-CTAAACTAATGAGTTGAATGGATTCTTAGCAG-3'。

the leech tyrosine sulfotransferase gene is applied to preparing leech tyrosine sulfotransferase in a protein expression system.

In the application, the expression method of the leech tyrosine sulfotransferase adopts escherichia coli (E.coli) (II)Escherichia coli) Or Pichia pastoris (Pichia pastoris) Expressing the gene sequence of the leech tyrosine sulfotransferase to obtain the leech tyrosine sulfotransferase, wherein the amino acid sequence of the leech tyrosine sulfotransferase is shown as SEQ ID number 1.

The leech tyrosine sulfotransferase gene is applied to the sulfonation of recombinant hirudin to form natural hirudin.

Furthermore, the recombinant hirudin is used as a substrate, 3 '-adenosine monophosphate-5' -phosphosulfate PAPS is used as a sulfonic acid group donor, and the natural hirudin is formed by catalysis of hirudin tyrosine aminotransferase; the catalytic reaction system of the leech tyrosine sulfotransferase is that 0.1-50 mM PAPS, 0.1-100 mu M leech tyrosine sulfotransferase and 0.1-50 mM recombinant hirudin are added into 1-200 mM Tris-HCl solution.

Further, the catalytic reaction temperature of leech tyrosine sulfotransferase is 10-50 ℃; the reaction pH is 5-9; the reaction time is 1-50 h.

Preferably, the catalytic reaction temperature of the leech tyrosine sulfotransferase is 37 ℃; the reaction pH was 7; the reaction time was 1 h.

Compared with the prior art, the invention has the beneficial effects that:

1) according to the application, a leech tyrosine sulfotransferase gene sequence is excavated from leech genome and transcriptome data, a leech tyrosine sulfotransferase gene is expressed through microbial cell recombination, leech tyrosine sulfotransferase is obtained, and on the basis, the hirudin is catalyzed and recombined to synthesize natural hirudin, so that the anticoagulant activity of the hirudin is effectively improved.

2) Compared with other tyrosine sulfotransferases, the leech tyrosine sulfotransferase obtained by the application has stronger substrate preference on recombinant hirudin, and is suitable for preparing natural hirudin on a large scale.

Drawings

FIG. 1 is an electrophoretogram of a leech cDNA library construction; in the figure, A is an electrophoresis analysis chart of total RNA extraction of hirudo nipponica; b is cDNA electrophoresis analysis picture of hirudo nipponica;

FIG. 2 is a schematic diagram of the cloning and identification of the gene of hirudo tyrosine sulfotransferase;

FIG. 3 is a schematic diagram of the construction of a recombinant E.coli cell factory for the synthesis of hirudin tyrosine sulfotransferase; in the figure, A is recombinant Escherichia coliE. coli BL21 pET28a (+)-TPSThe construction of (a); b is SDS-PAGE analysis of colibacillus cell engineeringSynthetic hirudin tyrosine sulfotransferase case, where Lane M represents marker,

Lane

1 and 2 recombinant E.coliE. coli BL21 pET28a (+)-TPSIntracellular supernatant sample, lane 3 recombinant E.coliE. coliBL21 pET28a (+) intracellular supernatant samples,

lanes

4 and 5 recombinant E.coliE. coli BL21 pET28a (+)-TPSIntracellular pellet sample, lane 6 recombinant E.coliE. coliBL21 pET28a (+) intracellular precipitation samples;

FIG. 4 is a schematic diagram of the construction of synthetic hirudo tyrosine sulfotransferase in recombinant Pichia cell factories; in the figure, A is recombinant pichia pastorisP. pastoris GS115 pPIC9K-TPSThe construction of (a); b is SDS-PAGE analysis of the synthesis of hirudin tyrosine sulfotransferase in Pichia pastoris cell factories, where lane M represents marker and lane 1 is recombinant Pichia pastorisP. pastorisGS115 pPIC9K intracellular sample, lane 2 recombinant Pichia pastorisP. pastorisGS115 pPIC9K extracellular sample,

lanes

3 and 5 recombinant Pichia pastorisP. pastoris GS115 pPIC9K-TPSIntracellular samples,

lanes

4 and 6 for recombinant Pichia pastorisP. pastoris GS115 pPIC9K-TPSAn extracellular sample;

FIG. 5 is a diagram showing the isolation of the product of the hydrolysis of natural hirudin by carboxypeptidase; a is a separation diagram of a product obtained by hydrolyzing natural hirudin 1 by carboxypeptidase; b is a separation diagram of a product obtained by hydrolyzing natural hirudin 2 by carboxypeptidase;

FIG. 6 is a diagram showing analysis of catalytic products of hirudin tyrosine sulfotransferase enzyme by MALDI-TOF-MS; wherein A is the MS map of the peptide segment consisting of the 1 st to 60 th amino acids of the recombinant hirudin 1, and B is the MS map of the peptide segment consisting of the 1 st to 60 th amino acids of the recombinant hirudin 2;

FIG. 7 is a diagram showing the identification of amino acid products of hydrolysis of hirudin by carboxypeptidase by HPLC-MS;

FIG. 8 is a graph showing the effect of different tyrosine sulfotransferases on the sulfonation efficiency of hirudin.

Detailed Description

The invention is further described with reference to specific examples.

Example 1 mining of leech tyrosine-sulfotransferase genes based on leech transcriptome database

1. Excavation of alternative leech tyrosine sulfotransferase gene

Leech tyrosine sulfotransferase alternative gene fragment EN-133k-group2100.contig1 is excavated by performing Blastx analysis on Japanese leech genome data. ORF program (http:// www.ncbi.nlm. nih. gov/orffinder) is adopted to analyze the open reading frame of the alternative gene, Protein Blast is adopted to carry out database homology comparison, and the sequence SEQ ID number 3 is determined to be the nucleotide sequence of the alternative leech tyrosine sulfotransferase through comparison, and SEQ ID number 1 is the amino acid sequence of the alternative leech tyrosine sulfotransferase.

2. Construction of a leech cDNA library

Feeding Hirudo nipponica with fresh duck blood for 2 h, and quickly freezing Hirudo with liquid nitrogen. After grinding with liquid nitrogen, total leech RNA (a in fig. 1) was extracted using a small RNA Extraction Kit (TaKaRa MiniBEST universal RNA Extraction Kit), and a leech cDNA Library (B in fig. 1) was obtained using a reverse transcription Kit (cDNA Library Construction Kit).

3. Leech tyrosine sulfotransferase alternative gene amplification

Designing a primer F1/R1 (ATGGCTGCTTTC ATAATCGAGATTATTG, CTAAACTAATGAGTTGAATGGATTCTTAGCAG) according to the sequence of the alternative gene fragment SEQ ID number 3, and amplifying the tyrosine sulfotransferase gene fragment by using high fidelity PCR polymerase Prime Star and taking a leech cDNA library as a template. After the fragment is recovered, dephosphorylation of the end of a vector pUC 1185 'is carried out, phosphorylation of the end of the recovered fragment 5' and connection of the phosphorylated fragment and a dephosphorylated vector are carried out by using a blunt-end Ligation Kit Blunting Ligation Kit (TaKaRa) according to the Kit instructions, and finally the conversion is carried out at 42 ℃ for 90 sE. coliJM109 obtains a recombinant cloning vector of the candidate gene, and the nucleotide sequence of the gene to be detected is identified through sequencing and is consistent with the sequence of the candidate gene fragment SEQ ID number 3 (figure 2).

Example 2 preparation of Hirudo tyrosine sulfotransferase Using microbial cells

1. Preparation of leech tyrosine sulfotransferase by recombinant escherichia coli

Considering the codon preference of microbial cells for protein expression, we will artificially synthesize codon-optimized leech tyrosine sulfotransferase alternative gene in order to obtain high-efficiency expression of leech tyrosine sulfotransferase geneTPSIntegrated by Gibbson assembly, homologous recombination into pET28a (+)NdeI andNoti site, construction of recombinant expression vector pET28a (+) -TPSWhereinTPSThe sequence of (a) is shown as SEQ. ID number 2. Transformation with the above recombinant expression vectorE. coliBL21 Escherichia coli recombinant expression strain for obtaining leech tyrosine sulfotransferaseE. coli BL21 pET28a (+)-TPSFor subsequent preparation of leech tyrosine sulfotransferase (a in fig. 3).

Selecting recombinant Escherichia coli expression strain on plateE. coli BL21 pET28a (+)-TPSA single colony was inoculated into 3 mL of LB medium (to which was added kanamycin to a final concentration of 50 mg/L), and cultured overnight at 37 ℃ and 200 rpm. Transfer the mixture to 50 mL of TB medium (supplemented with kanamycin to a final concentration of 50 mg/L) at a rate of 1% (v/v), and culture the mixture at 37 ℃ to OD₆₀₀0.6-0.8, adding IPTG with final concentration of 0.1 mM, inducing at 37 deg.C for 5 h, then 8000 rpm, centrifuging at 4 deg.C for 5 min, and collecting thallus. Washing with precooled Tris-HCl (pH7.0), resuspending the thallus to 50 mL, and carrying out ultrasonic treatment in ice bath under the conditions of: 400W, 2 s of work, 3 s of pause, 200 cycles until the bacterial liquid is clarified, 14000 rpm, 20 min of centrifugation at 4 ℃, and collecting supernatant, namely the crude enzyme liquid (B in figure 3).

2. Construction of recombinant pichia pastoris cell factory and preparation of leech tyrosine sulfotransferase

Artificially synthesized leech tyrosine sulfotransferase alternative geneTPSIntegrated into pPIC9K by Gibbson assembly, homologous recombinationEcoR I andNoti site, constructing a recombinant expression vector pPIC9K-TPSWhereinTPSThe sequence of (a) is shown as SEQ. ID number 3. Transforming pichia pastoris with the recombinant expression vector to obtain transformantP. patoris GS115 pPIC9K-TPSFor subsequent use of Pichia pastorisThe mother cell factory prepared leech tyrosine sulfotransferase (a in fig. 4).

Pichia transformants were picked from platesP. patoris GS115 pPIC9K-TPSA single colony was inoculated into 3 mL of YPD medium and cultured overnight at 30 ℃ and 200 rpm. 50 mL of BMMY medium (added to methanol at a final concentration of 0.5 g/L) was inoculated at 1% (v/v) and induced at 200 rpm for 5 days at 20 ℃. Centrifuging the above culture at 8000 rpm and 4 deg.C for 5 min, and collecting supernatant to obtain Hirudo tyrosine sulfotransferase crude enzyme solution (B in FIG. 4).

EXAMPLE 3 establishment of the catalytic System for leech tyrosine sulfotransferase

1. Purification of leech tyrosine sulfotransferase

After the Ni-NTA Agarose Fast Flow column was equilibrated with Buffer A (20 mM Tris-HCl, 0.5M NaCl, 20 mM imidazole, pH 7.4), the

crude enzyme solutions

1 and 2 of hirudin tyrosine sulfotransferase were passed through 0.22 μ M filters, respectively, and the supernatant was passed through the Ni-NTA Agarose Fast Flow column at a rate of 3 mL/min, combined for 10 min, and then Buffer B (20 mM Tris-HCl, 0.5M NaCl, 500 mM imidazole, pH 7.4) was eluted to collect the enzyme active fractions, which were concentrated and freeze-dried.

2. Preparation of natural hirudin

Taking recombinant hirudin 1 (Sigma) or recombinant hirudin 2 (Sigma) as a raw material, wherein a reaction solution comprises 1-200 mM Tris-HCl (pH5.0), 0.1-50 mM PAPS, 0.1-100 mu M hirudin tyrosine sulfotransferase and 0.1-50 mM recombinant hirudin, reacting at 10-50 ℃ for 1-50 h, and then heating at 70 ℃ for 5 min to terminate the reaction.

The method takes recombinant hirudin 1 (Sigma) or recombinant hirudin 2 (Sigma) as a raw material, reaction liquid comprises 20 mM Tris-HCl (pH7.0), 3 mM PAPS, 3 mu M hirudin tyrosine sulfotransferase and 3 mM recombinant hirudin, the reaction is terminated by heating at 70 ℃ for 5 min after reacting for 1 h at 37 ℃, and enzyme catalysis products of the recombinant hirudin 1 and enzyme catalysis products of the recombinant hirudin 2, namely natural hirudin 1 and natural hirudin 2, are respectively prepared.

Example 4 detection of Natural hirudin

After natural hirudin 1 and natural hirudin 2 are hydrolyzed by adopting the combined action of carboxypeptidase A and carboxypeptidase B, an amino acid fragment and the rest polypeptide residue are hydrolyzed by preparing liquid phase separation, an amino acid fragment obtained by enzyme hydrolysis is detected by utilizing high performance liquid chromatography-mass spectrometry (HPLC-MS), and the rest polypeptide residue structure is detected by MALDI-TOF-MS, wherein the hydrolysis reaction system of the carboxypeptidase is as follows: 20 mM Tris-HCl (pH7.0), 1 g/L carboxypeptidase A, 1 g/L carboxypeptidase B, 25 g/L natural hirudin; the enzyme catalysis condition is 24 hours at 25 ℃.

The chromatographic conditions for preparing the liquid phase high performance liquid chromatography are that a chromatographic column: inertsil ODS-34.6X 250 mm; mobile phase: 0.065% trifluoroacetic acid in water (a), 0.05% trifluoroacetic acid in acetonitrile (B); detection wavelength: 220 nm; flow rate: 1 mL/min. Elution procedure time (min) 0.01: pump a 95% + pump B5%, time (min) 25.00: pump a 35% + pump B65%, time (min) 25.01: pump a 35% + pump B65%, time (min) 27.00: pump A5% + pump B95%, time (min) 27.01: pump a 95% + pump B5%, time (min) 32.00: pump a 95% + pump B5%.

The HPLC-MS conditions used for high performance liquid chromatography are chromatographic column: agilent advanced Bio MS Spent Media 2.1X 150 mm; mobile phase: 10 mM ammonium acetate aqueous solution (A), 10 mM ammonium acetate acetonitrile solution (B); detection wavelength: flow rate: 0.25 mL/min. Elution procedure time (min) 0: pump 10% + pump B90%, time (min) 2: pump a 10% + pump B90%, time (min) 12: pump a 60% + pump B40%, time (min) 13: pump a 80% + pump B20%, time (min) 16: pump a 80% + pump B20%, time (min) 17: pump a 10% + pump B90%, time (min) 25: pump a 10% + pump B90%. Corresponding mass spectrometry conditions used: an ion source: ESI; flow rate of the atomizer: 1.5L/min; flow rate of drying gas: 5L/min; ion source temperature: 200 ℃; transmission line temperature: at 250 ℃ to obtain a mixture.

The mass spectrum conditions of MALDI-TOF-MS used were: mixing a matrix solution (6 g/L alpha-cyano-4-hydroxycinnamic acid and 2 g/L diammonium hydrogen citrate dissolved in 10 mL of 50% acetonitrile/water solution (containing 0.1% trifluoroacetic acid)) with a sample to be tested, dropping the mixture on a stainless steel target plate, naturally drying and crystallizing the stainless steel target plate, and testing the mixture. The mass spectrum conditions used in the method are that under a cation reflection mode, each spectrogram of a primary mass spectrum is accumulated for 800 times, and the secondary mass spectrum is accumulated for 1200 times, wherein the collision induced dissociation conditions are 1000 eV collision energy plus air collision (1 kV, Gas on), the source internal voltage is 8 kV, and the collision cell voltage is 7 kV. The database search conditions used were: the signal-to-noise ratio of the tandem mass spectrum Data is set to be 4, the plasmid errors of the primary mass spectrum and the secondary mass spectrum are both set to be 0.2 Da, the Data are analyzed through Mascot software, and the amino acid sequence of the polypeptide is identified by utilizing NCBI database retrieval or utilizing an Ion fragmentation calculator (Ion fragmentation calculator) and a Protein-Protein MS-Product program provided by Data Explorer for assistance in manual analysis.

The results show that separation of native hirudin 1 and native hirudin 2 catalyzed by carboxypeptidase A and carboxypeptidase B from the preparative liquid phase yielded 5 absorption peaks at 280 nm, respectively carboxypeptidase A, carboxypeptidase B, hirudin 1 fragment, hirudin 2 fragment and hirudin tyrosine sulfotransferase (FIG. 5). From the total ion spectrum of the hirudin 1 fragment (A in figure 6) and the hirudin 2 fragment (B in figure 6), the characteristic fragment peaks of the substances obtained after the action of carboxypeptidase A and B on natural hirudin 1 and natural hirudin 2 are respectively consistent with the N-terminal sequences of the substrate recombinant hirudin 1 and the substrate recombinant hirudin 2, which indicates that 60 amino acid residues at the N-terminal of the recombinant hirudin 1 and the recombinant hirudin 2 are not changed after the catalysis of hirudin tyrosine sulfonate transferase. Meanwhile, HPLC-MS measurement of 5 amino acid residues at the C-terminus by co-hydrolysis of carboxypeptidase A and carboxypeptidase B showed that the tyrosine at position 63 of recombinant hirudin 1 and recombinant hirudin 2 was sulfonate-modified (FIG. 7). Therefore, the leech tyrosine sulfotransferase gene is successfully excavated, and the sulfonation modification of the recombinant hirudin can be smoothly catalyzed.

Example 5 Effect of different sulfotransferases on the sulfonation efficiency of hirudin

According to the method employed in example 3, natural hirudin synthesis systems of acyl sulfotransferase, human tyrosine sulfotransferase, bovine tyrosine sulfotransferase and hirudin tyrosine sulfotransferase were constructed, respectively. The enzyme catalysis system is 20 mM Tris-HCl (pH7.0), 3 mM PAPS, 5 mM MnCl₂，5 mM MgCl₂And 3In an enzyme-catalyzed reaction system of 0 g/L recombinant hirudin 1 or

recombinant hirudin

2, 15 μ M of acylsulfotransferase, human-derived tyrosine sulfotransferase, bovine-derived tyrosine sulfotransferase or hirudin tyrosine sulfotransferase is respectively added, the reaction is terminated by heating at 70 ℃ for 5 min after 1 h of reaction at 37 ℃, and the catalytic rate of the enzyme-catalyzed reaction is detected by the method used in example 4.

The catalytic rate of the sulfotransferase is characterized by the conversion of recombinant hirudin, calculated as the sulfonation of tyrosine, by the method: and (3) converting the peak area of the sample to be detected in the liquid chromatogram with the peak area of the standard substance to obtain the concentration of the sulfotyrosine.

The results show that under the current catalytic conditions, compared with acyl sulfotransferase, bovine-derived sulfotransferase and human-derived sulfotransferase reported in the literature, the catalytic efficiency of the hirudin tyrosine sulfotransferase with recombinant hirudin 1 as a substrate is respectively increased by 29.6 times, 5.4 times and 5.8 times, and reaches 66.4 +/-0.5 nM/(h.mu M protein); when recombinant hirudin 2 was used as the substrate, the catalytic efficiency of hirudin tyrosine sulfotransferase was increased by 23.8, 5.7 and 5.3 times, respectively, to 63.6. + -. 0.8 nM/(h. mu.M protein), compared to the 3 sulfotransferases described above (FIG. 8).

Sequence listing

<110> institute of plant of Chinese academy of sciences of Jiangsu province

<120> leech tyrosine sulfotransferase gene and application thereof

<130> 1

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 244

<212> PRT

<213> Hirudo tyrosine sulfotransferase (Artificial)

<400> 1

Ala Ala Phe Ile Ile Glu Ile Ile Val Lys His Gly Glu Pro Ala Pro

1 5 10 15

Arg Leu Cys Asn Lys Asp Pro Phe Thr Leu Lys Tyr Met Val Glu Leu

20 25 30

Ala Asp Met Phe Pro Asn Ala Arg Phe Leu Leu Met Val Arg Asp Gly

35 40 45

Arg Ala Val Val His Ser Val Ile Ser Arg Lys Val Thr Ile Thr Gly

50 55 60

Phe Asp Leu Asp Asn Phe Glu Ala Ser Leu Gln Lys Trp Asn Ser Ile

65 70 75 80

Ile Ser Ser Met Tyr Thr Gln Cys Gln Lys Val Gly Pro Thr Lys Cys

85 90 95

Met Met Val Phe Tyr Glu Gln Leu Val Leu His Pro Ala Gln Val Met

100 105 110

Thr Asp Val Leu Lys Phe Leu Glu Ile Pro Trp Asp Glu Arg Val Met

115 120 125

His His Gln Asp Tyr Ile Asn Lys Pro Gly Gly Ile Ser Leu Ser Lys

130 135 140

Ser Glu Arg Ser Thr Asp Gln Val Val Lys Pro Val Asn Leu Glu Ala

145 150 155 160

Leu Thr Lys Trp Val Asp His Val Pro Asp Asn Val Lys Lys Asn Ile

165 170 175

Arg Arg Leu Ala Pro Met Leu Glu Lys Leu Gly Tyr Asp Pro Asp Gly

180 185 190

Tyr Pro Pro Asn Tyr Gly Ile Pro Asp Ala Ser Val Trp Asn Gln Thr

195 200 205

Asn Gln Val Leu Lys Asn Ser Gln Tyr Trp Ser Asn Lys Ala Ala Glu

210 215 220

Val His Ser Lys Ile Asp Lys Asn Ser Thr Ser Ala Lys Asn Pro Phe

225 230 235 240

Asn Ser Leu Val

<210> 2

<211> 738

<212> DNA

<213> Hirudo tyrosine sulfotransferase Gene (Artificial)

<400> 2

atggctgctt tcataatcga gattattgtc aagcatggag agccagctcc ccgtttgtgt 60

aataaagatc ccttcacctt gaaatacatg gtggaacttg cagacatgtt tccaaatgct 120

cgatttctgc tgatggtgcg tgatgggcgt gcagtcgtcc attctgttat atcaaggaag 180

gttaccatca caggttttga tttggacaac ttcgaggctt ctctacagaa gtggaattca 240

ataatttctt ccatgtacac tcaatgtcaa aaagtgggtc ccacaaaatg catgatggtt 300

ttctatgagc aattggttct tcatcctgct caagtcatga ctgatgtact caaatttctt 360

gaaattccat gggatgaaag agtgatgcat catcaagatt acatcaataa gcctggtgga 420

atttccttat ctaaatctga acgatcaact gatcaggtgg taaagcctgt gaatttggag 480

gctttaacaa agtgggttga tcatgtgcct gataatgtta aaaagaatat caggagactt 540

gcaccaatgc ttgagaaact tggctatgac cctgatgggt accctccaaa ttatggaatt 600

ccagatgcaa gtgtgtggaa ccaaaccaat caggttttga aaaattctca gtattggagc 660

aacaaggccg ctgaagtcca ctcaaagatc gacaagaaca gcacgtctgc taagaatcca 720

ttcaactcat tagtttaa 738

<210> 3

<211> 738

<212> DNA

<213> hirudin tyrosine sulfotransferase prediction sequence (Artificial)

<400> 3

atggctgctt tcataatcga gattattgtc aagcatggag agccagctcc ccgtttgtgt 60

aataaagatc ccttcacctt gaaatacatg gtggaacttg cagacatgtt tccaaatgct 120

cgatttctgc tgatggtgcg tgatgggcgt gcagtcgtcc attctgttat atcaaggaag 180

gttaccatca caggttttga tttggacaac ttcgaggctt ctctacagaa gtggaattca 240

ataatttctt ccatgtacac tcaatgtcaa aaagtgggtc ccacaaaatg catgatggtt 300

ttctatgagc aattggttct tcatcctgct caagtcatga ctgatgtact caaatttctt 360

gaaattccat gggatgaaag agtgatgcat catcaagatt acatcaataa gcctggtgga 420

atttccttat ctaaatctga acgatcaact gatcaggtgg taaagcctgt gaatttggag 480

gctttaacaa agtgggttga tcatgtgcct gataatgtta aaaagaatat caggagactt 540

gcaccaatgc ttgagaaact tggctatgac cctgatgggt accctccaaa ttatggaatt 600

ccagatgcaa gtgtgtggaa ccaaaccaat caggttttga aaaattctca gtattggagc 660

aacaaggccg ctgaagtcca ctcaaagatc gacaagaaca gcacgtctgc taagaatcca 720

ttcaactcat tagtttag 738

<210> 4

<211> 28

<212> DNA

<213> F1(Artificial)

<400> 4

atggctgctt tcataatcga gattattg 28

<210> 5

<211> 32

<212> DNA

<213> R1(Artificial)

<400> 5

ctaaactaat gagttgaatg gattcttagc ag 32

Claims

1. A leech tyrosine sulfotransferase gene has a nucleotide sequence shown as SEQ ID number 2 or SEQ ID number 3.

2. The protein expressed by the leech tyrosine sulfotransferase gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID number 1.

3. A recombinant expression vector containing the leech tyrosine sulfotransferase gene of claim 1.

4. A recombinant strain comprising the leech tyrosine sulfotransferase gene of claim 1.

5. The recombinant strain containing the leech tyrosine sulfotransferase gene of claim 4, wherein the strain is Escherichia coli or Pichia pastoris.

6. The use of the leech tyrosine sulfotransferase gene of claim 1 in the sulfonation of recombinant hirudin to form natural hirudin.

7. The use of claim 6, wherein the recombinant hirudin is used as a substrate, 3 '-adenosine monophosphate-5' -phosphosulfate PAPS is used as a sulfonic acid group donor, and the natural hirudin is formed by catalysis of hirudin tyrosine aminotransferase; the catalytic reaction system of the leech tyrosine sulfotransferase is that 0.1-50 mM 3 '-adenosine phosphate-5' -phosphosulfate PAPS, 0.1-100 mu M leech tyrosine sulfotransferase and 0.1-50 mM recombinant hirudin are added into 1-200 mM Tris-HCl solution.

8. The use of claim 7, wherein the leech tyrosine sulfotransferase is catalyzed at a temperature of 10-50 ℃; the reaction pH is 5-9; the reaction time is 1-50 h.

9. The use of claim 8, wherein the leech tyrosine sulfotransferase catalyzed reaction temperature is 37 ℃; the reaction pH was 7; the reaction time was 1 h.