CN1644697A

CN1644697A - Thioredoxin peroxidase gene of China Qingdao branchiostoma and use thereof

Info

Publication number: CN1644697A
Application number: CN 200410077542
Authority: CN
Inventors: 徐安龙; 廖剑; 孙孜孜; 王磊; 董美玲
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2004-12-24
Filing date: 2004-12-24
Publication date: 2005-07-27
Anticipated expiration: 2024-12-24
Also published as: CN1273599C

Abstract

This invention relates to thioredoxin peroxisome (TPx) gene and encoded protein of Qingdao branchiostoma as well as their use in preparation of medicine for treating cancers and preventing from senility. By RT-PCR method, TPx is separated from whole RNA of branchiostoma ovary. Its DNA sequence is shown in sequence table <400>1. Its amino acid sequence of gene-encoded protein is shown in sequence table <400>2. It has anti-oxidation effect, so as to be used for medicines.

Description

Thioredoxin peroxidase gene of Chinese Qingdao branchiostoma and application thereof

Technical Field

The invention relates to a thioredoxin peroxidase gene of Chinese Qingdao branchiostoma and a proteincoded by the same, and application of the protein in preparing anti-tumor and anti-aging medicaments.

Background

The Wenchang fish has red fish meat, glittering and translucent shape. The body is slender like a scalpel, so Western Europe calls its two sharps or festuca arundinacea. The number of the branchiostoma belcheri is 29. The method is distributed in tropical and subtropical shallow sea areas of 8-16 meters, especially in annular areas between 48 degrees of north latitude and 40 degrees of south latitude. Although amphioxus is not a vertebrate, the common denominator of vertebrate embryogenesis is represented by chords, neural tubes and pharyngeal gill clefts of amphioxus, and thus occupies an extremely important position in the animal clade. Thus, contemporary embryology still uses amphioxus embryogenesis as the basic model of vertebrate embryogenesis to date.

Peroxiredoxin Prx is a class of antioxidant proteases in organisms. The peroxiredoxin has received more and more attention since its discovery in Saccharomyces cerevisiae by K. Kim in 1988 for more than ten years. Peroxiredoxin enzymes are produced in vivo primarily by reduction of hydrogen peroxide and certain hydroperoxides ((II)) ) To realize the anti-oxidation effect of the self. Peroxiredoxins belong to the peroxidase family. The hydrogen donors (hydrogen donors) of peroxidase enzymes are mainly found in glutathione, thioredoxin, NADH and NADPH. The most obvious feature of peroxiredoxin compared to other peroxidases is that most peroxiredoxins are thioredoxin as the only hydrogen donor in vivo. As such, the peroxiredoxin reductase is also known as Thioredoxin Peroxidase (TPx). However, not all peroxiredoxins are thioredoxin as the only hydrogen donor.

Peroxiredoxins are ubiquitous in most organisms. Studies have shown that yeast, plants, animals, protozoa, parasites and even eubacteria and archaea contain this enzyme in vivo, suggesting that the enzyme peroxiredoxin is a very important gene. Peroxiredoxins are mainly located in the cytosol and are also distributed in small amounts in the mitochondria, chloroplasts, peroxisomes and nuclei. Experiments prove that the peroxiredoxin is abundantly expressed in organisms. Many organisms contain more than one isoform of a peroxiredoxin enzyme. At least six peroxiredoxins have been discovered in mammalian cells.

The major functions of the peroxiredoxin in vivo include two aspects: cell detoxification ability and resistance to oxidative stress; the second is to modulate signal transduction and immune responses mediated by hydrogen peroxide. Peroxiredoxin can be divided into two main classes according to the number of active cysteine residues they possess: 1-Cys and 2-Cys peroxide reductases. Further studies of structure and reaction mechanism led to the results that we can also classify 2-Cys peroxide reductases into two classes: typical and atypical 2-Cys peroxide reductases. The classical 2-Cys peroxiredoxin reductase, which we also refer to as thioredoxin peroxidase (TPx), since this class of enzyme relies in vivo only on thioredoxin as its sole hydrogen donor.

Studies have shown that parasite peroxiredoxin reductase plays an important role in the parasite's resistance to host immune responses and oxidative stress; in mammals, particularly humans, the protein is closely associated with disease, signaling, and immune responses. With the progress of research on peroxiredoxins, the importance of peroxiredoxins in organisms has been recognized more and more. The protein has good prospect in the prevention and treatment of parasites and the research of anti-tumor and anti-aging drugs.

Disclosure of Invention

The invention aims to provide a novel thioredoxin peroxidase gene of Chinese Qingdao branchiostoma and a protein Bbt-TPx coded by the same.

The invention also aims to provide the application of the protein of the gene in preparing anti-tumor and anti-aging medicaments.

The invention uses RT-PCR method to separate thioredoxin peroxidase gene TPx from the total RNA of the ovary of Wenchang fish in Qingdao, and the DNA sequence is shown as<400>1 sequence in the sequence table.

The amino acid sequence of the protein (recombinant amphioxus thioredoxin peroxidase Bbt-TPx) coded by the gene of the invention is shown as a<400>2 sequence in a sequence table; the isoelectric point of the protein is 7.30, and the molecular weight is 22150 daltons.

The protein Bbt-TPx was expressed in an intracellular soluble form in E.coli by the expression vector pET-32a (+) M-TPx.

The expression vector pET-32a (+) M-TPx is a high-efficiency expression vector which inserts an affinity tag site of His and a TPX gene sequence in the middle of a multiple cloning site of a plasmid pET-32a (+) M (purchased from Nobogen company), and the system enables TPX gene to be expressed in an intracellular soluble form in escherichia coli, and the expression amount can reach 600 mg/L.

The selected Wenchang belongs to Wenchang in Qingdao China (Branchiostoma belcheri tsingtauense) and is collected from Shaozuokou water area in Qingdao City in Shandong province in China.

Construction of ovary cDNA library of Wenchang fish in Qingdao China: firstly, separating the ovary of the Wenchang fish and extracting the total RNA. Taking total RNA to carry out reverse transcription to synthesize a first strand cDNA product. And then taking cDNAfor ligation reaction, respectively coating transformation liquid on plates, and shaking the rest for total library colonies. Picking monoclonal stock from plate

The invention obtains 1 new clone of coding Chinese Qingdao branchiostoma thioredoxin peroxidase by determining the large-scale sequence of the recombinant clone, and the clone is named as TPx (the DNA sequence of the TPx is shown as a<400>1 sequence in a sequence table). The novel gene codes a mature peptide with 198 amino acids (the amino acid sequence of the mature peptide is shown as a<400>2 sequence in a sequence table), the isoelectric point is 7.30, the molecular weight is 22, 150 daltons, the mature peptide has the characteristic of a typical primary structure of thioredoxin peroxidase, and two active cysteine functional domains are arranged in the molecule.

The invention designs a pair of primers, amplifies a new gene TPx for coding the thioredoxin peroxidase of the Chinese Qingdao branchiostoma belcheri from a T vector by a PCR method, clones the gene TPx to a prokaryotic fusion expression vector pET-32a (+) M to construct an expression plasmid pET-32a (+) M-TPx, and converts the expression plasmid into escherichia coli BL21(DE 3)/pLysS. The expression vector (pET-32a (+) M-TPx) uses T7 as a promoter, is expressed in a soluble form, and has a 6 XHis structure at the N-terminal, so that the purification can be conveniently carried out by utilizing immobilized metal ligand affinity chromatography. Through exploration and optimization of conditions such as culture time, induction time, temperature and the like, the expression amount of the recombinant Bbt-TPx protein can reach 600mg/L and is basically in a soluble state.

The invention also searches and optimizes the purification condition of the recombinant Bbt-TPx protein, and the ultrasonic lysate of the expression product is processed by Ni²⁺And (3) carrying out chromatography on the chemically synthesized Sepharose affinity chromatography to obtain a target protein with high purity, and carrying out further SP-Sepharose cation exchange chromatography on the target protein to obtain the recombinant Bbt-TPx protein with the purity of more than 95%.

Tests prove that the recombinant amphioxus thioredoxin peroxidase obtained by the invention has biological activity. The recombinant amphioxus thioredoxin peroxidase has an antioxidant effect. The recombinant branchiostoma thioredoxin peroxidase has reduction H₂O₂Activity of (2). When supercoiled DNA or thiol-group-sensitive proteins are damaged by external oxidation, the recombinant Bbt-TPx protein has a significant protective effect on it. Therefore, the recombinant thioredoxin peroxidase can be used for preparing anti-tumor and anti-aging medicaments.

The expression plasmid pET-32a (+) M-TPx containing the amphioxus thioredoxin peroxidase coding sequence is subjected to BaM HI/Xho I double enzyme digestion to obtain a 610bp fragment, namely the amphioxus thioredoxin peroxidase TPx coding sequence.

The replication method of the expression plasmid of the invention comprises the following steps: reference is made to Sambrook (Sambrook, et al 1989, Molecular cloning, Cold Spring Harbor laboratory Press, USA) method, in CaCl₂Method plasmids were transformed in E.Coli.DH5 α or BL21(DE3)/pLysS strains, bacteria were transformed in LB medium containing ampicillin (100. mu.g/mL), and plasmids were extracted by an alkaline method.

Drawings

FIG. 1 is a comparison of the results of the present invention for the sulfoximine peroxidase of amphioxus and other species of the protein, wherein bold and shaded indicate the same; the boxed representation is a functional domain of the protein.

FIG. 2 shows the result of electrophoresis of the PCR product of thioredoxin peroxidase gene TPx of Wenchakus in Qingdao. 1: TPx gene PCR product; 2: blank control without template; m: 1Kb DNA molecular weight standard.

FIG. 3 shows the restriction enzyme digestion and PCR identification of pET32a (+) M-TPx recombinant expression vector. M: a DNA molecular weight standard of 1 Kb; 1: carrying out double enzyme digestion on Bam HI and Xho I of the PET32a (+) M-TPx recombinant expression vector; 2: PCR identification of the PET32a (+) M-TPx recombinant expression vector.

FIG. 4 is an electrophoresis picture of recombinant Bbt-TPx protein induced expression and affinity chromatography. M: protein molecular weight standards; 1: the protein expression amount of the recombinant Bbt-TPx protein in total bacteria; 2: protein expression level of recombinant Bbt-TPx protein in supernatant; 3: 50mmol/L Tris-HCl (pH8.0), 500mmol/L NaCl elution peak of recombinant Bbt-TPx protein; 4: 50mmol/L Tris-HCl (pH8.0), 500mmol/L NaCl, 100mmol/L imidazole elution peak of recombinant Bbt-TPx protein; 5: 50mmol/L Tris-HCl (pH8.0), 500mmol/L NaCl and 150mmol/L imidazole elution peak of recombinant Bbt-TPx protein; 6: 50mmol/L Tris-HCl (pH8.0), 500mmol/L NaCl, 300mmol/L imidazole elution peak of recombinant Bbt-TPx protein.

FIG. 5 is an SDS-PAGE analysis of SP-Sepharose cation exchange chromatography of recombinant Bbt-TPx protein. M: protein molecular weight standards; 1: ni of recombinant Bbt-TPx protein²⁺Chelating chromatography 300mmol/L imidazole peak exchange buffer 50mM PB (pH6.3); 2: 50mM PB (pH6.3), 500mM NaCl elution peak of recombinant Bbt-TPx protein.

FIG. 6 is a schematic diagram of the construction of pET32a (+) M-TPx recombinant expression vector.

FIG. 7 shows the reduction of recombinant Bbt-TPx protein to H₂O₂Graph for measuring activity. Series 1: recombinant Bbt-TPx protein at a concentration of0 ug/ml; series 2: the concentration of the recombinant Bbt-TPx protein is 100 ug/ml; series 3: the concentration of the recombinant Bbt-TPx protein is 50 ug/ml; series 4: the concentration of the recombinant Bbt-TPx protein is 20 ug/ml. FIG. 8 is a diagram showing the result of agarose electrophoresis in an experiment of recombinant Bbt-TPx protein protected supercoiled DNA. 1: supercoil DNA of blank control; 2: only adding DTT; 3: adding only Fe³⁺(ii) a 4: addition of DTT and Fe only³⁺；5～9：DTT+Fe³⁺+500, 200, 100, 50, 25ug/ml TPx.SF: Superlipided Formof Plasmid, NF: Nickel Form of Plasmid.

FIG. 9 is a graph of the GS protein protecting activity of recombinant Bbt-TPx protein. Series 1: the reaction system has no DTT and Fe³⁺And TPx; series 2: the reaction system contains 100ug/ml TPx; series 3: the reaction system contains 20ug/ml TPx; series 4: the reaction system contains DTT and Fe³⁺No TPx.

Detailed Description

The following examples will assist one of ordinary skill in the art in further understanding the invention, but are not intended to limit the invention in any way.

The first embodiment is as follows: construction and EST analysis of ovary cDNA library of Wenchang fish in Qingdao China

Extraction of total RNA and cDNA synthesis: separating ovary of Wenchang fish in Qingdao China, extracting total RNA of ovary by guanidinium isothiocyanate method, and extracting with phenol/chloroform to remove protein to obtain 100 μ g total RNA of ovary. 1ug of total RNA was extracted with SMARTIII lignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII/3 'PCR primer (5' -ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)₃₀N_-1N-3') were reverse transcribed to synthesize the first strand, resulting in 10. mu. lcDNA first strand product.

Constructing and identifying an amphioxus ovary cDNA library: mu.l of cDNA was used for ligation reaction, 5. mu.l of the reaction system was transformed to 1. mu.l, 1/200, 1/100 and 2/100 volumes of transformation medium were plated separately, and the rest were used to shake the total pool colonies. Single clones were picked from the plates for seed retention and a certain number of single clones were randomly picked for sequencing and bioinformatics analysis.

Example two: determination and analysis of recombinant branchiostoma belcheri thioredoxin peroxidase gene sequence

Plasmid and sequence of the thioredoxin peroxidase gene of the branchiostoma belcheri are derived from the gene cluster numbered bbelo2001 in the ovary cDNA library of the branchiostoma belcheri of the example I. The bioinformatics analysis of the cDNA sequence shows that the length of the cDNA sequence is 1108 bp. The base sequence of the DNA tools 6.0 is analyzed by using tool software, and the protein with the maximum reading frame, the length of 597bp and the coding of 198 amino acid residues is obtained. It was found by BLASTX to have significant homology with human (Homo sapiens) peroxiredoxin 1(hPRx1) (accession number gi |4505591| ref | NP _002565.1|) (Identities 155/198 (78%), poistives 176/198 (88%)). Further analysis shows that the thioredoxin peroxidase gene of the Branchiostoma belcheri encodes a mature protein with 198 amino acids, the molecular weight of the protein is 22.15KD, and the isoelectric point is 7.30. The gene does not contain a signal peptide, a transmembrane region and a glycosylation site. The protein was a cytosolic protein as shown by subcellular localization analysis. Blast homology search analysis shows that the homology of the sulfoximine peroxidase of the branchiostoma belcheri and the peroxidase reductase with two active cysteine functional domains is quite high. It has more than 80% homology with both the first and second classes of peroxiredoxins (FIG. 1).

Example three: construction of recombinant amphioxus thioredoxin peroxidase expression plasmid

Synthesizing a pair of primers according to the two end sequences of the TPx gene, wherein the upstream primer contains a BamH I cutting site, and the downstream primer contains an Xho I cutting site.

An upstream primer: 5' -CGCGGA TCCTCT GCT GGA AAT GCC AAG-3’

BaM HI

A downstream primer: 5' -CCGCTC GAG TCA TTACTG CTT GGA GAA ATA TTC TTT G-3’；

Xho I Stop codon

The thioredoxin peroxidase TPx gene fragment (containing the enzyme cleavage site) is obtained by amplifying the library of the Branchiostoma belcheri in the second embodiment by using a PCR method, the length is about 610bp, and the size is consistent with the expected size (figure 2). The recovered and purified PCR product of TPx gene was digested with BamHI and Xho I, and inserted into a window of pETTRX, which was also digested with BamHI and Xho I, to construct expression vector pET-32a (+) M-TPx containing TPx gene fragment, as shown in FIG. 6. The recombinant plasmid was digested with BamHI and Xho I to cut out fragments that were consistent in size with the PCR product, as well as fragments that were consistent in size with the linear vector pET-32a (+) M (5.9 kb). A signature fragment corresponding to the expected size of TPx was also obtained by amplifying the recombinant plasmid with primers specific to the upstream and downstream TPx (FIG. 3). This indicates that the TPx gene has been inserted into the plasmid vector pET-32a (+) M. The purified pET-32a (+) M-TPx recombinant plasmid is subjected to reverse sequencing by taking T7 terminator (synthesized by Shanghai Boya bioengineering company) as a sequencing primer, the sequence of the recombinant plasmid is completely consistent with the sequencing result of the thioredoxin peroxidase TPx gene of the Branchiostoma belcheri, and the result shows that the synthesized primer has no error in PCR amplification and correct code reading frame.

Example four: expression of recombinant amphioxus thioredoxin peroxidase

pET-32a (+) M-TPx was transformed into E.coli BL21(DE 3)/pLysS. SDS-PAGE electrophoretic analysis of the supernatant of the ultrasonic lysis of the genetically engineered bacteria shows that the bacteria have obvious specific expression product band after induction, and the molecular weight is consistent with the theoretical value of 24.46kD predicted by software DNATOL 5.1 (figure 4).

After the investigation of conditions such as culture time, induction concentration, temperature and the like: the induction expression comparison is carried out after the thalli are subjected to amplification culture for different time, and the culture conditions of the genetic engineering bacteria are as follows: the single colony was inoculated in 50ml of ampicillin resistant LB medium and cultured overnight at 37 ℃ and 250rpm/min, 20ml of the overnight culture was inoculated in 2L of ampicillin resistant LB medium and cultured at 37 ℃ and 250rpm/min until OD600 became 0.6 (about 2h for amplification), 100mM IPTG and 20% glucose were added to final concentrations of 0.1mM and 0.2%, respectively, and after induction culture at 25 ℃ and 250rpm/min for 10h, the cells were harvested by centrifugation. SDS-PAGE analysis shows that the expression amount of the recombinant Bbt-TPx protein accounts for over 20%of the total protein of the thallus under the condition and is basically in a soluble state (figure 4).

Example five: purification of recombinant branchiostoma thioredoxin peroxidase

The cultured bacterial liquid after induction expression is centrifuged for 5 minutes at 6,000rpm/min at 4 ℃, the thalli are resuspended in TE buffer solution (pH8.0), centrifuged, and then precooled 50mmol/LTris-HCl (pH8.0) is used,washing thallus with 500mmol/LNaCl solution, ultrasonic treating, cracking supernatant, and passing through Ni²⁺The fusion protein was purified by chromatography on a Chelating Sepharose affinity column in one step and analyzed by SDS-PAGE and thin-layer scanning to show that the purity of the fusion protein reached 80% (FIG. 4). Culturing with 1L gene engineering bacteriaThe nutrient is the material, and about 600mg of purified recombinant protein is finally obtained.

In order to improve the yield and concentration of the recombinant protein, simplify the experimental steps and shorten the treatment time of the recombinant protein, when the recombinant protein is purified by Ni2+ affinity chromatography, a one-step method is adopted for purification. According to the characteristic of strong binding capacity of the foreign protein expressed by the vector plasmid pET-32a (+) M and the Ni2+ affinity column, simplified elution conditions are selected: 50mM Tris, 500mM NaCl, pH8.0 (solution A); 100mM imidazole, 50mM Tris, 500mM NaCl, pH8.0 (liquid B); 150mM imidazole, 50mM Tris, 500mM NaCl, pH8.0 (liquid C); 300mM imidazole, 50mM Tris, 500mM NaCl, pH6.0 (liquid D). Recombinant TPx protein was eluted at D (fig. 4). The fractions having the best separation effect were judged from the SDS-PAGE results.

The recombinant protein (PBS as eluent) is further purified by SP-Sepharose cation exchange chromatography, and the recombinant amphioxus thioredoxin peroxidase Bbt-TPx protein with the purity of more than 95 percent is obtained (figure 5).

Example six: recombinant Bbt-TPx protein reduction H₂O₂Activity assay

The activity test was performed by the method of Lim YS et al (Biochem Biophys Res Commun, 1994, 199 (1): 199-206.). The reaction system was 1ml and comprised 5mmol/L DTT, 50mmol/L HEPES-HCl (pH7.0) buffer and recombinant Bbt-TPx protein (0, 20ug/ml, 50ug/ml, 100 ug/ml). After standing at room temperature for 10 minutes, H was added₂O₂(200 ummol/L). Respectively standing at room temperature for reaction for 0, 2.5 min, 5min and 7.5 min, adding 100ul 100% (w/v) TCA after 10 min, mixing uniformly, and immediately adding 200ul 10mmol/L Fe (NH)₄)₂SO₄And 100ul 2.5mol/L KSCN, O.D was determined.₄₇₅. If one is not added with H₂O₂The system of (3) was used as a blank control (FIG. 7). As can be seen from FIG. 7, in the line to which the recombinant Bbt-TPx protein was addedIn the column, as the reaction time was extended, o.d.₄₇₅The absorption value decreases gradually, which shows that H₂O₂Is reduced by recombinant Bbt-TPx protein. And as the concentration of recombinant Bbt-TPx protein increases, O.D.₄₇₅The faster the absorption value decreases, indicating reduced H₂O₂The greater the amount. While series 1 with Bbt-TPx was not added, there was essentially no change in the absorption value. This experiment demonstrated that recombinant Bbt-TPx protein has reduced H₂O₂And the reducing power is in direct proportion to the concentration of recombinant Bbt-TPx protein.

Example seven: recombinant Bbt-TPx protein protected supercoiled DNA experiment

The activity test was performed by the method of Lim YS et al. The reaction system is50ul, comprising 10mmol/L DTT, 3ummol/LFeCl₃50mmol/L HEPES-HCl (pH7.0) buffer solution, 1ug of supercoiled DNA (pGAPZ α A) extracted by PEG method and recombinant Bbt-TPx protein (500ug/ml, 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml) were incubated at 37 ℃ for 2 hours₃And a recombinant protein; one system did not aggravate histone as a control. After the reaction, 10ul of running agarose was used for electrophoresis detection (gel concentration 0.8%), and the results were analyzed by an ultraviolet spectroscopic imaging system (FIG. 8). The results show that the plasmid DNA used as a control exists in only one conformation of supercoiled (Lane 1) when DTT and Fe are added simultaneously³⁺At this time, the supercoiled conformation of the plasmid is almost destroyed, forming another conformation (nickform) (lane 4). After adding recombinant Bbt-TPx protein to the reaction system, it can be seen that the supercoiled conformation of the plasmid is protected to different degrees (lane 5-9) according to the concentration of recombinant Bbt-TPx protein, and the degree of protection of the supercoiled conformation of the plasmid is proportional to the concentration of recombinant Bbt-TPx protein. This experiment demonstrates that recombinant Bbt-TPx protein has some protective effect on supercoiled DNA when some free radicals damage DNA.

Example eight: activity experiment of recombinant Bbt-TPx protein for protecting GS protein

The activity test was performed according to the method of Kim.K et al. The reaction system is 50ul,comprises 10ug/ml GS, 10mmol/LDTT, 3umol/L FeCl₃50mmol/L imidazole-HCl (pH7.0) and various concentrations of recombinant Bbt-TPx protein (20ug/ml and 100 ug/ml). The system comprises two parts, one part is 30ul and contains GS, imidazole and Bbt-TPx; another 20ul fraction containing DTT and FeCl₃. The two fractions were mixed and incubated at 30 ℃for different times (0, 2min, 8min, 15min, 25min), then 2ml of gamma-Glutamyltransferase Assay mix was added, and after incubation at 30 ℃ for 5min 1ml of Stop mix was added and O.D was determined.₅₄₀. In addition, recombinant Bbt-TPx protein, DTT and FeCl are not added in the preparation of the integrated system₃(ii) a One system did not include recombinant Bbt-TPx protein as a control. Gamma-Glutamyl-hydroxamate-Fe formed by transfer of glutamine and iron ions according to the remaining GS protein of the reaction³⁺O.d of compound.₅₄₀The absorbance was used to determine the ability of the recombinant Bbt-TPx protein to protect the GS protein activity. Reaction time is plotted as abscissa, o.d.₅₄₀The absorbance was plotted on the ordinate and the activity of the recombinant Bbt-TPx protein to protect the GS protein was observed (FIG. 9). As can be seen from FIG. 9, DTT and Fe were contained in the reaction system³⁺When no recombinant Bbt-TPx protein is present, the activity of the GS protein is rapidly reduced along with the prolonging of the reaction time, and when recombinant Bbt-TPx protein with different concentrations is added into the reaction system, the activity of the GS protein is protected to different degrees. When the concentration of the recombinant Bbt-TPx protein is 100ug/ml, the GS protein activity is hardly decreased. The experiment proves that the recombinant Bbt-TPx protein has a protective effect on some proteins which are sensitive to ROS in organisms, such as GS and the like.

Sequence listing

<110>Zhongshan university

<120>thioredoxin peroxidase gene of Wenchang fish in Qingdao China and application thereof

<160>2

<210>1

<211>597

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<213>Wenchang fish in Qingdao China (Branchiostoma belcheri tsingtauense)

<220>

<221>mat peptide

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<400>1

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Met Ser Ala Gly Asn Ala Lys Leu Gln His Pro Ala Pro Asn Phe Glu

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agc acg gct gta cta ccc tct ggg gag ttc aag acc ata aaa ctc tcg 96

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Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Val Glu

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Glu Phe Arg Lys Ile Asn Cys Glu Val Val Ala Cys Ser Thr Asp Ser

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caa ttc tcc cac ttg gcc tgg acg aac acc ccc aga aag cag ggt gga 288

Gln Phe Ser His Leu Ala Trp Thr Asn Thr Pro Arg Lys Gln Gly Gly

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ctg ggc cag atg aag atc cca atc ctg gcc gac aaa gcg atg acc ata 336

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Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly

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<213>Wenchang fish in Qingdao China (Branchiostoma belcheri tsingtauense)

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<400>2

Met Ser Ala Gly Asn Ala Lys Leu Gln His Pro Ala Pro Asn Phe Glu

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Ser Thr Aln Val Leu Pro Ser Gly Glu Phe Lys Thr Ile Lys Leu Ser

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Asp Tyr Lys Gly Lys Tyr Leu Val Ile Phe Phe Tyr Pro Leu Asp Phe

35 40 45

Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Val Glu

50 55 60

Glu Phe Arg Lys Ile Asn Cys Glu Val Val Ala Cys Ser Thr Asp Ser

65 70 75 80

Gln Phe Ser His Leu Ala Trp Thr Asn Thr Pro Arg Lys Gln Gly Gly

85 90 95

Leu Gly Gln Met Lys Ile Pro Ile Leu Ala Asp Lys Ala Met Thr Ile

100 105 110

Ser Arg Asp Tyr Gly Val Leu Met Glu Pro Glu Gly Ile Ala Phe Arg

115 120 125

Gly Leu Phe Ile Ile Asp Asp Lys Gly Thr Leu Arg Gln Ile Thr Ile

130 135 140

Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr Leu Arg Leu Val

145 150 155 160

Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly

165 170 175

Trp Lys Pro Gly Ala Asp Thr Ile Lys Pro Asp Val Lys Asn Ser Lys

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Glu Tyr Phe Ser Lys Gln

195

Claims

1. The DNA sequence of thioredoxin peroxidase gene TPx separated from the total RNA of the ovary of Wenchang fish in Qingdao of China by using the RT-PCR method is shown as<400>1 sequence in the sequence table.

2. The protein coded by the gene of claim 1, wherein the amino acid sequence of the protein is shown as<400>2 sequence in a sequence table; the isoelectric point of the protein is 7.30, and the molecular weight is 22150 daltons.

3. The protein of claim 2, wherein the protein is expressed in an intracellularly soluble form in E.coli by the expression vector pET-32a (+) M-TPx.

4. The protein as claimed in claim 3, wherein the expression vector pET-32a (+) M-TPx is a high-efficiency expression vector with His affinity tag site and TPX gene sequence inserted in the middle of the multiple cloning site of plasmid pET-32a (+) M, and the system can make TPx gene be expressed in Escherichia coli in soluble form, and the expression quantity can reach 600 mg/L.

5. Use of a protein according to claim 2, 3 or 4 for the preparation of a medicament for anti-tumour and anti-ageing.