CN102586202A - Thioredoxin and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of molecular biology, in particular to Cynoglossus semilaevis thioredoxin and a preparation method and application thereof. The thioredoxin is shown as an amino acid sequence in a sequence table SEQ ID No.1. The preparation method comprises the following steps of: constructing a plasmid pETrx, transforming the plasmid pETrx by using BL21(DE3) to obtain BL21(DE3)/pETrx, inducing by using isopropyl-beta-D-isopropylthiogalactoside, and purifying recombinant protein by using an affinity chromatography column to obtain the thioredoxin. The thioredoxin has remarkable reductase activity, a remarkable antioxidation effect and a remarkable cell growth promotion effect.

Description

A kind of Trx and preparation thereof and application

Technical field

The present invention relates to biology field, a kind of specifically Cynoglossus semilaevis Trx and preparation and application.

Background technology

(thioredoxin is the small molecules reduction albumen of about 12 kDa of a kind of molecular weight Trx) to Trx, extensively is present in various protokaryons and most eukaryotes.Trx has a catalytic site high conservative, the tool reducing activity, and its constitutional features is CXXC.Trx and the effect of various sulfydryl albumen are two sulfydryls with its disulfide bond reduction.Function whereby, Trx is regulated and control multiple important biomolecule process, like the keeping of redox state, genetic expression, immunoreation etc.The Trx of known higher animal has the various biological activity, comprises anti-oxidant activity, growth promoting function, anti-apoptotic, antitumor action etc.But the research of fish Trx seldom, and its effect and BA are neither very clear.

Summary of the invention

The object of the invention is to provide a kind of Trx and preparation and application.

For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:

A kind of Trx, Trx are shown in the aminoacid sequence among the sequence table SEQ ID No.1.

Said Trx is the recombinant protein that contains the eukaryotic expression recombination plasmid pETrx transformed into escherichia coli DH5 α acquisition of tabulation SEQ ID No.1.

The preparation method of Trx:

With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1, is connected with carrier pBS-T behind the PCR product purification, connects the mixed solution transformed into escherichia coli, gets plasmid pBSTrx;

Above-mentioned plasmid pBSTrx and plasmid pET259 are cut with restriction enzyme EcoRV enzyme, and endonuclease bamhi connects with the T4DNA ligase enzyme, connects liquid and is transformed into intestinal bacteria, gets plasmid pETrx;

Above-mentioned gained plasmid pETrx is transformed BL21 (DE3), and gained transformant BL21 (DE3)/pETrx purifying gained recombinant protein is the Trx shown in the aminoacid sequence among the sequence table SEQ ID No.1;

Said F1 is 5 '-GATATCATGGTTTACGAAGTGAAAG-3 '; R1 is 5 '-GATATCTTCTTTTTCATACTGCTTC-3 '.

The application of Trx, the amino acid sequences encoded Trx among the said sequence table SEQ ID No.1 is as the preparation of reductase activity, anti-oxidant or promoting growth of cell.

The present invention has following advantage: Trx preparation of the present invention is simple, has tangible reductase activity, antioxygenation and promoting growth of cell ability.

Description of drawings

(wherein, the Trx that obtains of purifying is a swimming lane 2 to the Trx electrophoretogram that the purifying that Fig. 1 provides for the embodiment of the invention obtains; Swimming lane 1: molecular weight marker.)。

The reductase activity detection figure of the Trx that Fig. 2 provides for the embodiment of the invention.

Embodiment

Below in conjunction with embodiment the present invention is described further.Embodiment is intended to the description of giving an example to the present invention, but not limits the invention in any form.

Involved in embodiments of the present invention routine property experimental technique all adopts following method:

1. plasmid extraction, DNA (PCR) product purification, dna fragmentation reclaim the corresponding reagent box that all uses " TIANGEN Biotech (Beijing) Co., Ltd. " from gel.

2. intestinal bacteria are with Hanahan method (Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001); Yeast conversion is pressed the method (Catalog no.V175-20) of Invitrogen company; Polyacrylamide gel electrophoresis (SDS-PAGE) is according to Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001.

3. all restriction enzymes and ligase enzyme are all available from " Niu Yinglun Bioisystech Co., Ltd ", Beijing.

Embodiment 1

Trx of the present invention is the aminoacid sequence among the sequence table SEQ ID No.1.Sequence table SEQ ID No.1 is:

MVYEVKDYNDFTQQLKRAGNKLVVVDFTATWCQPCKNISPVFEELANQYKNVVFLKVDVDEADDVSTECE?I?NCMPTFLFYRNEKRVHSFSGANSDTLRAAVKQYEKE

(a) sequence signature:

Length: 107

Type: aminoacid sequence

Chain: strand

Topological framework: linearity

(b) molecule type: protein

(c) suppose: not

(d) antisense: not

(e) initial source: Cynoglossus semilaevis

Constructional feature: this albumen expection contains a CXXC structural domain, and 35-38 constitutes by amino acid,

Embodiment 2

The preparation method of Trx:

1) structure of plasmid pETrx:

With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1.The PCR condition is: 94 ℃ of preparatory sex change template DNAs of 60s, 94 ℃ of 40s then, 48 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 58 ℃ of 60s, 72 ℃ of 60s, after 30 circulations again at 72 ℃ of extension 7-10min.Be connected 4-6 hour with carrier pBS-T (purchasing) in room temperature behind the PCR product purification in " TIANGEN Biotech (Beijing) Co., Ltd. "; On the LB substratum that contains peace card penicillium mould (100ug/ml), Xgal (40ug/ml), isopropyl-(24ug/ml), cultivated 18-24 hour after connecting mixed solution transformed into escherichia coli DH5 α; The screening transformant extracts plasmid, is plasmid pBSTrx.(plasmid pET259 building process is referring to Zheng, W.J., Hu with pBSTrx and plasmid pET259; Y.H., Sun, L.; 2010.Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus) .Fish.Shellfish.Immunol.28 829-836) cuts the back with restriction enzyme EcoRV enzyme and reclaims 0.3kb and 5.4kb fragment, and this two fragment is connected with the T4DNA ligase enzyme; Connect liquid and be transformed into bacillus coli DH 5 alpha; On the LB substratum that contains kantlex (30ug/ml), cultivated 18-24 hour, the screening transformant extracts plasmid, is pETrx.

Said LB moity is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium-chlor, 97.5% zero(ppm) water.Said F1 is 5 '-GATATCATGGTTTACGAAGTGAAAG-3 '; R1 is 5 '-GATATCTTCTTTTTCATACTGCTTC-3 '.

2) expression of Trx and preparation

The plasmid pETrx of step 1) is transformed BL21 (DE3) (purchasing in " TIANGEN Biotech (Beijing) Co., Ltd. "), on the LB substratum that contains kantlex (30ug/ml), cultivate, the screening transformant is BL21 (DE3)/pETrx.BL21 (DE3)/pETrx is cultured to OD in 37 ℃ in containing the LB substratum of kantlex ₆₀₀Be 0.6; Adding isopropyl-makes its final concentration reach 0.4mM; 30 ℃ were continued wave and culture 4-5 hour; Then use the nickel-nitrilotriacetic acid affinity column purification of recombinant proteins (chromatography condition: wash post twice with 4-5ml Wash buffer, wash post with 0.5-1ml Elution buffer subsequently, collect all elutriants) of GE Healthcare (U.S.) company.Recombinant protein in the elutriant is carried out mass spectroscopy, be confirmed that it is the Trx shown in the aminoacid sequence among the sequence table SEQ ID No.1.Recombinant protein is analyzed the molecular weight consistent (referring to Fig. 1) of sequence in finding its molecular weight and showing SEQ ID No.1 with polyacrylamide gel electrophoresis (SDS-PAGE).

Above-mentioned Wash buffer composition is: 50mM NaH ₂PO ₄, 300mM NaCl, 20mM imidazole, pH 8.0; Above-mentioned Elution buffer composition is: 50mM NaH ₂PO ₄, 300mM NaCl, 250mMimidazole, pH 8.0.

Embodiment 3

The reductase activity of Trx

The Trx Trx of purifying is added A buffer (0.1M PBS, 2mM DTT, 2mMEDTA-Na ₂, 1.25mg/ml insulin) to final concentration be 4uM.Control group adds isopyknic PBS.Measure light absorption value (A at 650nm behind 25 ℃ of insulation different times ₆₅₀).The result shows, the A of control group ₆₅₀Almost nil, and add the A that Trx organizes ₆₅₀Value prolongs along with soaking time and significantly strengthens, and explains that Trx has tangible reductase activity (referring to Fig. 2).

Said PBS moity is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na ₂HPO ₄.12H ₂O, 0.024% NaH ₂PO ₄, surplus is a water.

Embodiment 4

The protection cell antioxygenation of Trx

Cynoglossus semilaevis head-kidney lymphocyte (is purchased the HyClone in Thermo Scientific, Beijing, the dull and stereotyped cultivation in 96-hole China) in containing the L-15 substratum.The extraction of head-kidney lymphocyte, cultural method are referring to reference Hu Y; Chen L; Sun L.CXCL8 of Scophthalmus maximus:expression, biological activity, and immunoregulatory effect.Dev Comp Immunol.2011; 35:1030-7.15ul Trx or PBS (contrast) are mixed 25 ℃ of insulation 0.5h with 15ul20mM DTT.It is 3ug/ml that Trx after the insulation is added lymphocyte to final concentration, adds H subsequently ₂O ₂To final concentration be 200uM.22 ℃ of insulation 1h, (China), 22 ℃ of insulation 4h add 200 μ ldimethyl sulfoxide, are measuring light absorption value (A at 490nm subsequently for Sangon, Shanghai to add 20 μ l5mg/ml MTT ₄₉₀).The result shows, adds the A of the cell of Trx ₄₉₀Be 3 times of control cells, explain that Trx can protect cell resistance H ₂O ₂Oxygenizement.

Embodiment 5

The promoting growth of cell effect of Trx

With Trx Trx or PBS (contrast) add as in the lymphocyte of above-mentioned embodiment 4 said cultivations to final concentration be 1.5ug/ml.Cell in 22 ℃ of insulations 2 days, is added 20 μ l 5mg/mlMTT, and 22 ℃ of insulation 4h add 200 μ l dimethyl sulfoxide, are measuring light absorption value (A at 490nm subsequently ₄₉₀).The result shows, adds the A of the cell of Trx ₄₉₀Be 2.5 times of control cells, explain that Trx can effectively promote cell proliferation.

In sum, Trx of the present invention has tangible reductase activity, antioxygenation and promoting growth of cell ability.

Claims (4)

1. Trx, it is characterized in that: Trx is shown in the aminoacid sequence among the sequence table SEQ ID No.1.

2. by the described Trx of claim 1, it is characterized in that: said Trx is the recombinant protein that contains the eukaryotic expression recombination plasmid pETrx transformed into escherichia coli DH5 α acquisition of tabulation SEQ ID No.1.

3. the preparation method of the described Trx of claim 1 is characterized in that:

With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1, is connected with carrier pBS-T behind the PCR product purification, connects the mixed solution transformed into escherichia coli, gets plasmid pBSTrx;

Above-mentioned plasmid pBSTrx and plasmid pET259 are cut with restriction enzyme EcoRV enzyme, and endonuclease bamhi connects with the T4DNA ligase enzyme, connects liquid and is transformed into intestinal bacteria, gets plasmid pETrx;

Above-mentioned gained plasmid pETrx is transformed BL21 (DE3), and gained transformant BL21 (DE3)/pETrx purifying gained recombinant protein is the Trx shown in the aminoacid sequence among the sequence table SEQ ID No.1;

Said F1 is 5 '-GATATCATGGTTTACGAAGTGAAAG-3 '; R1 is 5 '-GATATCTTCTTTTTCATACTGCTTC-3 '.

4. the application of the described Trx of claim 1 is characterized in that: the amino acid sequences encoded Trx among the said sequence table SEQ ID No.1 is as the preparation of reductase activity, anti-oxidant or promoting growth of cell.

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