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Thioredoxin and preparation method and application thereof

A technology of thioredoxin and sulfur oxide, which is applied in the field of molecular biology, can solve problems such as unclear functions and biological activities, and achieve obvious reductase activity and simple preparation

Inactive Publication Date: 2012-07-18
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on fish thioredoxin, and its role and biological activity are not very clear

Method used

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  • Thioredoxin and preparation method and application thereof
  • Thioredoxin and preparation method and application thereof
  • Thioredoxin and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The thioredoxin of the present invention is the amino acid sequence in SEQ ID No.1 of the sequence table. The sequence listing SEQ ID No.1 is:

[0023] MVYEVKDYNDFTQQLKRAGNKLVVVDFTATWCQPCKNISPVFEELANQYKNVVFLKVDVDEADDVSTECE I NCMPTLFYRNEKRVHSFSGANSDTLRAAVKQYEKE

[0024] (a) Sequence features:

[0025] ·Length: 107

[0026] Type: amino acid sequence

[0027] Chain type: single chain

[0028] Topology: linear

[0029] (b) Molecule type: protein

[0030] (c) Assumption: No

[0031] (d) Antisense: no

[0032] (e) Original source: tongue sole

[0033] Structural features: The protein is expected to contain a CXXC domain consisting of amino acids 35-38,

Embodiment 2

[0035] The preparation method of thioredoxin:

[0036] 1) Construction of plasmid pETrx:

[0037] The cDNA of tongue sole was used as a template and PCR amplification was carried out with primers F1 and R1. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 94°C for 40s, 48°C for 60s, 72°C for 60s, after 5 cycles, change to 94°C for 40s, 58°C for 60s, 72°C for 60s, and then repeat at 72°C after 30 cycles. ℃ extension reaction 7-10min. After the PCR product was purified, it was ligated with the carrier pBS-T (purchased from "Tiangen Biochemical Technology (Beijing) Co., Ltd.") at room temperature for 4-6 hours, and the ligation mixture was transformed into E. , Xgal (40ug / ml), and isopropyl-β-D-thiogalactoside (24ug / ml) were cultured on LB medium for 18-24 hours, and the transformants were screened to extract the plasmid, which was the plasmid pBSTrx. PBSTrx and plasmid pET259 (see Zheng, W.J., Hu, Y.H., Sun, L., 2010. Cloning and analysis of a ferr...

Embodiment 3

[0043] Thioredoxin reductase activity

[0044] Add the purified thioredoxin Trx to A buffer (0.1M PBS, 2mM DTT, 2mM EDTA-Na 2 , 1.25mg / ml insulin) to a final concentration of 4uM. An equal volume of PBS was added to the control group. Absorbance was measured at 650nm after incubation at 25°C for different times (A 650 ). The results showed that A in the control group 650 Almost zero, while A who joined the Trx group 650 The value is significantly strengthened with the extension of incubation time, indicating that thioredoxin has obvious reductase activity (see figure 2 ).

[0045] The composition of PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

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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

technical field [0001] The invention relates to the field of molecular biology, in particular to a half-smooth tongue sole thioredoxin and its preparation and application. Background technique [0002] Thioredoxin (Trx) is a small molecule reducing protein with a molecular weight of about 12 kDa, which widely exists in various prokaryotic and eukaryotic organisms. Thioredoxin possesses a highly conserved, reductively active catalytic site characterized by CXXC. Thioredoxin interacts with various sulfhydryl proteins, reducing their disulfide bonds to dithiols. Through this function, thioredoxin regulates a variety of important biological processes, such as maintenance of redox state, gene expression, immune response, etc. It is known that thioredoxin in higher animals has a variety of biological activities, including antioxidant activity, growth-promoting effect, anti-apoptosis, anti-tumor effect and so on. However, there are few studies on fish thioredoxin, and its functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70A61P39/06A61P35/00
Inventor 孙黎李永鑫
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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