Trachinotus ovatus thioredoxin gene

A pomfret thioredoxin and oval technology, applied in the field of genetic engineering, can solve the problems of restricting sustainable development and high mortality, and achieve good reduction activity and reduction effect

Active Publication Date: 2015-04-01
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in recent years, the frequent occurrence of diseases of pomfret ovata has caused a high mortality rate and has become one of the technical bottleneck

Method used

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  • Trachinotus ovatus thioredoxin gene
  • Trachinotus ovatus thioredoxin gene
  • Trachinotus ovatus thioredoxin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Acquisition of thioredoxin cDNA gene of ovate pomfret

[0039] The full-length thioredoxin gene of the present invention is obtained by sequencing the transcriptome, and the thioredoxin cDNA sequence is specifically shown in SEQ ID NO.1. Liver total RNA of pompano ovata ( figure 1 Shown) According to the instructions of M-MLV reverse transcriptase, the cDNA obtained by reverse transcription is used as a template such as figure 2 , Reverse transcription reaction conditions: incubate at 70°C for 5 minutes, ice-bath for 5 minutes, add M-MLV reverse transcriptase master mix, incubate at 42°C for 90 minutes, inactivate at 70°C for 15 minutes.

[0040] The amino acid sequence of thioredoxin in pomfret was obtained by the software DNAStar7.1.

Embodiment 2

[0041] Example 2 Synthesis of thioredoxin gene from ovate pomfret

[0042]According to the complete cDNA sequence of the ovoid pomfret thioredoxin gene in Example 1, a pair of primers were designed and synthesized, and the upstream primer was to add a restriction enzyme site of BamH I before 22 bases from the 117th base and 3 protected bases (5'-GGTGGATCCATGGTCCGCGAAGTGTCAGATC-3'), a total of 31bp; the downstream primer is the first 22 bases from the 419th base plus the EcoRI restriction enzyme site and 2 protected bases (5'-GCGAATTCTCATTTATATTTCATCCAGTTTC-3'), a total of 30bp. Using the cDNA library of the pomfret ovata as a template, the DNA sequence corresponding to the amino acid sequence from the 1st to the 107th amino acid sequence of the pomfret ovata was amplified by PCR method, which is the corresponding DNA sequence of the mature peptide of the pomfret ovale thioredoxin Sequence, PCR amplification conditions are: pre-denaturation at 94°C for 2min; then denaturation ...

Embodiment 3

[0044] Example 3 Construction of an Escherichia coli expression vector containing the ovoid pompano thioredoxin gene

[0045] After the PCR product amplified in the above-mentioned Example 2 was digested with BamH I and EcoRI, the digested product was recovered with the PCR product purification kit of AxyGen Company, and the 324bp oval pomfret thioredoxin gene fragment was isolated and purified; the expression vector pRSET A was also digested with BamH I and EcoRI, and the digested product was subjected to agarose gel electrophoresis to separate and purify a large fragment of 2.9kb, which was mixed with the 324bp thioredoxin gene fragment of oval pomfret at a ratio of 1:3. After ligation with T4 ligase overnight at 16°C, transfer into Escherichia coli BL21 with standard calcium chloride transformation method, screen for ampicillin-resistant transformants, extract plasmids with standard methods, and screen for recombinants with a size of about 3.2 kb. Two fragments of 324bp and...

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Abstract

The invention discloses a cDNA of a trachinotus ovatus thioredoxin gene. The nucleotide of the cDNA is as shown in the SEQ ID NO.1 in the description. The invention further discloses an expression vector containing the trachinotus ovatus thioredoxin gene, a recombinant microorganism transformed by utilizing the vector, and a method for preparing the trachinotus ovatus thioredoxin gene by utilizing the microorganism.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a thioredoxin gene of ovoid pomfret, a carrier containing the gene, a recombinant bacterial strain, and the preparation of thioredoxin of ovoid pomfret by using the recombinant strain. Background technique [0002] Thioredoxin (Trx), also known as interleukin-1-like cytokine, is a small molecule reducing protein ubiquitous in organisms, with a molecular weight of about 12kD, and has common characteristics: ① All Trx have an exposed A highly conserved active center (CXXC) composed of two Cys residues; ②The secondary structure includes 4 α-helices and 5 β-sheets, and the β-sheets are surrounded by α-helices to form a compact spherical shape; ③The active center is located at the beginning of the α-helix and between the tail of the β sheet; ④The active center of the oxidized Trx has a disulfide bond (S-S) composed of Cys residues. Trx was originally an electro...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70
Inventor 张殿昌王龙郭华阳马振华张楠江世贵
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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