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Methionine sulfoxide reductase as well as coding gene, preparation method and application thereof

A technology of methionine sulfoxide and reductase, which is applied in the fields of genes, strains and their uses, sequence preparation methods, and eggs, can solve the problems of unachievable and high extraction costs of selenoprotein MsrA, and achieve anti-oxidation and large-scale production Industrialized mass production and the effect of reducing production costs

Active Publication Date: 2020-07-28
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the high extraction cost of the selenoprotein MsrA in the prior art and the inability to realize its mass production and expression, thereby providing a methionine sulfoxide reductase that can be used for genetic engineering expression

Method used

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  • Methionine sulfoxide reductase as well as coding gene, preparation method and application thereof
  • Methionine sulfoxide reductase as well as coding gene, preparation method and application thereof
  • Methionine sulfoxide reductase as well as coding gene, preparation method and application thereof

Examples

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Embodiment 1

[0055] This embodiment provides a method for cloning the methionine sulfoxide reductase gene of Haematococcus pluvialis, which specifically includes the following steps:

[0056] (1) extract the total RNA of Haematococcus pluvialis, utilize PCR cDNA Synthesis Kit (Clontech, USA) was used to reverse transcribe total RNA into cDNA.

[0057] (2) Using the cDNA of Haematococcus pluvialis as a template, the amplification primers HpMsrA-F and HpMsrA-R were used to amplify to obtain the cDNA amplification fragment of the methionine sulfoxide reductase gene in Haematococcus pluvialis. Wherein, the nucleotide sequence of HpMsrA-F is shown in SEQ ID NO.3, and the nucleotide sequence of HpMsrA-R is shown in SEQ ID NO.4. The PCR amplification conditions were: 95°C for 2min; (95°C for 30s, 58°C for 45s, 72°C for 1min, 35 cycles); 72°C for 10min.

[0058] (3) Carry out 5'-RACE amplification and 3'-RACE amplification to the cDNA fragment in step (2), wherein, the primers used in 5'-RACE ...

Embodiment 2

[0064] This embodiment provides a method for preparing methionine sulfoxide reductase, which specifically includes the following steps:

[0065] S1, constructing a recombinant expression vector expressing the methionine sulfoxide reductase of the amino acid sequence shown in SEQ ID NO.1, specifically:

[0066] At both ends of the cDNA fragment of the methionine sulfoxide reductase gene, XbaI and XhoI restriction sites were introduced, and pET-28a (purchased from Solarbio) was used as the carrier, and the restriction endonucleases of XbaI and XhoI were respectively used to pET-28a Carry out enzyme digestion with the gene fragment of MsrA, and recover the digested fragment by gel to obtain the linear plasmid fragment of 5'XbaI-pET-28a-3'XhoI and the gene fragment of 5'XbaI-MsrA-3'XhoI.

[0067] Add the recovered 5'XbaI-pET-28a-3'XhoI and 5'XbaI-MsrA-3'XhoI into 10 μL T4 ligase system, and ligate at 16°C overnight. The DH5a competent cells were transformed with the ligation prod...

Embodiment 3

[0072] This embodiment provides a recombinant expression vector and a recombinant strain comprising the methionine sulfoxide reductase gene, wherein the methionine sulfoxide reductase gene is specifically a cDNA fragment having the nucleotide sequence shown in SEQ ID NO.2.

[0073] Specifically, the recombinant expression vector is the recombinant expression vector pET-28a-MsrA constructed in Example 2 with pET-28a as the carrier and XbaI and XhoI as the cloning sites; the recombinant strain is the recombinant expression vector pET-28a- Recombinant transformants obtained by transforming MsrA into BL21 competent cells.

[0074] The recombinant expression vector pET-28a-MsrA and the recombinant strain provided in this example can be used to induce the expression of methionine sulfoxide reductase in vitro, so as to reduce the production cost of methionine sulfoxide reductase and realize large-scale industrial mass production.

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Abstract

The invention discloses a methionine sulfoxide reductase, wherein the methionine sulfoxide reductase is selected from any one of the following (a1)-(a3): (a1) is an amino acid sequence shown as SEQ IDNO. 1; (a2) is an amino acid sequence having at least 85% homology with the amino acid sequence shown in SEQ ID NO. 1; and (a3) is a homolog, derivative, fragment or mutant of the amino acid sequencerepresented by (a) or (b) without losing biological activity. The sequence of the methionine sulfoxide reductase of haematococcus pluvialis is provided for the first time, a foundation is laid for genetic engineering expression of selenoprotein MsrA, and large-scale industrial production of the methionine sulfoxide reductase with high catalytic activity is facilitated. The invention also discloses a gene, a recombinant expression vector, a recombinant cell line or a recombinant strain for coding the methionine sulfoxide reductase, which is beneficial to realizing in-vitro gene engineering expression of MsrA and reducing the acquisition cost of MsrA.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a methionine sulfoxide reductase, a methionine sulfoxide reductase gene, a recombinant expression vector, a recombinant cell line or a recombinant strain and its use, and the cDNA sequence of the methionine sulfoxide reductase gene Preparation method, preparation method of methionine sulfoxide reductase, detection primer and detection probe, chip or kit of methionine sulfoxide reductase. Background technique [0002] Oxygen in organisms undergoes incomplete reduction during normal metabolism to generate reactive oxygen species (ROS, ReactiveOxygen Species), mainly including superoxide anion radicals, hydrogen peroxide, peroxynitrite anions, hypochlorous acid, singlet oxygen and hydroxyl radicals, etc. ROS can interact with a series of biomolecules, causing oxidative damage or affecting many signal transduction processes, regulating cell proliferation, differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/10C12Q1/6876C12N15/11A61K38/44A61P39/06A61P25/28A61P27/12
CPCC12N9/0051C12N15/1003C12Q1/6876A61K38/44A61P39/06A61P25/28A61P27/12
Inventor 郑怡鸿胡章立陶明李泽黄观钦薛登峰
Owner SHENZHEN UNIV
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