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Structure of human recombinant methionine sulfoxide reductase A (MsrA) protein, and construction method of structure

A technology of methionine sulfoxide and construction method, which is applied in the field of structure and construction of human recombinant methionine sulfoxide reductase A protein, to improve the anti-As effect of HDL

Inactive Publication Date: 2019-12-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] But the problem encountered is: MsrA is an intracellular enzyme, and after functioning, the oxidized MsrA needs the intracellular thioredoxin and NADPH system to regenerate its reducing ability, and MsrA has no direct binding to HDL. How to make MsrA in the cell Targeted binding to HDL to play a role, and can make MsrA return to the cell to restore the function and then enter the circulation

Method used

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  • Structure of human recombinant methionine sulfoxide reductase A (MsrA) protein, and construction method of structure
  • Structure of human recombinant methionine sulfoxide reductase A (MsrA) protein, and construction method of structure
  • Structure of human recombinant methionine sulfoxide reductase A (MsrA) protein, and construction method of structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 of the present invention provides the structure of a secreted human recombinant methionine sulfoxide reductase A protein, including:

[0094] The structure contains a segment of human apoE signal peptide and EpK peptide, as shown in SEQ ID NO.4:

[0095] Met Lys Val Leu Trp AlaAla Leu Leu Val Thr Phe LeuAla Gly Cys GlnAlaHis His His His His His Leu Arg Lys LeuArg Lys Arg Leu Leu Arg Lys Lys LysLys Lys Lys Lys Gln Val Ala Glu Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln IleArg Leu Gln Ala Glu

[0096] Wherein, the complete structure of the EpK peptide is expressed as shown in SEQ ID NO.2:

[0097] LeuArg Lys LeuArg Lys Arg Leu LeuArg Lys Lys Lys Lys Lys Lys GlnValAla GluValArgAla Lys Leu Glu GlnAla Gln Gln IleArg Leu GlnAla Glu;

[0098] The structure of MsrA is a 213-amino acid chain without signal peptide, which is connected to form a complete EpK-MsrA fusion protein polypeptide chain through the coding amino acids of BamHI restriction endonuclease recogniti...

Embodiment 2

[0121] The embodiment of the present invention aims at the construction structure proposed in the embodiment 1, and elaborates on how to demonstrate its effect through experiments. Specifically, the experimental research is conducted on the in vivo function of the recombinant Epk-MsrA fusion protein. Including the following process:

[0122] Process 1. Packaging of recombinant lentiviral particles containing human Epk-MsrA

[0123] The packaging of the recombinant lentiviral particles containing human Epk-MsrA comprises the following steps:

[0124]Step 1. Extraction and identification of lentiviral vectors and packaging plasmids: Inoculate the pWPI-Epk-MsrA recombinant plasmid (pWPI-GFP as a control) and the DH5α strains of the three lentiviral packaging plasmids PLP1, PLP2, and pVSVG, respectively, into cells containing In 250ml of liquid LB medium with 100μg / ml ampicillin, shake at 37°C and 200-300rpm for 12-16h; the next day, centrifuge the bacterial culture solution a...

Embodiment 3

[0147] The embodiment of the present invention provides the construction of a recombinant human Epk-MsrA fusion protein expressed by prokaryotic cells, including:

[0148] The recombinant prokaryotic expression vector pET28a-EpK-MsrA was constructed by genetic engineering technology, and the recombinant EpK-MsrA fusion protein expressed in Escherichia coli was purified by NTA-Ni column affinity chromatography to obtain the purified EpK-MsrA fusion protein.

[0149] In the embodiment of the present invention, because the pET8a prokaryotic expression vector containing the His-tag and the Escherichia coli host bacteria are used, the signal peptide sequence of EpK and the 6-His sequence in the middle of the structure are no longer designed (as presented in Example 1) ), a typical basic structure of EpK-MsrA described in the embodiments of the present invention, as shown in SEQ ID NO.5:

[0150] Met Cys Leu Arg Lys Leu Arg Lys Arg Leu Leu Arg Lys Lys Lys Lys Lys Lys Gln Val Ala Glu...

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Abstract

The invention discloses a structure of a human recombinant methionine sulfoxide reductase A (MsrA) protein, and a construction method of the structure. The structure contains a section of compositionstructure of human EpK peptides; and the structure of the MsrA is 213 amino acid chains without signal peptides which are connected to form a complete human EpK-MsrA fusion protein polypeptide chain through encoded amino acids of a BamHI restriction enzyme identification sequence. The prepared recombinant EpK-MsrA fusion protein is highly expressed in the liver in a mode of infecting scavenger receptor B-group I-type gene knockout (SR-BI- / -) mice with slow viruses and can be secreted into blood; and the secreted EpK-MsrA fusion protein can be characteristically bound to high-density lipoprotein (HDL), thus the content of apolipoprotein (AI) and the content of paraoxonase 1 of HDL functional protein components are increased, the activity of the AI and the activity of the paraoxonase 1 of HDL functional protein components are improved, inflammatory components in plasma and the HDL are reduced, and the effect of resisting an atherosclerotic cardiovascular disease is achieved.

Description

【Technical field】 [0001] The invention relates to the technical field of medicine, in particular to the structure and construction method of a human recombinant methionine sulfoxide reductase A protein. 【Background technique】 [0002] Coronary heart disease, stroke and other important cardiovascular and cerebrovascular diseases caused by atherosclerosis (Atherosclerosis, abbreviated as As) have become the "number one killer" that seriously endangers human health. The occurrence and development of As are closely related to lipid metabolism disorder, oxidative stress, inflammation and other etiologies. Among them, lipid metabolism disorder is one of the most important causes. Abnormal deposition of lipids in cells can promote the increase of reactive oxygen species (Reactive Oxygen Species, abbreviated as: ROS) and the release of inflammatory factors, inducing oxidative stress and inflammation. , and the increase of ROS and inflammation levels aggravated the imbalance of lipi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N15/867C07K1/22
CPCC07K14/775C07K2319/02C07K2319/21C12N9/0051C12N15/70C12N15/86C12N2740/15043C12Y108/05003
Inventor 喻红曹佳徐延勇赵小杰刘梦婷杜芬李菲菲刘雨周瑞
Owner WUHAN UNIV
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