Recombinant human apoE peptide mimics, preparation method and application

A peptide segment and binding region technology, applied in the field of the preparation of apolipoprotein E peptidomimetics, can solve the problems of inter-immunogenicity and safety, organ specificity, low solubility, cytotoxic fragments, complex structure, etc. The effect of chemical expression and purification, high yield, and simple purification process

Inactive Publication Date: 2013-01-09
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, apoE (34kD) contains 299 amino acids, and it has not been successful to obtain apoE with a high expression yield. Secondly, there are still some other problems in the clinical application of full-length apoE as a therapeutic drug, including low solubility and easy to be absorbed by enzymes in vivo. Degradation produces cytotoxic fragments, which have a large molecular weight and complex structure when being developed as a drug. At the same time, the gene regulation of apoE by genetic engineering technology also has problems such as immunogenicity, safety and organ specificity among species.

Method used

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  • Recombinant human apoE peptide mimics, preparation method and application
  • Recombinant human apoE peptide mimics, preparation method and application
  • Recombinant human apoE peptide mimics, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Design of recombinant vector and purification and identification of EpK peptide

[0061] The present invention designs a derivative peptide (EpK) containing an N-terminal LDLR binding region and a C-terminal lipid-binding region of human apoE. This peptide contains a cysteine ​​(Cys) at the N-terminal and an apoE LDLR-binding region (the LRKLRKRLLR peptide segment at positions 141-150), followed by an apoE lipid binding region (the QVAEVRAKLEEQAQQIRLQAE peptide segment at positions 234-254) connected by 6 Lys residues.

[0062] The 38 amino acid sequence of EpK is as follows:

[0063] NH 2 -CLRKLRKRLLRKKKKKKQVAEVRAKLEEQAQQIRLQAE-COOH

[0064] The present invention uses genetic engineering technology to construct a recombinant vector, and the recombinant peptide expressed in bacteria undergoes two-step affinity chromatography of chitin beads and heparin Sepharose CL-6B to obtain a purified recombinant human apoE peptidomimetic (EpK). The specific steps are a...

Embodiment 2

[0076] Example 2: Characteristic detection of purified EpK peptide secondary structure and lipid-binding activity

[0077] ApoE has a very high α-helical structure. According to the designed EpK peptide sequence, it contains a part of the LDLR binding region and a lipid binding region of human apoE. Based on the prediction of the protein secondary structure prediction software (Network Protein Sequence Analysis), EpK may contain 89.74% In order to confirm the ability of EpK to form an α-helix, we analyzed the secondary structure of EpK by circular dichroism, and compared EpK with human full-length apoE3 by DMPC binding experiments to observe their binding to lipids. ability.

[0078] (1) Characterization of the secondary structure of the EpK peptide

[0079] Circular dichroism (CD) was used to measure the secondary structure of the EpK peptide, that is, at 37°C, a cuvette with a liquid layer thickness of 0.2cm was used to measure the spectrum of the sample in the far ultravio...

Embodiment 3

[0088] Example 3: EpK enhances macrophage cholesterol efflux and inhibits macrophage inflammation

[0089] Functional evaluation of purified EpK using primary mouse peritoneal macrophages. Examination of whether EpK mediates cholesterol efflux in cholesterol-loaded macrophages. Second, to detect whether EpK inhibits primary apoE - / - Inflammatory responses induced by LPS in mouse peritoneal macrophages. apoE - / - The mouse model (from Jackson Laboratory) was raised in the Animal Experiment Center (SPF grade) of Zhongnan Hospital of Wuhan University.

[0090] (1) Cell culture:

[0091] In order to obtain mouse macrophages, mice were injected intraperitoneally with 3ml 3% thioglycollate, and mice were euthanized 3 days later, and mouse peritoneal macrophages were collected by perfusion with 10ml PBS. Cells were seeded in 6-well or 24-well cell culture plates, cultured in Dulbecco's Modified Eagle Medium (DMEM) (both purchased from GIBCO) containing 10% fetal bovine serum (FBS...

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Abstract

The invention discloses a recombinant human apoE peptide mimics, a preparation method and an application, according to the invention, a separated recombinant apoE peptide mimics (EpK) has a sequence of an amino acid sequence shown in SEQ ID NO:1. The invention is characterized in that a prokaryotic expression system is used for expressing, and a two-step affinity chromatography method is used for preparing purified EpK, the yield is high, the cost is low, the purifying flow is simple, and the high purity protein without any label is obtained, and the scale expression and purification can be carried out to obtain pure peptide with high amount. The EpK has the functional characteristic that an alpha helical structure is provided, and is combined with lipid in vivo or in vitro, and is preferably combined with HDL. EpK and HDL combined with EpK can substantially enhance the mediation macrophage cholesterol outflow and inhibit the function of macrophage inflammation reaction, simultaneously, EpK can enhance the macrophage antioxidation activity. The invention provides the application of the recombinant human apoE peptide mimics in the medicines for treating or preventing cardio cerebrovascular diseases such as coronary heart disease, myocardial infarction, apoplexy and the like.

Description

technical field [0001] The present invention relates to the field of biotechnology medicine and its application, in particular to a recombinant human apolipoprotein E (apoE) mimetic active peptide, to a preparation method of apolipoprotein E (apoE) mimetic peptide, and to a Application of lipoprotein E (apoE) peptidomimetic in the treatment and / or prevention of atherosclerosis and its related cardiovascular and cerebrovascular diseases such as coronary heart disease, myocardial infarction and stroke. Background technique [0002] Atherosclerosis is a dysfunction of the vessel wall induced by a variety of complex etiologies (oxidative stress, inflammation, dyslipidemia, high glucose, etc.), characterized by lipid accumulation and inflammatory cell infiltration in the arterial wall subintimal, including Foaming of macrophages, proliferation of smooth muscle cells, massive accumulation of extracellular matrix, and finally the formation of atherosclerotic plaques, resulting in n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/22C12N15/12C12N15/70A61K38/17A61P9/10
Inventor 喻红樊大平杜芬商亮吴尧潘阳曹佳李小明
Owner WUHAN UNIV
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