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Recombinant protein function structure domain and use thereof in inhibition of macrophage tissue factor expression

A functional domain, macrophage technology, applied in coagulation/fibrinolytic factors, recombinant DNA technology, animal/human protein, etc.

Inactive Publication Date: 2016-06-08
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as the main scavenger receptor on the surface of macrophages, LOX-1 binds to oxLDL to mediate endocytosis and whether it mediates the upregulation of TF induced by oxLDL has not been reported yet.
[0006] The previous work showed that the β2-GPIDV expressed in yeast was combined with oxLDL through the oxLDL-negative phospholipid epitope structure, whether this combination interfered with the binding of oxLDL to the receptor LOX-1, and then inhibited the up-regulated expression of TF induced by oxLDL , it is not yet clear

Method used

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  • Recombinant protein function structure domain and use thereof in inhibition of macrophage tissue factor expression
  • Recombinant protein function structure domain and use thereof in inhibition of macrophage tissue factor expression
  • Recombinant protein function structure domain and use thereof in inhibition of macrophage tissue factor expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example loxLDL induces up-regulation of TF expression in mouse macrophage J774A.1 via LOX-1.

[0053] 1.1qRT-PCR detection of gene expression of LOX-1 and TF in mouse macrophages.

[0054] The mouse macrophages that were changed the day before were placed in a six-well plate. After 24 hours, the cells adhered to the wall and incubated with different concentrations of oxLDL (0, 2.5, 5, 10, 20, 50 μg / mL). After 4 hours, the cells were collected and used Trizol The total RNA of mouse macrophages was routinely extracted by the method, and the mRNA was reverse-transcribed into cDNA by a reverse transcription kit, and the specific operation was carried out with reference to the kit.

[0055] Further, three pairs of primers for β-actin, TF, and LOX-1 were designed to amplify the above cDNA as a template respectively, and the gene fragment was obtained through a qPCR kit, and the specific operation was carried out with reference to the kit. The expression of TF and LOX-1 genes...

Embodiment 2

[0072] Example 2 β2-GPIDV inhibits the expression of LOX-1 and TF in J774A.1 cells

[0073] 2.1 Induced expression and purification of target protein

[0074] (1) cell fermentation

[0075] 1) Inoculate Pichia pastoris X-33 engineered bacteria expressing human β2-GPIDV preserved in our laboratory into 10ml BMGY, and culture for 24 hours as a seed medium.

[0076] 2) Take an appropriate amount of bacterial solution from the seed solution and inoculate it into 1L of BMGY at a ratio of 1:200, and incubate for 24 hours.

[0077] 3) Discard the supernatant after centrifugation at 1500rpm, and add 100ml BMMY. Add 1% methanol every day. induced expression for 96h

[0078] (2) Pretreatment before protein purification

[0079] 1) Centrifuge the fermentation broth at 6000rpm for 20min to collect the supernatant.

[0080] 2) Adjust the pH of the supernatant to 8.0 with concentrated hydrochloric acid

[0081] 3) Centrifuge at 6000rpm for 20min to collect the supernatant.

[0082] 4)...

Embodiment 3

[0100] Example 3 A 293T cell model expressing LOX-1 was established, and its expression localization and biological activity were detected.

[0101] 3.1 PCR amplification of LOX-1 gene

[0102] The primers were synthesized by Dalian Bao Biology Co., Ltd., the upstream and downstream primers are as follows:

[0103] Upstream primer (primer 1):

[0104] 5′-CTA GCTAGC ATGACTTTTGATGACCTAAAG-3′

[0105] Downstream primer (primer 2):

[0106] 5′-TTG GAATTC TCACTGTGCTCTTAGGTTTGC-3′

[0107] Among them, the 5' end of the upstream primer contains a protective base, NheI restriction site (single dash) and the 5' end of LOX-1 part of the amino acid coding gene sequence; the 5' end of the downstream primer contains a protective base, EcoRI enzyme The cleavage site (single dash), the complementary sequence of the amino acid coding gene at the 3' end of LOX-1, and the stop codon.

[0108] Using the LOX-1 fragment as a template, PCR was carried out with primers 1 and 2 to obtain the ...

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Abstract

The invention relates to the field of biomedicines, and concretely relates to a recombinant protein function structure domain and a use thereof in inhibition of macrophage tissue factor expression. In the invention, up-regulation of TF (tissue factor) expression by oxLDL is confirmed, the up-regulation is realized through starting signal regulation under the mediation of LOX-1, a recombinant beta2-GPIDV can inhibit oxLDL induced TF and LOX-1 expression, and intervention of combination of the oxLDL and the LOX-1 is proved through a flow cytometer method by adopting an autonomously constructed expression human LOX-1 gene 293T cell model in order to block out expression of downstream TF and LOX-1. The combination of the OxLDL and the LOX-1 induces TF expression to initiate thrombus, and also initiates endothelial cell dysfunction to generate an inflammation medium, and is important to form and develop atherothrombosis, so the potential treatment effect of the recombinant beta2-GPIDV in the atherothrombosis diseases is displayed in the invention.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a β2-glycoprotein fifth domain (β2-GPIDV) which inhibits and intervenes the combination of oxLDL and LOX-1 to inhibit the expression of TF and LOX-1, showing its role in A possible potential therapeutic role in inhibiting atherosclerotic thrombosis disease. Background technique [0002] Atherosclerosis (AS) is an arterial disease characterized by the formation of intimal atheromatous plaque or fibrous plaque, mainly involving large and medium arteries, and its clinical manifestations mainly include angina pectoris, arrhythmia, myocardial infarction, and even sudden death . Oxidized low-density lipoprotein (oxLDL) is an independent risk factor for AS, which induces the occurrence of the disease through various ways. Among them, the recognition and endocytosis of oxLDL by scavenger receptors on the surface of macrophages leads to the transformation of monocytes and macrophages ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/745C12N15/85
CPCC07K14/745C12N15/85C12N2800/107
Inventor 刘庆平迟彦于田田李敬达王仁军
Owner DALIAN UNIV
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