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Recombinant PAI-1 inhibitor, composition containing recombinant PAI-1 inhibitor, and uses of recombinant PAI-1 inhibitor and composition in treatment and detection

A PAI-1, inhibitor technology, applied in the field of biomedicine, can solve the problems of easily induced thrombosis, inhibited local fibrinolytic activity, etc.

Active Publication Date: 2015-07-22
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other words, once the level of PAI-1 in the blood circulation increases or the activity of PA is inhibited, the local fibrinolytic activity will be inhibited, the blood will appear in a hypercoagulable state, and it is easy to induce thrombosis (3)

Method used

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  • Recombinant PAI-1 inhibitor, composition containing recombinant PAI-1 inhibitor, and uses of recombinant PAI-1 inhibitor and composition in treatment and detection
  • Recombinant PAI-1 inhibitor, composition containing recombinant PAI-1 inhibitor, and uses of recombinant PAI-1 inhibitor and composition in treatment and detection
  • Recombinant PAI-1 inhibitor, composition containing recombinant PAI-1 inhibitor, and uses of recombinant PAI-1 inhibitor and composition in treatment and detection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Cloning of embodiment one PAI-Trap gene, and expression and purification of PAI-Trap protein

[0103] (1) The PAI-Trap here is constructed by carrying out the S195A mutation of the uPA hydrolase domain (uPA-SPD, whose nucleotide sequence is SEQ ID NO.10), also referred to as uPA-S195A(I16-E244) or uPA-S195A (amino acids named as chymotrypsinogen number):

[0104] The uPA-SPD-pPicZαA plasmid (Invitrogen) was used as a template.

[0105] Primer design:

[0106] Sense primer: 5'-GCCAGGGAGACTCAGGGGGACC-3' (SEQ ID NO.6)

[0107] Antisense: 5'-GGTCCCCCCTGAGTCTCCCCTG-3' (SEQ ID NO.7)

[0108] PCR system:

[0109] wxya 2 O 32ul

[0110] 5HF buffer (Thermal scientific) 10ul

[0111] 2mM dNTP (Shanghai Sangong) 5ul

[0112] 20mM sense primer 1ul

[0113] 20mM antisense primer 1ul

[0114] Template (20ng)0.5ul

[0115] Phuison (Thermal scientific) 0.5ul

[0116] PCR conditions:

[0117] 98°C for 3 minutes;

[0118] 25 loops:

[0119] 98°C for 20 seconds,

[0120] 6...

Embodiment 2

[0166] Expression and purification of embodiment two PAI-1, uPA and tPA

[0167] (1) Expression and purification of PAI-1: The recombinant PAI-1 expression plasmid pT7-PL (a gift from Professor Zhou Aiwu of Shanghai Jiao Tong University) was transformed into BL21 Escherichia coli strain (Invitrogen). The recombinant expression strain was inoculated in LB (1 tryptone, 0.5% yeast extract powder, 1% NaCl, containing 100mg / LAmp) and cultured overnight at 37°C, then inoculated in fresh LB (1 tryptone, 0.5% yeast extract powder) at a ratio of 1:100 , 1% NaCl, containing 100mg / L Amp) at 37°C to OD600 of about 0.6, induced with 0.5mM IPTG at 20°C for 6 hours, centrifuged at 10,000rpm for 10 minutes, collected the bacteria, and washed with buffer A (25mM MESpH6.1 , 1M NaCl), ultrasonically crushed, and centrifuged at 10,000rpm for 30 minutes, the supernatant was combined with Ni-NTA (Qiagen) column for 2 hours, and gradient elution was performed with imidazole-containing buffer A, and ...

Embodiment 3

[0173] The activity measurement of embodiment three PAI-Trap

[0174] The enzyme activity test of PAI-1 was mainly carried out according to the reported Chromogenic Assay (Liang, A., et al., 2005). Briefly, in a 100 μL system (50mM Tris pH7.4, 150mM NaCl), different concentrations of PAI-Trap were pre-incubated with 5nM PAI-1 (final concentration) for 10 minutes, then 5nM uPA was added to mix and react at room temperature After 10 minutes, the luminescent substrate S2444 (Chromogenix) was added and immediately placed in a BioTek Synergy4 microplate reader at 405 nm, 15 seconds / reading for 10 minutes. Each test was repeated at least 3 times. IC of PAI-trap to PAI-1 50 The nonlinear regression (Sigmoidal) in Origin7.5 software was used for fitting.

[0175] The competitive inhibition of PAI-1 by a typical PAI-trapSEQ ID NO.4 is as follows image 3 shown. Inhibitory IC of PAI-trap on PAI-1 50 The results are shown in Table 1. These PAI-Traps all have strong inhibitory abili...

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Abstract

The present invention provides a recombinant PAI-1 inhibitor, which is an uPA hydrolase domain with S195A mutation or a PAI-1 hydrolase domain with S195A mutation, and has PAI-1 binding force. The present invention further provides a composition containing the inhibitor, and uses of the composition in preparation of treatment drugs and PAI-1 detection reagents. According to the present invention, the uPA or tPA hydrolase domain is modified to optimize the binding force with the PAI-1 so as to construct the recombinant PAI-1 inhibitor, wherein the recombinant PAI-1 inhibitor contains the least S195A point mutant.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant PAI-1 inhibitor, a composition containing it and a medical application thereof. Background technique [0002] Urokinase Plasminogen Activator System (uPA system) is composed of uPA, uPAR (uPA receptor) and two specific inhibitors PAI-1 (Plasminogen Activator Inhibitor-1) and PAI-2, which participate in the regulation of many important Physiological processes such as fibrinolysis, cell adhesion, infection and metastasis (1). In this system, uPA belongs to the serine protease (Serine Protease) family. After binding to uPAR on the cell membrane, uPA can specifically catalyze the conversion of inactive plasminogen (Plasminogen) into active plasminase (Plasmin), which can Degrades a variety of proteins in the extracellular matrix, including fibrin, to regulate many important physiological and pathological processes. [0003] PAI-1 is an important member of the serine proteas...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/58C12N15/63A61K38/49A61P7/02A61P3/10A61P35/00G01N33/573
Inventor 黄明东林忠辉龚利虎洪泽彬袁彩江龙光刘敏
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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