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Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof

A technology of tissue factor and inhibitors, which is applied in the field of functional determination and long-acting recombinant tissue factor pathway inhibitors, can solve the problems of short half-life and achieve the effects of prolonged half-life, simple preparation process and high-efficiency anticoagulation

Inactive Publication Date: 2004-09-15
海菲尔(辽宁)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The short half-life is the biggest shortcoming of TFPI when it plays a therapeutic role. It must be administered continuously for 72-96 hours to maintain the curative effect. Each course of treatment requires about 160-320mg. Such a large dose brings a huge economic burden to the patient.

Method used

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  • Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof
  • Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 The docking and analysis of TFPI and LRP on a computer workstation

[0018] Using Insight II software, TFPI and LRP were docked on a computer workstation, and the 269, 282, 288 and 289 lysines at the carboxy terminus of TFPI were found to be the key sites for the interaction between the two.

Embodiment 2

[0019] Design, preparation and characterization of embodiment 2 LTFPI

[0020] (1) Cloning and transformation of LTFPI gene and construction of prokaryotic expression plasmid rLTFPI-pET28

[0021] The design method of LTFPI is as follows: replace the lysine at 269, 282, 288 and 289 positions of the carboxy terminus of TFPI with alanine. The sequence was obtained by PCR point mutation, recombined with pUC19, positive clones were screened by enzymatic hydrolysis, and the nucleotide sequence analysis confirmed that the gene sequence was correct. Then the LTFPI gene was cut out and recombined with the prokaryotic expression vector pET28 to form the prokaryotic expression plasmid rLTFPI-pET28. Transform Escherichia coli JM109, extract the plasmid, identify with the corresponding restriction endonuclease, obtain characteristic fragments, and confirm that positive clones were obtained.

[0022] The method of obtaining the LTFPI gene is as follows:

[0023] Using the overlap extens...

Embodiment 3

[0062] Embodiment 3 LTFPI half-life determination

[0063] The half-life of TFPI in rats was measured by dPT method: rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg / kg), administered via femoral vein (1 mg / kg), intubated in the common carotid artery, and blood was collected at different times after administration. Anticoagulate with 109mM sodium citrate 1:9, centrifuge at 4°C for 15min, 4000r / min, absorb the upper layer of plasma, and measure dPT.

[0064] Dilute the standard solution of thromboplastin 500 times with normal saline, and this is the working solution of thromboplastin. Take 100 μL plasma, add 100 μL thromboplastin working solution, incubate at 37°C for 1 min, add 10 μL 250mmol / LCaCl 2 solution, start timing the plasma coagulation time. The average value of 3 experiments was recorded as the experimental result.

[0065] Taking wild-type TFPI as the control group, observe the half-life of LTFPI (K282A) and LTFPI (K269A, K282A, ...

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Abstract

The invention belongs to biology technology field, which concretely refers to controlled release tissue factor path inhibitor (LTFPI) and the manufacturing method, and the application. The invention analyzes and experiments the biology information science and structure molecular biology science of tissue factor path inhibitor (TFPI) and its acceptor low density lipoprotein acceptor correspondent protein (LRP), it ascertains the part where the TFPI carboxy end combines with LRP and is eliminated. It designs TFPI carboxy end mutant, which is recombined with primary nucleus or eukarya expressing carrier after constructing LTFPI gene through PCR location, it converts bacillus coli or bici yeast, sifts the high expression project fungus. The primary project fungus are yeasted and expanded, crushed, then they are centrifugated and collects the inclusion body, purifies the LTFPI through molecular sift and ion interchanging two-step method; the eukarya project fungus are carried on with two-step purifying directly. The half-life are prolonged, it has good pour-depressant function.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a long-acting recombinant tissue factor pathway inhibitor (LTFPI). More specifically, the present invention relates to determination of LTFPI mutation site and mutation, gene cloning, gene expression, protein purification and renaturation, and functional determination. Background technique [0002] Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor with a molecular weight of about 40kd. The precursor has 304 amino acids, and the mature protein has 276 amino acids. The latter consists of an amino terminal, three series of Kunitz-type The main functions are as follows: (1) domain 1 inhibits tissue factor (TF) and coagulation factor VII / VIIa, and produces anti-platelet aggregation by inhibiting TF; (2) domain 2 inhibits Coagulation factor X / Xa; (Broze GJ.Annu RevMed, 1995; 46:103-112); (3) Domain 3 inhibits the binding of endotoxin and CD14, resul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63
Inventor 马端宋后燕白浩张农
Owner 海菲尔(辽宁)生物科技有限公司
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