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Anticoagulation and thrombolytic thrombus target fusion mA5UKB

A dual-function, fusion protein technology, applied in the field of pharmaceutical bioengineering, can solve the problems of treatment failure, large amount of treatment, short half-life, etc.

Inactive Publication Date: 2004-12-22
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used clinical thrombolytic drugs are mainly plasminogen activators, such as streptokinase (SK), urokinase (UK), single-chain urokinase (scu-PA), tissue plasminogen activator (t -PA) etc., although these drugs have strong thrombolytic effect, there are the following problems: (1) bleeding: because these drugs not only activate the plasminogen in the fibrin clot, but also activate the plasminogen in the plasma Plasminogen increases the activity of plasmin in plasma, and plasma plasmin can hydrolyze blood coagulation factor VII, etc., resulting in the reduction of blood coagulation factors and causing bleeding
(2) The half-life in the body is short, so the therapeutic dosage is large
(3) Re-infarction: After the plasmin system is activated, platelets can be activated at the same time, so that fibrinogen and Von Willebrand factors can be aggregated to platelet GpIIb / IIIa receptors, thereby promoting thrombus formation, which is easy to form again in a short time after thrombolysis blood clots that make treatment fail

Method used

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  • Anticoagulation and thrombolytic thrombus target fusion mA5UKB
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  • Anticoagulation and thrombolytic thrombus target fusion mA5UKB

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 constructs mAnxA5 mutant gene

[0073] The gene sequence of human AnnexinA5 was obtained from human placenta tissue by RT-PCR method. In order to facilitate further operations such as amplification and enzyme digestion, this gene was inserted into plasmid pUC119 to obtain recombinant plasmid pUC119-AnxA5. (Referring to relevant chapters of "Molecular Cloning", Science Press, published in 1992).

[0074] 1. Mutate the "V-G-D" sequence at position 142-144 in the AnnexinA5 gene to the "R-G-D" sequence

[0075] In a 100μl PCR reaction system, add 2μl (15pmol / μl) of primers P1 and P2, 1μl (50ng / μl) of pUC119-AnxA5 template DNA, 15mM MgCl 2 10 μl, 3 units of high-fidelity pfu DNA polymerase, 5 μl of 10mM dNTP, 10 μl of 10×PCR buffer, supplemented with double distilled water to 100 μl. The PCR conditions are: the first cycle of denaturation at 94°C for 4 minutes, and the second cycle: denaturation at 94°C for 45 seconds; annealing at 55°C for 45 seconds; extens...

Embodiment 2

[0080] Example 2 Construction of Fusion Gene mA5UKB

[0081] 1. PCR amplification of urokinase B chain gene

[0082] The synthetic urokinase B chain gene was inserted into plasmid pUC119 to obtain recombinant plasmid pUC119-UKB. In 100μl PCR reaction system, add 15pmol / μl of P6, 2μl of P7 primers, 1μl (50ng) of template plasmid DNA pUC119-UKB, 5μl of 10mM dNTP, 10μl of 10×PCR buffer, 15mM MgCl 2 10 μl. The PCR conditions are: the first cycle of denaturation at 94°C for 4 minutes, and the second cycle: denaturation at 94°C for 45 seconds; annealing at 55°C for 45 seconds; extension at 72°C for 1 minute. A total of 30 cycles were performed, and the last cycle was extended at 72°C for 10 minutes. After the reaction, the product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered from the gel. The obtained PCR product was confirmed to be the urokinase B chain gene (UKB gene) by sequencing.

[0083] 2. Construction of fusion gene mA5UKB gene

[0...

Embodiment 3

[0085] Example 3 Construction of prokaryotic expression plasmid pET28a-mA5UKB and engineering bacteria BL21 (mA5UKB)

[0086] The invention adopts IPTG-inducible Escherichia coli expression vector pET-28a. The full length of the plasmid is 5396bp (base pairs), containing phage T7 promoter, Luc repressor gene LucI, plasmid replication origin (ori) and Kanna resistance gene (kan r ). The fusion gene mA5UKB obtained in Example 2 was double digested with Nco I and Sal I, and the digested fragments were recovered from the gel. Mix it with the vector pET28a (molecule number: 5:1) that was also digested with Nco I and Sal I, add T4 ligase to the 20ul ligation system, and connect overnight at 14°C to obtain the recombinant plasmid pET28a-mA5UKB ( figure 1 ).

[0087] Preparation of competent cells BL21(DE3): Pick a single colony of Escherichia coli BL21(DE3) and put it in 3ml LB medium (containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride, pH7.0) Incubate overnight a...

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Abstract

A thrombus target fusion mA5UKB with both anticoagulating and thrombolytic functions is prepared from the mutant mAnxA5 of AnnexinA5 having anti-coagulating function and the urokinase B chain (UKB) having thrombolytic function through fusing. It can be used to prepare the thrombolytic medicine.

Description

Technical field: [0001] The invention relates to the technical field of medical bioengineering, and is a fusion protein mA5UKB of a mutant mAnxA5 of annexin A5 (AnnexinA5) and urokinase B chain (UKB). Has anticoagulant effect. Background technique: [0002] Thrombosis, such as acute myocardial infarction (AMI) and venous thromboembolism, is a class of cardiovascular diseases that seriously endanger human health and life. In western countries, the death caused by thrombosis has accounted for the first place in the total death rate of the population. In my country, with economic development and population aging, the number of patients with thrombosis is increasing, and the demand for antithrombotic drugs is also increasing. At present, the commonly used clinical thrombolytic drugs are mainly plasminogen activators, such as streptokinase (SK), urokinase (UK), single-chain urokinase (scu-PA), tissue plasminogen activator (t -PA) etc., although these drugs have strong thrombol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61P7/02C07K19/00
Inventor 孙树汉颜宏利陈蕊雯
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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