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ADAMTS13-MDTCS fusion protein with function of prolonging half life in vivo and application thereof

A technology of fusion protein and protein, applied in the field of pharmaceutical bioengineering, can solve the problem that the biological activity consistency of protein drugs cannot be guaranteed, and achieve the effects of prolonged effect and safety, uniform substrate and better curative effect.

Active Publication Date: 2015-09-23
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology needs to be further optimized. The disadvantages include: a large number of drugs are implanted into the body at one time, and the patient bears the additional risk of drug toxicity from sudden release; protein drugs are encapsulated and implanted in the body for a long time, and the biological activity consistency of protein drugs cannot be guaranteed. Wait
[0022] At present, there is no clear report on how to effectively extend the half-life of ADAMTS13-MDTCS protein while ensuring the biological activity of the original protein ADAMTS13-MDTCS

Method used

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  • ADAMTS13-MDTCS fusion protein with function of prolonging half life in vivo and application thereof
  • ADAMTS13-MDTCS fusion protein with function of prolonging half life in vivo and application thereof
  • ADAMTS13-MDTCS fusion protein with function of prolonging half life in vivo and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0063] The construction of embodiment 1 ADAMTS13-MDTCS-HSA fusion protein and its plasmid

[0064] 1.1 Materials and methods

[0065] Reagent: HEK293-Free style cell line (Life tech);

[0066] Cell culture reagents: high sugar DMEM, Opti-MEM, 293-F expression Medium, FBS, 0.25% trypsin-EDTA solution, antibiotic G418 for cell resistance screening (Life tech); strain: Top10 (Tiangen Biochemical); vector : The pCEP4 vector is preserved by ourselves; kits: gel recovery kit; PCR purification kit, small amount of plasmid extraction kit, large amount of endotoxin-free plasmid extraction kit (Tiangen Biochemical);

[0067] Lipofectamine 2000 (Life tech) for transfection liposome; polymerase for PCR (TAKARA); restriction endonuclease (NEB); all sequencing was done by Life tech; HSA antibody for Western-blot (Santa Cruz); ADAMTS13 antibody , HRP secondary antibody (Abcam); Ultra-15 ultrafiltration tube (Millipore); Superdex purification column (GE health).

[0068] 1.2 Construction o...

Embodiment 2

[0086] Example 2 Transient expression and protein identification of ADAMTS13-MDTCS fusion protein

[0087] a) Inoculation of cells. The day before transfection, inoculate the cells into a 10cm plate (try not to use cells that grow to 90% in two days, if the growth is slow, please spread more cells). When transfecting, the cell confluence is required to be 90-95%;

[0088] b) For each plate, dilute 50 μl of LIP 2000 with 700 μl Opti-MEM Medium (the volume of LIP is not higher than 1 / 10 of the total volume), and incubate at room temperature for 5 minutes;

[0089] c) Dilute 70 μl of DNA with 680 μl Opti-MEM Medium per plate (the volume of DNA should not be higher than 1 / 10 of the total volume);

[0090] d) Add the diluted DNA to the diluted LIP2000 (1:1 mix), and incubate at room temperature for 20 minutes;

[0091] e) Carefully wash the prepared 10cm plate cells once with 3-5ml sterile PBS, gently add the Bath pipette along the wall, so as not to wash up the 293F cells, do n...

Embodiment 3

[0096] Example 3 Screening of cell lines stably expressing ADAMTS13-MDTCS HSA fusion protein

[0097] a) Inoculation of cells. Cells were seeded into 10 cm dishes one day before transfection. When transfecting, the cell confluence is required to be 90-95%;

[0098] b) For each plate, dilute 50 μl of LIP2000 with 700 μl Opti-MEM Medium (the volume of LIP is not higher than 1 / 10 of the total volume), and incubate at room temperature for 5 minutes;

[0099] c) Dilute 70 μl of DNA with 680 μl Opti-MEM Medium per plate (the volume of DNA should not be higher than 1 / 10 of the total volume);

[0100] d) Add the diluted DNA to the diluted LIP2000 (1:1 mix), and incubate at room temperature for 20 minutes;

[0101] e) Carefully wash the prepared 10cm plate cells once with 3-5ml sterile PBS, add the Bath pipette gently along the wall, so as not to wash up the 293F cells, and do not shake vigorously;

[0102] f) Add the incubated DNA+LIP2000 mixed solution, 1.5ml / dish, directly into ...

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Abstract

The invention belongs to the technical field of medical bioengineering technology, and discloses a mutant ADAMTS13-MDTCS fusion protein of human von willebrand factor lyase and the application of the mutant ADAMTS13-MDTCS fusion protein for preparing a drug for treating thrombotic thrombocytopenic purpura (TTP). According to the mutant ADAMTS13-MDTCS fusion protein, an ADAMTS13-MDTCS and human serum albumin are combined through a connecting peptide to form the fusion protein, the fusion protein ensures the biological activity of original ADAMTS13-MDTCS protein, the half-life period of the ADAMTS13-MDTCS is remarkably prolonged, the problem of existing ADAMTS13-MDTCS degradation is solved, and the mutant ADAMTS13-MDTCS fusion protein has the long-acting biological activity, is high in protein expression and can be used for industrial production.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a fusion protein of von Willebrand factor lyase mutant ADAMTS13-MDTCS and human serum albumin HSA, a preparation method and its use in the preparation and treatment of thrombotic thrombocytopenic purpura (TTP) application in medicine. Background technique [0002] 1.1 Thrombotic Thrombocytopenic Purpura [0003] Thrombotic thrombocytopenic purpura (TTP) is a microvascular thrombosis and hemorrhage syndrome, which is caused by the reduction of platelets due to a large number of consumption, and the formation of platelet thrombus in the blood microcirculation. The clinical feature is the formation of purpura on the skin surface. Microvascular hemolytic anemia and thrombocytopenia are the two core features of the disease, and may be accompanied by nervous system damage, kidney damage, and fever. Clinically, TTP is divided into congenital TTP (gene mutation) and acqu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/765C12N9/88C12N15/62A61K38/51A61K47/48A61P7/02A61K47/42
Inventor 于代冠朱泽尧吴朝霞张炳文米奇·托特雷拉
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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