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Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same

A tissue factor and inhibitor technology, applied in the field of genetic engineering, can solve the problems of cost limitation and inconvenience of medication, and achieve the effect of promoting application, reducing the number of administrations, and reducing the cost of treatment

Inactive Publication Date: 2009-02-11
INST OF BIOMEDICAL ENG CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, since TFPI is rapidly cleared in the blood circulation, a large dose (about 20 mg / kg / d) is required to achieve the purpose of clinical treatment, and the inconvenience and high cost of medication greatly limit its clinical application and therapeutic effect (Palmier MO , Hall LJ, Reisch CM, et al.Clearance of recombinant tissue factor pathway inhibitor(TFPI)in rabbits.Thromb Haemost, 1992.68(1):33-36.Warshawsky l, Bu G, Mast A, et al.The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo. J Clin Invest, 1995.95(4): 1773-1781. Ho G, Narita M, Broze GJ, Jr., et al. Recombi nantfull- length tissuefactor pathway inhibitor fails to bind to the cell surface: implications forcatabolism in vitro and in vivo. Blood, 2000.95(6): 1973-1978. Bai H, MaD, ZhangYG, et al. Molecular design and characterization of recombinant long half- life mutants of human ti sue factor pathway inhibitor. Thromb Haemost, 2005.93(6): 1055-1060.)

Method used

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  • Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same
  • Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same
  • Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Extraction of mTFPI-pPIC9 plasmid DNA

[0036] 100ul of fresh bacterial solution TB1 (containing mTFPI-pPIC9 plasmid) was inoculated in 10ml of LB medium at 37°C and cultured overnight at 250rpm; take 10 250ml Erlenmeyer flasks, add 100ml of medium to each bottle, and inoculate 1ml of the above bacterial solution at 37°C , culture overnight at 250rpm; centrifuge to take the precipitate, resuspend the precipitate with 120ml of GTE buffer, add 240mg of dissolving enzyme, shake vigorously to mix, and bathe in water at 30°C for 30min; add 240ml of lysate (freshly prepared with 0.2N NaOH 1% SDS) and gently invert Mix well and let stand for 5 minutes; add 180ml of 5M potassium acetate solution (pH 5.5; 3M potassium acetate, 2M acetic acid), invert and mix well, and ice-bath for 10 minutes. Centrifuge at 8000rpm for 15min, filter with two layers of gauze, take the supernatant, add an equal volume of isopropanol, mix well and centrifuge at 8000rpm for 20min, take the ...

Embodiment 2

[0037] Example 2 Construction of TFPI-containing mutant gene (m 0 TFPI) carrier

[0038] Using the plasmid DNA mTFPI-pPIC9 (constructed by the Genetic Engineering Laboratory of the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College) as a template, the mutation primer m 0 (5'AAA GGA GGC CTA ATT GAT GAC GAT GAC AAA AGA AAG AAG CAG AGA 3'), 3'Aox(5'GAC TGG TTC CAA TTG ACAAGC 3'); the first PCR amplification was performed for forward and reverse primers. After agarose gel electrophoresis, the PCR product was purified with a DNA gel recovery kit (Beijing Dingguo Biotechnology Development Center). Still using the plasmid DNA mTFPI-pPIC9 as a template, the PCR product above and 5'Aox (5'GCA AAT GGC ATT CTG ACA TCC 3') were used as primers for the second PCR amplification (see figure 1 , m in the figure 0 , m 1 , m 2 It is a mutation primer, containing a mutation site. 5'Aox and 3'Aox are general primers for Pichia pastori...

Embodiment 3

[0039] Example 3 Construction and Identification of Recombinant Yeast GS115

[0040] Get the plasmid DNA (m 0 TFPI-pPIC9) was digested with 50u Sac1 for 1 hour. After the plasmid was precipitated with absolute ethanol, it was dissolved in 20ul sterile water. Yeast competent cells were prepared by PEG1000 method. Add 50ug of DNA directly to the competent cells which are still in the frozen state, and put them in a water bath at 37°C for 5 minutes, then add 1.2ml of PEG solution (40% PEG 1000, 0.2M Bicine, pH 8.35), and put them in a water bath at 30°C for 1h. Centrifuge and resuspend the cells with 1.5ml NaCl solution (0.15M NaCl, 10mM Bicine, pH 8.35). Centrifuge again, resuspend the bacteria with 0.2ml NaCl solution (0.15M NaCl, 10mM Bicine, pH 8.35), spread on the RDB selection plate, and incubate at 30°C for 4-6 days (see figure 2 ). Pick the clones from the RDB selection plate and inoculate them in 10ml of YPD medium, and culture them at 30°C and 250rpm for 18-24 hou...

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Abstract

The invention discloses the m0TFPI, comprising the recombination carrier and host cell. The m0TFPI has nucleic acid sequence said by SEQ ID No.1 in sequence table. The recombination carrier m0TFPI-pPIC9 is made by slaking fragment of m0TFPI DNA and pPIC9 carrier with XhoI and EcoRIand, adding T4 ligases, and carrying out coupled reaction. On the base of the invention, the metabolism time of the TFPI recombination protein in blood circulation can be extended, the administer drug frequencies of TFPI can be reduced, and the therapeutic expense can be reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tissue factor pathway inhibitor mutant gene, a recombinant vector containing the gene, a recombinant yeast transformed with the vector and the expressed protein. Background technique [0002] Human tissue factor pathway inhibitor (Tissue Factor Pathway Inhibitor, TFPI) is an important inhibitor of tissue factor pathway, as a physiological inhibitor of TF:FVIIa complex, it has an important effect on the coagulation process. TFPI can inhibit thrombus formation and vascular smooth muscle cell proliferation, etc. Therefore, it can slow down the occurrence and development of vascular restenosis, atherosclerosis and myocardial infarction. In addition, animal experiments have proved that human TFPI recombinant protein (hrTFPI) has preventive effects on thrombosis, septic shock, vascular restenosis, endotoxemia and DIC (disseminated intravascular coagulation). ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/63C12N15/81
Inventor 冷希岗余波宋丽萍刘兰霞张海玲
Owner INST OF BIOMEDICAL ENG CHINESE ACAD OF MEDICAL SCI
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