Tissue factor pathway inhibitor (TFPI), preparation method thereof and application thereof

A technology of tissue factor and inhibitor, applied in the field of tissue factor pathway inhibitor and its preparation and application, can solve the problems such as unclear specific mechanism, and achieve the effect of inhibiting bacterial infection and highly effective antibacterial effect

Inactive Publication Date: 2011-05-25
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recent animal model studies have found that purified recombinant human TFPI-1 can alleviate the

Method used

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  • Tissue factor pathway inhibitor (TFPI), preparation method thereof and application thereof
  • Tissue factor pathway inhibitor (TFPI), preparation method thereof and application thereof
  • Tissue factor pathway inhibitor (TFPI), preparation method thereof and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] The tissue factor pathway inhibitor TFPI-1 is shown in the amino acid sequence of SEQ ID No. 1 in the sequence listing; the tissue factor pathway inhibitor TFPI-2 is shown in the amino acid sequence of SEQ ID No. 2 in the sequence listing.

[0030] The sequence listing SEQ ID No.1 is:

[0031] MAPVKWWILCAVLLCCVPRFGSCRRDRQADGPLPELFIFNELCALKDEPGPCKAIKDRFFFNVDTGHCELFEYGGCGGNA

[0032] NNFETLEDCEETCVVSDNKNPCHLAEAPGPCRGLVTRYFFDSGSQQCKHFYYGGCFGNANNFKSMAECQAKCQNPENPTK

[0033] APEVHTQPAGKSNVVQPTILTGEMTVSEPQVQTNETHKNPKVLRPTELCFDPVDRGTCQGSEKRFAYNPITKRCHAFSYS

[0034] GCGGNRNNFAYRKDCMIKCRRRKVHGPMIRIRKKNLDNILHRSI

[0035] (a) Sequence features:

[0036] ●Length: 284

[0037] ●Type: amino acid sequence

[0038] ●Chain type: single chain

[0039] ●Topological structure: linear

[0040] (b) Molecule type: protein

[0041] (c) Assumption: No

[0042] (d) Antisense: no

[0043] (e) Original source: American Redfish

[0044] Structural features: The most obvious structural fe...

Embodiment 2

[0060] Preparation method of tissue factor pathway inhibitor TFPI-1 and TFPI-2:

[0061] 1) Construction of plasmid pE15TFPI1:

[0062] Using American redfish cDNA as a template, PCR amplification was performed with primers TP1F1 and TP1R1. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 94°C for 40s, 52°C for 60s, 72°C for 60s, after 5 cycles, change to 94°C for 40s, 58°C for 60s, 72°C for 60s, after 25 cycles and then at 72°C. ℃ extension reaction 7-10min. After the PCR product was purified, it was ligated with the carrier pBS-T (purchased from "Tiangen Biochemical Technology (Beijing) Co., Ltd.") at room temperature for 2-8 hours, and the ligation mixture was transformed into E. ml Xgal and isopropyl-β-D-thiogalactoside (24ug / ml) were cultured on LB medium for 18-24 hours, and the transformants were screened to extract a plasmid, which was plasmid pBSTP1.

[0063] The above-mentioned plasmid pBSTP1 and plasmid pET259 (see Zheng, W.J., Hu, Y.H...

Embodiment 3

[0074] Application of Tissue Factor Pathway Inhibitors TFPI-1 and TFPI-2

[0075] 1) Preparation of Edwardsiella tarda. Cultivate Edwardsiella tarda TX1 (preserved in CGMCC, numbered as CGMCC No.2330) in LB medium to OD 600 0.5, and then centrifuged (5000g, 4°C) for 10min. Collect the cells and suspend them in PBS to a final concentration of 1x10 5 cfu / ml.

[0076] The composition of PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 .

[0077] 2) Bactericidal effects of tissue factor pathway inhibitors TFPI-1 and TFPI-2. Mix 20 ul of the bacteria solution from the above step 1) with 0.5 uM TFPI-1, TFPI-2 and PBS (control) prepared in Example 2 respectively, and incubate at 25° C. for 3-5 h. Then the mixture was spread on LB solid plates and cultured at 28°C for 32-48h. Colony counts revealed that TFPI-1 and TFPI-2 treatment resulted in about 90% reduction in bacterial numbers compared to PBS, ie about 90% of bacteria we...

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Abstract

The invention relates to the field of microbiology, in particular to a tissue factor pathway inhibitor (TFPI), a preparation method thereof and application thereof. A TFPI-1 is represented by the amino acid sequence SEQ ID No.1 in the sequence list; and a TFPI-2 is represented by the amino acid sequence SEQ ID No.2 in the sequence list. The preparation method comprises: converting colibacillus BL21 (DE3) by plasmids pE15TFPI1 and pE15TFP2 to obtain BL21/ pE15TFPI1 and BL21/ pE15TFPI2 respectively; inducing the obtained products with isopropyl-beta-D-thiogalactoside; and purifying recombinant proteins through affinity chromatography to obtain TFPI-1 and TFPI-2. The TFPI has a high-efficiency sterilization effect and therefore can resist to bacterial infection and be used for disease prevention and treatment. The TFPI provided by the invention can effectively crack many pathogenic bacteria so as to inhibit bacterial infection.

Description

technical field [0001] The invention relates to the field of molecular microbiology, in particular to a tissue factor pathway inhibitor (tissue factor pathway inhibitor, TFPI) and its preparation and application. Background technique [0002] Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that can inhibit the activation of coagulation mediated by tissue factor (TF). The mechanism of action of TFPI is that it can form a stable complex TFPI-Xa with factor Xa, thereby inactivating Xa. TFPI-Xa can also inhibit the complex of TF and factor VIIa, thereby hindering the production of Xa. TFPI includes two types, namely TFPI-1 and TFPI-2, both of which can inhibit blood coagulation. Studies have shown that human TFPI-1 is involved in innate immunity and inflammatory response processes, while TFPI-2 has serine protease inhibitor activity and plays an important role in tumor cell growth and metastasis. Recent animal model studies have found that purified r...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/63C12P21/02A61K38/17A61P31/04
Inventor 张敏孙黎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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