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Procoagulant polypeptide LGTX-F2 and application thereof

A LGTX-F2, coagulation-promoting technology, applied in the field of biomedicine, can solve the problems of insufficient understanding of important functions, lack of hemostatic drugs, and no in-depth research on active ingredients, etc., and achieve obvious effects

Active Publication Date: 2018-11-16
湖南生达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, no molecules that can specifically enhance the activity of blood coagulation factor II have been found, resulting in insufficient understanding of the important role of blood coagulation factor XII in the blood coagulation process, and there is also a lack of hemostatic drugs targeting blood coagulation factor XII
[0008] The format tarantula is a large spider distributed in the southwest of my country, and the active ingredients in its venom have not been studied in depth

Method used

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  • Procoagulant polypeptide LGTX-F2 and application thereof
  • Procoagulant polypeptide LGTX-F2 and application thereof
  • Procoagulant polypeptide LGTX-F2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Preparation of procoagulant polypeptide LGTX-F2

[0018] Construction of prokaryotic expression vector

[0019] Select the appropriate prokaryotic expression vector according to the size of the target protein to facilitate the subsequent separation and purification steps. Finally, it was determined to use the PET-32a prokaryotic expression vector to express the protein. Considering the high expression efficiency of the vector and the fusion protein (adapter protein 18KDa + target protein 7KDa) digested by TEV, the molecular weight difference between the adapter protein and the target protein is relatively large, which is easy for subsequent separation and purification. Finally, the double restriction site of the prokaryotic expression vector was determined to use EcoR I (-GG^AATTCC-) and Hind III (-CA^AGCTTG-) two restriction sites, and the rTEV restriction site (-ENLYFQ ^G-), the prokaryotic expression vector plasmid was synthesized by Nanjing GenScript. ...

Embodiment 2

[0056] Example 2: Procoagulant polypeptide LGTX-F2 shortens recalcification and APTT time

[0057] APTT experimental method:

[0058] Equilibrate the APTT reagent to room temperature, gently invert the APTT reagent, and at the same time, place the CaCl2 solution in a water bath or a constant temperature incubator to preheat to 37°C. The final concentrations of the incubated samples were 0nM, 30nM, 0.3 μM, 3μM.

[0059] APTT test method

[0060] Table 3-1 The method of APTT experiment

[0061]

[0062] Plasma recalcification test method:

[0063] Combine 20 μL of normal human plasma (plus 3% sodium citrate for anticoagulation) with 3 μL of samples (incubate samples with final concentration of 0 μM, 0.75 μM, 1.5 μM, 3 μM). Mix in Hepes buffer (150 mM NaCl, pH 7.5) to a final volume of 70 μL. Then incubate for 10 min in a constant temperature incubator at 37°C. After incubation, add 50 μL of CaCl preheated at 37°C 2 solution (25 mM) to initiate the reaction. The enzyma...

Embodiment 3

[0065] Example 3: Procoagulant polypeptide LGTX-F2 shortens tail bleeding time

[0066] Experimental method of mouse tail bleeding model:

[0067] Take 20-25g Kunming mice and divide them into negative control group (saline), dosing group (0.625mg / kg, 1.25mg / kg, 2.5mg / kg) and positive control group (5mg / kg EACA), each group 6, randomly assigned, half male and half male. 15 minutes before tail clipping, 100 μl saline, 100 μl sample (0.625 mg / kg, 1.25 mg / kg, 2.5 mg / kg) and 100 μl EACA (5 mg / kg) were injected into tail vein respectively. After adding the medicine, cut the tail 7mm with a razor blade and immediately place it in a 37-degree constant temperature saline solution. The timing starts when the tail bleeds, and the timing stops when the bleeding stops. If bleeding occurs again within 120 seconds, continue timing, and so on. Repeat until the bleeding stops. At the end of the experiment, only the cumulative total time of bleeding was recorded. Statistical analysis of exp...

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Abstract

The invention discloses application of a procoagulant polypeptide LGTX-F2 to preparation of coagulation drugs. The procoagulant polypeptide LGTX-F2 contains 65 amino acid residues, wherein the molecular weight of the procoagulant polypeptide LGTX-F2 is 7444.51 Da, the isoelectric point is 7.75 and the amino acid sequence of the procoagulant polypeptide LGTX-F2 is as shown in a SEQ ID: 1. The procoagulant polypeptide LGTX-F2 disclosed by the invention is expressed by escherichia coli, has very remarkable coagulant activity and can be applied to preparation of the coagulation drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of procoagulant polypeptide LGTX-F2 in the preparation of procoagulant drugs. Background technique [0002] Hemorrhage or excessive bleeding is one of the leading causes of death in clinical operations. Local hemostatic drugs often cannot solve the problem during deep or complex bleeding. It is necessary to stimulate the deep coagulation of the body to improve the efficiency of hemostasis. Statistics show that nearly 10 million patients with hemorrhagic disease are clinically treated in my country every year, mainly in surgical departments and some internal medicine departments. Hemostatic drugs are irreplaceable and necessary products in clinical practice, and have the characteristics of strong clinical compliance. With the improvement of surgical operation level and surgical treatment coverage in our country, and the shortage of organ transpla...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P7/04
CPCA61P7/04C07K14/43518A61K38/00
Inventor 容明强刘中华李朋朋
Owner 湖南生达生物科技有限公司
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