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Use of tissue fibre proteinogen

A technology of fibrinogen and fibrin, applied in the field of new application of tissue fibrinogen, can solve problems such as unclear mechanism

Active Publication Date: 2007-04-25
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, heat shock proteins do not exist in the circulatory system, and the mechanism by which the body protects protein molecules outside the cell is still unclear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Application of tissue fibrinogen in the refolding of denatured recombinant protein-endostatin

[0019] Tissue fibrinogen is prepared into a solution with a concentration of 3 mg / ml with phosphate buffered saline (PBS), and the high-concentration endostatin stock solution that has been completely reduced and denatured is diluted into the refolding solution in one step to make the endostatin The final concentration was 0.2 mg / ml. After 2 hours, centrifuge at 14,000 rpm (or 12,000 g) for 15 minutes to remove the precipitate, and use a nickel-affinity chromatography column (because recombinant endostatin contains a nickel-affinity histidine tag) to separate and purify the soluble endostatin . The results of oxidation electrophoresis of the product showed that disulfide bonds were formed; the retention time of HPLC analysis was consistent with that of the refolded standard; the fluorescence emission spectrum showed that the maximum emission wavelength was around ...

Embodiment 2

[0020] Example 2. Application of tissue fibrinogen in anti-amyloid deposition

[0021] Yeast prion is a molecule similar to mad cow disease protein, which can spontaneously undergo conformational changes in vitro to form amyloid deposits or their precursors. This process can be detected with a change in fluorescence specific for the dye Thioflavin T.

[0022] The yeast prion protein was made into a 0.5 μM solution with PBS buffer solution. The experimental group solution also contained 0.5 μM tissue fibrinogen, and the control group did not contain tissue fibrinogen; stand at room temperature, and sample 300 μl from the system at different time points , add the dye Thioflavin T with an equal volume concentration of 25 μM; detect the fluorescence, and finally draw a trend graph according to the fluorescence changes at different time points. It is found that with the extension of time, the control group without tissue fibrinogen can observe a significant increase in fluorescence...

Embodiment 3

[0023] Embodiment 3, the experiment of tissue fibrinogen protection citrate synthase thermal denaturation

[0024] Citrate synthase is a key enzyme in the tricarboxylic acid cycle, but the protein has poor thermal stability, and will quickly denature and aggregate at 43°C in vitro, and then form precipitates. This aggregation process can be reflected by changes in light scattering.

[0025] A fluorometer FL4500 (Hitachi) was used to detect the light scattering process, and the excitation light of the instrument was adjusted to 500 nm, the emitted light to 500 nm, and the slit width to 2.5 nm.

[0026] Dissolve citrate synthase in 40 mM HEPES buffer to make the final concentration 0.15 μM, and add tissue fibrinogen HEPES solution to the experimental group to make the final concentrations 0.05, 0.10, 0.15, 0.3, 0.45, 0.6, 0.75 μM, the control group was added with the same volume of HEPES solution, the experimental sample was placed in a 43°C water bath, and the light scattering...

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Abstract

Use of tissue fiber proteinogen achieves low cost. It can improve primary and secondary structure fold of denaturing recombinant protein, accessory genital recovery rate of occlusion body and recombinant protein renaturation. It has molecular mate activity and can be used to prevent deposition of protein starch sampler and thermal denaturation.

Description

technical field [0001] The present invention relates to a new application of tissue fibrinogen. Background technique [0002] Tissue fibrinogen exists in the blood and is an important protein in the coagulation process. It is also called coagulation factor I. Its molecular weight is about 340,000 Daltons. It consists of two subunits connected by disulfide bonds to form a dimer. Each subunit consists of three polypeptide chains intertwined. During the coagulation process, fibrin is digested by thrombin to form insoluble fibrin polymers, and blood fibers and platelets formed by fibrin polymers together form a firm hemostatic plug. The content of tissue fibrinogen in blood is about 1.5-4 mg / ml. Meanwhile, tissue fibrinogen is also a kind of stress protein. Its content is related to the immune status of the body and can reflect the risk of cardiovascular and cerebrovascular diseases. [0003] The expression product of recombinant protein in Escherichia coli is easy to be misfo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K14/475A61K39/395A61P43/00
Inventor 罗永章唐华东雷清新付彦
Owner TSINGHUA UNIV
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