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Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit

A tissue factor and detection reagent technology, applied in the detection field, can solve the problems of different sensitivities, different concentrations, poor sensitivity, etc., and achieve the effect of high sensitivity, obvious effect, and reduced deviation

Active Publication Date: 2020-02-11
BEIJING SICCEEDER TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different companies have different sources of thromboplastin, different concentrations, and different sensitivities, resulting in problems such as poor product quality, instability, and poor sensitivity.

Method used

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  • Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit
  • Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit
  • Buffer composition for purifying tissue factor, purification preparation, PT detection composition and PT detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 culture medium and each buffer formula

[0053] LB medium: Dissolve 10g of tryptone, 5g of yeast extract, and 10g of NaCl in 1L of pure water, and adjust the pH to about 7.2.

[0054] Breaking buffer: 25mM Tris-HCl, 150mM NaCl, 25mM imidazole, adjust pH to 8.0. (No Triton).

[0055] 10% (v / v) Triton Disruption Buffer: Dilute with Disruption Buffer ie add 1 part of 100% Triton to 9 parts of Disruption Buffer.

[0056] Loading buffer: dilute with breaking buffer, add 1% Triton solution (dilute to 1% Triton solution with prepared 10% (v / v) Triton breaking buffer).

[0057] Equilibrium buffer: 25mM Hepes, 150mM NaCl, 25mM imidazole, 0.5% Triton (dilute to 0.5% Triton solution with prepared 10% (v / v) Triton breaking buffer), adjust pH to 8.0.

[0058] Elution buffer: 25mM Hepes, 150mM NaC, 250mM imidazole, 0.5% Triton (diluted into 0.5% Triton solution with prepared 10% (v / v) Triton breaking buffer), adjust pH to 8.0.

Embodiment 2

[0059] Embodiment 2 fermentation culture

[0060] Construction of recombinant glycerol bacteria: The human tissue factor gene sequence was obtained from Genbank, NCBI Reference Sequence: NM_001993.4, and the obtained sequence information was submitted to a third-party organization (the third-party organization is the Institute of Biological Products) for recombinant plasmid construction.

[0061] The method is as follows: the human tissue factor gene sequence comes from Genbank, NCBI Reference Sequence: NM-001993.4, the sequence information of the extracellular region and the transmembrane region is selected, and the extracellular region and the transmembrane region are synthesized by Shanghai Sangon Biotechnology Co., Ltd. by designing primers The fragments were cloned into the Pet48b plasmid by restriction enzyme digestion and transformed into DE3 (BL21) for subsequent strain fermentation and protein purification.

[0062] Take out a glycerol bacterium (transformed DE3(BL21)...

Embodiment 3

[0063] Example 3 protein purification

[0064] The crushed supernatant that was stirred overnight was filtered with a suction filter pump, and the precipitate was removed by centrifugation. Ready for purification.

[0065] Surfactants are added prior to protein purification. Only by adding a surfactant can the structure of the protein be opened and the TF recombinant protein exhibit activity, and this surfactant can be TritonX-100 or NP40 (ethyl phenyl polyethylene glycol), and the addition amount is 0.5-2%. (v / v). And keep the surfactant throughout the purification process.

[0066] Use a prepacked nickel column for purification, and keep the purification flow rate at 5mL / min in each step. The nickel column is first equilibrated with 5 column volumes of loading buffer, and then loaded. After loading, it is equilibrated with 10 column volumes of equilibration buffer, and finally The sample was eluted with 3 column volumes of elution buffer, and the eluted sample was collec...

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Abstract

The invention relates to the field of detection, in particular to a buffer composition for purifying a tissue factor, a purification preparation, a PT (prothrombin time) detection composition and a PTdetection kit. A key raw material, namely tissue thromboplastin, is prepared by a genetic engineering mode; high-purity r-TF (recombinant tissue factor) can be obtained on a large scale by a fermentation technology and a purification technology, and the problems that tissues such as rabbit brains and placentas are difficult in source, big in difference between batches and low in sensitivity are solved. A high-sensitivity prothrombin PT diagnosis kit based on r-TF liquid type has high stability, small difference between batches and high sensitivity, is mainly applied to screening of a clinicalextrinsic coagulation pathway, monitoring of oral warfarin medicines and auxiliary diagnosis of diseases such as liver disease, and can be traced back to an international standard product PT reagent,so that detection comparability of reagents among different reference laboratories and different manufacturers is achieved.

Description

technical field [0001] The invention relates to the detection field, in particular to a buffer composition for purifying tissue factor, a purification preparation, a PT detection composition and a PT detection kit. Background technique [0002] The four blood coagulation items belong to one of the clinical inspection items of the laboratory department, and belong to routine screening examinations, mainly for the detection of exogenous blood coagulation factors. The detection principle of PT is: add excessive tissue thromboplastin and calcium ions to the tested plasma to convert prothrombin into thrombin, which converts fibrinogen into fibrin. The time to detect plasma clotting is the prothrombin time. Prothrombin time (PT) is one of the four coagulation routines, and the PT kit is used for screening of extrinsic coagulation pathways, monitoring of warfarin, and auxiliary diagnosis of other diseases. [0003] Its most important application is that the International Normaliz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64G01N33/86
CPCC12N9/6424G01N33/86G01N2333/7454
Inventor 丁重辉李博华胡晓娟
Owner BEIJING SICCEEDER TECH CO LTD
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