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Method for constructing, expressing and purifying human recombination factor and application

A tissue factor and expression method technology, applied in chemical instruments and methods, biochemical equipment and methods, applications, etc., can solve the problems of low biological activity, reduced soluble protein expression level, low uniformity of TF structure and activity, etc. The effect of simplifying the process and improving the expression rate

Inactive Publication Date: 2005-10-26
山西省博奥特医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In the invention patent application of Fudan University, the obtained r-sTF is only the extracellular part of TF, which is different from the natural full-length TF or TF 243 Compared with the truncated type (containing transmembrane structure), its biological activity is lower
In addition, the expression of r-sTF in E. coli is induced by increasing the fermentation temperature to form inclusion body precipitates, while higher culture temperatures often reduce the expression level of soluble proteins, and inclusion body precipitates need to undergo protein denaturation and complexation. Therefore, the purification process of r-sTF is cumbersome, and the uniformity of TF structure and activity is low

Method used

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  • Method for constructing, expressing and purifying human recombination factor and application
  • Method for constructing, expressing and purifying human recombination factor and application
  • Method for constructing, expressing and purifying human recombination factor and application

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Experimental program
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Effect test

Embodiment 1

[0038] rhTF 243 Construction of expression plasmid vector

[0039] Total RNA was obtained from human placenta tissue, and PCR upstream primer P1 and downstream primer P2 were designed according to the known TF nucleotide sequence.

[0040] Upstream primer P1: 5'-ACCAGCCATGGCTCAGGCACTACAAATACTGTGGC-3'

[0041] Downstream primer P2: 5'-GGATCCTCAGTGTAGAGATATAGCCAGGATG-3'

[0042] The P1 primer contains several TF protein N-terminal amino acids and NcoI restriction sites, and the P2 primer contains several TF protein C-terminal amino acids, termination codes and BamHI restriction sites.

[0043] Using total RNA as a template and P1 and P2 as primers, the TF target gene fragment was obtained by conventional RT-PCR method; the collected rhTF 243 The gene fragment was treated with restriction enzymes NcoI and BamHI, and recombined with the phoA plasmid to construct the TF expression plasmid vector pTF 243 .

[0044] TF expression plasmid vector pTF 243 Transform into Escherichi...

Embodiment 2

[0046] rhTF 243 Fermentation expression of

[0047] 1. Preparation of fermentation medium

[0048] Composition: LB medium 1000mL, glucose 50g, inorganic salt solution 10mL, 0.01% ampicillin. The inorganic salt solution consists of 0.1% sodium dihydrogen phosphate, 0.5% dipotassium hydrogen phosphate, 0.01% ferric chloride, 0.03% zinc sulfate, 0.01% cobalt chloride, 0.03% copper sulfate, 0.5% ammonium sulfate and 0.05% magnesium sulfate composition.

[0049] After sterilizing the mixed solution of LB medium, glucose and inorganic salt at 121° C. for 20-30 minutes, ampicillin was added to prepare the fermentation medium.

[0050] 2. Shake flask fermentation culture

[0051] Take 2mL of the genetically recombined engineering bacteria liquid obtained by screening and add it to a conical flask containing 1000mL of fermentation medium, put it in a constant temperature shaker at 30°C, and cultivate it at a speed of 200r / min for 16h-20h, until the OD value at 600nm is measured at ...

Embodiment 3

[0059] rhTF 243 protein purification

[0060] 1. Preparation of TP monoclonal antibody immunoaffinity chromatography column

[0061] Weigh 15 mg of purified TF monoclonal antibody, 1 g of CNBr-activated Sepharose matrix, 100 mL of 0.4 mol / L NaHCO 3 Coupling buffer (pH8.3), 11.7 g NaCl, mixed at 25°C and packed into the column, shaken slowly for 2h, drained the liquid; then blocked with 0.1mol / L Tris buffer (pH8.2) for 2h, static at 25°C Set aside, drain the liquid; wash with 0.1 mol / L sodium acetate solution at pH 3.0 and 0.1 mol / L Tris buffer at pH 8.3 in sequence, and finally store with 5 times the column volume of PBS buffer at 4°C to prepare TP monoclonal antibody Immunoaffinity chromatography columns.

[0062] 2. rhTF 243 protein purification

[0063] 1) Bacterial fragmentation

[0064] Dissolve 20 g of the obtained fermented bacteria in 100 mL of 20 mmol / L Tris-EDTA buffer at 2 °C to 8 °C, add 1% Triton X-100, shake and mix, and use a high-pressure homogenizer to p...

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Abstract

The present invention relates to a human recombinant tissue factor, its construction, expression and purification method and application. Said method includes the following steps: recombining TF gene fragment obtained from human placenta with phoA plasmid, constructing plasmid vector pTF243, transforming it into colibacillus MM294, screening and obtaining engineering strain, inoculating said strain into culture medium to directly make fermentation and expression to obtain bacterial liquor, then using Q-Sepharose Fast Flow matrix to make the bacterial liquor under the process of ion exchange chromatography, then pass through TF monoclonal antibody immunoaffinity chromatographic column to make adsorption and elution so as to obtain the rhTF243 protein.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to human recombinant tissue factor, in particular to a method for constructing, expressing and purifying human recombinant tissue factor. The present invention also relates to the application of the human recombinant tissue factor. Background technique [0002] In the early 20th century, Schmid and Moranitz first discovered the initiation factor of blood coagulation in tissues and named it tissue factor (Tissue factor, TF). By the 1980s, tissue factor was confirmed to be the most important initiating factor in the physiological coagulation process. With the help of modern molecular biology techniques such as recombinant DNA, artificial mutagenesis and gene knockout, people have further studied TF at the molecular and gene levels, and found that TF is related to systemic inflammatory response syndrome and shock, DIC, thrombotic disease, transplant rejection, malignant The oc...

Claims

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Application Information

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IPC IPC(8): C07K14/43
Inventor 焦进安赵邑郝建平李晋川谢红赵峰梅
Owner 山西省博奥特医学检验有限公司
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