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Preparation technology of high-purity recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

A preparation process and high-purity technology, applied in the field of preparation of high-purity recombinant TRAIL, can solve the problems of changes in the culture system, affecting the yield and purity of TRAIL, and achieve good chromatographic separation, increase yield and purity, and increase separation. rate effect

Inactive Publication Date: 2020-06-26
南京市检捷生物信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the preparation of high-purity recombinant TRAIL, high-density fermentation of Escherichia coli is required, but due to the metabolism of Escherichia coli cells, the absorption of nutrients, the discharge of toxic by-products, and the absorption of oxygen will all lead to changes in the culture system, thereby affecting Yield and Purity of TRAIL

Method used

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  • Preparation technology of high-purity recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

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Effect test

Embodiment 1

[0043] Step 1: Add 5% of the frozen culture solution into the medium shake flask, and stir at 180 rpm and 37°C for 1 hour.

[0044] Wherein, the culture solution, in terms of mass concentration, includes the following components: tryptone 27g / L; yeast extract 9g / L; acid hydrolyzed casein 9g / L; glucose 10g / L; calcium chloride 0.5g / L Sodium chloride 0.8g / L; Sodium dihydrogen phosphate 8 g / L; Ferrous acetate 0.035g / L; Defoamer 1g / L; Proper amount of acetic acid, adjust the pH to 6.5.

[0045] Step 2: Inoculate the Escherichia coli containing the recombinant plasmid of the recombinant TRAIL gene at a ratio of 1% into the obtained culture medium shake flask; rotate at 220 rpm and culture at 36° C. for fermentation.

[0046] Wherein, the Escherichia coli preparation process of the recombinant TRAIL gene fragment includes the following steps: Step 1, the construction of the target gene, using human pancreatic tissue cDNA as a template to perform PCR amplification from the tryptophan ...

Embodiment 2

[0051] Step 1: Inject the frozen culture solution into the medium shake flask at a ratio of 1%, and stir at 100 rpm and 30° C. for 1 hour.

[0052] Wherein, the culture solution includes the following components in terms of mass concentration: tryptone 20g / L; yeast extract 8g / L; acid hydrolyzed casein 12 g / L; glucose 8 g / L; calcium chloride 0.01 g / L; sodium chloride 0.5 g / L; sodium dihydrogen phosphate 5 g / L; ferrous acetate 0.02 g / L; defoamer 1 g / L; appropriate amount of acetic acid, adjust the pH to 6.8.

[0053] Step 2: Inoculate the Escherichia coli containing the recombinant plasmid of the recombinant TRAIL gene at a ratio of 0.5% into a culture medium shake flask; ferment and culture at 200 rpm and a temperature of 30°C.

[0054] Wherein, the Escherichia coli preparation process of the recombinant TRAIL gene fragment includes the following steps: Step 1, the construction of the target gene, using human pancreatic tissue cDNA as a template to perform PCR amplification fr...

Embodiment 3

[0059] Step 1: Add 5% of the frozen culture solution into the medium shake flask, and stir at 200 rpm and 40°C for 0.5 hours.

[0060] Wherein, the culture solution includes the following components in terms of mass concentration: tryptone 40g / L; yeast extract 12g / L; acid hydrolyzed casein 8g / L; glucose 12 g / L; calcium chloride 0.5 g / L L; sodium chloride 1.0 g / L; sodium dihydrogen phosphate 10 g / L; ferrous acetate 0.05 g / L; defoamer 1 g / L; appropriate amount of acetic acid, adjust the pH to 5.5.

[0061] Step 2: Inoculate Escherichia coli containing the recombinant plasmid of the recombinant TRAIL gene at a rate of 10% into a culture medium shake flask; ferment culture at 250 rpm and 40°C.

[0062]Wherein, the Escherichia coli preparation process of the recombinant TRAIL gene fragment includes the following steps: Step 1, the construction of the target gene, using human pancreatic tissue cDNA as a template to perform PCR amplification from the tryptophan promoter, gene h-TRAIL...

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Abstract

The invention discloses a preparation technology of high-purity recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and belongs to the field of genetic recombination. The preparation technology comprises five steps including thallus inoculation, fermentation culture, material supplementing culture, crushing and collection and chromatographic separation. According to the preparation technology, a promoter structure for gene recombination is studied, casein acid hydrolysate is selected as an induction nutrient component, and the yield of the recombinant TRAIL is increased; when ferrous ions are used as chelation ions, a better chromatographic separation effect can be realized, and negative effects on growth of Escherichia coli are avoided; the pH value of the nutrientcomponent is reasonably controlled by studying the material supplementing manner, and the condition that the ferrous ions are converted into toxic substances to affect bioactivity of the recombinantTRAIL; and thallus crushing and TRAIL collection are further optimized, silochrom is selected as an adsorption medium, and the separation rate f the TRAIL is increased. The yield and purity of the TRAIL are improved.

Description

technical field [0001] The invention belongs to the field of gene recombination, in particular to a preparation process of high-purity recombinant TRAIL. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (Tumor necrosis factor-relatedapoptosis-inducing ligand, TRAIL), as a potential anticancer drug, consists of 281 amino acids, is a type II transmembrane protein, and has the same amino acid sequence as TNF-a and FasL It has homology and can induce tumor cell apoptosis, but TNF-a and FasL have toxic side effects on cells and cannot be used in clinical practice. Studies have shown that the 114-281 amino acid extracellular region fragment of TRAIL is soluble and can significantly inhibit multiple The apoptosis of tumor cell lines is less toxic to normal tissues and cells. By binding to death receptors, cascade signaling pathways are activated, Caspases are activated, and a series of apoptosis characteristics are caused, such as chromosome con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K1/22C12N15/12C12N15/70C12N1/21C12R1/19
CPCC07K14/70575C12N15/70
Inventor 余根芳
Owner 南京市检捷生物信息科技有限公司
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