Tripolymer TRAIL protein and application thereof

A trimer and protein technology, applied in the direction of peptide/protein components, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve problems such as difficult formation of trimer structures, and reduce immunogens Sex, increasing solubility and stability, improving the effect of enzymatic hydrolysis resistance

Inactive Publication Date: 2016-06-22
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult for TRAIL to form a trimer structure under natural conditions, and there are still many problems to be solved in the clinical application of TRAIL.

Method used

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  • Tripolymer TRAIL protein and application thereof
  • Tripolymer TRAIL protein and application thereof
  • Tripolymer TRAIL protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A trimeric TRAIL recombinant protein, such as figure 1 The schematic diagram of the preparation of PEG-HZ-TRAIL is shown, the inducing ligand is tumor necrosis factor-related apoptosis-inducing ligand TRAIL, and the preparation method is a chemical modification method, including the following steps:

[0048] Step 1: Preparation of HZ-TRAIL: Gene sequence 114-281 in human TRAIL protein was cloned using pET expression vector to prepare recombinant gene with N-terminal histidine tag and isoleucine zipper motif. TRAIL recombinant protein of pET23dwpET23dw-His-ILZ-hTRAIL114-281, referred to as HZ-TRAIL, such as figure 2 The schematic diagram showing the preparation of HZ-TRAIL;

[0049] Step 2: Extraction and purification of HZ-TRAIL: Transform the HZ-TRAIL prepared in Step 1 into Escherichia coli, and in LB culture medium containing 1mmol / L isopropylthiogalactopyranoside (IPTG) at 25°C , amplified for 9 hours, and then recombinantly expressed HZ-TRAIL in Escherichia coli...

Embodiment 2

[0053] A trimeric TRAIL recombinant protein, such as figure 1 The schematic diagram of the preparation of PEG-HZ-TRAIL is shown, the inducing ligand is tumor necrosis factor-related apoptosis-inducing ligand TRAIL, and the preparation method is a chemical modification method, including the following steps:

[0054] Step 1: Preparation of HZ-TRAIL: Use the pET expression vector to clone the 114-281 gene sequence of the human TRAIL protein, and prepare the recombinant gene pET23dw-His with N-terminal histidine tag and isoleucine zipper motif - TRAIL recombinant protein of ILZ-hTRAIL114-281, referred to as HZ-TRAIL, such as figure 2 The schematic diagram showing the preparation of HZ-TRAIL;

[0055] Step 2: Extraction and purification of HZ-TRAIL: Transform the HZ-TRAIL prepared in Step 1 into Escherichia coli, and in LB culture medium containing 1mmol / L isopropylthiogalactopyranoside (IPTG) at 28°C , propagated for 8 hours, and then recombinantly expressed HZ-TRAIL in Escheri...

Embodiment 3

[0059] A trimeric TRAIL recombinant protein, such as figure 1 The schematic diagram of the preparation of PEG-HZ-TRAIL is shown, the inducing ligand is tumor necrosis factor-related apoptosis-inducing ligand TRAIL, and the preparation method is a chemical modification method, including the following steps:

[0060] Step 1: Preparation of HZ-TRAIL: Use the pET expression vector to clone the 114-281 gene sequence in the human TRAIL protein, and prepare a recombinant gene with an N-terminal histidine tag and an isoleucine zipper motif, pET23dw- The TRAIL recombinant protein of His-ILZ-hTRAIL114-281, referred to as HZ-TRAIL, such as figure 2 The schematic diagram showing the preparation of HZ-TRAIL;

[0061] Step 2: Extraction and purification of HZ-TRAIL: Transform the HZ-TRAIL prepared in step 1 into Escherichia coli, and in LB culture medium containing 1mmol / L isopropylthiogalactopyranoside (IPTG) at 30°C , multiplied for 6 hours, and then recombinantly expressed HZ-TRAIL in...

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Abstract

The invention belongs to the biotechnological field, and particularly relates to tripolymer TRAIL protein and application thereof.The tripolymer TRAIL recombination protein is prepared through the steps that a 114-281 amino acid sequence of TRAIL and a vector PET are subjected to gene cloning to prepare TRAIL recombination protein (HZ-TRAIL for short) with an N-terminal histidine label and an isoleucine zipper motif recombination gene pET23dw-His-ILZ-hTRAIL114-281; the HZ-TRAIL is modified by polyethylene glycol to enable PEG to be coupled to the N-terminal amino of the HZ-TRAIL protein through a covalent bond, and then the polyethylene glycol-modified TRAIL recombination protein, that is, the PEG-HZ-TRAIL protein is obtained.The TRAIL recombination gene is improved into the novel PEG-HZ-TRAIL through a PEGylation technique to produce the high-activity TRAIL protein of a tripolymer, and the tripolymer TRAIL protein has the advantage of being high in pharmacological efficacy on resisting tumors and rheumatoid arthritis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a trimeric TRAIL protein and its application. Background technique [0002] In recent years, due to the development of biopharmaceutical technology, mass production of protein or polypeptide drugs has become possible, but polypeptide drugs have a short half-life in the body circulation, are easily destroyed by proteolytic enzymes in the gastrointestinal tract, poor water solubility, and strong antigenicity. Allergies, and the disadvantages of easily causing the production of neutralizing antibodies limit its application. Therefore, in the prior art, the technology of performing PEG site-directed modification on proteins or polypeptides with medical effects to make them move toward pharmaceutical applications has been extensively studied. Polyethylene glycol (PEG) modification of proteins or polypeptides (pegylation) is to chemically couple activated PEG to protein or pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/17A61K47/48A61P35/00A61P29/00A61P19/02
Inventor 金成浩孙虎男金颖华鲁文赓臧延青刘畅
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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