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Novel gene engineering recombinant TRAIL fusion protein, and preparation method and use thereof

A fusion protein and genetic engineering technology, applied in the field of new genetic engineering recombinant TRAIL fusion protein and preparation, can solve the problems of weak biological activity, poor stability, short half-life of TRAIL, etc., achieve high purity, simple method, good anti-tumor or The effect of cancer cell activity

Active Publication Date: 2016-11-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a novel genetically engineered recombinant TRAIL fusion protein and its preparation method and use in order to solve the problems of TRAIL itself with short half-life, poor stability and weak biological activity

Method used

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  • Novel gene engineering recombinant TRAIL fusion protein, and preparation method and use thereof
  • Novel gene engineering recombinant TRAIL fusion protein, and preparation method and use thereof
  • Novel gene engineering recombinant TRAIL fusion protein, and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of Adenovirus Fiber-TRAIL Fusion Protein Prokaryotic Expression Vector

[0032] 1. Obtaining the target gene

[0033] PCR was carried out using the plasmid pDC316-TRAIL (human, aa95-281) with the TRAIL gene stored in the laboratory as a template, and the reaction conditions were: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C 1min, 30 cycles; 72°C extension for 10min. PCR products were identified by 1.2% agarose gel electrophoresis. Agarose gel recovery kit for purification of PCR products. Use Vector NTI Suited 6 software according to the human TRAIL gene sequence TRAIL primer that Genebank provides as follows:

[0034] Upstream primer sequence: 5'- GCTAGC ATGACCTCTGAGGAAACCATTTCTAC-3', containing NheI restriction site.

[0035] Downstream primer sequence: 5'- AAGCTT TTAGCCAACTAAAAAGGCCC-3', containing Hind IIl restriction site.

[0036] 2. Constructio...

Embodiment 2

[0046] Example 2 Construction of Adenovirus Fiber-TRAIL Fusion Protein Prokaryotic Expression Vector

[0047]1. Prokaryotic expression

[0048] The above three prokaryotic expression plasmids were transformed into Escherichia coli BL21 (DE3) competent cells (Dalian Bao Biology), spread on LB solid medium containing kanapenicillin (100ug / ml), and placed in a 37°C incubator Incubate overnight. Pick a single colony from the above plate and inoculate it in 10mL LB liquid medium for overnight culture. The obtained bacterial liquid was inoculated into 1L LB liquid medium, and cultured in large quantities by shaking at 37°C. When the OD600 value of the bacteria reached 0.8, the inducer isopropyl-β-D-thiogalactoside (IPTG, Beijing Dingguo) was added at a final concentration of 1 mmol / L, and the expression was induced at 20°C for 16 hours. SDS-PAGE electrophoresis was performed on the bacterial cell samples before and after induction, such as image 3 .

[0049] 2. Protein purific...

Embodiment 3

[0055] Example 3 Detection of the Killing Effect of Fiber-TRAIL Fusion Protein on Tumor Cells

[0056] Tumor cell lines:

[0057] Breast cancer cells: ZR-75-30, MCF7; lung cancer cells: A549; liver cancer cells: SMMC-7721; colon cancer cells: SW480; cervical cancer cells: Hela; normal breast cells: MCF-10A; normal liver cells: Chang Liver .

[0058] 1) The killing effect of FA1FT and HA5ST on tumor cells

[0059] Experimental method: Cells were cultured in a medium containing 10% calf serum, 100μg / ml streptomycin and 100U / ml penicillin at 37°C, 5% CO 2 cultivated under conditions. Will 1×10 4 3 cells were inoculated in 96-well plates, adhered to the wall overnight, then the culture medium was changed to the medium containing 2% calf serum, and the mixture obtained in Example 2 with different concentration gradients (0.01, 0.1, 1, 10 and 100 nM) was added. fusion protein. After reacting overnight, 20 μl of MTT solution was added, and after 4 hours of reaction, the superna...

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Abstract

The invention discloses a recombinant protein with improved stability and activity, obtained by using adenovirus fibrin fragments as a trimer motif and adopting TRAIL fusion expression and used as an antitumor therapy drug. The invention also provides a method for expression, purification and activity detection of the fusion protein. The method concretely comprises the following steps: 1, designing and constructing fusion gene, and carrying out low-temperature inducible expression by using an Escherichia coli system; 2, purifying the target protein by using a nickel column affinity chromatography technology, and removing an His label by using a thrombin digestion site to obtain the final target product; 3, detecting the killing activity of the fusion protein to tumor cells and the toxicity of the fusion protein to normal cells in vitro; 4, detecting the stability of the fusion protein under different conditions; and 5, detecting the in vivo antitumor activity of the fusion protein in a nude mouse model. Two adenovirus fibrin fragments are used as the TRAIL fusion protein of the trimer motif. The invention provides a method for simply and rapidly obtaining the high-stability TRAIL protein. The method can be applied to antitumor researches of the TRAIL protein. The abuses of poor stability and low activity of the TRAIL protein are solved.

Description

technical field [0001] The present invention relates to a fusion protein, its preparation method and application, in particular to a novel genetic engineering recombinant TRAIL fusion protein, its preparation method and application. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TNF related apoptosis-inducing ligand, TRAIL) is a member of the TNF superfamily, has a broad-spectrum anti-cancer function, and it is almost non-toxic to normal tissues and cells. The active TRAIL is in a trimer state, which is maintained by chelation between the zinc ion in the center of the trimer and the cysteine ​​on each monomer. However, TRAIL itself has short half-life, poor stability, and weak biological activity, and some studies have shown that the structural order of soluble TRAIL trimer will change after the introduction of tags, resulting in hepatotoxicity. Contents of the invention [0003] The object of the present invention is to provide a n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/17A61K38/16A61P35/00
CPCA61K38/00C07K14/005C07K14/70575C07K2319/00C12N2710/10022
Inventor 于彬于湘晖孔维闫晶怡王真
Owner JILIN UNIV
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