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TRAIL double target mutant protein MuR5S4TR, and preparation method and application thereof

A mutant protein and dual-target technology, applied in the fields of peptide/protein components, chemical instruments and methods, animal/human proteins, etc., can solve problems such as non-pharmaceutical realization methods, achieve a wide range of biological activities, significant amplification potential, and purification The effect of cost reduction

Active Publication Date: 2017-02-22
CHENGDU HUACHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the development process of small-molecule Smac agonist drugs, subject to the safety of small-molecule drugs, the difference between the two types of drugs and TRAIL, the convenience of single use, the stability of clinical efficacy and other factors, it is not an optimal The best way to achieve pharmaceuticals

Method used

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  • TRAIL double target mutant protein MuR5S4TR, and preparation method and application thereof
  • TRAIL double target mutant protein MuR5S4TR, and preparation method and application thereof
  • TRAIL double target mutant protein MuR5S4TR, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Sequence and primer design of TRAIL dual-target mutein

[0073]The present invention is based on the sequence of TRAIL-penetrating peptide-like mutant TRAIL-MuR5 (PCT / CN2015 / 073504: TRAIL-penetrating peptide-like mutant MuR5, preparation method and application) and selectively converts the second to The 7th to 10th amino acid sequence after the 6th RRRRR (R5) is mutated from proline, glutamine, arginine, valine (PQRV) sequence to the N-terminal 4 peptide of Smac protein, namely alanine, valine Acid, proline, isoleucine (AVPI) sequence, with 4 mutation sites, making the N-terminus of the mutant protein become 5 consecutive arginines (RRRRR) followed by the N-terminal 4-peptide alanine of the Smac protein Acid, valine, proline, isoleucine (AVPI) coding sequence. The new mutant protein has both the properties of a penetrating peptide-like mutant and the activity of a second mitochondria-derived activator of caspase. We named the new mutant protein TRAIL dual-target mutan...

Embodiment 2

[0085] The MuR5S4TR gene fragment was amplified by two-step PCR, and the fragment was directly ligated with the expression vector pET32a cut with the same restriction enzyme after double enzyme digestion, and a single colony of the ligation product was picked and identified.

[0086] See Example 1 for primer design, and the pET32a / MuR5TR template comes from patent PCT / CN2015 / 073504.

[0087] Specifically, the pET32a / MuR5TR cDNA sequence is shown in SEQ ID No:3.

[0088] Experimental procedure

[0089] 1. PCR amplification of the target fragment of MuR5S4TR

[0090] 1. Use the MuR5S4TR-2 / TR-Eco-R primer pair to amplify the MuR5S4TR-1 target fragment, prepare a reaction system according to Table 1, and the reaction system is 50 μl.

[0091] 2. Vortex to mix, centrifuge briefly, and collect the solution at the bottom of the tube.

[0092] 3. See Table 2 for the PCR amplification reaction conditions.

[0093] 4. Electrophoresis and photography.

[0094] 5. The target fragment...

Embodiment 3

[0159] Expression test of plasmid pET32a-MuR5S4TR

[0160] The plasmid with correct sequencing obtained in Example 2 was transformed into competent Escherichia coli BL21(DE3), and a single bacterium was picked for expression test to investigate the expression effect.

[0161] Experimental procedure

[0162] 1. Plasmid transformation and strain preservation

[0163] 1. Prepare 100ml of LB medium and sterilize at 121°C for 20min.

[0164] 2. Take 1 μl of pET32a-MuR5S4TR plasmid and add it to 100 μl BL21(DE3) competent cells, and keep it in ice bath for 30 minutes.

[0165] 3. Heat shock in a water bath at 42°C for 90 seconds.

[0166] 4. Incubate on ice for 3 minutes.

[0167] 5. Take 20 μl of transformed competent cells and smear them all on LB solid medium containing Amp, and culture overnight at 37°C.

[0168] 6. After colonies grow on the plate the next day, pick two single bacteria on the plate and add 50ml LB (Amp +) Incubate overnight at 37°C.

[0169] 7. Preserve ...

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PUM

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Abstract

The invention belongs to the field of genetically engineered drugs and provides a mutant protein MuR5S4TR of TRAIL, and a preparation method and use thereof. The 2-10th bits of an N-terminal amino acid sequence of the mutant protein are composed of a transmembrane peptide sequence RRRRR (R5) and a binding sequence AVPI of an apoptosis inhibitor XIAP, and the 11-169th bits are TRAIL protein peptide segments (123-281aa). The specific sequence is shown in SEQ ID NO:2.

Description

technical field [0001] The present invention relates to the field of genetic engineering drugs, in particular to a TRAIL dual-target mutant protein MuR5S4TR, its preparation method and application. The TRAIL dual-target mutant protein MuR5S4TR in the present invention has superior therapeutic effects on various types of tumors, and is A new generation of highly effective drug for inducing tumor cell apoptosis with great potential. Background technique [0002] 1. The progress and significance of Apo 2L / TRAIL in tumor therapy [0003] Tumor necrosis factor-related apoptosis-inducing ligand (Tumor necrosis factor-related apoptosis-inducing ligand, TRAIL) is a member of the tumor necrosis factor (Tumor necrosis factor, TNF) superfamily. In 1996, it was independently cloned by Pitti et al., who named it apoptin 2 ligand (Apo 2Ligand, Apo 2L). Later studies confirmed that Apo 2L and TRAIL are essentially the same protein, so it can be customarily called Apo 2L / TRAIL. The funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705A61K38/17A61P35/00
CPCC07K14/70578A61K38/00A61K38/17C07K14/525C07K19/00C12N15/70A61P35/00C07K14/70575
Inventor 陈守春徐琦闫娟黄先洲魏利佳胡海洋
Owner CHENGDU HUACHUANG BIOTECH CO LTD
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