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trail membrane-penetrating peptide-like mutant mur6, preparation method and application

A mutant and membrane-penetrating peptide technology, which is applied in the field of preparation of TRAIL membrane-penetrating peptide-like mutant MuR6, can solve the problems of low possibility of new drugs, existence of toxic and side effects, and inconspicuous advantages, so as to promote aggregation and have little impact on protein structure , enhance the effect of transduction

Inactive Publication Date: 2019-07-23
NANJING HUACHUANG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. Defects and Countermeasures of Apo2L / TRAIL in Tumor Therapy
[0009] Although the drug development process of TRAIL and its receptor agonistic monoclonal antibody has been temporarily frustrated, with the complete elucidation of the apoptosis signaling pathway and the complete revealing of the mutual conversion relationship between apoptosis and tolerance, the targeting of apoptosis signaling pathway The development of anticancer drugs has not stopped
At present, more researches are on the combined application of TRAIL and cytotoxic drugs, but most experiments show that this combination can only produce obvious synergistic and synergistic effects on tumor cells that are relatively sensitive to TRAIL, but cannot completely reverse a variety of different tumor cells. drug resistance
Since TRAIL and cytotoxic drugs belong to two different types of drugs, there are differences and differences in the dosage, route of administration, and mode of action of the drug species. It is less likely to develop a single, stable, and controllable new drug, and TRAIL and cytotoxic drugs After the combination of similar drugs, its toxic and side effects still exist, so its advantages are not obvious

Method used

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  • trail membrane-penetrating peptide-like mutant mur6, preparation method and application
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  • trail membrane-penetrating peptide-like mutant mur6, preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Sequence and primer design of TRAIL penetrating peptide-like mutant

[0059] Selectively transform the 114th to 119th amino acid coding sequence of the extracellular segment of the TRAIL wild-type protein from VRERGP to RRRRRR, that is, the 114th position is mutated from valine to arginine, and the 116th position is mutated from glutamic acid to sperm Amino acid, the 118th position is mutated from glycine to arginine, the 119th position is mutated from proline to arginine, and there are 4 mutation sites, making the N-terminal of the mutant protein a coding sequence of 6 consecutive arginines , becoming a protein containing a penetrating peptide-like structure.

[0060] The cDNA encoding the mutant is shown as SEQ ID NO:1, and the amino acid of the mutant is shown as SEQ ID NO:2.

[0061] Primers were synthesized as follows:

[0062] The upstream primer MuR6-TR-NdeI is shown in SEQ ID NO: 3;

[0063] The downstream primer TR-Eco-R is shown in SEQ ID NO:4.

Embodiment 2

[0065] The TRAIL-MuR6 fragment was amplified by PCR and ligated with pET32a, and the single colony picking and identification of the ligated product

[0066] The TRAIL-MuR6 fragment was amplified by PCR mutation using the pMD19 / TRAIL plasmid as a template. The target fragment of TRAIL-MuR6 and the vector pET32a were digested with NdeI and EcoRI respectively. The TRAIL-MuR6 fragment was connected with the vector pET32a with the excised Trx fusion tag sequence, transformed into Top10 competent cells, single clones were picked, and identified by double digestion with XbaI and EcoRI. See Example 1 for primer design. The original sequence of the pMD19 / TRAIL plasmid was derived from NCBI Reference Sequence: NM_003810.3, and the vector pET32a was derived from Invitrogen.

[0067] Experimental procedure

[0068] 1. PCR amplification of TRAIL-MuR6 target fragment

[0069] 1. Using the pMD19 / TRAIL plasmid as a template and the MuR6-TR-NdeI / TR-Eco-R primer pair to amplify the TRAIL-Mu...

Embodiment 3

[0128] pET32a / TRAIL-MuR6 expression test

[0129] 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.

[0130] Experimental procedure

[0131] 1. Plasmid transformation and strain preservation

[0132] 1. Prepare 100ml of LB medium and sterilize at 121°C for 20min.

[0133] 2. Add 1 μl of pET32a / TRAIL-MuR6 plasmids to 100 μl of BL21(DE3) competent cells, and ice-bath for 30 minutes.

[0134] 3. Heat shock in a water bath at 42°C for 90 seconds.

[0135] 4. Incubate on ice for 3 minutes.

[0136] 5. Take 20 μl of transformed competent cells and smear them all on LB solid medium containing Amp, culture at 37°C overnight.

[0137] 6. After colonies grow on the plate the next day, pick a single bacterium on the plate and add 50ml LB (Amp + ) at 37°C overnight.

[0138] 7. Preserve 20 glycerol bacteria, the final concent...

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Abstract

Provided is a TRAIL membrane-penetrating peptide-like mutant MuR6, its preparation method and application. The mutant is obtained by selectively transforming the 114th to 119th amino acid coding sequence of the extracellular segment of the TRAIL wild-type protein from VRERGP to RRRRRR, Become a protein containing a penetrating peptide structure. The TRAIL mutants can be used to treat tumors.

Description

technical field [0001] The invention relates to the field of genetic engineering drugs, in particular to a TRAIL membrane-penetrating peptide-like mutant MuR6, a preparation method and application. Background technique [0002] 1. The progress and significance of Apo2L / 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 (Apo2Ligand, Apo2L). Later studies confirmed that Apo2L and TRAIL are essentially the same protein, so it can be customarily called Apo2L / TRAIL. The function of TRAIL is firstly as a regulator of the organism's innate or acquired immunity, and secondly, it plays an anti-tumor role as an immune surveillance in the exogenous cell apoptosis pathway. The biggest advantage of TRAIL is that it can s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C12N15/12C12N15/70C12N15/10A61K38/17A61P35/00
CPCA61K38/00A61P35/00C07K14/525C12N15/10C12N15/70
Inventor 陈守春闫娟徐琦黄先洲魏利佳胡海洋
Owner NANJING HUACHUANG BIOTECHNOLOGY CO LTD
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