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PDGFR beta targeted TRAIL (TNF-related apoptosis-inducing ligand) variant as well as preparation method and application thereof

A technology of apoptosis-inducing ligand and tumor necrosis factor, which is applied in the direction of tumor necrosis factor, antineoplastic drugs, pharmaceutical formulations, etc., can solve the problem of poor therapeutic effect, poor anti-tumor effect, and lack of targeting of hepatic stellate cells And other issues

Active Publication Date: 2019-03-01
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) Since decoy receptors are widely expressed in normal tissues, TRAIL is consumed by normal tissues after entering the body, and the amount reaching the tumor site is small, so the anti-tumor effect is not good
However, due to the lack of targeting of TRAIL to hepatic stellate cells, the therapeutic effect is not good

Method used

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  • PDGFR beta targeted TRAIL (TNF-related apoptosis-inducing ligand) variant as well as preparation method and application thereof
  • PDGFR beta targeted TRAIL (TNF-related apoptosis-inducing ligand) variant as well as preparation method and application thereof
  • PDGFR beta targeted TRAIL (TNF-related apoptosis-inducing ligand) variant as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Molecular design and cloning construction of embodiment 1Z-hTRAIL variant

[0042] 1. Molecular design of Z-hTRAIL variants

[0043] Z PDGFRβ Consists of 58 amino acids (Table 1). hTRAIL is a fragment consisting of amino acids 114-281 of the extracellular domain of human TRAIL (see Table 1). Pass (G4S) 3 Linker will Z PDGFRβ Linked to the N-terminus of hTRAIL to construct fusion protein Z PDGFRβ -(G4S) 3 -hTRAIL (Z-hTRAIL for short)( figure 1 ).

[0044] 2. Construction of Z-TRAIL variant expression vector

[0045]According to the amino acid sequence of ZPDGFRβ, the initial coding gene was designed, and after optimization by nucleic acid analysis software, the gene sequence was synthesized by Nanjing GenScript. During synthesis, EcoRI / BamHI restriction endonuclease sites (EcoRI: gaattc / BamHI ggatcc) were added to both ends of the sequence. By double digestion and ligation, the gene sequence of ZPDGFRβ was loaded on the expression plasmid of pQE30-hTRAIL (the n...

Embodiment 2

[0049] Protein expression and separation and purification of embodiment 2Z-TRAIL variant

[0050] Pick the single clone of the expression strain M15-pQE30-Z-hTRAIL (prepared in Example 1) and inoculate it into double-resistant (containing ampicillin 100 μg / ml, kanamycin 30 μg / ml) LB liquid medium, 37 ° C Shaking culture, when the bacterial solution concentration A 600 When the temperature reaches about 0.8, add 0.05mM isopropyl-β-D-thiogalactopyranoside (Isopropylβ-D-1-thiogalactopyranoside, IPTG), and induce culture at 26°C for 14-16 hours. The cells were collected by centrifugation (7000g, 10min), resuspended with Lysis buffer (50mM phosphate buffer, pH8.0; 300mM NaCl; 20mM imidazole; 10mM β-mercaptoethanol), and added phenylmethanesulfonyl fluoride (PMSF ) to a final concentration of 1 mM, and ultrasonically disrupt the bacteria in an ice bath (power 300W, work for 10s, interval 30s, 40min in total). After breaking the bacteria, centrifuge (4°C, 25000g, 10min), repeat 4 t...

Embodiment 3

[0052] Example 3 Z-TRAIL variant binds to pericytes and kills surrounding tumor cells

[0053] After incubation with PDGFRβ-specific antibody, pericytes were analyzed by flow cytometry. The result is as Figure 4 As shown in A, PDGFRβ is highly expressed on the surface of pericytes. Pericytes were co-incubated with FAM-labeled Z-hTRAIL (Z-hTRAIL prepared in Example 2, FAM-labeled) or hTRAIL, and then analyzed by flow cytometry, it was found that Z-hTRAIL could bind to pericytes, and this combination Can be blocked by PDGFRβ-specific antibody, indicating fusion Z PDGFRβ Allows hTRAIL to bind to pericytes. In order to detect whether Z-hTRAIL combined with pericytes still has tumor cell killing function, pericytes were pre-incubated with Z-hTRAIL (prepared in Example 2) (1 μM) for 1 h, washed with PBS and then mixed with tumor cells (LS174T and HCT116, 1.5*10 4 ;COLO205, 2*10 4 ) were co-cultured overnight, and CCK-8 was used to detect cell viability.

[0054] The result i...

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Abstract

The invention discloses a TRAIL (TNF-related apoptosis-inducing ligand) variant. The variant is fusion protein of TRAIL and ZPDGFR beta, wherein ZPDGFR beta is linked to the N end or the C end of theTRAIL through a linker. The invention further discloses a nucleotide sequence as well as a recombinant vector and a recombinant bacterium which comprise the nucleotide sequence, and further disclosesa preparation method and an application of the variant. The TRAIL variant protein Z-hTRAIL has good tumor killing activity and definite curative effect on hepatic fibrosis-renal tubular ectasia syndrome, and has good clinical application prospects.

Description

technical field [0001] The present invention relates to the field of biotechnology drugs, in particular to a PDGFRβ-targeted tumor-promoting apoptosis-inducing ligand variant and its preparation method and use. Background technique [0002] Tumor necrosis factor apoptosis-related inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family, and its C-terminal 114-281 amino acids can be hydrolyzed by proteases into soluble extracellular segments, forming homotrimers, and having body binding ability. Membrane receptors for TRAIL include death receptors (DR4 and DR5) and decoy receptors (DcR1 and DcR2). Death receptors DR4 and DR5 molecules contain death domains, which can transmit death signals into cells after combining with TRAIL, and induce cell apoptosis. On the contrary, the decoy receptor DcR1 molecule does not contain a death domain, and the death domain of DcR2 is incomplete, both of which can bind to TRAIL, but cannot transmit death signals, and thu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/19A61K47/64A61P35/00A61P37/02A61P1/16
CPCA61K38/191A61K47/64A61P1/16A61P35/00A61P37/02C12N15/70C07K14/525C07K2319/01A61K38/16A61P37/06C07K19/00C12N15/09
Inventor 陶泽杨浩卢晓风
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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