Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof

A fusion protein, coding gene technology, applied in antitumor drugs, peptide/protein components, hybrid peptides, etc., can solve the problems of TRAIL sensitivity difference, TRAIL limitation, etc., and achieve the effect of inhibiting proliferation, inhibiting growth and low drug toxicity. Effect

Active Publication Date: 2015-05-20
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although early clinical trials are very encouraging, human recombinant TRAIL (Human recombinant TRAIL, hrTRAIL) is only limited to patients with TRAIL-sensitive tumors, and even tumors from the same tissue have different sensitivities to TRAIL
Therefore, the insensitivity of different tumor tissues to TRAIL limits the clinical application of TRAIL.

Method used

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  • Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof
  • Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof
  • Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Construction of fusion protein Ec-LDP-TRAIL recombinant expression plasmid pET30-Ec-LDP-TRAIL

[0139] Recombinant plasmid pET30a(+)-Ec-ldp-Hr (Guo Xiaofang et al., A bispecific enediyne-energized fusion protein containing ligand-based and antibody-based oligopeptides against epidermal growth factor receptor and human epidermal growth factor receptor2shows potent Cactivator Research, 2010, 16.7:2085-2094) and pET30a(+)-TRAIL (Jie Yang, TRAIL combined with LDM and mechanism of action and expression of TRAIL protein. MS thesis. Peking Union Medical College, 2010.) contain Ec-LDP gene and TRAIL functional gene are preserved by our laboratory. Two restriction sites, NdeI and XhoI, were introduced by overlapping PCR technology, and the primers were synthesized by Invitrogen.

[0140] P1 (SEQ ID NO:8): 5'-TATA CATATG AACTGTGTGGTGGGCTATATTGG-3' (the underline is the Nde I restriction site, followed by the Ec-ldp start sequence)

[0141] P2 (SEQ ID NO:9): 5'-CTCAC TGAACC...

Embodiment 2

[0146] fusion protein Ec-LDP-TRAIL in Escherichia coli BL21 (DE3) star TM inducible expression

[0147] The identified recombinant plasmid pET30-ec-ldp-trail was transformed into Escherichia coli BL21(DE3)star TM (Invitrogen company product), pick the recombinant transformant (this strain is Escherichia coli), which has been preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on September 17, 2013 (referred to as CGMCC, address : No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), strain preservation number CGMCC NO.8200) inoculated into 3ml LB liquid medium containing 50μg / ml kanamycin, 37℃ Incubate overnight with shaking. The next day, they were inoculated at a ratio of 5%, cultured with shaking at 37°C until the OD600 was 0.8, added IPTG to a final concentration of 0.2mM, and induced for 8 hours. An appropriate amount of bacterial liquid was taken, an...

Embodiment 3

[0148] fusion protein Ec-LDP-TRAIL affinity chromatography purification and separation preparation

[0149] 3.1 Extraction of inclusion body protein

[0150]The culture of recombinant bacteria induced by IPTG was centrifuged at 10000 g for 10 min, the supernatant was removed as much as possible, and the bacteria were collected. Resuspend the cells with 20ml of 20mM Tris-HCl per 1L of culture volume. Sonication disrupts cells. Centrifuge at 10,000 g at 4°C for 15 min to collect inclusion bodies and cell fragment proteins, and discard the supernatant. Resuspend the pellet with 40 ml of urea-free 1× binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 8.0). Centrifuge at 10,000 g for 15 min at 4°C, discard the supernatant, and collect the precipitate. Repeat the above steps, resuspend the pellet with 50ml of 1× binding buffer (20mM Tris-HCl, 0.5M NaCl, 20mM imidazole, 8M urea, pH8.0), and ice-bath for 1h to completely dissolve the inclusion body protein. Centrifug...

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Abstract

The invention provides an anti-tumor fusion protein, which has the following primary amino acid sequence structure: EGFR-specific targeting oligopeptide-connecting peptide-lidamycin apoprotein-connecting peptide-TRAIL functional peptide fragment, wherein the EGFR-specific targeting oligopeptide has an amino acid sequence shown in a SEQ ID NO: 1, the lidamycin apoprotein has an amino acid sequence shown in an SEQ ID NO: 2, and the TRAIL functional peptide fragment has an amino acid sequence shown in an SEQ ID NO: 3. The fusion protein is an EGFR and DR4/DR5 targeted bispecific fusion protein, and contains an EGFR targeted ligand oligopeptide, and the sensitivity of tumor cells to TRAIL and the natural drug resistance of reverse tumor cells to TRAIL are enhanced by an antagonistic EGFR signaling pathway. The invention also provides a coding gene of the fusion protein, an intensive fusion protein assembled with a lidamycin chromophore, and a pharmaceutical application of the intensive fusion protein.

Description

technical field [0001] The invention belongs to the technical field of medicinal protein. Specifically, the present invention relates to an anti-tumor fusion protein, its coding gene, a drug with the fusion protein as an active ingredient and its pharmaceutical use. Background technique [0002] TRAIL (TNF-related apoptosis-inducing ligand, tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine synthesized by the body itself, and its physiological function is mainly to regulate the body's immune response. TRAIL mainly induces apoptosis through the extracellular apoptotic pathway, but also involves the mitochondrial pathway in the later stage. The TRAIL signaling pathway refers to the use of TRAIL as a ligand to initiate apoptosis signals by binding to death receptors, and finally induce cell apoptosis. Studies have shown that TRAIL and agonist antibodies targeting TRAIL receptors can selectively induce tumor cell apoptosis, while normal tissues are not sens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P35/00
Inventor 朱大强陈淑珍王晓飞商悦李毅李良
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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