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Fusion protein TpG, encoding gene thereof, recombinant plasmid, strain and application

A fusion protein and recombinant plasmid technology, applied in the field of protein genetic engineering, can solve the problems of limited biomedical applications, short half-life, lack of structural domains, etc.

Pending Publication Date: 2021-09-10
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, gelonin lacks a membrane-binding domain, has a short half-life, and has poor stability, which greatly limits its biomedical applications.

Method used

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  • Fusion protein TpG, encoding gene thereof, recombinant plasmid, strain and application
  • Fusion protein TpG, encoding gene thereof, recombinant plasmid, strain and application
  • Fusion protein TpG, encoding gene thereof, recombinant plasmid, strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Preparation and characterization of embodiment 1, TpG

[0026] 1. Preparation of TpG

[0027] 1.1 Synthesis of pHLIP-Gelonin

[0028] The nucleic acid sequence of pHLIP-Gelonin (as shown in the 478th to 1344th positions of SEQ ID NO.2) was obtained through literature review, and then the gene pHLIP-Gelonin was synthesized by Shanghai Sangon Biological Co., Ltd. and ligated in pUC57 On the empty plasmid, the bacterial solution containing the pUC57-pHLIP-Gelonin recombinant plasmid was obtained.

[0029] 1.2 Construction of plasmids

[0030] (1) Extraction of pUC57-pHLIP-Gelonin and pET32a plasmids: Inoculate the pUC57-pHLIP-Gelonin and pET32a strains in 5 mL LB medium containing Amp (100 ng / μL) respectively, and culture in a constant temperature shaker at 37°C for 12- After 16 hours, the cells were collected by centrifugation at 3500 rpm for 5 minutes. The pET32a and pUC57-pHLIP-Gelonin plasmids were extracted by the SanPrep column plasmid DNA mini-extraction kit, an...

Embodiment 2

[0067] Embodiment 2, the in vitro antitumor activity evaluation of TpG

[0068] 1. Cell culture

[0069] Both SKOV3 cells and HCT-8 cells were cultured using RPMI 1640 medium, which was composed of serum-free RPMI 1640 medium, fetal bovine serum (FBS) and antibiotics (penicillin / streptomycin) at 89:10: 1 ratio for configuration. A549 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics (penicillin / streptomycin). All cells were kept at 37°C with 5% CO 2 cultured in a constant temperature cell incubator. The fresh medium was replaced in time according to the growth of the cells. When the cells grew to 80%, they were digested with 0.25% trypsin (without EDTA), and passed on to new cell culture flasks.

[0070] 2. FITC-labeled Gelonin and TpG

[0071] (1) Dissolve FITC-SCN in PBS buffer and prepare 1 mg / mL FITC labeling solution.

[0072] (2) Mix 1 mg of TpG (stored in PBS buffer) with FITC at a molar ratio of 1:2, make up to 1 mL with PBS buffer, and pla...

Embodiment 3

[0121] Embodiment 3, the in vivo antitumor activity evaluation of TpG

[0122] 1. Establishment of tumor-bearing nude mouse model

[0123] Female athymic BALB / c nude mice (4 weeks old) were purchased from the China Institute for Food and Drug Control, and were raised in the SPF animal room of the China Institute of Radiation Protection, and fed with sterile standard feed. The animal experiments were approved by the Experimental Animal Management and Use Ethics Committee of the Chinese Institute of Radiation Protection, and the approval number is CIRP-IACUC-(R)2020021. SKOV3 cells were suspended in saline, and each mouse was injected with 100 μL (containing 1×10 7 cells), the cell suspension was inoculated into the axilla of the right forelimb of nude mice by subcutaneous injection, and a xenograft model of human ovarian cancer SKOV3 nude mice was established.

[0124] 2. In vivo tumor inhibition experiment

[0125] The average tumor volume reaches 200mm 3 (defined as the 0...

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Abstract

The invention belongs to the field of gene engineering of proteins, and particularly relates to a fusion protein TpG, an encoding gene thereof, a recombinant plasmid, a strain and an application. The amino acid sequence of the fusion protein TpG is as shown in SEQ ID NO. 1. The fusion protein TpG is composed of a thioredoxin tag (Trx), a low-pH insertion peptide (pHLIP) and plant toxin Gonin. The plant toxin Gonin (TpG) with tumor acidity response is designed and successfully subjected to prokaryotic expression for the first time, and the plant toxin Gonin (TpG) with tumor acidity response is systematically and comprehensively characterized. Under a weak acidic condition, TpG can be more effectively internalized and enter tumor cells, and tumor cell growth is inhibited by inducing tumor cell apoptosis and inhibiting intracellular protein synthesis. In-vivo experiments show that compared with Gelonin, TpG can significantly inhibit tumor growth. The research can lay theoretical and experimental foundations for biomedical application of Gelonin.

Description

technical field [0001] The invention belongs to the field of protein genetic engineering, and specifically relates to a fusion protein TpG, its coding gene, recombinant plasmid, strain and application. Background technique [0002] Phytotoxin Gelonin is a protein derived from the seeds of Euphorbia plant Gelonium muhiflorum. Its molecular weight is about 30kDa and it belongs to type I ribosome inactivating protein. Due to its N-glycosidase activity, Gelonin can effectively inhibit protein translation, and thus has efficient cell killing, and is an effective anti-tumor molecule. However, gelonin lacks a membrane-binding domain, has a short half-life, and has poor stability, which greatly limits its biomedical applications. Contents of the invention [0003] One of the objectives of the present invention is to overcome the deficiencies of the prior art and provide a fusion protein TpG, the amino acid sequence of which is shown in SEQ ID NO.1. [0004] Further, the fusion p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/16A61K47/64A61P35/00C12R1/19
CPCC07K14/415C12N15/70A61K47/64A61P35/00C07K2319/35C07K2319/10A61K38/00
Inventor 丁国斌朱晨晨李卓玉武庚风李彬春
Owner SHANXI UNIV
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