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Fusion protein of ribonuclease and toxin membrane translocation domain and its preparation method and use

A fusion protein and protein technology, applied in the field of DNA recombination technology and biomedicine, can solve the problems of limiting the scope of application of Onc and different sensitivities.

Active Publication Date: 2016-01-20
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, further studies have found that different tumor cells have different sensitivities to Onc (Ulrich Arnold & Renate Ulbrich-Hofmann. Natural and engineered ribonuclease sa potential cancer therapeutics. Biotechnol Lett (2006) 28: 1615-1622)
This greatly limits the scope of application of Onc in tumor therapy

Method used

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  • Fusion protein of ribonuclease and toxin membrane translocation domain and its preparation method and use
  • Fusion protein of ribonuclease and toxin membrane translocation domain and its preparation method and use
  • Fusion protein of ribonuclease and toxin membrane translocation domain and its preparation method and use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1Onc-DT 203-390 Synthesis of fusion gene and construction of expression plasmid in Escherichia coli

[0121] To facilitate Onc-DT 203-390 Released from the endosome to the cytoplasm, connect Onc and DT with a 16-amino acid linker peptide containing a furin restriction site. The DNA sequence of furin-DT is a whole gene synthesis. In order to facilitate PCR, Onc and furin- The DT sequences were connected, and 25 Onc terminal base sequences were added to the 5' end of furin during the whole gene synthesis. See SEQ ID NO: 3 for the synthetic sequence of the whole gene. The following pair of primers were designed and synthesized according to the gene sequences of Onc and DT:

[0122] Onc-DT-U (SEQ ID NO: 4):

[0123] 5'CCCAGGACTGGCTGACTTTCCA3';

[0124] Onc-DT-L (SEQ ID NO: 5):

[0125] 5'GGGTCGACTTATTCCGGACCACCAGAAGC3'.

[0126] Wherein, Onc-DT-U is the coding sequence of the N-terminal of the Onc mature protein; Onc-DT-L is the reverse complementary seque...

Embodiment 2

[0128] Embodiment 2Onc-DT 203-390 Expression of fusion protein

[0129] The constructed expression plasmid was transformed into Escherichia coli BL21(DE3), and a single clone was picked overnight in 50ml LB medium and cultured overnight at 37°C. The next day, transfer to 1LTB medium at a ratio of 1:20, and culture at 37°C until A 600 =2.0, add IPTG concentration to 0.5mM to induce for 3.5 hours, collect the bacterial cells by centrifugation at 4°C, 6000rpm×10min, and the bacterial cells are used for the next step of inclusion body collection and purification.

Embodiment 3

[0130] Example 3 Inclusion Body Collection and Washing

[0131] Suspend the collected cells in buffer SSB_A (20mM Tris-HCl, 10mM EDTA, pH8.0), break the cells with an ultrasonic breaker; centrifuge at 12000rpm×10min at 4°C to collect inclusion bodies; 1 M guanidine hydrochloride, 65 mM DTT, 2 mM EDTA, pH 8.0) to suspend the inclusion bodies, stir overnight, collect the inclusion bodies at 4° C., 12000 rpm×10 min.

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Abstract

The invention provides a fusion protein of ribonuclease (Onc) and bacterial toxin membrane translocation domain, a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, a host cell containing the vector, and preparing the fusion protein by genetic engineering Methods of Fusion Proteins. The fusion protein of the present invention not only retains the activity of the original Onc, but also has a stronger ability to kill tumor cells, and can be used for anti-tumor therapy in a wider spectrum.

Description

technical field [0001] The invention belongs to the field of DNA recombination technology and biomedicine. More specifically, the present invention relates to a fusion protein of ribonuclease and bacterial toxin (toxin) membrane translocation domain, a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, a host cell containing the vector, and the use of a gene A method for preparing the fusion protein by engineering technology, and the application of the fusion protein in tumor therapy. Background technique [0002] Ribonuclease (Onconase, also known as: P30 protein, Ranpirnase, Onc for short) is a nuclease isolated from the oocyte and early embryo of the northern leopard frog (Ranapipiens), belonging to the RNaseA superfamily. Studies have shown that Onc enters mammalian cells through endocytosis, selectively degrades tRNA in the cytoplasm, inhibits protein biosynthesis, thereby inhibiting cell proliferation and causing cell apoptosis. In vitro ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P21/02A61K38/16A61P35/00
CPCA61K38/00A61P35/00C07K14/195C12N9/22
Inventor 王庆诚沈如凌孙瑞林费俭李俊
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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