Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating liver ischemia-reperfusion injury

A technology of reperfusion injury and fusion protein, which is applied in the field of recombinant protein, can solve the problems of transmitting apoptosis signals, etc., and achieve broad application prospects, unique effect and enhanced curative effect

Pending Publication Date: 2021-03-05
SHENZHEN ZHONGKE AMSHENN MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although sDR5 maintains the activity of binding to TRAIL ligands, it cannot transmit apoptosis signals to cells and can block the TRAIL-DR5-mediated apoptosis response

Method used

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  • Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating liver ischemia-reperfusion injury
  • Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating liver ischemia-reperfusion injury
  • Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating liver ischemia-reperfusion injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Design and recombination of recombinant human DR5-Fc expression sequence

[0046] After long-term accumulation of experience, the inventor constructed a recombinant fusion protein and fused human DR5 with Fc in various ways. The results of mass spectrometry analysis showed that most of the target proteins were cleaved at the N-terminal 11 amino acids (ITQQDLAPQQR). In order to express N The target protein with a relatively uniform terminal was finally screened to remove the intermediate connection sequence and 18 amino acids at the N-terminus on the basis of the commonly used signal peptide of the fusion protein, and named it sDR5-Fc (ZJ501-5). The plasmid was transiently transfected, and the expression supernatant was purified for mass spectrometry and activity analysis.

[0047] DNA sequence of ZJ501-5:

[0048] atgggtgtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatgtccagcccctcagagggattgtgtccacctggacaccatatctcagaagacggtagagattgcatctcctg...

Embodiment 2

[0052] Example 2: Expression of Human sDR5-Fc Recombinant Protein and Detection of Physicochemical Properties

[0053] The sDR5-Fc / pL101 plasmid was transiently transfected into CHO.K1 cells with Lipofectamine2000, and the supernatant was collected for Dot Blot detection after 48 hours. The result was positive, see figure 2 .

[0054] To recover CHO.K1-S cells, press 5×10 5 / ml 10ml after resuspension, use FreeStyle TM CHO Expression Medium serum-free medium, and add Glutamine at a final concentration of 8mmol / L for culture, placed at 37°C in 8% CO 2 Shake the incubator at 120rpm for cultivation. When the number of cells>1×10 6 Subculture and add liquid to 30ml, maintain cell number 2-5×10 5 / ml, each subsequent passage and maintain the density to 2-5×10 5 / ml, CHO.K1-S needs to be subcultured more than three times.

[0055] The day before transfection, adjust the cell density to 5-6×10 5 / ml 100ml, placed at 37°C 8% CO 2 Shake the incubator at 120rpm for culture, a...

Embodiment 3

[0080] Example 3: Identification of biological activity of ZJ501-5

[0081] (1) Establish a commercial Trail killing activity detection method

[0082] Collect the Jurkat cells in the logarithmic growth phase, resuspend the cells with 10% FCS RPMI-1640 / DMEM after counting, and adjust the cell density to 8*10 4 / ml, 100ul / well was added to a 96-well cell culture plate, and placed in a 37°C, 8% carbon dioxide incubator for 20-24 hours.

[0083] Resuspend the commercial Trail in the above-mentioned complete culture solution containing actinomycin D (final concentration is 0.03ug / ml) at a final concentration of 500-1000ng / ml, and use the complete culture solution containing actinomycin D for 2 times Specific dilution commercial Trail, a total of 15-20 concentration gradients. The diluted sample was added to a 96-well cell culture plate at 100ul / well, and placed in an incubator for 18-22 hours. Add 20ul / well of freshly prepared 20:1 mixed MTS / PMS chromogenic solution, continue t...

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Abstract

The invention discloses application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating liver ischemia-reperfusion injury. The nucleotide sequence for encoding the sDR5-Fc recombinant fusion protein has a) a base sequence shown in SEQ ID NO: 1, or b) a complementary pairing sequence with SEQ ID NO: 1, or c) a sequence which encodes the same sequence as the nucleotide sequence of a or b but is different from the nucleotide sequence of a or b due to the degeneracy of genetic codes; or the amino acid sequence of the sDR5-Fc recombinant fusion protein isshown in SEQ ID NO: 2, or the amino acid sequence is shown in SEQ ID NO: 2, and one or more amino acids are substituted, but the biological activity is not changed; or the amino acid sequence of thesDR5-Fc recombinant fusion protein is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

Description

technical field [0001] The invention relates to a recombinant protein, in particular to the application of a human sDR5-Fc recombinant fusion protein in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury. Background technique [0002] Hepatic ischemia reperfusion injury (hepatic ischemia reperfusion injury, HIRI) refers to the pathological phenomenon that the blood supply to the liver is blocked for a period of time during liver surgery, and the blood supply is restored, and the dysfunction and structural damage of the liver are not alleviated, but aggravated . [0003] my country is a country with a high incidence of liver diseases. Hepatic ischemia reperfusion injury (HIRI) is involved in hemorrhagic shock, liver tumor resection, liver transplantation and other diseases and operations. Liver ischemia-reperfusion injury (hepatic ischemia / reperfusion injury, HI / RI) often causes remote organ damage, and severe HIRI may lead to liver failur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K47/68A61P9/10C07K19/00C12N15/62
CPCA61K38/1793A61K31/704C07K14/70578A61K47/68A61K9/0019A61P9/10C07K2319/30A61K2300/00
Inventor 万晓春沈恩允张青梅陈倩胡永强
Owner SHENZHEN ZHONGKE AMSHENN MEDICINE CO LTD
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