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Small molecule peptides inhibitor of human heparinase

A technology of human heparanase and heptapeptide, which is applied in the field of biomedicine, can solve the problems of inapplicability in clinical practice and heterogeneous structure, and achieve the effect of potential medical value

Inactive Publication Date: 2010-12-29
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other inhibitors include sulfated laminarin, chitin sulfate, SdC28, Ca2SP, suramin, etc., but they cannot be used clinically due to molecular size, toxicity, heterogeneous structure, and multiple biological effects.

Method used

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  • Small molecule peptides inhibitor of human heparinase
  • Small molecule peptides inhibitor of human heparinase
  • Small molecule peptides inhibitor of human heparinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Cloning and expression of human heparanase 8kD small subunit

[0052] 1. Construct the expression vector:

[0053] Apply the PCR method (primer P1: GGAATTC CATATG CAGGACGTCGTGGACCT (introduction of Ned I restriction site) (SEQ ID No.21) and P2: CCG CTCGAG TTCCTTCTTGGGATCG (introduction of Xho I restriction site) (SEQ ID No. 22).

[0054] reaction system:

[0055] 2×GC Buffer I 25μl

[0056] TaKaRa LA Taq 0.5 μl

[0057] dNTP Mixture 8μl

[0058] Template 1μl

[0059] Upstream primer 1.5μl

[0060] Downstream primer 1.5μl

[0061] Sterilized water to make up volume to 50 μl

[0062] Reaction conditions: 94°C pre-mutation for 5 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, extension at 72°C for 1 minute, and final extension at 72°C for 10 minutes). DE3) The engineered strains were constructed and preserved by our laboratory, among which the pGEX-2PK vector is a product of GE Healthcare, and the...

Embodiment 2

[0079] Example 2: Screening of Human HPA Small Subunit Inhibitors

[0080] 1. On the basis of obtaining the human HPA small subunits whose purity and concentration meet the requirements, use the human HPA small subunits as the target molecular screening phage display random cyclic heptapeptide library (New England Biolabs company kit product), after five rounds screening, the recovery rate increased by about 10 3 times. From the plate of the last round of screening, 150 phage plaques were randomly picked and placed in the ER2738 culture in the logarithmic growth phase (New England Biolabs kit product), cultured with vigorous shaking at 37°C for 4.5h, and then centrifuged at 4°C. Clear, save the original phage species, and then prepare 150 corresponding monoclonal samples, select positive clones by ELISA detection, extract phage single-stranded DNA, and send it for sequencing. According to the DNA sequencing results, the amino acid sequences of the corresponding binding peptide...

Embodiment 3

[0094] Example 3: Detection of C7-1 and C7-5 activities using HepG-2 cells in vitro reconstituted basement membrane invasion assay

[0095] 1. Method

[0096] 1. Digest HepG-2 cells in the logarithmic growth phase (gifted by Mr. Gong Xin, Institute of Bioengineering, Academy of Military Medical Sciences) with trypsin (product of Amresco), add serum-containing medium, centrifuge, discard the culture medium, and wash with PBS for 1 resuspend with 0.1% BSA-serum-free DMEM medium (product of HyClone Company), and adjust the final concentration of HepG-2 cells to 1×10 6 / mL. Add 10% FBS-DMEM medium to the 24-well plate (lower chamber), 600 μL / well; use sterile tweezers to transfer the cell culture pool covered with Matrigel gel into the 24-well plate (lower chamber).

[0097] 2. Take 160 μL of HepG-2 cell suspension and add it to the cell culture pool; add different concentrations of C7-1 and 0.25 mg / mL C7-5 respectively, and add an equal volume of 0.1% BSA-serum-free DMEM medium...

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Abstract

The invention discloses an amino acid sequence of a human heparanase micromolecule polypeptide inhibitor and a group of small peptide with the binding activity of human heparanase small subunit (8kD), which belongs to the field of biomedicine. The amino acid sequence of the small peptide for inhibiting the activity of the human heparanase has a sequence table SEQIDNO.1 from an N extremity to a C extremity. On the basis of expressing and purifying the human heparanase small subunit, the human heparanase small subunit is used as a target to screen phage display random-ring 7 peptide library to obtain a unanimous amino acid sequence; the small peptide has the activity of inhibiting the invasion in vitro of highly metastatic tumor cells. In addition, a group of small peptide with the binding activity of human heparanase small subunit is further obtained. The amino acid sequence and the small peptide of the invention have the advantage that the small peptide has potential medical value in the targeting therapy of tumors.

Description

technical field [0001] The invention relates to a small molecule polypeptide inhibitor of human heparanase and its application in tumor treatment, belonging to the field of biomedicine. Background technique [0002] Tumor invasion and metastasis are the main causes of death in cancer patients. The metastasis of cancer cells needs to break through barriers such as extracellular matrix and basement membrane surrounding blood vessels. These barriers are mainly composed of proteins embedded in a fiber network of heparan sulfate carbohydrates. Heparanase (HPA) is the key enzyme involved in the degradation of extracellular matrix and basement membrane to help cancer cells metastasize. The role of HPA in tumor metastasis can be summarized as follows: (1) degrade extracellular matrix and basement membrane, destroy the barrier of cell invasion; (2) degrade endothelial basement membrane, promote angiogenesis; growth factors that promote tumor cell growth. [0003] HPA is to cleave ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K7/64C12N15/12A61K47/42A61P35/00A61K38/08A61K38/12
Inventor 刘刚程慧君陈惠鹏汤国营戴红梅
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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