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Preparation method of polypeptide for inhibiting gelatin enzyme A activity and its application

A technology of gelatinase and inhibition, which is applied in the field of polypeptides that inhibit the activity of gelatinase A and its preparation and application, and can solve the problems that the mechanism of action is not completely clear

Inactive Publication Date: 2006-11-29
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other inhibitors of MMP-2, such as: Kazal motif reverse-induced cysteine-rich protein (RECK), tissue factor pathway inhibitor-2 (TFPI-2), etc. can be used as inhibitors of MMP-2, and have been in vitro Expression, but because its mechanism of action is still not completely clear, it produces other side effects while inhibiting MMP-2 activity (Andrew H.Baker, Dylan R.Edwards and Gillian Murphy.Metalloproteinasein inhibitors: biological actions and therapeutic opportunities.Journal of cell science .2002, 115(19): 3719.)

Method used

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  • Preparation method of polypeptide for inhibiting gelatin enzyme A activity and its application
  • Preparation method of polypeptide for inhibiting gelatin enzyme A activity and its application
  • Preparation method of polypeptide for inhibiting gelatin enzyme A activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Screening method for polypeptide M204C4

[0031] experiment material

[0032] (1) The phage 12 peptide library and the recipient strain ER2738 were purchased from NEW ENGLAND Biolabs.

[0033] (2) Recombinant human MMP-2 was purchased from American R&D Company.

[0034] (3) MMP-2 Fluorescent Analysis Drug Screening Kit, purchased from Biomol, USA.

[0035] experiment method

[0036] (1) Amplification of phage Take 10 μl from the phage eluate from the first and second rounds of screening, add 20 mL and cultivate to absorbance A 600 =0.5 in ER2738 bacteria, place at room temperature for 15min, shake culture at 37°C 250r / min for 4.5h, centrifuge at 10000rpm at 4°C for 10min, separate the supernatant, then centrifuge the supernatant for 5min, add 1 / 6 volume of PEG8000NaCl ( 20% W / V PEG8000, 2.5mol / L NaCl) solution, overnight at 4°C; the next day, centrifuge the precipitated phage solution at 10,000rpm at 4°C for 15min, discard the supernatant, add 1mL TBS to ...

Embodiment 2

[0039] Example 2. The inhibitory effect and IC of polypeptide M204C4 on MMP-2 50 calculation

[0040] experiment method

[0041] According to the MMP-2 fluorescence detection kit method, neutralizing peptide M204C4 (synthesized by Xi’an Huachen Co., Ltd.) and MMP-2 were added to the 96-well detection plate, so that the final concentrations of M204C4 were 0, 10, 20, 50, 100, 200nmol / L, incubate at 37°C for 45min, add the substrate, immediately measure the fluorescence value at the wavelength of 328 / 393nm, and calculate the percentage of enzyme activity inhibition. Application of SPSS10.0 software to calculate IC 50 .

[0042] Experimental results

[0043] As shown in Table 1, adding different concentrations of M204C4 to the reaction system has different degrees of inhibitory effects on MMP-2, and with the increasing concentration of M204C4, the inhibitory effect on MMP-2 activity is gradually enhanced, showing a dose-dependent sex. IC of neutralizing peptide M204C4 inhibi...

Embodiment 3

[0045] Example 3. In vitro inhibition of pancreatic cancer cell line invasion by polypeptide M204C4

[0046] experiment material

[0047] (1) M204C4 polypeptide: synthesized by Xi'an Huachen Co., Ltd., dissolved in PBS buffer at pH 7.2.

[0048] (2) Human pancreatic cancer cell lines PANC-1 and CFPAC-1 were purchased from Shanghai Institute of Cells, Chinese Academy of Sciences. Cells were cultured in DMEM medium containing 10% FBS, 100ug / ml penicillin and 100ug / ml streptomycin in 5% CO 2 , Culture at 37°C. When the cell density reaches 80%-90%, it is digested and passaged with 0.25% trypsin.

[0049] experiment method

[0050] Inoculate PANC-1 and CFPAC-1 in culture flasks overnight. PGE with a final concentration of 10 μmol / L 2 The cells were treated for 8 hours, trypsinized, neutralized with serum-free DMEM containing 0.5% BSA, centrifuged, and the cells were collected. Cells were resuspended in serum-free DMEM, and the cell density was adjusted (PANC-1: 1.0×10 5 / m...

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Abstract

A polypeptide M204C4 for suppressing the activity of gelatinase A (MMP-2) is composed of the amino acid sequence shown by HNWTRWLLHPDRGGGS is disclosed. It can also suppress the invasion and transfer of external pancreas cancer cells PANC01 and CFPAC-1. Its preparing process includes such steps as affinity screening and reproduction of bacteriophage several times to obtain the bacteriophage with specific binding to MMP-2, coating, cloning and monoclonal screening with fluorescent screening reagent kit.

Description

1. Technical field [0001] The present invention relates to the preparation and application of biochemical polypeptides, specifically to the process of using phage display technology to screen and obtain the coding cDNA sequence of gelatinase A (MMP-2) inhibitory polypeptide M204C4, which has the ability to MMP-2 enzyme activity The inhibitory effect and the effect of inhibiting tumor cell invasion and metastasis in vitro; therefore, the polypeptide may be applied to the prevention and treatment of human tumor cell invasion. 2. Background technology [0002] Tumor has become a major killer threatening human life after cardiovascular disease. The tumor process includes: changes in certain genes in normal cells, formation of a malignant phenotype, invasion of adjacent normal tissues, and distant and systemic tissue spread (metastasis). According to clinical statistics, more than 80% of cancer patients died of tumor invasion and metastasis, so research on tumor cell invasion an...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/04C12N7/00C12Q1/68
Inventor 韩晓郑马庆孙玉洁
Owner NANJING MEDICAL UNIV
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