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
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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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