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Pegylated F polypeptide as well as preparation method and application thereof

A PEGylation and polypeptide technology, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of increasing the biological inhibitory activity of polypeptides, and achieve the effect of strong inhibitory activity of HeLa cells.

Pending Publication Date: 2022-05-06
阳光生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report that PEG modification can increase the bioinhibitory activity of polypeptides

Method used

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  • Pegylated F polypeptide as well as preparation method and application thereof
  • Pegylated F polypeptide as well as preparation method and application thereof
  • Pegylated F polypeptide as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the synthesis method of pegylated F polypeptide

[0054] Because there are multiple amide groups on the F polypeptide sequence, NHS-PEG with amide group reactivity was selected. 4 -Azide molecules carry out the PEGylation reaction of F polypeptide, the specific steps are as follows:

[0055] 1) A certain amount (1mg) of NHS-PEG 4 - Azide was dissolved in dimethyl sulfoxide (20 μL) to prepare a 250 mM stock solution;

[0056] 2) Dissolving 5 mg of F1 or F3 polypeptide in PBS to prepare a 5 mg / mL solution;

[0057] 3) Fully mix the polypeptide solution prepared in 2) with the PEG solution in 1) at a molar ratio of 1:20, and then react the mixture at room temperature for 1 hour;

[0058] 4) Then add 1M Tris-HCl (trishydroxymethylaminomethane hydrochloride) buffer solution to quench until the final concentration of Tris-HCl is 50-100mM, and incubate the reaction at room temperature for 5 minutes;

[0059] 5) adding 10% trifluoroacetic acid to the product m...

Embodiment 2

[0061] Embodiment 2: Purification and separation by high performance liquid chromatography

[0062] The PEGylated F polypeptide was subjected to continuous fractionation on an ultra-high performance liquid chromatography (Agilent 1290 Infinity II). The column used is AdvanceBio Peptide Mapping 2.1x150mm, 2.7μm. Two solvents were used: 0.1% trifluoroacetic acid in ultrapure water (A) and 0.1% trifluoroacetic acid in 100% acetonitrile (B), with a flow rate of 0.4 mL / min and a chromatographic signal wavelength of 210 nm. The specific linear gradients are shown in Tables 1 and 2 below. The collected fractions (see figure 1 , 2 ) was lyophilized with a SpeedVac vacuum concentrator (Thermofisher Scientific) and stored at -20°C until use.

[0063] Table 1 Method for separation of PEGylated F1 by high performance liquid chromatography

[0064] Time(min) A(％) B(％) 0.00 97.00 3.00 2.00 97.00 3.00 4.00 34.40 65.60 7.75 32.30 67.70 14.00 32....

Embodiment 3

[0069] Embodiment 3: product structure characterization

[0070] The lyophilized active fraction was redissolved in 50 μL of a mixed solution of 0.1% formic acid + 2% acetonitrile + 98% ultrapure water to make a 0.1 μg / μL peptide solution, and was analyzed by quadrupole time-of-flight mass spectrometry (SCIEX, Concord , Canada) for analysis. Samples were analyzed by LC-MS / MS on an ExionLC liquid chromatography system coupled to a QTOF X500R mass spectrometer (SCIEX, Concord, Canada) equipped with an electrospray ionization source. 15 μL of the sample was injected onto a 100 mm x 1.7 μm Aeris PEPTIDE XB-C18 100 uHPLC column (Phenomenex, Sydney, Australia) equipped with a SecurityGuard column for mass spectrometry. A linear gradient of 5-35% solvent B over 10 min at a flow rate of 400 μL / min, followed by a steeper gradient from 35% to 80% solvent B in 2 min and from 80% to 95% solvent B in 1 min with Eluted with peptide. Solvent B was held at 95% for 1 minute to clean the col...

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Abstract

The invention discloses a pegylated F polypeptide as well as a preparation method and application thereof. According to the technical scheme, pegylation modification is carried out on F polypeptide, that is, azide tetrapolyethylene glycol is modified on amino of the F polypeptide, so that the pegylation F polypeptide has remarkable human cervical cancer HeLa cell inhibition activity. Wherein the inhibition activity of the azido tetrapolyethylene glycol F1 polypeptide on HeLa cells is improved by about 3.7 times compared with that of the F1 polypeptide before modification; the inhibitory activity of the azido tetrapolyethylene glycol F3 polypeptide on HeLa cells is improved by about 6 times compared with that of the unmodified F3 polypeptide.

Description

technical field [0001] The field of biological polypeptides of the present invention relates to a PEGylated F polypeptide, in particular to a PEGylated F polypeptide and its preparation method and application. The PEGylated F polypeptide has significant inhibitory activity on human cervical cancer HeLa cells . Background technique [0002] Studies have found that F1 and F3 polypeptides have significant inhibitory effects on the proliferation of various cancer cell lines in vitro, including TC-1, HeLa and B16 (see patent PCT / CN2017 / 000744, 201811161509.9). After the successful in vitro test, the F polypeptide was tested in vivo to test whether it can induce host immune response in tumor-bearing mice, thereby serving as an adjuvant for cancer vaccines. Mice implanted with TC-1 cells intraperitoneally were tested using tumor local injection of F polypeptide alone or in combination with E6 / 7 antigen. The combination of F polypeptide with E6 / 7 antigen significantly increased th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K1/107A61K47/60A61K38/16A61P35/00
CPCC07K14/001C07K1/1077A61K47/60A61P35/00A61K38/00
Inventor 倪国颖王天放
Owner 阳光生物科技有限公司
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