Regrouped anticancer peptide, producing method and application of the same

A technology of anti-cancer peptides and recombinant plasmids, which is applied in the direction of peptides, anti-tumor drugs, drug combinations, etc., can solve the problems of restricting the application of anti-cancer drugs, difficulty in synthesis, and low content of peptides, so as to facilitate artificial synthesis and industrialization The effects of normalization, inhibition of invasion and metastasis

Inactive Publication Date: 2008-04-02
HEBEI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, the content of the currently isolated peptides is very low, and their synthesis

Method used

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  • Regrouped anticancer peptide, producing method and application of the same
  • Regrouped anticancer peptide, producing method and application of the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Recombination, identification, purification and collection of anticancer peptides of the present invention.

[0020] Using the IMPACT-TWIN system of NEB (New England Biolabs), the preparation of recombinant cyclic small peptides was realized through chitin bead affinity chromatography and intein self-splicing function.

[0021] 1. Construction of recombinant plasmid pTWIN2-RGD

[0022] The vector map of pTWIN2 is shown in Figure 1.

[0023] The partial sequence of the pTWIN2 vector is as follows:

[0024] Polylinker Region: pTW IN2

[0025] 5’...AC TGG GAC TCC ATC GTT TCT ATT ACG GAG ACT GGA GTC GAA GAG GTT TTT

[0026] Ssp DnaB Intein Forward Primer→

[0027] ←Intein

[0028] ...Ssp DnaB Intein... Val Ala Asn Asp Ile Ile Val His Asn

[0029] GAT TTG ACT GTG CCA GGA CCA CAT AAC TTT G TC GCG A AT GAC ATC ATT GTA CAC AAC

[0030] ...

Embodiment 2

[0088] Example 2 The inhibitory effect of the anticancer peptide of the present invention (hereinafter referred to as recombinant 13 peptide) on tumor cell migration.

[0089] Cells were seeded on glass slides, and after the cells grew to 100% confluence, the slides were taken out and scratched on the slides with a sterile tip. After the scraped cells were washed with PBS, the slides were placed in a place containing 13 peptide (10 , 20μg / ml) culture solution, continue to incubate for 24 hours, take out, fix and HE staining. Observe the cell wound healing under a low power microscope. Take 3 fields of view randomly, count the number of migrating cells, and express the cell density Migration activity. Blank medium was used as negative control. See Table 1 for detailed results.

[0090] Table 1: Inhibitory effect of recombinant 13 peptide on cell migration (take 20 μg / ml as an example)

[0091] cell type

negative control group

13 peptide treatment groups

...

Embodiment 3

[0094] Example 3 Inhibitory effect of recombinant 13 peptide on tumor cell invasion

[0095]Cells were seeded on a microporous filter membrane with a diameter of 5 microns, and the filter membrane was placed between the upper and lower layers of a Boyden chamber (inoculated cells face up), and the culture solution containing 13 peptides (10, 20 μg / ml) was added to the upper chamber (Negative control as above), add medium containing 10% calf serum to the lower chamber, incubate at 37°C for 6 hours, scrape off the cells on the inoculated surface with a cotton swab after taking it out, then fix the membrane, and take 3 fields of view under the microscope , counting the number of cells that penetrated to the back of the membrane, which indicates the invasion ability of the cells. See Table 2 for detailed results.

[0096] Table 2:

[0097] cell type

negative control group

13 peptide treatment groups

Vascular smooth muscle cells

MCF-7

OVCAR

HT2...

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Abstract

The present invention discloses an anti-cancer peptide with an amino acid sequence of SEQ ID NO.1, the expression body, the preparation method and the application in preparing anti-cancer drugs. The method comprises: a. the plasmid pTWIN2 carrier is used; a 13-peptide containing RGD sequence of OPN is inserted into the viscous end after treatment of endonuclease BspQI; the homocysteine TGC is added in designing of primer; the primer and the carrier after treatment of endonuclease are connected for a night at a temperature of 16 DEG C according to a ratio of 1 to 10 (molar ratio), through the denaturalization and annealing method of the primer; the recombinant plasmid pTWIN2-RGD can be prepared; b. the recombinant plasmid is digested by colicin and transferred into the colicin or Escherichia to induce and collect purified protein. The anti-cancer peptide of the present invention has good anti-cancer activity and is easy for synthesis.

Description

technical field [0001] The present invention relates to a recombinant amino acid and its preparation method and application, in particular to a recombinant polypeptide, its preparation method and its application. Background technique [0002] With the rapid development of tumor molecular science and peptide combinatorial chemistry, the role of peptides and peptide derivatives in tumor therapy has become a hot research topic. At present, many small molecule peptides or cyclic peptides with anticancer activity have been isolated from marine organisms. For example, Pettir, Arizona Cancer Institute, etc. isolated a series of small molecular peptides (dolastatins1--15) with anticancer activity from Dolabella auriculara sea hare (see Pettit GR, Kamano Y, Herald L L. et al. The isolation and structure of a remarkable marine animal antmeoplastic constituent.J Am Chem Soc.1987,109(22):6883-6885; literature 1-3); Rinerhart et al. isolated five species with anticancer properties from ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/00
CPCY02A50/30
Inventor 韩梅温进坤
Owner HEBEI MEDICAL UNIVERSITY
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