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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 is difficult, which severely restricts their application in the preparation of anticancer drugs.

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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