Telomeric protein polypeptide fragment with tumor cell killing activity and application thereof

A tumor cell and protein technology, applied in the field of telomere protein polypeptide fragments, can solve problems such as unclear mechanism of action

Active Publication Date: 2013-02-27
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of how Pot1 inhibits ATR at telomeres is unclear

Method used

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  • Telomeric protein polypeptide fragment with tumor cell killing activity and application thereof
  • Telomeric protein polypeptide fragment with tumor cell killing activity and application thereof
  • Telomeric protein polypeptide fragment with tumor cell killing activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, the preparation of recombinant adenovirus vector Adv-Pot1C100 / EGFP and Adv-EGFP

[0050] Recombinant adenoviral vectors Adv-Pot1C100 / EGFP and Adv-EGFP are constructed based on the Ad-MAX packaging system and prepared by Wujiahe Company. The specific process is as follows:

[0051] 1. Construction of shuttle plasmids for packaging recombinant adenoviruses

[0052] For the convenience of research and to achieve visual markers, the gene encoding the EGFP protein was introduced into the shuttle plasmid, as follows: the gene encoding the EGFP protein (sequence 3 in the sequence listing) was used as a template, and the primers EGFP-up and EGFP-down were used to carry out PCR amplification to obtain a DNA fragment containing restriction endonuclease recognition sites EcoR I and Bgl II at both ends, after double digestion with EcoR I and Bgl II, and the pDC316 vector (this element positive Yang Company, PD-01-28) large fragments were connected, and the obtained r...

Embodiment 2

[0073] Example 2, Effects of Adv-Pot1C100 Infecting Tumor Cells on Cell Growth and Apoptosis

[0074] In this example, the recombinant adenovirus vector Adv-Pot1C100 / EGFP prepared in Example 1 was used to infect telomerase-positive tumor cell lines (HeLa, HepG2, SMMC7721, BEL-7402, A549 and A498) and telomerase-negative tumor cell lines respectively. Tumor cell lines (U2os) and normal human diploid cell lines (WI-38, 2BS and BJ), the effects of Adv-Pot1C100 / EGFP infection on tumor cell growth and apoptosis were detected. The experiment was divided into three groups, one was the blank control group not infected with adenovirus Adv-Pot1C100 / EGFP; the other was the virus control group infected with Adv-EGFP; the third was the experimental group infected with Adv-Pot1C100 / EGFP. Experiments were repeated three times. The specific operation is as follows:

[0075] 1. Growth curve measurement:

[0076] (1) Digest cells with trypsin, neutralize with DMEM medium containing 10% serum...

Embodiment 3

[0087] Example 3, Detection of anti-tumor effect of recombinant adenovirus vector Adv-Pot1C100 / EGFP

[0088] In this example, telomerase-positive tumor cell lines (HeLa, HepG2, SMMC7721 and BEL-7402) were first used to construct a mouse tumor model; then the recombinant adenovirus vector Adv-Pot1C100 / EGFP or Adv-EGFP, observe the change of tumor size, so as to detect the anti-tumor effect of Adv-Pot1C100 / EGFP. The specific operation is as follows:

[0089] 1. Tumor formation experiment in nude mice

[0090] will be 5×10 6 -1×10 7 Two tumor cells (HeLa, HepG2, SMMC7721 or BEL-7402) were subcutaneously injected into the axillae of both sides of female SPF grade athymic nude mice at the age of 4-6 weeks, and the nude mouse tumor formation model was established. Inject 6 mice per cell

[0091] The results showed that mice in SMMC7721 group formed tumors 10 days after injection of tumor cells; mice in BEL-7402 group formed tumors 7 days after injection of tumor cells; mice in...

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Abstract

The invention discloses a telomeric protein polypeptide fragment with tumor cell killing activity and an application thereof. The polypeptide provided by the invention is (a) or (b) as follows: (a) polypeptide composed of amino acid sequence as shown in sequence 1 of a sequence table; and (b) polypeptide which is derived from sequence 1 relative to the tumor cell killing activity and replacing and/or missing and/or adding one or more amino acid residues for the amino acid sequence of the sequence 1. The experiment shows that the recombinant adenovirus which expresses the polypeptide provided by the invention can inhibit the growth of the tumor cell telomerase positive, induces the apoptosis of the tumor cell and inhibits the growth of tumors in vitro and vivo. The polypeptide provided by the invention has broad spectrum and specific telomerase positive tumor cell killing activity; and meanwhile, the polypeptide does not affect the growth and survival of normal human cell; therefore, the telomeric protein polypeptide fragment and the application thereof have significant meaning on the research of novel broad spectrum specific anticancer drugs.

Description

technical field [0001] The invention relates to a telomere protein polypeptide fragment with tumor cell killing activity and application thereof. Background technique [0002] Telomere is the end mark of cell chromosome, which is composed of telomere DNA and telomere protein. Telomere proteins include Pot1, TPP1, Tin2, TRF1, TRF2 and RAP1. These six proteins interact on telomere DNA to form various forms of telomere protein complexes, which play an important role in protecting telomere DNA from being misrecognized by cells as chromosomal damaged ends and triggering DNA damage responses at chromosomal ends. The functional structure of the telomeric protein complex is regulated by the length of the telomeric DNA sequence. Due to the lack of telomerase activity in normal human cells, the length of telomere DNA gradually shortens with the increase in the number of cell divisions, which eventually leads to the loss of the functional structure of the telomere protein complex, tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/11C12N15/62C12N15/63C12N5/10C12N15/861A61K38/16A61K48/00A61P35/00
Inventor 黄君健金蕊白云秀马航航徐晓玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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