Novel eukaryotic recombinant protein with tumor inhibition effect and preparation method thereof

A technology of tumor suppression and recombinant protein, which is applied in the field of medical bioengineering, can solve the problem of not being able to effectively inhibit tumor stem cells, and achieve the effect of promoting sensitivity and inhibiting tumor stem cells

Inactive Publication Date: 2016-11-09
WUXI PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the above-mentioned problems, the present invention provides a novel eukaryotic recombinant protein with tumor suppressing effect and its preparation method, so as to overcome the inability of small molecule drugs in the prior art to block TCF4 and other oncogenes except β-catenin. The combination leads to the problem that the tumor stem cells cannot be effectively inhibited, so that it can block the combination of TCF4 and the proto-oncogene in an all-round way, effectively inhibit the tumor stem cells, and promote the sensitivity of chemotherapy drugs

Method used

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  • Novel eukaryotic recombinant protein with tumor inhibition effect and preparation method thereof
  • Novel eukaryotic recombinant protein with tumor inhibition effect and preparation method thereof
  • Novel eukaryotic recombinant protein with tumor inhibition effect and preparation method thereof

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Effect test

Embodiment 1

[0049] The novel eukaryotic recombinant protein with tumor suppressing effect provided by Example 1 of the present invention, the 80 amino acid sequence of the amino terminal (N terminal) of the transcription factor TCF4 that can bind to β-catenin is named N240, at the N terminal of N240 Connect the nuclear localization peptide NLS, connect the transmembrane signal peptide TAT at the C-terminus of N240, add the His-FLAG tag between NLS and N240, and the functional region code of the new eukaryotic recombinant protein with tumor inhibitory effect is: NLS-His-FLAG -N240-TAT; the novel eukaryotic recombinant protein with tumor suppressive effect has a total length of 132 amino acids, of which 1-25 are NLS sequences, 26-39 are His-FLAG tag sequences, 42-121 are N240 sequences, and 122-132 are The TAT sequence, the His-FLAG tag sequence and the N240 sequence are connected by two amino acids of RS; the amino acid sequence of the novel eukaryotic recombinant protein with tumor suppres...

Embodiment 2

[0072]The cultured COS-7 cells were added with 500 μM new eukaryotic recombinant protein NLS-N240 with tumor suppressive effect, random scrambled polypeptide NLS-N240sc, and N240 eukaryotic recombinant protein without NLS sequence, after 6 hours with 4% paraformaldehyde Fixed for subsequent immunofluorescence staining, wherein, NLS-N240sc is a new type of eukaryotic recombinant protein with tumor suppressive effect, and the N240 sequence is replaced by the scrambled control sequence (SKIDLGDGFGLEGNEDQDEA) at the first 8-27 amino acids of the N-terminal of the TCF4 protein, other links The scheme remains the same, because the small sequence is not convenient for expression through plasmids, so the fusion protein is directly synthesized by the polypeptide synthesis method;

[0073] Double immunofluorescent staining using anti-His mouse monoclonal antibody and anti-tubulin rabbit polyclonal antibody, see Figure 4 , Figure 4 It is a schematic diagram of the cell localization sh...

Embodiment 3

[0075] Human glioma cells U251 and A172 were cultured with neural stem cell culture medium (D / F12+EGF+bFGF+N2), and NLS-N240, a novel eukaryotic recombinant protein with tumor suppressive effect, and control protein NLS-N240sc were added at the same time, at a concentration of 500μM, and a solvent control group was set at the same time. When the solvent control group formed stem cell spheres with a diameter of about 100μm after culturing for 5 days, the cell spheres suspended in the culture medium were collected, and some cells were used to extract RNA for PCR detection, and some cells were used for protein extraction. The expression level of the target gene c-Myc was detected by Western blot, wherein, NLS-N240sc is a new type of eukaryotic recombinant protein with tumor suppressive effect, and N240 was replaced by the scrambled control sequence (SKIDLGDGFGLEGNEDQDEA) of the first 8-27 amino acids at the N-terminal of the TCF4 protein Sequence, other linking schemes remain unch...

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Abstract

The invention relates to a novel eukaryotic recombinant protein with a tumor inhibition effect and a preparation method thereof and belongs to the field of medical bioengineering. The novel eukaryotic recombinant protein with a tumor inhibition effect comprises 132 amino acids, 1th-25th amino acids form an NLS sequence, 26th-39th amino acids form a His-FLAG label sequence, 42th-121th amino acids form an N240 sequence, 122th-132th amino acids form a TAT sequence and the His-FLAG label sequence is bonded with the N240 sequence through two RS amino acids. The novel eukaryotic recombinant protein solves the problem that the existing small molecular drug cannot obstruct bonding of TCF4 and other cancer genes but beta-catenin so that the existing small molecular drug cannot effectively inhibit tumor stem cells, realizes comprehensive blocking of bonding of TCF4 and a protooncogene, effectively inhibits tumor stem cells and improves chemotherapeutic drug sensitivity.

Description

technical field [0001] The invention relates to the field of medical bioengineering, in particular to a novel eukaryotic recombinant protein with tumor suppressing effect and a preparation method thereof. Background technique [0002] In recent years, the theory of tumor stem cells (TSCs) has attracted more and more attention. At present, tumor stem cells have been successfully isolated from brain tumors, lung cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer, skin cancer and other malignant tumors. The tumor stem cell hypothesis holds that tumor recurrence and metastasis after drug treatment are closely related to the survival of tumor stem cells. The proposal of cancer stem cells provides a reasonable explanation for the mechanism of tumor occurrence and recurrence, and is of great significance for the development of more effective anti-tumor drugs and the research of stem cells and cell biology. With the further research on TSCs, it is found that T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N15/85A61P35/00
CPCC07K14/47C07K2319/43C07K2319/10C07K2319/21C07K2319/01
Inventor 邹健殷莹平锋锋蒋孟军陈越新
Owner WUXI PEOPLES HOSPITAL
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