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Metastatic tumor deletion protein inhibitor polypeptide

A technology for protein inhibitors and metastatic tumors, applied in the field of preparation and application of recombinant plasmids for protein inhibitors of metastatic tumors, polypeptides, polypeptides and polynucleotides, can solve the problem that the physiological function of Mtss1 and the impact of tumor progression on its signaling pathway have not been fully elucidated and other problems, to achieve the effect of inhibiting tumor cell internalization

Inactive Publication Date: 2012-12-12
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the current data imply that abnormal expression of Mtss1 gene is associated with tumor progression, the physiological function of Mtss1 and the influence of its signaling pathway on tumor progression have not been fully elucidated

Method used

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  • Metastatic tumor deletion protein inhibitor polypeptide
  • Metastatic tumor deletion protein inhibitor polypeptide
  • Metastatic tumor deletion protein inhibitor polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of GST-tagged polypeptide-encoding DNA plasmid.

[0029] The mouse-derived recombinant DNA plasmid pMtss1-Myc-His was used as a template, and the polynucleotides of 5'-CAGTTAGGATCCACCATCAGCGAC-3' and 5'-CAGTTAGTCGACCTAGATGCTCCGGTG-3' sequences were used as primers for PCR amplification. The amplification reaction conditions are: 50μl reaction system, using PfuUltra TM II fusion HS DNA polymerase and its matching 10x buffer, 250 μM of dNTP, 20 ng / μl of template DNA, and 0.5 μM of primer. React 30 cycles on a Mastercycler-type DNA gradient thermal cycler according to the following conditions: 95°C, 20s; 67.4°C, 20s; 72°C, 15s.

[0030] The PCR product was confirmed to have a molecular weight of about 250bp by agarose gel electrophoresis and ultraviolet imaging. It was extracted with a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), and NaAc and Mix with 2 times the amount of absolute ethanol, centrifuge at high speed for 5 minutes, w...

Embodiment 2

[0033] Example 2 Expression and detection of the inhibitor polypeptide represented by sequence number 2 and its polynucleotide recombinant plasmid in eukaryotic / prokaryotic cells.

[0034] Human embryonic kidney cell 293T cells were cultured using DMEM high-glucose medium containing 10% calf serum BCS, adding penicillin at a final concentration of 100 units / ml and streptomycin at a final concentration of 100 μg / ml. Before DNA transfection, 1.5x10 6 Cells were seeded in 100mm cell culture dishes using normal culture medium without antibiotics at 37°C and 5% CO 2 conditions overnight. Then prepare the transfection mixture: 10 μg green fluorescent protein GFP-tagged inhibitor polypeptide plasmid DNA, 300 μl DMEM, 60 μl SuperFect. After the transfection mixture was incubated at room temperature for 5-10 minutes, add 3ml of normal culture medium and mix well to replace the cell culture medium and culture the cells for 2-3 hours, then replace it with normal culture medium and cult...

Embodiment 3

[0037] Example 3 Detection of the binding ability of the inhibitor polypeptide represented by sequence number 2 bound to the GST column to the Mtss1 protein.

[0038] Transfect the pMtss1-Myc-His plasmid into 293T cells, add different concentrations of GST column-bound polypeptides to the expressed cell lysate, incubate at 30 rpm at 4°C for 2 hours, take supernatant samples, and dissolve them with 8% dodecylsulfonate Sodium phosphate (SDS)-polyacrylamide gel electrophoresis for separation. Using the Western Blot method, mouse anti-Myc antibody (9E10) and goat anti-mouse IgG were used to treat respectively, and then developed with chemiluminescence substrate and exposed for imaging to obtain binding ability data, and β-actin was used as an internal reference to determine the binding ability Sample volume (such as image 3 ). The smaller the amount of Mtss1-Myc in the sample, the stronger the binding ability of the GST column to bind the polypeptide.

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Abstract

The invention provides a kind of metastatic tumor deletion protein inhibitor polypeptide, which contains an amino acid sequence indicated by SEQ ID NO.2. The invention also provides polynucleotide for coding the polypeptide and a polynucleotide recombinant vector. According to the metastatic tumor deletion protein inhibitor polypeptide, an effect for inhibiting tumor cell introjection, interfering the form variation of the cell membrane and interfering the transferring of relevant signals of the tumor can be realized by inhibiting the dimerization effect of Mtss1 proteins, and the in-vitro combination dissociation constant and the inhibitory activity of the polypeptide-protein are in a nanomolar level.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a metastatic tumor deletion protein inhibitor polypeptide, and also relates to the preparation and application of the polypeptide and polynucleotide recombinant plasmid and the biological activity of the polypeptide substance for inhibiting Metastasis suppressor 1 dimerization. Background technique [0002] The expression product protein of the Inverse BAR (I-BAR) family gene can bind to the cell membrane, and can regulate the morphological changes of the cell membrane and the state of the cell under the action of extracellular stimuli [Frost, A. et al. (2009) Cell 137( 2): 191-196.]. [0003] Metastasis suppressor 1 (hereinafter referred to as Mtss1) [Lee, Y.G. et al. (2002) Neoplasia 4 (4): 291-294.] gene is an important member of the I-BAR family, due to its abnormal expression in metastatic cells, and It has regulatory functions on membrane movement, cell polarity, in...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/63A61K38/17A61P35/00
Inventor 詹熙吉民曹萌
Owner SOUTHEAST UNIV
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