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An antitumor protein peptide that inhibits Foxm1

An anti-tumor and protein peptide technology, applied in the fields of genetic engineering and oncology, can solve the problem of inability to obtain induced pluripotent stem cells

Active Publication Date: 2021-06-01
XINSHENG KANGYUAN BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, FOXM1 is involved in the maintenance of cell stemness (Nucleic Acids Res 2010. 38: 8027-8038). Inhibition of FOXM1 leads to the inability to obtain induced pluripotent stem cells, which is an essential factor in the reprogramming process of induced pluripotent stem cells (PLoS One 2014 .9:e92304)

Method used

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  • An antitumor protein peptide that inhibits Foxm1
  • An antitumor protein peptide that inhibits Foxm1
  • An antitumor protein peptide that inhibits Foxm1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, Construction of prokaryotic expression vector pHis-M1-138-R9.

[0036] 1. Amplification of the His-M1-138-R9 gene fragment. Design primers to amplify the gene fragment by PCR. The primer sequence is: Primer 1 (upstream primer): GCG CCC ATG GTG CAT CAC CAT CAC CAT CAC ATG AAA ACT AGC CCC CGTCG, primer 2 (downstream primer): GCG GGA TCC CTA CCT TCT CCT TCT CCT TCT CCT TCT CCT CAGGGT CAC TTC TGT C. Using pcDNA3.1-FOXM1 as a template, under the guidance of primers 1 and 2, the reaction system for PCR amplification of M1-138 was as follows: clone plasmid pcDNA3.1-FOXM1 (80ng / μL) 1 μL, 10X PCR Buffer (ThermoScientific) 5 μL, dNTPs (2mM each) 5μL, MgSO 4 Solution (25mM) 4μL, KOD-Plus-Neo (1.0U / μL) 1μL, primer 1 (100μM) 1μL, primer 2 (100μM) 1μL, DMSO 2μL, add deionized water to supplement the reaction system to 50μL. PCR reaction conditions: first 94°C for 5min; then 95°C for 30sec, 57°C for 30sec, 68°C for 50sec, a total of 30 cycles; then 68°C for 10min, 4°C fo...

Embodiment 2

[0039] Example 2, large-scale purified M1-138 recombinant protein.

[0040] 1. Induced expression of M1-138 recombinant protein. The prokaryotic expression vector pHis-M1-138-R9 was transformed into Escherichia coli Rostta DE3 competent cells, cultured at 37°C overnight (12-16hr), randomly selected single clones, and inoculated into 5mL LB medium (containing 25μg / mL ampicillin and 25μg / mL chloramphenicol), shake culture at 37℃ for 6-8hr. Add the bacteria solution to 100 mL of LB culture solution (containing 25 μg / mL ampicillin and 25 μg / mL chloramphenicol) and shake at 37°C overnight (12-16hr), take the bacteria solution to detect the OD 600 value, adjust OD 600 When the value reached 0.8-1, IPTG inducer (final concentration 8 μM) was added, and shaking culture was induced at 20°C for 20 hr. Centrifuge at 4000rpm for 20min to collect the bacteria, and use 15mL Binding Buffer (20mM Na 3 PO 4 , 500mM NaCl, 20mMimidazole, pH 7.4) to resuspend the bacteria, and sonicate for 4...

Embodiment 3

[0042] Example 3, M1-138 recombinant protein inhibits lung cancer cell line A549.

[0043] In order to verify the inhibitory effect of M1-138 on the proliferation of lung cancer cells, lung cancer A549 cells were selected, and the commercial Cell Counting Kit (CCK) kit was used to investigate the effect of M1-138 on the proliferation of tumor cells. Seed the cell suspension (100 μL, 4000 cells / well) in a 96-well plate. Place the culture plate in the incubator (37°C, 5% CO 2) after pre-incubation for 12 hr, the cells were treated with different concentrations of M1-138 (0.5, 1, 2, 4, 8, 16, 20, 24, 28, 32, 36 μM) and cultured for another 12 hr. Add 10 μL of CCK WST-8 reagent to each well, incubate in the incubator for 1-4hr, measure the absorbance at 450nm with a microplate reader, and calculate the cell viability of the cells to be tested, cell viability*(%)=[A( Added drug)-A (blank)] / [A (no drug added)-A (blank)] ×100, where, A (medicated): absorbance of cells, CCK solution...

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Abstract

The invention discloses a protein peptide segment derived from the FOXM1 protein and comprising the sequence of 1-138 amino acid residues at its nitrogen terminal, and provides a candidate for inhibiting the function of FOXM1 and developing protein and polypeptide anti-tumor drugs. The protein peptide has one of the following amino acid residue sequences: 1) SEQ ID NO: 1 in the sequence listing; 2) replacing, deleting, inserting and / or adding one, Two or several amino acid residues, realize the protein peptide that inhibits or reduces the activity and function of FOXM1. According to the amino acid sequence of SEQ ID NO: 1 in the sequence list, the recombinant protein with membrane penetrating ability prepared by the present invention exhibits inhibitory effect on various tumor cells.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and oncology, and relates to the expression of an anti-tumor protein peptide that inhibits FOXM1 and its application. Background technique [0002] From the gene expression analysis of various human tumor samples, it was found that the expression of transcription factor FOXM1 was increased in tumor cells, and the detection of its expression level has been used in the diagnosis and prognosis of various tumors (US7056674, 7081340, 7308364, 7526387, 7531300, CN201510355817 .5). From the perspective of gene function, FOXM1 was first identified as a protein regulating cell cycle and cell proliferation (Mol Cell Biol 1997. 17: 1626-1641). In the process of cell proliferation, FOXM1 is involved in regulating the transcription of multiple genes related to the cell cycle, thereby controlling the process of cell DNA replication and mitosis (Mol CellBiol 1999. 19: 8570-8580, Proc Natl Acad Sci US A 2002....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N15/63C12N15/62A61K38/17A61P35/00
CPCA61K38/00C07K14/4748C07K2319/10C12N15/63C12N15/70
Inventor 谭拥军张振旺余景卫
Owner XINSHENG KANGYUAN BIOPHARML
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