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Cancer inhibition polypeptide 127aa and expression vector and application thereof

A 127aa, eukaryotic expression vector technology, applied in the field of tumor suppressor polypeptide 127aa and its expression vector, to achieve the effects of small molecular weight, less drug resistance, and easy penetration of tumor cells

Inactive Publication Date: 2016-06-22
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In cells with PLDLS mutations in E1A (PLDLS mutant E1A cannot bind to CtBP), no E1A response to p16 was found Ink4a Therefore, it is speculated that E1A exerts this function by binding to the C-terminal PLDLS-binding region of CtBP, and whether E1A inhibits p16 through the C-terminal PLDLS-binding region of CtBP2 in ovarian cancer Ink4a The expression of

Method used

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  • Cancer inhibition polypeptide 127aa and expression vector and application thereof
  • Cancer inhibition polypeptide 127aa and expression vector and application thereof
  • Cancer inhibition polypeptide 127aa and expression vector and application thereof

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Experimental program
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Embodiment 1

[0023] The 127aa polypeptide recombinant expression plasmid of the present invention connects the coding DNA sequence of the 127aa polypeptide into the pCMV-(DYKDDDDK)-N eukaryotic expression vector, and the vector map is shown in figure 1 , the amino acid sequence of the polypeptide is shown in SEQ ID NO.1 and the corresponding coding DNA sequence is shown in SEQ ID NO.2. The main process is as follows:

[0024] 1) According to the cDNA sequence of the 127aa polypeptide, use PrimerPrimer5.0 software to design primers, and add restriction endonuclease BglII and KpnI recognition sequences and linkers before the upstream primer and downstream primer respectively, and the upstream primer 5'-GGAAGATCTCTGCCCTTGTGGATAAGCACAAAGT-3' , the downstream primer 5'-CGGGGTACCCTACGGGATGTTGCACACGGC-3', using the full length of Flag-CtBP2 as a template, the 127aa polypeptide DNA sequence was amplified by PCR, and the 50μL reaction system consisted of: 5.0μL 10×Taq polymerase buffer, 4.0μL dNTPs...

Embodiment 2

[0029] 1) CCK8 experiment proves the anti-tumor effect of recombinant peptide at the cellular level

[0030] Taking ovarian cancer as an example, ovarian cancer cells HO-8910 were divided into 5×10 3 The density of cells / well was seeded in 96-well culture plate, and the 127aa polypeptide eukaryotic expression vector and other control groups were transiently transfected into HO-8910 cells using Lipofectamin2000kit. After 0, 6, 12, 24 and 48 hours, the medium was discarded, and each Add 10 μL of 5 mg / mL CCK8 (CCK8 is prepared with sterile 1640 basal culture, ready-to-use), and react in the dark for 2 hours, then measure the absorbance at 450 nm. The absorption value reflects the cell activity. According to the experimental data, time is used as the abscissa and the absorption value is plotted as the ordinate. Such as Figure 5 As shown, the results of CCK8 showed that compared with the empty control group, the 127aa polypeptide eukaryotic expression group significantly inhibited...

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Abstract

The invention discloses a cancer inhibition polypeptide 127aa and an expression vector and application thereof. A segment of a specific amino acid sequence on CtBP2 serves as the polypeptide, a DNA sequence corresponding to the polypeptide is transferred to a pCMV-(DYKDDDDK)-N eukaryotic expression vector through genetic recombination of a bioengineering technology, and then a competitive inhibitor for E1A-combined CtBP2 is obtained; in-vitro tests show that the fused polypeptide can enable the level of p16<Ink4a> protein in tumor cells to be lowered, and then proliferation of the tumor cells is inhibited. Cytology shows that the recombinant polypeptide has the important anti-cancer function of inhibiting the activity of the tumor cells. The cancer inhibition polypeptide supplies experimental bases to new target development in tumor treatment and has the very important development and application prospect on application of the cancer inhibition polypeptide in clinical tumor treatment.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to tumor suppressor polypeptide 127aa and its expression vector and application. Background technique [0002] The mortality rate of ovarian cancer ranks first among female malignant tumors, and the 5-year survival rate of the most common advanced epithelial ovarian cancer stays at about 20-30% for a long time. Early treatment, poor prognosis. Therefore, it is of great significance to strengthen the research on the treatment of tumors and explore new therapeutic approaches. Peptide drugs have attracted widespread attention due to their advantages such as small molecules, easy synthesis, easy structure-activity relationship research, easy modification, strong pertinence, and obvious curative effects. In recent years, with the continuous improvement of biotechnology and peptide synthesis technology, peptide drugs have high purity, low toxicity and side effects, almost no immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/79A61K38/16A61P35/00
CPCC07K14/00A61K38/00
Inventor 沈爱国成佳琳茅国新刘蓉
Owner NANTONG UNIVERSITY
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