Ku70 protein mutant with tumor cell proliferation inhibition function, and gene and application thereof

A technology for tumor cell proliferation and protein mutants, applied in the field of genes and applications, Ku70 protein mutants, which can solve the problems of patient sensitivity, serious toxic and side effects, and dose dependence.

Active Publication Date: 2020-06-26
南京华方生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Interferon and interleukin are commonly used protein drugs in clinical tumor treatment, but the efficacy

Method used

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  • Ku70 protein mutant with tumor cell proliferation inhibition function, and gene and application thereof
  • Ku70 protein mutant with tumor cell proliferation inhibition function, and gene and application thereof
  • Ku70 protein mutant with tumor cell proliferation inhibition function, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Mu4 protein expression vector pBICEP-N-Flag-Mu4 clone construction

[0019] Deletion mutation primers were designed according to the complete sequence of the Ku70 protein gene (NM_001288977) to construct a mutant Ku70 protein with a deletion of 37 amino acids at the C-terminal. The sequences of the mutation primers are shown in Table 1.

[0020] Table 1 Mutation Primers

[0021]

[0022] The pBICEP-N-Flag-Ku70 plasmid preserved in the laboratory, this plasmid is to insert wild-type full-length Ku70 between the multiple cloning site MIu I and BamH I of the commercialized plasmid pBICEP-CMV-3 (purchased from Sigma Company) Gene sequence (see plasmid map figure 1 ).

[0023] Using the pBICEP-N-Flag-Ku70 plasmid as a template, the DNA fragment of the pBICEP-N-Flag-Mu4 plasmid was obtained by mutation PCR. The PCR reaction system is shown in Table 2.

[0024] Table 2 PCR amplification system

[0025] template DNA 1μl Upstream primer (10μM) ...

Embodiment 2

[0032] Example 2: Detection of the inhibitory activity of Mu4 protein on the proliferation of ShKu70 / HeLa cells

[0033] (1) Screening of ShKu70 / HeLa cells

[0034] The Ku70 ShRNA plasmid was transferred into HeLa cells by lipofection method. The Ku70ShRNA plasmid was purchased from Jikai Company, and the plasmid map (GV248 plasmid) is as follows: figure 2 As indicated, the coding sequence of Ku70 ShRNA (as shown in SEQ ID No.5) was inserted into the multiple cloning site. The plasmid is transfected into cells, and ShRNA targeting Ku70mRNA can be expressed, thereby inhibiting the expression of Ku70 and reducing the level of Ku70 protein in cells.

[0035] After 24 hours of transfection, 1 μg / ml of puromycin was added to the culture medium to select HeLa cells that integrated the transfected plasmid DNA in the genome. After one month of screening, ShKu70 / HeLa cells stably inhibiting the expression of endogenous Ku70 were obtained by limiting dilution method and monoclonal s...

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Abstract

The invention discloses a Ku70 protein mutant with a tumor cell proliferation inhibition function, and a gene and application thereof. The amino acid sequence of the Ku70 protein mutant is shown as SEQ ID NO.1. According to the invention, influence of a Ku70 protein on tumor cell proliferation is utilized, and the variant Ku70 protein Mu4 which can effectively inhibit tumor proliferation is constructed through artificial modification. In-vitro anti-tumor activity detection and animal experiments show that the modified protein shows good anti-tumor proliferation characteristics, can be used forresearching the action mechanism of Ku70 participating in tumor cell proliferation, and provides a theoretical basis and model for developing anti-tumor drugs. Therefore, the Ku70 protein mutant hasa certain application prospect in the field of medicines.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a Ku70 protein mutant, gene and application with the function of inhibiting tumor cell proliferation. Background technique [0002] Interferon and interleukin are commonly used protein drugs in clinical tumor treatment, but the curative effect of these drugs is generally only sensitive to some patients, and the toxicity and side effects are serious and dose-dependent. With the development of technology, more and more exogenous proteins are used in clinical tumor therapy. [0003] Ku protein is a kind of widely distributed nuclear protein, which is a heterodimer composed of two subunits, Ku70 and Ku80. In recent years, studies at home and abroad have shown that Ku70 is related to cell cycle regulation, cell differentiation, nuclear morphology and double-strand break DNA repair. Studies have found that the reason why Ku70 can catalyze the repair of broken DNA is that its conn...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12A61K48/00A61K38/17A61P35/00
CPCA61K38/00A61K48/005A61P35/00C07K14/47
Inventor 贾静王屹喆汤湛贾振宇郑晓亮王孝举
Owner 南京华方生物工程有限公司
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