A kind of ku70 protein mutant with the function of inhibiting tumor cell proliferation, gene and application

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

Active Publication Date: 2021-10-08
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 of these drugs is generally only sensitive to some patients, and the side effects are serious and dose-dependent

Method used

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  • A kind of ku70 protein mutant with the function of inhibiting tumor cell proliferation, gene and application
  • A kind of ku70 protein mutant with the function of inhibiting tumor cell proliferation, gene and application
  • A kind of ku70 protein mutant with the function of inhibiting tumor cell proliferation, gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: MU4 protein expression vector pbicep-n-flag-mu4 cloning construction

[0019] The lack of anti-transcripts are designed according to the gene full sequence (NM_001288977) of the Ku70 protein, and a variant Ku70 protein for constructing a deletion C-terminal 37 amino acids, and the sequence of mutant primers is shown in Table 1.

[0020] Table 1 mutation primer

[0021]

[0022] The PbiceP-N-Flag-Ku70 plasmid preserved in the laboratory is a full length of the wild type in the multi-clon site of commercial plasmid pBiceP-CMV-3 (purchased from Sigma). U70 Gene sequence (granular map figure 1 ).

[0023] The PbiceP-N-Flag-Ku70 plasmid was a template, and the PbiceP-N-Flag-MU4 plasmid DNA fragment was obtained by mutation PCR method, and the PCR reaction system was shown in Table 2.

[0024] Table 2 PCR amplification system

[0025] Template DNA 1μL Upstream primer (10μm) 1μL Downstream primer (10μm) 1μL DREAMTAQ PCR MASTER MIX (2 ×) 25μL ...

Embodiment 2

[0032] Example 2: Mu4 protein on SHKU70 / HELA cell proliferation inhibitory activity

[0033] (1) Screening Shku70 / HeLA cells

[0034] The Ku70 shRNA plasmid was transferred to HeLa cells using liposome transfection. Ku70ShRNA plasmids from Jikai Company, plasmid map (GV248 plasmid) figure 2 As shown, the coding sequence of the Ku70 Shrna is inserted into the polyclon position (as shown in SEQ ID NO 5). The plasmid transfected into the cells, which expressed ShRNA targeted to Ku70 mRNA, thereby inhibiting the expression of Ku70, reducing the Ku70 protein level within the cell.

[0035] After 24 hours of transfection, 1 μg / ml of puromycin was added to the medium to screen the Hele cells which were integrated into the plasmid DNA in the genome. After screening for 1 month, a limited dilution method and monoclonal picking method were used to obtain SHKU70 / HELA cells expressed in endogenous Ku70.

[0036] (2) Liposome transfection method to shku70 / heLA cells into pBiceP-N-Fla...

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Abstract

The invention discloses a Ku70 protein mutant, gene and application with the function of inhibiting tumor cell proliferation. The amino acid sequence of the Ku70 protein mutant is shown in SEQ ID NO.1. The present invention utilizes the effect of Ku70 protein on tumor cell proliferation, and through artificial transformation, constructs mutant Ku70 protein Mu4 that can effectively inhibit tumor proliferation. In vitro anti-tumor activity testing and animal experiments have shown that the modified protein exhibits good anti-tumor proliferation properties, which can be used to study the mechanism of Ku70 involved in tumor cell proliferation, and provide a theoretical basis and model for the development of anti-tumor drugs. It has a certain application prospect in the field of medicine.

Description

Technical field [0001] The present invention relates to the field of biomedical technology, and more particularly to a Ku70 protein mutant, gene and application having a proliferation function of tumor cell proliferation. Background technique [0002] Interferon, interleukin is a common protein drug in clinical tumor treatment, but these drugs are generally only sensitive to some patients, and the poison side effects will also have dose dependence. With the development of technology, more and more exogenous proteins are used in clinical tumor treatment. [0003] Ku proteins are a wide range of nuclear proteins, and is a heterologous dimer consisting of two subunits of Ku70 and Ku80. In recent years, research has shown that KU70 is related to cell cycle regulation, cellular differentiation, nuclear morphology, and double strand break DNA repair. The study found that the KU70 can catalyze the fracture DNA repair is because it is a non-sequence-dependent manner, and the DNA-dependen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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