Novel application of fusion protein TAT-DCF1

A technology of TAT-DCF1 and fusion protein, applied in peptide/protein components, medical preparations containing active ingredients, drug combinations, etc., can solve the problems of slow development of new drugs

Inactive Publication Date: 2017-07-04
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, due to the influence of the blood-brain barrier and blood-brain tumor barrier, the development of new drugs is relatively slow

Method used

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  • Novel application of fusion protein TAT-DCF1
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  • Novel application of fusion protein TAT-DCF1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of recombinant plasmids

[0029] The experimental steps of subcloning culture and transformation of Escherichia coli refer to the standard experimental steps in the molecular cloning textbook. Using the pEGFP-N2-DCF1 plasmid as a template, DCF1 target genes of different lengths (full-length DCF1, intracellular DCF1(Cy), extracellular DCF1(Ex), autophagy fragment DCF1( 19)) and the EGFP gene, the target fragment of TAT is obtained by primer annealing. All PCR products and pET30a vector were digested with restriction enzymes Nde I and xho I performed double digestion and ligation, so that the C-terminals of the five recombinant plasmids constructed had 6xHis tags. The recombinant plasmids were named pET30a-TAT-EGFP, pET30a-TAT-DCF1, pET30a-TAT-DCF1(Cy), pET30a-TAT-DCF1(Ex), pET30a-TAT-DCF1(19)-EGFP, all plasmids were Contains (His)6 tag. The positive clones of each recombinant were selected for preliminary PCR identification and double enz...

Embodiment 2

[0030] Example 2: Induced expression and soluble identification of fusion protein

[0031] 1. Transform the recombinant plasmid into RosettaTM2 (DE3) competent cells, and select transformants on LB solid culture plates containing kanamycin resistance. Pick a single colony and inoculate it in 3ml of LB liquid medium containing 50ug / ml kanamycin, and culture overnight at 37°C and 210r / min in a shaker. Draw 2.5ml of the cultured bacterial solution and inoculate it into 250ml of LB liquid medium, culture with shaking at 37°C until the OD600 in the logarithmic growth phase is about 0.6, add IPTG inducers to the solution respectively, so that the final concentrations are 0.2 and 0.4 respectively , 0.6, 0.8, 1mM, and induced for 4 hours at different temperatures (18°C, 25°C, 28°C, 37°C) to optimize the induction expression conditions. In order to analyze the induction efficiency, it is necessary to set up an uninduced control group at the same time. After the induction, centrifuge ...

Embodiment 3

[0034] Example 3: Purification of Fusion Protein

[0035] Use Ni 2+ The fusion protein was purified by affinity chromatography. Briefly, the column was equilibrated with 5 column volumes of PBS, and the protein solution was combined with the His filler at 4°C for 2h. The column was washed sequentially with PBS washing buffer containing different concentrations of imidazole, and then the target protein was sequentially eluted with PBS elution buffer containing different concentrations of imidazole, and the purified protein was quantified with the BCA protein quantification kit. The purity of the purified protein was checked by PAGE electrophoresis stained with Coomassie blue ( image 3 ).

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Abstract

The invention relates to a novel application of fusion protein TAT-DCF1. By judging dcf1-induced U251 cell line apoptosis by virtue of such methods as immuno-electron microscope, atomic force microscope, CCK8 proliferation assay, JC-1 dyeing, nude mouse tumor heterotopic transplantation and the like, results show that dcf1, which is positioned in mitochondria, can cause pathological change of the mitochondria structure and result in drop in membrane potential, so that expression amounts of related genes are changed, and the apoptosis is finally induced. Nude mouse experiments show that them dcf1 can obviously diminish tumor volume of glioma, so that tumor growth is inhibited.

Description

technical field [0001] The invention relates to a new application of fusion protein TAT-DCF1. Background technique [0002] Malignant gliomas are the most aggressive, deadly, and prevalent brain malignancies in adults. Even with advances in surgical techniques, radiotherapy, and chemotherapy, the mid-term survival of glioma patients is still only 14 months. Although gene therapy is a promising therapeutic approach, the development of safe and effective gene delivery systems remains problematic. In addition, on the way of these treatments, even if the treatment effect is not good, the patients will still suffer more pain, so it is necessary to develop drugs with low side effects. [0003] Dendritic cytokine factor (DCF1), also known as transmembrane protein 59 (TMEM59), is a membrane protein that was previously found to be involved in the differentiation of neural stem cells. Recent studies have shown that the complete dcf1 Gene overexpression of DCF1 protein in glioma ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K47/64A61P35/00A61P35/04
CPCA61K38/1709
Inventor 文铁桥王倩王娇周方方李洁
Owner SHANGHAI UNIV
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