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TAT (Trans-activating factor) kringle domain-modified nenurogenin2 fusion protein, as well as preparation method thereof and application thereof

A fusion protein and structural domain technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, peptide/protein components, etc., to achieve the effect of low preparation cost and high activity

Inactive Publication Date: 2012-09-12
XIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no molecular biological methods to obtain a large amount of neural transcription factor neurognin2 and use it in the treatment of central nervous system injury and degenerative diseases.

Method used

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  • TAT (Trans-activating factor) kringle domain-modified nenurogenin2 fusion protein, as well as preparation method thereof and application thereof
  • TAT (Trans-activating factor) kringle domain-modified nenurogenin2 fusion protein, as well as preparation method thereof and application thereof
  • TAT (Trans-activating factor) kringle domain-modified nenurogenin2 fusion protein, as well as preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Preparation of the TAT-neurognin2 fusion protein of the present invention

[0033] 1. Obtain the pTAT-neurognin2 recombinant expression plasmid vector;

[0034] (1) Select the enzyme containing restriction endonuclease Nco I and xho I site, and the pTAT-HA expression vector containing the protein transduction domain PTD;

[0035] (2) Obtain the neurognin2 gene sequence from Genebank, and then optimize the gene sequence through prokaryotic expression;

[0036] (3) Synthesize the neurognin2 target gene fragment after prokaryotic expression optimization, and the two ends of the sequence contain Nco I and xho I restriction site, and loaded into the pUC57 vector to form pUC57-neurognin2 plasmid vector;

[0037] (4) use Nco I and xho I Two enzymes cut the pTAT-HA expression vector and the pUC57-neurognin2 target fragment plasmid vector into the same cohesive ends with double enzymes. Enzyme digestion reaction conditions: 37 ℃ 1h-16h, 65 ℃ ...

Embodiment 2

[0066] Example 2: Identification of the transduction function of the TAT-neurognin2 fusion protein of the present invention

[0067] 1. Identification of transmembrane transduction of TAT-neurognin2 fusion protein (such as image 3 shown);

[0068] 2. Identification of TAT-neurognin2 fusion protein crossing the blood-brain barrier (such as Figure 4 shown);

[0069] In the present invention, the TAT-neurognin2 protein is injected intraperitoneally into the mouse, and after 6 hours, the brain of the anesthetized mouse is taken, and the frozen section is used to analyze the transduction effect of the TAT-neurognin2 fusion protein through the blood-brain barrier by immunofluorescence histochemistry. The results show that TAT- The neurognin2 fusion protein can pass through the blood-brain barrier and enter the brain parenchyma, while the brain tissues of mice injected with the No-TAT-neurognin2l fusion protein showed negative reactions, indicating that TAT can mediate protein ...

Embodiment 3

[0070] Example 3: The effect of the TAT-neurognin2 fusion protein of the present invention on inducing AS cells to differentiate into neurons in vitro

[0071] 1. Culture of mouse astrocytes

[0072] Take out the cerebral cortex of Kunming mice 7 days after birth, add it to the culture dish of D-Hanks solution, cut the tissue repeatedly with iris scissors until it becomes minced, suck the tissue into a 15ml centrifuge tube, add 0.125% trypsin (Sigma) 37 ℃ water bath for 20 minutes, add serum to the centrifuge tube to stop the digestion, blow gently with a round-tip dropper, let stand for 10 minutes, carefully draw the supernatant, add the supernatant to the pre-packed with poly-lysine (25μg / ml) Add DMEM / f12 containing 10% serum (Gibico) to the culture flask, change half of the medium on the third day, and then change the medium every three days, 12-15 days, when the cells are almost full, put the cells in the shaker Purify in the bed at 300 rpm for 15-19 hours, add new cu...

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Abstract

The invention provides a TAT (trans-activating factor) kringle domain-modified nerve transcription factor nenurogenin2 fusion protein, as well as a preparation method and application thereof. Specifically, the fusion protein TAT-nenurogenin2 formed by the nerve transcription factor nenurogenin2 is modified by the protein transduction kringle domain of the human immunodeficiency virus trans-form activation transduction protein TAT. The preparation of the fusion protein provided by the invention comprises the following steps of: (1) selecting a TAT protein transduction domain-containing pTAT-HA expression vector; (2) synthesizing the nerve transcription factor nenurogenin2 target gene segment optimized by prokaryotic expression; (3) connecting T4 ligase with the enzyme-digested product to obtain a pTAT-nenurogenin2 recombinant expression plasmid vector by means of cloning and screening; and (4) transforming the recombinant expression plasmid vector into colon bacillus DE3 competent cell, and inducing protein expression. According to the invention, a large number of TAT-nenurogenin2 fusion protein which is low in cost, high in activity, and capable of inducing the astrocyte to be transformed into the nerve cell with biological activity can be firstly prepared, and the TAT-nenurogenin2 is easy to penetrate through the blood brain barrier (BBB), so that the TAT kringle domain-modified nenurogenin2 fusion protein is wide in the prospect in the clinical treatment of the brain injury disease.

Description

technical field [0001] The invention relates to a fusion protein for treating central nervous system (central nervous system, CNS) damage and degenerative diseases and a preparation method thereof. Background technique [0002] Trauma, stroke, cerebral hemorrhage, spinal cord injury, and neurodegenerative diseases (such as PD, AD, etc.) have the characteristics of high mortality and high disability rate. How to reduce the degree of neurological damage and improve the function of damaged neurons, Improving the quality of life of patients is of great significance to patients, their families and society, and is a worldwide problem. At present, there is no effective treatment for neurological dysfunction caused by CNS injury and degenerative diseases. Therefore, finding new and effective drugs for the treatment of CNS injury and degenerative diseases has become one of the research hotspots. [0003] In the past, it was generally believed that the main reason why the neurologic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/12C12N15/49C12N15/63A61K38/17A61K47/48A61P25/00A61P25/28
Inventor 苟兴春弥曼闫爱丽景晓红陈海
Owner XIAN MEDICAL UNIV
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