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Recombinant TAT-MANF (trans-activator of transcription-mesencephalic astrocyte derived neurotrophic factor) fusion protein and application thereof

A fusion protein and sequence technology, applied in recombinant DNA technology, drug combination, peptide/protein components, etc., to achieve the effect of good nerve cell protection, simple construction method and good druggability

Inactive Publication Date: 2013-08-21
SUZHOU RES INST OF TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no molecular biological method to obtain the neurotrophic factor MANF that can pass through the blood-brain barrier and use it for the treatment of central nervous system injuries and degenerative diseases such as Parkinson's disease.

Method used

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  • Recombinant TAT-MANF (trans-activator of transcription-mesencephalic astrocyte derived neurotrophic factor) fusion protein and application thereof
  • Recombinant TAT-MANF (trans-activator of transcription-mesencephalic astrocyte derived neurotrophic factor) fusion protein and application thereof
  • Recombinant TAT-MANF (trans-activator of transcription-mesencephalic astrocyte derived neurotrophic factor) fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction of recombinant TAT-MANF fusion protein expression vector

[0039] (1) Cloning of human midbrain astrocyte-derived neurotrophic factor MANF:

[0040] The Blunt-MANF vector containing the human MANF gene was identified by Invitrogen sequencing, and the sequencing result was consistent with the human MANF gene registered on GenBank, indicating that this plasmid can be used to amplify the MANF gene. Design and synthesize PCR primers P1 and P2 (synthesized by Shanghai Invitrogen Company) according to the human MANF gene sequence registered on GenBank, and add TAT sequence, His tag and appropriate restriction enzyme sites to the upstream and downstream primers, respectively. Using Blunt-MANF as a template, the designed and synthesized P1 and P2 primers were used to amplify the target fragment of about 531bp.

[0041] PCR reaction system

[0042] Reagent name

Volume (μl)

upstream primer

2.5

downstream primer

2.5

10×buffe...

Embodiment 2

[0051] Expression, Identification and Purification of Recombinant TAT-MANF Fusion Protein Expression Vector

[0052] (1) Integration and induced expression of recombinant TAT-MANF fusion protein gene in BL21(DE3) expression strain

[0053] Transform E.coli BL21(DE3) with the constructed vector pET22b-TAT-MANF recombinant plasmid, spread on solid LB medium containing ampicillin, and culture overnight (12-16h) in a 37°C incubator. Pick a single colony and put it in 100mL liquid LB medium containing ampicillin. When the OD value reaches 0.6-1.0 (about 3-5h), the temperature is lowered to 16°C and pre-cultured for 1h, then add IPTG to make the final concentration 0.2-1.6 mM, induced overnight (16-18h) at 16°C. Centrifuge at 8000 rpm at 4°C for 5 minutes to collect the cells, and wash with 0.2M PBS for 1-3 times.

[0054] (2) Purification and identification of recombinant TAT-MANF fusion protein

[0055] The lysate was added to the above-mentioned induced expression bacterial so...

Embodiment 3

[0057] Detecting the Transduction Function of Recombinant TAT-MANF Fusion Protein by Fluorescent Laser Confocal Microscopy in Immune Cells

[0058] Cerebral vascular endothelial cells are one of the components of the blood-brain barrier. First, we used cerebrovascular endothelial cells B-Endo3 to detect whether TAT-MANF can enter the cells through the cell membrane. will be 5×10 5 B-Endo3 cells were seeded into 6-well culture plates and cultured in DMEM medium containing 10% fetal bovine serum, 5% CO2, at 37°C. After the cells adhered to the wall, 1 μg / mL FITC fluorescently labeled fusion protein TAT-MANF was added, washed 3 times with 1×PBS every 2 hours, observed and photographed under a confocal laser microscope (Zeiss, S100 microscope).

[0059] In the present invention, the fusion protein TAT-MANF labeled with FITC fluorescence is added to the cultured cerebrovascular endothelial cell B-Endo3, and after incubation for 6 hours, the immunofluorescence detection shows brigh...

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Abstract

The invention relates to a recombinant TAT-MANF (trans-activator of transcription-mesencephalic astrocyte derived neurotrophic factor) fusion protein and an application thereof. A sequence of the recombinant TAT-MANF fusion protein comprises a TAT sequence positioned at an amino terminal and an MANF sequence positioned at a carboxyl terminal. The recombinant TAT-MANF fusion protein is used for nervous system diseases. The fusion protein can enter cells after striding cell membranes, can pass through a blood brain barrier, has a nerve cell protection effect and can be used for relieving neuron lesions related to Parkinson disease.

Description

technical field [0001] The invention belongs to the field of fusion protein and its construction method and application, in particular to a recombinant TAT-MANF fusion protein and its application. Background technique [0002] Parkinson's disease (PD) is a common neurodegenerative disease in the middle-aged and elderly. Its main pathological change is the progressive death of dopaminergic neurons in the substantia nigra compacta, leading to the depletion of the neurotransmitter dopamine in the striatum. There are about 500,000 specialized dopaminergic nerve cells in the substantia nigra compacta of normal people. When the dopaminergic innervation is destroyed by 75-80%, the symptoms of Parkinson's disease will appear. Clinical manifestations include tremor, rigidity, postural instability, and akinesia / bradykinesia. There are as many as 3 million PD patients in my country. At present, the treatment of PD is still based on drug symptomatic treatment, which can temporarily imp...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/18A61K47/48A61P25/16A61P25/28A61P25/00A61P9/10A61K47/42
Inventor 房健民郭佳李晨
Owner SUZHOU RES INST OF TONGJI UNIV
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