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Recombinant adeno-associated virus expressing BDNF-HA2TAT, and construction method thereof

A construction method and virus technology, applied in the field of recombinant adeno-associated virus and its construction, can solve unreported problems and achieve the effect of preventing and treating depression and stress disorders

Inactive Publication Date: 2013-05-08
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But about a BDNF-HA expression 2 The recombinant adeno-associated virus of TAT and its construction method have not been reported yet

Method used

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  • Recombinant adeno-associated virus expressing BDNF-HA2TAT, and construction method thereof
  • Recombinant adeno-associated virus expressing BDNF-HA2TAT, and construction method thereof
  • Recombinant adeno-associated virus expressing BDNF-HA2TAT, and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of recombinant adeno-associated virus for packaging and expressing BDNF

[0035] Clone the BDNF cDNA fusion gene, connect the PCR product into the pGEM-T Easy vector, and obtain the pGEM-T Easy / BDNF transformed recipient strain E. Coli DH5α, the recombinant plasmid DNA was extracted by alkaline lysis, identified by enzyme digestion; the BDNF gene with cohesive ends was obtained by enzyme digestion, and the BDNF gene was inserted into the vector plasmid carrying the penetrating peptide TAT to construct the recombinant vector plasmid pSSCMV-BDNF-HA 2 TAT, recombinant plasmid pSSCMV-BDNF-HA 2 TAT was digested with EcoRI and BamHI to prepare BDNF-HA 2 The TAT fragment was connected to the PUC19 plasmid for sequencing; under the action of calcium phosphate, the recombinant vector plasmid pSSCMV-BDNF-HA 2 HEK293 cells were co-transfected with TAT, packaging plasmid pAAV / Ad and helper plasmid pFG140 to prepare and express BDNF-HA 2 Recombinant adeno...

Embodiment 2

[0277] Example 2 Expression of BDNF-HA 2 Animal experiment of recombinant adeno-associated virus of TAT

[0278] Recombinant adeno-associated virus expressing brain-derived neurotrophic factor enters the brain through the nasal-brain pathway to produce antidepressant-like effects: C57 BL / 6 female and male adult mice were divided into nasal instillation of normal saline and BDNF-HA 2 TAT / AAV two treatment groups, the two treatments were divided into 1d administration group, 5 consecutive days administration group and 10 consecutive days administration group; the 1d administration group underwent behavioral study on 1, 2, and 3 days after administration respectively. Test (forced swimming test (forced swimming test, FST) and open field exploration test (open-field exploration test, OPF), 5 consecutive days of administration group and 10 consecutive days of administration group respectively at 1, 3, 7 days after administration Behavioral tests (FST and OPF) were performed ...

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Abstract

The present invention relates to recombinant adeno-associated virus expressing BDNF-HA2TAT, and a construction method thereof. According to the present invention, an adeno-associated virus vector BDNF-HA2TAT / AAV carrying a fusion gene BDNF-HA2TAT is constructed, the recombinant adeno-associated virus expressing BDNF-HA2TAT can secrete and express macromolecule protein BDNF, the expressed BDNF can pass through blood brain barrier, and a BDNF gene sequence is introduced into the AAV vector, such that continuous BDNF secretion expression can be achieved so as to solve the difficult problem of repeated administration required by BDNF; and anti-depression efficacy screening experiment results show that the recombinant adeno-associated virus capable of secreting and expressing BDNF-HA2TAT has an anti-depression effect, and a method for effectively preventing and treating depressive disorders, stress disorders and other mental disorders is established.

Description

technical field [0001] The present invention relates to a kind of recombinant vector, specifically, a kind of expression BDNF-HA 2 Recombinant adeno-associated virus of TAT and its construction method. Background technique [0002] Depressive disorders have neuropathological changes such as hippocampal neuron atrophy, accelerated apoptosis, and neuronal regeneration disorders. How to effectively protect hippocampal neurons has always been a hot and difficult point in the study of depression. Brain derived neurotrophic factor (BDNF) is an important member of the neurotrophic factor family, and the down-regulation of BDNF may be involved in the structural and functional changes of the hippocampus in depression induced by chronic stress. Injecting exogenous BDNF into the hippocampal dentate gyrus of a depressed rat model of forced swimming and learned helplessness produced antidepressant-like activity, suggesting that central administration of BDNF is effective for depression...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/12C12N15/63
Inventor 高成阁马现仓党永辉杨广笑李强陈策杨小波郭丽阳
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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