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Nucleic acid encoding BDNF and application of nucleic acid

A nucleic acid and encoding technology, which is applied in the nucleic acid encoding BDNF and its application fields, can solve problems such as difficulty in crossing the blood-brain barrier and unsatisfactory therapeutic effects

Active Publication Date: 2020-12-04
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Animal experiments have shown that BDNF has a certain protective effect on the damaged optic nerve, but because BDNF is a biological macromolecule, it is difficult to cross the blood-brain barrier, so the therapeutic effect is not very ideal

Method used

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  • Nucleic acid encoding BDNF and application of nucleic acid
  • Nucleic acid encoding BDNF and application of nucleic acid
  • Nucleic acid encoding BDNF and application of nucleic acid

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preparation example Construction

[0059] The present invention also provides a preparation method of the adeno-associated virus expressing the brain-derived nerve growth factor, which comprises: transfecting the host cell with the plasmid combination, and obtaining the adeno-associated virus of the brain-derived nerve growth factor through purification. In the transfection step, the molar ratio of the recombinant vector of the present invention, pAdHelper, and pAAV-r2c5 in the plasmid combination is 1:1:1.

[0060] The adeno-associated virus expressing brain-derived nerve growth factor prepared by the preparation method of the present invention.

[0061] The application of the recombinant vector of the present invention, or the plasmid combination, or the adeno-associated virus in the preparation of medicines for preventing and treating eye diseases.

[0062] The eye disease is an eye disease related to optic nerve damage; the optic nerve damage includes acute optic nerve damage or chronic optic nerve damage. ...

Embodiment 1

[0070] Example 1 Sequence Optimization and Vector Screening

[0071] 1.1 Sequence optimization

[0072] Based on the amino acid sequence (SEQ ID NO:1) and the natural coding sequence (SEQ IDNO:2) of the BDNF protein, in this example, multiple optimized BDNF coding sequences were designed and analyzed for the optimized BDNF coding sequences and test screening. As a result, it was found that, compared with the natural BDNF coding sequence, the specially optimized DNA coding sequence, as shown in SEQ ID NO: 3-SEQ ID NO: 6, can significantly improve the expression efficiency of BDNF protein.

[0073] 1.2 Vector construction

[0074] Add two restriction sites of BamH I and Not I to the original fragment and the optimized sequence respectively, or use the product amplified by PCR with the primers designed for the new gene and the pAAV2 plasmid vector to carry out BamH I and Not I double enzymes respectively The digested products were recovered, ligated overnight with T4 DNA Ligas...

Embodiment 2

[0080] Example 2. Preparation of scAAV2 / 2-coBDNF virus

[0081] 2.1 pscAAV-coBDNF recombinant adeno-associated virus coating

[0082] HEK293 cells (seeded at 225cm 2 Cell culture flasks), the cells were harvested after 48 hours. Cells were resuspended in PBS and freeze-thawed 3 times.

[0083] 2.2 Purification and concentration of scAAV2 / 2-coBDNF virus

[0084] Three steps of chloroform treatment-PEG / NaCl precipitation-chloroform extraction were used to separate, concentrate and purify to obtain rAAV2 / 2-BDNF_native, rAAV2 / 2-BDNF_opt1, rAAV2 / 2-BDNF_opt2, rAAV2 / 2-BDNF_opt3 and rAAV2 / 2 - BDNF_opt4 virus. Total recovery = number of virus particles in the final product / number of virus particles in the starting material.

[0085] 2.3 Real-time quantitative PCR method to detect the physical titer of scAAV2 / 2-coBDNF

[0086] Experimental materials: SYBRⅡ (takara); target fragment primer (20uM); target plasmid for packaging virus (known concentration); virus to be tested; PCR eig...

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Abstract

The invention relates to the technical field of gene engineering, and in particular to nucleic acid for encoding BDNF and application of the nucleic acid. According to the invention, a BDNF fragment which is efficiently expressed in vivo is obtained through codon optimization, so that an AAV vector for efficiently expressing BDNF, an adeno-associated virus and a medicine for treating eye diseasesare constructed. Experiments show that the AAV virus carrying the BDNF gene can directly infect retinal ganglion cells and continuously express BDNF protein for protecting and promoting optic nerve regeneration in target cells, so that retinal nerve cells after optic nerve injury are protected.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to nucleic acid encoding BDNF and its application. Background technique [0002] The optic nerve belongs to the central conduction bundle, and optic nerve injury is a disease of conduction dysfunction caused by a certain cause in the optic nerve conduction pathway. Common causes include trauma, ischemia, poisoning, demyelination, tumor compression, inflammation, metabolism, etc. After optic nerve injury, a large number of retinal ganglion cells (RGCs) die and axon regeneration is difficult, which eventually leads to optic atrophy and impaired visual function. Currently, there is no treatment for visual impairment caused by optic nerve damage. [0003] Neurotrophins are a family of proteins that induce neuronal survival, development and function. Among them, brain-derived nerve growth factor BDNF is a secreted dimeric growth factor that exists in most human tissues, in...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/864A61K48/00A61K38/18A61P27/02A61P27/06
CPCC07K14/475C12N15/86A61K48/0008A61K48/005A61K48/0075A61K38/185A61P27/02A61P27/06C12N2750/14143C12N2800/107
Inventor 李斌刘婷
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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