Pim-1 gene-containing adeno-associated virus 2 and application thereof to repairing of injured optic nerve

A pim-1 and gene technology is applied in the application field of preparing a drug for treating and repairing damaged optic nerve, and can solve the problems that the research on Pim-1 gene and repairing damaged optic nerve, the lack of research on the nervous system, etc.

Active Publication Date: 2017-05-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, most of the researches on Pim-1 focus on tumors, myocardial protection and regeneration, and there are few studies on the nervous system. There is no related research on Pim-1 gene and the repair of damaged optic nerves, and there is no literature report on the construction of a Recombinant adeno-associated virus 2 vector carrying Pim-1 gene for treatment of damaged optic nerve and other nerve injuries

Method used

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  • Pim-1 gene-containing adeno-associated virus 2 and application thereof to repairing of injured optic nerve
  • Pim-1 gene-containing adeno-associated virus 2 and application thereof to repairing of injured optic nerve
  • Pim-1 gene-containing adeno-associated virus 2 and application thereof to repairing of injured optic nerve

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Obtaining the target gene fragment

[0049] The sequence of Pim-1 gene was queried from GenBank, primers were designed with VectorNTI software, and the cDNA clone of CDS region of Pim-1 gene was used as a template to amplify the Pim-1 gene fragment by PCR.

[0050] PCR amplification of the target gene: Use the high-fidelity PrimeSTAR enzyme to amplify the target gene. The reaction system and conditions are as follows:

[0051] Table 1: Amplification system using Pim-1 gene cDNA clone as template

[0052]

[0053] 2. Construction of Pim-1 recombinant adeno-associated virus 2 expression vector

[0054] (1) Enzyme digestion of adeno-associated virus 2 expression vector and PCR product of target gene

[0055] The pAOV-CAGMINI-EGFP-2A-MCS-3FLAG vector (purchased from Addgene) was digested with NheI, and the PCR product was digested with NheI endonuclease, and the vector and purpose were recovered after identification by agarose gel electrophoresis. genetic DNA.

[...

Embodiment 2

[0118] 1. Establishment of rat optic nerve crush injury model

[0119]After routine disinfection of experimental animals in each group, each rat was anesthetized by intraperitoneal anesthesia with 10% chloral hydrate (0.4ml / 100mg). Use microscissors to cut the bulbar conjunctiva at the limbus, and then bluntly dissect the bulbar conjunctiva with microtweezers until the posterior bulbar. During the operation, do not damage blood vessels and other tissues to avoid bleeding, fully expose the optic nerve, and 2mm at the retrobulbar. Temporary aneurysm clipping was used for 5s (for the specific operation, please refer to the published literature of the inventor [31] Wang R, Xu J, Xie J, et al.Hyperbaric oxygen preconditioningpromotes survival of retinal ganglion cells in a rat model of optic nervecrush.J Neurotrauma. 2010(27): 763-770.) simulates clinical incomplete optic nerve injury (eg figure 1 shown).

[0120] 2. Grouping of experimental animals

[0121] (1) Sham operation g...

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Abstract

The invention relates to the technical field of genetic engineering, aims to solve the major clinical medical problem of how to effectively treat an injured optic nerve with low intrinsic retinal ganglion cell regeneration capability which is the key factor for recovery of the visual function, and provides a Pim-1 gene-containing recombinant adeno-associated virus 2 vector and a construction method for the same. The adeno-associated virus 2 vector is a DNA (deoxyribonucleic acid) sequence containing a Pim-1 gene shown as SEQ ID NO:1. The invention also provides the application of the Pim-1 gene-containing recombinant adeno-associated virus 2 vector to preparation of a medicament for treating and repairing the injured optic nerve. Novel powerful means and a theoretical foundation are provided for clinical treatment of optic nerve injuries, and the Pim-1 gene-containing recombinant adeno-associated virus 2 vector is significant for treatment of CNS (central nervous system) diseases and injuries.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of an adeno-associated virus 2 carrying a Pim-1 gene in the preparation of a medicine for treating and repairing damaged optic nerve. Background technique [0002] After central nervous system (CNS) disease and injury, nerve regeneration is very difficult, and how to effectively treat it has always been a major problem in clinical treatment. The optic nerve is part of the CNS. After optic nerve injury, the main reasons for the difficulty of axonal regeneration include the massive death of retinal ganglion cells (RGCs), the low intrinsic regeneration ability of RGCs, the lack of cells that guide axonal regeneration, the microenvironment that inhibits regeneration, and the lack of neurotrophic factors. The low intrinsic regenerative capacity of RGCs after optic nerve injury is a key factor in optic nerve atrophy and blindness due to regeneration failure...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/66C12N15/65A61K48/00A61P27/02A61P25/02
CPCA61K48/0008C12N15/65C12N15/66C12N15/86C12N2750/14143
Inventor 许家军张守梅王栋刘芳黄婷婷
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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