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Recombinant gland-related virus AAV-gas6 and application

A technology of aav-gas6 and virus, applied in the direction of recombinant DNA technology, virus, application, etc., can solve the problems of JE patients without specific and effective means, and achieve the effect of enhancing integrity, maintaining steady state, and stable expression

Pending Publication Date: 2021-03-19
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific and effective method for the treatment of JE patients, and the mortality rate of clinical cases can reach 10-15%.

Method used

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  • Recombinant gland-related virus AAV-gas6 and application
  • Recombinant gland-related virus AAV-gas6 and application
  • Recombinant gland-related virus AAV-gas6 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Construction of recombinant adeno-associated virus AAV-gas6

[0024] 1. Determine the CDS region of the mouse gas6 gene sequence;

[0025] 2. Extract total RNA from mouse brain tissue, reverse transcribe it into cDNA as template DNA, design and synthesize the upstream and downstream primers of the gas6 gene coding region (as shown in SEQ ID NO: 2-3), and introduce restriction enzymes Restriction site BglⅡ and xhoⅠ, PCR amplification to obtain mouse gas6 expression gene, namely the nucleic acid sequence shown in SEQ ID NO: 1;

[0026] The PCR amplification system is: template DNA 1 μL, upstream primer: 1 μL, downstream primer R: 1 μL, dNTP: 2 μL, Q5 ultra-fidelity DNA polymerase: 0.5 μL, Buffer: 2 μL, ddH2O: 12.5 μL;

[0027] The amplification program is: 98°C for 30s; 98°C for 10s, 63°C for 15s, 72°C for 60s, a total of 30 cycles; 72°C for 2min; 4°C hold. PCR products were subjected to 1% nucleic acid electrophoresis, and the target band was recovered from the gel.

...

Embodiment 2

[0032] Recombinant adeno-associated virus AAV-gas6 intracerebroventricular injection and virus challenge verification

[0033] 1. The prepared AAV-gas6 virus is used for intracerebroventricular injection after verification of in vitro cell infection experiments.

[0034]2. Mice were 3-4 weeks old, using a stereotaxic instrument and a microinjector, and the injection site: 0.6mm behind the bregma site, 1.2mm next to the midline, and 2.3mm under the dura mater, using a microinjection injection volume of 4 μl (experimental group AAV-gas6, control group AAV-GFP), slow injection, about 5min / time. At 1, 2, and 3 weeks after injection, gas6 in the brain tissue of each group was collected to detect changes in the expression of gas6 in the brain tissue. At the 4th week after the intracerebroventricular injection, different virus doses were used to challenge the plantar injection of JEV, and the survival rate of the mice in each group was recorded.

[0035] The result is as Figure 1...

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Abstract

The invention relates to the technical field of recombinant gland-related viruses and particularly relates to a recombinant gland-related virus AAV-gas6, a preparation method therefor and applicationof the recombinant gland-related virus AAV-gas6. The invention provides the recombinant gland-related virus AAV-gas6 containing a nucleic acid sequence represented by SEQ ID NO: 1. The invention further provides the preparation method for the recombinant gland-related virus AAV-gas6. The recombinant gland-related virus AAV-gas6 provided by the invention is advantageously applied to preparation ofdrugs for treating or relieving JEV-induced viral encephalitis.

Description

technical field [0001] The invention relates to the technical field of recombinant adeno-associated virus, in particular to the recombinant adeno-associated virus AAV-gas6 and its preparation method and application. Background technique [0002] Viral encephalitis has a high mortality rate and disability rate, and is one of the major threats to human life and health. In Asia, Japanese encephalitis virus (JEV), the epidemic Japanese encephalitis virus, is one of the main pathogens causing viral encephalitis. In 1935, the virus was first isolated from the brain tissue of a dead patient by Japanese scholars, so it was named Japanese encephalitis virus. JEV belongs to the Flaviviridae Flaviviridae single-stranded positive-sense RNA virus. The encephalitis caused by JEV is mainly transmitted by the Culex tritaeniorhynchus carrying the virus through biting humans. In my country, it is called epidemic Japanese encephalitis or Japanese encephalitis ( Japanese encephalitis, JE), is ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/864A61K35/76A61P31/14C12R1/93
CPCC12N7/00C12N15/86C07K14/47A61K35/76A61P31/14C12N2750/14121C12N2750/14152C12N2750/14143Y02A50/30
Inventor 边培育雷迎峰
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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