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ADAR1 (Adenosine Deaminases Acting on RNA1) over-expressed virus vector as well as construction method and application thereof

A virus vector and construction method technology, applied in the field of genetic engineering, can solve the problems of undisclosed ADAR1 application and the like

Inactive Publication Date: 2019-01-11
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of them disclose the ADAR1 overexpression vector and its application in the preparation of sepsis therapeutic drugs

Method used

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  • ADAR1 (Adenosine Deaminases Acting on RNA1) over-expressed virus vector as well as construction method and application thereof
  • ADAR1 (Adenosine Deaminases Acting on RNA1) over-expressed virus vector as well as construction method and application thereof
  • ADAR1 (Adenosine Deaminases Acting on RNA1) over-expressed virus vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The construction of embodiment 1 mouse ADAR1 expression vector

[0052] Due to the possible "off-target" phenomenon in RNAi experiments, we synonymously mutated the wild-type ADAR1 gene according to the RNAi target sequence, specifically cloning the sequence of mouse ADAR1 overexpressing adenovirus (Ad+O / E-ADAR1) : NM_001146296.1, ADAR1 low-expression adenovirus sequence (Ad-shADAR1): CCATGAACCTCGATTTAAA and negative control sequence (Ad-NC): TTCTCCGAACGTGTCACGT, carry out synonymous mutation on ADAR1 overexpression adenovirus sequence, and ADAR1 overexpression adenovirus sequence The sequence of the virus, the sequence of the ADAR1 low-expression adenovirus and the negative control sequence were respectively constructed with the lentiviral PCDH-CMV-MCS-EF1-copGFP plasmid as the backbone to construct the ADAR1 lentiviral expression vector.

Embodiment 2

[0053] Embodiment 2 establishes cecal ligation and puncture (CLP) sepsis mouse model

[0054] 1. Experimental animals

[0055] Select 6-week-old male C57BL / 6 mice, weighing 20-25g, and culture them under standard laboratory conditions.

[0056] 2. The experimental steps are as follows:

[0057] (1) On the morning of the operation day, the mice began to fast, and were anesthetized by intraperitoneal injection of 4% chloric acid hydrate. They were placed on the experimental operating table in a supine position, and after the abdominal skin was sterilized with 75% alcohol, a wound about 1 cm long was cut along the middle of the abdomen, and opened. The abdominal cavity finds the cecum and squeezes out the gas distal to the cecum.

[0058] (2) According to the design requirements of each group, a 3-0 line was used to ligate at 1 cm from the blind end or at the base of the cecum to avoid intestinal ischemia and necrosis caused by ligation of mesenteric vessels, and a 12-gauge nee...

Embodiment 3

[0061] Example 3 Protective effect of ADAR1 overexpression virus vector on septic mice

[0062] 1. Experimental animal: the CLP sepsis mouse model prepared in Example 2.

[0063] 2. Experimental scheme: Divide the mouse models into five groups, put them in the fixer, expose the tail, and inject 100 microliters of ADAR1 overexpression adenovirus vector (containing 1×10 8 PFU ADAR1 overexpression virus), the same dose of low expression virus vector, the plasmid group without ADAR nucleic acid sequence, the same dose of PBS (sham model group, model group)---CLP+Ad-NC group. Mice were anesthetized on the first and third day after injection, and small intestinal samples were collected for histological analysis.

[0064] 3. Experimental results

[0065] CLP induced sepsis in mice, respectively overexpressed / disturbed ADAR1 in the small intestine, and H&E staining was used to evaluate the histomorphology of the small intestine of septic mice. figure 1 The results showed that after...

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Abstract

The invention provides an ADAR1 over-expressed virus vector, a preparation method of the virus vector, and application of the ADAR1 over-expressed virus vector in aspects of protecting the intestinalstructure integrity, maintaining the intestinal steady-state, improving the activity of intestinal macrophages, inhibiting the level of inflammatory cytokines and preparing products for treating sepsis. The ADAR1 over-expressed virus vector disclosed by the invention provides novel potential therapeutic targets for treatment of sepsis.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an ADAR1 overexpression virus vector, in particular to an ADAR1 overexpression virus vector, its construction method and its ability to protect the integrity of the small intestine structure and maintain intestinal homeostasis; improve intestinal macrophages activity; inhibiting the level of inflammatory cytokines and its application in the preparation of products for the treatment of sepsis. Background technique [0002] Sepsis is a systemic inflammatory response syndrome caused by infection and is a common complication of severe trauma, burns and infectious diseases. When sepsis progresses and circulatory failure develops, the condition progresses to septic shock and multiple organ dysfunction syndrome. The pathogenesis of sepsis is complex and the mortality rate is high, which is a prominent problem in wartime trauma and emergency critical care medicine. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N5/10A61K38/50A61P29/00A61P31/00
CPCA61K38/50A61P29/00A61P31/00C12N5/0645C12N15/66C12N15/86C12N2710/10043C12Y305/04004
Inventor 李俊杰刘善收谢建刚赵彬尹文
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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