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AQP5 (Aquaporin 5) recombination overexpression vector and construction method and application thereof

A technology of overexpression vectors and recombinant vectors, which is applied in the field of construction and evaluation of therapeutic effects, and can solve the problems of lack of effective treatment methods and methods for heart failure

Active Publication Date: 2019-02-12
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The problem in the prior art is the lack of means and methods that can effectively treat heart failure

Method used

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  • AQP5 (Aquaporin 5) recombination overexpression vector and construction method and application thereof
  • AQP5 (Aquaporin 5) recombination overexpression vector and construction method and application thereof
  • AQP5 (Aquaporin 5) recombination overexpression vector and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Adeno-associated virus vector construction and transformation

[0054] 1, pHBAAV-TBG-MCS-P2A-zsgreen carrier (structure such as figure 1) with EcoRI and MluI, the enzyme digestion system is as follows: 40 μl enzyme digestion system: about 2 hours at 37 degrees

[0055]

[0056] 2. The carrier is recovered after digestion.

[0057] 3. AQP5 fragment PCR recovery

[0058] The AQP5 sequence was amplified by PCR, and the system (50 μl) was shown in the table below, wherein the sequence of the forward primer was shown in SEQ ID NO.2, and the sequence of the reverse primer was shown in SEQ ID NO.3.

[0059] Table 2

[0060]

[0061]

[0062] PCR program: Table 3

[0063]

[0064] Use gel electrophoresis to obtain the target fragment, wherein the PCR product such as image 3 shown. It can be seen that the band of the target fragment is clear.

[0065] 4. Reaction system (20 μl) for ligation of the processed target fragment and carrier:

[0066] Tab...

Embodiment 2

[0073] Embodiment 2 virus packaging

[0074] 1. Adeno-associated vectors, packaging cells and strains

[0075] Virus packaging system: three-plasmid system, pAAV-RC, pHelper, pHBAAV-m-AQP5-GFP.

[0076] Packaging cell line: AAV-293 cells, long-term preservation by Hanbio Biotechnology Co., Ltd., the culture conditions are DMEM+10%FBS, 37°C, 5%CO 2 , relative humidity 95%.

[0077] Strains: E. coli strain Stbl3, used to amplify the adeno-associated vector and helper packaging vector plasmids.

[0078] 2. Experimental principle and steps

[0079] Prepare the adeno-associated virus shuttle plasmid and its auxiliary packaging vector plasmid. The three plasmid vectors were extracted with high purity and no endotoxin respectively (see the appendix for the large-scale extraction steps of the plasmid), co-transfected into AAV-293 cells, and replaced 6 hours after transfection After 72 hours, collect AAV-293 cells rich in adeno-associated virus particles. After repeated freezing an...

Embodiment 3

[0093] Example 3 Injection of adeno-associated virus highly expressing AQP5 into heart failure model mice improves the symptoms of heart failure in mice

[0094] 1. Experimental materials

[0095] C57BL / 6 mice (the plasmid can be purchased from Shanghai Slack Experimental Animal Co., Ltd.).

[0096] 2. Experimental method

[0097] 2.1 Modeling of heart failure in mice

[0098] 24 mice were taken, among which 12 mice were opened with the right anterior chest incision, and the ascending aorta was exposed. The ascending aorta was constricted with a constriction ring at 1 cm above the aorta. After 4 weeks, the Mice with heart failure, and the other 12 mice as a sham operation group (Sham) only underwent thoracotomy but no aortic coarctation surgery.

[0099] 2.2 Take 12 heart failure model operated mice and 12 sham-operated control mice in the above step, and inject the recombinant adenovirus AAV-NTCP prepared in Example 1 and the control adeno-associated virus AAV-GFP (not exp...

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Abstract

The invention provides an AQP5 (Aquaporin 5) recombination overexpression vector and a construction method and application thereof. The recombination vector is obtained in a way that a target gene AQP5 is inserted into an adeno-associated virus vector, and the adeno-associated virus vector at least comprises the sequence elements which can be operably connected: 5' tip inverse repetitive sequence,a TBG (thyroxine-binding globulin) promoter sequence, an MCS (Multiple Cloning Site) area, a P2A sequence, a ZsGreen gene sequence and a 3' tip inverse repetitive sequence. An inventor deeply researches and finds that the AQP5 recombination overexpression vector can alleviate the heart failure symptom of a mouse, the adeno-associated virus which carries the gene is injected into the mouse with heart failure, and the symptom of heart failure of the mouse can be effectively alleviated. Each heart function of the mouse and other indexes can be effectively improved, which means that the technicalscheme is suitable for treating heart failure diseases.

Description

technical field [0001] The invention relates to an AQP5 recombinant overexpression vector and its construction method and use, belonging to the field of biotechnology, in particular to the construction of an adeno-associated virus vector for treating cardiovascular diseases and the evaluation of the therapeutic effect. Background technique [0002] 1. Adeno-associated virus [0003] Adeno-associated virus (AAV) is a member of the Parvoviridae family. Members of this family are a class of tiny, non-enveloped, icosahedral viruses. It encodes cap and rep genes in two terminal inverted repeats (ITRs). The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. The diameter of virus particles is between 20 and 26 nm, and contains a linear single-stranded DNA genome with a size between 4.7 and 6 kb. Parvoviruses have been isolated from insects to humans. AAV virus belongs to the category of dependent viruses (De...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N5/10C12N15/66A61K48/00A61K38/17A61P9/04
CPCA61K38/1709A61K48/0025A61K48/005A61P9/04C12N15/66C12N15/86C07K14/47C12N2750/14143
Inventor 陈意雄蔡晓龙邹杰朱鹏飞白易鑫
Owner 汉恒生物科技(上海)有限公司
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