Blood vessel growth hormone gene recombined gland relative vrius carrier and its preparation process and application thereof

An angiogenin and gene recombination technology, applied in the field of genetic medicine, can solve the problems of adenovirus contamination, application, obstacles, etc., and achieve the effects of low equipment requirements, immune response avoidance, and wide application range

Inactive Publication Date: 2008-05-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional preparation of recombinant adeno-associated virus is to use adenovirus-assisted transfection method, but the problem of adenovirus contamination is an obstacle to its application

Method used

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  • Blood vessel growth hormone gene recombined gland relative vrius carrier and its preparation process and application thereof
  • Blood vessel growth hormone gene recombined gland relative vrius carrier and its preparation process and application thereof
  • Blood vessel growth hormone gene recombined gland relative vrius carrier and its preparation process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation (packaging) of rAAV-ANG of the present invention:

[0031] According to the ANG gene sequence and the structure of plasmid pAAV-MCS, design primers for the angioglossin gene with restriction enzymes BamHI and EcoRI restriction sites, and amplify the angiogenin ring deoxyribonucleic acid fragment (398bp) by polymerase chain reaction ), connected to the plasmid pAAV-MCS to construct the plasmid pAAV-ANG, restriction enzyme digestion and sequencing to identify the insert direction and sequence are correct (seefigure 1 ). Aspirate 10μg each of plasmids pAAV-ANG, pAAV-RC and pHelper, and use calcium phosphate co-precipitation method to transfect 293 cells to construct recombinant angiogenin gene adeno-associated virus vector (rAAV-ANG); at the same time establish a positive control, namely pAAV-LacZ 10μg each of pAAV-RC and pHelper was co-transfected into 293 cells to construct a recombinant reporter gene LacZ virus vector (rAAV-LacZ). The recombinant vir...

Embodiment 2

[0032] Example 2: Recombinant virus rAAV-ANG is used to study the capillary density, left ventricular remodeling and cardiac function of normal rat myocardial tissue:

[0033] Rats were given general anesthesia by intraperitoneal injection of chloral hydrate (400mg / kg), taken supine position, cut the trachea, connected to the animal ventilator, opened the chest cavity, and exposed the heart. Pipette 120μL of rAAV-ANG (about 1.2×10 9 IU) was injected at four points on the anterior wall of the left ventricle, 30 μL at each site. Close the chest cavity and remove the ventilator. After the rats were awake, they were fed with ordinary feed in a clean environment for four weeks. The results showed that rAAV-ANG efficiently transfected normal rat myocardial tissue and significantly up-regulated the expression of angiogenin protein in normal rat myocardial tissue (see figure 2 ), increase the capillary density in the myocardial tissue (see image 3 ). But it did not affect the ventricular...

Embodiment 3

[0034] Example 3: Research on the use of recombinant virus rAAV-ANG for gene therapy in rats with myocardial infarction:

[0035] Rats were given general anesthesia by intraperitoneal injection of chloral hydrate (400mg / kg), taken supine position, cut the trachea, connected to the animal ventilator, opened the chest cavity, and exposed the heart. Open the pericardial wall, quickly squeeze the heart out of the chest cavity, and ligate the left anterior descending coronary artery with an 8-0 silk thread between the left atrial appendage and the right ventricular outflow tract, resulting in acute myocardial infarction. Then quickly put the heart back into the chest cavity. Pipette 120μL of rAAV-ANG (about 1.2×10 9 IU) Intramyocardial injection was performed at four points on the edge of the infarcted area of ​​the left ventricular anterior wall. Close the chest cavity and remove the ventilator. After the rats are awake, they are fed with ordinary feed in a clean environment. Four wee...

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Abstract

The invention discloses blood vessel auxin gene recombination gland correlation viral carrier. It is recombination gland correlation virus. Its manufacturing includes following steps: amplifying gene fragment; forming and identifying recombinant plasmid pAAV-ANG; collecting, purifying and titer testing for gene recombination gland correlation viral carrier. It can be applied to ischemic heart disease and peripheral tissue ischemic disease therapy medicines, culture in vitro cell and whole experiment animal or patient. Its application is extensive. Infection efficiency is high. The physicochemical property of the gland correlation viral carrier is stable. It has a lot of similar traditional medicines condition, and is fit for popularization and application.

Description

Technical field [0001] The invention relates to a gene medicine, in particular to an angiogenin (ANG) gene recombinant adeno-associated virus vector (rAAV-ANG). It can be applied to gene therapy and related research of ischemic heart disease including angina pectoris, acute myocardial infarction, ischemic cardiomyopathy and peripheral ischemic diseases. The invention also provides a method for preparing the recombinant adeno-associated virus vector. Background technique [0002] With the development of the Human Genome Project and the gradual deepening of molecular biology research, the clarification of the molecular mechanisms of many diseases and the continuous discovery of pathogenic genes, gene therapy has developed from the treatment of single-gene inherited diseases to the treatment of common and frequently-occurring diseases with multiple genes , Especially in the treatment of cardiovascular and cerebrovascular diseases and various tumors. The three key issues in gene ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/12A61K48/00A61P9/10
Inventor 胡申江赵晓燕李江
Owner ZHEJIANG UNIV
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