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Method for constructing pathogen infection animal model

An animal model, sensitive technology, applied in the field of experimental animals and medical research, can solve problems such as affecting the basic indicators of animal models, major changes in the immune system, etc., to achieve the effect of helping research and drug development, and improving susceptibility

Active Publication Date: 2019-01-25
北京华阜康生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the systematic genetic engineering operation is carried out, the immune system of the animal itself changes significantly, which may affect the basic indicators of the animal model

Method used

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  • Method for constructing pathogen infection animal model
  • Method for constructing pathogen infection animal model
  • Method for constructing pathogen infection animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A gene silencing vector targeting the Ager gene was constructed, and the silencing targets included the positions shown in Table 1 below. Design and synthesize the neck loop sequence for each sequence, and then clone it into the shRNA expression vector.

[0035] Table 1 Silencing targets

[0036]

[0037]

[0038] Carry out DNA synthesis for the above sequence, and synthesize double-stranded DNA, use the enzyme-cleaved cloning vector, and connect it into the gene silencing vector. The information of the vector is as follows: figure 1 shown. The vector was sequenced correctly, and the purified plasmid was used for subsequent transfection and virus packaging.

[0039] Culture NIH 3T3 cells until the monolayer density reaches 80%, and replace the medium for transfection. Add the transfection reagent containing the transfection plasmid to the cell culture for transient transfection. Assess transfection efficiency using fluorescent plasmids. Compared with the inte...

Embodiment 2

[0042]The in vivo experiment was carried out with the silencing plasmid obtained by mixing the 6th and 7th sites with high silencing efficiency in equal proportions in Example 1. The lentivirus is packaged, and the packaging vector is a defective virus vector (pLV[shRNA]-EGFP, VectorBuilder) of the lentivirus background, and is co-transfected with the helper plasmid pVSV-G into the packaging cell HEK293T cells, and the virus is packaged in the cell. Collect the cell culture for 72 hours, freeze and thaw five times from -80°C to 37°C, and obtain the virus. The virus liquid is centrifuged at a differential speed of 5000rpm-30000rpm to remove the cell components and enrich the virus, so that the titer of the virus reaches 1×10 8 More than pfu / mL. Adjust the concentration of the packaged virus liquid to 5×10 7 pfu / mL, and add Polybrene, a cationic transfection auxiliary reagent, with a final concentration of 5 μg / mL, and inject it into the tail vein of mice with a dose of 100 μL....

Embodiment 3

[0045] BALB / c mice with down-regulated expression of Ager lung tissue constructed above were infected with a certain amount of PVM virus at the same time as control mice of the same age, same sex, and same strain without gene silencing operations. for 10 4 pfu / mL, the inoculated virus volume was 30 μL.

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Abstract

The invention belongs to the experimental animal and medical research field, and discloses a method for constructing a pathogen infection sensitive animal model, which inhibits the expression of an animal Ager gene through a gene silencing technology. This method utilizes gene silencing technique to establish the animal model of innate immune receptor Ager gene expression down-regulation, which can be used to carry out pathogen infection experiment and improve the susceptibility of animals to pathogen, and has no significant effect on general immune index. Therefore, it is helpful to study theinfection mechanism of infectious diseases and drug development.

Description

technical field [0001] The invention belongs to the field of experimental animals and medical research, and in particular relates to a method for constructing an animal model, in particular to a method for constructing an animal model sensitive to pathogenic infection. Background technique [0002] Experimental animals are an important carrier of medical research, especially the study of infectious diseases. The establishment of experimental animal infection animal models of various pathogens is an important process for research on the pathogenic mechanism of infectious diseases and drug development. Among them, mice, as a relatively clear model animal such as genetic background, have become the first choice for the study of various pathogenic infections. However, humans or other animals, compared with mice, have evolutionary distances due to the evolution of their immune systems. Differences in the immune system make mice less susceptible to some pathogens. This phenomen...

Claims

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Application Information

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IPC IPC(8): C12N15/867A01K67/027
CPCA01K67/0275A01K2207/05A01K2227/105A01K2267/03C12N15/86C12N2740/15043C12N2800/107
Inventor 向志光佟巍徐艳峰郭智李欣悦
Owner 北京华阜康生物科技股份有限公司
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