Animal model for expressing human ACE2 and application of animal model

An animal model and human-derived technology, applied in the field of animal models expressing human ACE2, can solve the problems of long breeding cycle, non-obvious tissue specificity of phenotype, etc.

Active Publication Date: 2021-04-20
GUANGZHOU PACKGENE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages are long screening period, long breeding period, random integration is prone to phenotypic instability, and the tissue specificity of phenotype is not obvious
[0007] In addition to the above disadvantages, traditional animal models such as non-human primates and traditional transgenic animals have problems such as mild and unobvious symptoms after coronavirus challenge during the modeling process, and it is difficult to achieve severe or critically ill models of new coronary pneumonia. modeling

Method used

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  • Animal model for expressing human ACE2 and application of animal model
  • Animal model for expressing human ACE2 and application of animal model
  • Animal model for expressing human ACE2 and application of animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Establishment of a mouse model of lung disease expressing human ACE2

[0040] 1. Construction of recombinant adeno-associated virus backbone vector

[0041] Search through the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore) to obtain the mouse ACE2 gene (NCBI Reference Sequence: NM_001130513.1) signal peptide sequence, and obtain the human ACE2 gene (NCBI Reference Sequence : NM_001371415.1) mature peptide sequence, the sequence was combined and codon-optimized (SEQ.ID.NO.1), and the cloning vector, PUC-hACE2, was synthesized by Suzhou Jinweizhi Technology Co., Ltd.

[0042] Primers F:5'-tccaccggtcgccaccatgtccagctcctcc-3'(SEQ.ID.NO.2), R:5'-atctcgagcggaattctcagaaactcgtttg-3'(SEQ.ID.NO.3) were designed to amplify hACE2 sequence using PUC-hACE2 as template .

[0043] Cut the adeno-associated virus vector pAAV-GFP with restriction endonucleases AgeI and EcoRI, use agarose gel electrophoresis, and cut the large fragment of the DNA product after electrophore...

Embodiment 2

[0050] Embodiment 2, compare different administration modes and carry out modeling

[0051] In Step 3 of Example 1, the mice were given hACE2 modeling agent AAV-hACE2 (1 ×10 10 vg / μL, the administration volume is 100 μL), and the combination group is 50 μL sprayed through the lung cannula and 50 μL injected into the tail vein.

[0052] image 3 It is shown that after 2 weeks of modeling, three mice were selected in each group, and the lung tissues of the mice were collected and ground, and protein lysate was added for Western Blot detection. Model test results showed that tail vein injection could not make mice express human ACE2 gene in lung. The expression level of the lung intubation jet and tail vein injection group was not better than that of the lung intubation jet group.

Embodiment 3

[0053] Embodiment 3, using SARS-Cov-2 to challenge hACE2 model mice

[0054] Using 1×10 5 PFU SARS-Cov-2 virus carries out nasal drop infection to lung intubation injection group mice (the model mouse that embodiment 1 makes) and control group AAV-GFP mice. The weight of the mice in the lung intubation injection group began to drop after infection with SARS-Cov-2, and the body weight dropped to 80% of the control group on the 7th day, and continued until the 12th day; then the body weight gradually recovered, and at the end of the experiment 21 Days, the body weight recovered to 90% of the control group, such as Figure 4 As shown in A.

[0055] The hACE2 model mice and control mice were tested for SARS-COV-2 neutralizing antibodies on the 10th and 21st days respectively, and the hACE2 model mice were higher than the control group on the 10th and 21st days, Such as Figure 4 Shown in B.

[0056] The N and ORF1 gene expressions of SARS-Cov-2 in the lung tissue were signifi...

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Abstract

The invention discloses an animal model for expressing human ACE2 and application of the animal model. A mouse AC E2 gene signal peptide sequence and a human ACE2 gene mature peptide sequence are combined and subjected to codon optimization and amplification to obtain an hACE2 sequence, and a recombinant adeno-associated virus AAV-hACE2 is further prepared; and recombinant adeno-associated virus AAV-hACE2 is sprayed to the lung of the mouse by intubation for at least two weeks to obtain a mouse model with lung and brain tissue highly expressing human ACE2. According to the invention, the modeling speed for constructing the novel coronapneumonia virus susceptible mouse model is relatively high, and modeling can be succeeded only in two weeks. The animal model fully reveals pathological characteristics of AAV-hACE2 mice infected with novel coronavirus and application of the AAV-hACE2 mice in novel coronavirus vaccines, so that the model is clear to be suitable for pharmacodynamic evaluation of the novel coronavirus vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an animal model expressing human ACE2 (angiotensin-converting enzyme 2) and its application. Background technique [0002] Novel coronavirus pneumonia COVID-19 has become a pandemic worldwide. It is extremely important and urgent to understand the mechanism of virus infection and develop therapeutic drugs and vaccines. [0003] The SARS-COV-2 novel coronavirus spike (Spike) glycoprotein invades the body by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor of cells in the human body. [0004] However, the major key problem facing the research of new coronary pneumonia virus is the lack of ideal animal models. Although non-human primates (such as monkeys) are closer to humans, they are not an ideal choice for animal models due to problems such as high price, long test period, and inconvenient operation. [0005] Mice are potentially ideal animal models,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/864C12N7/01A01K67/027A61K49/00
CPCY02A50/30
Inventor 周泽鑫李华鹏张钰张超王薇薇李舸
Owner GUANGZHOU PACKGENE BIOTECH CO LTD
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