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ACE2 cell humanized mouse model as well as construction method and application thereof

A mouse model and construction method technology, applied in the field of animal genetic engineering, can solve the problems of unfavorable reconstruction of the immune system, hindering the study of viruses and immune cells, long modeling cycle, etc., to reduce background pollution, avoid xenograft rejection, shorten the The effect of the experimental cycle

Pending Publication Date: 2020-09-04
湖南昭泰生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ACE2 transgenic mice need to cultivate embryonic stem cells, and the establishment and cultivation of embryonic stem cell lines are more complicated and the modeling period is longer
At the same time, the operation process of the ACE2 knockout mouse construction method is relatively complicated, and the success rate is not high
Moreover, conventional transgenic mice or knockout mice contain the mouse's own immune cells, such as T cells, B cells, and NK cells, which are not conducive to rebuilding the humanized immune system and hinder research on humanized ACE2 mice The relationship between viruses and immune cells
[0004] Due to the fact that there are relatively few animal models of the new coronavirus at present, the construction time is long, and they cannot be reproduced in large quantities, which limits the progress of research on new coronavirus vaccines or immunization methods

Method used

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  • ACE2 cell humanized mouse model as well as construction method and application thereof
  • ACE2 cell humanized mouse model as well as construction method and application thereof
  • ACE2 cell humanized mouse model as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The present invention provides a mouse model of ACE2 cell humanization, and its construction method is as follows:

[0055] (1) Construction of BGC823-ACE2-GFP stable cell line:

[0056] ①Use lentiviral vectors to construct plasmids overexpressing ACE2 receptor and green fluorescent protein (GFP), and then use 293T cells to prepare lentiviral supernatant suspension;

[0057] ② After centrifuging and filtering the lentiviral supernatant, use Jurkat cells to detect virus titer; among them, titer (TU / mL) = cell number × fluorescence percentage × 10 3 / Volume of virus stock solution (μL);

[0058] ③ Taking the multiplicity of infection (MOI) as 10, every 1×10 6 Add 10 to each BGC823 cell 7 TU lentivirus for infection;

[0059] ④Change the medium after 6 hours of infection, and continue to culture; 48 hours later, take the infected cells and test the infection efficiency by flow cytometry; the infection efficiency test results are as follows: figure 1 As shown, among th...

Embodiment 2

[0070] This embodiment is beneficial to step (1) in Example 1 to construct cells overexpressing ACE2 receptors with Jurkat, NALM6, Huh7, A549 and H1299 as parent cells;

[0071] The infection efficiency and the infection efficiency after sorting are shown in Table 1.

[0072] Table 1

[0073] cell name infection efficiency Infection efficiency after sorting ACE2-A549 cells 90％ --- ACE2-Huh7 cells 90％ --- ACE2-H1299 cells 44％ 90％ ACE2-Jurkat cells 50％ 90％ ACE2-Nalm6 cells 17％ 90％

[0074] It can be seen from the above table that when A549 and Huh7 are used as mother cells, the infection efficiency can reach 90%. Although the infection efficiency of the other three types of cells does not reach 90%, the infection efficiency can reach 90% after sorting.

Embodiment 3

[0076] In this example, the distribution of ACE2-positive cells in the lungs of mice was observed by pathological detection methods.

[0077] After the mice were killed, the lungs of the mice were taken and photographed under a microscope to observe the GFP signal; then a part of the lungs was used to prepare a single cell suspension, and the proportion of BGC823 cells in the lungs of the mice was detected by flow cytometry, and the other A part was used to prepare paraffin sections for HE staining and immunofluorescence detection.

[0078] The results of flow cytometry were as Figure 5 As shown, it is shown that BGC823-ACE2-GFP cells account for 27.3% of the total number of cells in the lungs of mice; wherein the negative control group is a blank mouse (no injection of cells), wherein the GFP content is 2.81%;

[0079] HE staining results as Figure 6 As shown, it shows that after the injection of BGC823-ACE2-GFP cells, tumor lesions were found in the lungs of the mice, an...

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Abstract

The invention provides an ACE2 cell humanized mouse model as well as a construction method and application thereof. The mouse model adopts an immunodeficient mouse as a parent and the body of the mouse contains humanized cells for overexpressing an ACE2 receptor. The mouse model can overexpress the ACE2 receptor in human cells in the body of the immunodeficient mouse, the infection efficiency of acoronavirus taking the ACE2 receptor as an infection medium is improved, meanwhile, xenograft rejection can be avoided, and background pollution is reduced. In addition, the mouse model can also be used for transplantation and immune reconstruction of human immune cells, and is of great significance for further research on the killing effect of the human immune system on the virus, screening of vaccines and evaluation of immune treatment means.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to an ACE2 cell humanized mouse model and its construction method and application. Background technique [0002] Novel coronavirus (serve acute respiratory syndrome coronavirus 2, SARS-CoV-2), SARS coronavirus and MERS coronavirus belong to the genus Coronaviridae β. At the same time, the virus is also listed by the World Health Organization as the seventh virus to infect humans Coronavirus. The binding of SARS-nCOV-2 to the specific receptor of angiotensin-converting enzyme 2 (ACE2) on the surface of human cells is the first step in causing human infection with SARS-nCOV-2. Among them, ACE2 is the receptor of coronaviruses such as SARS and SARS-nCOV-2. The ACE2 gene is located on the human X chromosome, encodes 805 amino acid residues, belongs to type I transmembrane glycoprotein, and has a relative molecular mass of 120kDa. [0003] Studies have fo...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/867C12N5/10C12N15/65A01K67/027
CPCC12N15/8509C12N15/86C12N15/65C12N9/485A01K67/0271C12Y304/17023C12N2740/15043A01K2207/12A01K2227/105A01K2267/0337
Inventor 不公告发明人
Owner 湖南昭泰生物医药有限公司
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