CN111621523A - ACE2 cell humanized mouse model and construction method and application thereof - Google Patents
ACE2 cell humanized mouse model and construction method and application thereof Download PDFInfo
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Abstract
The invention provides an ACE2 cell humanized mouse model and a construction method and application thereof. The mouse model takes an immunodeficiency mouse as a parent, and human cells over expressing ACE2 receptors are contained in the body of the mouse model. The mouse model can over-express an ACE2 receptor in human cells in an immunodeficient mouse body, so that the infection efficiency of coronavirus taking the ACE2 receptor as an infection medium is improved, meanwhile, the generation of xenograft rejection can be avoided, and the background pollution is reduced. In addition, the mouse model can also be used for transplantation and immune reconstruction of human immune cells, and has important significance for further researching the killing effect of the human immune system on viruses, screening vaccines and evaluating immunotherapy means.
Description
Technical Field
The invention belongs to the technical field of animal genetic engineering, and particularly relates to an ACE2 cell humanized mouse model and a construction method and application thereof.
Background
The novel coronavirus (SARS-CoV-2), SARS coronavirus and MERS coronavirus belong to the family Coronaviridae, and the virus is classified as the seventh coronavirus infecting human by the world health organization. The first step of SARS-nCOV-2 infection in human is the combination of SARS-nCOV-2 and Angiotensin converting enzyme 2 (ACE 2) on human cell surface. Wherein, ACE2 is receptor of coronavirus such as SARS, SARS-nCOV-2, ACE2 gene is located in X chromosome of human, codes 805 amino acid residues, belongs to type I transmembrane glycoprotein, and has relative molecular mass of 120 kDa.
It was found that wild type mice are not naturally infected with COVID-19 virus at present, although their own ACE2 is also present in mice, and therefore researchers have been working on developing ACE2 transgenic mice and ACE2 knockout mice, among others. However, ACE2 transgenic mice require embryonic stem cell culture, and the process of establishing and culturing embryonic stem cell lines is complicated, and the modeling period is long. Meanwhile, the construction method of the ACE2 knockout mouse is complex in operation process and low in success rate. Furthermore, conventional transgenic or knockout mice contain immune cells of the mouse itself, such as T cells, B cells and NK cells, which are detrimental to the reconstitution of the humanized immune system and prevent the study of the relationship between virus and immune cells on humanized ACE2 mice.
Because the animal model of the novel coronavirus is less, the construction time is longer, and the novel coronavirus cannot be propagated in large quantity at present, the process of researching the novel coronavirus vaccine or the immunization method is limited. Therefore, the mouse model with short construction time and high infection efficiency of the novel coronavirus is of great significance for preventing and treating the novel coronavirus.
Disclosure of Invention
In view of the problems in the prior art, the invention provides an ACE2 cell humanized mouse model and a construction method and application thereof. The construction period of the ACE2 cell humanized mouse model is short, infection of novel coronavirus is facilitated, and effective help can be provided for further research and evaluation of anti-coronavirus immune cell therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an ACE2 cell humanised mouse model which is an immunodeficient mouse maternal and contains in vivo human cells overexpressing the ACE2 receptor. Wherein the immunodeficient mouse is a commercial mouse and can be purchased from a conventional biological laboratory equipment manufacturer.
The mouse model provided by the invention can over-express the ACE2 receptor in human cells in an immunodeficient mouse body, and can improve the infection efficiency of coronavirus (such as SARS-nCOV-2) taking the ACE2 receptor as a cell infection medium; meanwhile, human cells expressing human ACE2 receptors are taken as virus hosts, so that the basis for evaluating the antiviral effect of immune cells such as NK (natural killer) and the like is provided, the occurrence of xenograft rejection can be avoided, the background pollution is reduced, and the effect and the advantage of the mouse cells expressing the human ACE2 receptors cannot be replaced.
The immune deficient mice can be used for transplantation and immune reconstruction of human immune cells, and provide a foundation for development and screening of vaccines and immunotherapy. The ACE2 cell humanized mouse model provided by the invention is an important host animal model of coronavirus taking an ACE2 receptor as a cell infection vector, and the infection efficiency is very high and can reach 66%.
As a preferred embodiment of the present invention, the immunodeficient mice comprise NSI and/or NSIN mice.
Preferably, the immunodeficient mice comprise NOD/SCID IL2rg-/-Foxn 1-/-type and/or NOD/SCID 2 rg-/-type mice.
As a preferable technical scheme of the invention, the human source cell over expressing ACE2 receptor takes any one of BCG823, A549, Huh7, Jurkat, Nalm6, 293T or H1299 as a mother cell, and BCG823 is preferable.
In the invention, human cells expressing ACE2 receptors are taken as virus hosts, which is the basic for evaluating the antiviral effect of immune cells such as NK (natural killer) and the like, and cannot be replaced by mouse cells expressing human ACE2 receptors in the prior art, because the immune cells have xenograft rejection to cells of different species, background infection can be formed, and whether the immune effect is caused by xeno rejection or virus sensitive killing cannot be judged.
Wherein, when BCG823 is taken as a mother cell, ACE2 can be uniformly distributed in mouse lung, and is more obvious in alveolar tissue, which is beneficial to virus infection.
Preferably, the human cells overexpressing human ACE2 receptor are prepared by the following method: and mixing the lentivirus vector carrying the ACE2 gene with a packaging cell to prepare a lentivirus suspension, and mixing the lentivirus suspension with a mother cell to obtain the ACE2 over-expressing cell.
As a preferred technical scheme of the invention, the ACE2 gene is coupled with a Luciferase (Luciferase) gene. In the invention, when ACE2 is over-expressed, luciferase gene can be coupled, and in vivo tracing, infection and treatment condition evaluation can be carried out in immunodeficient mice.
Preferably, the ACE2 gene is coupled to the Green Fluorescent Protein (GFP) gene. In the invention, if the selected immunodeficient mouse is NOD/SCID IL2rg-/-Foxn1-/- (NSIN strain) mouse, a green fluorescent protein gene is coupled on an ACE2 gene, ACE2 and GFP are jointly over-expressed, and the immunodeficient mouse can also trace in vivo in a non-body-hair NSIN mouse.
The nude mouse used in the experiment of the invention does not contain T cells, B cells and NK cells, has extremely high immunodeficiency degree, and can improve the living body imaging effect of GFP and other autofluorescence due to the hairless characteristic of the nude mouse compared with a white mouse, thereby facilitating experimental research. However, the mouse usable for constructing the mouse model according to the present invention is not limited to the nude mouse.
Preferably, the mouse model also includes human immune cells in vivo. The human immune cells include NK immune cells or NK-like immune cells.
The novel coronavirus infects human cells through ACE2 receptor, thereby causing phenotype change of the human cells, and expresses ligand of NK cell killing receptor, thereby causing killing of NK cells. Human immune cells are implanted in a mouse model body, a humanized immune system can be reconstructed, and the method can be used for research and development of novel vaccines and treatment means of coronavirus, particularly cell treatment and provides an important model.
In a second aspect, the present invention provides a method for constructing a mouse model with humanized ACE2 cells according to the first aspect, wherein an immunodeficient mouse is used as a parent, and human cells over expressing human ACE2 receptors are transferred into the immunodeficient mouse, so as to obtain the mouse model with humanized ACE2 cells.
The preparation method provided by the invention is to implant a cell line over-expressing human ACE2 receptor into an immunodeficiency mouse body, so that an ACE2 cell humanized mouse model is quickly established, the experimental period is greatly shortened, and mass propagation can be carried out.
As a preferred technical scheme of the invention, the construction method comprises the following steps:
(1) mixing a lentiviral vector carrying an ACE2 gene with a packaging cell to prepare a lentiviral suspension, and mixing the lentiviral suspension with a mother cell to obtain the human cell over expressing an ACE2 receptor;
(2) transferring the human cells over expressing ACE2 receptor into an immunodeficiency mouse body by tail vein injection, and culturing to obtain the ACE2 cell humanized mouse model.
Preferably, the ratio of the amount of the mother cell to the lentivirus used in step (1) is 1 (8-12) TU, such as 1: 8TU, 1: 8.2TU, 1: 8.5TU, 1: 9TU, 1: 9.5TU, 1: 10TU, 1: 10.5TU, 1: 11TU, 1: 11.5TU or 1: 12TU, etc. in the present invention, the number of infection (MOI) is 10, and each 1 × 10 MOI is 106Adding 10 cells into each mother cell7TU total virus amount.
Preferably, the injection concentration of the human cells overexpressing ACE2 receptor in step (2) is (1 × 10)3～1×104) μ L, which may be, for example, 1 × 103mu.L, 2 × 103mu.L, 3 × 103mu.L, 4 × 103mu.L, 5 × 103mu.L, 6 × 103mu.L, 7 × 103mu.L, 8 × 103Mu L, 9 × 103mu.L or 1 × 104mu.L, etc., preferably 5 × 103mu.L in the present invention, human cells overexpressing ACE2 receptor were dispensed, 1 × 10 per tube6One, 200 μ L in volume, was injected into each mouse through the tail vein in 1 tube.
Preferably, the construction method further comprises the step of transferring human immune cells into the immunodeficient mouse.
Illustratively, the mouse model of the ACE2 cell humanization according to the present invention can be prepared by the following method:
(1) construction of human cells overexpressing the ACE2 receptor
Constructing a plasmid for over-expressing ACE2 receptor and Green Fluorescent Protein (GFP) by using a lentiviral vector, preparing a lentiviral supernatant suspension by using 293T cells, centrifuging and filtering the lentiviral supernatant, and detecting the virus titer by using Jurkat cells, wherein the titer (TU/mL) is equal to the cell number × fluorescence percentage × 103Volume of virus stock (μ L);
the multiplicity of infection (MOI) is 8-12, each 1 × 106BGC823 cells were added (8 × 10)6～1×107) Tu lentivirus infection; changing the culture solution after a period of infection, and continuing culturing; after 36-60 h, taking the infected cells to detect the infection efficiency by a flow cytometer;
and continuously carrying out amplification culture, and sorting GFP positive cells by using a flow cytometer to obtain the human cells over-expressing the ACE2 receptor.
(2) Construction of ACE2 cell humanized mouse model
Preparing a single cell suspension by taking a proper amount of the human cells over expressing the ACE2 receptor, and subpackaging the single cell suspension into sterile clean PCR tubes, wherein the injection concentration of the human cells over expressing the ACE2 receptor is (1 × 10)3～1×104) mu/L; the tail of the mouse is fixed, and the human source cells over expressing ACE2 receptor are injected into the mouse body through tail vein injection to obtain the mouse model of the ACE2 cell humanization.
Based on the construction method of the ACE2 cell humanized mouse model, the invention also provides a device for constructing the ACE2 cell humanized mouse model. The device comprises a transfection unit, an introduction unit and a feeding unit, wherein the transfection unit is used for mixing a lentivirus vector and a packaging plasmid to co-transfect packaging cells, the introduction unit is used for mixing a lentivirus solution and human cells and transferring the human cells into a mouse body, and the feeding unit is used for culturing the mouse, providing nutrition and medicines required by the mouse and finally culturing to obtain the ACE2 cell humanized mouse model.
In a third aspect, the present invention also provides the use of a mouse model of the humanization of ACE2 cells according to the first aspect for studying a novel coronavirus pathogenesis, screening immunotherapeutic methods, or assessing the effectiveness of immunotherapy.
The mouse model of ACE2 cell humanization provided by the invention has wider application, and can be used for the research of SARS-nCOV-2 pathogenesis, vaccine identification, drug screening and other non-diagnosis and treatment aspects.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the mouse model constructed by the invention selects an immune-deficient mouse, and a cell line over expressing a human ACE2 receptor is transplanted into the mouse body, so that the mouse model for ACE2 cell humanization is quickly established, the experimental period is greatly shortened, and a better biological model is provided for screening and evaluating an immunotherapy method; meanwhile, compared with primate animal models such as cynomolgus monkeys, the mouse model provided by the invention has the advantages of short construction period, low experiment cost and the like;
(2) in the invention, an immunodeficiency mouse is selected to highly express an ACE2 receptor in human cells in a mouse body, so that the generation of xenotransplantation rejection is avoided, background pollution is reduced, and virus infection efficiency is improved, experiments prove that when BGC823 is taken as a cell line for over-expressing the ACE2 receptor, BGC823 cells can be gathered in the lung of the mouse, so that the infection of viruses is facilitated, and the final result shows that the infection rate of SARS-nCOV-2 to the mouse model provided by the invention can reach 66%; meanwhile, the vaccine can also be used for transplantation and immune reconstruction of human immune cells, and has important significance for further researching the killing effect of the human immune system on viruses and screening and evaluating vaccines and immunotherapy means.
Drawings
FIG. 1 is a graph showing the infection efficiency of BGC823 cells with lentiviruses detected by the flow cytometer in example 1.
FIG. 2 is an immunoblot assay of BGC823-GFP and ACE2 protein in BGC823-ACE2-GFP cells in example 1.
FIG. 3 is an in vivo imaging assay of the BGC823-GL mouse model of example 1.
FIG. 4 is an in vivo imaging assay of the BGC823-ACE2-GFP mouse model in example 1.
FIG. 5 is a graph showing the results of flow cytometry on the number of BGC823-ACE2-GFP cells in the mouse lung in example 3.
FIG. 6 is a graph showing the results of HE staining of the lungs of the mouse in example 3.
FIG. 7 is a graph showing the results of immunofluorescence staining of the lungs of the mouse in example 3.
FIG. 8 is a histogram of the amount of viral RNA detected by fluorescent quantitative PCR in the mouse model after the administration of rhinovirus of example 4.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
In the following examples, unless otherwise specified, the experimental methods or detection methods used are those conventional in the art.
Example 1
The invention provides an ACE2 cell humanized mouse model, which is constructed by the following steps:
(1) constructing a BGC823-ACE2-GFP stable cell strain:
firstly, constructing plasmids of over-expression ACE2 receptor and Green Fluorescent Protein (GFP) by using a lentiviral vector, and preparing a lentiviral supernatant suspension by using 293T cells;
② will be slowAfter centrifugation and filtration of the virus supernatant, virus titer detection is carried out by using Jurkat cells, wherein the titer (TU/mL) is the cell number × fluorescence percentage × 103Volume of virus stock (μ L);
③ multiplicity of infection (MOI) 10, every 1 × 10610 was added to each of BGC823 cells7Tu lentivirus infection;
fourthly, changing the liquid after 6 hours of infection, and continuing to culture; after 48 hours, taking the infected cells to detect the infection efficiency by a flow cytometer; the results of the infection efficiency test are shown in FIG. 1, in which the GFP content was 42.9%.
Continuously carrying out amplification culture, sorting GFP positive cells by using a flow cytometer, and detecting the ACE2 protein level in the sorted GFP positive cells by using immunoblotting; the results are shown in FIG. 2, which shows that BGC823 wild type (i.e., BGC823-GFP) does not express ACE2, while BGC823-ACE2-GFP is capable of high expression of ACE 2.
Thus, the resulting positive cells overexpress the ACE2 receptor and GFP, abbreviated BGC823-ACE2-GFP cells.
(2) Construction of mouse chimeric humanized ACE2 lung model
The nude mice used in this example were from Hunan Shoutai biomedical Co., Ltd (patent No: CN110951787A), and were T-cell-, B-cell-and NK-cell-free immunodeficient mice having an extremely high degree of immunodeficiency. Compared with a mouse, the nude mouse can improve the living imaging effect of GFP and other autofluorescence due to the hairless characteristic, and is convenient for experimental research.
① A single cell suspension was prepared from the BGC823-ACE2-GFP cells constructed in step (1) and aliquoted into sterile clean PCR tubes, 1 × 10 per tube6Volume of 200 μ L, 1 tube per mouse;
meanwhile, BGC823-GFP-Luciferase (abbreviated as BGC823-GL, and the construction method is the same as the step (1) by overexpressing GFP and Luciferase, and the group is used as a positive control group)
Fixing the tail of the mouse, and injecting the tumor cell BGC823-ACE2-GFP into the mouse body through tail vein injection;
thirdly, after modeling for 1 week, respectively imaging through GFP and Luciferase channels of living body imaging;
the results are shown in FIG. 3, where the signal represents the GFP-Luciferase signal, and after one week of modeling by BGC823-GL, a stronger Luciferase signal was detected in the mouse model lung; meanwhile, as shown in FIG. 4, the signal in the figure indicates ACE2-GFP signal, i.e., it was shown that GFP signal was also successfully detected in mice injected with BGC823-ACE2-GFP cells, but the GFP signal was not significant.
Example 2
This example facilitated the step (1) in example 1 of constructing ACE2 receptor overexpressing cells that are blast cells, with Jurkat, NALM6, Huh7, a549, and H1299 as mother cells;
the infection efficiency and the infection efficiency after sorting are shown in table 1.
TABLE 1
| Cell name | Efficiency of infection | Infection efficiency after sorting |
| ACE2- |
90％ | --- |
| ACE2- |
90％ | --- |
| ACE2-H1299 cells | 44％ | 90％ |
| ACE2-Jurkat cells | 50％ | 90％ |
| ACE2-Nalm6 cell | 17％ | 90％ |
As can be seen from the above table, when a549 and Huh7 were used as the mother cells, the infection efficiency was up to 90%, and the infection efficiency of the remaining three cells was not up to 90%, but the infection efficiency after sorting was up to 90%.
Example 3
In the embodiment, the distribution of ACE2 positive cells in the lung of a mouse is observed by a pathological detection method.
After the mice are killed, taking the lungs of the mice to take a picture under a microscope, and observing GFP signals; and then, one part of the lung is used for preparing single cell suspension, the BGC823 cell proportion of the mouse lung is detected by using a flow cytometer, and the other part of the lung is used for preparing paraffin sections for HE staining and immunofluorescence detection.
The flow cytometry results are shown in FIG. 5, which shows that BGC823-ACE2-GFP cells account for 27.3% of the total cells in the mouse lung; wherein the negative control group is a blank mouse (without injected cells), and the GFP content is 2.81%;
the HE staining results are shown in FIG. 6, which indicates that after BGC823-ACE2-GFP cells are injected, tumor foci are found in the lung of the mouse, and tumor accumulation is found in the alveoli; over-expression of ACE2 in mouse lung is beneficial to infection of virus and improves infection efficiency.
The immunofluorescent staining results are shown in FIG. 7, wherein the results from left to right are respectively the results of the immunofluorescent staining of BGC823-ACE2-GFP intracellular ACE2, DNA binding with DAPI (4', 6-diamidino-2-phenylindole) and a combination of the two, which are observed under different channels, and the results show that the ACE2 is uniformly distributed in mouse lungs and is more obvious in alveolar tissues.
By combining the embodiment 1 and the embodiment 3, the mouse lung model of the chimeric humanized ACE2 can be quickly and effectively established, and the human cells over expressing ACE2 can be gathered in mouse lungs, particularly alveolar tissues, so that the infection of viruses is facilitated, and the infection efficiency is improved.
Example 4
In this example, a novel coronavirus was used to infect a humanized ACE2 mouse model to explore the infection efficiency.
The humanized ACE2 mouse model constructed in example 1 is sent to a P3 laboratory, infected with the novel coronavirus SARS-nCOV-2 by nasal drip, and the mouse is dissected after 24h, and lung tissue is taken out for fluorescent quantitative PCR detection.
The results are shown in FIG. 8, and the viral RNA was detected in 2 of 3 experimental mice infected with the novel coronavirus, indicating that 2 mice successfully infected with the novel coronavirus and the infection efficiency was 66%. In the experiment, due to the particularity of the novel coronavirus, although 3 mice are only adopted to detect the infection rate, the infection efficiency can still be reflected to be higher.
In conclusion, the ACE2 gene can be transfected into various human cell lines such as BGC823, Jurkat, NALM6, Huh7, A549 and H1299 by using a lentiviral vector, and the transfection efficiency is high. Meanwhile, when the obtained BGC823-ACE2-GFP is transplanted into a mouse body, the cells are only gathered in the lung of the mouse, ACE2 expression is detected in the alveoli of the lung of the mouse, and the lung overexpression is beneficial to infection of novel coronavirus; the mouse model provided by the invention is an immune deficient mouse, is beneficial to reconstructing a humanized immune system, and further provides a basis for researching the relationship between the novel coronavirus and the immune system.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. A mouse model of ACE2 cell humanization, wherein the mouse model is generated from an immunodeficient mouse and comprises in vivo human cells overexpressing ACE2 receptor.
2. The mouse model of claim 1, wherein the immunodeficient mouse comprises an NSI and/or NSIN type immunodeficient mouse;
preferably, the immunodeficient mice comprise NOD/SCID IL2rg-/-Foxn 1-/-type and/or NOD/SCID 2 rg-/-type mice.
3. The mouse model according to claim 1 or 2, wherein the human cell overexpressing the ACE2 receptor is a mother cell selected from any one of BCG823, a549, Huh7, Jurkat, Nalm6, 293T, or H1299, preferably BCG 823.
4. The mouse model of any one of claims 1 to 3, wherein the human cells overexpressing the ACE2 receptor are prepared by the following method:
mixing a lentiviral vector carrying an ACE2 gene with a packaging cell to prepare a lentiviral suspension, and mixing the lentiviral suspension with a mother cell to obtain the human cell over expressing an ACE2 receptor;
preferably, the ACE2 gene is coupled to a luciferase gene;
preferably, the ACE2 gene is coupled to the green fluorescent protein gene.
5. The mouse model of any one of claims 1 to 4, further comprising human immune cells in vivo.
6. A construction method of the ACE2 cell humanized mouse model as claimed in any one of claims 1 to 5, characterized in that an immunodeficiency mouse is used as a parent, and a human cell over expressing ACE2 receptor is transferred to the immunodeficiency mouse to obtain the ACE2 cell humanized mouse model.
7. The construction method according to claim 6, characterized in that it comprises the steps of:
(1) mixing a lentiviral vector carrying an ACE2 gene with a packaging cell to prepare a lentiviral suspension, and mixing the lentiviral suspension with a mother cell to obtain the human cell over expressing an ACE2 receptor;
(2) transferring the human cells over expressing ACE2 receptor into an immunodeficiency mouse body by tail vein injection, and culturing to obtain the ACE2 cell humanized mouse model.
8. The method for constructing a recombinant human embryonic stem cell of claim 6 or 7, wherein the ratio of the use amount of the blast cells to the use amount of the lentiviruses in the step (1) is 1 (8-12) TU;
preferably, the injection concentration of the human cells overexpressing ACE2 receptor in step (2) is (1 × 10)3～1×104) mu.L/L.
9. The method of construction according to any one of claims 6 to 8, further comprising the step of transferring human immune cells into the immunodeficient mouse.
10. Use of a mouse model of ACE2 cell humanization according to any one of claims 1 to 5 for studying a novel coronavirus pathogenesis, screening immunotherapeutic methods, or assessing the effectiveness of immunotherapy.
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