WO2021225384A1 - Animal model transgenic pig having increased susceptibility to severe acute respiratory syndrome coronavirus 2, and production method therefor - Google Patents

Abstract

The present invention relates to a transgenic cell line and a transgenic pig which are produced by preparing and using a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2) in pigs. According to the present invention, the transgenic cell line and transgenic pig express human ACE2 and have high susceptibility to SARS-CoV-2, and thus can be effectively used as a non-clinical animal model for researching SARS-CoV-2 or for developing prophylactic or therapeutic agents for infection of the virus.

Description

Transgenic pig for animal model with increased susceptibility to type 2 severe acute respiratory syndrome coronavirus and manufacturing method thereof

The present invention has increased sensitivity to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), a trait that can be used for non-clinical evaluation of SARS-CoV-2 infectious diseases It relates to a converted pig and a method for preparing the same.

Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a virus first discovered in Wuhan City, Hubei Province, China in 2019. belongs to the coronavirus (Coroviridae) family and is a virus similar to SARS-CoV (severe acute respiratory syndrome coronavirus) and MERS coronavirus (MERS-CoV, Middle East respiratory syndrome coronavirus). is known Although the source and route of infection have not been confirmed yet, it has been reported that infection is highly likely through contact with bats in the Wuhan area and transmission is possible through close person-to-person contact.

However, until now, no treatment has been developed that shows a clear therapeutic effect on SARS-CoV-2 infection, so the treatment is focused on symptoms. Therefore, research on the prevention or treatment of SARS-CoV-2 infection is being actively conducted.

On the other hand, SARS-CoV-2 has a spike protein protruding like a crown on the outside, and the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) gene and acts as a cell entrapment. It has been reported to cause infection by endocytosis (Daniel Wrapp et al., Science, 2020). In humans and other species, ACE2 genes have similar amino acid sequences, but the corona virus dendritic protein binds specifically to the human ACE2 gene. Therefore, it is recommended to use a transgenic model in which human ACE2 is inserted in order to show sensitivity similar to that of humans in non-clinical testing of SARS-CoV-2 infection using animal models.

Therefore, although a mouse animal model expressing human ACE2 has been developed, in the animal disease model, the pathology and symptoms of the animal disease model are very different from those observed in humans, so clinical trials based on the results from the rodent disease model There have been many problems when implementing it.

Therefore, the need for a disease animal model using a species closer to humans has been raised due to the clear differences in dimenzone, reproduction, lifespan, and behavior from humans. In the case of primates, there are restrictions that can be used for disease research only in extremely limited fields due to their scarcity and cost and difficulty of breeding management. As a model animal, the demand for use in the biomedical field is increasing.

Pigs have been recognized anatomically and physiologically similar to humans, and are already being used in research for histopathological mechanisms and treatment of various diseases. Ethical problems can be avoided, a stable breeding system is established, and the gestation period is short, so maintenance and management are easy when developing experimental animal models.

Therefore, in order to develop an effective preventive or therapeutic agent for SARS-CoV-2 infection, the development of an animal model using pigs is required.

The present inventors were studying for the development of an animal model that increased sensitivity to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), human angiotensin converting enzyme 2 (Angiotensin- The present invention was completed by preparing a transgenic cell line expressing converting enzyme 2, ACE2) and a transgenic pig, and confirming that the sensitivity of the transgenic cell line and the transgenic pig to SARS-CoV-2 was increased compared to that of the wild type.

Therefore, an object of the present invention is to provide a recombinant vector for making an animal model with increased sensitivity to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2).

Another object of the present invention is to provide a transformed cell line with increased sensitivity to SARS-CoV-2.

Another object of the present invention is to provide a nuclear transfer embryo for the production of transgenic pigs for animal models with increased sensitivity to SARS-CoV-2.

Another object of the present invention is to provide a transgenic pig for animal models with increased sensitivity to SARS-CoV-2.

Another object of the present invention is to provide a method for producing a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2.

Another object of the present invention is to provide a screening method for preventing or treating a SARS-CoV-2 infectious disease.

In order to achieve the above object, the present invention is human angiotensin-converting enzyme 2 (Angiotensin-converting enzyme 2, ACE2) gene, including an enhancer and a promoter, type 2 severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, Provided is a recombinant vector for making an animal model with increased sensitivity to SARS-CoV-2).

In addition, in order to achieve the above another object, the present invention provides a transformed cell line with increased sensitivity to SARS-CoV-2, prepared by transforming a somatic cell with the recombinant vector according to the present invention.

In addition, in order to achieve the above another object, the present invention is a transgenic pig production for an animal model with increased sensitivity to SARS-CoV-2, in which the transgenic cell line according to the present invention is introduced into a fertilized porcine egg to be nuclear-transferred. Provides nuclear transfer embryos for

In addition, in order to achieve the above another object, the present invention provides a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transgenic cell line according to the present invention.

In addition, in order to achieve the above another object, the present invention provides a nuclear transfer step of preparing a reconstituted egg by transplanting the transformed cell line according to the present invention into a denucleated egg; and transplanting the reconstituted egg into the fallopian tube of a surrogate mother.

In addition, in order to achieve the above another object, the present invention provides a transgenic cell line with increased sensitivity to SARS-CoV-2 or a transgenic pig for animal models with increased sensitivity to SARS-CoV-2 according to the present invention. Provided is a screening method for a preventive or therapeutic agent for SARS-CoV-2 infection.

The present invention relates to a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2), a transgenic cell line expressing human ACE2 using the same, a nuclear transfer egg for producing a transgenic pig, a transgenic pig, and the above-mentioned trait A method for producing a transgenic pig and its use, wherein the transgenic pig expressing human ACE2 shows a high sensitivity to SARS-CoV-2, which can be used in a study on SARS-CoV-2 or the virus-infected disease. It can be usefully used as a non-clinical animal model for the development of prophylactic or therapeutic agents.

1 is a schematic diagram of a recombinant vector for expressing human angiotensin converting enzyme 2 (Angiotensin-converting enzyme 2, ACE2) according to the present invention.

Figure 2 shows whether the human ACE2 vector according to the present invention was transduced into female and male pig ear fibroblasts, and the transduction was confirmed. FLAG protein expression was confirmed, and Figure 2c is a confirmation of the ACE2 protein expression of each cell.

3 is a result of confirming the expression of human ACE2 in the transformed cell line according to the present invention.

Figure 4 confirms the expression of human ACE2 in the transformed cell line according to the present invention, Figure 4a confirms the expression of the human ACE2 protein, Figure 4b is the expression of the FLAG protein tagged to the human ACE2 gene (WT: fiber) blast cell line, TG: transformed cell line).

5 shows the sensitivity to SARS-CoV-2 of 4 transformed cell lines according to the present invention (WT: fibroblast cell line, TG: transformed cell line).

6 is a photograph of cloned eggs obtained by substituting a somatic cell nucleus in a transformed cell line according to the present invention with a donor cell.

7 shows the results of genetic analysis of transgenic cloned pigs produced using the cloned embryos (PC: donor cells, NC: wild-type Yucatan mini-pigs, #1 to #7: transgenic cloned pigs).

8 shows the GFP fluorescence of ear fibroblasts of wild-type pigs and transgenic cloned pigs according to the present invention.

9 shows the chromosomes of a wild-type pig and a transgenic cloned pig according to the present invention. FIGS. 9a and 9b are results of chromosome analysis of two cloned transgenic pigs, and FIG. 9c is a chromosome analysis result of a wild-type pig.

10 shows the results of confirming the expression of FLAG tagging protein and human ACE2 protein in ear fibroblasts derived from wild-type pigs and transgenic cloned pigs according to the present invention (PC: donor cells, NC: wild-type pig-derived fibroblasts, TG1) and TG2: ear fibroblasts derived from transgenic cloned pigs).

11 shows the results of confirming the expression of human ACE2 protein in the organs of wild-type pigs and transgenic cloned pigs according to the present invention (PC: donor cells, WT (wild-type), TG (transgenic cloned pigs).

12 is a comparison of SARS-CoV-2 clinical symptoms of wild-type pigs and transgenic cloned pigs according to the present invention. (Control: wild-type pigs, ACE1 and ACE2: transgenic cloned pigs).

13 is a result of confirming the release of virus from the nasal passages of wild-type pigs and transgenic cloned pigs according to the present invention, FIG. 13a is the result of confirming Egene RNA, and FIG. Pigs, ACE1 and ACE2: transgenic cloned pigs, D1: 1 day after inoculation, D2: 2 days after inoculation, D3: 3 days after inoculation, D4: 4 days after inoculation, D5: 5 days after inoculation).

14 is a comparison of histopathological symptoms in lung tissues of wild-type pigs and transgenic cloned pigs according to the present invention (WT: wild-type pig, hACE2#1 and hACE2#2: transgenic cloned pig).

Hereinafter, the present invention will be described in detail.

The present invention is human angiotensin-converting enzyme 2 (Angiotensin-converting enzyme 2, ACE2) gene, including an enhancer and promoter, type 2 severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) It provides a recombinant vector for the production of animal models with increased sensitivity to .

In the present invention, the "human angiotensin-converting enzyme 2 (Angiotensin-converting enzyme 2, ACE2)" is one of metallo-carboxypeptidase, a type 1 transmembrane protein homologous to angiotensin converting enzyme, It is found in eukaryotes and bacteria. Angiotensin-converting enzyme 2 plays an important role in the renin-angiotensin-aldosterone system (RAAS) that regulates water and blood pressure in the body, and is expressed in the heart, lungs, kidneys, vascular endothelium, and digestive system.

In the present invention, the human angiotensin converting enzyme 2 (ACE2) gene acts as a receptor binding domain (RBD) of the spike-protein of SARS-CoV-2. .

In the present invention, the sequence encoding the human angiotensin converting enzyme 2 is preferably represented by the nucleotide sequence of SEQ ID NO: 1. In addition, the recombinant vector for making an animal model with increased sensitivity to SARS-CoV-2 may include all or part of the sequence of human angiotensin converting enzyme 2 represented by the nucleotide sequence of SEQ ID NO: 1, and a functional equivalent thereof may include The "functional equivalent" means at least 70% or more, preferably 80% or more, of the base sequence of SEQ ID NO: 1 as a result of deletion, substitution, or insertion of a base, more preferably It refers to a polynucleotide having substantially the same physiological activity as the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 as having sequence homology of 90% or more, more preferably 95% or more. The "% of sequence homology" for a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion of additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (ie, gaps) compared to (not including).

In the present invention, the "vector" refers to a gene construct comprising the nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host, wherein the regulatory sequence is a capable promoters, optional operator sequences for regulating such transcription, and sequences regulating the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it can replicate within a cell, and any vector known in the art may be used, for example, a plasmid, cosmid, phage particle, or viral vector.

In the present invention, the "recombinant vector" is an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell when the coding gene of the target polypeptide to be expressed is operably linked. can be used, and the recombinant vector can be expressed in a host cell. The host cell may preferably be a eukaryotic cell, and an expression control sequence such as a promoter, terminator, enhancer, etc., a sequence for membrane targeting or secretion, etc. is appropriately selected according to the type of host cell. and can be combined in various ways depending on the purpose.

In the present invention, the "enhancer" is a part that induces a structural change of a DNA template so that transcription occurs more actively. The enhancer is represented by a unique nucleotide sequence for each gene, and has a feature of promoting transcription at any position in the gene.

In the present invention, "promoter" refers to a DNA sequence site to which transcriptional regulators bind, and is intended to induce overexpression of a target gene. Examples of the promoter include a Pribnow box, a TATA box, and the like.

In the present invention, the promoter may be any one exogenous promoter selected from the group consisting of CAG promoter, CMV promoter, EF1α promoter, ICAM2 promoter, SV40 promoter, PGK1 promoter, Ubc promoter and β-Act promoter. , but is not limited thereto. In the present invention, the human ACE2 gene may be introduced into the host by a random integration or knock-in method using the exogenous promoter, but is not limited thereto.

In the present invention, the recombinant vector has a vector map described in the following figure, and is not limited thereto, as long as it is a vector capable of expressing human ACE2 of the present invention.

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In addition, in the present invention, the recombinant vector preferably includes a sequence represented by the nucleotide sequence of SEQ ID NO: 2, but this is only an example and the present invention is not limited thereto. SEQ ID NO: 2 includes a tag protein and a sequence encoding hACE2.

According to an embodiment of the present invention, a recombinant vector containing the human ACE2 gene of SEQ ID NO: 1, CAGGS promoter and EGFP fluorescent protein in pCMV-5' FLAG vector, represented by the nucleotide sequence of SEQ ID NO: 3 was used (Fig. 1 see).

In the present invention, the promoter may be any one endogenous promoter selected from the group consisting of GGTA1 (alpha 1,3-galactosyltransferase) promoter, ROSA26 promoter and AAVS1 (PPP1R12C locus) promoter, but is limited thereto no.

The GGTA1 is a gene unique to pigs that is well expressed in whole organs and blood, that is, a foreign gene is inserted into the sequence encoding the GGTA1 protein between the left arm and the right arm, and the GGTA1 It is intended to be expressed using a unique promoter of the gene. The ROSA26 or AAVS1 is a locus in which a gene is safely and stably well expressed when a foreign gene is inserted. is the location

In the present invention, “left arm” and “right arm” refer to regions in which homologous recombination occurs.

In the present invention, "homologous recombination" refers to a gene recombination that occurs through exchange at a locus with homology, and is used for the production of a transgenic animal having an allele with a loss of function.

In the present invention, the human ACE2 gene may be introduced into the host by a knock-in method using the endogenous promoter, but is not limited thereto.

In the present invention, the recombinant vector has a vector map described in the following figure, and is not limited thereto, as long as it is a vector capable of expressing human ACE2 of the present invention.

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The vector map of the figure is a vector containing an endogenous promoter, and is characterized in that human ACE2 is expressed using its own promoter.

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The vector map of the figure shows a vector including an exogenous promoter for the knock-in system.

The vector of the present invention is not particularly limited as long as it can replicate within an individual or cell, and any vector known in the art may be used, and the vector that can be used in the present invention includes a viral vector, a plasmid vector. , cosmid vector, fosmid vector, phage vector, BAC (bacterial artificial chromosome) vector, bacteriopharge vector, PAC (P1-based artificial chromosome) vector and It may be one or more selected from the group consisting of YAC (yeast artificial chromosome) vectors.

In addition, the viral vector is not limited thereto, but infection using any one selected from the group consisting of lentivirus, adenovirus, retrovirus, poxvirus, herpes virus, alphavirus, pomivirus and adeno-associated virus (infection) The human ACE2 gene can be introduced through

The present invention is for the preparation of an animal model for research or non-clinical testing of SARS-CoV-2, and the term "animal" means any mammal other than humans. Such animals include animals of all ages, including embryos, fetuses, newborns and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (eg, mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (eg, chickens, turkeys, ducks, geese), and primates (eg, chimpanzees, monkeys, rhesus monkeys). It is most preferred that the pigs have high anatomical and physiological similarities to humans.

In addition, the present invention provides a transformed cell line with increased sensitivity to SARS-CoV-2, prepared by transforming the recombinant vector according to the present invention into a somatic cell.

In the present invention, the "cell line" refers to each individual of the cell line when cells are isolated, pure culture, and subcultured, and at this time, the cell line can be distinguished from other cell lines by genetic traits, and the original cell traits even in subculture It says to keep

In the present invention, the cell line may be an oocyte line, a fibroblast line or a kidney cell line, preferably a fibroblast cell line. The cell line can more specifically use a fetal-derived cell line, and at the same time a primary cell line can be used. It is most preferred to use a primary fibroblast line.

In the present invention, the "transformation" refers to a change in the genetic properties of an organism by DNA given from the outside, and is also referred to as transformation, transformation or transformation. That is, "transformation" means introducing a gene into a host cell so that it can be expressed in the host cell.

The method of transforming by introducing the recombinant vector for preparing an animal model with increased sensitivity to SARS-CoV-2 of the present invention into a cell line may be introduced into a host cell using a method known in the art. Methods for introducing the recombinant vector according to the present invention into a host cell include, but are not limited to, nucleofection, transient transfection, microinjection, transduction, cell fusion, calcium phosphate Precipitation method, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, PEG (polyethylenglycol) It may be a precipitation method, particle gun bombardment, sonication, electroporation, or heat shock. In an embodiment of the present invention, transformation was performed by a lipofection method using a cationic lipid.

In the present invention, the transformed cell line was deposited with the Korea Cell Line Research Foundation (KCLRF) on May 04, 2021, and was given an accession number KCLRF-BP-00512.

The transformed cell line may be propagated and cultured according to methods known in the art to which the present invention pertains. A suitable medium is any use that can be developed for the culture of animal cells and, in particular, mammalian cells, or prepared in the laboratory with appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and/or micronutrients. Any available medium may be used.

The medium may be any basic medium suitable for animal cell growth, as a non-limiting example, as a basic medium generally used for culture is MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memory Institute Medium). ), K-SFM (Keratinocyte Serum Free Medium), and in addition, any medium used in the industry may be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM/F12 medium, PCM medium, M199/F12 (mixture) (GIBCO) and MSC expansion medium (Chemicon).

To this basal medium may be added anabolic sources of carbon, nitrogen and micronutrients, including but not limited to, serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources. It will be apparent that a person skilled in the art to which the present invention pertains can appropriately culture by a known method by selecting or combining the most suitable medium for stem cells derived from various tissue origins.

In addition, it is apparent that culture can be performed while controlling conditions such as a suitable culture environment, time, and temperature based on common knowledge in this field.

In addition, the present invention provides a nuclear transfer egg for the production of a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2, which is nuclear-transferred by introducing the transformed cell line according to the present invention into a denucleated fertilized swine egg.

In the present invention, the "nuclear transfer" refers to a genetic manipulation technique that artificially combines other cells or nuclei to a denucleated egg to have the same trait. "Nuclear transfer embryo" refers to an egg into which a nuclear donor cell is introduced or fused, and the nuclear transfer embryo may refer to a cloned egg.

In the present invention, the "denucleated egg" means an egg from which the nucleus has been removed.

In the present invention, the "cloning" is a genetic manipulation technique to create a new individual having the same gene set as that of one individual. It refers to having a nuclear DNA sequence substantially identical to a nuclear DNA sequence. The present invention utilizes the technique of cloning pigs using nuclear transfer technology. In particular, somatic cell nuclear transfer technology is a technology that can give birth to offspring without going through meiotic division and haploid chromosome-bearing germ cells, which are generally performed in the reproductive process. It is a method of generating a new individual by producing and transplanting the fertilized egg into a living body.

In the present invention, the "nuclear donor cell" refers to a cell or nucleus of a cell that transfers a nucleus to a nuclear receptor, a nuclear recipient. "Oocyte" preferably refers to a mature egg that has reached the middle stage of secondary meiosis. In the present invention, as the nuclear donor cells, somatic cells or stem cells of pigs may be used.

The “somatic cell” is a cell other than a germ cell among cells constituting a multicellular organism, a differentiated cell that is specialized for a certain purpose and does not become a cell other than that, and a cell having several different functions cells having the ability to differentiate into

The present invention also provides a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transgenic cell line according to the present invention.

In the present invention, the pig may use any known kind that can be appropriately selected and used by those skilled in the art. The pig (scientific name: Susscrofadomestica) belongs to the family Sua, Jinshu, supra, and oxi (including cattle and goats), and the boar family, and the number of chromosomes is 2n=38. Originally, pigs are omnivores raised as livestock for meat, but in recent years, due to the fact that their physiological and anatomical findings are similar to those of humans, and with high interest in animal welfare, they are also receiving great attention as experimental animals. Pigs are anatomically similar in size to humans, so they are suitable as a heterogeneous organ model, but also have many physiological and genetic similarities to humans. In addition, since it has a high reproductive ability, it is suitable for the development of new biological materials for production and treatment, as well as a good animal model for toxicity and safety evaluation.

In the present invention, a mini-pig (representative example: Pitman, Moore) having a maximum weight of 60-70 kg close to that of a human is used, or a small pig (adult weight: 30-40 kg) that is easier to use in experiments Göttingen type, Yucatan-based mini-pigs, micro-pigs, etc. can also be used. Alternatively, inbred mini-pigs (NIH-based) with clear characteristics as well as a small body type can be used. In an embodiment of the present invention, a Yucatan mini pig was used.

In addition, the present invention provides a nuclear transfer step of preparing a reconstituted egg by transplanting the transformed cell line according to the present invention into a denucleated egg; and transplanting the reconstituted egg into the fallopian tube of a surrogate mother.

In this regard, the human ACE2 gene may be transformed using any known method, and preferably, somatic cell nuclear transfer (SCNT) may be used. That is, it is possible to produce transgenic pigs for animal models with increased sensitivity to SARS-CoV-2 of the present invention by using a somatic cell nuclear transfer (SCNT) method using a transgenic cell line expressing the human ACE2 gene.

According to an embodiment of the present invention, the transgenic cell line expressing human ACE2 according to the present invention or the transgenic pig expressing human ACE2 showed higher sensitivity compared to the wild type when infected with SARS-CoV-2.

Therefore, the transgenic cell line expressing human ACE2 or the transgenic pig expressing human ACE2 according to the present invention can be usefully utilized as a non-clinical model for research on SARS-CoV-2 or for the prevention or development of therapeutic agents for the virus-infected disease. can

Therefore, the present invention is a method of SARS-CoV-2 infection using a transgenic cell line with increased sensitivity to SARS-CoV-2 or a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2 according to the present invention. A method for screening a prophylactic or therapeutic agent is provided.

More specifically, the method comprises the steps of:

1) inoculating SARS-CoV-2 into a transgenic cell line with increased sensitivity to SARS-CoV-2 or a transgenic pig for animal model with increased sensitivity to SARS-CoV-2 according to the present invention;

2) administering a candidate substance for the prevention or treatment of SARS-CoV-2 infection disease to the transgenic cell line or transgenic pig inoculated with SARS-CoV-2; and

3) Comparing the group to which the candidate substance was administered and the control group to which the candidate substance was not administered, selecting a candidate substance that significantly changes the symptom or histopathological indicator value of SARS-CoV-2 infection.

The symptom or histopathological indicator value may be a known clinical symptom or histopathological symptom appearing during SARS-CoV-2 infection. Although not limited thereto, the clinical symptoms may be any one or more selected from the group consisting of fever, dyspnea, headache, sore throat, hemoptysis, nausea, cough, sputum, diarrhea, loss of smell, malaise, anorexia, muscle pain and cognitive impairment. .

In addition, the histopathological index value may be any one or more selected from the group consisting of secretion of inflammatory cytokines, edema, local bleeding, and infiltration of inflammatory cells, but is not limited thereto.

In addition, the histopathological indicator value may be evaluated in various tissues affected by SARS-CoV-2 infection or tissues from which SARS-CoV-2 can be released, but is not limited thereto, but is not limited to lung, liver, brain, It may be evaluated in any one or more tissues selected from the group consisting of nasal passages, scalp, eyes, kidneys, heart and intestines.

In addition, the present invention may provide a method for producing human ACE2 protein using the transformed cell line expressing human ACE2 according to the present invention.

In addition, the present invention comprises the steps of culturing a transformed cell line expressing human ACE2 according to the present invention; And it may provide a method for producing human ACE2 protein comprising the step of obtaining the human ACE2 protein expressed from the cultured medium or the transformed cell line expressing the cultured human ACE2.

In the present invention, the "cultivation" means to grow cells in an artificially controlled environment. The cells can be grown in a conventional medium. The medium contains nutrients required by the cells to be cultured, that is, to be cultured in order to culture specific cells, and may be mixed with a material for a specific purpose additionally added. The medium is also referred to as an incubator or culture medium, and is a concept including all of natural medium, synthetic medium, or selective medium.

The above-described contents of the present invention are applied equally to each other unless contradictory to each other, and those skilled in the art with appropriate modifications are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited only to the following examples.

Example 1. Preparation of recombinant vector for expression of human angiotensin-converting enzyme 2 (ACE2)

After analyzing the nucleotide sequence for human ACE2 gene expression, the human cDNA library was amplified as a template strand. More specifically, cloning was performed so that the EGFP fluorescent protein could be expressed by the human ACE2 gene of SEQ ID NO: 1 and the CAGGS promoter using restriction enzymes in the pCMV-5' FLAG vector. The schematic diagram of the vector is shown in FIG. 1, and the complete vector sequence analysis was performed to confirm whether or not the gene was inserted (performed by Solgent). The entire vector sequence is shown in SEQ ID NO:3.

Example 2. Construction of a transgenic cell line expressing ACE2

The recombinant vector prepared in Example 1 was transduced into wild-type female and male Yucatan mini-pig ear fibroblasts using lipofectamine 3000 (Invitrogen, CA, USA). More specifically, the ears of healthy wild-type Yucatan mini-pigs were wiped with 70% ethanol and then ear tissues were obtained using a bivalve. After transporting it to the laboratory in DPBS containing 1X anti-anti (antibiotic, Gibco, NY, USA), attach the tissue to a 6-well multidish, and PEF) in DMEM (Welgene, Korea) medium containing 15% fetal bovine serum (Hyclone, Utah, USA) and 1X penicillin/streptomycin (Gibco, NY, USA) Separately cultured. The day before transduction, 1 × 10 ⁶ cells were transferred to a 10 cm cell culture dish and transduced the next day according to the protocol of Lipofectamine 3000. The introduced cells were sorted by a fluorescence activated cell sorter (FACS AriaIII, BDbioscience, CA, USA) using FLAG tagging protein, and the results are shown in FIGS. 2A to 2C .

As a result, as shown in FIGS. 2a to 2 , it was confirmed that the FLAG protein was well expressed in the transduced cells, and it was confirmed that the human ACE2 protein was also well expressed. The isolated cells were proliferated through single cell line culture and then subjected to FACS analysis to confirm protein expression. As a result, as shown in FIG. 3 , it was confirmed that human ACE2 protein was well expressed in the four selected cell lines (#2, #27, #33, #37). In addition, Western blot was performed to additionally confirm protein expression. More specifically, the constructed cell line was treated with RIPA buffer (Biosesang, Korea) containing a proteinase inhibitor, and then incubated on ice for 10 minutes. After sonication, it was incubated again on ice for 10 minutes and centrifuged at 4°C at a speed of 13000 rpm for 20 minutes. The supernatant was transferred to a new tube, loaded on a 10% acrylamide SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane. It was stirred for 1 hour in TBST (Tris Buffered Saline with Tween 20) containing 5% skim milk. Thereafter, human ACE2 antibody (Santacruz, CA, USA) and FLAG (Siama, MO, USA) antibody were each treated at a concentration of 1:1000 and reacted at 4°C overnight. This was washed 3 times for 10 minutes using TBST buffer, then treated with HRP-conjugated secondary antibody for each antibody at a concentration of 1:1000 in TBST containing 5% skim milk, and reacted at room temperature for 1 hour. Then, after washing 3 times for 10 minutes with TBST buffer, color development was confirmed using ECL (enhanced chemiluminescence) solution. As a result, as shown in FIG. 4 , bands of the ACE2 protein ( FIG. 4a ) and the FLAG protein tagged with the human ACE2 gene ( FIG. 4b ) were identified at the same size (130 kDa). Therefore, it was confirmed that human ACE2 protein was well expressed in the transformed cell line constructed in Examples of the present invention based on the present results.

Example 3. Verification of sensitivity to SARS-CoV-2 of transformed cell lines

After infecting the four transformed cell lines and the wild-type cell line (fibroblast line) constructed in Example 2 with SARS-CoV-2, their sensitivity was confirmed. Infected cells were amplified by the realtime reverse transcription polymerase chain method (Realtime RT-PCR) for the ORF1b gene and RdRp gene of SARS-CoV-2 to examine the presence of virus, and the results are shown in FIG. Korea).

As a result, it was confirmed that high sensitivity to SARS-CoV-2 was observed in the four transformed cell lines expressing human ACE2 protein compared to the wild type.

Among the transformed cell lines TG#2 (named hACE2-TG), TG#27, TG#33 and TG#37, TG#2 (hACE2-TG) was entrusted to the Korea Cell Line Research Foundation as KCLRF-BP-00512.

Example 4. Production of transgenic cloned pigs

4-1. Preparation of oocytes, production of nuclear transfer eggs and transplantation of surrogate sows

Using the transformed cell line constructed in Example 2, a nuclear transfer egg was prepared by slightly modifying the known method (refer to Theriogenology. 127 80-87, 2019), and it was transplanted into a surrogate sow. First, ovaries of immature sows were obtained, and then placed in a 0.9% NaCl solution maintained at 35 °C and transported to the laboratory. Cumulus-oocyte complexes (COCs) were aspirated from immature follicles with a diameter of 2-6 mm using an 18-gauge needle fixed in a 10 mL disposable syringe. The COCs were treated with 0.1% polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg/ml luteinizing hormone. hormone, LH, (Sigma)), 0.5 μg/ml follicle stimulating hormone (FSH, (Sigma)), 10 ng/mL epithelial growth factor (EGF, (Sigma)), 75 μg/ It was washed 3 times with TCM-199 (Gibco) containing ml penicillin G and 50 μg/ml streptomycin (Gibco). About 50 to 60 COCs were transferred to a 4-well multi-dish covered with mineral oil, 500 μL of the same medium was added, _{and incubated at 5% CO 2} and 39° C. conditions. After 42 to 44 hours, the cultured COCs were strongly vortexed in TL-HEPES containing 0.1% PVA and 0.2% hyaluronidase for 4 minutes to separate oocytes from cumulus cells. Aspirate the first polar body and adjacent cytoplasm using a fine glass pipette in TCM-199 containing 0.3% bovine serum albumin (BSA, (Sigma)) and 7.5 μg/ml cytochalasin B By doing so, the cell nucleus was removed from the oocyte. Prior to somatic cell nuclear transfer (SCNT), the donor cells prepared in Example 2 were cultured in DMEM medium containing 0.5% FBS for 3 days for serum starvation. Donor cells were placed in the periviteline space of the oocyte in contact with the oocyte membrane. The inoculated oocytes were placed between two platinum electrodes with a diameter of 0.2 mm at an interval of 1 mm in a medium consisting of 0.3 M mannitol, 1.0 mM CaCl ₂ , 0.1 mM MgCl _{2 and 0.5 mM HEPES.} Fusion/activation was induced by applying two consecutive DC pulses of 1.1 kV/cm for 30 μs (BTX, USA). Then, 20 to 30 reconstructed embryos were transferred to a 4-well multidish covered with mineral oil, and NCSU (North Carolina State University)-23 medium supplemented with 500 mL of 0.4% BSA was added. After 7 days of culture, the embryos were observed under a fluorescence microscope, and as shown in FIG. 6 , it was confirmed that the GFP fluorescence was well expressed in the nuclear-transferred embryos. The reconstructed embryos were surgically transplanted into the fallopian tubes of sows, the first day of standing estrus. Pregnancy status was confirmed with an ultrasound scanner (Mysono 201, Medison Co. LTD, Korea).

4-2. Validation of transgenic cloned pigs

Genetic analysis was performed using the umbilical cords of 7 transgenic cloned pigs produced in Example 4-1. After obtaining the produced piglet-derived tail tissue, genomic DNA was isolated according to the protocol using Dneasy Blood & Tissue Kits (Qiagen, GmbH, Germany). Using the isolated genomic DNA as a template, forward primer (5'-GTACATCTACGTATTAGTCATCGC-3' (SEQ ID NO: 4)), reverse primer (5'-GTTCAACCGTTTGCTCTTGTCTTC-3' (SEQ ID NO: 5)), Profi PCR premix (Bioneer, Korea) After mixing, initial denaturation at 95 °C for 5 minutes, then at 95 °C for 40 seconds, at 60 °C for 40 seconds, and at 72 °C for 1 minute, repeated 35 times, and finally PCR (Applied Biosystems) was performed at 72 °C for 7 minutes. After the completed PCR product was loaded on a 1% agarose TAE gel, the band was confirmed, and the result is shown in FIG. 7 . As a result, it was confirmed that the human ACE2 expression vector was well inserted in the transgenic saphenous pigs #1, #2, #3 and #6. In addition, the selected individual-derived ear tissue fibroblasts were obtained as described in Example 2, and the GFP fluorescent protein inserted into the vector was confirmed using a fluorescence microscope (Olympus, Japan). As a result, as shown in FIG. 8 , it was confirmed that GFP fluorescence was well exhibited in the transgenic pig-derived ear fibroblasts into which the human ACE2 expression vector was inserted. Also, chromosome analysis was performed using individual-derived ear fibroblasts through karyotyping and FISH (Fluorescence In-situ hybridization) (Gendix, Korea), and the results are shown in FIG. 9 . As a result, it was confirmed to have a normal pig chromosome (18 pairs of homologous chromosomes + sex chromosomes), and as a result of analysis after synthesizing a human ACE2-specific probe, it was confirmed that both individuals were inserted into the pig chromosome 17 (see arrow).

Example 5. Characterization of transgenic cloned pigs

5-1. Protein Expression Analysis

Western blotting was performed in the same manner as in Example 2 using the transgenic cloned pig-derived ear fibroblasts into which the human ACE2 expression vector produced in Example 4 was inserted. As a result, as shown in FIG. 10, it was confirmed that FLAG-tagged protein and human ACE2 protein bands were well generated at the same size as PC (positive control, donor cells). It was confirmed that the human ACE2 protein band was well generated in the results using the heart, lung, and small intestine.

5-2. SARS-CoV-2 inoculation test

A SARS-CoV-2 challenge inoculation test was performed using the transgenic cloned pigs and wild-type pigs (Yucatan mini-pigs) produced in Example 4 (performed by the Zoological Infectious Diseases Institute). After intranasal inoculation of SARS-CoV-2 to an individual, body weight and body temperature were measured daily. As a result, as shown in FIG. 12a, the weight increase rate was decreased in the human ACE2-expressing transgenic cloned pig compared to the wild type, and body temperature increased as shown in FIG. 12b. . This is a common clinical symptom of SARS-CoV-2 infection. In particular, Yucatan mini-pigs showed high fever exceeding normal body temperature (39.2 ℃ ±0.5, sinclair) after challenge inoculation, and symptoms of lethargy were also confirmed. As a result of performing E gene and RdRP gene gene analysis of each individual 3 days after inoculation (using seegene's kit, Korea), virus shedding was confirmed in the nasal cavity of the transgenic cloned pig compared to the wild type as shown in FIG. 13 . In addition, after euthanizing each individual 5 days after inoculation, histopathological examination was performed on the tissues. As a result of the analysis, as shown in FIG. 14, local bleeding with intraalveolar edematous changes was confirmed in a part of the lungs of human ACE2-expressing transgenic cloned pigs compared to the wild-type, and mild vascular and peribronchiolar infiltration of inflammatory cells was observed in more than half of the lung lobes. It was confirmed and mainly infiltrated cells were identified as mononuclear inflammatory cell lineage.

Therefore, it was confirmed that the transgenic cloned pig expressing human ACE2 according to the present invention exhibited high sensitivity to SARS-CoV-2.

Overall, the present invention is to prepare a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2), and to prepare a transgenic cell line and a transgenic pig using this, the transgenic cell line and transformation Transgenic pigs express human ACE2 and show high sensitivity to SARS-CoV-2, which is useful as a non-clinical animal model for research on SARS-CoV-2 or for the prevention or treatment of viral infections. can be utilized

[Accession Number]

Name of depositary institution: Korea Cell Line Research Foundation

Accession number: KCLRF-BP-00512

Deposit date: 20210504

Claims (17)

Human angiotensin-converting enzyme 2 (ACE2) gene represented by the nucleotide sequence of SEQ ID NO: 1, including an enhancer and a promoter, type 2 severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS) Recombinant vector for making animal models with increased sensitivity to -CoV-2). The method of claim 1, Recombinant vector for animal model production with increased sensitivity to SARS-CoV-2, characterized in that the animal model is a pig. The method of claim 1, The promoter is any one exogenous promoter selected from the group consisting of CAG promoter, CMV promoter, EF1α promoter, ICAM2 promoter, SV40 promoter, PGK1 promoter, Ubc promoter and β-Act promoter, SARS- Recombinant vector for making animal models with increased sensitivity to CoV-2. The method of claim 1, The recombinant vector is a recombinant vector for animal model production with increased sensitivity to SARS-CoV-2, characterized in that it is represented by the following vector map.

The method of claim 1, The recombinant vector is a recombinant vector for animal model production with increased sensitivity to SARS-CoV-2, characterized in that it comprises a sequence represented by the nucleotide sequence of SEQ ID NO: 2. The method of claim 1, The promoter is any one endogenous promoter selected from the group consisting of GGTA1 (alpha 1,3-galactosyltransferase) promoter, ROSA26 promoter and AAVS1 (PPP1R12C locus) promoter. Recombinant vector for making animal models with increased sensitivity. The method of claim 1, The recombinant vector is a recombinant vector for animal model production with increased sensitivity to SARS-CoV-2, characterized in that it is represented by the following vector map.

A transformed cell line with increased sensitivity to SARS-CoV-2, prepared by transforming the recombinant vector of claim 1 into a somatic cell. 9. The method of claim 8, The somatic cell is a transformed cell line with increased sensitivity to SARS-CoV-2, characterized in that it is a porcine fibroblast. 9. The method of claim 8, The transformed cell line, characterized in that the accession number KCLRF-BP-00512, a transformed cell line with increased sensitivity to SARS-CoV-2. A nuclear transfer embryo for the production of transgenic pigs for animal models with increased sensitivity to SARS-CoV-2, which is nuclear-transferred by introducing the transgenic cell line of claim 8 into a fertilized porcine egg. A transgenic pig for animal models with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transgenic cell line of claim 8. A nuclear transfer step of preparing a reconstituted egg by transplanting the transformed cell line of claim 8 into a denucleated egg; and A method for producing a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2, comprising the step of transplanting the reconstituted egg into the fallopian tube of a surrogate mother. SARS-CoV using the transgenic cell line with increased sensitivity to SARS-CoV-2 according to claim 8 or a transgenic pig for animal model with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transgenic cell line -2 Screening method for preventing or treating an infectious disease. 15. The method of claim 14, The method comprises the following steps, a screening method for preventing or treating a SARS-CoV-2 infectious disease: 1) The transgenic cell line with increased sensitivity to SARS-CoV-2 of claim 8 or SARS-CoV-2 in transgenic pigs with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer inoculating CoV-2; 2) administering a candidate substance for the prevention or treatment of SARS-CoV-2 infection disease to the transgenic cell line or transgenic pig; and 3) Comparing the group to which the candidate substance was administered and the control group to which the candidate substance was not administered, selecting a candidate substance that significantly changes the symptom or histopathological indicator value of SARS-CoV-2 infection. 16. The method of claim 15, The symptoms are fever, shortness of breath, headache, sore throat, hemoptysis, nausea, cough, sputum, diarrhea, loss of smell, malaise, anorexia, muscle pain and cognitive impairment, characterized in that at least one selected from the group consisting of, SARS-CoV -2 Screening method for preventing or treating an infectious disease. 16. The method of claim 15, The histopathological index value is any one or more selected from the group consisting of inflammatory cytokine secretion, edema, local bleeding, and inflammatory cell infiltration.

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