KR20230075800A - Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection - Google Patents

Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection Download PDF

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KR20230075800A
KR20230075800A KR1020210162333A KR20210162333A KR20230075800A KR 20230075800 A KR20230075800 A KR 20230075800A KR 1020210162333 A KR1020210162333 A KR 1020210162333A KR 20210162333 A KR20210162333 A KR 20210162333A KR 20230075800 A KR20230075800 A KR 20230075800A
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rheumatoid arthritis
covid
infection
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조미라
박성환
이선영
이아람
이채림
우진석
이연수
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가톨릭대학교 산학협력단
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
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    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/50—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for simulation or modelling of medical disorders
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/80—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for detecting, monitoring or modelling epidemics or pandemics, e.g. flu

Abstract

본 발명은 코로나바이러스 감염동반 류마티스 관절염 질환 모사 모델과 스크리닝 플랫폼에 관한 것으로서, 본 발명의 COVID-19 감염동반 류마티스 관절염 동물모델은, COVID-19 spike의 감염으로, 관절염 지수 및 발병율 더욱 증가하고, 관절 염증 지수 및 골부식 지수 또한, 더욱 증가한 것을 확인하였다. 또한, COVID-19의 감염과 관련된 면역세포 및 류마티스 관절염과 관련된 면역세포가 증가한 것을 확인하고, COVID-19 혈전 마커인 PF4 및 Anti-pholipid antibody가 증가하며, COVID-감염 및 류마티스 관절염과 관련된 염증인자들의 발현이 증가한 것을 확인하여, 본 발명의 동물모델이 COVID-19 감염동반 류마티스 관절염을 효과적으로 반영하는 것을 확인하였다.The present invention relates to a rheumatoid arthritis disease accompanying coronavirus infection model and screening platform. Inflammation index and bone erosion index were also confirmed to be further increased. In addition, it was confirmed that immune cells related to COVID-19 infection and immune cells related to rheumatoid arthritis increased, COVID-19 blood clot markers PF4 and Anti-pholipid antibody increased, and inflammatory factors related to COVID-infection and rheumatoid arthritis By confirming that their expression increased, it was confirmed that the animal model of the present invention effectively reflects rheumatoid arthritis accompanying COVID-19 infection.

Description

코로나바이러스 감염동반 류마티스 관절염 질환 모사 모델과 스크리닝 플랫폼{Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection}Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection}

본 발명은 코로나바이러스 감염동반 류마티스 관절염 질환 모사 모델과 스크리닝 플랫폼에 관한 것이다.The present invention relates to a rheumatoid arthritis disease accompanying coronavirus infection model and screening platform.

코로나바이러스는 사람, 조류, 설치류, 및 포유류 등 숙주범위가 넓은 RNA 바이러스로서 유전체의 크기가 약 30kb에 이르며, 모든 RNA 바이러스 중에서 가장 큰 바이러스이다. 바이러스 표면의 단백질 돌기가 태양광환(colona) 혹은 왕관(crown) 모양과 비슷해 코로나바이러스라고 명명되었다.Coronavirus is an RNA virus with a wide range of hosts, such as humans, birds, rodents, and mammals, with a genome size of about 30 kb, and is the largest among all RNA viruses. The protein projections on the surface of the virus are similar to the shape of a colona or crown, hence the name coronavirus.

사람에게 감염되어 질병을 유발하는 코로나바이러스는 7종이 알려져 있으며, 이중 4종(HCoV-229E, HCoV-OC43, HCoV-NL63, HKU1)은 지역사회 감염 호흡기 사람코로나바이러스(community-acquired respiratory HCoV, CAR HCoV)라고도 하는데, 전 세계 어디에서나 온도 및 기후 측면에서 종종 여름, 가을보다 겨울, 봄에 빈번하게 발생하고 성인의 상기도 감염의 10~30%의 원인이 되는 것으로 알려져 있다. 이외에, 2003년 유행했던 중증급성호흡기증후군(Severe Acute Respiratory Syndrome, SARS) 및 2012년 유행했던 중동호흡기증후군(Middle East Respiratory Syndrome, MERS)을 유발한 코로나바이러스(SARS-CoV 및 MERS-CoV)는 동물에서 사람으로 전파된 종으로 하기도 감염을 통해 심각한 호흡기 증후군을 유발하는 것으로 알려져 있다. 최근 2019년 12월에 중국 후베이성 우한시에서 발생한 폐렴환자에서 신종 코로나바이러스가 확인되어 처음에 2019년 신종 코로나바이러스(2019 novel coronavirus, 2019-nCoV)로 불렸고 이후 중증급성호흡기증후군 코로나바이러스 2(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)라는 정식명칭으로 명명되었으며, 이 균주가 유발한 감염증은 세계보건기구(WHO)에 의해 Coronavirus disease 2019(COVID-19)로 명명되었다(한국에서는 한글명칭 ‘코로나바이러스감염증-19’과 그 약칭 ‘코로나-19’로 명명됨).There are seven known coronaviruses that infect humans and cause disease, and four of them (HCoV-229E, HCoV-OC43, HCoV-NL63, HKU1) are community-acquired respiratory human coronaviruses (community-acquired respiratory HCoV, CAR). HCoV), which is known to cause 10-30% of upper respiratory tract infections in adults, occurs more frequently in winter and spring than in summer and fall in terms of temperature and climate everywhere in the world. In addition, the coronaviruses (SARS-CoV and MERS-CoV) that caused the Severe Acute Respiratory Syndrome (SARS) epidemic in 2003 and the Middle East Respiratory Syndrome (MERS) epidemic in 2012 are It is a species that has spread from humans to humans and is known to cause severe respiratory syndrome through infection of the lower respiratory tract. Recently, in December 2019, a novel coronavirus was identified in a pneumonia patient in Wuhan, Hubei Province, China, and was first called 2019 novel coronavirus (2019-nCoV), then severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2). Respiratory syndrome coronavirus 2, SARS-CoV-2), and the infection caused by this strain was named Coronavirus disease 2019 (COVID-19) by the World Health Organization (in Korea, the Korean name '' Named 'COVID-19' and its abbreviation 'COVID-19').

SARS-CoV-2는 SARS 및 MERS 코로나바이러스와 같이 박쥐에서 기원한 베타코로나바이러스계통이며, 해당 바이러스의 유전자 염기서열분석결과, 박쥐 유래 유사 코로나바이러스와 가장 높은 상동성(89.1%)을 갖는 것으로 확인되었다. COVID-19는 주로 호흡기를 통해 감염되며 증상이 거의 없는 감염 초기에 전염성이 강한 특징을 보이며, 감염 후에는 인후통, 고열, 기침, 호흡곤란 등의 증상을 거쳐 폐렴으로 발전한다. COVID-19가 전세계적으로 확산하자 세계보건기구(WHO)는 2020년 3월 11일에 이 질환에 대해 범유행(팬데믹)을 선언하였고, 2020년 5월 기준 전세계적으로 COVID-19 환자수 약 470만명, 사망자수 약 30만명에 달하여 COVID-19가 급속도로 확산하고 있다.SARS-CoV-2 is a betacoronavirus lineage originating in bats like SARS and MERS coronaviruses, and as a result of genetic sequencing of the virus, it was confirmed to have the highest homology (89.1%) with bat-derived similar coronaviruses. It became. COVID-19 is mainly transmitted through the respiratory tract and shows a highly contagious characteristic in the early stages of infection when there are few symptoms. After infection, symptoms such as sore throat, high fever, cough, and shortness of breath develop into pneumonia. As COVID-19 spread worldwide, the World Health Organization (WHO) declared the disease a pandemic on March 11, 2020, and as of May 2020, the number of COVID-19 patients worldwide With about 4.7 million deaths and about 300,000 deaths, COVID-19 is spreading rapidly.

COVID-19를 치료하기 위하여 개발되고 있는 COVID-19 치료제는 작용기전에 따라 크게 ‘항바이러스제’와 ‘면역조절제’로 나누어진다. 항바이러스제는 감염의 원인이 되는 바이러스를 제거하는 의약품으로 가장 일반적으로 개발되고 있으며, 대표적으로는 최근 허가를 받은 ‘렘데시비르’가 이에 속한다. 면역조절제는 주요 기전에 따라 항염증제와 면역증강제가 있다. 항염증제는 COVID-19 감염으로 발생하는 과도한 면역작용을 조절하여 염증반응을 억제한다. 바이러스에 의한 감염으로 면역반응이 과도하게 일어나면 염증을 유발하는 물질인 사이토카인이 과도하게 분비(사이토카인 폭풍, cytokine storm)되고, 염증반응이 비정상적으로 증가하여 폐 조직 등 정상 세포를 손상시키고, 급성호흡곤란 증후군으로 이어져 체내 산소부족으로 장기 등이 손상되기에, 항염증제는 이러한 과도한 염증반응을 일으키는 사이토카인을 억제하여 정상 세포의 손상을 보호하게 된다. 반면, 적절한 면역반응은 바이러스 증식을 억제하고 바이러스에 감염된 세포를 제거하는 등 이롭게 작용하며, 일부 면역력이 떨어진 환자의 경우 바이러스 침투에 취약하다고 보고되어졌다. 면역력이 떨어진 환자의 경우에는, 적절히 자가 면역을 높이는 면역증강제가 감염의 진행을 막거나, 회복하는데 도움을 줄 수 있어, 면역증강제를 이용한 COVID-19의 치료도 고려되고 있다.COVID-19 therapeutic agents being developed to treat COVID-19 are largely divided into 'antiviral agents' and 'immunomodulators' according to their mechanism of action. Antiviral drugs are most commonly developed as medicines that remove the virus that causes infection, and 'Remdesivir', which has recently been approved, is a representative example. Immunomodulators include anti-inflammatory agents and immunostimulants depending on the main mechanism. Anti-inflammatory drugs suppress the inflammatory response by controlling the excessive immune response caused by COVID-19 infection. When the immune response is excessive due to infection with a virus, cytokine, a substance that induces inflammation, is secreted excessively (cytokine storm), and the inflammatory response abnormally increases, damaging normal cells such as lung tissue, and acute Since organs are damaged due to lack of oxygen in the body leading to respiratory distress syndrome, anti-inflammatory drugs protect normal cells from damage by suppressing cytokines that cause such excessive inflammatory reactions. On the other hand, it has been reported that an appropriate immune response works beneficially, such as inhibiting virus proliferation and removing virus-infected cells, and some patients with weakened immunity are vulnerable to virus penetration. In the case of patients with weakened immunity, immunosuppressants that properly increase autoimmunity can prevent the progression of infection or help recover, so treatment of COVID-19 using immunosuppressants is also being considered.

한편, 이러한 COVID-19의 대유행으로, 류마티스 관절염 환자는, COVID-19의 감염 위험이 증가하고, 류마티스 관절염을 포함한 자가면역질환 환자에서는 COVID-19 감염으로, 비-자가면역질환 환자와 비교하여, 사망률이 높다는 것이 보고되었으며, 염증성 관절염이 아닌 결합 조직 질환(류마티스 관절염, 루푸스 등) 환자에서 COVID-19가 감염되었을 경우, 예후가 더욱 불안하다는 것이 보고되었다. 특히 미국 재향군인회에서 공유한 데이터베이스에서는, 류마티스 관절염이 동반된 COVID-19 환자의 입원 또는 사망에 대하여, 일반적인 COVID-19 환자와 비교하여, 더 큰 위험성이 있다는 것을 공론화 하였다.On the other hand, with this COVID-19 pandemic, rheumatoid arthritis patients have an increased risk of COVID-19 infection, and COVID-19 infection in patients with autoimmune diseases, including rheumatoid arthritis, compared to patients with non-autoimmune diseases, It has been reported that the mortality rate is high, and it has been reported that the prognosis is more unstable when COVID-19 is infected in patients with connective tissue diseases (rheumatoid arthritis, lupus, etc.) other than inflammatory arthritis. In particular, in the database shared by the American Veterans Association, it was publicized that there is a greater risk of hospitalization or death for COVID-19 patients with rheumatoid arthritis compared to COVID-19 patients in general.

그러나, COVID-19가 동반된 류마티스 관절염 환자에서는, 항염증제 또는 면역조절제의 효과를 입증 연구 결과는 아직까지 보고되지 않았다. COVID-19의 감염으로 류마티스 관절염 환자의 체내에서, 사이토카인의 과발현이나 자가면역 반응성에 대하여 항염증제 또는 면역조절제의 효과나 반응을 확인한 대한 연구는 미비한실정이며, 류마티스 관절염 환자에서, COVID-19 감염으로 인한 합병증을 개선하고, 감염 이후의 후유증을 완화시키기 위한 효과적인 약물은 보고된바 없다. COVID-19가 동반된 류마티스 관절염 치료제의 개발이 부족한 대표적인 원인은, COVID-19가 동반된 류마티스 관절염의 병태를 반영하는 모델이 부족하고, 일반적인 COVID-19 환자보다 예후가 불량한 COVID-19가 동반된 류마티스 관절염 환자의 면역 상태를 확인할 수 있는 방법이 부족하여, 효과적인 약물을 스크리닝 하기 어렵기 때문이다.However, in rheumatoid arthritis patients with COVID-19, the results of studies proving the effectiveness of anti-inflammatory or immunomodulatory agents have not been reported yet. Studies confirming the effect or response of anti-inflammatory drugs or immunomodulators on cytokine overexpression or autoimmune reactivity in patients with rheumatoid arthritis due to COVID-19 infection are incomplete. No effective drugs have been reported to improve the complications caused by the infection and alleviate the sequelae after infection. A representative cause of the lack of development of treatment for rheumatoid arthritis with COVID-19 is the lack of a model that reflects the condition of rheumatoid arthritis with COVID-19, and patients with COVID-19 who have a poorer prognosis than general COVID-19 patients. This is because it is difficult to screen effective drugs due to a lack of methods for confirming the immune status of patients with rheumatoid arthritis.

이에, 본 발명자들은, COVID-19가 동반된 류마티스 동물모델을 제작하고, 제작된 동물모델이 COVID-19가 동반된 류마티스 질환의 여러 가지 인자 및 지표를 반영하는 것을 확인하여, COVID-19가 동반된 류마티스 동물모델 및 이를 이용한 치료제를 스크리닝하는 방법을 확립하여, 본 발명을 완성하였다.Accordingly, the present inventors produced a rheumatic animal model accompanied by COVID-19, confirmed that the produced animal model reflected various factors and indicators of rheumatoid disease accompanied by COVID-19, and confirmed that COVID-19 was accompanied by The present invention was completed by establishing a rheumatic animal model and a method for screening a therapeutic agent using the same.

본 발명의 목적은 류마티스 관절염이 유도된 마우스에 코로나바이러스 유래의 spike 단백질이 주입된, COVID-19 감염동반 류마티스 관절염 동물모델을 제공하는 것이다.An object of the present invention is to provide an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which rheumatoid arthritis-induced mice are injected with coronavirus-derived spike protein.

본 발명의 다른 목적은, 마우스에 류마티스 관절염을 유도하는 단계; 및Another object of the present invention is to induce rheumatoid arthritis in mice; and

상기 류마티스 관절염이 유도된 마우스에, 코로나바이러스 유래의 spike 단백질을 주입하는 단계;를 포함하는 COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법을 제공하는 것이다.To provide a method for manufacturing an animal model of rheumatoid arthritis accompanied by COVID-19 infection, including the step of injecting coronavirus-derived spike protein into the rheumatoid arthritis-induced mouse.

본 발명의 또 다른 목적은, 상기의 COVID-19 감염동반 류마티스 관절염 동물모델에, 후보 물질을 처리하는 단계;를 포함하는, COVID-19 감염동반 류마티스 관절염의 치료 물질의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a therapeutic substance for rheumatoid arthritis accompanied by COVID-19 infection, including the step of treating the candidate substance in the animal model of rheumatoid arthritis accompanied by COVID-19 infection.

상기 목적을 달성하기 위하여, 본 발명은 류마티스 관절염이 유도된 마우스에 코로나바이러스 유래의 spike 단백질이 주입된, COVID-19 감염동반 류마티스 관절염 동물모델을 제공한다.In order to achieve the above object, the present invention provides an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which a coronavirus-derived spike protein is injected into a mouse in which rheumatoid arthritis is induced.

또한, 본 발명은, 마우스에 류마티스 관절염을 유도하는 단계; 및In addition, the present invention comprises the steps of inducing rheumatoid arthritis in mice; and

상기 류마티스 관절염이 유도된 마우스에, 코로나바이러스 유래의 spike 단백질을 주입하는 단계;를 포함하는 COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법을 제공한다.It provides a method for producing an animal model of rheumatoid arthritis accompanied by COVID-19 infection, including the step of injecting coronavirus-derived spike protein into the rheumatoid arthritis-induced mouse.

또한, 본 발명은, 상기의 COVID-19 감염동반 류마티스 관절염 동물모델에, 후보 물질을 처리하는 단계;를 포함하는, COVID-19 감염동반 류마티스 관절염의 치료 물질의 스크리닝 방법을 제공한다.In addition, the present invention provides a screening method for a therapeutic substance for rheumatoid arthritis accompanied by COVID-19 infection, comprising the step of treating the candidate substance in the animal model of rheumatoid arthritis accompanied by COVID-19 infection.

본 발명의 COVID-19 감염동반 류마티스 관절염 동물모델은, COVID-19 spike의 감염으로, 관절염 지수 및 발병율 더욱 증가하고, 관절 염증 지수 및 골부식 지수 또한, 더욱 증가한 것을 확인하였다. 또한, COVID-19의 감염과 관련된 면역세포 및 류마티스 관절염과 관련된 면역세포가 증가한 것을 확인하고, COVID-19 혈전 마커인 PF4 및 Anti-pholipid antibody가 증가하며, COVID-감염 및 류마티스 관절염과 관련된 염증인자들의 발현이 증가한 것을 확인하여, 본 발명의 동물모델이 COVID-19 감염동반 류마티스 관절염을 효과적으로 반영하는 것을 확인하여, 관련 산업에 유용하게 이용할 수 있다.In the animal model of rheumatoid arthritis accompanied by COVID-19 infection of the present invention, it was confirmed that the infection of the COVID-19 spike further increased the arthritis index and incidence rate, and the joint inflammation index and bone erosion index also increased. In addition, it was confirmed that immune cells related to COVID-19 infection and immune cells related to rheumatoid arthritis increased, COVID-19 blood clot markers PF4 and Anti-pholipid antibody increased, and inflammatory factors related to COVID-infection and rheumatoid arthritis By confirming that their expression increased, it was confirmed that the animal model of the present invention effectively reflects rheumatoid arthritis accompanying COVID-19 infection, and can be usefully used in related industries.

도 1은 본 발명의 COVID-19 감염동반 류마티스 동물모델에서의, 관절염 지수 및 발병율을 확인한 도이다.
도 2는 본 발명의 COVID-19 감염동반 류마티스 동물모델에서 관절 손상 정도를 H&E 염색으로 확인한 도이다(A: H&E 염색 결과, B: 관절염 지수 및 골부식 지수 정량화).
도 3은 본 발명의 COVID-19 감염동반 류마티스 동물모델에서의 면역세포 변화를 유세포 분석으로 확인한 도이다(A: 비장 내 면역세포 변화 확인, B: 혈액 및 비장 내 IFNa 확인).
도 4는 본 발명의 COVID-19 감염동반 류마티스 동물모델의 혈청에서 COVID-19 혈전 마커인 PF4 및 Anti-pholipid antibody의 발현을 ELISA로 확인한 도이다(A: PF4 확인, B: Anti-pholipid antibody 확인).
도 5는 COVID-19 spike가 감염된 관절 활막 세포 및 이의 배양액에서, 염증과 관련된 사이토카인의 유전자 및 단백질 발현을 분석한 도이다(A: 세포 내 사이토카인 발현 확인, B: 배양액 내 IL-6 발현 확인).1 is a diagram confirming the arthritis index and incidence in the rheumatoid animal model accompanying COVID-19 infection of the present invention.
Figure 2 is a diagram confirming the degree of joint damage in the COVID-19 infection-accompanied rheumatic animal model of the present invention by H&E staining (A: H&E staining result, B: arthritis index and bone erosion index quantification).
Figure 3 is a diagram confirming immune cell changes in the COVID-19 infection-accompanied rheumatic animal model of the present invention by flow cytometry (A: confirmation of immune cell changes in the spleen, B: confirmation of IFNa in blood and spleen).
Figure 4 is a diagram confirming the expression of COVID-19 blood clot marker PF4 and anti-pholipid antibody in the serum of the rheumatoid animal model accompanying COVID-19 infection of the present invention by ELISA (A: PF4 confirmation, B: anti-pholipid antibody confirmation) ).
5 is a diagram illustrating the analysis of gene and protein expression of cytokines related to inflammation in joint synovial cells infected with the COVID-19 spike and their culture (A: confirmation of intracellular cytokine expression, B: expression of IL-6 in the culture medium). check).

본 발명은 류마티스 관절염이 유도된 마우스에 코로나바이러스 유래의 spike 단백질이 주입된, COVID-19 감염동반 류마티스 관절염 동물모델을 제공한다.The present invention provides an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which a coronavirus-derived spike protein is injected into a mouse in which rheumatoid arthritis is induced.

본 발명의 “관절염”은 인체의 뼈와 뼈사이의 연골, 관절낭, 활막, 인대 힘줄, 근육으로 구성된 관절부에서 나타나는 염증성 질환으로서, 자가면역이 원인인 것으로 이해되는 ‘만성 관절 류마티스(류마티스 관절염)’과 관절을 구성하는 연골세포에 노화 등의 퇴행이 발생하여 나타나는 ‘퇴행성 관절염’으로 분류된다. 류마티스 관절염은 자가 면역 반응에 따른 염증 전달 물질의 증가로 인해, 활막의 염증과 신생혈관 형성, 관절 연골 및 뼈 손상을 특징으로 하는 질환으로, 유전적 요인, 감염 등 환경적 요인에 의해 면역 체계의 이상을 초래하는 면역 매개성 질환으로, 발병원인이 아직 밝혀지지 않은 질환이다. 한편, 퇴행성 관절염의 일종인 골 관절염은 특정한 기질적 원인이 없이 주로 노년에 많이 발생되며, 만성적으로 진행되면 관절구조의 변형으로 보행 장애 등의 운동 장애를 수반하고, 관절연골(cartilage)의 점진적인 손상이나 퇴행성 변화로 인해 관절을 이루는 뼈와 인대 등에 손상이 일어나서 염증과 통증이 발생하는 질환이다.."Arthritis" of the present invention is an inflammatory disease occurring in joints composed of cartilage, joint capsule, synovium, ligament, tendon, and muscle between bones of the human body, and 'chronic joint rheumatism (rheumatoid arthritis)', which is understood to be caused by autoimmunity It is classified as 'degenerative arthritis' that occurs when degeneration, such as aging, occurs in cartilage cells constituting joints. Rheumatoid arthritis is a disease characterized by synovial inflammation, neovascularization, articular cartilage and bone damage due to an increase in inflammatory transmitters following an autoimmune response. It is an immune-mediated disease that causes abnormalities, and the cause is not yet known. On the other hand, osteoarthritis, a type of degenerative arthritis, occurs mainly in old age without a specific organic cause. When it progresses chronically, it is accompanied by movement disorders such as gait disturbance due to deformation of joint structure, and gradual damage to articular cartilage. It is a disease that causes inflammation and pain due to damage to bones and ligaments that make up joints due to degenerative changes.

본 발명의 용어 “spike 단백질”은 전자현미경을 통해 볼 수 있는 바이러스 외피(viral capsid 또는 viral envelope)에서 바깥으로 돌출된 돌기형태의 단백질을 말하며, 바이러스가 숙주세포의 수용체와 결합할 때 사용하는 단백질을 뜻하며, 단백질 도메인을 통해 세포막에 걸쳐 있는 바이러스 막 단백질로 구성되며, spike 단백질의 바깥 도메인은 숙주세포의 표면에 위치한 수용체들과의 결합을 통해, 바이러스가 세포 내로 침입할 수 있도록 하며, spike 단백질의 바깥쪽 항원 결정기는 바이러스의 혈청학적 속성과 숙주 항체와의 상호작용을 결정하는 역할을 한다.The term "spike protein" of the present invention refers to a protrusion-shaped protein that protrudes outward from the viral envelope (viral capsid or viral envelope), which can be seen through an electron microscope, and is a protein used by viruses to bind to receptors in host cells. It is composed of viral membrane proteins that span the cell membrane through the protein domain, and the outer domain of the spike protein enables the virus to enter the cell through binding to receptors located on the surface of the host cell. The outer epitope of is responsible for determining the serological properties of the virus and its interaction with host antibodies.

본 발명의 일실시예에 따르면, 상기 코로나바이러스는 SARS-Cov-2(Severe acute respiratory syndrome coronavirus 2)인 것일 수 있다.According to one embodiment of the present invention, the coronavirus may be Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2).

본 발명의 일실시예에 따르면, 상기 spike 단백질은 서열번호 1의 염기서열로 이루어진 벡터로 발현되는 것일 수 있다.According to one embodiment of the present invention, the spike protein may be expressed as a vector consisting of the nucleotide sequence of SEQ ID NO: 1.

본 발명의 일실시예에 따르면, 상기 동물모델은, 관절염 지수 및 발병율이 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to one embodiment of the present invention, in the animal model, the arthritis index and incidence rate may be increased compared to the reference value of the control group.

본 발명의 일실시예에 따르면, 상기 동물모델은, 비장 내 면역 관련 세포가 대조군의 기준치와 비교하여 증가된 것일 수 있고, 상기 면역 관련 세포는 Th1, Th17 또는 Treg인 것일 수 있다.According to one embodiment of the present invention, in the animal model, the number of immune-related cells in the spleen may be increased compared to a reference value of a control group, and the immune-related cells may be Th1, Th17, or Treg.

본 발명의 일실시예에 따르면, 상기 동물모델은, 혈액 또는 비장 내 IFNa의 발현이 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to one embodiment of the present invention, in the animal model, the expression of IFNa in the blood or spleen may be increased compared to the reference value of the control group.

본 발명의 일실시예에 따르면, 상기 동물모델은, 혈액 내 혈전 마커인 PF4 또는 Anti-pholipid antibody의 양이 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to one embodiment of the present invention, in the animal model, the amount of PF4 or anti-pholipid antibody, which is a blood clot marker, may be increased compared to the reference value of the control group.

본 발명의 일실시예에 따르면, 상기 동물모델은, 염증성사이토카인인 IFN-γ, TNF-α, IL-6, IL-1β 및 MCP-1으로 이루어진 군에서 선택된 사이토카인의 발현이, 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to one embodiment of the present invention, the animal model, the expression of a cytokine selected from the group consisting of inflammatory cytokines IFN-γ, TNF-α, IL-6, IL-1β and MCP-1, in the control group It may be increased compared to the reference value.

또한, 본 발명은, 마우스에 류마티스 관절염을 유도하는 단계; 및In addition, the present invention comprises the steps of inducing rheumatoid arthritis in mice; and

상기 류마티스 관절염이 유도된 마우스에, 코로나바이러스 유래의 spike 단백질을 주입하는 단계;를 포함하는 COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법을 제공한다.It provides a method for producing an animal model of rheumatoid arthritis accompanied by COVID-19 infection, including the step of injecting coronavirus-derived spike protein into the rheumatoid arthritis-induced mouse.

본 발명에서 상기 COVID-19 감염동반 류마티스 관절염 동물모델에서, 마우스에 류마티스 관절염을 유도하는 방법은 통상적으로 관절염 동물모델을 제조하기 위해 사용하는 방법이라면 모두 사용할 수 있으며, 또한, 종래 방법을 변형하여 관절염을 유발할 수 있는 어떠한 방법도 사용될 수 있다. 바람직하게는 2형 콜라겐을 투여하는 방법을 사용할 수 있으며, 더욱 바람직하게는 2형 콜라겐을 CFA(complete freud's adjuvant) 또는 IFA(incomplete freud's adjuvant)와 혼합한 혼 합용액을 투여하는 방법을 사용할 수 있다.In the present invention, the method of inducing rheumatoid arthritis in mice in the animal model of rheumatoid arthritis accompanied by COVID-19 infection can be any method conventionally used to prepare an animal model of arthritis, and also, by modifying the conventional method, arthritis Any method that can cause can be used. Preferably, a method of administering type 2 collagen can be used, and more preferably, a method of administering a mixed solution obtained by mixing type 2 collagen with complete freud's adjuvant (CFA) or incomplete freud's adjuvant (IFA) can be used. .

상기 제2형 콜라겐은 1차 및 2차로 반복 투여할 수 있는데, 보다 구체적으로는 2형 콜라겐을 동량의 CFA(complete freud's adjuvant)와 혼합한 혼합용액을 비만 마우스에 1차 접종한 다음, 마우스를 일정기간 사육시키고, 이후, 2형 콜라겐을 동량의 IFA(incomplete freud's adjuvant)와 혼합한 혼합용액을 2차 접종하여 마우스에 류마티스 관절염을 유발시킬 수 있다.The type 2 collagen may be repeatedly administered first and secondly. More specifically, a mixed solution obtained by mixing type 2 collagen with an equal amount of CFA (complete freud's adjuvant) is firstly inoculated into obese mice, and then the mice are inoculated. After breeding for a certain period of time, rheumatoid arthritis can be induced in mice by secondary inoculation of a mixed solution in which type 2 collagen is mixed with an equal amount of incomplete freud's adjuvant (IFA).

또한, 관절염 유발 약물을 동물모델에 투여할 경우, 상기 약물의 투여방법으로는 이에 제한되지는 않으나, 경구 투여법, 피내 주사법, 피하 주사법, 근육내 주사법, 복강내 주사법, 정맥내 주사법 등 실험조건에 따라 약물을 효율적으로 전달할 수 있는 적절한 방법을 선택하여 사용할 수 있으며, 본 발명에서는 피내 주사법으로 투여하였으나, 이에 제한되는 것은 아니다.In addition, when administering an arthritis-inducing drug to an animal model, the method of administering the drug is not limited thereto, but is not limited thereto. Depending on the method, an appropriate method for efficiently delivering the drug can be selected and used. In the present invention, intradermal injection was administered, but is not limited thereto.

또한, 본 발명에 따른 관절염 마우스 동물모델의 제조를 위해 사용할 수 있는 실험용 마우스의 종류로는 이에 제한되지는 않으나, 계통별 분류상 폐쇄군(Closed Colony)에 속하는 ICR 또는 DDY; 근교계(Inbred)에 속하는 BALB/cA, C57BL/6N, C3H/HeN, DBA/2N 또는 CBA/N; 교잡군(hybrid)에 속하는 BDF1(C57BL/6 x DBA/2), CDF1(CBA/N x DBA/2) 또는 B6C3F1(C57BL/6 x C3H/HeN); 돌연변이계(Mutant)에 속하는 BALB/c-nu 또는 C,B-17SCID 등 실험용 마우스로 사용되고 있는 것이라면 모두 이용할 수 있다. 바람직하게는 DBA 마우스를 사용 할 수 있다. 상기 “DBA” 마우스는 류마티스 관절염의 모델로 이용되는 마우스로서, 제2형 콜라겐으로 면역화 하면 자가 면역 반응에 의해 매개되는, 류마티스 관절염, 다발성 관절염이 유도되는 마우스이며, 인간의 상태와 유사하게 콜라겐으로 유도된 관절염에서, 활액막염과 연골 및 뼈의 손상/소실을 나타내는 마우스이다.In addition, the type of experimental mouse that can be used for the preparation of the arthritic mouse animal model according to the present invention is not limited thereto, but ICR or DDY belonging to the closed colony in classification by lineage; BALB/cA, C57BL/6N, C3H/HeN, DBA/2N or CBA/N belonging to inbred; BDF1 (C57BL/6 x DBA/2), CDF1 (CBA/N x DBA/2) or B6C3F1 (C57BL/6 x C3H/HeN) belonging to a hybrid; Any mouse used as an experimental mouse, such as BALB/c-nu or C,B-17SCID belonging to the Mutant family, may be used. Preferably, a DBA mouse may be used. The “DBA” mouse is a mouse used as a model for rheumatoid arthritis, and when immunized with type 2 collagen, rheumatoid arthritis and polyarthritis mediated by an autoimmune reaction are induced, and similar to the human condition, collagen Mice exhibiting synovitis and damage/loss of cartilage and bone in induced arthritis.

본 발명의 방법에 따라 제조된 COVID-19 감염동반 류마티스 관절염 동물모델은 통상적으로 사용되는 류마티스 관절염 마우스 동물모델과 비교하여, 관절염 지수와 관절염 발생비율이 증가되며, 류마티스 관절염과 COVID-19 감염에 따른 인자들의 발현이 모두 증가하는 특징이 있다.Compared to the commonly used rheumatoid arthritis mouse animal model, the rheumatoid arthritis animal model with COVID-19 infection prepared according to the method of the present invention has an increased arthritis index and arthritis incidence rate, and is associated with rheumatoid arthritis and COVID-19 infection. It is characterized by increased expression of all factors.

본 발명의 일실시예에 따르면, 상기 류마티스 관절염은, 제 2형 콜라겐(Type 2 collagen)로 유도되는 것일 수 있다.According to one embodiment of the present invention, the rheumatoid arthritis may be induced by type 2 collagen.

또한, 본 발명은, 상기의 COVID-19 감염동반 류마티스 관절염 동물모델에, 후보 물질을 처리하는 단계;를 포함하는, COVID-19 감염동반 류마티스 관절염의 치료 물질의 스크리닝 방법을 제공한다.In addition, the present invention provides a screening method for a therapeutic substance for rheumatoid arthritis accompanied by COVID-19 infection, comprising the step of treating the candidate substance in the animal model of rheumatoid arthritis accompanied by COVID-19 infection.

본 발명의 일실시예에 따르면, 상기 후보물질은, 관절염 지수 및 발병율을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the candidate substance may reduce the index and incidence of arthritis.

본 발명의 일실시예에 따르면, 상기 후보물질은, 비장 내 Th1 또는 Th17의 발현을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the candidate substance may decrease the expression of Th1 or Th17 in the spleen.

본 발명의 일실시예에 따르면, 상기 후보물질은, 혈액 또는 비장의 IFNa의 발현을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the candidate substance may decrease the expression of IFNa in blood or spleen.

본 발명의 일실시예에 따르면, 상기 후보물질은, 혈액 내 혈전 마커인 PF4 또는 Anti-pholipid antibody의 양을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the candidate substance may be one that reduces the amount of PF4 or anti-pholipid antibody, which is a thrombus marker, in the blood.

본 발명의 일실시예에 따르면, 상기 후보물질은, 염증성사이토카인인 IFN-γ, TNF-α, IL-6, IL-1β 및 MCP-1으로 이루어진 군에서 선택된 사이토카인의 발현을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the candidate substance is to reduce the expression of a cytokine selected from the group consisting of inflammatory cytokines IFN-γ, TNF-α, IL-6, IL-1β and MCP-1. can

이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<실시예 1> COVID-19 감염동반 류마티스 관절염 동물모델 제작<Example 1> Production of COVID-19 infection-accompanied rheumatoid arthritis animal model

본원 발명의 COVID-19 감염동반 류마티스 관절염 치료제 스크리닝 플랫폼을 구축하기 위하여, COVID-19 감염동반 류마티스 관절염 동물모델을 제작하고자 하였다. 구체적으로, 7주령의 DBA 마우스의 꼬리에서, 경피 내로 주사를 통해 제2형 콜라겐(Type 2 collagen) 및 완전 프로인트 항원보강제(complete Freund's adjuvant, CFA)를 1차 주사하였고, 2주 뒤 동일한 방법으로 불완전 프로인트 항원보강제(Incomplete Freund’s Adjuvant, IFA)를 주사하여 류마티스 관절염 마우스 모델링을 진행하였다. COVID-19의 감염을 동반하기 위하여, COVID-19 plasmid spike를 포함하는 벡터(pcDNA3.1-SARS2-Spike, www.addgene.org/145032/., 서열번호 1)를 정맥주사를 통해 주 1회 100 μg/mice의 양으로 주입하였다. 류마티스 관절염 평가는, 관절염 지수로 평가하였으며, 류마티스 관절염 지수는 하기 표 1의 방법으로 평가하였다. 대조군으로는 관절염이 동반된 마우스에 생리식염수를 주입한 Vehicle 군을 이용하였다.In order to establish a screening platform for the treatment of rheumatoid arthritis accompanying COVID-19 infection of the present invention, an animal model of rheumatoid arthritis accompanying COVID-19 infection was intended. Specifically, in the tail of 7-week-old DBA mice, type 2 collagen and complete Freund's adjuvant (CFA) were first injected through intradermal injection, and 2 weeks later, the same method was used. Incomplete Freund's Adjuvant (IFA) was injected to proceed with rheumatoid arthritis mouse modeling. To accompany COVID-19 infection, a vector containing the COVID-19 plasmid spike (pcDNA3.1-SARS2-Spike, www.addgene.org/145032/., SEQ ID NO: 1) is injected intravenously once a week. It was injected in an amount of 100 μg/mice. Evaluation of rheumatoid arthritis was evaluated by an arthritis index, and the rheumatoid arthritis index was evaluated by the method shown in Table 1 below. As a control group, a vehicle group in which physiological saline was injected into mice with arthritis was used.

점수score 평가기준Evaluation standard 0점0 points 부종이나 종창이 없다.No edema or swelling. 1점1 point 발 또는 발목관절에 국한된 경한 부종과 발적Mild swelling and redness localized to the foot or ankle joint 2점2 points 발목관절에서 족근골 (Metatarsal bone)에 걸친 경한 부종과 발적Mild swelling and redness from the ankle joint to the metatarsal bone 3점3 points 발목관절에서 족근골에 걸친 중등도의 부종과 발적Moderate swelling and redness from the ankle joint to the tarsal bone 4점4 points 발목에서 다리 전체에 걸쳐 부종과 발적이 있는 경우Swelling and redness from the ankle to the entire leg

그 결과, 도 1에 나타낸 바와 같이, COVID-19 spike가 주입된 마우스에서는 관절염 지수가 더욱 증가하였으며, 관절염 발병율 또한 증가한 것을 확인하였다.As a result, as shown in Figure 1, it was confirmed that the arthritis index was further increased in the mice injected with the COVID-19 spike, and the incidence of arthritis was also increased.

<실시예 2> 관절 및 연골 손상 확인<Example 2> Confirmation of joint and cartilage damage

상기 실시예 1의 COVID-19 감염동반 류마티스 동물모델에서, COVID-19의 감염 동반으로 류마티스 관절염이 영향을 받는지 확인하고자 하였다. 구체적으로 상기 실시예 1의 COVID-19 감염동반 류마티스 마우스를 관절염 유도 10주차에 인도적으로 희생하고 관절부를 수득하였다. 이를 절편으로 제작하고, 헤막토실린 & 에오신(Hematoxylin & Eosin, H&E)으로 염색하여, 관절 조직 및 연골 손상을 확인하였다.In the rheumatoid animal model with COVID-19 infection of Example 1, it was attempted to determine whether rheumatoid arthritis is affected by infection with COVID-19. Specifically, the COVID-19 infection-accompanied rheumatoid mouse of Example 1 was humanely sacrificed at 10 weeks after arthritis was induced, and joints were obtained. It was made into sections and stained with Hematoxylin & Eosin (H&E) to confirm joint tissue and cartilage damage.

그 결과, 도 2에 나타낸 바와 같이, COVID-19 감염동반 류마티스 관절염 마우스에서는, Vehicle 그룹보다 염증세포의 침윤이 증가하여, 관절 염증 지수 및 뼈 부식 지수가 유의적으로 증가한 것을 확인하였다.As a result, as shown in FIG. 2, in rheumatoid arthritis mice with COVID-19 infection, it was confirmed that the infiltration of inflammatory cells increased more than the Vehicle group, and the joint inflammation index and bone erosion index significantly increased.

<실시예 3> 면역세포 분석<Example 3> Immune cell analysis

본 발명의 COVID-19 감염동반 류마티스 관절염 마우스에서, 면역 관련 세포의 변화를 확인하고자 하였다. 구체적으로, 상기 실시예 1의 마우스 모델의 희생시점에서, 비장을 수득하고, 이를 유세포 분석을 이용하여, 면역세포의 변화를 확인하였다.In the rheumatoid arthritis mice with COVID-19 infection of the present invention, changes in immune-related cells were confirmed. Specifically, at the time of sacrifice of the mouse model of Example 1, the spleen was obtained, and changes in immune cells were confirmed using flow cytometry.

그 결과, 자가면역질환과 관련된 것으로 알려진, Th17 및 Treg는 유의한 차이가 없어, 두 동물모델에서, 류마티스 관절염이 제대로 유도된 것을 확인하였으며, 체내에서, anti-virus defence로 기능하는 것으로 알려진 IFN(Th1)은 COVID-19 spike가 주입된 군에서는, vehicle 군과 비교하여, 유의적으로 증가한 것을 확인하였다(도 3A).As a result, there was no significant difference between Th17 and Treg, which are known to be related to autoimmune diseases, confirming that rheumatoid arthritis was properly induced in the two animal models, and in vivo, IFN (known to function as anti-virus defense) Th1) was confirmed to significantly increase in the group injected with the COVID-19 spike compared to the vehicle group (Fig. 3A).

또한, COVID-19 감염 환자에서 증가하는 것으로 알려진 IFNa는 COVID-19 spike가 주입된 군에서는, vehicle 군과 비교하여, 혈액 세포(blood) 및 비장 세포(splenocyte) 모두에서 유의적으로 증가한 것을 확인하여(도 3B), 본 발명의 COVID-19 감염동반 동물모델이, 효과적으로 COVID-19 감염 및 류마티스 관절염을 반영하는 것을 확인하였다.In addition, IFNa, known to increase in COVID-19 infected patients, was significantly increased in both blood cells and splenocytes in the group injected with the COVID-19 spike compared to the vehicle group. (FIG. 3B), it was confirmed that the COVID-19 infection companion animal model of the present invention effectively reflects COVID-19 infection and rheumatoid arthritis.

<실시예 4> 연골 보호 및 연골 손상 억제 확인<Example 4> Confirmation of cartilage protection and inhibition of cartilage damage

COVID-19 백신의 부작용으로 가장 많이 알려진 부작용은 혈전 증상으로서, 혈전의 생성 여부를 확인하는 마커를 이용하여, 본 발명의 COVID-19 감염동반 류마티스 관절염 마우스가, COVID-19의 감염을 효과적으로 반영하는지 확인하고자 하였다. 구체적으로, 상기 실시예 1의 마우스를, 희생 시점에서 혈액을 분리하고, 분리한 혈액으로부터 혈청을 수득하였다. 수득된 혈청으로부터, 현재 COVID-19 환자에서 혈전 증상의 마커로 사용되는 PF4 및 Anti-pholipid antibody의 양을 ELISA로 측정하였다.The most well-known side effect of the COVID-19 vaccine is thrombosis, and whether the rheumatoid arthritis mice accompanied by COVID-19 infection of the present invention effectively reflect COVID-19 infection by using a marker to determine whether or not a blood clot is generated. wanted to check. Specifically, blood was separated from the mouse of Example 1 at the time of sacrifice, and serum was obtained from the separated blood. From the obtained serum, the amount of PF4 and anti-pholipid antibody, currently used as a marker of thrombotic symptoms in COVID-19 patients, was measured by ELISA.

그 결과, COVID-19 spike가 주입된 군에서는, 야생형 마우스 및 Vehicle 군과 비교하여, 혈청 내의 PF4 및 Anti-pholipid antibody의 양이 유의적으로 증가한 것을 확인하였다(도 4A 및 도 4B).As a result, in the group injected with the COVID-19 spike, it was confirmed that the amount of PF4 and anti-pholipid antibody in serum significantly increased compared to the wild-type mouse and vehicle groups (FIGS. 4A and 4B).

<실시예 5> 염증 변화 확인<Example 5> Confirmation of inflammatory changes

본 발명의 COVID-19 spike가 COVID-19 감염에서의 염증 및 류마티스 관절염에서의 염증 상태를 반영하는지 확인하고자 하였다. 구체적으로, 관절 활막 세포(FLS)에 Spike lentivirus sup를 감염시켜, 관절 활막 세포의 염증 변화를 관찰하고자 염증성 사이토카인의 mRNA 수준을 측정하였다. 또한, 관절 활막 세포를 배양했던 배지를 이용하여 IL-6를 측정하였다.We wanted to confirm whether the COVID-19 spike of the present invention reflects the inflammation in COVID-19 infection and the inflammatory state in rheumatoid arthritis. Specifically, by infecting joint synovial cells (FLS) with Spike lentivirus sup, mRNA levels of inflammatory cytokines were measured to observe inflammatory changes in joint synovial cells. In addition, IL-6 was measured using a medium in which articular synovial cells were cultured.

그 결과, COVID-19 감염에 의해 발생하는 것으로 알려진 cytokine storm의 종류인 INF-γ, TNF-α, IL-6가 대조군과 비교하여, Spike 감염시 유의적으로 증가한 것을 확인하였으며, 류마티스 관절염 발생시 증가하는 IL-1β 및 MCP-1 또한 유의적으로 증가하였다(도 5A). 또한, 바이러스 감염에 대표적으로 증가한다고 알려진 IL-6의 양이, spike 감염 관절 활막 세포의 배양액에서 크게 증가한 것을 확인하였다(도 5B).As a result, it was confirmed that INF-γ, TNF-α, and IL-6, which are types of cytokine storm known to be caused by COVID-19 infection, increased significantly during Spike infection compared to the control group, and increased during rheumatoid arthritis. IL-1β and MCP-1 also significantly increased (FIG. 5A). In addition, it was confirmed that the amount of IL-6, which is known to be typically increased by viral infection, increased significantly in the culture medium of spike-infected joint synovial cells (FIG. 5B).

따라서, 본 발명의 COVID-19 감염동반 류마티스 관절염 동물모델은, COVID-19 spike의 감염으로, 관절염 지수 및 발병율 더욱 증가하고, 관절 염증 지수 및 골부식 지수 또한, 더욱 증가한 것을 확인하였다. 또한, COVID-19의 감염과 관련된 면역세포 및 류마티스 관절염과 관련된 면역세포가 증가한 것을 확인하고, COVID-19 혈전 마커인 PF4 및 Anti-pholipid antibody가 증가하며, COVID-감염 및 류마티스 관절염과 관련된 염증인자들의 발현이 증가한 것을 확인하여, 본 발명의 동물모델이 COVID-19 감염동반 류마티스 관절염을 효과적으로 반영하는 것을 확인하였다.Therefore, the COVID-19 infection-accompanied rheumatoid arthritis animal model of the present invention confirmed that the infection of the COVID-19 spike further increased the arthritis index and incidence rate, and the joint inflammation index and bone erosion index also increased. In addition, it was confirmed that immune cells related to COVID-19 infection and immune cells related to rheumatoid arthritis increased, COVID-19 blood clot markers PF4 and Anti-pholipid antibody increased, and inflammatory factors related to COVID-infection and rheumatoid arthritis By confirming that their expression increased, it was confirmed that the animal model of the present invention effectively reflects rheumatoid arthritis accompanying COVID-19 infection.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection <130> PN2110-493 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 9202 <212> DNA <213> Artificial Sequence <220> <223> pcDNA3.1-SARS2-Spike <400> 1 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa 60 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt 120 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg 180 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 240 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 300 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 360 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 420 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 480 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 540 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 600 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 660 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 720 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac 780 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcatgtttgt 840 cttcctggtc ctgctgcctc tggtctcgtc tcagtgcgtg aacctgacta ctagaaccca 900 gctgcctcct gcctatacta actccttcac ccgcggcgtg tactacccag acaaggtgtt 960 ccgcagctcc gtgctgcact ccacccagga tctgttcctg cccttcttca gcaacgtgac 1020 ctggttccac gccatccacg tgagcggcac caatggcacc aagcggttcg acaatcccgt 1080 gctgccattc aacgatggcg tgtacttcgc ctccaccgag aagagcaaca tcatccgcgg 1140 ctggatcttc ggcaccaccc tggactccaa gacccagagc ctgctgatcg tgaacaatgc 1200 caccaacgtg gtcatcaagg tgtgcgagtt ccagttctgc aatgatccat tcctgggcgt 1260 gtactaccac aagaacaata agtcctggat ggagagcgag ttccgcgtgt acagctccgc 1320 caacaattgc accttcgagt acgtgtccca gcccttcctg atggacctgg agggcaagca 1380 gggcaatttc aagaacctgc gcgagttcgt gttcaagaat atcgatggct acttcaagat 1440 ctactccaag cacaccccca tcaacctggt gcgcgacctg ccacagggct tcagcgccct 1500 ggagccactg gtggatctgc caatcggcat caacatcacc aggttccaga ccctgctggc 1560 cctgcaccgc agctacctga ccccaggcga cagctccagc ggatggaccg ctggagctgc 1620 tgcctactac gtgggctacc tgcagccccg caccttcctg ctgaagtaca acgagaatgg 1680 caccatcacc gacgccgtgg attgcgccct ggatccactg tccgagacaa agtgcaccct 1740 gaagagcttc accgtggaga agggcatcta ccagacctcc aatttccgcg tgcagccaac 1800 cgagagcatc gtgcgcttcc ccaatatcac caacctgtgc ccattcggcg aggtgttcaa 1860 cgctaccagg ttcgccagcg tgtacgcttg gaatcgcaag cgcatctcca actgcgtggc 1920 cgactacagc gtgctgtaca actccgccag cttctccacc ttcaagtgct acggcgtgtc 1980 ccccaccaag ctgaatgatc tgtgcttcac caacgtgtac gccgatagct tcgtgatcag 2040 gggcgacgag gtgcgccaga tcgctccagg acagaccggc aagatcgctg actacaatta 2100 caagctgccc gacgatttca ccggctgcgt gatcgcctgg aactccaaca atctggatag 2160 caaagtgggc ggcaactaca attacctgta ccgcctgttc cgcaagtcca atctgaagcc 2220 attcgagcgc gacatctcca ccgagatcta ccaggctgga agcaccccat gcaatggagt 2280 ggagggcttc aactgctact tccccctgca gagctacggc ttccagccaa ccaacggagt 2340 gggataccag ccatacaggg tggtggtgct gtccttcgag ctgctgcacg ctccagctac 2400 cgtgtgcgga ccaaagaaga gcaccaatct ggtgaagaac aagtgcgtga acttcaattt 2460 caacggcctg accggaaccg gcgtgctgac cgagtccaac aagaagttcc tgccattcca 2520 gcagttcgga agggacatcg ctgataccac cgacgccgtg cgcgacccac agaccctgga 2580 gatcctggat atcaccccat gctccttcgg cggcgtgagc gtgatcaccc caggaaccaa 2640 taccagcaac caggtggccg tgctgtacca ggacgtgaat tgcaccgagg tgccagtggc 2700 tatccacgct gatcagctga ccccaacctg gcgcgtgtac agcaccggat ccaacgtgtt 2760 ccagacccgc gccggatgcc tgatcggagc tgagcacgtg aacaattcct acgagtgcga 2820 catcccaatc ggagctggaa tctgcgccag ctaccagacc cagaccaact ccccaaggag 2880 ggctcgcagc gtggccagcc agtccatcat cgcctacacc atgtccctgg gcgccgagaa 2940 tagcgtggcc tacagcaaca attccatcgc catcccaacc aacttcacca tctccgtgac 3000 caccgagatc ctgcccgtgt ccatgaccaa gaccagcgtg gactgcacca tgtacatctg 3060 cggcgattcc accgagtgca gcaacctgct gctgcagtac ggcagcttct gcacccagct 3120 gaatcgcgcc ctgaccggaa tcgctgtgga gcaggataag aacacccagg aggtgttcgc 3180 ccaggtgaag cagatctaca agaccccccc aatcaaggac ttcggcggct tcaatttcag 3240 ccagatcctg cccgatccaa gcaagccctc caagcgcagc ttcatcgagg acctgctgtt 3300 caacaaggtg accctggccg atgccggctt catcaagcag tacggcgatt gcctgggcga 3360 catcgctgcc cgcgacctga tctgcgccca gaagttcaat ggcctgaccg tgctgccacc 3420 actgctgacc gatgagatga tcgctcagta cacctccgcc ctgctggccg gaaccatcac 3480 cagcggatgg accttcggcg ctggagccgc cctgcagatc cccttcgcca tgcagatggc 3540 ctaccgcttc aacggcatcg gcgtgaccca gaatgtgctg tacgagaacc agaagctgat 3600 cgccaatcag ttcaactccg ccatcggcaa gatccaggac tccctgtcca gcaccgccag 3660 cgccctgggc aagctgcagg atgtggtgaa tcagaacgcc caggccctga ataccctggt 3720 gaagcagctg tccagcaact tcggcgccat ctccagcgtg ctgaatgata tcctgagccg 3780 cctggacaag gtggaggctg aggtgcagat cgataggctg atcaccggcc gcctgcagtc 3840 cctgcagacc tacgtgaccc agcagctgat cagggctgct gagatcaggg ccagcgccaa 3900 tctggctgct accaagatgt ccgagtgcgt gctgggacag agcaagaggg tggacttctg 3960 cggcaagggc taccacctga tgtccttccc acagagcgcc ccacacggag tggtgttcct 4020 gcacgtgacc tacgtgccag cccaggagaa gaacttcacc accgctccag ctatctgcca 4080 cgatggcaag gctcacttcc cacgcgaggg cgtgttcgtg tccaacggca cccactggtt 4140 cgtgacccag cgcaatttct acgagcccca gatcatcacc accgacaata ccttcgtgag 4200 cggcaactgc gacgtggtca tcggaatcgt gaacaatacc gtgtacgatc ccctgcagcc 4260 agagctggac tccttcaagg aggagctgga taagtacttc aagaatcaca ccagccccga 4320 cgtggatctg ggcgacatct ccggcatcaa tgccagcgtg gtgaacatcc agaaggagat 4380 cgaccgcctg aacgaggtgg ccaagaatct gaacgagtcc ctgatcgatc tgcaggagct 4440 gggcaagtac gagcagtaca tcaagtggcc atggtacatc tggctgggct tcatcgccgg 4500 cctgatcgcc atcgtgatgg tgaccatcat gctgtgctgc atgacctcct gctgcagctg 4560 cctgaagggc tgctgctcct gcggcagctg ctgcaagttc gatgaggacg atagcgagcc 4620 cgtgctgaag ggcgtcaaac tgcactatac aggctccacc gagacatccc aggtcgctcc 4680 cgcttagctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 4740 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4800 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4860 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 4920 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 4980 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5040 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5100 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5160 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 5220 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5280 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5340 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5400 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 5460 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 5520 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 5580 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 5640 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 5700 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 5760 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 5820 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 5880 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 5940 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 6000 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 6060 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 6120 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 6180 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 6240 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 6300 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 6360 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 6420 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 6480 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 6540 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 6600 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 6660 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 6720 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 6780 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 6840 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 6900 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 6960 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 7020 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 7080 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 7140 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 7200 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 7260 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 7320 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 7380 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 7440 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 7500 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 7560 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 7620 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 7680 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 7740 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 7800 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 7860 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 7920 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 7980 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 8040 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 8100 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 8160 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 8220 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 8280 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 8340 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 8400 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 8460 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 8520 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 8580 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 8640 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 8700 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 8760 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 8820 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 8880 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 8940 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 9000 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 9060 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 9120 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac 9180 aatctgctct gatgccgcat ag 9202 <110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection <130> PN2110-493 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 9202 <212> DNA <213> artificial sequence <220> <223> pcDNA3.1-SARS2-Spike <400> 1 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa 60 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt 120 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg 180 actagttat aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 240 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 300 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 360 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 420 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 480 tacatgacct tatgggactt tcctacttgg cagtacatct acgtatagt catcgctatt 540 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 600 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 660 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 720 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac 780 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcatgtttgt 840 cttcctggtc ctgctgcctc tggtctcgtc tcagtgcgtg aacctgacta ctagaaccca 900 gctgcctcct gcctatacta actccttcac ccgcggcgtg tactacccag acaaggtgtt 960 ccgcagctcc gtgctgcact ccacccagga tctgttcctg cccttcttca gcaacgtgac 1020 ctggttccac gccatccacg tgagcggcac caatggcacc aagcggttcg acaatcccgt 1080 gctgccattc aacgatggcg tgtacttcgc ctccaccgag aagagcaaca tcatccgcgg 1140 ctggatcttc ggcaccaccc tggactccaa gacccagagc ctgctgatcg tgaacaatgc 1200 caccaacgtg gtcatcaagg tgtgcgagtt ccagttctgc aatgatccat tcctgggcgt 1260 gtactaccac aagaacaata agtcctggat ggagagcgag ttccgcgtgt acagctccgc 1320 caacaattgc accttcgagt acgtgtccca gcccttcctg atggacctgg agggcaagca 1380 gggcaatttc aagaacctgc gcgagttcgt gttcaagaat atcgatggct acttcaagat 1440 ctactccaag cacaccccca tcaacctggt gcgcgacctg ccacagggct tcagcgccct 1500 ggagccactg gtggatctgc caatcggcat caacatcacc aggttccaga ccctgctggc 1560 cctgcaccgc agctacctga ccccaggcga cagctccagc ggatggaccg ctggagctgc 1620 tgcctactac gtgggctacc tgcagccccg caccttcctg ctgaagtaca acgagaatgg 1680 caccatcacc gacgccgtgg attgcgccct ggatccactg tccgagacaa agtgcaccct 1740 gaagagcttc accgtggaga agggcatcta ccagacctcc aatttccgcg tgcagccaac 1800 cgagagcatc gtgcgcttcc ccaatatcac caacctgtgc ccattcggcg aggtgttcaa 1860 cgctaccagg ttcgccagcg tgtacgcttg gaatcgcaag cgcatctcca actgcgtggc 1920 cgactacagc gtgctgtaca actccgccag cttctccacc ttcaagtgct acggcgtgtc 1980 ccccaccaag ctgaatgatc tgtgcttcac caacgtgtac gccgatagct tcgtgatcag 2040 gggcgacgag gtgcgccaga tcgctccagg acagaccggc aagatcgctg actacaatta 2100 caagctgccc gacgatttca ccggctgcgt gatcgcctgg aactccaaca atctggatag 2160 caaagtgggc ggcaactaca attacctgta ccgcctgttc cgcaagtcca atctgaagcc 2220 attcgagcgc gacatctcca ccgagatcta ccaggctgga agcaccccat gcaatggagt 2280 ggagggcttc aactgctact tccccctgca gagctacggc ttccagccaa ccaacggagt 2340 gggataccag ccatacaggg tggtggtgct gtccttcgag ctgctgcacg ctccagctac 2400 cgtgtgcgga ccaaagaaga gcaccaatct ggtgaagaac aagtgcgtga acttcaattt 2460 caacggcctg accggaaccg gcgtgctgac cgagtccaac aagaagttcc tgccattcca 2520 gcagttcgga agggacatcg ctgataccac cgacgccgtg cgcgacccac agaccctgga 2580 gatcctggat atcaccccat gctccttcgg cggcgtgagc gtgatcaccc caggaaccaa 2640 taccagcaac caggtggccg tgctgtacca ggacgtgaat tgcaccgagg tgccagtggc 2700 tatccacgct gatcagctga ccccaacctg gcgcgtgtac agcaccggat ccaacgtgtt 2760 ccagacccgc gccggatgcc tgatcggagc tgagcacgtg aacaattcct acgagtgcga 2820 catcccaatc ggagctgggaa tctgcgccag ctaccagacc cagaccaact ccccaaggag 2880 ggctcgcagc gtggccagcc agtccatcat cgcctacacc atgtccctgg gcgccgagaa 2940 tagcgtggcc tacagcaaca attccatcgc catcccaacc aacttcacca tctccgtgac 3000 caccgagatc ctgcccgtgt ccatgaccaa gaccagcgtg gactgcacca tgtacatctg 3060 cggcgattcc accgagtgca gcaacctgct gctgcagtac ggcagcttct gcacccagct 3120 gaatcgcgcc ctgaccggaa tcgctgtgga gcaggataag aacacccagg aggtgttcgc 3180 ccaggtgaag cagatctaca agaccccccc aatcaaggac ttcggcggct tcaatttcag 3240 ccagatcctg cccgatccaa gcaagccctc caagcgcagc ttcatcgagg acctgctgtt 3300 caacaaggtg accctggccg atgccggctt catcaagcag tacggcgatt gcctgggcga 3360 catcgctgcc cgcgacctga tctgcgccca gaagttcaat ggcctgaccg tgctgccacc 3420 actgctgacc gatgagatga tcgctcagta cacctccgcc ctgctggccg gaaccatcac 3480 cagcggatgg accttcggcg ctggagccgc cctgcagatc cccttcgcca tgcagatggc 3540 ctaccgcttc aacggcatcg gcgtgaccca gaatgtgctg tacgagaacc agaagctgat 3600 cgccaatcag ttcaactccg ccatcggcaa gatccaggac tccctgtcca gcaccgccag 3660 cgccctgggc aagctgcagg atgtggtgaa tcagaacgcc caggccctga ataccctggt 3720 gaagcagctg tccagcaact tcggcgccat ctccagcgtg ctgaatgata tcctgagccg 3780 cctggacaag gtggaggctg aggtgcagat cgataggctg atcaccggcc gcctgcagtc 3840 cctgcagacc tacgtgaccc agcagctgat cagggctgct gagatcaggg ccagcgccaa 3900 tctggctgct accaagatgt ccgagtgcgt gctgggacag agcaagaggg tggacttctg 3960 cggcaagggc taccacctga tgtccttccc acagagcgcc ccacacggag tggtgttcct 4020 gcacgtgacc tacgtgccag cccaggagaa gaacttcacc accgctccag ctatctgcca 4080 cgatggcaag gctcacttcc cacgcgaggg cgtgttcgtg tccaacggca cccactggtt 4140 cgtgacccag cgcaatttct acgagcccca gatcatcacc accgacaata ccttcgtgag 4200 cggcaactgc gacgtggtca tcggaatcgt gaacaatacc gtgtacgatc ccctgcagcc 4260 agagctggac tccttcaagg aggagctgga taagtacttc aagaatcaca ccagccccga 4320 cgtggatctg ggcgacatct ccggcatcaa tgccagcgtg gtgaacatcc agaaggat 4380 cgaccgcctg aacgaggtgg ccaagaatct gaacgagtcc ctgatcgatc tgcaggagct 4440 gggcaagtac gagcagtaca tcaagtggcc atggtacatc tggctgggct tcatcgccgg 4500 cctgatcgcc atcgtgatgg tgaccatcat gctgtgctgc atgacctcct gctgcagctg 4560 cctgaagggc tgctgctcct gcggcagctg ctgcaagttc gatgaggacg atagcgagcc 4620 cgtgctgaag ggcgtcaaac tgcactatac aggctccacc gagacatccc aggtcgctcc 4680 cgcttagctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 4740 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4800 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgataggtg 4860 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 4920 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 4980 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5040 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5100 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5160 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 5220 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5280 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5340 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5400 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 5460 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 5520 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 5580 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 5640 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 5700 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 5760 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 5820 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 5880 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 5940 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 6000 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 6060 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 6120 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 6180 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 6240 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 6300 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 6360 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 6420 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 6480 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 6540 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 6600 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 6660 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 6720 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 6780 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 6840 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 6900 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 6960 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 7020 cacacacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 7080 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 7140 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 7200 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 7260 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 7320 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 7380 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 7440 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 7500 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 7560 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 7620 agctgggctg tgtgcacgaa ccccccgttc agccccgaccg ctgcgcctta tccggtaact 7680 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 7740 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 7800 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 7860 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 7920 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 7980 tctttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 8040 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 8100 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 8160 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 8220 agataactac gatacggggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 8280 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 8340 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 8400 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 8460 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 8520 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 8580 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 8640 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 8700 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 8760 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 8820 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 8880 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 8940 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 9000 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 9060 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 9120 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac 9180 aatctgctct gatgccgcat ag 9202

Claims (19)

류마티스 관절염이 유도된 마우스에 코로나바이러스 유래의 spike 단백질이 주입된, COVID-19 감염동반 류마티스 관절염 동물모델.An animal model of rheumatoid arthritis accompanied by COVID-19 infection by injecting coronavirus-derived spike protein into rheumatoid arthritis-induced mice. 제 1항에 있어서,
상기 코로나바이러스는 SARS-Cov-2(Severe acute respiratory syndrome coronavirus 2)인 것을 특징으로 하는, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
Characterized in that the coronavirus is SARS-Cov-2 (Severe acute respiratory syndrome coronavirus 2), an animal model of rheumatoid arthritis accompanied by COVID-19 infection. 제 1항에 있어서,
상기 spike 단백질은 서열번호 1의 염기서열로 이루어진 벡터로 발현되는 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
The spike protein is an animal model of rheumatoid arthritis accompanied by COVID-19 infection, which is expressed as a vector consisting of the nucleotide sequence of SEQ ID NO: 1. 제 1항에 있어서,
상기 동물모델은, 관절염 지수 및 발병율이 대조군의 기준치와 비교하여 증가된 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
The animal model is an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which the arthritis index and incidence rate are increased compared to the reference value of the control group. 제 1항에 있어서,
상기 동물모델은, 비장 내 면역 관련 세포가 대조군의 기준치와 비교하여 증가된 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
The animal model is an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which immune-related cells in the spleen are increased compared to the reference value of the control group. 제 5항에 있어서,
상기 면역 관련 세포는 Th1, Th17 또는 Treg인 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 5,
The immune-related cells are Th1, Th17 or Treg, COVID-19 infection-accompanied rheumatoid arthritis animal model. 제 1항에 있어서,
상기 동물모델은, 혈액 또는 비장 내 IFNa의 발현이 대조군의 기준치와 비교하여 증가된 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
The animal model is an animal model of rheumatoid arthritis accompanied by COVID-19 infection, in which the expression of IFNa in the blood or spleen is increased compared to the reference value of the control group. 제 1항에 있어서,
상기 동물모델은, 혈액 내 혈전 마커인 PF4 또는 Anti-pholipid antibody의 양이 대조군의 기준치와 비교하여 증가된 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
The animal model is a COVID-19 infection-accompanied rheumatoid arthritis animal model in which the amount of PF4 or Anti-pholipid antibody, which is a blood clot marker, is increased compared to the reference value of the control group. 제 1항에 있어서,
상기 동물모델은, 염증성사이토카인인 IFN-γ, TNF-α, IL-6, IL-1β 및 MCP-1으로 이루어진 군에서 선택된 사이토카인의 발현이, 대조군의 기준치와 비교하여 증가된 것인, COVID-19 감염동반 류마티스 관절염 동물모델.According to claim 1,
In the animal model, the expression of cytokines selected from the group consisting of inflammatory cytokines IFN-γ, TNF-α, IL-6, IL-1β and MCP-1 is increased compared to the reference value of the control group, An animal model of rheumatoid arthritis with COVID-19 infection. 마우스에 류마티스 관절염을 유도하는 단계; 및
상기 류마티스 관절염이 유도된 마우스에, 코로나바이러스 유래의 spike 단백질을 주입하는 단계;를 포함하는 COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법.inducing rheumatoid arthritis in mice; and
A method of manufacturing an animal model of rheumatoid arthritis accompanied by COVID-19 infection, comprising the step of injecting coronavirus-derived spike protein into the rheumatoid arthritis-induced mouse. 제 10항에 있어서,
상기 류마티스 관절염은, 제 2형 콜라겐(Type 2 collagen)로 유도되는 것인, COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법.According to claim 10,
The method of producing an animal model of rheumatoid arthritis accompanied by COVID-19 infection, wherein the rheumatoid arthritis is induced by type 2 collagen. 제 10항에 있어서,
상기 코로나바이러스는, SARS-Cov-2(Severe acute respiratory syndrome coronavirus 2)인, COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법.According to claim 10,
The coronavirus is SARS-Cov-2 (Severe acute respiratory syndrome coronavirus 2), a method for producing an animal model of rheumatoid arthritis accompanied by COVID-19 infection. 제 10항에 있어서,
상기 spike 단백질은, 서열번호 1의 염기서열로 이루어진 벡터로 발현되는 것인, COVID-19 감염동반 류마티스 관절염 동물모델의 제작 방법.According to claim 10,
The spike protein is expressed as a vector consisting of the nucleotide sequence of SEQ ID NO: 1, a method for producing an animal model of rheumatoid arthritis accompanied by COVID-19 infection. 제 1항의 COVID-19 감염동반 류마티스 관절염 동물모델에, 후보 물질을 처리하는 단계;를 포함하는, COVID-19 감염동반 류마티스 관절염의 치료 물질의 스크리닝 방법.A method for screening a substance for treating rheumatoid arthritis accompanied by COVID-19 infection, comprising the step of processing a candidate substance in the animal model of claim 1 of COVID-19 infection. 제 14항에 있어서,
상기 후보물질은, 관절염 지수 및 발병율을 감소시키는 것인, 방법.According to claim 14,
The method of claim 1, wherein the candidate substance reduces the index and incidence of arthritis. 제 14항에 있어서,
상기 후보물질은, 비장 내 Th1 또는 Th17의 발현을 감소시키는 것인, 방법.According to claim 14,
The method of claim 1, wherein the candidate substance reduces the expression of Th1 or Th17 in the spleen. 제 14항에 있어서,
상기 후보물질은, 혈액 또는 비장의 IFNa의 발현을 감소시키는 것인, 방법.According to claim 14,
The method, wherein the candidate substance reduces the expression of IFNa in blood or spleen. 제 14항에 있어서,
상기 후보물질은, 혈액 내 혈전 마커인 PF4 또는 Anti-pholipid antibody의 양을 감소시키는 것인, 방법.According to claim 14,
The candidate material is to reduce the amount of PF4 or Anti-pholipid antibody, which is a thrombotic marker in the blood, method. 제 14항에 있어서,
상기 후보물질은, 염증성사이토카인인 IFN-γ, TNF-α, IL-6, IL-1β 및 MCP-1으로 이루어진 군에서 선택된 사이토카인의 발현을 감소시키는 것인, 방법.According to claim 14,
Wherein the candidate substance reduces the expression of a cytokine selected from the group consisting of inflammatory cytokines IFN-γ, TNF-α, IL-6, IL-1β and MCP-1, the method.
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