KR102099366B1 - Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein - Google Patents
Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein Download PDFInfo
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- KR102099366B1 KR102099366B1 KR1020180060589A KR20180060589A KR102099366B1 KR 102099366 B1 KR102099366 B1 KR 102099366B1 KR 1020180060589 A KR1020180060589 A KR 1020180060589A KR 20180060589 A KR20180060589 A KR 20180060589A KR 102099366 B1 KR102099366 B1 KR 102099366B1
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- prrsv
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Abstract
본 발명은 돼지생식기호흡기증후군 바이러스 Nsp1 단백질의 면역 증강 용도에 관한 것으로, 구체적으로 본 발명은 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1) 유전자가 도입된 도 1의 개열지도를 갖는 재조합 발현벡터, 상기 재조합 발현벡터로 형질전환된 Nsp1 단백질을 과발현하는 형질전환 세포주, Nsp1 (Non-structural protein 1) 단백질; 상기 단백질을 암호화하는 유전자 서열을 포함하는 재조합 벡터; 또는 상기 재조합 벡터로 형질전환된 세포주를 유효성분으로 포함하는 면역증강용 조성물 및 상기 조성물을 이용한 면역 증강 방법에 관한 것이다. The present invention relates to the immune enhancing use of the porcine reproductive and respiratory syndrome virus Nsp1 protein, specifically, the present invention has a cleavage map of FIG. 1 in which the porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) gene is introduced. A recombinant expression vector, a transformed cell line overexpressing the Nsp1 protein transformed with the recombinant expression vector, and a non-structural protein 1 (Nsp1) protein; A recombinant vector comprising a gene sequence encoding the protein; Or it relates to a composition for enhancing immunity comprising the cell line transformed with the recombinant vector as an active ingredient and a method for enhancing immunity using the composition.
Description
본 발명은 돼지생식기호흡기증후군 바이러스 Nsp1 단백질을 유효성분으로 포함하는 면역증강용 조성물에 관한 것이다. The present invention relates to a composition for enhancing immunity comprising a porcine reproductive and respiratory syndrome virus Nsp1 protein as an active ingredient.
돼지생식기호흡기증후군(PRRS) 바이러스는 현재 전 세계적으로 양돈 산업에 가장 큰 피해를 주는 질병이다. 이 질병이 처음 발생한 당시에는 번식 및 분만 시 손실이 가장 뚜렷했으나, 지속적인 연구가 진행되면서 PRRS 바이러스는 돼지호흡기복합증후군 (Porcine Respiratory Disease Comple, PRDC) 또는 이유후전신성소모성증후군을 유도하는 요인으로서도 매우 중요하게 인식되고 있다. 돼지생식기호흡기증후군을 유발하는 PRRS 바이러스는 크게 북미형과 유럽형으로 나뉘며, 변이가 심해 수많은 종들이 존재한다. 또한 폐포 대식세포를 파괴하여 다른 병원체의 2차 감염이 일어나기 쉽도록 하고, 직접적으로 폐에 간질성 폐렴을 일으키며, 모돈에 급성 감염되면 고열을 동반하여 유산을 일으키기도 한다.The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is currently the most damaging disease in the swine industry worldwide. At the time of the first occurrence of this disease, loss during reproduction and delivery was most pronounced, but as continuous research progressed, the PRRS virus was also very important as a factor inducing Porcine Respiratory Disease Comple (PRDC) or post-weaning systemic wasting syndrome. Is recognized. The PRRS virus, which causes swine reproductive and respiratory syndrome, is largely divided into the North American type and the European type. In addition, by destroying alveolar macrophages, secondary infections of other pathogens are likely to occur, and interstitial pneumonia is directly caused in the lungs.
PRRS 바이러스는 돼지의 비강, 근육, 경구, 자궁, 그리고 질 등을 통해서 돼지생식기호흡기증후군을 유발한다. 일반적으로 돼지들은 경구 감염보다는 비 경구감염에 더 큰 감수성을 가진다. PPRS 바이러스의 감염의 유형에는 비 간접적인 전파, 수직 감염, 지속 감염, 농장 내에서의 전파, 농장 사이의 전파가 있다. 비 간접적인 전파는 장비, 도구, 의복, 물, 음식, 매개곤충 (모기, 파리), 공기 중의 비말, 고기의 육즙 등을 통해서 일어날 수 있으며, PRRS 바이러스에 급성 감염된 돼지를 접촉한 후 60분 이내의 작업자의 작업복, 장화, 손 등에서도 PPRS 바이러스가 검출된다. 특히 겨울철 영하의 추운 날씨에서 PRRS 바이러스의 간접 전파가 더 촉진될 수 있다. The PRRS virus causes swine reproductive and respiratory syndrome through the pig's nasal cavity, muscles, oral cavity, uterus, and vagina. Pigs are generally more susceptible to non-oral infections than oral infections. Types of infection of the PPRS virus include non-indirect transmission, vertical infection, persistent infection, transmission within the farm, and transmission between farms. Non-indirect transmission can occur through equipment, tools, clothing, water, food, insects (mosquitoes, flies), airborne droplets, meat gravy, etc., within 60 minutes of contact with a pig infected with the PRRS virus. PPRS virus is also detected in workers' work clothes, boots, and hands. In particular, in cold weather in subzero winter, the indirect transmission of PRRS virus can be further promoted.
PRRS 바이러스의 임상증상은 바이러스의 종, 감염된 돼지의 면역 상황과 감수성, 복합 감염되는 질병, 그리고 다른 관리 요인들에 의해서 다양하게 나타난다. 현재 PRRS 바이러스의 진단은 PCR을 이용한 항원 검출과 ELISA를 이용한 항체 측정 그리고 혈청 항체 검사가 주를 이루고 있으나, 신속한 진단이 이루어지지 않고 있으며 바이러스 감염에 의한 질병을 효과적으로 치료할 수 있는 치료제의 개발도 진행되지 못하고 있는 실정이다.The clinical symptoms of PRRS virus vary according to the species of virus, the immune status and susceptibility of infected pigs, the disease of multiple infections, and other management factors. Currently, the diagnosis of PRRS virus mainly consists of antigen detection using PCR, antibody measurement using ELISA, and serum antibody testing, but rapid diagnosis has not been made, and the development of therapeutic agents that can effectively treat diseases caused by viral infection is not underway. It is not being done.
따라서 PRRS 바이러스를 포함하여 각종 바이러스 감염에 의한 질병을 예방하고 치료할 수 있는 근본적인 방법으로 면역 체계를 증진시킬 수 있는 새로운 기술 개발이 필요하다. Therefore, it is necessary to develop new technologies that can improve the immune system as a fundamental method to prevent and treat diseases caused by various viral infections, including PRRS virus.
이에 본 발명자들은 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1)가 대식세포의 식세포 작용을 증진시켜 궁극적으로 면역증진 효과가 있음을 규명하였고, 보다 구체적으로 Nsp1의 단백질을 과발현시키면 Smad4의 발현은 감소시키고 Rac1의 발현은 증진시켜 돼지 대식세포의 식세포 작용 증진을 통해 면역활성을 증진시킬 수 있음을 확인하였다. Accordingly, the present inventors have confirmed that the porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) enhances the phagocytic action of macrophages and ultimately has an immune-enhancing effect, and more specifically, when overexpressing the protein of Nsp1, Smad4 It was confirmed that it is possible to increase the expression of Rac1 and enhance the expression of Rac1, thereby enhancing the immune activity through enhancing the phagocytic action of porcine macrophages.
따라서 본 발명의 목적은 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1) 유전자가 도입된 도 1의 개열지도를 갖는, 재조합 발현벡터를 제공하는 것이다.Accordingly, an object of the present invention is to provide a recombinant expression vector having a cleavage map of FIG. 1 in which a porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) gene is introduced.
본 발명의 다른 목적은 상기 재조합 발현벡터로 형질전환된 Nsp1 단백질을 과발현하는 형질전환 세포주를 제공하는 것이다.Another object of the present invention is to provide a transformed cell line that overexpresses the Nsp1 protein transformed with the recombinant expression vector.
본 발명의 또 다른 목적은 Nsp1 (Non-structural protein 1) 단백질; 상기 단백질을 암호화하는 유전자 서열을 포함하는 재조합 벡터; 또는 상기 재조합 벡터로 형질전환된 세포주를 유효성분으로 포함하는, 면역증강용 조성물을 제공하는 것이다.Another object of the present invention is Nsp1 (Non-structural protein 1) protein; A recombinant vector comprising a gene sequence encoding the protein; Or to provide a composition for enhancing immunity, comprising the cell line transformed with the recombinant vector as an active ingredient.
본 발명의 또 다른 목적은 인간을 제외한 포유동물에 제5항의 조성물을 투여하는 단계를 포함하는, 면역 증강 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing immunity, comprising administering the composition of
상기와 같은 목적을 달성하기 위해 본 발명은, 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1) 유전자가 도입된 도 1의 개열지도를 갖는, 재조합 발현벡터를 제공한다.In order to achieve the above object, the present invention provides a recombinant expression vector having a cleavage map of FIG. 1 in which a porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) gene is introduced.
본 발명의 일실시예에 있어서, 상기 Nsp1(Non-structural protein 1) 유전자는 서열번호 1의 염기서열로 이루어진 것일 수 있다.In one embodiment of the present invention, the Nsp1 (Non-structural protein 1) gene may be composed of the nucleotide sequence of SEQ ID NO: 1.
또한 본 발명은 상기 재조합 발현벡터로 형질전환된 Nsp1 단백질을 과발현하는 형질전환 세포주를 제공한다. In addition, the present invention provides a transformed cell line that overexpresses the Nsp1 protein transformed with the recombinant expression vector.
본 발명의 일실시예에 있어서, 상기 Nsp1 단백질은 서열번호 2의 아미노산 서열로 이루어진 것일 수 있다. In one embodiment of the present invention, the Nsp1 protein may be composed of the amino acid sequence of SEQ ID NO: 2.
또한, 본 발명은 Nsp1 (Non-structural protein 1) 단백질; 상기 단백질을 암호화하는 유전자 서열을 포함하는 재조합 벡터; 또는 상기 재조합 벡터로 형질전환된 세포주를 유효성분으로 포함하는, 면역증강용 조성물을 제공한다. In addition, the present invention Nsp1 (Non-structural protein 1) protein; A recombinant vector comprising a gene sequence encoding the protein; Or it provides a composition for enhancing immunity, comprising the cell line transformed with the recombinant vector as an active ingredient.
본 발명의 일실시예에 있어서, 상기 Nsp1 단백질은 서열번호 2의 아미노산 서열로 이루어진 것이고, 상기 유전자는 서열번호 1의 염기서열로 이루어진 것이며, 상기 재조합 벡터는 도 1의 개열지도를 갖는 것일 수 있다.In one embodiment of the present invention, the Nsp1 protein consists of the amino acid sequence of SEQ ID NO: 2, the gene consists of the nucleotide sequence of SEQ ID NO: 1, and the recombinant vector may have a cleavage map of FIG. 1. .
본 발명의 일실시예에 있어서, 상기 조성물은 Smad4의 발현을 감소시키고, Rac1의 발현은 증가시키며, 대식세포의 식세포 작용을 증진시키는 활성을 갖는 것일 수 있다.In one embodiment of the present invention, the composition may decrease the expression of Smad4, increase the expression of Rac1, may have an activity that enhances the phagocytic action of macrophages.
나아가 본 발명은 인간을 제외한 포유동물에 제5항의 조성물을 투여하는 단계를 포함하는, 면역 증강 방법을 제공한다. Furthermore, the present invention provides a method for enhancing immunity, comprising administering the composition of
본 발명에 따른 Nsp1 (Non-structural protein 1) 단백질은 유비퀴틴-프로테아좀에 의한 단백질 분해과정을 통해 Smad4의 발현 감소를 매개시키고 반면 식세포 작용을 유도하는 Rac1의 발현은 증가시켜 궁극적으로 대식세포의 식세포 작용 증진을 통해 면역증강 효과를 유도할 수 있다. 따라서 본 발명에 따른 Nsp1 (Non-structural protein 1) 단백질은 새로운 면역 증강제로서 사용할 수 있으며 나아가 감염성 질환의 예방 또는 치료를 위한 백신 등에 유용하게 이용될 수 있다.The Nsp1 (Non-structural protein 1) protein according to the present invention mediates the reduction of Smad4 expression through proteolysis by ubiquitin-proteasome, while increasing the expression of Rac1, which induces phagocytosis, ultimately increases the expression of macrophages. Immune enhancement effect can be induced by promoting phagocytosis. Therefore, the Nsp1 (Non-structural protein 1) protein according to the present invention can be used as a new immune enhancer and further useful for vaccines for the prevention or treatment of infectious diseases.
도 1은 본 발명의 일실시예에서 PRRSV Nsp1 유전가자 도입된 재조합 벡터의 모식도를 나타낸 것이다.
도 2는 PRRSV Nsp1 유전가자 도입된 재조합 벡터를 대식세포인 3D4/31 세포주에 도입시킨 후, Nsp1의 발현을 RT-PCR 분석을 통해 확인한 것을 나타낸 것이다.
도 3은 PRRSV Nsp1이 과발현된 돼지 대식세포 세포주에서 Smad4 의 발현 수준을 웨스턴 블럿으로 확인한 결과로서, 3a는 Nsp1 단백질이 과발현된 돼지 대식세포 세포주의 전체 세포, 세포질 및 핵내에서 발현된 Smad4의 발현수준을 확인한 것이고, 3b는 사이클로헥사마이드를 시간별로 처리한 후 Smad4의 발현수준을 확인한 것이며, 3c는 MG132를 시간별로 처리한 후 Smad4의 발현수준을 확인한 것이다.
도 4는 PRRSV Nsp1 유전자가 과발현된 돼지 대식세포 세포주에서의 Rac1의 발현수준을 RT-PCR 분석을 통해 확인한 것을 나타낸 것이다.
도 5는 PRRSV Nsp1 유전자가 과발현된 돼지 대식세포 세포주의 식세포작용을 확인한 결과를 나타낸 것이다. 1 is a schematic diagram of a recombinant vector introduced with a PRRSV Nsp1 gene in an embodiment of the present invention.
2 shows that the expression of Nsp1 was confirmed through RT-PCR analysis after introducing the PRRSV Nsp1 gene introduced recombinant vector into macrophage 3D4 / 31 cell line.
Figure 3 is a result of confirming the expression level of Smad4 in the porcine macrophage cell line overexpressed PRRSV Nsp1 by Western blot, 3a is the expression level of Smad4 expressed in whole cells, cytoplasm and nucleus of the porcine macrophage cell line overexpressed with Nsp1 protein 3b is the cyclohexamide treated hourly, and then the expression level of Smad4 was confirmed. 3c was the treatment of MG132 hourly, and then the expression level of Smad4 was confirmed.
Figure 4 shows that the expression level of Rac1 in the porcine macrophage cell line overexpressed with PRRSV Nsp1 gene was confirmed through RT-PCR analysis.
Figure 5 shows the results confirming the phagocytosis of the PRRSV Nsp1 gene overexpressed porcine macrophage cell line.
본 발명은 새로운 면역 증강제로서 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1) 단백질의 용도를 제공함에 특징이 있다.The present invention is characterized by providing a use of a porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) protein as a new immune enhancer.
본 발명자들은 면역 증강제로서 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1)의 역할을 규명하기 위해, Nsp1을 과발현시킬 수 있는 재조합 발현 벡터를 제조하였고, 상기 벡터를 돼지의 대식세포에 도입시켜 과발현시킨 후, Nsp1 유전자가 도입된 세포와 유전자를 도입하지 않은 세포주에서 Smad4 및 Rac1 단백질의 발현 수준을 측정하였다.The present inventors prepared a recombinant expression vector capable of overexpressing Nsp1 to characterize the role of porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) as an immune enhancer, and the vector was added to the macrophages of pigs. After introduction and overexpression, the expression levels of Smad4 and Rac1 proteins were measured in cells into which the Nsp1 gene was introduced and cell lines in which the gene was not introduced.
그 결과, Nsp1의 과발현은 유비퀴틴-프로테오좀에 의한 단백질 분해과정을 통해 Smad4의 발현 감소를 매개함을 확인할 수 있었고 대식세포의 식세포 작용을 증진시키는 Rac1의 발현은 증가시킬 수 있음을 확인하였다.As a result, it was confirmed that the overexpression of Nsp1 mediates a decrease in the expression of Smad4 through the proteolytic process by ubiquitin-proteosome, and the expression of Rac1 that enhances the phagocytic action of macrophages can be increased.
상기 돼지생식기호흡기증후군(PRRS)의 원인체인 PRRS 바이러스(PRRSV)는 양성 단일 가닥 RNA 외피 바이러스로 Arteriviridae family에 속한다. 바이러스의 유전체는 15kb로 적어도 10개의 ORFs (Open reading frames)를 가지고 있다. ORF1a와 ORF1b가 전체 유전체의 75%를 지니고 있으며, 이로부터 발현된 단백질이 14개의 Nsp 단백질들을 만들어낸다. PRRSV ORF1a로부터 만들어지는 Nsp1은 383개의 아미노산으로 이루어져있고, Nsp1α과 Nsp1β의 두 개의 소단위체로 이루어져 있다. 또한 zinc finger motif를 가지고 있어 PPRSV 복제 시기에 subgenomic mRNAs의 전사에도 필요하다고 알려져 있다.The PRRS virus (PRRSV), which is the cause of the porcine reproductive and respiratory syndrome (PRRS), is a positive single-stranded RNA envelope virus and belongs to the Arteriviridae family. The genome of the virus is 15 kb and has at least 10 ORFs (Open reading frames). ORF1a and ORF1b have 75% of the total genome, and the expressed protein produces 14 Nsp proteins. Nsp1 made from PRRSV ORF1a consists of 383 amino acids and consists of two subunits, Nsp1α and Nsp1β. In addition, it has a zinc finger motif and is known to be necessary for transcription of subgenomic mRNAs during PPRSV replication.
또한, Smad4는 TGF-β 경로에서 신호 전달에 관여하여 Smad 경로의 한 성분이며, 단백질로 포유류에서 유일하게 존재하는 Co-smad (commone regulator smad)이다. Smad2/3과 결합하여 이형삼중 복합체(heterotrimeric complex)를 이루어 핵 내 이동(nuclear translocation)에 관여한다. 이 복합체는 DNA에 존재하는 SBE (Smad-binding element)에 결합하여 인테그린, E-카드헤린(cadherin), 콜라겐과 같은 유전자를 조절하는 것으로 알려져 있다. 한편, 본 발명에서는 Nsp1의 과발현이 유비퀴틴-프로테아좀에 의한 Smad4의 단백질 분해 작용을 매개하여 Smad4의 발현을 감소시킴을 확인하였다.In addition, Smad4 is a component of the Smad pathway involved in signal transduction in the TGF-β pathway, and is the only protein present in mammals, which is Co-smad (commone regulator smad). Combined with Smad2 / 3, it forms a heterotrimeric complex and is involved in nuclear translocation. This complex is known to regulate genes such as integrin, E-cadherin and collagen by binding to SBE (Smad-binding element) present in DNA. On the other hand, in the present invention, it was confirmed that overexpression of Nsp1 reduces the expression of Smad4 by mediating the proteolytic action of Smad4 by ubiquitin-proteasome.
Rac1은 대식세포의 식세포작용에 관여하며, 세포성장, 세포골격 재구성, 항 미생물 독성 등에 관여하는 Rho family에 속하는 GTPase이다. Rac1은 액틴네트워크를 통한 세포 이동성에 관여하며, 대식세포에서 활성화되면 막 리모델링(membrane remodeling)을 통해 식세포 작용을 유발하는 것으로 알려져 있다. Rac1 is a GTPase belonging to the Rho family, which is involved in the phagocytosis of macrophages, and is involved in cell growth, cytoskeletal reconstruction, and antimicrobial toxicity. Rac1 is involved in cell mobility through the actin network, and when activated in macrophages, it is known to induce phagocytosis through membrane remodeling.
또한, 대식세포(macrophage)는 우리 몸을 구성하는 중요한 선천면역세포 중 하나로, 모든 조직에 다양한 형태로 분포하며 정상상태에서는 침입한 외부 병원체 및 독성물질에 대한 식세포 작용을 통해 몸을 보호하는 역할을 수행한다. 또한, 이러한 대식세포의 작용은 적응면역, 상처 치료, 염증 반응 등 주요 면역반응들에도 매우 중요하다고 알려져 있다. In addition, macrophage (macrophage) is one of the important congenital immune cells that make up our body, distributed in various forms in all tissues, and under normal conditions, protects the body through phagocytosis of invading external pathogens and toxic substances. Perform. In addition, it is known that the action of macrophages is also important for major immune responses such as adaptive immunity, wound healing, and inflammatory responses.
또한, 대식세포는 우리 몸에 침입한 항원을 잡아먹는 식세포 작용(phagocytosis)을 할 뿐만 아니라 분해한 항원 조각을 세포 표면에 표시하여 T 세포에게 알리는 항원지시 세포로서의 역할을 하며, 다양한 사이토카인을 분비하여 염증 반응을 유도하고 면역세포를 활성화 시키는 작용을 한다.In addition, macrophages not only perform phagocytosis that eats the antigens that have invaded our bodies, but also serve as antigen-indicating cells to notify T cells by displaying the fragments of dissociated antigens on the cell surface and secrete various cytokines. By inducing an inflammatory response and activating immune cells.
이러한 점에서 본 발명의 Nsp1 과발현은 Rac1의 발현 증가 효과를 유도할 수 있음을 실험을 통해 확인하였고, 나아가 돼지 대식세포주의 식세포 작용을 증가시킬 수 있어 궁극적으로 면역증강 활성을 유도할 수 있음을 확인하였다.In this regard, it was confirmed through experiments that the overexpression of Nsp1 of the present invention can induce the effect of increasing the expression of Rac1, and furthermore, it is possible to increase the phagocytosis of the porcine macrophage cell line, ultimately inducing immunopotentiation activity. Did.
그러므로 본 발명은 돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1(Non-structural protein 1) 유전자가 도입된 재조합 발현벡터를 제공할 수 있으며, 바람직하게 상기 발현벡터는 도 1의 개열지도를 갖는 것일 수 있다. Therefore, the present invention can provide a recombinant expression vector into which the pig reproductive and respiratory syndrome virus (PRRSV) Nsp1 (Non-structural protein 1) gene is introduced, and preferably, the expression vector may have a cleavage map of FIG. 1.
또한, 본 발명은 상기 본 발명의 재조합 발현벡터로 형질전환된 Nsp1 단백질을 과발현하는 형질전환 세포주를 제공한다. In addition, the present invention provides a transformed cell line that overexpresses the Nsp1 protein transformed with the recombinant expression vector of the present invention.
본 발명에 있어 상기 세포주는 당업계에서 통상적으로 사용되는 형질전환방법에 의하여 제조 가능하다. 당분야의 기술적 범주에서 통상의 분자 생물학, 미생물학, 면역학 및 재조합 DNA 기술 등을 사용할 수 있다. In the present invention, the cell line can be prepared by a transformation method commonly used in the art. Conventional molecular biology, microbiology, immunology and recombinant DNA techniques can be used in the technical scope of the art.
본 발명에 있어서, 상기 세포주는 본 발명에 의한 Nsp1을 과발현시킬 수 있다면 특별한 제한이 있는 것은 아니나, 본 발명의 일실시예에는 레트로바이러스로 패키징하는 세포주인 293GPG 세포(Ory et al., 1996)를 이용하였다. 상기 세포주를 이용하면 발현 효율을 높일 수 있는 이점이 있다.In the present invention, the cell line is not particularly limited as long as it can overexpress Nsp1 according to the present invention. In one embodiment of the present invention, 293GPG cells (Ory et al., 1996), which are cell lines packaged with retrovirus, are used. Was used. The use of the cell line has the advantage of increasing the expression efficiency.
또한, 본 발명은 Nsp1 (Non-structural protein 1) 단백질; 상기 단백질을 암호화하는 유전자 서열을 포함하는 재조합 벡터; 또는 상기 재조합 벡터로 형질전환된 세포주를 유효성분으로 포함하는 면역증강용 조성물을 제공할 수 있다. In addition, the present invention Nsp1 (Non-structural protein 1) protein; A recombinant vector comprising a gene sequence encoding the protein; Alternatively, it is possible to provide a composition for enhancing immunity comprising a cell line transformed with the recombinant vector as an active ingredient.
바람직하게 상기 Nsp1 단백질은 서열번호 2의 아미노산 서열로 이루어진 것이고, 상기 유전자는 서열번호 1의 염기서열로 이루어진 것이며, 상기 재조합 벡터는 도 1의 개열지도를 갖는 것일 수 있다.Preferably, the Nsp1 protein is composed of the amino acid sequence of SEQ ID NO: 2, the gene is composed of the nucleotide sequence of SEQ ID NO: 1, and the recombinant vector may have a cleavage map of FIG. 1.
본 발명에 있어, 면역증강용 조성물은 백신들 또는 접종물들에 대한 특이적 면역 반응들을 증진시키는 약제일 수 있다. 면역증강용 조성물은 백신 또는 접종물 내에 통합된 경우, 일반적으로 또는 특이적으로 그 제조 중의 면역원성 물질들에 대한 특이적 면역반응성을 가속, 연장 또는 그 품질을 증진시키는 작용을 하는, 임의의 물질 또는 제제로서 정의될 수 있다. In the present invention, the composition for enhancing immunity may be a drug that enhances specific immune responses to vaccines or inoculants. Immunity-enhancing compositions, when incorporated into a vaccine or inoculum, generally or specifically any substance that acts to accelerate, prolong or enhance the quality of the specific immune response to immunogenic substances during its manufacture. Or as a formulation.
바람직한 형태로서, 본 발명의 면역증강용 조성물은 당업자에게 공지된 추가 성분을 포함할 수 있고, 하나 이상의 수의학적으로 허용되는 담체를 포함할 수 있다. "수의학적으로 허용 가능한 담체"는 임의의 모든 용매, 분산매질, 코팅제, 보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장제, 흡수지연제 등을 포함한다. In a preferred form, the composition for enhancing immunity of the present invention may include additional components known to those skilled in the art, and may include one or more veterinary acceptable carriers. “Veterinary acceptable carrier” includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like.
통상 면역증강용 조성물은 전형적으로, 약학적으로 허용가능한 희석제 중에 면역보강제를 유효량으로 용해 또는 분산된 면역증강용 조성물로서 사용된다. 사용량은, 사용된 T-세포 자극 면역원성 폴리펩티드 부분 및 사용된 구축물에 따라, 상이한 숙주 동물들에서 광범위하게 변경될 수 있다. 전형적인 양은 숙주 체중 kg 당 Nsp1 단백질 약 1마이크로그램(㎍)(㎍/kg) 내지 숙주 체중 kg 당 약 1밀리그램(mg) (mg/kg)이다. 보다 일반적인 양은 약 5㎍/숙주체중 kg 내지 약 0.5mg/숙주체중 kg이다. 상기 희석제는 전형적으로 수계이며, 하나 이상의 추가의 면역보강제들, 버퍼들, 염들 및 점도 개선제들을 포함할 수 있다. 희석제의 성분들은 백신 또는 접종물에 종종 존재하는 물질들이다.Typically, the composition for adjuvants is typically used as a composition for adjuvants in which an adjuvant is dissolved or dispersed in an effective amount in a pharmaceutically acceptable diluent. The amount used can vary widely in different host animals, depending on the T-cell stimulated immunogenic polypeptide portion used and the construct used. Typical amounts are about 1 microgram (μg) (μg / kg) of Nsp1 protein per kg of host body weight to about 1 milligram (mg) (mg / kg) per kg of host body weight. A more common amount is about 5 μg / kg of host body to about 0.5 mg / kg of host body. The diluent is typically aqueous and may include one or more additional adjuvants, buffers, salts and viscosity improvers. The components of the diluent are substances that are often present in vaccines or inoculants.
본 발명의 상기 면역증강용 조성물은 경구 혹은 비경구적으로 투여할 수 있다. 본 발명의 상기 면역증강용 조성물의 적합한 투여량은 제제화 방법, 투여방식, 대상 동물의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 당업자는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.The composition for enhancing immunity of the present invention can be administered orally or parenterally. Suitable dosages of the composition for enhancing the immunity of the present invention include factors such as formulation method, mode of administration, age of the target animal, weight, sex, severity of disease symptoms, food, administration time, route of administration, rate of excretion and response sensitivity. It is varied by, and a skilled artisan can easily determine and prescribe a dose effective for treatment or prevention.
본 발명에 의한 Nsp1의 유효량은 Nsp1이 면역반응을 유도하거나 유도할 수 있는 항원의 양을 의미하지만, 이에 국한되는 것은 아니며, 유효량은 적어도 10주, 바람직하게는 적어도 12주, 더욱 바람직하게는 적어도 20주의 면역 지속시간(DOI)을 부여하는 항원의 양으로 정의되는 것이 바람직하다. The effective amount of Nsp1 according to the present invention refers to the amount of antigen that Nsp1 can induce or induce an immune response, but is not limited thereto, and the effective amount is at least 10 weeks, preferably at least 12 weeks, more preferably at least It is preferably defined as the amount of antigen that confers a 20-week immune duration (DOI).
일반적으로, 상기 면역증강용 조성물에 포함되는 세포주는 필요에 따라 적정한 농도로 인간 또는 포유동물에게 접종될 수 있으며, 바람직하게는 인간을 제외한 포유동물에 접종할 수 있다. 예를들어, OD 600에서 OD값을 측정한 후 대상 동물에 약 0.5 ~ 1.5 x 10^10 CFU/ml의 값으로 접종할 수 있다. 그러나, 투여량은 이에 한정되지 않고, 제형의 종류, 투여 방법, 연령이나 체중, 증상 등을 고려하여 최종적으로 수의사의 판단에 의해 적절히 결정할 수 있다. In general, the cell line included in the composition for enhancing immunity can be inoculated to humans or mammals at an appropriate concentration, if necessary, and preferably inoculated to mammals other than humans. For example, after measuring the OD value at OD 600, the target animal may be inoculated with a value of about 0.5 to 1.5 x 10 ^ 10 CFU / ml. However, the dosage is not limited to this, and may be appropriately determined by the veterinarian's judgment in consideration of the type of dosage form, the method of administration, age, weight, and symptoms.
본 발명에 의한 상기 세포주 또는 Nsp1 단백질은 Smad4의 발현 감소 및 Rac1의 발현 증가 효과가 있고, 대식세포주의 식세포 작용 증진을 통해 강력한 면역반응을 유도할 수 있는 이점이 있다.The cell line or Nsp1 protein according to the present invention has the effect of reducing the expression of Smad4 and increasing the expression of Rac1, and has the advantage of inducing a strong immune response through enhancing phagocytosis of macrophage lines.
나아가 본 발명은 인간을 제외한 포유동물에 본 발명에 따른 면역증강용 조성물을 투여하는 단계를 포함하는, 면역 증강 방법을 제공할 수 있다.Furthermore, the present invention can provide a method for enhancing immunity, comprising administering a composition for enhancing immunity according to the present invention to a mammal other than a human.
또한, 본 발명은 인간을 제외한 포유동물에 본 발명에 따른 면역증강용 조성물을 투여하는 단계를 포함하는, 감염성 질환의 예방 또는 치료 방법을 제공할 수 있다.In addition, the present invention can provide a method for preventing or treating an infectious disease, comprising administering a composition for enhancing immunity according to the present invention to a mammal other than a human.
상기 감염성 질환은 PRRSV 등과 같은 각종 바이러스, 박테리아 및 세균 등을 포함하는 유해 물질 감염으로 유발되는 질환을 포함할 수 있다. 특히 감염성 질환의 예방에 있어서 가장 중요한 것은 보다 효율적인 백신의 개발이며, 백신의 경우 새로운 면역 증진제 또는 면역 보강제의 개발이 시급한데, 그러한 이유는 동일한 efficacy의 백신이라도 어떤 면역보강제를 사용하는냐에 따라 동일한 항원에 반응하는 면역반응을 보다 효율적으로 증진시킬 수 있기 때문이다. The infectious disease may include a disease caused by infection of harmful substances including various viruses, bacteria, and bacteria, such as PRRSV. In particular, the most important thing in the prevention of infectious diseases is the development of more efficient vaccines, and in the case of vaccines, the development of new immune enhancers or adjuvants is urgent, which is why the same antigens are used depending on which adjuvants are used for vaccines of the same efficacy. This is because it is possible to more effectively enhance the immune response in response to.
이러한 점에서 본 발명은 면역반응의 시작이되는 주요항원제시세포(antigen presenting cell)인 대식세포나 수지상세포에 Nsp1 단백질이나 유전자를 도입하여 대식세포의 식세포 작용을 활성화시킬 수 있어 면역반응을 보다 효율적으로 증진시킬 수 있다.In this regard, the present invention can activate macrophage phagocytosis of macrophages by introducing Nsp1 protein or gene into macrophage or dendritic cells, which are the main antigen presenting cells that initiate the immune response. Can promote.
그러므로 본 발명의 면역증강용 조성물은 면역증진을 유도할 수 있고 감염성 질환을 예방 또는 치료할 수 있다.Therefore, the composition for enhancing immunity of the present invention can induce immunity enhancement and prevent or treat infectious diseases.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
돼지생식기호흡기증후군 바이러스(PRRSV) Nsp1 유전자가 도입된 재조합 발현벡터의 제조Preparation of a recombinant expression vector into which the porcine reproductive and respiratory syndrome virus (PRRSV) Nsp1 gene is introduced
돼지생식기호흡기증후군 바이러스(PRRSV)의 Nsp1 cDNA 구축을 위해 유전자 데이터베이스(NCBI genebank)에서 얻은 염기서열에 따라 Nsp1 서열을 합성하였다. 이 후 상기 유전자 데이터베이스에서 얻은 염기서열에 따라 프라이머를 제작한 후, 역전사 중합효소 연쇄반응(Reverse transcription Polymerase Chain Reaction, RT-PCR)을 통하여 PRRSV Nsp1 cDNA를 수득하였고, 이후 제한효소인 EcoRI과 SalI을 이용하여 레트로바이러스 벡터(pBabe-pruo)에 제한효소로 처리한 PRRSV Nsp1 cDNA 유전자를 삽입하였다. 상기 과정을 통해 구축한 PRRSV Nsp1 과발현 재조합 벡터의 벡터맵은 도 1에 나타내었으며, 상기 실험에 사용한 프라이머 서열을 하기 표 1에 나타내었고, PRRSV Nsp1 cDNA의 유전자 서열을 서열번호 1에 나타내었다. In order to construct Nsp1 cDNA of porcine reproductive and respiratory syndrome virus (PRRSV), Nsp1 sequence was synthesized according to the nucleotide sequence obtained from the gene database (NCBI genebank). Then, after preparing the primers according to the nucleotide sequence obtained from the gene database, PRRSV Nsp1 cDNA was obtained through reverse transcription polymerase chain reaction (RT-PCR), and then the restriction enzymes EcoRI and SalI were obtained. The PRRSV Nsp1 cDNA gene treated with restriction enzyme was inserted into a retroviral vector (pBabe-pruo). The vector map of the PRRSV Nsp1 overexpression recombinant vector constructed through the above process is shown in Figure 1, the primer sequences used in the experiment are shown in Table 1, and the gene sequence of PRRSV Nsp1 cDNA is shown in SEQ ID NO: 1.
PRRSV Nsp1
U87392
<< 실시예Example 2> 2>
PRRSV Nsp1 단백질이 과발현되는 형질전환 세포주의 제조Preparation of a transformed cell line in which the PRRSV Nsp1 protein is overexpressed
상기 실시예 1에서 PRRSV Nsp1 cDNA가 삽입된 pBabe-puro 벡터를 테트라사이클린 오프 시스템(tetracycline off system)이 가동되는 293GPG 세포주를 이용하여 돼지의 대식세포주(3D4/31 세포주)에서 과발현 시켰다. 우선 293GPG 세포를 조직배양 플레이트에 5 x 106 개의 세포가 되도록 분주하고 10% 우태아 혈청을 함유한 DMEM 배지에서 배양하였다. 세포가 24시간 후 웰 표면의 70~80% 정도 채울 정도로 자라면, 혈청이 없는 DMEM 배지에 상기 실시예 1에서 제조한 PRRSV Nsp1 단백질 발현 벡터 DNA 1μg을 6μg의 Lipofectamine plus(Invitrogen)와 혼합한 후, 293GPG 세포에 각각 처리하였다. 이후 293GPG 세포로부터 생성된 PRRSV Nsp1 유전자를 함유하는 바이러스가 포함된 배양 상층액을 수득하고 이를 숙주세포인 3D4/31 세포에 처리하여 5시간 배양 후, 혈청이 10% 함유된 배지로 교체한 다음 4시간 더 배양하였고 이러한 과정을 3회 반복하였다.In Example 1, the pBabe-puro vector into which PRRSV Nsp1 cDNA was inserted was overexpressed in a pig macrophage cell line (3D4 / 31 cell line) using a 293GPG cell line running a tetracycline off system. First, 293GPG cells were divided into 5 x 10 6 cells in a tissue culture plate and cultured in DMEM medium containing 10% fetal calf serum. After the cells grow to fill 70-80% of the surface of the well after 24 hours, 1 μg of PRRSV Nsp1 protein expression vector DNA prepared in Example 1 in DMEM medium without serum is mixed with 6 μg of Lipofectamine plus (Invitrogen). , 293GPG cells. Thereafter, a culture supernatant containing a virus containing the PRRSV Nsp1 gene generated from 293GPG cells was obtained, and treated with 3D4 / 31 cells, which are host cells, and cultured for 5 hours, and then replaced with a medium containing 10% serum, followed by 4 Cultured for an additional hour and this process was repeated 3 times.
그런 뒤, PRRSV Nsp1이 형질 도입된 세포를 분리하기 위해 퓨로마이신(puromycin) 항생제 1.5 μg/ml를 처리한 후, 상기 항생제에 저항성을 갖는 PRRSV Nsp1 단백질이 발현되는 세포를 수득함으로써 PRRSV Nsp1 단백질이 과발현되는 형질전환 세포주를 얻었다. 이 후 트리졸(Trizol, Invitrogen) 1 ml 당 200 μl의 클로로포름(sigma)이 함유된 용액을 세포주에 처리하여 세포주로부터 전체 RNA를 분리하였고, 이때 추출한 1μg의 mRNA를 주형으로 Superscript III (Invitrogen) 역전사 효소 (Reverse transcriptase)를 이용하여 역전사 연쇄중합반응에 의해 cDNA를 합성하였다. 이 후, 상기 cDNA를 주형으로 하여 하기 표 2의 프라이머를 사용하여 역전사 중합효소 연쇄반응을 수행하였고, PRRSV Nsp1 유전자의 발현 정도를 측정하였다. 여기서 대조군은 PRRSV Nsp1 유전자가 삽입되지 않은 pBabe-puro 벡터만을 발현시킨 3D4/31 세포주(mock)의 mRNA를 사용하였다.Subsequently, PRRSV Nsp1 protein is overexpressed by obtaining cells expressing PRRSV Nsp1 protein resistant to the antibiotic after treating with 1.5 μg / ml of a puromycin antibiotic to isolate cells transfected with PRRSV Nsp1. Transformed cell line. Thereafter, a solution containing 200 μl of chloroform (sigma) per 1 ml of Trizol (Trizol, Invitrogen) was treated in the cell line to separate total RNA from the cell line, and the superscript III (Invitrogen) reverse transcription was performed using the extracted 1 μg mRNA as a template. CDNA was synthesized by reverse transcription chain polymerization using an enzyme (Reverse transcriptase). Subsequently, a reverse transcription polymerase chain reaction was performed using the cDNA as a template and using the primers in Table 2 below, and the expression level of the PRRSV Nsp1 gene was measured. Here, as a control, mRNA of a 3D4 / 31 cell line (mock) expressing only the pBabe-puro vector without the PRRSV Nsp1 gene was used.
PRRSV Nsp1
pGAPDH
분석결과, 도 2에 나타낸 바와 같이 본 발명에서 제조한 PRRSV Nsp1 유전자가 도입된 3D4/31 세포주에서 Nsp1 유전자가 과발현 되고 있음을 확인할 수 있었다. As a result of the analysis, it was confirmed that the Nsp1 gene is overexpressed in the 3D4 / 31 cell line into which the PRRSV Nsp1 gene prepared in the present invention was introduced, as shown in FIG. 2.
<실시예 3><Example 3>
PRRSVPRRSV Nsp1Nsp1 과발현된 Overexpressed 3D43D4 /31 세포주에서 / 31 in cell line Smad4의Smad4 발현 감소 Decreased expression
PRRSV Nsp1 단백질이 과발현되는 돼지 대식세포(3D4/31)에서 Smad4 단백질의 발현정도를 웨스턴블로팅(Western blotting)을 통해 확인하였다. 먼저 상기 실시예 2에서 제조된 PRRSV Nsp1 단백질이 과발현되는 3D4/31 세포를 프로테아제 억제제 칵테일(proteinase inhibitor cocktail)이 포함된 RIPA 용해 버퍼 (RIPA lysis buffer, 50 mM Tris, 150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS)로 용해시킨 후, 10% SDS 폴리아크릴아마이드 겔(polyacrylamide gel)에 전기 영동한 후, Hybond-P PVDF 막(GE Healthcare, Buckinghamshire, UK)으로 단백질들을 이동시켰다. 이후 돼지 C1qBP 분자에 특이적으로 결합하는 항 Smad4 항체(Abcam)를 멤브레인(membrane)에 처리한 후 ECL 시스템(GE healthcare,Buckinghamshire, UK)을 이용하여 발색시켜 Smad4 단백질의 발현 여부를 측정하였으며 그 결과를 도 3a에 나타내었다. 이때 내적 대조군(Internal control)으로 β-actin을 이용하였다.The expression level of Smad4 protein in porcine macrophages (3D4 / 31) overexpressing PRRSV Nsp1 protein was confirmed by Western blotting. First, 3D4 / 31 cells overexpressing the PRRSV Nsp1 protein prepared in Example 2 are RIPA lysis buffers containing proteinase inhibitor cocktail (RIPA lysis buffer, 50 mM Tris, 150 mM NaCl, 1.0% NP- After dissolving with 40, 0.5% deoxycholate, 0.1% SDS), electrophoresis was performed on a 10% SDS polyacrylamide gel, and then proteins were transferred to a Hybond-P PVDF membrane (GE Healthcare, Buckinghamshire, UK). . Subsequently, the anti-Smad4 antibody (Abcam) that specifically binds to the pig C1qBP molecule was treated on a membrane, and then developed using an ECL system (GE healthcare, Buckinghamshire, UK) to measure the expression of Smad4 protein. It is shown in Figure 3a. At this time, β-actin was used as an internal control.
분석 결과, PRRSV Nsp1 단백질이 과발현되는 돼지 대식세포(3D4/31)의 경우 핵, 사이토졸 및 세포 전체에서 Smad4 단백질의 발현이 감소됨을 알 수 있었다. 즉, 이러한 결과는 PRRSV Nsp1 단백질이 Smad4 단백질의 발현 또는 활성을 억제하는 작용이 있음을 의미한다. As a result of the analysis, it was found that in the case of porcine macrophages (3D4 / 31) overexpressing the PRRSV Nsp1 protein, the expression of Smad4 protein was decreased in the nucleus, cytosol, and the entire cell. That is, these results indicate that the PRRSV Nsp1 protein has an action of inhibiting the expression or activity of the Smad4 protein.
나아가 본 발명자들은 PRRSV Nsp1 단백질에 의한 Smad4의 발현 감소의 기작을 구체적으로 알아보기 위해, 먼저 단백질 합성 억제제인 사이클로헥사마이드(Cycloheximide) 100μg/ml를 세포주에 각각 시간별로 처리한 후, 위와 같이 웨스턴 블럿 방법으로 Smad4의 발현 정도를 분석하였고, 또한 유비퀴닌화된 단백질을 매개하여 단백질을 분해하는 프로테오좀 억제제인 MG132(20μM)을 시간별로 처리한 후, 동일한 방법을 수행하여 Smad4의 발현 정도를 분석하였다. Furthermore, the present inventors first examined the mechanism of PRRSV Nsp1 protein by reducing the expression of Smad4, first treated with the protein synthesis inhibitor Cycloheximide 100μg / ml for each cell line hourly, and then Western blot as above. The expression level of Smad4 was analyzed by the method, and MG132 (20 μM), a proteosome inhibitor that mediates ubiquininated protein to decompose the protein, was processed hourly, and the same method was performed to analyze the expression level of Smad4. Did.
그 결과, 사이클로헥사마이드를 처리한 세포주에서는 Smad4의 발현이 세포질 및 핵 모두에서 감소하고 있는 것으로 나타났고, 특히 PRRSV Nsp1 단백질이 과발현된 돼지 대식세포 세포주에서 Smad4의 발현이 더 두드러지게 감소하고 있는 것으로 나타났다(도 2b 참조). 한편, MG132를 처리한 경우, PRRSV Nsp1 단백질이 과발현된 돼지 대식세포 세포주에서는 사이클로헥사마이드에 의한 Smad4의 발현감소가 억제되고 있는 양상을 확인할 수 있었다. 즉, PRRSV Nsp1 단백질이 과발현된 돼지 대식세포 세포주에서는 사이클로헥사마이드 처리 시 Smad4의 발현이 감소되는 것으로 나타났으나, 프로테오좀 억제제인 MG132를 처리한 군은 MG132를 처리하지 않은 군에 비해 Smad4의 발현이 증가되어 있는 것으로 나타났다(도 3c 참조).As a result, it was found that the expression of Smad4 was decreased in both the cytoplasm and nucleus in the cell line treated with cyclohexamide, and in particular, the expression of Smad4 was more significantly decreased in the porcine macrophage cell line overexpressing the PRRSV Nsp1 protein. Appeared (see Figure 2b). On the other hand, when treated with MG132, the PRRSV Nsp1 protein was overexpressed in the pig macrophage cell line, it was confirmed that the pattern is inhibited by the reduction of the expression of Smad4 by cyclohexamide. That is, in the porcine macrophage cell line overexpressing the PRRSV Nsp1 protein, the expression of Smad4 was decreased upon cyclohexamide treatment, but the group treated with the proteosome inhibitor MG132 was compared to the group not treated with MG132. It was shown that the expression was increased (see Fig. 3c).
<실시예 4><Example 4>
PRRSVPRRSV Nsp1Nsp1 과발현된 Overexpressed 3D43D4 /31 세포주에서 / 31 in cell line Rac1Rac1 의 발현 감소 Decreased expression of
PRRSV Nsp1 과발현 돼지 대식세포주에서 돼지 Rac1 유전자의 발현정도를 확인하기 위하여 Real-time PCR을 진행하였다. 이를 위해 Mock/3D4/31 세포(대조군)와 PRRSV Nsp1/3D4/31 세포(Nsp1 과발현 세포주)를 수집하여 상기 실시예에서 기술된 방법으로 RNA를 분리하고 cDNA를 합성하여 Real-time PCR을 수행하였다.Real-time PCR was performed to confirm the expression level of porcine Rac1 gene in the PRRSV Nsp1 overexpressing porcine macrophage cell line. To this end, Mock / 3D4 / 31 cells (control) and PRRSV Nsp1 / 3D4 / 31 cells (Nsp1 overexpressing cell line) were collected to isolate RNA by the method described in the above example, and synthesized cDNA to perform real-time PCR. .
그 결과, 도 4에 나타낸 바와 같이, PRRSV Nsp1이 과발현되는 세포주에서는 Rac1 유전자의 발현이 증가되어 있는 것으로 나타났다. 따라서 PRRSV Nsp1은 Rac1 유전자의 발현은 증가시키는 반면, Smad4의 발현은 감소시키는 활성이 있음을 알 수 있었다.As a result, as shown in FIG. 4, it was shown that the expression of the Rac1 gene is increased in the cell line in which PRRSV Nsp1 is overexpressed. Therefore, it was found that PRRSV Nsp1 increases Rac1 gene expression while reducing Smad4 expression.
<< 실시예Example 5> 5>
PRRSVPRRSV Nsp1Nsp1 과발현에 따른 대식세포인 Macrophages according to overexpression 3D43D4 /31 세포주의 식세포 활성분석/ 31 Phagocytic activity analysis of cell lines
나아가 본 발명자들은 본 발명의 PRRSV Nsp1이 대식세포의 식세포 활성 증가를 통해 면역증진 작용이 있는지 확인하였다. 식세포는 체내의 이물질 또는 외부에서 침입한 세균 등을 섭취하여 이들을 제거하는 역할을 한다. Furthermore, the present inventors confirmed that PRRSV Nsp1 of the present invention has an immune-enhancing effect through an increase in macrophage activity of macrophages. Phagocytes take in foreign substances in the body or bacteria that have invaded from the outside to remove them.
이러한 식세포 작용 분석을 위해 먼저, PRRSV NSP1 단백질 과발현 돼지 대식세포를 6 웰 플레이트에 웰 당 5x105세포가 되도록 접종하고 10% 송아지 혈청과 1% 페니실린/스트렙토마이신, 10mM HEPES, 1mM sodium pyruvate 및 4.5g/L glucose를 포함하고 있는 RPMI 1640 배지에서 배양하였다. 24시간 후 세포가 웰 표면의 40~60%정도를 덮을 정도로 자라면, yellow-green fluorescent microsphere (size 2.0 μm, FluoSpheres carboxylate modified microspheres, Invitrogen) 형광비드를 1ml의 배지에 1:500으로 희석하여 처리한 후, 37℃의 조건에서 4시간 배양하였다. 이 후, 세포에서 식세포 작용을 통해 흡수한 형광비드를 유세포분석기(Flowcytometry)로 분석하였다.For this phagocytosis analysis, first, PRRSV NSP1 protein overexpressing pig macrophages were inoculated to 5x10 5 cells per well in a 6-well plate, 10% calf serum and 1% penicillin / streptomycin, 10mM HEPES, 1mM sodium pyruvate and 4.5g It was cultured in RPMI 1640 medium containing / L glucose. After 24 hours, if the cells grow to cover about 40-60% of the surface of the well, the yellow-green fluorescent microsphere (size 2.0 μm, FluoSpheres carboxylate modified microspheres, Invitrogen) fluorescent beads are diluted 1: 500 in 1 ml of medium and treated. After that, the cells were cultured at 37 ° C for 4 hours. Subsequently, the fluorescent beads absorbed from the cells through phagocytosis were analyzed by flow cytometry.
그 결과, 도 5에 나타낸 바와 같이 PRRSV Nsp1 단백질이 과발현된 돼지 대식세포 세포주는 대조군에 비해 식세포 작용이 월등하게 증가되어 있는 것으로 나타났다. As a result, as shown in Figure 5, the PRRSV Nsp1 protein overexpressed porcine macrophage cell line was found to have a significantly increased phagocytic action compared to the control group.
이러한 결과들을 통해 본 발명자들은 본 발명의 PRRSV Nsp1 단백질은 대식세포의 식세포 작용을 촉진시켜 면역반응을 효과적으로 유도할 수 있는 바, 면역증강을 위한 보조제로 활용할 수 있으며, 나아가 면역저하에 의해 유발될 수 있는 감염질환의 예방 또는 치료를 위한 치료제 및 백신보조제로 이용할 수 있음 확인하였다.Through these results, the present inventors PRRSV Nsp1 protein of the present invention can effectively induce an immune response by promoting the phagocytic action of macrophages, and can be used as an adjuvant for enhancing immunity, and may also be caused by immunosuppression. It was confirmed that it can be used as a therapeutic agent and vaccine supplement for the prevention or treatment of infectious diseases.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.
<110> Industry Academic Cooperation Foundation of Korea University <120> Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein <130> NPDC69125 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1146 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for PRRSV Nsp1 <400> 1 atgtctggga tacttgatcg gtgcacgtgt acccccaatg ccagggtgtt tatggcggag 60 ggccaagtct actgcacacg atgcctcagt gcacggtctc tccttcccct gaacctccaa 120 gtttctgagc tcggggtgct aggcctattc tacaggcccg aagagccact ccggtggacg 180 ttgccacgtg cattccccac tgttgagtgc tcccccgccg gggcctgctg gctttctgca 240 atctttccaa tcgcacgaat gaccagtgga aacctgaact tccaacaaag aatggtacgg 300 gtcgcagctg agctttacag agccggccag ctcacccctg cagtcttgaa ggctctacaa 360 gtttatgaac ggggttgccg ctggtacccc attgttggac ctgtccctgg agtggccgtt 420 ttcgccaatt ccctacatgt gagtgataaa cctttcccgg gagcaactca cgtgttgacc 480 aacctgccgc tcccgcagag acccaagcct gaagactttt gcccctttga gtgtgctatg 540 gctactgtct atgacattgg tcatgacgcc gtcatgtatg tggccgaaag gaaagtctcc 600 tgggcccctc gtggcgggga tgaagtgaaa tttgaagctg tccccgggga gttgaagttg 660 attgcgaacc ggctccgcac ctccttcccg ccccaccaca cagtggacat gtctaagttc 720 gccttcacag cccctgggtg tggtgtttct atgcgggtcg aacgccaaca cggctgcctt 780 cccgctgaca ctgtccctga aggcaactgc tggtggagct tgtttgactt gcttccactg 840 gaagttcaga acaaagaaat tcgccatgct aaccaatttg gctaccagac caagcatggt 900 gtctctggca agtacctaca gcggaggctg caagttaatg gtctccgagc agtaactgac 960 ctaaacggac ctatcgtcgt acagtacttc tccgttaagg agagttggat ccgccatttg 1020 aaactggcgg gagaacccag ctactctggg tttgaggacc tcctcagaat aagggttgag 1080 cctaacacgt cgccattggc tgacaaggaa gaaaaaattt tccggtttgg cagtcacaag 1140 tggtac 1146 <210> 2 <211> 383 <212> PRT <213> Artificial Sequence <220> <223> Amino acid for PRRSV Nsp1 <400> 2 Met Ser Gly Ile Leu Asp Arg Cys Thr Cys Thr Pro Asn Ala Arg Val 1 5 10 15 Phe Met Ala Glu Gly Gln Val Tyr Cys Thr Arg Cys Leu Ser Ala Arg 20 25 30 Ser Leu Leu Pro Leu Asn Leu Gln Val Ser Glu Leu Gly Val Leu Gly 35 40 45 Leu Phe Tyr Arg Pro Glu Glu Pro Leu Arg Trp Thr Leu Pro Arg Ala 50 55 60 Phe Pro Thr Val Glu Cys Ser Pro Ala Gly Ala Cys Trp Leu Ser Ala 65 70 75 80 Ile Phe Pro Ile Ala Arg Met Thr Ser Gly Asn Leu Asn Phe Gln Gln 85 90 95 Arg Met Val Arg Val Ala Ala Glu Leu Tyr Arg Ala Gly Gln Leu Thr 100 105 110 Pro Ala Val Leu Lys Ala Leu Gln Val Tyr Glu Arg Gly Cys Arg Trp 115 120 125 Tyr Pro Ile Val Gly Pro Val Pro Gly Val Ala Val Phe Ala Asn Ser 130 135 140 Leu His Val Ser Asp Lys Pro Phe Pro Gly Ala Thr His Val Leu Thr 145 150 155 160 Asn Leu Pro Leu Pro Gln Arg Pro Lys Pro Glu Asp Phe Cys Pro Phe 165 170 175 Glu Cys Ala Met Ala Thr Val Tyr Asp Ile Gly His Asp Ala Val Met 180 185 190 Tyr Val Ala Glu Arg Lys Val Ser Trp Ala Pro Arg Gly Gly Asp Glu 195 200 205 Val Lys Phe Glu Ala Val Pro Gly Glu Leu Lys Leu Ile Ala Asn Arg 210 215 220 Leu Arg Thr Ser Phe Pro Pro His His Thr Val Asp Met Ser Lys Phe 225 230 235 240 Ala Phe Thr Ala Pro Gly Cys Gly Val Ser Met Arg Val Glu Arg Gln 245 250 255 His Gly Cys Leu Pro Ala Asp Thr Val Pro Glu Gly Asn Cys Trp Trp 260 265 270 Ser Leu Phe Asp Leu Leu Pro Leu Glu Val Gln Asn Lys Glu Ile Arg 275 280 285 His Ala Asn Gln Phe Gly Tyr Gln Thr Lys His Gly Val Ser Gly Lys 290 295 300 Tyr Leu Gln Arg Arg Leu Gln Val Asn Gly Leu Arg Ala Val Thr Asp 305 310 315 320 Leu Asn Gly Pro Ile Val Val Gln Tyr Phe Ser Val Lys Glu Ser Trp 325 330 335 Ile Arg His Leu Lys Leu Ala Gly Glu Pro Ser Tyr Ser Gly Phe Glu 340 345 350 Asp Leu Leu Arg Ile Arg Val Glu Pro Asn Thr Ser Pro Leu Ala Asp 355 360 365 Lys Glu Glu Lys Ile Phe Arg Phe Gly Ser His Lys Trp Tyr *** 370 375 380 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Forward primer <400> 3 ccggaattca tgtctgggat acttgat 27 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Reverse primer <400> 4 acgcgtcgac tcagtaccac ttgtgactgc c 31 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Forward primer <400> 5 gcctcagtgc acggtctctc ctt 23 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Reverse primer <400> 6 ggctgtgaag gcgaacttag a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH Forward primer <400> 7 atgaccacag tccatgccat c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH Reverse primer <400> 8 cctgcttcac caccttcttg 20 <110> Industry Academic Cooperation Foundation of Korea University <120> Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein <130> NPDC69125 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1146 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for PRRSV Nsp1 <400> 1 atgtctggga tacttgatcg gtgcacgtgt acccccaatg ccagggtgtt tatggcggag 60 ggccaagtct actgcacacg atgcctcagt gcacggtctc tccttcccct gaacctccaa 120 gtttctgagc tcggggtgct aggcctattc tacaggcccg aagagccact ccggtggacg 180 ttgccacgtg cattccccac tgttgagtgc tcccccgccg gggcctgctg gctttctgca 240 atctttccaa tcgcacgaat gaccagtgga aacctgaact tccaacaaag aatggtacgg 300 gtcgcagctg agctttacag agccggccag ctcacccctg cagtcttgaa ggctctacaa 360 gtttatgaac ggggttgccg ctggtacccc attgttggac ctgtccctgg agtggccgtt 420 ttcgccaatt ccctacatgt gagtgataaa cctttcccgg gagcaactca cgtgttgacc 480 aacctgccgc tcccgcagag acccaagcct gaagactttt gcccctttga gtgtgctatg 540 gctactgtct atgacattgg tcatgacgcc gtcatgtatg tggccgaaag gaaagtctcc 600 tgggcccctc gtggcgggga tgaagtgaaa tttgaagctg tccccgggga gttgaagttg 660 attgcgaacc ggctccgcac ctccttcccg ccccaccaca cagtggacat gtctaagttc 720 gccttcacag cccctgggtg tggtgtttct atgcgggtcg aacgccaaca cggctgcctt 780 cccgctgaca ctgtccctga aggcaactgc tggtggagct tgtttgactt gcttccactg 840 gaagttcaga acaaagaaat tcgccatgct aaccaatttg gctaccagac caagcatggt 900 gtctctggca agtacctaca gcggaggctg caagttaatg gtctccgagc agtaactgac 960 ctaaacggac ctatcgtcgt acagtacttc tccgttaagg agagttggat ccgccatttg 1020 aaactggcgg gagaacccag ctactctggg tttgaggacc tcctcagaat aagggttgag 1080 cctaacacgt cgccattggc tgacaaggaa gaaaaaattt tccggtttgg cagtcacaag 1140 tggtac 1146 <210> 2 <211> 383 <212> PRT <213> Artificial Sequence <220> <223> Amino acid for PRRSV Nsp1 <400> 2 Met Ser Gly Ile Leu Asp Arg Cys Thr Cys Thr Pro Asn Ala Arg Val 1 5 10 15 Phe Met Ala Glu Gly Gln Val Tyr Cys Thr Arg Cys Leu Ser Ala Arg 20 25 30 Ser Leu Leu Pro Leu Asn Leu Gln Val Ser Glu Leu Gly Val Leu Gly 35 40 45 Leu Phe Tyr Arg Pro Glu Glu Pro Leu Arg Trp Thr Leu Pro Arg Ala 50 55 60 Phe Pro Thr Val Glu Cys Ser Pro Ala Gly Ala Cys Trp Leu Ser Ala 65 70 75 80 Ile Phe Pro Ile Ala Arg Met Thr Ser Gly Asn Leu Asn Phe Gln Gln 85 90 95 Arg Met Val Arg Val Ala Ala Glu Leu Tyr Arg Ala Gly Gln Leu Thr 100 105 110 Pro Ala Val Leu Lys Ala Leu Gln Val Tyr Glu Arg Gly Cys Arg Trp 115 120 125 Tyr Pro Ile Val Gly Pro Val Pro Gly Val Ala Val Phe Ala Asn Ser 130 135 140 Leu His Val Ser Asp Lys Pro Phe Pro Gly Ala Thr His Val Leu Thr 145 150 155 160 Asn Leu Pro Leu Pro Gln Arg Pro Lys Pro Glu Asp Phe Cys Pro Phe 165 170 175 Glu Cys Ala Met Ala Thr Val Tyr Asp Ile Gly His Asp Ala Val Met 180 185 190 Tyr Val Ala Glu Arg Lys Val Ser Trp Ala Pro Arg Gly Gly Asp Glu 195 200 205 Val Lys Phe Glu Ala Val Pro Gly Glu Leu Lys Leu Ile Ala Asn Arg 210 215 220 Leu Arg Thr Ser Phe Pro Pro His His Thr Val Asp Met Ser Lys Phe 225 230 235 240 Ala Phe Thr Ala Pro Gly Cys Gly Val Ser Met Arg Val Glu Arg Gln 245 250 255 His Gly Cys Leu Pro Ala Asp Thr Val Pro Glu Gly Asn Cys Trp Trp 260 265 270 Ser Leu Phe Asp Leu Leu Pro Leu Glu Val Gln Asn Lys Glu Ile Arg 275 280 285 His Ala Asn Gln Phe Gly Tyr Gln Thr Lys His Gly Val Ser Gly Lys 290 295 300 Tyr Leu Gln Arg Arg Leu Gln Val Asn Gly Leu Arg Ala Val Thr Asp 305 310 315 320 Leu Asn Gly Pro Ile Val Val Gln Tyr Phe Ser Val Lys Glu Ser Trp 325 330 335 Ile Arg His Leu Lys Leu Ala Gly Glu Pro Ser Tyr Ser Gly Phe Glu 340 345 350 Asp Leu Leu Arg Ile Arg Val Glu Pro Asn Thr Ser Pro Leu Ala Asp 355 360 365 Lys Glu Glu Lys Ile Phe Arg Phe Gly Ser His Lys Trp Tyr *** 370 375 380 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Forward primer <400> 3 ccggaattca tgtctgggat acttgat 27 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Reverse primer <400> 4 acgcgtcgac tcagtaccac ttgtgactgc c 31 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Forward primer <400> 5 gcctcagtgc acggtctctc ctt 23 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRRSV Nsp1 Reverse primer <400> 6 ggctgtgaag gcgaacttag a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH Forward primer <400> 7 atgaccacag tccatgccat c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH Reverse primer <400> 8 cctgcttcac caccttcttg 20
Claims (8)
시험관내 대식세포의 식세포 작용 증진용 조성물.Nsp1 (Non-structural protein 1) protein consisting of the amino acid sequence of SEQ ID NO: 2; A recombinant vector comprising an Nsp1 gene sequence consisting of the nucleotide sequence of SEQ ID NO: 1 encoding the protein; Or the cell line transformed with the recombinant vector; containing as an active ingredient,
Composition for enhancing macrophage action of macrophages in vitro.
상기 조성물은 Smad4의 발현을 감소시키고, Rac1의 발현은 증가시키는 것을 특징으로 하는, 시험관내 대식세포의 식세포 작용 증진용 조성물.The method of claim 5,
The composition is characterized in that to reduce the expression of Smad4, Rac1 expression is increased, a composition for enhancing the phagocytic action of macrophages in vitro.
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| Beura, LK. et al., Journal of Virology (2010) 84(3):1574-1584* |
| GenBank FJ175689.1 < Porcine respiratory and reproductive syndrome virus isolate PRRSV03, complete genome > (2016.07.26.)* |
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