KR101438542B1 - Immunologic adjuvant using ORF3 encoded by PCV2 - Google Patents
Immunologic adjuvant using ORF3 encoded by PCV2 Download PDFInfo
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- KR101438542B1 KR101438542B1 KR1020120115098A KR20120115098A KR101438542B1 KR 101438542 B1 KR101438542 B1 KR 101438542B1 KR 1020120115098 A KR1020120115098 A KR 1020120115098A KR 20120115098 A KR20120115098 A KR 20120115098A KR 101438542 B1 KR101438542 B1 KR 101438542B1
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Abstract
본 발명은 돼지 써코바이러스2(PCV2) ORF3 유전자가 도입된 도 1의 개열지도를 가지는 재조합 발현벡터 및 이를 포함하는 면역보강제에 관한 것이다. 본 발명에 의하면, PCV2 ORF3 단백질 과발현은 세포내에서 염증성 사이토카인인 IL-6와 IL-8의 과다 발현을 유발하여 더욱 강한 면역반응을 일으킬 수 있어 PCV2에 의한 PMWS을 포함하는 감염성 질환 또는 암의 치료 또는 예방에 유용하게 이용할 수 있다. The present invention relates to a recombinant expression vector having a cleavage map of FIG. 1 into which a porcine circovirus 2 (PCV2) ORF3 gene has been introduced, and a immunoconjugate containing the recombinant expression vector. According to the present invention, overexpression of PCV2 ORF3 protein can induce overexpression of IL-6 and IL-8, which are inflammatory cytokines in the cell, resulting in a stronger immune response. Therefore, it is possible to prevent the infectious disease or cancer including PCW2- Can be usefully used for treatment or prevention.
Description
본 발명은 돼지 서코바이러스 제2형의 ORF3를 이용하는 면역보강제에 관한 것이다. 더욱 상세하게, 본 발명은 돼지 서코바이러스 제2형(PCV2)의 ORF3(open reading frame 3) 단백질을 과발현하여 유비퀴틴-프로테오좀에 의한 단백질 분해과정을 통해 RGS16 분자의 발현 감소를 매개하며, 이에 따라 염증성 사이토카인(pro-inflammatory cytokine)인 인터루킨-6(IL-6; interleukin-6)와 인터루킨-8(IL-8; interleukin-8)의 과다 발현을 유도할 수 있는 돼지 서코바이러스 제2형의 ORF3를 포함하는 새로운 면역 보강제에 관한 것이다.
The present invention relates to an immunostimulant using porcine circovirus type 2 ORF3. More specifically, the present invention overexpresses the ORF3 (open reading frame 3) protein of porcine circovirus type 2 (PCV2), mediates the reduction of the expression of RGS16 molecule through protein degradation by ubiquitin-proteasome, (IL-6) and interleukin-8 (IL-8), which are proinflammatory cytokines, are involved in the pathogenesis of porcine circovirus type 2 Lt; RTI ID = 0.0 > ORF3. ≪ / RTI >
돼지 서코바이러스 2형(PCV2)은 단일 가닥의 닫힌 원형의 게놈을 보유하는 작은 비-외피 바이러스이며 1768개의 염기서열을 갖는다(Rose et al., 2012). 돼지 서코바이러스는 돼지에게 질병을 일으키지 못하는 제 1형과 이유후 전신 소모성 증후군(Post-weaing Multisystemic Wasting Syndrome, PMWS)의 병인학적 요인이 되는 제 2형으로 나눌 수 있다(Tischer et al., 1995; Allan et al., 1999). Swine circovirus type 2 (PCV2) is a small non-enveloped virus carrying a single-stranded closed circular genome and has 1768 nucleotide sequences (Rose et al., 2012). The porcine circovirus can be divided into
PMWS는 말초 림프관의 확장, 림프구의 감소, 점진적인 체중감소, 호흡기 장애(respiratory dysfunction), 설사 등의 증상을 가지며 다양한 원인에 의해 발병한다(Allan and Ellis, 2000; Segales et al., 2005). PMWS is caused by a variety of causes including peripheral lymphatic dilatation, lymphocyte depletion, progressive weight loss, respiratory dysfunction, and diarrhea (Allan and Ellis, 2000; Segales et al., 2005).
PMWS 이외에, PCV2가 연관된 감염 질병으로는 돼지 생식기 및 호흡기 증후군(porcine reproductive and respiratory syndrome, PRRS), 가성 광견병(pseudorabies), 글래서씨병(Glasser's disease), 화농성 기관지폐렴(suppurative bronchopneumonia) 등이 있다.In addition to PMWS, PCV2-associated infectious diseases include porcine reproductive and respiratory syndrome (PRRS), pseudorabies, Glasser's disease, and suppurative bronchopneumonia.
PCV2 감염으로 인한 피해는 매우 심각하다. PCV2 감염으로 인한 피해로 인해 2010년 정부가 300억 원의 예산을 조성, 농가에 관납백신을 공급할 정도로 PCV2 백신의 중요성은 이미 다 알려진 사실이다. 그러나 PCV2 불활화 백신의 문제점은 아직 백신에 의해 항체 형성이 미약하고 백신접종 이후에도 여전히 돼지의 체내에 바이러스가 감염되어 있는 상태를 유지하여 면역 스트레스나 기타 다른 감염원에 의해 쉽게 증상을 발생시키고 있다는 점이다. 따라서, 양돈 산업계에 미치는 이러한 강력한 영향으로 인해, PCV2에 대한 효과적인 백신 및 백신에 의한 면역반응을 효율적으로 증진시킬 수 있는 면역보강제의 개발이 점차 중요해지고 있다. The damage caused by PCV2 infection is very serious. The importance of the PCV2 vaccine is already known to the extent that the government will provide 30 billion won for the budget in 2010 and supply the vaccine to farmers due to the damage caused by PCV2 infection. However, the problem with PCV2 inactivated vaccine is that the antibody formation is weak by the vaccine and the virus still remains in the body of the pig after the vaccination, which is easily caused by immune stress or other infectious agents . Thus, due to this strong impact on the swine industry, the development of immunoconjugants that can effectively enhance effective vaccines and vaccine-mediated immune responses against PCV2 is becoming increasingly important.
PCV2는 ORF1 단백질과 ORF2의 주된 두 가지의 단백질로 구성된다. ORF1은 바이러스의 복제를 담당하는 단백질을 암호화하고 ORF2는 약 30kDa의 분자량을 갖는 당단백질로서 바이러스의 유전체 핵산을 둘러싸는 캡시드를 암호화하고 있다(Mankertz et al., 1998; Nawagitgul et al., 2000). 비교적 새로운 단백질인 ORF3 단백질은 PCV2의 복제에는 연관이 없다고 알려져 있으나 생체 내, 외에서 PCV2의 병원성에 관련이 있다고 보고되어 있다(Liu et al., 2005 and 2006). 더욱이, 바이러스가 유도하는 세포자멸사(apoptosis)는 ORF3 단백질과 돼지 p53의 E3 유비퀴틴 연결효소와의 상호작용을 통해서 p53의 단백질 발현을 높임으로써 중개된다고 보고되어 있다(Liu et al., 2007). PCV2 consists of two major proteins, ORF1 and ORF2. ORF1 encodes a protein responsible for replication of the virus and ORF2 is a glycoprotein with a molecular weight of about 30 kDa that encodes a capsid that surrounds the viral genomic nucleic acid (Mankertz et al., 1998; Nawagitgul et al., 2000) . The ORF3 protein, a relatively new protein, is known to be unrelated to the replication of PCV2, but it has been reported to be related to the virulence of PCV2 in vitro and in vivo (Liu et al., 2005 and 2006). Furthermore, virus-induced apoptosis has been reported to be mediated by enhancing protein expression of p53 through interaction of ORF3 protein with E3 ubiquitin ligating enzyme in porcine p53 (Liu et al., 2007).
박테리아 투 하이브리드 시스템(Bacterial two hybrid system)을 이용하여 찾아낸 ORF3와 상호작용하는 또 다른 단백질로는 G 단백질 신호조절 인자인 RGS16(Regulators of G protein signaling 16)이 있다(Timmusk et al., 2006). RGS 단백질은 large G 단백질의 활성화된 알파 소단위와 결합함으로써, 신호전달의 종결을 가속화하여 신호전달을 억제하는 역할을 한다고 알려져 있고(Willars, 2006), 따라서 RGS 단백질의 조절은 신경전달물질이나 호르몬, 키모카인(chemokine) 등 다양한 자극에 대한 세포의 반응에 영향을 미칠 수 있다(Jean-Baptiste et al., 2006). Another protein interacting with ORF3 found using the bacterial two hybrid system is Regulators of G protein signaling 16 (Timmusk et al., 2006). RGS protein is known to play a role in signal transduction by accelerating the termination of signal transduction by binding to the activated alpha subunit of large G protein (Willars, 2006) Chemokine, etc. (Jean-Baptiste et al., 2006).
한편, 인터루킨-6(IL-6; interleukin-6)는 면역반응에 의해 T 세포, B 세포, 대식세포(macrophage), 상피세포(epithelial cell) 등 다양한 세포로부터 분비되는 사이토카인(cytokine)이다. IL-6는 B 세포의 항체 생산, 림프구(lymphocyte)의 활성 자극 등의 다양한 작용을 한다(Kishimoto, 1989). 인터루킨-8(interleukin-8)은 대식세포나 상피세포, 내피세포(endothelial cell) 등 다양한 세포에서 생성되는 키모카인(chemokine)이다. IL-8은 호중구(neutrophil)를 포함한 과립구(granulocyte), 대식세포, 비만세포(mast cell) 등의 표적세포들을 감염장소로 이동시키고 활성화를 자극하는 작용을 한다(Baggiolini and Clark-Lewis, 1992). Interleukin-6 (IL-6), on the other hand, is a cytokine secreted from various cells such as T cells, B cells, macrophages, and epithelial cells by an immune response. IL-6 acts in a variety of ways, including antibody production of B cells and stimulation of lymphocyte activation (Kishimoto, 1989). Interleukin-8 is a chemokine produced by various cells such as macrophages, epithelial cells and endothelial cells. IL-8 acts to stimulate activation of target cells such as neutrophils, including granulocytes, macrophages, and mast cells (Baggiolini and Clark-Lewis, 1992) .
본 발명의 배경이 되는 기술로, 대한민국 특허공보 10-1030792(201.04.27)에는 돼지 써코바이러스2(PCV2) ORF2 유전자의 표면발현용 벡터 및 이로 형질전환된 살모넬라 백신 균주에 대해 기재되어 있고, 국제공개 WO2006/132598 A1(2006.12.14)에는 PCV2의 ORF3 및 이의 apoptosis의 작용을 확인하고, 적어도 부분적으로 비정상 ORF3 발현으로 인하여 약독화된 PVC2 바이러스 백신에 관해 기재되어 있다. 하지만 현재까지 PCV2 ORF3와 RGS16의 상호작용이 어떠한 효과가 있는지 정확히 확인된 사실이 없다.As a background of the present invention, Korean Patent Publication No. 10-1030792 (Apr. 21, 201) describes a vector for surface expression of a porcine circovirus 2 (PCV2) ORF2 gene and a salmonella vaccine strain transformed with the same, Published WO2006 / 132598 A1 (Dec. 16, 2006) describes the action of ORF3 of PCV2 and its apoptosis, and at least in part the attenuation of the abnormal ORF3 expression in relation to PVC2 virus vaccines. However, it is not known exactly how the interaction of PCV2 ORF3 and RGS16 has been so far.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 상기 종래기술의 문제점을 해결하기 위해, 연구한 결과 PCV2 ORF3의 유전자를 돼지 상피세포에 도입한 결과, PCV2 ORF3가 RGS16 분자의 발현 감소를 유발한다는 것을 밝혀냈다. 이러한 RGS16 분자의 발현 감소는 PCV2 ORF3가 RGS16 분자의 유비퀴틴-프로테오좀에 의한 단백질 분해과정을 촉진함으로써 유발된다. 또한 RGS16 분자의 발현 감소에 의해 상피세포에서 IL-6와 IL-8이 과다 발현하게 됨을 확인하였다. 따라서, PCV2 ORF3 단백질에 의한 돼지 RGS16 분자의 발현 감소 기작을 통한 염증성 사이토카인의 과다 발현을 이용하여 더욱 강한 면역반응을 일으킬 수 있는 새로운 면역 보강제를 제공할 수 있다. The inventors of the present invention found that PCV2 ORF3 induces a decrease in expression of RGS16 molecules by introducing the gene of PCV2 ORF3 into porcine epithelial cells. This reduction in the expression of the RGS16 molecule is induced by the PCV2 ORF3 promoting the proteolytic process by the ubiquitin-proteasome of the RGS16 molecule. In addition, IL-6 and IL-8 were overexpressed in epithelial cells by decreased expression of RGS16 molecules. Therefore, it is possible to provide a new immunoconjugate which can induce a stronger immune response by using overexpression of inflammatory cytokine through a mechanism of reducing the expression of porcine RGS16 molecule by PCV2 ORF3 protein.
따라서, 본 발명의 목적은 돼지 서코바이러스 제2형의 ORF3를 포함하는 재조합 발현벡터 및 이를 포함하는 PCV2 ORF3 단백질에 의한 돼지 RGS16 분자의 발현 감소 기작을 통한 염증성 사이토카인의 과다 발현을 이용하여 더욱 강한 면역반응을 일으킬 수 있는 새로운 면역보강제를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a recombinant expression vector comprising the ORF3 of porcine circovirus type 2 and a PCV2 ORF3 protein comprising the same, wherein the expression of the porcine RGS16 molecule is reduced by the overexpression of an inflammatory cytokine And to provide a new adjuvant that can cause an immune response.
본 발명의 다른 목적은 상기 면역 보강제를 이용한 감염성 질환 및 암의 치료 또는 예방 방법을 제공하는 것이다. It is another object of the present invention to provide a method for the treatment or prevention of infectious diseases and cancer using the immunoconjugate.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 더욱 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 돼지 써코바이러스2(PCV2) ORF3 유전자가 도입된 도 1의 개열지도를 가지는 재조합 발현벡터를 제공한다.According to one aspect of the present invention, there is provided a recombinant expression vector having the cleavage map of FIG. 1 into which a porcine circovirus 2 (PCV2) ORF3 gene has been introduced.
본 발명의 다른 양태에 따르면, 본 발명은 상기 재조합 발현벡터로 형질전환된 ORF3 유전자를 과발현하는 세포주를 제공한다.According to another aspect of the present invention, there is provided a cell line overexpressing the ORF3 gene transformed with said recombinant expression vector.
본 발명의 또 다른 양태에 따르면, 본 발명은 돼지 써코바이러스2(PCV2) ORF3 유전자가 도입된 재조합 발현벡터로 형질전환된 세포주를 포함하는 백신용 또는 항암치료용 면역보강제를 제공한다.According to still another aspect of the present invention, there is provided a vaccine or chemotherapeutic adjuvant comprising a cell line transformed with a recombinant expression vector into which a porcine circovirus 2 (PCV2) ORF3 gene is introduced.
본 발명의 또 다른 양태에 따르면, 본 발명은 돼지 써코바이러스2(PCV2) ORF3 단백질을 포함하는 백신용 또는 항암치료용 면역보강제를 제공한다.According to yet another aspect of the present invention, there is provided a vaccine or anticancer therapy adjuvant comprising a porcine circovirus 2 (PCV2) ORF3 protein.
본 발명의 또 다른 양태에 따르면, 본 발명은 인간을 제외한 동물에서 상기 세포주 또는 ORF3 단백질을 포함하는 면역보강제를 투여하는 단계를 포함하는, 감염성 질환 또는 암의 치료 또는 예방 방법을 제공한다.
According to another aspect of the present invention, the present invention provides a method for treating or preventing an infectious disease or cancer, comprising administering the cell line or an adjuvant comprising the ORF3 protein in an animal other than a human.
이하, 본 발명에 대해서 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 PCV2 ORF3 유전자를 세포에 과발현시킨 후, PCV2 ORF3 유전자를 과발현시키지 않은 세포와 비교하여 염증성 사이토카인인 IL-6와 IL-8의 발현을 측정한 결과, PCV2 ORF3 유전자를 세포에 과발현시킨 세포주에서는 IL-6와 IL-8의 발현이 증가하는 것을 확인하였다. 이러한 PCV2 ORF3에 의한 IL-6와 IL-8의 과발현은 PCV2 ORF3 단백질이 RGS16 분자의 유비퀴틴-프로테오좀에 의한 단백질 분해과정을 촉진함으로써 RGS16 분자의 발현 감소를 유도하기 때문임을 확인하고 본 발명을 완성하였다. The present inventors measured the expression of IL-6 and IL-8, which are inflammatory cytokines, in comparison with cells not overexpressing PCV2 ORF3 gene after overexpressing PCV2 ORF3 gene. As a result, PCV2 ORF3 gene was overexpressed The expression of IL-6 and IL-8 was increased in the cell line. This overexpression of IL-6 and IL-8 by PCV2 ORF3 confirmed that the PCV2 ORF3 protein induced protein degradation by ubiquitin-proteasome of RGS16 molecule, leading to a decrease in the expression of RGS16 molecule. Completed.
따라서, 본 발명의 일 양태에 따르면, 본 발명은 돼지 써코바이러스2(PCV2) ORF3 유전자가 도입된 도 1a의 개열지도를 가지는 재조합 발현벡터를 제공한다.Accordingly, in accordance with one aspect of the present invention, there is provided a recombinant expression vector having the cleavage map of FIG. 1A into which the porcine circovirus 2 (PCV2) ORF3 gene has been introduced.
본 발명에 있어서, PCV2 ORF3 유전자의 전체 DNA 염기서열은 도 2(서열번호 1)에 나타나 있다. 본 발명에 있어, 상기 PCV2 ORF3 유전자 서열은 일반적으로 공지된 방법을 통해 벡터에 삽입할 수 있다. 통상적으로, 하나 이상의 제한효소로 DNA 서열 및 벡터를 절단하고, 단편들을 함께 결찰하여 최종적으로 발현될 DNA 서열을 벡터에 결합시킨다. 본 발명에 있어, PCV2 ORF3 cDNA는 XhoI와 NotI site를 이용하여 PLNCX2 벡터에 삽입되었다(도 1a). 이렇게 제조된 재조합 벡터는 발현 효율을 높일 수 있는 이점이 있다.
In the present invention, the entire DNA sequence of the PCV2 ORF3 gene is shown in Fig. 2 (SEQ ID NO: 1). In the present invention, the PCV2 ORF3 gene sequence can be inserted into a vector through a generally known method. Typically, the DNA sequence and vector are cut with one or more restriction enzymes, and the fragments are ligated together to bind the DNA sequence to be finally expressed to the vector. In the present invention, the PCV2 ORF3 cDNA was inserted into the PLNCX2 vector using XhoI and NotI sites (Fig. 1A). The recombinant vector thus produced has an advantage of increasing expression efficiency.
본 발명의 다른 양태에 따르면, 본 발명은 상기 재조합 발현벡터로 형질전환된 ORF3 유전자를 과발현하는 재조합 세포주를 제공한다. 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. According to another aspect of the present invention, there is provided a recombinant cell line which overexpresses the ORF3 gene transformed with said recombinant expression vector. In the present invention, the cell line can be produced by a transformation method commonly used in the art. Conventional molecular biology, microbiology, immunology, recombinant DNA technology, and the like can be used in the technical scope of the art.
본 발명에 있어서, 상기 세포주는 본 발명에 의한 ORF3 유전자를 과발현시킬 수 있다면 특별한 제한이 있는 것은 아니나, 본 발명의 일 실시예에는 레트로바이러스로 packaging하는 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 the ORF3 gene according to the present invention. In an embodiment of the present invention, 293GPG cells (Ory et al., 1996) Respectively. When the cell line is used, there is an advantage that the expression efficiency can be increased.
본 발명의 또 다른 양태에 따르면, 본 발명은 돼지 써코바이러스2(PCV2) ORF3 유전자가 도입된 재조합 발현벡터를 포함하는 세포주 또는 돼지 써코바이러스2(PCV2) ORF3 단백질을 포함하는 백신용 또는 항암치료용 면역보강제를 제공한다.According to another aspect of the present invention, the present invention provides a method for producing a recombinant expression vector comprising a recombinant expression vector into which a porcine circovirus 2 (PCV2) ORF3 gene is introduced or a porcine circovirus 2 (PCV2) ORF3 protein Provide an adjuvant.
본 발명에 있어, 면역보강제는 백신들 또는 접종물들에 대한 특이적 면역 반응들을 증진시키는 약제이다. 면역보강제는, 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, adjuvants are agents that promote specific immune responses to vaccines or vaccines. An adjuvant is any substance or substance that, when incorporated into a vaccine or inoculum, generally or specifically acts to accelerate, prolong or enhance its specific immunoreactivity to the immunogenic material during its production, or Can be defined as an agent.
바람직한 형태로서, 본 발명의 면역보강제는 당업자에게 공지된 추가 성분을 포함할 수 있고, 하나 이상의 수의학적으로 허용되는 담체를 포함할 수 있다. "수의학적으로 허용가능한 담체"는 임의의 모든 용매, 분산매질, 코팅제, 보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장제, 흡수지연제 등을 포함한다.In a preferred form, the adjuvant of the present invention may comprise additional ingredients known to those skilled in the art and may include one or more veterinarily acceptable carriers. "Veterinarily 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 면역보강제는 전형적으로, 약학적으로 허용가능한 희석제 중에 면역보강제-유효량으로 용해 또는 분산된 면역보강제 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 ORF3 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, adjuvants are typically used as adjuvant compositions dissolved or dispersed in an adjuvant-effective amount in a pharmaceutically acceptable diluent. The amount used can vary widely in different host animals, depending on the T-cell stimulating immunogenic polypeptide portion used and the construct used. A typical amount is about 1 microgram (占 퐂) (占 퐂 / kg) of ORF3 protein per kg body weight of the host to about 1 milligram (mg) (mg / kg) per kg body weight of the host. A more general amount is about 5 μg / kg of host body weight to about 0.5 mg / kg of host body weight. The diluent is typically aqueous, and may include one or more additional adjuvants, buffers, salts, and viscosity modifiers. The ingredients of the diluent are substances that are often present in vaccines or inoculations.
본 발명의 상기 면역보강제는 경구 혹은 비경구적으로 투여할 수 있다. The adjuvant of the present invention can be administered orally or parenterally.
본 발명의 상기 면역보강제의 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. A suitable dose of the adjuvant of the present invention may be determined by factors such as the formulation method, administration method, age, body weight, sex, disease symptom, food, administration time, administration route, excretion rate, Those skilled in the art will readily be able to determine and prescribe dosages effective for treatment or prophylaxis.
본 발명에 의한 PCV2 ORF3의 유효량은 PCV2 ORF3가 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.An effective amount of PCV2 ORF3 according to the present invention means an amount of an antigen capable of inducing or inducing an immune response by PCV2 ORF3, but is not limited thereto, and an effective amount is at least 10 weeks, preferably at least 12 weeks, Is defined as the amount of antigen that confers an immunity duration (DOI) of at least 20 weeks.
일반적으로, 상기 면역보강제에 포함되는 세포주는 필요에 따라 적정한 농도로 동물에게 접종될 수 있다. 예를 들어, 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.Generally, the cell line contained in the immunostimulant can be inoculated into an animal at an appropriate concentration as required. For example, an OD value at OD 600 may be measured and then inoculated to a subject at a value of about 0.5 to 1.5 x 10 ^ 10 CFU / ml. However, the dosage is not limited to this, and can be appropriately determined according to the judgment of the veterinarian ultimately considering the type of formulation, the administration method, age, weight, symptoms, and the like.
본 발명에 의한 상기 세포주 또는 ORF3 단백질은 RGS16 단백질의 발현을 감소시키고 면역보강제가 투여된 동물의 IL-6 및 IL-8의 발현을 증가시켜 강력한 면역반응을 유도할 수 있는 이점이 있다.
The cell line or ORF3 protein according to the present invention has an advantage of decreasing the expression of the RGS16 protein and increasing the expression of IL-6 and IL-8 in the animal to which the adjuvant is administered, thereby inducing a strong immune response.
본 발명의 또 다른 양태에 따르면, 본 발명은 인간을 제외한 동물에서 상기 세포주 또는 ORF3 단백질을 포함하는 면역보강제를 투여하는 단계를 포함하는, 감염성 질환 또는 암의 치료 또는 예방 방법을 제공한다. According to another aspect of the present invention, the present invention provides a method for treating or preventing an infectious disease or cancer, comprising administering the cell line or an adjuvant comprising the ORF3 protein in an animal other than a human.
본 발명에 있어서, 동물은 이에 한정되는 것은 아니나, 바람직하게 돼지, 또는 새끼돼지를 포함한다. In the present invention, animals include, but are not limited to, pigs or piglets.
본 발명에 의한 상기 세포주 또는 ORF3 단백질을 동물에 투여하게 되면, RGS16 단백질의 발현을 감소시키고 투여된 동물의 IL-6 및 IL-8의 발현을 증가시켜 강력한 면역반응을 유도할 수 있다. 따라서, PMWS을 일으키는 PCV2 감염에 의한 질환을 포함한 감염성 질환 또는 암을 치료 또는 예방할 수 있다. Administration of the cell line or ORF3 protein according to the present invention to an animal can reduce the expression of RGS16 protein and increase the expression of IL-6 and IL-8 in the administered animal to induce a strong immune response. Accordingly, an infectious disease or cancer including a disease caused by PCV2 infection causing PMWS can be treated or prevented.
감염성 질환의 예방에 있어서 가장 중요한 것은 보다 효율적인 백신의 개발이다. 백신의 경우 새로운 면역 보강제의 개발이 시급한데, 그러한 이유는 동일한 efficacy의 백신이라도 어떤 면역보강제를 사용하는냐에 따라 동일한 항원에 반응하는 면역반응을 보다 효율적으로 증진시킬 수 있기 때문이다. 본 발명은 면역반응의 시작이 되는 주요항원제시세포(antigen presenting cell)인 대식세포나 수지상세포에 PCV2 ORF3 단백질이나 유전자를 도입하여 염증성 사이토카인(pro-inflammatory cytokine)인 인터루킨-6(IL-6; interleukin-6)와 인터루킨-8(IL-8; interleukin-8)의 발현을 증가시키는 것을 원리로 하기 때문에, 동일한 항원에 반응하는 면역반응을 보다 효율적으로 증진시킬 수 있다. The most important thing in the prevention of infectious diseases is the development of more efficient vaccines. In the case of vaccines, the development of new adjuvants is urgent because even the same efficacy vaccine can improve the immune response to the same antigen more efficiently depending on which adjuvant is used. The present invention relates to a pro-inflammatory cytokine such as interleukin-6 (IL-6, IL-6, or IL-6) by introducing a PCV2 ORF3 protein or gene into macrophages or dendritic cells which are major antigen- , interleukin-6 (IL-8), and interleukin-8 (IL-8), the immune response to the same antigen can be more efficiently promoted.
항암 치료 중에 면역세포의 활성을 증진시켜 항암 치료하는 방법이 있으며, 이 때 면역반응의 시작이 되는 주요항원제시세포(antigen presenting cell)인 대식세포나 수지상세포에 암세포에서 발현하는 항원을 제시해 주어, 이 항원을 발현하는 암세포를 죽이는 방법이 있다. 이 때 대식세포나 수지상세포에 PCV2 ORF3 단백질이나 유전자를 도입하여 염증성 사이토카인(pro-inflammatory cytokine)인 인터루킨-6(IL-6; interleukin-6)와 인터루킨-8(IL-8; interleukin-8)의 발현을 증가시키는 것을 원리로 하기 때문에, 암에서 발현하는 항원에 반응하는 대식세포나 수지상세포의 활성을 보다 효율적으로 증진시킬 수 있다.
There is a method of chemotherapy by enhancing the activity of immune cells during chemotherapy. At this time, antigens expressed in cancer cells are expressed in macrophages or dendritic cells, which are antigen presenting cells that are the beginning of immune response, There is a way to kill cancer cells that express this antigen. In this case, PCV2 ORF3 protein or gene was introduced into macrophages or dendritic cells to induce pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) ), It is possible to more efficiently enhance the activity of macrophages or dendritic cells that respond to an antigen expressed in cancer.
상술한 바와 같이, 본 발명에 따른 PCV2 ORF3 단백질은 RGS16 분자의 유비퀴틴-프로테오좀에 의한 단백질 분해과정을 촉진함으로써 RGS16 분자의 발현 감소를 유도하는 것을 특징으로 한다. 본 발명의 상기 PCV2 ORF3 단백질은 세포내에서 염증성 사이토카인인 IL-6와 IL-8의 과다 발현을 유발한다.As described above, the PCV2 ORF3 protein according to the present invention is characterized by inducing a decrease in the expression of RGS16 molecule by promoting the protein degradation process by the ubiquitin-proteasome of the RGS16 molecule. The PCV2 ORF3 protein of the present invention induces overexpression of the inflammatory cytokines IL-6 and IL-8 in the cells.
따라서, 본 발명에 따른 PCV2 ORF3 단백질은 보다 강한 면역반응을 일으킬 수 있는 새로운 면역 보강제를 제공하여, PCV2에 의한 질환인 PMWS용 백신이나 항암치료 등에 유용하게 이용될 수 있다.
Therefore, the PCV2 ORF3 protein according to the present invention provides a new immunoconjugate capable of inducing a stronger immune response, and thus can be usefully used for a vaccine for PMWS, which is a disease caused by PCV2, or an anti-cancer treatment.
도 1a은 본 발명에 의한 ORF3 단백질을 과발현하는 세포 구축을 위한 PCV2 ORF3 / PLNCX2 모식도를 나타낸다.
도 1b는 본 발명에 의한 PCV2 ORF3 분자가 도입된 PK15 세포에서 ORF3 유전자의 과발현을 확인한 결과를 나타낸다. NC., 음성대조군(negative control)을 나타낸다.
도 2는 본 발명에 의한 PCV2 ORF3 분자의 전체 DNA 서열을 나타낸다.
도 3a은 본 발명에 의한 PCV2 ORF3 분자가 도입된 PK15 세포에서 돼지 RGS16 단백질의 발현을 확인한 결과를 나타낸다.
도 3b는 본 발명에 의한 PCV2 ORF3 분자가 도입된 PK15 세포에서 단백질 생합성 억제제(CHX, cycloheximide)를 처리하였을 때, 돼지 RGS16 단백질의 양 변화를 확인한 결과를 나타낸다.
도 3c는 본 발명에 의한 PCV2 ORF3 분자가 도입된 PK15 세포에서 프로테오좀 억제제(MG132)를 처리하였을 때, 돼지 RGS16 단백질의 유비퀴틴화(ubiquitination) 수준의 증가를 나타낸다.
도 4는 본 발명에 의한 돼지 RGS16 단백질의 상위 리간드인 PAF(platelet activating factor)를 처리하였을 때, 염증성 사이토카인인 IL-6, 및 IL-8의 유전자 발현을 확인한 결과를 나타낸다.FIG. 1A is a schematic diagram of PCV2 ORF3 / PLNCX2 for constructing cells overexpressing the ORF3 protein according to the present invention. FIG.
FIG. 1B shows the result of confirming overexpression of the ORF3 gene in PK15 cells into which PCV2 ORF3 molecules according to the present invention are introduced. NC., And negative control.
Figure 2 shows the entire DNA sequence of the PCV2 ORF3 molecule according to the invention.
FIG. 3A shows the results of confirming expression of porcine RGS16 protein in PK15 cells into which PCV2 ORF3 molecules according to the present invention are introduced.
FIG. 3B shows the results of confirming the change in the amount of the porcine RGS16 protein when the protein biosynthesis inhibitor (CHX, cycloheximide) was treated in PK15 cells into which the PCV2 ORF3 molecule according to the present invention was introduced.
Figure 3c shows an increase in the level of ubiquitination of the porcine RGS16 protein when treated with the proteosome inhibitor (MG132) in PK15 cells transfected with PCV2 ORF3 molecules according to the present invention.
FIG. 4 shows the results of confirming gene expression of inflammatory cytokines IL-6 and IL-8 when treated with PAF (platelet activating factor), which is a higher ligand of porcine RGS16 protein according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example 1: One: PCV2PCV2 ORF3ORF3 과발현 돼지 Overdose pig 신장상피세포Kidney epithelial cells ( ( PK15PK15 ) 구축 ) build
PCV2의 ORF3를 클로닝하기 위해, 돼지 비장조직에서 genomic DNA를 추출하고, 연쇄중합반응에 의해 전체 ORF 부위 DNA 서열을 동정하였다. 그 후 전체 ORF 부위 DNA를 제한효소인 XhoI 과 NotI site를 이용하여 T easy vector에 삽입하였다. 그 후 ORF3 부위에 특이적인 프라이머를 사용하여 ORF3의 cDNA를 연쇄중합반응에 의해 클로닝하였고, XhoI과 NotI site를 이용하여, retroviral vector(PLNCX2)에 삽입하였다(도 1a). 이때 사용한 프라이머는 아래와 같다(표 1).In order to clone ORF3 of PCV2, genomic DNA was extracted from porcine spleen tissues and the DNA sequence of the whole ORF region was identified by chain polymerization. The whole ORF region DNA was then inserted into T easy vector using restriction enzymes XhoI and NotI site. Then, the cDNA of ORF3 was cloned by chain polymerization using a primer specific to the ORF3 site, and inserted into a retroviral vector (PLNCX2) using XhoI and NotI sites (Fig. 1A). The primers used were as follows (Table 1).
그 후 PCV2 ORF3 cDNA가 삽입된 PLNCX2 벡터를 사용하여 Tetracycline off system의 293GPG 세포를 이용하여 돼지 신장상피세포(PK15)에 PCV2 ORF3 cDNA를 과발현시켰다. 우선 293GPG 세포를 6 well 조직 배양 플레이트에 well 당 5 x 105 세포가 되도록 접종하고 10% 송아지 혈청을 함유한 DMEM 배지에서 배양하였다. 세포가 24시간 후 well 표면의 70-80% 정도를 덮을 정도로 자라면, 혈청이 없는 DMEM 배지에서 1 ㎍의 ORF3 유전자 발현 벡터 DNA를 6 ㎍의 Lipofectamine plus (Invitrogen)를 사용하여, 도입된 발현 벡터를 레트로바이러스로 packaging하는 세포주인 293GPG 세포에 도입시켜 24시간 배양하였다. 293GPG 세포로부터 생성된 ORF3 단백질 유전자를 함유하는 바이러스가 포함된 배양 상층액을 이용하여 숙주세포를 4-5시간 배양 후 혈청이 10% 함유된 배지로 바꿔서 4시간 배양하였으며 이 과정을 3회 반복하였다. 이 때 사용된 숙주세포는 PK15 세포주이며, ORF3 형질 도입된 세포를 얻기 위해 G418 항생제 1.5 mg/ml를 처리하여 ORF3 유전자가 발현되는 세포를 얻었다.
PCV2 ORF3 cDNA was overexpressed in pig kidney epithelial cells (PK15) using 293GPG cells of Tetracycline off system using PLNCX2 vector with PCV2 ORF3 cDNA inserted. First, 293GPG cells were inoculated on a 6-well tissue culture plate at a density of 5 × 10 5 cells per well and cultured in DMEM medium containing 10% calf serum. If the cells grew to cover about 70-80% of the well surface after 24 hours, 1 μg of ORF3 gene expression vector DNA was transfected into 6 μg of Lipofectamine plus (Invitrogen) in serum-free DMEM medium, Was introduced into 293GPG cells, a cell line packaged with retrovirus, and cultured for 24 hours. The host cells were cultured for 4-5 hours using a culture supernatant containing the virus containing the ORF3 protein gene generated from 293GPG cells, and then cultured for 4 hours in a medium containing 10% serum. This procedure was repeated three times . The host cell used was a PK15 cell line. To obtain the ORF3 transduced cells, 1.5 mg / ml of G418 antibiotic was treated to obtain cells expressing the ORF3 gene.
PCV2 ORF3 단백질이 과발현된 PK15 세포주는 10% 송아지 혈청과 1% 페니실린/스트렙토마이신을 포함하고 있는 DMEM 배지에서 세포 배양하였다. 그 후, 이 세포주에서 RT-PCR로 ORF3 cDNA 유전자의 과발현을 확인하였다. 우선 RT-PCR을 수행하기 위해, ORF3 단백질이 과발현된 PK15 세포주를 트리졸(Trizol, Invitrogen) 1ml 당 200 ㎕의 chloroform (sigma)을 넣고 전체 RNA를 분리하였고, 이때 추출한 5 ㎍의 mRNA를 주형으로 Superscript III (Invitrogen) 역전사 효소를 이용하여 역전사 연쇄중합반응에 의해 cDNA를 합성한 후, ORF3 단백질과 GAPDH 프라이머를 이용하여 연쇄중합반응을 실시하였다. 이때 사용한 중합 효소는 Taq polymerase (Gene craft)이며 사용한 프라이머는 아래와 같다.PCV2 ORF3 protein-overexpressed PK15 cell lines were cultured in DMEM medium containing 10% calf serum and 1% penicillin / streptomycin. Thereafter, overexpression of the ORF3 cDNA gene was confirmed by RT-PCR in this cell line. In order to perform RT-PCR, the PK15 cell line overexpressing the ORF3 protein was subjected to isolation of total RNA by adding 200 μl of chloroform (Sigma) per 1 ml of Trizol (Invitrogen). The extracted 5 μg of mRNA was used as a template CDNA was synthesized by reverse transcription polymerase using Superscript III (Invitrogen) reverse transcriptase, and sequential polymerization was performed using ORF3 protein and GAPDH primer. The polymerase used was Taq polymerase (Gene craft) and the primers used were as follows.
이때 PCR 조건은 denaturation 95도에서 30초, anealing 60도에서 30초, extension 72도에서 30초의 온도, 시간 조건으로 35 사이클로 진행하였다.The PCR conditions were 30 cycles at 95 ° C for denaturation, 30 seconds at 60 ° anealing, and 35 cycles at temperature of 72 ° C for 30 seconds.
또한, 이때 사용된 음성대조군(N.C., negative control)은 ORF3 유전자가 삽입되지 않은 G418 항생제 저항성 유전자를 발현시키는 벡터를 발현시킨 PK15 세포주의 mRNA가 사용되었다. RT-PCR 결과 183 bp의 돼지 서코바이러스 2형 ORF3 단백질 유전자의 mRNA transcript 산물이 ORF3를 도입한 세포에서만 발견됐다(도 1b 참조). 내적 대조군(Internal control)인 GAPDH 산물(271 bp)은 음성대조군(N.C.)과 ORF3를 도입한 세포 모두에서 발견되었다(도 1b 참조).
In addition, the negative control (NC) used herein was mRNA of a PK15 cell line expressing a vector expressing the G418 antibiotic resistance gene without the ORF3 gene inserted therein. RT-PCR showed that the mRNA transcript product of the 183 bp porcine circovirus type 2 ORF3 protein gene was found only in cells transfected with ORF3 (see FIG. 1B). The internal control GAPDH product (271 bp) was found in both negative control (NC) and cells transfected with ORF3 (see FIG. 1b).
실시예Example 2: 2: PCV2PCV2 ORF3ORF3 과발현 돼지 Overdose pig 신장상피세포Kidney epithelial cells ( ( PK15PK15 )에서 )in RGS16RGS16 의 발현 감소 측정 및 기전 분석Of expression and mechanism of expression
PCV2 ORF3 단백질이 상호작용한다고 알려진 돼지 RGS16의 발현 정도를 측정하기 위하여, 웨스턴블로팅(Western blotting)을 통해 돼지 RGS16의 단백질 양을 측정하였다. 먼저 ORF3 단백질이 과발현된 PK15 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 용해 (lysis) 시킨 후, 12% SDS 폴리아크릴아마이드 겔(polyacrylamide gel)에 전기 영동시켜 Hybond-P PVDF membrane (GE Healthcare, Buckinghamshire, UK)에 트랜스퍼(transfer)하였다. 그 후, 돼지 RGS16 분자에 특이적으로 결합하는 항 RGS16 항체(Santacruz)를 멤브레인(membrane)에 처리한 후 ECL system (GE healthcare, Buckinghamshire, UK)을 이용하여 발색시켜 RGS16 단백질의 발현 정도를 측정하였다. To measure the expression level of porcine RGS16, which is known to interact with the PCV2 ORF3 protein, the amount of protein of porcine RGS16 was measured by Western blotting. First, PK15 cells overexpressing ORF3 protein were incubated with RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS) containing a proteinase inhibitor cocktail , And then transferred to a Hybond-P PVDF membrane (GE Healthcare, Buckinghamshire, UK) by electrophoresis on a 12% SDS polyacrylamide gel. Thereafter, an anti-RGS16 antibody (Santacruz) specifically binding to the porcine RGS16 molecule was treated on the membrane and then developed using an ECL system (GE healthcare, Buckinghamshire, UK) to measure the expression level of the RGS16 protein .
그 결과, RGS16 분자는 LPS (Lipopolysaccharide) 1 ㎍/ml와 Poly I:C (Polyinosinic:polychtidylic acid) 10 ㎍/ml과 같은 면역 자극제(immunostimulant)의 48시간 처리에 관계없이 RGS16의 발현 정도가 ORF3 단백질이 과발현된 PK15 세포주에서 감소한 것을 확인할 수 있었다(도 3a 참조). As a result, the expression level of RGS16 in the RGS16 molecule was significantly higher than that of the ORF3 protein regardless of treatment with an immunostimulant such as 1 μg / ml of LPS (Lipopolysaccharide) and 10 μg / ml of Poly I: C (Polyinosinic: polychidylic acid) Was decreased in the overexpressed PK15 cell line (see Fig. 3A).
ORF3에 의한 RGS16 분자의 발현 감소가 유비퀴틴화 (Ubiquitination)에 의해 매개되는 프로테오좀 (Proteasome) 단백질 분해 기작에 의한 것인지 알아보기 위해, 먼저 단백질 합성을 억제하는 억제제인 사이클로헥사마이드 (Cycloheximide) 100 ㎍/ml를 시간별로 처리한 후 위와 같은 방법으로 RGS16 분자의 밴드를 관찰하여 안정성을 확인하였고, co-immunoprecipitation을 통해 RGS16의 유비퀴틴화 정도를 측정하였다. ORF3 단백질이 과발현된 PK15 세포주의 co-immunoprecipitation 실험을 위해 프로테오좀을 억제할 수 있는 MG132 (10 μM)를 4시간 처리하고 프로테아제 억제제 칵테일(proteinase inhibitor cocktail)이 포함된 NP-40 용해 버퍼 (NP-40 lysis buffer, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1.0% NP-40)로 용해(lysis) 시킨 후, 항 RGS16 항체를 이용하여 면역 침전시켰다. 위와 같은 방법으로 멤브레인(membrane)에 트랜스퍼(transfer) 시킨 후, 항 유비퀴틴 항체를 이용하여 RGS16 단백질의 유비퀴틴화 정도를 측정하였다.To investigate whether the decrease in the expression of RGS16 molecule by ORF3 is due to the proteasome proteolytic mechanism mediated by ubiquitination, 100 μg of cycloheximide, an inhibitor of protein synthesis inhibition, / ml was treated with time, and the stability of the band was observed by observing the band of RGS16 molecule in the same manner as above. The degree of ubiquitination of RGS16 was measured by co-immunoprecipitation. For the co-immunoprecipitation of the ORF3 protein-overexpressed PK15 cell line, MG132 (10 μM), which can inhibit proteosomes, was treated for 4 hours and NP-40 lysis buffer containing proteinase inhibitor cocktail (NP -40 lysis buffer, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1.0% NP-40), followed by immunoprecipitation using an anti-RGS16 antibody. After transferring to a membrane by the above method, the degree of ubiquitination of RGS16 protein was measured using an anti-ubiquitin antibody.
그 결과, ORF3 단백질이 과발현된 PK15 세포주에서 RGS16 분자의 발현 정도가 훨씬 빨리 감소함을 확인할 수 있었고(도 3b), ORF3 단백질이 과발현된 PK15 세포주에 MG132를 처리하였을 경우 유비퀴틴화 정도가 증가하는 것을 확인하였다 (도 3c 참조). 이때 사용된 멤브레인(membrane)은 항 RGS16 항체를 처리하여 위와 같은 방법으로 RGS16 분자의 밴드를 확인하여 면역침전 반응 여부를 확인하였다. ORF3 단백질이 과발현된 PK15 세포주에 MG132 처리하였을 경우, RGS16 분자의 발현이 증가하는 양상을 확인할 수 있었다. 내적 대조군(Internal control)은 항 β-actin 항체에 의한 β-actin의 발현량으로, 항 RGS16 항체를 이용하여 면역침전하기 전의 모든 샘플에서 동일한 β-actin이 검출되어 동일한 양의 단백질을 가지고 면역침전 반응을 수행한 것임을 확인하였다.
As a result, it was confirmed that the expression level of the RGS16 molecule was significantly reduced in the PK15 cell line overexpressing the ORF3 protein (FIG. 3b), and the degree of ubiquitination was increased when the MG153 was overexpressed in the PK15 cell line overexpressing the ORF3 protein (See FIG. 3C). At this time, the membrane used was treated with anti-RGS16 antibody, and the band of RGS16 molecule was identified by the above method to confirm immunoprecipitation reaction. The expression of RGS16 molecule was found to increase when MG132 treatment was applied to PK15 cell line overexpressing ORF3 protein. The internal control is the amount of β-actin expressed by the anti-β-actin antibody. The same β-actin was detected in all the samples before immunoprecipitation using the anti-RGS16 antibody, Reaction was carried out.
실시예Example 3: 3: PCV2PCV2 ORF3ORF3 과발현 돼지 Overdose pig 신장상피세포Kidney epithelial cells ( ( PK15PK15 )에서 )in ILIL -6와 -6 and ILIL -8의 발현 증가 측정 Increased expression of -8
돼지 RGS16 분자의 발현 정도의 감소가 세포의 면역작용에 있어 어떤 영향을 미치는지 알아보기 위해 염증반응에 중요한 역할을 하고 있는 대표적인 염증성 사이토카인인 IL-6와 IL-8의 mRNA 발현 정도를 정량적 실시간 중합효소 연쇄반응 (Quantitative real-time PCR)을 이용하여 알아보았다. In order to investigate the effect of decreasing the expression level of the porcine RGS16 molecule on the immune function of the cells, the degree of mRNA expression of IL-6 and IL-8, which are typical inflammatory cytokines, And quantitative real-time PCR.
RGS16 분자가 조절할 수 있는 신호를 전달하여 역할을 더욱 확실하게 드러내기 위해 large G 단백질 수용체인 혈소판활성화인자수용체(Platelet activating factor receptor, PAFR)를 자극할 수 있는 혈소판활성화인자(PAF) 100nM을 ORF3 단백질이 과발현된 PK15 세포주에 6시간 처리하였다. 이 후, 상기 실시예 1에 나타낸 것과 같이 트리졸(Trizol, Invitrogen) 1 ml 당 200 ㎕의 클로로포름 (sigma)을 넣고 전체 RNA를 분리하였고, 이때 추출한 1 ㎍의 mRNA를 주형으로 Superscript III (Invitrogen) 역전사 효소를 이용하여 역전사 연쇄중합반응에 의해 cDNA를 합성하였다. 이 후, 상기 cDNA를 주형으로 하여 유전자 데이터베이스에서 얻은 염기서열에 따라 프라이머를 제작한 후, 정량적 실시간 중합효소 연쇄반응(Quantitative real-time PCR)을 이용하여 각각의 유전자를 얻었다.In order to more clearly demonstrate the role of the RGS16 molecule in regulating signals, 100 nM of platelet activating factor (PAF) capable of stimulating the platelet activating factor receptor (PAFR), which is a large G protein receptor, Were treated for 6 hours with overexpressed PK15 cell line. Thereafter, as shown in Example 1, 200 μl of chloroform (Sigma) was added per 1 ml of Trizol (Invitrogen), and total RNA was isolated. Supernatant (Invitrogen) CDNA was synthesized by reverse transcription polymerase using reverse transcriptase. After that, primers were prepared according to the nucleotide sequence obtained from the gene database using the cDNA as a template, and then each gene was obtained using a quantitative real-time PCR.
*housekeeping gene * housekeeping gene
GAPDH : Glyceraldehyde 3-phosphate dehydrogenase
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase
연쇄중합반응에서는 TaqMan probe (Roche)와 LC480 probe master mix (Roche)를 사용하였다. 상기 사이토카인 발현 전사 결과는 도 4에 나타내었다. 도 4에 나타낸 것과 같이, ORF3 단백질이 과발현된 PK15 세포주에서 IL-6 및 IL-8의 전사량이 증가하는 것을 관찰할 수 있으며, PAF를 처리 시에는 차이가 더욱 극명하게 나타나는 것을 확인할 수 있다. 상기 GAPDH는 세포내에서 발현량이 세포의 조건에 따라 잘 변하지 않는 유전자로써, 본 발명의 PK15 세포주의 사이토카인 발현량 조사에서 세포내 기준으로 사용하였다.
For the chain polymerization, TaqMan probe (Roche) and LC480 probe master mix (Roche) were used. The result of transcription of the cytokine expression is shown in Fig. As shown in FIG. 4, it can be seen that the transcription amount of IL-6 and IL-8 increases in the PK15 cell line overexpressing the ORF3 protein, and it can be confirmed that the difference is more apparent in the treatment of PAF. The GAPDH gene was used as an intracellular reference for the expression level of cytokines in the PK15 cell line of the present invention as a gene whose expression amount in cells did not change well depending on the conditions of the cells.
이상으로 본 발명의 특정한 부분을 상세히 기술하였으나, 당업계의 통상의 지식을 가진 자에게 있어 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 균등물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.
<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Immunologic adjuvant using ORF3 encoded by PCV2 <130> P11-121004-01 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 315 <212> DNA <213> PCV2 <400> 1 atggtaacca tcccaccact tgtttctagg tggtttccag tatgtggttt ccgggtctgc 60 aaaattagca gcccatttgc ttttaccaca cccaggtggc cccacaatga cgtgtacatt 120 ggtcttccaa tcacgcttct gcattttccc gctcactttc aaaagttcag ccagcccgcg 180 gaaatttctg acaaacgtta cagggtgctg ctctgcaacg gtcaccagac tcccgctctc 240 caacaaggta ctcacagcag tagacaggtc actccgttgt ccttgagatc gaggagctcc 300 acattcaata agtaa 315 <110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Immunologic adjuvant using ORF3 encoded by PCV2 <130> P11-121004-01 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 315 <212> DNA <213> PCV2 <400> 1 atggtaacca tcccaccact tgtttctagg tggtttccag tatgtggttt ccgggtctgc 60 aaaattagca gcccatttgc ttttaccaca cccaggtggc cccacaatga cgtgtacatt 120 ggtcttccaa tcacgcttct gcattttccc gctcactttc aaaagttcag ccagcccgcg 180 gaaatttctg acaaacgtta cagggtgctg ctctgcaacg gtcaccagac tcccgctctc 240 caacaaggta ctcacagcag tagacaggtc actccgttgt ccttgagatc gaggagctcc 300 acattcaata agtaa 315
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| KR20190135296A (en) | 2018-05-28 | 2019-12-06 | 고려대학교 산학협력단 | Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein |
| KR20190135297A (en) | 2018-05-28 | 2019-12-06 | 고려대학교 산학협력단 | Immunosuppressive composition comprising porcine circovirus type 2 open reading frame 5 |
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| KR20080027289A (en) * | 2005-06-07 | 2008-03-26 | 테마섹 라이프 사이언스 래보러토리 리미티드 | Porcine circovirus type 2 vaccines |
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| KR20190135296A (en) | 2018-05-28 | 2019-12-06 | 고려대학교 산학협력단 | Composition for immune enhancement comprising porcine reproductive and respiratory syndrome virus Nsp1 protein |
| KR20190135297A (en) | 2018-05-28 | 2019-12-06 | 고려대학교 산학협력단 | Immunosuppressive composition comprising porcine circovirus type 2 open reading frame 5 |
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