KR20230153047A - Recombinant Vaccinia virus vaccines comprising antigen ROP4 of Toxoplasma gondii - Google Patents
Recombinant Vaccinia virus vaccines comprising antigen ROP4 of Toxoplasma gondii Download PDFInfo
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- KR20230153047A KR20230153047A KR1020220052767A KR20220052767A KR20230153047A KR 20230153047 A KR20230153047 A KR 20230153047A KR 1020220052767 A KR1020220052767 A KR 1020220052767A KR 20220052767 A KR20220052767 A KR 20220052767A KR 20230153047 A KR20230153047 A KR 20230153047A
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- rop4
- toxoplasma
- vaccinia virus
- vaccine
- leu
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- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
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Abstract
본 발명은 톡소포자충의 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 포함하는 재조합 백시니아 바이러스 백신에 관한 것으로서, 구체적으로, ROP4 항원 유전자를 이용하여 재조합 백시니아 바이러스를 제조하였으며, 동물모델에서 효능을 관찰하였다. 본 발명의 재조합 백시니아 바이러스 백신은 경구 경로를 통해 접종된 마우스를 톡소플라즈마 원충으로 감염시켜 점막 및 전신 면역을 유도하는 것을 확인하였다. 이에 본 발명의 재조합 백시니아 바이러스 백신은 효과적인 톡소포자충 백신으로서 기술적인 파급 효과를 야기할 수 있다. The present invention relates to a recombinant vaccinia virus vaccine containing the Toxoplasma antigen rhoptry protein 4 (ROP4). Specifically, the recombinant vaccinia virus was prepared using the ROP4 antigen gene, and was administered in an animal model. Efficacy was observed. The recombinant vaccinia virus vaccine of the present invention was confirmed to induce mucosal and systemic immunity by infecting mice inoculated via the oral route with Toxoplasma parasites. Accordingly, the recombinant vaccinia virus vaccine of the present invention can cause technological ripple effects as an effective Toxoplasma vaccine.
Description
본 발명은 톡소포자충의 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 포함하는 재조합 백시니아 바이러스 백신에 대한 것이다.The present invention relates to a recombinant vaccinia virus vaccine containing the Toxoplasma antigen rhoptry protein 4 (ROP4).
톡소포자충은 세포 내 기생충(obligare intercellular parasite)으로서 전 세계적으로 분포하는 인간 및 동물에 대한 톡소포자충증(Toxoplasmosis)을 야기하는 원인 원충이다. 전 세계 인구의 약 3분의 1 이상이 톡소포자충에 감염된 것으로 추정되며, 국내에서도 그룹에 따라 2 내지 25%의 감염율을 나타내는 것으로 보고된다.Toxoplasma gondii is an obligare intercellular parasite that causes toxoplasmosis in humans and animals and is distributed worldwide. It is estimated that more than one-third of the world's population is infected with Toxoplasma gondii, and in Korea, the infection rate is reported to be between 2 and 25% depending on the group.
톡소포자충은 산모를 감염시켰을 때 톡소포자충의 영양형(trophozoite)은 태반을 거쳐 태아를 감염시킬 수 있다. 톡소포자충의 감염에 의해 초기의 태아는 유산 또는 사산에 이를 수 있으며, 중후기의 태아는 정상적 분만에도 불구하고 시력손상, 수두증, 정신박약 등의 선천적 기형이 야기될 수 있다. 또한, 건강한 개체를 감염시킨 톡소포자충은 면역계 세포, 망상내피계 세포 등을 파괴시켜 림프선염, 망막맥락막염, 뇌척수염 등의 질병을 유발할 수 있으며, 숙주의 면역부전시 뇌에서 증식된 낭포(Cyst)가 활성화 되어 뇌수막염 또는 망막맥락막염을 일으킬 수 있다.When Toxoplasma infects a mother, the trophozoite form of Toxoplasma can infect the fetus through the placenta. Toxoplasma infection can cause miscarriage or stillbirth in early-stage fetuses, and congenital deformities such as vision impairment, hydrocephalus, and mental retardation in mid- to late-stage fetuses, despite normal delivery. In addition, Toxoplasma that infects a healthy individual can destroy immune system cells, reticuloendothelial cells, etc., causing diseases such as lymphadenitis, retinochoroiditis, and encephalomyelitis, and when the host's immune system fails, cysts (Cysts) proliferate in the brain. can become activated and cause meningitis or retinochoroiditis.
톡소포자충 감염증은 보통 스피라마이신(spiramycin) 또는 피리메타민(pyrimethamine)을 단독으로 투여하거나, 말라리아의 예방약으로 개발된 피리메타민과 설파(sulfa)제를 병용 투여함으로써 치료한다. 그러나, 스피라마이신은 약효가 크지 않아 예방용으로 쓰이고 있으며, 피리메타민은 임신 중에는 치료 효과가 잘 나타나지 않는다. 또한, 피리메타민과 설파메톡사졸(sulfamethoxazole) 등의 설파제를 병용 투여시 골수 억제를 일으켜 혈소판 수를 감소시킬 수 있으며, 엽산의 병용 투여로 인해 알러지 반응, 신장 장애, 혈액 장애, 오심(惡心), 구토 등의 부작용을 유발할 수 있다. 더욱이, 현재 가장 널리 사용되는 항톡소포자충제인 피리메타민이나 설파디아진(sulfadiazine)은 톡소포자충의 내약성 증가로 인하여 치료 효과가 점차 감소하고 있어, 톡소포자충 감염증에 대한 효과적인 인간 백신이 개발되어 있지 않은 실정이다.Toxoplasma infection is usually treated by administering spiramycin or pyrimethamine alone, or by administering a combination of pyrimethamine and sulfa drugs, which were developed as preventive drugs for malaria. However, spiramycin does not have much efficacy and is therefore used for prevention, and pyrimethamine does not show much of a therapeutic effect during pregnancy. In addition, co-administration of sulfa drugs such as pyrimethamine and sulfamethoxazole may cause bone marrow suppression and decrease the number of platelets, and co-administration of folic acid may cause allergic reactions, kidney failure, blood disorders, and nausea. ), may cause side effects such as vomiting. Moreover, the therapeutic effectiveness of the currently most widely used anti-Toxoplasma drugs, pyrimethamine or sulfadiazine, is gradually decreasing due to increased tolerance of Toxoplasma gondii, and no effective human vaccine against Toxoplasma infection has been developed. This is the situation.
톡소포자충에 대한 DNA, 아단위 또는 단백질 백신이 연구된 바 있으나, 이들은 성공하지 못하였다. DNA 백신을 이용한 면역화는 낮은 면역원성을 보인 한편, 아쥬반트 IL-18을 포함하는 DNA 백신은 체액성 및 세포성 면역 반응을 유도하여 제한적인 보호를 유도할 수 있었다. 따라서, 톡소포자충 감염증에 대해 매우 효과적인 신규한 백신을 발견하는 것은 연구자들에게 여전히 큰 과제로 남아있으므로, 대안적인 방법을 사용하여 톡소포자충 감염증으로부터 개체를 보호할 수 있는 효과적인 백신의 개발이 필수적이다.DNA, subunit or protein vaccines against Toxoplasma have been studied, but these have not been successful. While immunization with DNA vaccines showed low immunogenicity, DNA vaccines containing adjuvant IL-18 were able to induce humoral and cellular immune responses, leading to limited protection. Therefore, discovering a new, highly effective vaccine against Toxoplasma infection remains a major challenge for researchers, and it is essential to develop an effective vaccine that can protect individuals from Toxoplasma infection using alternative methods.
본 발명의 목적은 톡소포자충 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 발현하는 재조합 발현 벡터가 형질감염된 재조합 백시니아 바이러스(Vaccinia virus)를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 조성물 및 이의 제조방법을 제공하는데 있다.The object of the present invention is to provide a method for inducing an immune response against Toxoplasma gondii containing a recombinant Vaccinia virus transfected with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4). The object is to provide a vaccine composition and a method for manufacturing the same.
상기 목적을 달성하기 위하여, 본 발명은 톡소포자충 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 발현하는 재조합 발현 벡터가 형질감염된 재조합 백시니아 바이러스(Vaccinia virus)를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 조성물을 제공한다.In order to achieve the above object, the present invention provides an immune response against Toxoplasma gondii comprising a recombinant vaccinia virus transfected with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4). Provides a vaccine composition capable of inducing.
또한, 본 발명은 톡소포자충 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 발현하는 재조합 발현 벡터로 백시니아 바이러스(Vaccinia virus) 세포를 형질 감염시키는 단계; 및 상기 형질감염된 백시니아 바이러스(Vaccinia virus) 세포를 배양하여 ROP4를 발현시키는 단계를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 제조 방법을 제공한다.In addition, the present invention includes the steps of transfecting vaccinia virus cells with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4); and culturing the transfected vaccinia virus cells to express ROP4. It provides a method for producing a vaccine capable of inducing an immune response against Toxoplasma gondii.
본 발명은 톡소포자충의 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 포함하는 재조합 백시니아 바이러스 백신에 관한 것으로서, 구체적으로, ROP4 항원 유전자를 이용하여 재조합 백시니아 바이러스를 제조하였으며, 동물모델에서 효능을 관찰하였다. 본 발명의 재조합 백시니아 바이러스 백신은 경구 경로를 통해 접종된 마우스를 톡소플라즈마 원충으로 감염시켜 점막 및 전신 면역을 유도하는 것을 확인하였다. 이에 본 발명의 재조합 백시니아 바이러스 백신은 효과적인 톡소포자충 백신으로서 기술적인 파급 효과를 야기할 수 있다. The present invention relates to a recombinant vaccinia virus vaccine containing the Toxoplasma antigen rhoptry protein 4 (ROP4). Specifically, the recombinant vaccinia virus was prepared using the ROP4 antigen gene, and was administered in an animal model. Efficacy was observed. The recombinant vaccinia virus vaccine of the present invention was confirmed to induce mucosal and systemic immunity by infecting mice inoculated via the oral route with Toxoplasma parasites. Accordingly, the recombinant vaccinia virus vaccine of the present invention can cause technological ripple effects as an effective Toxoplasma vaccine.
도 1은 톡소포자충 ROP4 재조합 백시니아 바이러스의 특성을 확인한 결과를 나타낸다.
도 2는 톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 경구 접종시킨 후 수집한 혈청 및 점막 조직에서 항체반응을 확인한 결과를 나타낸다.
도 3은 톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시킨 후, 항체 분비 세포(ASC) 반응을 통해 톡소포자충 특이적 IgG 및 IgA 항체 생산을 확인한 결과를 나타낸다.
도 4 및 도 5는 톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시킨 후, 유세포 분석기를 통해 면역세포 활성을 확인한 결과를 나타낸다.
도 6은 톡소포자충 ROP4 재조합 백시니아 바이러스 백신 방어효능 결과를 나타낸다.
도 7은 톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시키고 Toxoplasma gondii (ME49 strain) 치사량의 50배 이상 마우스에 감염시킨 후 뇌 조직에서 톡소포자충 cyst의 수를 측정하고, 마우스의 몸무게 및 생존율을 확인한 결과를 나타낸다.Figure 1 shows the results of confirming the characteristics of Toxoplasma ROP4 recombinant vaccinia virus.
Figure 2 shows the results of confirming antibody responses in serum and mucosal tissues collected after oral inoculation of Toxoplasma ROP4 recombinant vaccinia virus vaccine into mice.
Figure 3 shows the results of confirming the production of Toxoplasma-specific IgG and IgA antibodies through antibody secreting cell (ASC) reaction after inoculating mice with Toxoplasma ROP4 recombinant vaccinia virus vaccine.
Figures 4 and 5 show the results of confirming immune cell activity through flow cytometry after inoculating mice with Toxoplasma ROP4 recombinant vaccinia virus vaccine.
Figure 6 shows the protective efficacy results of Toxoplasma ROP4 recombinant vaccinia virus vaccine.
Figure 7 shows the results of measuring the number of Toxoplasma cysts in brain tissue after inoculating mice with the Toxoplasma ROP4 recombinant vaccinia virus vaccine and infecting the mice with Toxoplasma gondii (ME49 strain) at more than 50 times the lethal dose, and measuring the body weight and survival rate of the mice. Indicates the confirmed result.
본 발명은 톡소포자충 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 발현하는 재조합 발현 벡터가 형질감염된 재조합 백시니아 바이러스(Vaccinia virus)를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 조성물을 제공한다. The present invention provides a vaccine composition capable of inducing an immune response against Toxoplasma gondii comprising a recombinant vaccinia virus transfected with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4). provides.
바람직하게는, 상기 백신 조성물은 경구 또는 비강 투여할 수 있으나, 이에 한정되는 것은 아니다.Preferably, the vaccine composition can be administered orally or nasally, but is not limited thereto.
본 발명에서 “톡소포자충(Toxoplasma gondii)”는 콕시디아아강(subclass of coccidia)에 속하는 첨복포자충이다. 톡소포자충은 세포 내 기생충(obligare intercellular parasite)으로서 전 세계적으로 분포하는 인간 및 동물에 대한 톡소포자충증(Toxoplasmosis)을 야기하는 원인 원충이다. 톡소포자충은 크게 난포낭(oocyst, 오시스트), 영양형(tachyzoit, 타키조이트), 감염형(bradyzoit, 브래디조이트), 분열체(schizont, 스키존트) 및 생식모체(gametocyte, 감마토사이트) 단계의 5가지 발육 단계를 거친다. 톡소포자충은 종숙주인 고양이 분변의 난포낭(oocyst)에 의해 오염된 물 또는 야채를 섭취함으로써 감염되거나, 중간숙주인 돼지, 양, 소 등의 육류에 낭포로 존재하는 톡소포자충을 섭식할 때 감염될 수 있다. 예컨대, 상기 톡소포자충 원충은 Toxoplasma gondii ME49 strain일 수 있으나 이에 제한되는 것은 아니다.In the present invention, “ Toxoplasma gondii” is a species of Toxoplasma gondii belonging to the subclass of coccidia. Toxoplasma gondii is an obligare intercellular parasite that causes toxoplasmosis in humans and animals and is distributed worldwide. Toxoplasma is largely divided into oocyst, vegetative form (tachyzoit), infective form (bradyzoit), schizont, and gametocyte. It goes through five stages of development. Toxoplasma gondii can be infected by consuming water or vegetables contaminated with oocysts from cat feces, which is the definitive host, or by consuming Toxoplasma cysts present as cysts in meat such as pigs, sheep, and cattle, which are intermediate hosts. You can. For example, the Toxoplasma parasite is Toxoplasma gondii It may be the ME49 strain, but is not limited thereto.
구체적으로 상기 ROP4는 서열번호 1의 아미노산 서열로 이루어질 수 있다. Specifically, ROP4 may consist of the amino acid sequence of SEQ ID NO: 1.
예컨대, 상기 ROP4는 Genebank Accession No. ABU24469.1로 표현되는 아미노산 서열일 수 있다.For example, ROP4 is Genebank Accession No. It may be the amino acid sequence represented by ABU24469.1.
또한, 상기 ROP4는 상기 서열번호 1의 아미노산 서열로 이루어진 단백질의 기능적 동등물을 포함한 것일 수 있다.Additionally, the ROP4 may contain a functional equivalent of the protein consisting of the amino acid sequence of SEQ ID NO: 1.
본 발명에서 용어 “기능적 동등물"은 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1의 아미노산 서열과 적어도 70% 이상, 구체적으로는 80% 이상, 더욱 구체적으로는 90% 이상, 가장 구체적으로는 95% 이상의 서열 상동성을 가지는 것으로, 서열번호 1의 아미노산 서열과 실질적으로 동질의 생리활성을 가지는 단백질을 의미한다.In the present invention, the term “functional equivalent” refers to an amino acid sequence that is at least 70%, more specifically 80%, more specifically 90% or more, and most specifically 90% or more of the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. This refers to a protein having a sequence homology of 95% or more and having substantially the same physiological activity as the amino acid sequence of SEQ ID NO: 1.
본 발명에서 용어 "실질적으로 동등한 생리활성"은 상기 ROP4와의 구조적, 기능적 상동성으로 인해 톡소포자충에 대한 특이적인 면역 반응을 유도할 수 있는 활성을 의미한다.In the present invention, the term “substantially equivalent physiological activity” refers to an activity that can induce a specific immune response against Toxoplasma gondii due to structural and functional homology with ROP4.
보다 구체적으로 상기 ROP4는 서열번호 2의 핵산 서열에 의해 암호화될 수 있다. 예컨대, 상기 ROP4는 Genebank Accession No. EU047558.1로 표현되는 유전자일 수 있다.More specifically, ROP4 may be encoded by the nucleic acid sequence of SEQ ID NO: 2. For example, ROP4 is Genebank Accession No. It may be a gene expressed as EU047558.1.
본 발명에서 “백신 조성물”은 백신 효과를 가지는 단백질 항원을 투여함으로써 이후의 병원균 감염을 예방할 수 있는 조성물을 의미한다. 상기 백신 조성물은 톡소포자충에 대한 항원성을 나타내는 조성물일 수 있다.In the present invention, “vaccine composition” refers to a composition that can prevent subsequent pathogen infection by administering a protein antigen having a vaccine effect. The vaccine composition may be a composition that exhibits antigenicity against Toxoplasma gondii.
구체적으로, 상기 백신 조성물은 약학적으로 허용되는 담체, 보조제, 면역 자극제 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나를 포함하는 것일 수 있다.Specifically, the vaccine composition may include any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immune stimulants, and combinations thereof.
상기 약학적으로 허용되는 담체는 당업계에 공지되어있으며, 단백질, 설탕 등을 포함한다. 상기 담체는 수용액 또는 비-수용액, 현탁액 및 에멀전일 수 있다. 비-수용액 담체는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식용유 예컨대 올리브 오일 및 주사 가능한 유기 에스테르 예컨대 에틸 올리에이트를 포함한다. 수용액 담체는 식용수 및 완충배지를 포함하는 물, 알코올/수용액, 에멀전 또는 현탁액을 포함한다. 비경구 담체는 염화나트륨 용액, 링거 덱스트로오스, 덱스트로오스 및 염화나트륨, 유산처리 링거 또는 고정 오일을 포함한다. 정맥주사용 담체는 링거 덱스트로오스를 기본으로 하는 것과 같은 전해질 보충제, 액체 및 영양 보충제 등을 포함한다. 방부제 및 기타 첨가제로서 항미생물제제, 항산화제, 킬레이트제, 불활성 가스 등과 같은 것을 추가로 포함할 수 있다. 방부제로는 포르말린 티메로살, 네오마이신, 폴리믹신 B 및 암포테리신 B를 포함할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutically acceptable carriers are known in the art and include proteins, sugars, etc. The carrier may be aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/aqueous solutions, emulsions or suspensions, including drinking water and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Carriers for intravenous injection include electrolyte supplements, liquid and nutritional supplements, such as those based on Ringer's dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. may be additionally included. Preservatives may include, but are not limited to, formalin thimerosal, neomycin, polymyxin B, and amphotericin B.
상기 백신 조성물은 보조제를 추가로 포함할 수 있다. 상기 보조제는 면역반응의 향상 및/또는 접종 후 흡수 속도를 촉진하는 화합물 또는 혼합물을 칭하는 것으로 임의의 흡수-촉진제를 포함한다. 허용가능한 보조제는 프로인트 완전보존제, 프로인트 불완전보존제, 사포닌, 미네랄 젤 예컨대 수산화 알루미늄, 계면활성제 예컨대 리소레시틴, 플투론 폴리올, 다가음이온, 펩타이드, 오일 또는 탄화수소 에멀전, 키홀림펫 헤모시아닌, 디니트로페놀 등을 포함하나, 이에 제한되는 것은 아니다.The vaccine composition may further include adjuvants. The adjuvant refers to a compound or mixture that enhances the immune response and/or accelerates the rate of absorption after vaccination and includes any absorption-enhancing agent. Acceptable auxiliaries include Freund's complete preservative, Freund's incomplete preservative, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, plutonic polyol, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, D It includes, but is not limited to, nitrophenol.
상기 백신 조성물은 면역 자극제를 추가로 포함할 수 있다. 상기 면역 자극제는 인공적으로 합성된 레바미솔, 이소프레노신 및 사이토카인을 포함할 수 있다. 사이토카인의 일 예로서 인터페론-α, 인터류킨-2, GM-CSF, GCSF 등이 있다. 상기 백신 조성물은 상기 바이러스-유사입자 또는 이의 농축액을 포함하는 형태이거나, 형질전환 숙주 세포 자체 또는 형질전환 세포의 건조 분말 형태로 사용할 수 있다. 또한, 상기 백신 조성물은 다른 식품 또는 식품 성분과 함께 사용할 수 있으며 통상적인 방법에 따라 적절하게 사용될 수 있다.The vaccine composition may further include an immune stimulant. The immune stimulants may include artificially synthesized levamisole, isoprenosine, and cytokines. Examples of cytokines include interferon-α, interleukin-2, GM-CSF, and GCSF. The vaccine composition can be used in the form of the virus-like particles or a concentrate thereof, or in the form of a dried powder of the transformed host cells themselves or the transformed cells. Additionally, the vaccine composition can be used together with other foods or food ingredients and can be used appropriately according to conventional methods.
상기 백신 조성물은 안정제, 유화제, 수산화알루미늄, 인산알루미늄, pH 조정제, 계면활성제, 리포솜, 이스콤(iscom) 보조제, 합성 글리코펩티드, 증량제, 카복시폴리메틸렌, 세균 세포벽, 세균 세포벽의 유도체, 세균백신, 동물 폭스바이러스 단백질, 바이러스 일부(subviral) 입자 보조제, 콜레라 독소, N, N-디옥타데실-N',N'-비스(2-하이드록시에틸)-프로판디아민, 모노포스포릴 지질 A, 디메틸디옥타데실-암모늄 브로마이드 및 이의 혼합물로 구성된 군에서 선택된 하나 이상의 제2보조제를 더 포함할 수 있다.The vaccine composition includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, bacterial cell walls, derivatives of bacterial cell walls, bacterial vaccines, Animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldiamine It may further include one or more second auxiliaries selected from the group consisting of octadecyl-ammonium bromide and mixtures thereof.
상기 백신 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형 및 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 제제화할 경우에는 통상적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제가 함께 사용될 수 있다.The vaccine composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and sterile injectable solutions according to conventional methods. When formulating, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used together.
경구투여를 위한 고형제제는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 레시틴 유사 유화제에 적어도 하나 이상의 부형제 예컨대, 전분, 칼슘카보네이트calcium carbonate), 수크로오스 또는 락토오스, 젤라틴 등이 함께 사용될 수 있다. 또한 상기 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제가 사용될 수도 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the lecithin-like emulsifier with at least one excipient such as starch, calcium carbonate, sucrose or lactose, and gelatin. etc. can be used together. In addition to the above excipients, lubricants such as magnesium styrate talc may also be used.
경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 사용될 수 있으며, 통상적으로 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다.Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. there is.
비경구투여를 위한 제제에는 멸균된 수용액, 비수용성제, 현탁제, 유제, 동결건조제제가 사용될 수 있다. 비수용성제제, 현탁제는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous preparations, suspensions, emulsions, and freeze-dried preparations. Non-aqueous preparations and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
본 발명에서 용어 “벡터”는 연결되어 있는 핵산 단편을 운반하는데 이용되는 핵산 분자를 의미한다. 상기 벡터는 박테리아, 플라스미드, 파지, 코스미드, 에피솜, 바이러스 및 삽입가능한 DNA 단편(즉, 상동재조합에 의해 숙주 세포 게놈 안으로 삽입 가능한 단편)이 사용될 수 있으나, 이에 제한되지 않는다. 상기 플라스미드는 벡터의 일종으로서 내부에 추가적으로 DNA 단편을 연결시킬 수 있는 고리형의 이중 가닥 DNA 루프를 의미한다. 또한, 바이러스 벡터는 추가적인 DNA를 바이러스 게놈 안에 연결시킬 수 있다.In the present invention, the term “vector” refers to a nucleic acid molecule used to transport linked nucleic acid fragments. The vector may include, but is not limited to, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments (i.e., fragments that can be inserted into the host cell genome by homologous recombination). The plasmid is a type of vector and refers to a circular double-stranded DNA loop within which additional DNA fragments can be linked. Additionally, viral vectors can incorporate additional DNA into the viral genome.
본 발명에서 용어 “발현 벡터”는 작동 가능하도록 연결된 목적 단백질을 암호화하는 유전자의 발현을 지시할 수 있는 벡터를 의미한다. 일반적으로, 재조합 DNA 기술의 사용에서 발현 벡터는 플라스미드 형태이므로, 용어 플라스미드 및 벡터가 상호 교환적으로 사용될 수 있다. 그러나, 바이러스 벡터와 같이 동일한 기능을 수행하는 다른 형태의 발현 벡터들도 포함할 수 있다.In the present invention, the term “expression vector” refers to a vector that can direct the expression of a gene encoding an operably linked target protein. Generally, in the use of recombinant DNA technology, expression vectors are in the form of plasmids, so the terms plasmid and vector can be used interchangeably. However, other types of expression vectors that perform the same function, such as viral vectors, may also be included.
상기 발현 벡터는 숙주 세포 내로 도입되고, 상기 도입된 벡터에 의해 형질 전환된 숙주 세포는 상기 바이러스-유사입자를 생산할 수 있다. 이 때, 상기 벡터는 숙주 생물체에 의해 인지되는 프로모터를 포함할 수 있다.The expression vector is introduced into a host cell, and the host cell transformed by the introduced vector can produce the virus-like particle. At this time, the vector may contain a promoter recognized by the host organism.
상기 ROP4를 암호화하는 핵산 서열은 상기 프로모터 서열과 작동 가능하게 연결될(operably linked) 수 있다. 상기 "작동 가능하게 연결된(operably linked)"은 하나의 핵산 단편이 다른 핵산 단편과 결합되어 그의 기능 또는 발현이 다른 핵산 단편에 의해 영향을 받는 것을 의미한다. 즉, 상기 바이러스-유사입자를 암호화하는 유전자는 벡터 내에 있는 프로모터와 작동 가능하게 연결되어 발현이 조절될 수 있다.The nucleic acid sequence encoding ROP4 may be operably linked to the promoter sequence. The term “operably linked” means that one nucleic acid fragment is linked to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. In other words, the gene encoding the virus-like particle can be operably linked to a promoter in the vector to control its expression.
또한, 상기 발현 벡터는 형질 전환된 숙주 세포를 선별하는데 필요한 적절한 마커 유전자를 포함할 수 있다. 상기 마커 유전자는 항생제 저항성 유전자 또는 형광 단백질 유전자일 수 있으며, 상기 항생제 저항성 유전자는 히그로마이신 저항성 유전자, 카나마이신 저항성 유전자, 클로람페니콜 저항성 유전자 및 테트라사이클린 저항성 유전자로 구성된 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다. 상기 형광 단백질 유전자는 효모-강화 녹색 형광 단백질(yeast-enhanced green fluorescent protein, yEGFP) 유전자, 녹색 형광 단백질(green fluorescent protein, GFP) 유전자, 청색 형광 단백질(blue fluorescent protein, BFP) 유전자 및 적색 형광 단백질(red fluorescent protein, RFP) 유전자로 구성된 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.Additionally, the expression vector may contain an appropriate marker gene necessary for selecting transformed host cells. The marker gene may be an antibiotic resistance gene or a fluorescent protein gene, and the antibiotic resistance gene may be selected from the group consisting of a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene, but is limited thereto. That is not the case. The fluorescent protein genes include the yeast-enhanced green fluorescent protein (yEGFP) gene, green fluorescent protein (GFP) gene, blue fluorescent protein (BFP) gene, and red fluorescent protein. (red fluorescent protein, RFP) may be selected from the group consisting of genes, but is not limited thereto.
본 발명에서 “형질 감염”은 상기 벡터를 미생물 또는 특정 세포 내로 운반하는 방법을 의미하며, 형질 감염 대상 세포가 원핵세포인 경우에는, CaCl2 방법, 하나한 방법 및 전기 천공 방법 등에 의해 실시될 수 있다. 형질 감염 대상 세포가 진핵세포인 경우에는, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법 및 유전자 밤바드먼트 등을 이용하여 실시할 수 있으나, 이에 제한되는 것은 아니다. 효모와 같은 진균의 형질 감염의 경우에는, 일반적으로 리튬 아세테이트 및 열 충격을 이용한 형질 감염법과 전기천공법에 의해 실시될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, “transfection” refers to a method of transporting the vector into a microorganism or a specific cell. If the target cell for transfection is a prokaryotic cell, it can be carried out by the CaCl2 method, Hanhan method, electroporation method, etc. . If the cell to be transfected is a eukaryotic cell, the transfection can be carried out using microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. It is not limited. In the case of transfection of fungi such as yeast, it can generally be carried out by transfection and electroporation using lithium acetate and heat shock, but is not limited thereto.
또한, 본 발명은 톡소포자충 항원 곤봉체 단백질 4(rhoptry protein 4; ROP4)를 발현하는 재조합 발현 벡터로 백시니아 바이러스(Vaccinia virus) 세포를 형질 감염시키는 단계; 및 상기 형질감염된 백시니아 바이러스(Vaccinia virus) 세포를 배양하여 ROP4를 발현시키는 단계를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 제조 방법을 제공한다.In addition, the present invention includes the steps of transfecting vaccinia virus cells with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4); and culturing the transfected vaccinia virus cells to express ROP4. It provides a method for producing a vaccine capable of inducing an immune response against Toxoplasma gondii.
바람직하게는, 상기 ROP4는 서열번호 1의 아미노산 서열로 이루어질 수 있다. Preferably, ROP4 may consist of the amino acid sequence of SEQ ID NO: 1.
이하에서는, 본 발명을 한정하지 않는 실시예에 따라 본 발명을 상세히 설명한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 따라서, 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. Below, the present invention will be described in detail according to examples that do not limit the present invention. Of course, the following examples of the present invention are only intended to embody the present invention and do not limit or limit the scope of the present invention. Accordingly, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is interpreted to fall within the scope of the rights of the present invention.
<< 실시예Example 1> 톡소포자충( 1> Toxoplasma gondii ( ToxoplasmaToxoplasma gondiigondii ) ) 곤봉체Club body 단백질 4( Protein 4 ( rhoptryrhoptry protein 4; ROP4)를 발현하는 백시니아 바이러스 백신 제조 protein 4; Manufacturing of vaccinia virus vaccine expressing ROP4)
톡소포자충(Toxoplasma gondii) tachyzoite를 마우스에 IP 경로로 감염시킨 후 4-5일째 복강에서 대량의 tachyzoite를 수집하였다. Invitrogen사 RNA 추출키트로 톡소포자충 RNA를 추출하고 톡소포자충 항원 곤봉체 단백질(rhoptry protein 4; ROP4)을 RT-PCR 방법으로 증폭시켰다. 증폭된 ROP4를 pRB21 벡터에 삽입 후 Cellfectine II 시약 (Invitrogen Waltham MA USA)을 이용해 CV-1 세포에 형질감염 시켰으며 감염된 세포의 형태학적 변화를 확인하였다. 스크레퍼로 감염된 세포를 수확하였으며 원심분리기로 세포를 펠렛화 후 상층액을 제거하였다. 세포를 37℃와 -80℃에서 동결 및 해동을 3회 반복하였다. 이 절차를 거친 후 세포를 초음파 처리하고 6000rpm으로 20분, 4℃로 원심분리하였다. 상등액을 제거하고 36% 수크로스(Sucrose) 위에 밀도구배를 통해 20000rpm으로 30분, 4℃에서 원심분리하여 바이러스 밴드를 확인하였다. 생성된 재조합 백시니아 바이러스 역가를 측정하고 동물실험에 사용하였다. Toxoplasma gondii ) tachyzoites were infected in mice by IP route, and a large amount of tachyzoites were collected from the abdominal cavity 4-5 days later. Toxoplasma RNA was extracted using an RNA extraction kit from Invitrogen, and the Toxoplasma antigen rhoptry protein 4 (ROP4) was amplified using RT-PCR. The amplified ROP4 was inserted into the pRB21 vector and transfected into CV-1 cells using Cellfectine II reagent (Invitrogen Waltham MA USA), and morphological changes in the infected cells were confirmed. Infected cells were harvested with a scraper, the cells were pelleted using a centrifuge, and the supernatant was removed. Cells were frozen and thawed three times at 37°C and -80°C. After this procedure, the cells were sonicated and centrifuged at 6000 rpm for 20 minutes at 4°C. The supernatant was removed, and the virus band was confirmed by centrifugation at 4°C for 30 minutes at 20,000 rpm through a density gradient on 36% sucrose. The titer of the generated recombinant vaccinia virus was measured and used in animal experiments.
실험방법: T. gondii ROP4 유전자는 제한 효소(StuI, HindIII)로 절단되었다. ROP4 유전자와 pRB21 벡터는 각각 1761 bp 및 5537 bp로 확인되었다(M 마커 Lane 1 pRB21 Lane 2 pRB21 Lane 3 ROP4)(도 1A). ROP4-rVV 바이러스 역가는 Vero 세포에서 역가를 측정하였다. 플라크는 37℃, 5% CO2에서 4일간 배양 후 현미경에서 시각화하였다. 100 배율에서 세 번 반복하여 측정하였다(도 1B).Experimental method: The T. gondii ROP4 gene was cut with restriction enzymes (StuI, HindIII). The ROP4 gene and pRB21 vector were identified as 1761 bp and 5537 bp, respectively (M marker Lane 1 pRB21 Lane 2 pRB21 Lane 3 ROP4) (Figure 1A). ROP4-rVV virus titer was measured in Vero cells. Plaques were cultured at 37°C and 5% CO 2 for 4 days and then visualized under a microscope. Measurements were repeated three times at 100x magnification (Figure 1B).
실험결과: 전기영동을 통해 재조합 플라스미드를 제한효소를 이용해 ROP4 유전자와 pRB21 벡터가 삽입됨을 확인하였다. 희석 배수가 높을수록 플라크 수는 반비례로 감소하였다. Control 그룹에선 플라크가 관측되지 않았다.Experiment results: Through electrophoresis, it was confirmed that the ROP4 gene and pRB21 vector were inserted into the recombinant plasmid using restriction enzymes. As the dilution factor increased, the number of plaques decreased in inverse proportion. No plaques were observed in the Control group.
<실시예 2> 동물모델에서 백신효능 검증<Example 2> Verification of vaccine efficacy in animal model
톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 경구 접종시킨 후 수집한 혈청 및 점막 조직에서 항체반응을 확인하였다.Antibody responses were confirmed in serum and mucosal tissues collected after oral inoculation of Toxoplasma ROP4 recombinant vaccinia virus vaccine into mice.
실험방법: 각 점막 샘플 획득은 면역화 4주 후 톡소포자충을 치사량의 약 50배로 감염시킨 후 16dpi에 희생되었다. Serum은 헤파린 튜브를 이용해 마우스 안와 채혈을 통해 수집하고 한 시간 상온에서 반응 후 5000rpm 10분간 원심분리기를 통해 상등액을 수집하였다. Intestine은 마우스 위에서 약 1-2cm 아래에서부터 5cm 기준으로 잘라 1× PBS 500 μl가 담긴 튜브에 수집하였다. Vagainal 샘플은 1× PBS 200 μl로 질관을 반복적으로 세척 후 튜브에 수집하였다. 수집된 점막 샘플은 37℃ 인큐베이션을 1시간 반응시킨 후 5000rpm 10분간 원심분리기를 통해 상등액을 얻었다. 수집된 점막 샘플은 ELISA를 이용해 측정하였다. 96well 플레이트에 ME49 항원을 (4 μg/ml)로 코팅하여 4℃ 16시간 반응하였다. Brain은 희석하지 않았다. Serum 1:100, intestine 1:100, vagainal 1:20로 1× PBS로 희석하였으며 1차 항체로 37℃ 한 시간 반응되었다. 2차 항체로 HRP conjugated goat anti-mouse IgG와 IgA(1:2000 희석)를 한 시간 37℃에서 반응 후 ELISA 리더기를 이용해 490nm로 측정하였다.Experimental method: Each mucosal sample was obtained 4 weeks after immunization, infected with Toxoplasma gondii at approximately 50 times the lethal dose, and then sacrificed at 16dpi. Serum was collected through mouse orbital blood collection using a heparin tube, reacted at room temperature for one hour, and then centrifuged at 5000 rpm for 10 minutes to collect the supernatant. Intestine was cut into 5cm sections starting from approximately 1-2cm above the mouse and collected in a tube containing 500 μl of 1×PBS. Vagainal samples were collected in tubes after repeatedly washing the vaginal canal with 200 μl of 1×PBS. The collected mucosal samples were incubated at 37°C for 1 hour and then centrifuged at 5000 rpm for 10 minutes to obtain a supernatant. Collected mucosal samples were measured using ELISA. A 96-well plate was coated with ME49 antigen (4 μg/ml) and reacted at 4°C for 16 hours. Brain is undiluted. Serum 1:100, intestine 1:100, and vagainal 1:20 were diluted in 1×PBS and reacted with primary antibody at 37°C for one hour. As secondary antibodies, HRP conjugated goat anti-mouse IgG and IgA (1:2000 dilution) were reacted at 37°C for one hour and then measured at 490 nm using an ELISA reader.
실험결과: 획득된 혈청 및 점막 샘플은 면역 유도를 평가하기 위해 마우스의 점막 조직에서 항체 생산을 평가하였다(도 2). A 및 B : T. gondii 특이적 혈청 IgG와 IgA 반응은 Boost 이후 높게 측정되었으며, 특히 IgA 반응이 높게 측정되었다. 점막 조직 항체반응은 16dpi에서 면역화되지 않은 비 면역화 감염 대조군(Naive+Cha) 그룹보다 백신 접종된 그룹 높게 반응하였다. C 및 D : Brain 조직에서 ROP4-rVV로 마우스를 면역화하면 IgG와 IgA 항체가 더 많이 생성되었다. E 및 F : Intestine 조직에서 ROP4-rVV로 마우스를 면역화하면 IgG에 대해 경구 경로 면역 시 Naive + Cha 그룹 보다 높은 수준의 T. gondii 특이적 IgG가 유도되었으며 점막 항체반응인 IgA 항체가 높게 수준으로 유도되었다. G 및 H: ROP4-rVV로 마우스를 면역화하면 vaginal 조직에서 IgG 및 IgA 항체반응이 증가하였다(도 2). Experimental results: The obtained serum and mucosal samples were evaluated for antibody production in mucosal tissues of mice to evaluate immunity induction (Figure 2). A and B: T. gondii Specific serum IgG and IgA responses were measured to be high after Boost, especially IgA responses. The mucosal tissue antibody response was higher in the vaccinated group than in the non-immunized infection control group (Naive+Cha) at 16 dpi. C and D: Immunization of mice with ROP4-rVV in brain tissue produced more IgG and IgA antibodies. E and F: Immunization of mice with ROP4-rVV in the intestine tissue induced a higher level of T. gondii -specific IgG and a higher level of IgA antibody, which is a mucosal antibody response, than in the Naive + Cha group during oral route immunization to IgG. It has been done. G and H: Immunization of mice with ROP4-rVV increased IgG and IgA antibody responses in vaginal tissue (Figure 2).
톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시킨 후 항체 분비 세포(ASC) 반응을 통해 톡소포자충 특이적 IgG 및 IgA 항체 생산을 확인하였다.After the Toxoplasma ROP4 recombinant vaccinia virus vaccine was inoculated into mice, the production of Toxoplasma-specific IgG and IgA antibodies was confirmed through antibody secreting cell (ASC) reaction.
실험방법: 항체 분비 세포(ASC) 반응 유도를 평가하기 위해 비장 및 장간막 림프절(MLN)을 수집하였다. 마우스에서 비장세포(1×106 세포/마우스) 및 림프절 세포(1×105 세포/마우스) 및 림프절(MLN) 세포의 단일 세포 현탁액을 T. gondii ME49 항원(4g/mL)으로 코팅된 96 well Immuno plate에 각 well에 넣고 37℃, 5% CO2 인큐베이터에서 5일 동안 배양하였다. 원심분리기를 이용해 상층액을 제거하고 2차 항체로 HRP conjugated goat anti-mouse IgG와 IgA(1:2000 희석)를 한 시간 37℃에서 반응 후 ELISA 리더기를 이용해 490nm로 측정하였다.Experimental Methods: Spleen and mesenteric lymph nodes (MLN) were collected to evaluate the induction of antibody-secreting cell (ASC) responses. Single-cell suspensions of splenocytes (1×10 6 cells/mouse) and lymph node cells (1×10 5 cells/mouse) and lymph node (MLN) cells from mice were coated with T. gondii ME49 antigen (4 g/mL)96. It was placed in each well of a well immunoplate and cultured in an incubator at 37°C and 5% CO 2 for 5 days. The supernatant was removed using a centrifuge and reacted with secondary antibodies of HRP conjugated goat anti-mouse IgG and IgA (1:2000 dilution) at 37°C for one hour and measured at 490 nm using an ELISA reader.
실험결과: 16dpi에서 마우스를 희생시켜 비장에서 T. gondii 특이적 IgG 및 IgA를 이용해 항체 분비 세포의 수준을 평가하였다. ROP4-rVV 백신으로 면역화된 마우스의 경우 비장(A, B)과 림프절(C, D)에서 IgG와 IgA의 항체반응이 비 면역화 감염 대조군(Naive +Cha)보다 항체반응이 높게 유도되었다(도 3).Experimental results: Mice were sacrificed at 16dpi and the level of antibody-secreting cells was evaluated in the spleen using T. gondii- specific IgG and IgA. In mice immunized with the ROP4-rVV vaccine, higher IgG and IgA antibody responses were induced in the spleen (A, B) and lymph nodes (C, D) than the non-immunized infected control group (Naive +Cha) (Figure 3 ).
톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시킨 후 유세포 분석기를 통해 면역세포 활성을 확인하였다.Toxoplasma ROP4 recombinant vaccinia virus vaccine was inoculated into mice and immune cell activity was confirmed through flow cytometry.
실험방법: 비장 세포 및 장간막 림프절 세포는 유세포 분석을 위해 비장 세포 (1×106 세포/마우스) 및 림프절 세포(1×105 세포/마우스)의 단일 세포 현탁액을 2시간 동안 5% CO2에서 37℃에서 T. gondii ME49 항원 (4 μg/ml)로 자극시켰다. 이후 마우스의 비장 및 장간막 림프절(Mesenteric lymp node)의 Germinal center B 세포의 증식을 평가하기 위해 유세포 분석을 수행하였다. 각 항체는 BD Biosciences(Franklin Lakes NJ USA) 및 Invitrogen(Waltham MA USA)에서 구입한 다음 형광 접합 항체로 염색하였다. 염색된 세포는 Accuri C6 유세포 분석기를 사용하여 획득하고 C6 Accuri 소프트웨어(BD Biosciences Franklin Lakes NJ USA)로 분석하였다.Experimental Method: For spleen cells and mesenteric lymph node cells, single cell suspensions of spleen cells (1×10 6 cells/mouse) and lymph node cells (1×10 5 cells/mouse) were grown for 2 hours in 5% CO 2 for flow cytometric analysis. Stimulation was performed with T. gondii ME49 antigen (4 μg/ml) at 37°C. Afterwards, flow cytometry was performed to evaluate the proliferation of germinal center B cells in the spleen and mesenteric lymph nodes of mice. Each antibody was purchased from BD Biosciences (Franklin Lakes NJ USA) and Invitrogen (Waltham MA USA) and then stained with fluorescently conjugated antibodies. Stained cells were acquired using an Accuri C6 flow cytometer and analyzed with C6 Accuri software (BD Biosciences Franklin Lakes NJ USA).
실험결과: ROP4-rVV 면역화는 강력한 Germinal center B 세포 반응을 유도하였다. 비 면역화 감염 대조군(Naive+Cha)의 경우 비장 세포에서 13.4%이며 ROP4-rVV 백신으로 면역화된 마우스는 21.3%로 7.9% 높은 세포가 측정되었다. 림프절 세포에서 비 면역화 그룹은 13.8%이며 ROP4-rVV 백신 그룹은 25.3%으로 11.5% 높게 측정되었다(도 4).Experimental results: ROP4-rVV immunization induced a strong germinal center B cell response. In the case of the non-immunized infected control group (Naive+Cha), spleen cells were measured at 13.4%, and in mice immunized with the ROP4-rVV vaccine, 21.3%, which is 7.9% higher, were measured. Lymph node cells were measured at 13.8% in the non-immunized group and 25.3% in the ROP4-rVV vaccine group, which is 11.5% higher (Figure 4).
실험방법: 유세포 분석은 림프절 세포(1×105 세포/마우스)의 단일 세포 현탁액을 2시간 동안 5% CO2에서 37℃에서 T. gondii ME49 항원(4 μg/ml)로 자극하였다. 이후 CD4+ 및 CD8+ T 세포를 측정하였다. 각 항체는 BD Biosciences(Franklin Lakes NJ USA) 및 Invitrogen(Waltham MA USA)에서 구입한 다음 형광 접합 항체로 염색하였다. 염색된 세포는 Accuri C6 유세포 분석기를 사용하여 획득하고 C6 Accuri 소프트웨어(BD Biosciences Franklin Lakes NJ USA)로 분석하였다.Experimental method: For flow cytometry, single cell suspensions of lymph node cells (1×10 5 cells/mouse) were stimulated with T. gondii ME49 antigen (4 μg/ml) at 37°C in 5% CO 2 for 2 hours. Then, CD4 + and CD8 + T cells were measured. Each antibody was purchased from BD Biosciences (Franklin Lakes NJ USA) and Invitrogen (Waltham MA USA) and then stained with fluorescently conjugated antibodies. Stained cells were acquired using an Accuri C6 flow cytometer and analyzed with C6 Accuri software (BD Biosciences Franklin Lakes NJ USA).
실험결과: 림프절에서 ROP4-rVV 백신의 면역화는 CD4+ 및 CD8+ T 세포 반응을 유도하였다. 비 면역화 감염 대조군(Naive+Cha)의 경우 CD4+ T 세포에서 42.9%이며 ROP4-rVV 백신으로 면역화된 마우스는 47.5%로 4.6% 높은 세포가 측정되었다. CD8+ T 세포에서 Naive+Cha 그룹은 16%이며 ROP4-rVV 백신 그룹은 24%로 8% 높게 측정되었다(도 5). Results: Immunization with ROP4-rVV vaccine induced CD4 + and CD8 + T cell responses in lymph nodes. In the case of non-immunized infection control group (Naive+Cha), CD4 + T cells were measured at 42.9%, and in mice immunized with ROP4-rVV vaccine, 47.5%, which is 4.6% higher cells. CD8 + T cells were measured at 16% in the Naive+Cha group and 24% in the ROP4-rVV vaccine group, which is 8% higher (Figure 5).
<< 실시예Example 3> 톡소포자충 재조합 3> Toxoplasma recombination 백시니아vaccinia 바이러스 백신 방어효능 Antivirus defense effectiveness
실험방법: 16 dpi에서 마우스의 뇌 조직을 500 μl의 1× PBS에 넣어 개별적으로 균질화 이후 원심분리기를 통해 상득액을 수집하였다. 전 염증성 사이토카인 IFN-gamma 및 IL-6는 BD OptEIA ELISA 키트(BD Biosciences Franklin Lakes NJ USA)를 사용하여 T. gondii에 감염된 마우스의 뇌 상등액을 이용해 570nm로 ELISA 측정기를 이용하여 생성된 표준곡선을 사용하여 사이토카인 농도를 계산하였다.Experimental method: At 16 dpi, mouse brain tissue was placed in 500 μl of 1×PBS, homogenized individually, and the supernatant was collected through a centrifuge. The pro-inflammatory cytokines IFN-gamma and IL-6 were measured using the BD OptEIA ELISA kit (BD Biosciences Franklin Lakes NJ USA) using brain supernatants from mice infected with T. gondii . A standard curve was generated using an ELISA meter at 570 nm. Cytokine concentration was calculated using
실험결과: 전 염증성 사이토카인 IFN-gamma 및 IL-6 생산량을 결정하기 위해 치사량의 톡소포자충을 감염시킨 후 16 dpi에 뇌의 상등액을 수집하여 염증반응을 확인하였다(A 및 B). 비 면역화 감염 대조군(Naive+Cha)과 비교하였을 때, ROP4-rVV로 면역화된 그룹(Immunization)은 뇌 상등액에서 현저히 낮은 수준의 IFN-gamma가 검출되었다. 전 염증성 사이토카인 IL-6는 비 면역화 감염 대조군 (Naive+Cha)와 비교 시 백신 접종그룹은 낮은 수준이 검출되는 것을 확인하였다(도 6).Experimental results: To determine the production of pro-inflammatory cytokines IFN-gamma and IL-6, brain supernatants were collected at 16 dpi after infection with a lethal dose of Toxoplasma gondii to confirm the inflammatory response (A and B). Compared to the non-immunized infection control group (Naive+Cha), significantly lower levels of IFN-gamma were detected in the brain supernatant of the ROP4-rVV-immunized group (Immunization). It was confirmed that the pro-inflammatory cytokine IL-6 was detected at a lower level in the vaccinated group compared to the non-immunized infection control group (Naive+Cha) (Figure 6).
톡소포자충 ROP4 재조합 백시니아 바이러스 백신을 마우스에 접종시키고 Toxoplasma gondii (ME49 strain) 치사량의 50배 이상 마우스에 감염시킨 후, 뇌 조직에서 톡소포자충 cyst의 수를 측정하고, 마우스의 몸무게 및 생존율을 확인하였다.After inoculating mice with the Toxoplasma ROP4 recombinant vaccinia virus vaccine and infecting the mice with more than 50 times the lethal dose of Toxoplasma gondii (ME49 strain), the number of Toxoplasma cysts was measured in brain tissue, and the body weight and survival rate of the mice were confirmed. .
실험방법: 16 dpi에서 마우스 뇌 조직을 1× PBS로 균질화 이후 44% percoll과 혼탁하여 67% percoll 위에 쌓아 밀도구배 원심분리법을 이용해 12100rpm, 4℃, 20분을 사용 후 3개의 층에서 가운데 층만 획득하여 RBC 용액을 이용해 적혈구를 제거하였다. 이후 1× PBS를 넣어 원심분리기를 통해 6000rpm, 20분간 사용해 다른 용액은 제거하고 톡소포자충만 획득하였다. 수집된 cyst는 현미경(Leica DMi8, Leica,Wetzlar, Germany)을 통해 슬라이드글라스에서 5개의 다른 영역에서 수를 측정 후 평균을 계산하여 측정하였다. 몸무게와 생존율은 톡소포자충 감염 이후 5일 간격으로 측정하였으며, 비 면역화 감염 대조군의 몸무게가 75% 이하로 체중이 손실되었을 때 동물실험을 진행하였다.Experimental method: At 16 dpi, mouse brain tissue was homogenized with 1 Then, red blood cells were removed using RBC solution. Afterwards, 1×PBS was added and centrifuged at 6000 rpm for 20 minutes to remove other solutions and obtain only Toxoplasma gondii. The collected cysts were measured by measuring the number in five different areas on a slide glass through a microscope (Leica DMi8, Leica, Wetzlar, Germany) and then calculating the average. Body weight and survival rate were measured at 5-day intervals after Toxoplasma infection, and animal experiments were conducted when the non-immunized infected control group lost less than 75% of their body weight.
실험결과: 비 면역화 마우스와 ROP4-rVV 백신 면역화 마우스에 치사량은 선행연구보다 50배 이상으로 매우 높은 양의 톡소포자충을 감염시킨 후 뇌를 수집하여 cyst 수를 확인하였다(A). ROP4-rVV 백신을 접종한 그룹은 비 면역화 감염 대조군(Naive + Cha)은 약 6000개 이상의 cyst 수가 검출되었다. 그러나 백신 접종 그룹은 cyst 수는 약 2000개로 3배 낮은 cyst 수를 확인하였다. 그러므로 몸무게 측정 시 16일 차에 감염 대조군은 75%로 체중 손실이 높았다. 반면에 백신 접종 그룹의 체중감소율은 약 90% 보호되었다. 생존율 결과에서 백신 접종그룹은 100%에 생존율을 나타냈다. 그러나 비 면역화 감염 대조군의 생존율은 0%이었다(도 7). Experimental results: Non-immunized mice and ROP4-rVV vaccine-immunized mice were infected with a very high dose of Toxoplasma gondii (the lethal dose was more than 50 times higher than in previous studies), and then their brains were collected to check the number of cysts (A). In the group vaccinated with the ROP4-rVV vaccine, the number of cysts exceeding 6,000 was detected in the non-immunized infection control group (Naive + Cha). However, the number of cysts in the vaccinated group was approximately 2,000, which was three times lower. Therefore, when measuring body weight, the infected control group had a high weight loss of 75% on day 16. On the other hand, the weight loss rate of the vaccinated group was protected by about 90%. The survival rate results showed that the vaccinated group had a survival rate of 100%. However, the survival rate of the non-immunized infected control group was 0% (Figure 7).
이러한 결과는 ROP4-rVV 백신은 치사량의 50배가 넘는 톡소포자충으로 감염시킴에도 불구하고 ROP4-rVV가 매우 우수한 수준으로 톡소포자충에 대한 높은 백신 보호 효과를 나타낸다는 것을 뒷받침한다. These results support that ROP4-rVV shows a very high vaccine protection effect against Toxoplasma gondii at an excellent level, even though the ROP4-rVV vaccine infects the vaccine with more than 50 times the lethal dose of Toxoplasma gondii.
<110> University-Industry Cooperation Group of Kyung Hee University <120> Recombinant Vaccinia virus vaccines comprising antigen ROP4 of Toxoplasma gondii <130> ADP-2022-0112 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 578 <212> PRT <213> Toxoplasma gondii <400> 1 Met Gly His Pro Thr Ser Phe Gly Gln Pro Ser Cys Leu Val Trp Leu 1 5 10 15 Ala Ala Ala Phe Leu Val Leu Gly Leu Cys Leu Val Gln Gln Gly Ala 20 25 30 Gly Arg Gln Arg Pro His Gln Trp Lys Ser Ser Glu Ala Ala Leu Ser 35 40 45 Val Ser Pro Ala Gly Asp Ile Val Asp Lys Tyr Ser Arg Asp Ser Thr 50 55 60 Glu Gly Glu Asn Thr Val Ser Glu Gly Glu Ala Glu Gly Ser Arg Gly 65 70 75 80 Gly Ser Trp Leu Glu Gln Glu Gly Val Glu Leu Arg Ser Pro Ser Gln 85 90 95 Asp Ser Gln Thr Gly Thr Ser Thr Ala Ser Pro Thr Gly Phe Arg Arg 100 105 110 Leu Leu Arg Arg Leu Arg Phe Trp Arg Arg Gly Ser Thr Arg Gly Ser 115 120 125 Asp Asp Ala Ala Glu Val Ser Arg Arg Thr Arg Val Pro Leu His Thr 130 135 140 Arg Leu Leu Gln His Leu Arg Arg Val Ala Arg Ile Ile Arg His Gly 145 150 155 160 Val Ser Ala Ala Ala Gly Arg Leu Phe Gly Arg Val Arg Gln Val Glu 165 170 175 Ala Glu Arg Pro Gln Pro Val Phe Thr Glu Gly Asp Pro Pro Asp Leu 180 185 190 Glu Thr Asn Ser Leu Tyr Tyr Arg Asp Lys Val Pro Gly Gln Gly Ile 195 200 205 Ile Gln Glu Ile Leu Arg Gln Lys Pro Gly Ile Ala His His Pro Glu 210 215 220 Ser Phe Ser Val Val Ala Ala Asp Glu Arg Val Ser Arg Thr Leu Trp 225 230 235 240 Ala Glu Gly Gly Val Val Arg Val Ala Ser Glu Leu Gly Gln Pro Gly 245 250 255 Arg Val Leu Val Arg Gly Arg Arg Ile Gly Leu Phe Arg Pro Gly Met 260 265 270 Gln Phe Glu Ala Thr Asp Gln Ala Thr Gly Glu Pro Met Thr Ala Leu 275 280 285 Val Gly His Thr Val Leu Glu Ala Thr Ala Arg Asp Val Asp Ser Met 290 295 300 Arg Asn Glu Gly Leu Ala Val Gly Leu Phe Gln Lys Val Lys Asn Pro 305 310 315 320 Tyr Leu Ala Asn Arg Tyr Leu Arg Phe Leu Ala Pro Phe Asp Leu Val 325 330 335 Thr Ile Pro Gly Lys Pro Leu Val Gln Lys Ala Lys Ser Arg Asn Glu 340 345 350 Val Gly Trp Val Lys Asn Leu Leu Phe Leu Leu Pro Pro Thr His Val 355 360 365 Asp Met Glu Thr Phe Val Asp Glu Ile Gly Arg Phe Pro Gln Glu Asp 370 375 380 Arg Pro Leu Ala Asp Ala Ala Arg Leu Tyr Leu Thr Val Gln Ala Val 385 390 395 400 Arg Leu Val Ala His Leu Gln Asp Glu Gly Val Val His Gly Lys Ile 405 410 415 Met Pro Asp Ser Phe Cys Leu Lys Arg Glu Gly Gly Leu Tyr Leu Arg 420 425 430 Asp Phe Gly Ser Leu Val Arg Ala Gly Ala Lys Val Val Val Pro Ala 435 440 445 Glu Tyr Asp Glu Tyr Thr Pro Pro Glu Gly Arg Ala Ala Ala Arg Ser 450 455 460 Arg Phe Gly Ser Gly Ala Thr Thr Met Thr Tyr Ala Phe Asp Ala Trp 465 470 475 480 Thr Leu Gly Ser Val Ile Phe Leu Ile Trp Cys Ser Arg Ala Pro Asp 485 490 495 Thr Lys Ser Gly Tyr Glu Tyr Ser Val Glu Phe Phe Phe Ser Arg Cys 500 505 510 Arg Arg Val Pro Glu Asn Val Lys Leu Leu Val Tyr Lys Leu Ile Asn 515 520 525 Pro Ser Val Glu Ala Arg Leu Leu Ala Leu Gln Ala Ile Glu Thr Pro 530 535 540 Glu Tyr Arg Glu Met Glu Glu Gln Leu Ser Ala Ala Ser Arg Leu Tyr 545 550 555 560 Ser Gly Asp Gly Thr Leu Thr Gly Gly Asp Asp Asp Met Pro Pro Leu 565 570 575 Glu Thr <210> 2 <211> 2039 <212> DNA <213> Toxoplasma gondii <400> 2 tccggcatgg aaaaaatgcg tctagctaac cgcgggcccc aatgtcgcac ggccttggtc 60 aagggacggc gcgacctgca gagacgggaa tccgagccaa gcgctgagtg tccttgcttc 120 tggctgagcc gccatcctgc ggcaccgtcc atgactgcgc acagcatccc gggagtatgg 180 tgattgtgtc agtcttattt cttttgaagg cacttcagat gtgtacttca gaatgttgtt 240 acgataattg cggatgcagc tgccacctct gaacggttga tttggttgtg agtcctccca 300 acatggggca ccctacctct ttcggacagc cgtcgtgtct tgtctggcta gctgcggcat 360 tccttgttct ggggctttgc ctcgtccagc aaggcgctgg cagacagcgg cctcaccagt 420 ggaagagctc ggaagccgct ttgagtgtca gtccggcagg agacatcgtg gacaagtatt 480 ctcgtgattc cactgaggga gaaaacactg tctcagaggg ggaagctgag ggcagtcggg 540 ggggttcatg gctggagcag gagggggttg aattaaggtc gccttcacag gacagccaga 600 caggaacctc gaccgcgtcc cccacaggct tcagaaggtt actcaggcgt ttgcgttttt 660 ggcgtcgtgg gagtacacgc ggatccgatg atgcagcaga agtttccagg aggactcggg 720 ttcctctgca tacccgtctg cttcagcatt tgcggcgagt agcgagaatc atccgacacg 780 gggtttcagc ggccgctggc agattgttcg ggcgagttcg gcaagttgag gcggaacgtc 840 cacaacccgt tttcactgaa ggtgatcctc cggatctcga gacgaattcc ttgtattatc 900 gcgacaaggt tccaggacag gggataatcc aggagatcct caggcagaag ccgggaatcg 960 cacaccaccc cgagtcattt agtgtggttg ctgctgacga gagagtatca cggactctgt 1020 gggctgaggg cggggttgtg agagttgctt cagaattggg ccaacctggg agagtgctcg 1080 tgagggggag acgaatcggt ttgtttcgcc caggaatgca gtttgaagca acagaccagg 1140 cgacaggaga accgatgact gcgcttgttg gccatactgt gcttgaggcc acggcaagag 1200 acgtagattc gatgcgcaac gaaggattag ctgttgggtt gtttcaaaag gtgaagaacc 1260 catatctagc taacaggtat ctaagatttc tggccccgtt cgatttggtg acgatcccgg 1320 ggaagccatt agttcagaaa gctaagtcac gtaatgaggt tggctgggtc aaaaatctgt 1380 tgtttctgct tcctccaact catgtcgata tggaaacgtt tgtcgacgag attggtcgat 1440 ttccacaaga ggaccgcccc ctagctgatg ccgctcgatt gtatcttact gttcaggcgg 1500 tgaggttggt agcgcacctc caagacgaag gagtggtgca cgggaagata atgcctgatt 1560 cgttttgttt gaagcgtgag gggggcctgt acttgcgtga ctttggctct ctggtgagag 1620 cgggcgcaaa agtggtggtg cccgcggaat atgatgagta tacgccgcca gaaggtcgtg 1680 cggccgcccg aagtcgtttc ggctctgggg cgaccacgat gacgtacgca ttcgacgcct 1740 ggacactggg ttcggttata tttttaattt ggtgctctcg agctcccgat acaaaatcgg 1800 ggtacgaata cagtgtcgaa ttcttttttt cgaggtgccg gcgggtgcca gagaacgtga 1860 aacttctggt ttacaagttg attaaccctt ctgtcgaggc ccgcctgctc gccttgcaag 1920 cgatagagac tccggaatac agggagatgg aagaacagct gtccgctgct tcgcgcctgt 1980 actcaggtga tgggacgttg acagggggag acgatgacat gccaccactg gaaacgtga 2039 <110> University-Industry Cooperation Group of Kyung Hee University <120> Recombinant Vaccinia virus vaccines comprising antigen ROP4 of Toxoplasma gondii <130> ADP-2022-0112 <160> 2 <170>CopatentIn 2.0 <210> 1 <211> 578 <212> PRT <213> Toxoplasma gondii <400> 1 Met Gly His Pro Thr Ser Phe Gly Gln Pro Ser Cys Leu Val Trp Leu 1 5 10 15 Ala Ala Ala Phe Leu Val Leu Gly Leu Cys Leu Val Gln Gln Gly Ala 20 25 30 Gly Arg Gln Arg Pro His Gln Trp Lys Ser Ser Glu Ala Ala Leu Ser 35 40 45 Val Ser Pro Ala Gly Asp Ile Val Asp Lys Tyr Ser Arg Asp Ser Thr 50 55 60 Glu Gly Glu Asn Thr Val Ser Glu Gly Glu Ala Glu Gly Ser Arg Gly 65 70 75 80 Gly Ser Trp Leu Glu Gln Glu Gly Val Glu Leu Arg Ser Pro Ser Gln 85 90 95 Asp Ser Gln Thr Gly Thr Ser Thr Ala Ser Pro Thr Gly Phe Arg Arg 100 105 110 Leu Leu Arg Arg Leu Arg Phe Trp Arg Arg Gly Ser Thr Arg Gly Ser 115 120 125 Asp Asp Ala Ala Glu Val Ser Arg Arg Thr Arg Val Pro Leu His Thr 130 135 140 Arg Leu Leu Gln His Leu Arg Arg Val Ala Arg Ile Ile Arg His Gly 145 150 155 160 Val Ser Ala Ala Ala Gly Arg Leu Phe Gly Arg Val Arg Gln Val Glu 165 170 175 Ala Glu Arg Pro Gln Pro Val Phe Thr Glu Gly Asp Pro Pro Asp Leu 180 185 190 Glu Thr Asn Ser Leu Tyr Tyr Arg Asp Lys Val Pro Gly Gln Gly Ile 195 200 205 Ile Gln Glu Ile Leu Arg Gln Lys Pro Gly Ile Ala His His Pro Glu 210 215 220 Ser Phe Ser Val Val Ala Ala Asp Glu Arg Val Ser Arg Thr Leu Trp 225 230 235 240 Ala Glu Gly Gly Val Val Arg Val Ala Ser Glu Leu Gly Gln Pro Gly 245 250 255 Arg Val Leu Val Arg Gly Arg Arg Ile Gly Leu Phe Arg Pro Gly Met 260 265 270 Gln Phe Glu Ala Thr Asp Gln Ala Thr Gly Glu Pro Met Thr Ala Leu 275 280 285 Val Gly His Thr Val Leu Glu Ala Thr Ala Arg Asp Val Asp Ser Met 290 295 300 Arg Asn Glu Gly Leu Ala Val Gly Leu Phe Gln Lys Val Lys Asn Pro 305 310 315 320 Tyr Leu Ala Asn Arg Tyr Leu Arg Phe Leu Ala Pro Phe Asp Leu Val 325 330 335 Thr Ile Pro Gly Lys Pro Leu Val Gln Lys Ala Lys Ser Arg Asn Glu 340 345 350 Val Gly Trp Val Lys Asn Leu Leu Phe Leu Leu Pro Pro Thr His Val 355 360 365 Asp Met Glu Thr Phe Val Asp Glu Ile Gly Arg Phe Pro Gln Glu Asp 370 375 380 Arg Pro Leu Ala Asp Ala Ala Arg Leu Tyr Leu Thr Val Gln Ala Val 385 390 395 400 Arg Leu Val Ala His Leu Gln Asp Glu Gly Val Val His Gly Lys Ile 405 410 415 Met Pro Asp Ser Phe Cys Leu Lys Arg Glu Gly Gly Leu Tyr Leu Arg 420 425 430 Asp Phe Gly Ser Leu Val Arg Ala Gly Ala Lys Val Val Val Pro Ala 435 440 445 Glu Tyr Asp Glu Tyr Thr Pro Pro Pro Glu Gly Arg Ala Ala Ala Arg Ser 450 455 460 Arg Phe Gly Ser Gly Ala Thr Thr Met Thr Tyr Ala Phe Asp Ala Trp 465 470 475 480 Thr Leu Gly Ser Val Ile Phe Leu Ile Trp Cys Ser Arg Ala Pro Asp 485 490 495 Thr Lys Ser Gly Tyr Glu Tyr Ser Val Glu Phe Phe Phe Ser Arg Cys 500 505 510 Arg Arg Val Pro Glu Asn Val Lys Leu Leu Val Tyr Lys Leu Ile Asn 515 520 525 Pro Ser Val Glu Ala Arg Leu Leu Ala Leu Gln Ala Ile Glu Thr Pro 530 535 540 Glu Tyr Arg Glu Met Glu Glu Gln Leu Ser Ala Ala Ser Arg Leu Tyr 545 550 555 560 Ser Gly Asp Gly Thr Leu Thr Gly Gly Asp Asp Asp Met Pro Pro Leu 565 570 575 Glu Thr <210> 2 <211> 2039 <212> DNA <213> Toxoplasma gondii <400> 2 tccggcatgg aaaaaatgcg tctagctaac cgcgggcccc aatgtcgcac ggccttggtc 60 aagggacggc gcgacctgca gagacgggaa tccgagccaa gcgctgagtg tccttgcttc 120 tggctgagcc gccatcctgc ggcaccgtcc atgactgcgc acagcatccc gggagtatgg 180 tgattgtgtc agtcttattt cttttgaagg cacttcagat gtgtacttca gaatgttgtt 240 acgataattg cggatgcagc tgccacctct gaacggttga tttggttgtg agtcctccca 300 acatggggca ccctacctct ttcggacagc cgtcgtgtct tgtctggcta gctgcggcat 360 tccttgttct ggggctttgc ctcgtccagc aaggcgctgg cagacagcgg cctcaccagt 420 ggaagagctc ggaagccgct ttgagtgtca gtccggcagg agacatcgtg gacaagtatt 480 ctcgtgattc cactgaggga gaaaacactg tctcagaggg ggaagctgag ggcagtcggg 540 ggggttcatg gctggagcag gagggggttg aattaaggtc gccttcacag gacagccaga 600 caggaacctc gaccgcgtcc cccacaggct tcagaaggtt actcaggcgt ttgcgttttt 660 ggcgtcgtgg gagtacacgc ggatccgatg atgcagcaga agtttccagg aggactcggg 720 ttcctctgca tacccgtctg cttcagcatt tgcggcgagt agcgagaatc atccgacacg 780 gggtttcagc ggccgctggc agattgttcg ggcgagttcg gcaagttgag gcggaacgtc 840 cacaacccgt tttcactgaa ggtgatcctc cggatctcga gacgaattcc ttgtattatc 900 gcgacaaggt tccaggacag gggataatcc aggagatcct caggcagaag ccgggaatcg 960 cacaccaccc cgagtcattt agtgtggttg ctgctgacga gagagtatca cggactctgt 1020 gggctgaggg cggggttgtg agagttgctt cagaattggg ccaacctggg agagtgctcg 1080 tgagggggag acgaatcggt ttgtttcgcc caggaatgca gtttgaagca acagaccagg 1140 cgacaggaga accgatgact gcgcttgttg gccatactgt gcttgaggcc acggcaagag 1200 acgtagattc gatgcgcaac gaaggattag ctgttgggtt gtttcaaaag gtgaagaacc 1260 catatctagc taacaggtat ctaagatttc tggccccgtt cgatttggtg acgatcccgg 1320 ggaagccatt agttcagaaa gctaagtcac gtaatgaggt tggctgggtc aaaaatctgt 1380 tgtttctgct tcctccaact catgtcgata tggaaacgtt tgtcgacgag attggtcgat 1440 ttccacaaga ggaccgcccc ctagctgatg ccgctcgatt gtatcttact gttcaggcgg 1500 tgaggttggt agcgcacctc caagacgaag gagtggtgca cgggaagata atgcctgatt 1560 cgttttgttt gaagcgtgag gggggcctgt acttgcgtga ctttggctct ctggtgagag 1620 cgggcgcaaa agtggtggtg cccgcggaat atgatgagta tacgccgcca gaaggtcgtg 1680 cggccgcccg aagtcgtttc ggctctgggg cgaccacgat gacgtacgca ttcgacgcct 1740 ggacactggg ttcggttata tttttaattt ggtgctctcg agctcccgat acaaaatcgg 1800 ggtacgaata cagtgtcgaa ttcttttttt cgaggtgccg gcgggtgcca gagaacgtga 1860 aacttctggt ttacaagttg attaaccctt ctgtcgaggc ccgcctgctc gccttgcaag 1920 cgatagagac tccggaatac agggagatgg aagaacagct gtccgctgct tcgcgcctgt 1980 actcaggtga tgggacgttg acagggggag acgatgacat gccaccactg gaaacgtga 2039
Claims (6)
상기 형질감염된 백시니아 바이러스(Vaccinia virus) 세포를 배양하여 ROP4를 발현시키는 단계를 포함하는 톡소포자충에 대한 면역 반응을 유도할 수 있는 백신 제조 방법.Transfecting Vaccinia virus cells with a recombinant expression vector expressing the Toxoplasma antigen rhoptry protein 4 (ROP4); and
A method for producing a vaccine capable of inducing an immune response against Toxoplasma gondii, comprising culturing the transfected vaccinia virus cells to express ROP4.
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