KR20230111815A - Manufacturing method of single domain vhh antibody for preventing or treating early mortality syndrome - Google Patents
Manufacturing method of single domain vhh antibody for preventing or treating early mortality syndrome Download PDFInfo
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- KR20230111815A KR20230111815A KR1020220007736A KR20220007736A KR20230111815A KR 20230111815 A KR20230111815 A KR 20230111815A KR 1020220007736 A KR1020220007736 A KR 1020220007736A KR 20220007736 A KR20220007736 A KR 20220007736A KR 20230111815 A KR20230111815 A KR 20230111815A
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- shrimp
- vhh antibody
- antigen
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Abstract
본 발명은 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체를 제조하기 위하여 낙타과 동물을 면역화 시키는 단계에서 항원과 함께 플라젤린(flagellin)을 낙타과 동물에 주입하는 단계를 포함하는 것을 특징으로 하는 단일 도메인 VHH 항체의 제조방법에 관한 것으로, 상기 방법은 다양한 항체들 중 항원특이적인 IgG2 및 IgG3의 생성을 선택적으로 증가시킬 수 있다.The present invention relates to a method for producing a single domain VHH antibody, comprising the step of injecting flagellin together with an antigen into a camelid animal in the step of immunizing a camelid animal to prepare a single domain VHH antibody for the prevention or treatment of shrimp premature death syndrome. The method can selectively increase the production of antigen-specific IgG 2 and IgG 3 among various antibodies.
Description
본 발명은 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체의 제조방법에 관한 것으로, 단일 도메인 VHH 항체를 제조하기 위하여 낙타과 동물을 면역화 시키는 단계에서 새우 조기 폐사 증후군의 원인균 또는 바이러스 항원과 함께 플라젤린(flagellin)을 낙타과 동물에 주입함으로써 다양한 항체들 중 항원 특이적인 IgG2 및 IgG3의 생성을 선택적으로 증가시킬 수 있는 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체의 제조방법에 관한 것이다.The present invention relates to a method for producing a single domain VHH antibody for preventing or treating shrimp premature mortality syndrome, and in the step of immunizing camelids to produce a single domain VHH antibody, flagellin together with a causative agent of shrimp premature death syndrome or a virus antigen is injected into camelid animals, thereby producing a single domain VHH antibody for preventing or treating premature shrimp death syndrome, which can selectively increase the production of antigen-specific IgG 2 and IgG 3 among various antibodies. .
나노바디는 단일 도메인 항체(single domain antibody, SDA)에서 가변영역(variable region of a heavy chain antibody, VHH)으로서, 낙타과 동물로부터 단리된 단일 도메인 항체, 소위 VHH 항체를 일컫는다. VHH 항체는 단일 도메인 분자이기 때문에 4개의 사슬, 즉 2개의 경쇄 및 2개의 중쇄로 이루어진 항체와는 달리 초분자 조립체 없이 세포에서 발현되며, 일반 항체보다 더 안정적이고 강력하다. 또한, VHH 항체는 약 15kDa의 매우 작은 분자이기 때문에 기존 항체로 접근 할 수 없는 숨겨진 에피토프에 결합가능하며, 더불어 치료용으로 적용할 경우, 작은 분자량에 의해 효율적인 침투 및 빠른 클리어런스(clearance)가 가능하다는 장점이 있다. 게다가, 동일한 세포 내외의 폴딩에 접합할 가능성이 더 높기 때문에 세포 내 프로빙을 위한 후보물질(intrabody)로 대두되고 있다.A nanobody is a variable region of a heavy chain antibody (VHH) in a single domain antibody (SDA), and refers to a single domain antibody isolated from a camelid animal, a so-called VHH antibody. Since VHH antibodies are single-domain molecules, they are expressed in cells without supramolecular assemblies, unlike antibodies consisting of four chains, namely two light chains and two heavy chains, and are more stable and potent than normal antibodies. In addition, since the VHH antibody is a very small molecule of about 15 kDa, it can bind to a hidden epitope that is inaccessible to conventional antibodies, and when applied for treatment, efficient penetration and rapid clearance are possible due to its small molecular weight. Moreover, it is emerging as a candidate for intracellular probing because it is more likely to bind to the same intracellular and extracellular folds.
VHH 항체는 항원으로 면역화 시킨 낙타과 동물로부터 B 세포를 수득한 다음, 파지 디스플레이(Phage display) 방법으로 항체를 선별하여 제조한다. 이 때, 낙타과 동물을 면역화 시키기 위해 항원을 낙타에 주입하면 낙타의 B 세포에서는 다양한 항체들(IgG, IgA, IgD, IgM, IgE 등)이 생성된다. 특히 낙타는 IgG1, IgG2, IgG3로 구성된 3개의 IgG 동형상(isotype)을 생성하는데, 이 중 IgG2 및 IgG3는 경쇄 및 CH1 도메인이 없는 구조를 갖는 항체이기 때문에 이로부터 VHH 항체를 생산한다. 그러나 면역화된 낙타가 생성하는 다양한 항체들 중, IgG2와 IgG3의 생성량이 현저히 낮아 VHH 항체를 제조하는데 어려움을 겪고 있다. 따라서 IgG2와 IgG3의 생성량을 선택적으로 증가시켜 VHH 항체를 효율적으로 제조하기 위한 방법의 개발이 필요하다.VHH antibodies are prepared by obtaining B cells from camelids immunized with an antigen, and then selecting antibodies using a phage display method. At this time, when an antigen is injected into a camel to immunize camelids, various antibodies (IgG, IgA, IgD, IgM, IgE, etc.) are produced in the camel's B cells. In particular, camels produce three IgG isotypes composed of IgG 1 , IgG 2 , and IgG 3 . Among them, IgG 2 and IgG 3 are antibodies having a structure without a light chain and a CH1 domain, from which VHH antibodies are produced. However, among various antibodies produced by immunized camels, production of IgG 2 and IgG 3 is remarkably low, making it difficult to produce VHH antibodies. Therefore, it is necessary to develop a method for efficiently producing VHH antibodies by selectively increasing the amount of IgG 2 and IgG 3 produced.
플라젤린(flagellin)은 세균에 있는 편모 섬유를 구성하고 있는 구형 단백질로서, 분자량은 균종에 따라 크게 다르며(30,000~70,000), 아미노산 조성에 있어 시스테인과 트립토판은 나타나지 않는 것이 특징이다. 이러한 플라젤린은 병원균과 관련된 분자 패턴을 인지하는 수용체인 TLR-5(Toll-like receptor-5)의 수용체에 결합하여 면역 조절 효과를 유도할 수 있음이 알려지면서 백신의 아쥬반트(adjuvant)로서의 가능성이 입증되었으며, 이는 특허 문헌에도 개시되어 있다. 구체적으로, 한국등록특허 제0795839호는 세균의 편모 구성인자 플라젤린을 유효성분으로 함유하는 백신 보조제에 관한 것으로, 플라젤린 구성 단백질이 상피세포에서 인터루킨-8의 생산을 자극하고 수지상 세포의 성숙을 촉진하며 항원-특이적 면역 반응을 증가시키는 것이 개시되어 있다. 그러나, 종래에는 플라젤린이 상기와 같이 백신의 아쥬반트로서의 기능성만 입증되었을 뿐, 플라젤린이 낙타과 동물에서 항원과 함께 주입될 경우, 다양한 항체들 중에서 IgG2 및 IgG3의 생성을 선택적으로 향상시키는 기능은 언급된 바 없다.Flagellin is a globular protein constituting flagellar fibers in bacteria, and its molecular weight varies greatly depending on the species of bacteria (30,000 to 70,000), and it is characterized by the absence of cysteine and tryptophan in its amino acid composition. As it is known that flagellin can induce an immunomodulatory effect by binding to the receptor of Toll-like receptor-5 (TLR-5), a receptor that recognizes molecular patterns associated with pathogens, its potential as an adjuvant for vaccines has been proven, and this is also disclosed in patent literature. Specifically, Korean Patent Registration No. 0795839 relates to a vaccine adjuvant containing flagellin, a component of bacterial flagella, as an active ingredient. It is disclosed that the flagellin component protein stimulates the production of interleukin-8 in epithelial cells, promotes the maturation of dendritic cells, and increases antigen-specific immune responses. However, in the past, only the function of flagellin as an adjuvant for vaccines has been demonstrated, and when flagellin is injected with an antigen in a camelid animal, the function of selectively enhancing the production of IgG 2 and IgG 3 among various antibodies has not been mentioned.
한편, 질병에 대한 방어기전으로서 사람뿐만 아니라 여러 포유류 및 조류도 감염성 질병으로부터 자신을 보호하거나 자손을 보호하기 위해 만들어내는 물질에 항체가 포함되어 있다. 포유류는 대부분 사람과 유사하게 약 150kDa의 IgG를 생산한다. 종래에는 여러 병원성 세균의 항체를 제조하기 위해 토끼, 소, 염소 등의 포유동물을 면역 처리하여 얻어진 혈청으로부터 병원성 세균 특이항체를 생산하는 방법이 광범위하게 사용되어 왔다. 그러나 이러한 방법을 통해 채취된 항체는 약 150kDa의 큰 분자이기 때문에 치료용으로 적용하는 경우에는 침투가 효율적이지 못하고 빠른 클리어런스가 일어나기 어렵다는 단점이 있다. 따라서 질병을 효율적으로 치료하기 위하여 더 작은 항체-유래 분자를 사용하는 것이 필요하다.On the other hand, as a defense mechanism against disease, antibodies are included in substances produced to protect not only humans but also various mammals and birds from infectious diseases to protect themselves or their offspring. Most mammals, similar to humans, produce IgG of about 150 kDa. Conventionally, a method of producing pathogenic bacteria-specific antibodies from serum obtained by immunizing mammals such as rabbits, cows, and goats has been widely used to prepare antibodies against various pathogenic bacteria. However, since the antibody collected through this method is a large molecule of about 150 kDa, it has disadvantages in that penetration is not efficient and rapid clearance is difficult to occur when applied for treatment. Therefore, there is a need to use smaller antibody-derived molecules to efficiently treat disease.
최근 전 세계적으로 성인병 예방을 위한 식생활 개선에 대한 관심이 급증함에 따라, 수산물의 수요가 지속적으로 증가되어 어류 양식의 생산량이 급증하고 있다. 따라서 양식업은 많은 나라에서 주요한 경제적 수단이 되고 있으며, 양식 어류의 질병발생은 경제적 발전에 커다란 영향을 미치고 있는 실정이다. 양식어류 중, 새우에 질병을 유발시키는 주요 원인으로 비브리오균과 흰반점 바이러스가 알려져 있다. 비브리오균(Vibrio genus)은 그람 음성형의 간균으로, 한쪽 끝에 1개, 종에 따라 2개 이상의 극모(極毛)를 지녀 활발하게 운동하고, 대표적인 병원균으로 콜레라를 일으키는 비브리오 콜레라(Vibrio colera)가 있다. 새우 질병의 원인으로는 장염 비브리오라 불리는 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum)이 있는데, 새우가 이러한 균에 감염될 경우, 24시간 내지 72시간 이내에 폐사하게 된다. 또한, 흰반점 증후군 바이러스(White spot syndrome virus, WSSV)는 꼬리와 유사한 부착물을 갖고 있고, 막대(rod) 형태의 캡시드(capsid) 및 피막(envelop)이 존재하는 형태이다. 새우가 흰반점 바이러스에 감염될 경우, 새우의 갑피(carapace), 외지(appendage) 및 큐티클에 흰반점이 나타나고, 간췌장(hepatopancreas)이 붉은색을 띄게 되며, 감염 후 3~5일이 경과하면 폐사하게 된다. 따라서 상기와 같이 다양한 균 및 바이러스에 의한 조기 폐사를 예방 및 치료하기 위한 항체의 대량생산이 필요한 실정이다.Recently, as interest in improving dietary habits for preventing adult diseases has rapidly increased worldwide, the demand for aquatic products has continuously increased, and thus the production of fish aquaculture is rapidly increasing. Therefore, aquaculture has become a major economic means in many countries, and disease outbreaks in farmed fish have a great impact on economic development. Among farmed fish, Vibrio bacteria and white spot virus are known as the main causes of disease in shrimp. Vibrio genus is a gram-negative bacillus, which has one at one end and two or more cirrus depending on the species and moves actively. The causes of shrimp disease include Vibrio parahaemolyticus , Vibrio harveyi , and Vibrio anguillarum , which are called enteritis vibrio. When shrimp are infected with these bacteria, they die within 24 to 72 hours. In addition, the white spot syndrome virus (WSSV) has an attachment similar to a tail, and has a rod-shaped capsid and an envelope. When a shrimp is infected with the white spot virus, white spots appear on the shrimp's carapace, appendage and cuticle, the hepatopancreas turns red, and 3 to 5 days after infection, it dies. Therefore, there is a need for mass production of antibodies for preventing and treating premature mortality caused by various bacteria and viruses as described above.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 새우 조기 폐사 증후군을 예방 또는 치료하기 위한 VHH 항체를 제조하기 위하여 플라젤린을 이용하는 경우, IgG2 및 IgG3의 생성을 선택적으로 증가시켜 효율적으로 VHH를 제조할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, as a result of intensive research efforts to overcome the problems of the prior art, the present inventors have confirmed that VHH can be efficiently produced by selectively increasing the production of IgG 2 and IgG 3 when flagellin is used to prepare VHH antibodies for preventing or treating premature shrimp death syndrome, thereby completing the present invention.
따라서, 본 발명의 주된 목적은 항원 특이적인 IgG2 및 IgG3의 생성을 선택적으로 증가시킴으로써 VHH 항체를 효율적으로 제조할 수 있는 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체의 제조방법, 이의 제조방법을 통해 제조된 항체 및 이의 용도를 제공하는 데 있다.Therefore, the main object of the present invention is the antigen-specific IgG 2 and IgG 3 It is to provide a method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome, which can efficiently produce VHH antibodies by selectively increasing production, antibodies produced through the production method, and uses thereof.
본 발명의 한 양태에 따르면, 본 발명은 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체를 제조하는 방법에 있어서, 새우 조기 폐사 증후군의 원인균 또는 바이러스 항원과 플라젤린(flagellin)의 혼합물을 낙타과 동물에 주입하여 면역화 시키는 단계를 포함하는 것을 특징으로 하는 단일 도메인 VHH 항체의 제조 방법을 제공한다.According to one aspect of the present invention, the method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome provides a method for producing a single domain VHH antibody comprising the step of injecting and immunizing a camelid animal with a mixture of a causative bacteria or virus antigen of shrimp premature death syndrome and flagellin.
나노바디는 낙타과 동물로부터 단리된 단일 도메인 항체, 소위 VHH 항체를 일컫는다. VHH 항체는 약 15kDa의 매우 작은 분자이기 때문에 이를 기반으로한 항체 치료제에 대한 기술들이 개발되고 있다. 일반적으로, VHH 항체는 항원으로 면역화 시킨 낙타과 동물로부터 B 세포를 수득 및 선별하여 제조하는데, 이 때, 면역화된 낙타과 동물의 B 세포에서는 다양한 항체들(IgG, IgA, IgD, IgM, IgE 등) 뿐만 아니라 IgG2 및 IgG3가 생성되며, 이 중에서 IgG2 및 IgG3는 경쇄 및 CH1 도메인이 없는 구조를 갖기 때문에 이로부터 VHH 항체를 생산한다. 그러나, 생성되는 IgG2와 IgG3는 생성량이 현저히 낮아 VHH 항체를 제조하는데 어려움을 겪고 있다. 이에, 본 발명자들은 낙타를 면역화 시킬 때 항원과 함께 플라젤린을 주입할 경우, IgG2와 IgG3의 생성량이 선택적으로 증가되어 새우 조기 폐사 증후군을 예방 또는 치료하기 위한 VHH 항체를 효율적으로 제조할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Nanobodies refer to single domain antibodies, so-called VHH antibodies, isolated from camelids. Since the VHH antibody is a very small molecule of about 15 kDa, technologies for antibody therapeutics based thereon are being developed. In general, VHH antibodies are prepared by obtaining and selecting B cells from camelid animals immunized with an antigen. At this time, various antibodies (IgG, IgA, IgD, IgM, IgE, etc.) as well as IgG 2 and IgG 3 are produced in the B cells of immunized camelid animals. Among them, IgG 2 and IgG 3 have a structure without a light chain and a CH1 domain, from which VHH antibodies are produced. However, the IgG 2 and IgG 3 produced It is difficult to produce VHH antibodies because the amount produced is remarkably low. Accordingly, the inventors of the present invention have confirmed that, when flagellin is injected together with an antigen when immunizing a camel, the production of IgG 2 and IgG 3 is selectively increased, thereby efficiently producing a VHH antibody for preventing or treating premature shrimp death syndrome. The present invention was completed.
본 발명의 단일 도메인 VHH 항체의 제조 방법에 있어서, 상기 새우 조기 폐사 증후군의 원인균은 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)로 이루어진 군에서 선택되는 하나 이상의 균이고, 바이러스는 흰반점 바이러스(white spot syndrome virus, WSSV)인 것을 특징으로 한다.In the method for producing a single domain VHH antibody of the present invention, the causative agent of the shrimp premature death syndrome is one or more bacteria selected from the group consisting of Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum, and the virus is a white spot syndrome virus (WSSV).
본 발명의 단일 도메인 VHH 항체의 제조 방법에 있어서, 상기 플라젤린은 살모넬라균(Salmonella typhimurium), 비브리오 패혈증균(Vibrio vulnficus) 및 리스테리아균(Listeria monocytogenes)으로 이루어진 군에서 선택된 세균에서 유래된 것을 특징으로 한다.In the method for producing a single domain VHH antibody of the present invention, the flagellin is derived from a bacterium selected from the group consisting of Salmonella typhimurium , Vibrio vulnficus and Listeria monocytogenes .
본 발명의 단일 도메인 VHH 항체의 제조 방법에 있어서, 상기 외막 단백질 또는 항원과 플라젤린의 혼합 비율은 5~20 : 1일 수 있으며, 바람직하게는 10:1인 것을 특징으로 한다. 예컨대, 외막 단백질 또는 항원의 함량이 1mg일 때 플라젤린은 0.05~0.2mg 일 수 있다. 상기 혼합 비율에서 플라젤린의 비율이 높아질수록 항원 특이적인 IgG2 및 IgG3의 생성이 선택적으로 이루어지지 않아 단일 도메인 VHH 항체의 제조가 효율적으로 이루어지기 어렵다.In the method for producing a single domain VHH antibody of the present invention, the mixing ratio of the outer membrane protein or antigen and flagellin may be 5 to 20:1, preferably 10:1. For example, when the content of outer membrane protein or antigen is 1 mg, flagellin may be 0.05 to 0.2 mg. As the ratio of flagellin increases in the mixing ratio, the antigen-specific IgG 2 and IgG 3 It is difficult to efficiently produce a single domain VHH antibody because it is not selectively produced.
본 발명의 일 실험예에 따르면, 외막 단백질 또는 항원과 플라젤린의 혼합에 따른 IgG2 및 IgG3의 생성을 확인한 결과, 외막 단백질 또는 항원과 플라젤린을 혼합하여 알파카를 면역화 시킬 경우(실시예), 외막 단백질 또는 항원만을 주입하여 알파카를 면역화 시킨 경우(비교예)보다 항원 특이적 IgG2 및 IgG3의 생성이 선택적으로 증가한 것을 확인하였다(실험예 1 참조). 이러한 결과는 단일 도메인 VHH 항체를 제조하기 위하여 알카파를 면역화시킴에 있어서, 플라젤린을 이용할 경우 VHH 항체를 생산하기 위한 IgG2 및 IgG3의 생성을 선택적으로 증가시킬 수 있으므로, VHH 항체를 효율적으로 제조할 수 있음을 시사한다.According to an experimental example of the present invention, as a result of confirming the production of IgG 2 and IgG 3 according to the mixing of the outer membrane protein or antigen and flagellin, it was confirmed that when alpaca was immunized by mixing the outer membrane protein or antigen with flagellin (Example), the production of antigen-specific IgG 2 and IgG 3 was selectively increased compared to the case where alpaca was immunized by injecting only the outer membrane protein or antigen (Comparative Example) (see Experimental Example 1). These results suggest that, in immunization with Alcapa to prepare a single domain VHH antibody, when flagellin is used, the production of IgG 2 and IgG 3 for VHH antibody production can be selectively increased, so that VHH antibody can be produced efficiently.
본 발명의 다른 한 양태에 따르면, 본 발명은 새우 조기 폐사 증후군의 원인균 또는 바이러스 항원과 플라젤린(flagellin)의 혼합물을 낙타과 동물에 주입하여 면역화 시키는 단계를 포함하는 제조방법으로 제조된 서열번호 1의 아미노산 서열로 표시되는 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체를 제공한다.According to another aspect of the present invention, the present invention provides a single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 1 prepared by a manufacturing method comprising injecting and immunizing a mixture of a causative bacteria or virus antigen and flagellin of shrimp premature death syndrome into a camelid animal.
본 발명의 다른 한 양태에 따르면, 본 발명은 새우 조기 폐사 증후군의 원인균 또는 바이러스 항원과 플라젤린(flagellin)의 혼합물을 낙타과 동물에 주입하여 면역화 시키는 단계를 포함하는 제조방법으로 제조된 서열번호 2의 아미노산 서열로 표시되는 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체를 제공한다.According to another aspect of the present invention, the present invention provides a single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 2 prepared by a manufacturing method comprising the step of injecting and immunizing a mixture of a causative bacteria or virus antigen and flagellin of shrimp premature death syndrome into a camelid animal.
본 발명의 서열번호 1의 아미노산 서열로 표시되는 단일 도메인 VHH 항체는 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)으로 이루어진 군에서 선택되는 하나 이상의 균, 특히 비브리오 파라해모라이티쿠스에 대하여 특이적 결합능을 가지며, 본 발명의 서열번호 2의 아미노산 서열로 표시되는 단일 도메인 VHH 항체는 흰반점 바이러스(white spot syndrome virus, WSSV)에 대하여 특이적 결합능을 갖는다.The single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 of the present invention has specific binding ability to one or more bacteria selected from the group consisting of Vibrio parahaemolyticus , Vibrio harveyi and Vibrio anguillarum, in particular, Vibrio parahaemolyticus, and has an amino acid sequence of SEQ ID NO: 2 of the present invention. Domain VHH antibodies have specific binding ability to white spot syndrome virus (WSSV).
본 명세서에서 비브리오 파라해모라이티쿠스, 비브리오 하베이 및 비브리오 앵길라룸을 언급하면서 사용되는 용어 ‘단일 도메인 VHH 항체’는 비브리오 파라해모라이티쿠스, 비브리오 하베이 및 비브리오 앵길라룸에 대한 특이 항체이고, 흰반점 바이러스를 언급하면서 사용되는 용어 ‘단일 도메인 VHH 항체’는 흰반점 바이러스에 대한 특이 항체로서, CDR이 단일 도메인 폴리펩티드의 일부인 항체이며, 중쇄 항체, 경쇄가 자연적으로 없는 항체, 종래의 4-쇄 항체에서 유래한 단일 도메인 항체, 조작된 항체 및 항체에서 유래된 것 외의 단일 도메인 스캐폴드를 포함한다.The term 'single domain VHH antibody' used herein while referring to Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum is a specific antibody to Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, and the term 'single domain VHH antibody' used while referring to white spot virus is a specific antibody to white spot virus, the CDRs being part of a single domain polypeptide, heavy chain antibodies, light This includes antibodies naturally devoid of chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
단일 도메인 항체는 마우스, 사람, 낙타, 라마, 염소, 토끼 또는 소 등의 종으로부터 유래될 수 있으나, 본 발명에서의 단일 도메인 항체는 낙타과 동물, 예컨대 낙타, 단봉낙타, 라마, 알파카 및 야생라마 등에서 유래한 것이다. 본 발명에 이용되는 단일 도메인 항체는 경쇄가 없는 중쇄 항체로 알려진 자연 발생 단일 도메인 항체이다. 좀 더 상세하게는 경쇄가 자연적으로 없는 중쇄 항체에서 유래된 이 가변성 영역은 종래의 4-쇄 면역글로불린의 VH와 구별하기 위하여 VHH 또는 나노바디(nanobody)로 불리어 진다.The single domain antibody may be derived from species such as mouse, human, camel, llama, goat, rabbit or cow, but the single domain antibody in the present invention is derived from camelids such as camels, dromedaries, llamas, alpacas and wild llamas. The single domain antibody used in the present invention is a naturally occurring single domain antibody known as a heavy chain antibody without a light chain. More specifically, this variable region derived from a heavy chain antibody naturally devoid of a light chain is called a VHH or nanobody to distinguish it from the VH of a conventional 4-chain immunoglobulin.
본 발명의 단일 도메인 VHH 항체는 비브리오 파라해모라이티쿠스, 비브리오 하베이 및 비브리오 앵길라룸, 또는 흰반점 바이러스를 특이적으로 인식할 수 있는 범위 내에서 첨부한 서열목록에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 개선시키기 위하여 항체의 아미노산 서열에 변화를 줄 수 있다. 이러한 변형은, 예를 들어 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다.The single domain VHH antibody of the present invention may include variants of the amino acid sequences described in the attached sequence listing within the range of specifically recognizing Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, or white spot virus. For example, changes may be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 단일 도메인 VHH 항체를 코딩하는 서열번호 3으로 표시되는 핵산 분자를 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid molecule represented by SEQ ID NO: 3 encoding a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 2의 아미노산 서열로 표시되는 단일 도메인 VHH 항체를 코딩하는 서열번호 4로 표시되는 핵산 분자를 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid molecule represented by SEQ ID NO: 4 encoding a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2.
본 명세서에서 용어 “핵산 분자”는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 갖으며, 핵산 분자에서 기본 구성 단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체 (analogue)도 포함한다. As used herein, the term "nucleic acid molecule" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, include not only natural nucleotides, but also sugar or base site modified analogs (analogues).
전술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 단일 도메인 VHH 항체를 코딩하는 핵산분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기 한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 배열(align)하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 배열된 서열을 분석한 경우에, 최소 61%의 상동성, 바람직하게는 70%의 상동성, 보다 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다.Considering the mutations having the aforementioned biological equivalent activity, the nucleic acid molecule encoding the single domain VHH antibody of the present invention is construed to include a sequence showing substantial identity with the sequences listed in the Sequence Listing. The above substantial identity means a sequence showing at least 61% homology, preferably 70% homology, more preferably 80% homology, and most preferably 90% homology, when the sequence of the present invention and any other sequence are aligned so as to correspond as much as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 3으로 표시되는 핵산 분자를 포함하는 재조합 벡터를 제공한다.According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule represented by SEQ ID NO: 3.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 4로 표시되는 핵산 분자를 포함하는 재조합 벡터를 제공한다.According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule represented by SEQ ID NO: 4.
본 명세서에서 용어 “벡터”는 숙주 세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터, 파아지미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터 같은 바이러스 벡터 등을 포함한다.As used herein, the term “vector” is a means for expressing a gene of interest in a host cell and includes a plasmid vector, a phagemid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and a viral vector such as an adeno-associated virus vector.
본 발명의 바람직한 구현예에 따르면, 본 발명의 벡터에서 단일 도메인 VHH 항체를 코딩하는 핵산 분자는 프로모터와 작동적으로 결합(operatively linked)되어 있다.According to a preferred embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a single domain VHH antibody is operatively linked to a promoter.
본 명세서에서, 상기 용어 “작동적으로 결합된”은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절 인자 결합 위치의 어레이)과 다른 핵산 서열 사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다.As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and/or translation of the other nucleic acid sequence.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 3으로 표시되는 핵산 분자를 포함하는 재조합 벡터로 형질전환된 숙주세포를 제공한다.According to another aspect of the present invention, the present invention provides a host cell transformed with a recombinant vector containing the nucleic acid molecule represented by SEQ ID NO: 3.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 4로 표시되는 핵산 분자를 포함하는 재조합 벡터로 형질전환 된 숙주세포를 제공한다.According to another aspect of the present invention, the present invention provides a host cell transformed with a recombinant vector containing the nucleic acid molecule represented by SEQ ID NO: 4.
본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주 세포는 당업계에 공지된 어떠한 숙주 세포도 이용할 수 있으며, 예컨대, 에스케리치아 콜라이(Escherichia coli), 바실러스(Bacillus) 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)와 같은 원핵 숙주 세포를 포함하나, 이에 제한되는 것은 아니다.Any host cell capable of stably cloning and expressing the vector of the present invention can be used as any host cell known in the art, for example, Escherichia coli, Bacillus strain, Streptomyces , Pseudomonas , Proteus mirabilis or Staphylococcus cus ), but is not limited thereto.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 및 서열번호 2의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 중 중 하나 이상을 유효성분으로 포함하는 새우 조기 폐사 증후군 예방 또는 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating premature death syndrome of shrimp comprising at least one of the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
본 발명에 따른 단일 도메인 VHH 항체는 비브리오 파라해모라이티쿠스, 비브리오 하베이 및 비브리오 앵길라룸과 같은 균주, 또는 흰반점 바이러스에 대한 항원과 특이적으로 결합하므로 단일 도메인 VHH 항체 단독 또는 통상의 약제학적으로 허용되는 담체와 함께 상기 항원들에 의해 발병하는 새우 폐사 증후군을 예방 또는 치료하기 위한 약학적 조성물로 사용가능하다.Since the single domain VHH antibody according to the present invention binds specifically to antigens for strains such as Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, or white spot virus, the single domain VHH antibody alone or in combination with a conventional pharmaceutically acceptable carrier can be used as a pharmaceutical composition for preventing or treating shrimp mortality syndrome caused by the antigens.
본 발명의 약학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 알긴산, 젤라틴, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, alginic acid, gelatin, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
본 발명의 약학적 조성물을 새우에 투여하는 방법으로는 새우에게 투여 가능한 어떠한 방식도 이용가능하며, 바람직하게는 서열번호 1의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 및 서열번호 2의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 중 하나 이상을 사료첨가제 원료 및 부형제를 혼합한 후, 상기 혼합물을 압축 펠렛팅화(extrusion pelleting)하여 사료로 제조하여 경구투여 방식으로 급이할 수 있으나, 이에 제한되지 않는다.As a method of administering the pharmaceutical composition of the present invention to shrimp, any method capable of administering to shrimp can be used, preferably, a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 After mixing at least one of the feed additive raw materials and excipients, the mixture may be compressed into pellets to prepare a feed and fed by oral administration, but is not limited thereto.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 및 서열번호 2의 아미노산 서열로 표시되는 단일 도메인 VHH 항체 중 중 하나 이상을 유효성분으로 포함하는 새우 조기 폐사 증후군용 사료첨가제 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a feed additive composition for shrimp premature death syndrome comprising at least one of the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
본 발명의 상기 사료첨가제는 원형 그대로 사용하거나 또는 추가적으로 가축에 허용되는 곡류 및 그 부산물 등의 공지된 담체, 안정제 등을 가할 수 있으며, 필요에 따라 구연산, 후말산, 아디픽산, 젖산, 사과산 등의 유기산이나 인산나트륨, 인산칼륨, 산성 피로인산염, 폴리인산염(중합인산염) 등의 인산염이나, 폴리페놀, 카테킨, 알파-토코페롤, 로즈마리 추출물, 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제, 항생물질, 항균제 및 기타의 첨가제 등을 가할 수도 있으며, 그 형상으로서는 분체, 과립, 펠릿, 현탁액 등의 적당한 상태일 수 있다.The feed additive of the present invention may be used in its original form or additionally known carriers such as cereals and their by-products acceptable for livestock, stabilizers, etc. may be added. If necessary, organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, sodium phosphate, potassium phosphate, acidic pyrophosphate, polyphosphates (polyphosphates), polyphenols, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice Extracts, natural antioxidants such as chitosan, tannic acid, and phytic acid, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granule, pellet, or suspension.
전술한 바와 같이, 본 발명에 따른 새우 조기 폐사 증후군의 예방 또는 치료용 단일 도메인 VHH 항체의 제조 방법은 낙타과 동물에서 생성되는 다양한 항체들 중에서 항원 특이적인 IgG2 및 IgG3의 생성을 선택으로 증가시킬 수 있다. 이에 따라 다양한 항체들보다 선택적으로 증가된 IgG2 및 IgG3를 단일 도메인 VHH 항체 제조에 이용할 수 있으므로, 단일 도메인 VHH 항체를 보다 효율적으로 제조할 수 있다.As described above, the method for preparing a single domain VHH antibody for preventing or treating premature shrimp death syndrome according to the present invention can selectively increase the production of antigen-specific IgG 2 and IgG 3 among various antibodies produced in camelid animals. Accordingly, since IgG 2 and IgG 3 selectively increased than various antibodies can be used for preparing single domain VHH antibodies, single domain VHH antibodies can be produced more efficiently.
도 1 및 2는 시험예 1에 따라 IgG2 및 IgG3의 생성량을 확인한 결과이다(도 1; 비브리오 파라해모라이티쿠스 외막 단백질, 도 2; 흰반점 바이러스).
도 3 및 도 4는 시험예 3에 따라 패닝(panning)에 의해 특이적인 항체들을 선별한 결과이다(도 3; 대조군, 도 4; 실시예).
도 5 및 도 6은 실험예 6에 따라 단일 도메인 VHH 항체의 특이성을 확인한 결과이다(도 5a: 비브리오 파라해모라이티쿠스에 대한 반응성 결과, 도 5b: 비브리오 하베이에 대한 반응성 결과, 도 6 흰반점 바이러스의 VP28 항원에 대한 반응성 결과).
도 7 내지 도 9는 실험예 7에 따라 새우 조기 폐사 증후군에 대한 억제 효과를 확인한 결과이다(도 7: 비브리오 파라해모라이티쿠스에 대한 억제 효과 결과, 도 8 및 9: 흰반점 바이러스의 VP28 항원에 대한 억제 효과 결과).1 and 2 are results confirming the amount of IgG 2 and IgG 3 produced according to Test Example 1 (FIG. 1; Vibrio parahaemolyticus outer membrane protein; FIG. 2; white spot virus).
3 and 4 are results of screening specific antibodies by panning according to Test Example 3 (FIG. 3; control, FIG. 4; Example).
5 and 6 are results confirming the specificity of the single domain VHH antibody according to Experimental Example 6 (FIG. 5a: Reactivity to Vibrio parahaemolyticus, FIG. 5b: Reactivity to Vibrio harvey, FIG. 6 Reactivity to VP28 antigen of white spot virus).
7 to 9 are results confirming the inhibitory effect on shrimp premature death syndrome according to Experimental Example 7 (Fig. 7: inhibitory effect on Vibrio parahaemolyticus, Figs. 8 and 9: inhibitory effect on VP28 antigen of white spot virus).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.
실시예: 낙타과 동물 면역화용 혼합물의 제조(1)Example: Preparation of a mixture for immunization of camelid animals (1)
낙타과 동물을 면역화 시키기 위한 혼합물을 표 1의 성분 및 함량으로 제조하였으며, 구체적인 실험 방법은 아래와 같다.A mixture for immunizing camelid animals was prepared with the ingredients and contents in Table 1, and the specific experimental method is as follows.
1) 세균성 항원 생산1) Bacterial antigen production
비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus)의 외막에 존재하는 외막 단백질 (outer membrane protein, 이하 OMP라 칭함)을 항원으로 이용하기 위해, 비브리오 파라해모라이티쿠스에서 OMP를 추출하였다. 비브리오 파라해모라이티쿠스는 전용배지(1.5 L)에서 37℃에서 24시간 배양하였다. 집락 형성을 확인 후 배양액을 원심분리(8,000 g, 4℃, 10 min)하여 균체를 회수한다. 회수한 균체는 35ml lysis buffer(20 mM Tris-HCl, 1 mM PMSF, pH 8)에 재부유시켜 초음파 분해하였다. 초음파 분해 후 원심분리 (10,000 g, 10 min, 4℃)하여 상층액을 얻는다. 상층액을 초고속원심분리기로 원심(20,000 g, 4℃, 1 h)하여 펠렛을 얻었다. 펠렛을 2% 사르코실(sarkosyl)을 포함하는 20 mM Tris-HCl (pH 8) 10 ml에 재부유시켜 상온에서 1시간 방치하였고, 초고속원심분리기로 원심 (20,000 g, 4C, 1 h) 하여 OMP 항원을 회수하였다. OMP 항원은 30 ml PBS로 2회 세척하여 알파카 면역접종 항원으로 이용하였다.In order to use the outer membrane protein (hereinafter referred to as OMP) present in the outer membrane of Vibrio parahaemolyticus as an antigen, OMP was extracted from Vibrio parahaemolyticus. Vibrio parahaemolyticus was cultured for 24 hours at 37 ° C. in a dedicated medium (1.5 L). After confirming colony formation, the culture medium is centrifuged (8,000 g, 4° C., 10 min) to recover cells. The recovered cells were resuspended in 35 ml lysis buffer (20 mM Tris-HCl, 1 mM PMSF, pH 8) and sonicated. After sonication, centrifugation (10,000 g, 10 min, 4 ℃) to obtain a supernatant. The supernatant was centrifuged (20,000 g, 4° C., 1 h) using an ultra-high-speed centrifuge to obtain a pellet. The pellet was resuspended in 10 ml of 20 mM Tris-HCl (pH 8) containing 2% sarkosyl, left at room temperature for 1 hour, and centrifuged (20,000 g, 4C, 1 h) in an ultra-high-speed centrifuge to recover the OMP antigen. The OMP antigen was washed twice with 30 ml PBS and used as an alpaca immunization antigen.
2) 바이러스성 항원 생산2) production of viral antigens
흰반점 바이러스(white spot syndrome virus, WSSV)는 숙주세포를 이용하여 배양하였다. 상기 바이러스를 감염시킨 후 2일경 세포병변이 확인되면 얼린 후 녹이는 과정을 3회 반복하여 바이러스를 분리시키고, 원심분리를 통하여 바이러스가 포함된 상층액을 회수하였다. 회수한 바이러스를 불활화하기 위해 바이러스 배양액을 60℃에서 30분간 열처리하여 불활화하였고, 추가적으로 초음파 분쇄를 진행하여 바이러스 항원 생산하였다.White spot syndrome virus (WSSV) was cultured using host cells. When cell lesions were confirmed about 2 days after infection with the virus, the freezing and thawing process was repeated three times to isolate the virus, and the supernatant containing the virus was recovered through centrifugation. In order to inactivate the recovered virus, the virus culture medium was inactivated by heat treatment at 60 ° C. for 30 minutes, and additionally ultrasonicated to produce viral antigens.
3) 플라젤린 생산3) Production of flagellin
살모넬라 엔테리티디스(Salmonella enteritidis)의 표준 균주에서 플라젤린을 분리하였다. 구체적으로 균주를 LB 배지 1.5 L 당 1%씩 접종하여 배양하였다. 37℃에서 24시간 동안 배양하였고, 4℃에서 원심분리 (6,000 g, 30 min)하여 균주를 회수하였다. 회수된 균주를 300 ml PBS로 부유 시킨 후 2회 세척하였고, 균주를 PBS로 재부유 시킨 후 염산으로 pH를 2.0으로 조정하고, 30분간 교반하였다. 교반 후 4℃에서 15분간 원심분리 (14,000 g)하여 상층액을 회수하고, 1 M 수산화나트륨으로 pH 7.4로 적정한 후 상층액에 65% 암모늄 설페이트가 되도록 가하여 4 ℃에서 하룻밤 동안 방치하였다. 상층액은 다시 원심분리 (20,000 g, 15 min)하였다. 원심분리 후 침전된 펠렛은 40 ㎖의 PBS로 재부유 시킨 후 투석망 (dialysis tube)에 넣어 PBS 상에서 48시간 투석하여 플라젤린을 생산하였다.Flagellin was isolated from a standard strain of Salmonella enteritidis . Specifically, the strain was inoculated and cultured at 1% per 1.5 L of LB medium. It was cultured at 37°C for 24 hours, and strains were recovered by centrifugation (6,000 g, 30 min) at 4°C. The recovered strain was suspended in 300 ml PBS and washed twice, and the strain was resuspended in PBS, the pH was adjusted to 2.0 with hydrochloric acid, and stirred for 30 minutes. After stirring, the supernatant was collected by centrifugation (14,000 g) at 4°C for 15 minutes, titrated to pH 7.4 with 1 M sodium hydroxide, added to the supernatant to make 65% ammonium sulfate, and left at 4°C overnight. The supernatant was centrifuged again (20,000 g, 15 min). After centrifugation, the precipitated pellet was resuspended in 40 ml of PBS, put in a dialysis tube, and dialyzed on PBS for 48 hours to produce flagellin.
4) 알파카 면역화를 위한 항원 제조4) Preparation of antigen for alpaca immunization
상기 1) 에서 제조한 OMP 항원 및 상기 2) 에서 제조한 바이러스 항원 1 mg씩을 항원 용액의 동일량의 adjuvant (Titer Max®)와 혼합 후 초음파 균질화하여 알파카 면역화용 혼합물을 제조하였다. 상기 혼합물에 플라젤린을 혼합하여 알파카 면역화를 위한 항원 제조를 완료하였다.Mixtures for alpaca immunization were prepared by mixing 1 mg each of the OMP antigen prepared in 1) and the virus antigen prepared in 2) above with the same amount of adjuvant (Titer Max®) as the antigen solution and then ultrasonically homogenized. The mixture was mixed with flagellin to complete antigen preparation for alpaca immunization.
비교예: 낙타과 동물 면역화용 혼합물의 제조(1)Comparative Example: Preparation of a mixture for immunization of camelid animals (1)
낙타과 동물을 면역화 시키기 위한 혼합물을 표 2의 성분 및 함량으로 포함하도록 제조하였다. 플라젤린을 포함하지 않는 것을 제외하고는 실시예와 제조방법은 동일하다.A mixture for immunizing camelid animals was prepared to include the ingredients and contents in Table 2. Except for not including flagellin, the Example and the manufacturing method are the same.
실험예 1: 낙타에서의 면역반응 유도, 분리된 혈액으로부터 IgGExperimental Example 1: Induction of immune response in camels, IgG from separated blood 22 및 IgG and IgG 33 의 생성 확인Check the creation of
실시예에서 제조한 혼합물을 낙타에 2 내지 4군데 분산하여 피하투여 하였다. 이 후, 2주 간격으로 5차례 같은 양의 혼합물을 같은 방법으로 투여하였다. 최종 투여 2주일 후, 항응고제가 들어있는 수혈팩에 혈액을 채취하였고, 원심분리 (6,000 g, 30 min)하여 혈장을 분리하였다. 혈장에서 IgG1, IgG2 그리고 IgG3를 분리하기 위해 Protein A와 Protein G column (GE Healthcare)을 이용하였다.The mixture prepared in Example was dispersed in 2 to 4 places on camels and administered subcutaneously. Thereafter, the same amount of the mixture was administered in the same manner 5 times at intervals of 2 weeks. Two weeks after the final administration, blood was collected in transfusion packs containing anticoagulants, and plasma was separated by centrifugation (6,000 g, 30 min). Protein A and Protein G columns (GE Healthcare) were used to separate IgG 1 , IgG 2 and IgG 3 from plasma.
항원 특이적 IgG1, IgG2 그리고 IgG3를 확인하기 위하여, PBS에서 희석된 각각의 바이러스성 항원과 세균성 항원 100 ng을 96-well plate에 하룻밤 4℃에서 코팅하여 ELISA 기반의 항원-항체 반응을 확인하였다. 항원을 코팅한 plate는 PBS-T (0.05% Tween 20)로 3회 세척하였고, 이후 3% BSA로 보충된 PBS로 상온에서 1시간 동안 blocking하였다. BSA blocking 후 plate를 PBS-T로 3회 세척한 이후 각각의 항체를 10 nM씩 가하여 상온에서 1시간 항원-항체 반응을 유도하였다. Plate를 PBS-T로 세척하고, 이후 anti-alpaca IgG (rabbit polycolnal antibody, 1:5,000)를 처리하였다. Plate를 세척하고, anti-rabbit IgG-HRP (goat antibody, 1:10,000)를 상온에서 1시간 반응시켰다. PBS-T로 세척한 plate를 TMB 기질을 첨가 이후 stop solution을 각각 well에 첨가하였다. 흡광도 평판 판독기를 이용하여 450 nm에서 측정하여 항원-항체 반응을 확인하였으며, 그 결과를 도 1 및 도 2에 나타내었다.In order to identify antigen-specific IgG 1 , IgG 2 and IgG 3 , 100 ng of each of the viral and bacterial antigens diluted in PBS was coated on a 96-well plate overnight at 4° C., and an ELISA-based antigen-antibody reaction was confirmed. The antigen-coated plate was washed three times with PBS-T (0.05% Tween 20), and then blocked for 1 hour at room temperature with PBS supplemented with 3% BSA. After BSA blocking, the plate was washed three times with PBS-T, and each antibody was added at 10 nM each to induce an antigen-antibody reaction at room temperature for 1 hour. The plate was washed with PBS-T, and then treated with anti-alpaca IgG (rabbit polycolnal antibody, 1:5,000). The plate was washed and reacted with anti-rabbit IgG-HRP (goat antibody, 1:10,000) at room temperature for 1 hour. Plates washed with PBS-T were added with TMB substrate and then stop solution was added to each well. The antigen-antibody reaction was confirmed by measuring the absorbance at 450 nm using a plate reader, and the results are shown in FIGS. 1 and 2 .
그 결과 도 1과 도 2에서 확인할 수 있듯이, 알파카를 면역화 시키기 위하여 항원과 플라젤린을 혼합하여 주입한 경우, 항원만 주입한 경우보다 항원 특이적 IgG2 및 IgG3의 생성이 선택적으로 증가하였다.As a result, as can be seen in FIGS. 1 and 2, when the mixture of antigen and flagellin was injected to immunize alpaca, the production of antigen-specific IgG 2 and IgG 3 was selectively increased compared to when only the antigen was injected.
실험예 2: 면역화된 낙타로부터 분리한 백혈구를 이용한 cDNA 합성 및 단일 도메인 항체 라이브러리의 제조Experimental Example 2: Synthesis of cDNA using leukocytes isolated from immunized camels and preparation of a single domain antibody library
알파카 면역화 후 혈액 100 ml를 채취한 다음 피콜 (Ficoll) 용액을 이용하여 말초혈액단핵구 (PBMC)를 분리하였다. 분리한 PBMC는 RNAiso Plus(TAKARA BIO)를 이용하여 total RNA를 추출하였다. 회수한 total RNA를 SuperScriptⅢ (Invitrogen)를 이용하여 cDNA를 합성하였다.After alpaca immunization, 100 ml of blood was collected and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll solution. Total RNA was extracted from the isolated PBMC using RNAiso Plus (TAKARA BIO). cDNA was synthesized from the recovered total RNA using SuperScript III (Invitrogen).
중합효소 연쇄 반응(Polymerase Chain Reaction, PCR)을 이용하여 VHH 유전자를 증폭하였다. IgG2 및 IgG3의 Forward primer 서열은 5′-AGKTGCAGCTCGTGGAGTCNGGNGG-3′(서열번호 5)이고, IgG2의 Reverse primer 서열은 5′-GGGGTCTTCGCTGTGGTGCG-3′(서열번호 6)이며, IgG3의 Reverse primer는 5′-TTGTGGTTTTGGTGTCTTGGG-3′(서열번호 7)이다.The VHH gene was amplified using polymerase chain reaction (PCR). The forward primer sequence of IgG 2 and IgG 3 is 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO: 5), the reverse primer sequence of IgG 2 is 5'-GGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 6), and the reverse primer of IgG 3 is 5'-TTGTGGTTTTGGTGTCTTGGG-3' (SEQ ID NO: 7).
중합효소 연쇄 반응은 구체적으로 합성한 cDNA를 템플릿으로하여 상기의 primer를 이용하여 진행하였으며, PCR 반응은 95℃에서 2 min, 96℃에서 30 sec, 58℃에서 30 sec, 72℃에서 1 min, 72℃에서 5 min 그리고 30 cycle의 조건에서 알파카 VHH 유전자를 증폭하였다.The polymerase chain reaction was carried out using the above primers using the specifically synthesized cDNA as a template, and the PCR reaction was 95 ° C for 2 min, 96 ° C for 30 sec, 58 ° C for 30 sec, 72 ° C for 1 min, 72 ° C. The alpaca VHH gene was amplified under conditions of 5 min and 30 cycles.
PCR 반응으로 증폭한 VHH 유전자는 forward primer(SfiⅠ 제한효소 사이트 추가, 5´-TGCTCCTCGCGGCCCAGCCGGCCATGGCTCAGGTGCAGCTCGTGGAGTCTGG-3´)(서열번호 8)와 reverse primer(SpeⅠ 제한효소 사이트 추가, 5´-ATGATGATGTGCACTAGTTTGTGGTTTTGGTGTCTTGGG-3´(서열번호 9) (IgG3), 5´-ATGATGATGTGCACTAGTGGGGTCTTCGCTGTGGTGCG-3´(서열번호 10) (IgG2)를 이용하여 PCR(95℃에서 2 min, 96℃에서 30 sec, 58℃에서 30 sec, 72℃에서 1 min 그리고 72℃에 5 min을 20 cycle)을 수행하여 제한효소 사이트를 부가하였다. 제한 효소 사이트를 부가한 VHH 유전자 단편을 파지미드벡터 (pKSTV03)에 삽입하였다. VHH 유전자가 삽입된 파지미드벡터는 파지 감염이 가능한 호스트인 E. coli TG-1 cell (Lucigen)에 형질전환 하였다. 상기 형질 전환된 E. coli TG-1의 적당량을 2YT-AG (2YT medium, 100 μg/ml ampicillin 2% glucose) 500 ml에 접종하고 배양 (37℃, 1.5 h) 후 M13KO7 헬퍼 파지 (Invitrogen, m.o.i = 10)에 감염시키고 16시간 배양 하였다. 배양한 배양액을 원심 분리하여 상층에 부유한 파지를 회수하기 위해 PEG (polyethylene glycol)/NaCl 침전법을 이용하여 파지를 침전시키고 회수하였다.The VHH gene amplified by PCR reaction was forward primer (SfiⅠ Restriction enzyme site addition, 5´-TGCTCCTCGCGGCCCAGCCGGCCATGGCTCAGGTGCAGCTCGTGGAGTCTGG-3´) (SEQ ID NO: 8) and reverse primer (SpeⅠ Restriction enzyme site added, 5′-ATGATGATGTGCACTAGTTTGTGGTTTTGGTGTCTTGGG-3′ (SEQ ID NO: 9) (IgG3), 5'-ATGATGATGTGCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 10) (IgG2) was used to perform PCR (20 cycles of 2 min at 95 ° C, 30 sec at 96 ° C, 30 sec at 58 ° C, 1 min at 72 ° C and 5 min at 72 ° C) to add a restriction enzyme site. A VHH gene fragment to which a restriction enzyme site was added was inserted into a phagemid vector (pKSTV03). The phagemid vector into which the VHH gene is inserted is a host capable of infecting phage.E. coli TG-1 cells (Lucigen) were transformed. the transformedE. coli An appropriate amount of TG-1 was inoculated into 500 ml of 2YT-AG (2YT medium, 100 μg/ml ampicillin 2% glucose), cultured (37°C, 1.5 h), infected with M13KO7 helper phage (Invitrogen, m.o.i = 10), and cultured for 16 hours. In order to collect the phage floating in the upper layer by centrifuging the culture medium, the phage were precipitated and recovered using a PEG (polyethylene glycol)/NaCl precipitation method.
실험예 3: 패닝(panning)에 의한 특이적인 항체들의 선별Experimental Example 3: Selection of specific antibodies by panning
상기에서 회수한 파지를 이용하여 패닝(panning) 실험을 진행하여 항원에 특이적으로 결합하는 파지를 증가시켰다. 구체적으로 4 μg의 항원을 2 ml의 PBS에 가한 다음 immunotube(Nunc)에 넣고 4℃에서 하룻밤 방치하였고, 다음날 PBS-T로 3회 세척하고 PBS로 희석한 3% skim milk를 사용하여 상온에서 2시간 동안 blocking하였다. 이후, PBS-T로 3회 세척하였다. 항원에 결합하는 파지를 선별하기 위해 파지가 포함된 용액 3 ml를 가하여 2시간 동안 반응시켰고, 이후 시험관 안의 용액을 버리고 PBS-T로 10회 세척 하였다. 항원에 결합한 파지를 용리하여 수거하기 위해 0.1 M glycine/HCl (pH 2) 1 ml을 넣고 상온에서 10분간 방치하여 파지를 용리시켰다. 이후, 2M Tris-base 50 μl를 넣어 중화시켰다. 이와 같은 패닝 (panning) 과정을 반복함으로써 항원에 특이적으로 결합하는 파지를 획득 및 증가시켰다.A panning experiment was conducted using the phages recovered above to increase the number of phages specifically binding to the antigen. Specifically, 4 μg of antigen was added to 2 ml of PBS, then placed in an immunotube (Nunc) and left overnight at 4 ° C. The next day, washed three times with PBS-T and blocked for 2 hours at room temperature using 3% skim milk diluted in PBS. Then, it was washed 3 times with PBS-T. In order to select phages that bind to the antigen, 3 ml of the phage-containing solution was added and allowed to react for 2 hours. Then, the solution in the test tube was discarded and washed 10 times with PBS-T. To elute and collect phages bound to the antigen, 1 ml of 0.1 M glycine/HCl (pH 2) was added and allowed to stand at room temperature for 10 minutes to elute the phages. Thereafter, 50 μl of 2M Tris-base was added to neutralize the mixture. By repeating the panning process, phages specifically binding to the antigen were obtained and increased.
도 4와 같이 알파카 항원 + 플라젤린 면역화를 통해 VHH 유전자 획득 후 패닝 기법을 이용하여 항원 특이적인 항체 선별 시, 대조군(도 3, 알파카 항원 단독 면역화)과 비교 시 패닝 라운드 2회에서 충분한 양성 파지를 획득할 수 있다.As shown in FIG. 4, when selecting antigen-specific antibodies using a panning technique after obtaining the VHH gene through immunization with alpaca antigen + flagellin, sufficient positive phage can be obtained in two rounds of panning compared to the control group (FIG. 3, immunization with alpaca antigen alone).
실험예 4: 양성 클론(clone)의 선별 및 확인Experimental Example 4: Selection and confirmation of positive clones
96-well plate의 well에 각각 200 μl의 2YT-AG 배지을 넣고, VHH 유전자가 포함된 E. coli TG-1 단일 콜로니를 멸균된 이쑤시게를 이용하여 각각의 well에 접종하고 배양 (37 ℃, 4 시간) 후 M13KO7 헬퍼 파지 (m.o.i = 5)를 넣어 감염시켰다. 헬퍼 파지 감염 후 대장균을 원심 분리하여 감염되지 않은 헬퍼 파지를 제거한 후 침전된 대장균을 2YT-AK 250 μl에 현탁하였다. 이후 37℃에서 16 시간 배양하였다. 배양액을 원심 분리하여 상층에 부유한 파지 용액을 회수하여 ELISA를 시행하였다.200 μl of 2YT-AG medium was added to each well of a 96-well plate, and a single colony of E. coli TG-1 containing the VHH gene was inoculated into each well using a sterilized toothpick. After incubation (37 ° C, 4 hours), M13KO7 helper phage (moi = 5) was added and infected. After infection with helper phages, E. coli was centrifuged to remove uninfected helper phages, and precipitated E. coli was suspended in 250 μl of 2YT-AK. Then, it was incubated for 16 hours at 37°C. The culture solution was centrifuged to recover the phage solution floating in the upper layer, and ELISA was performed.
항원에 결합하는 파지 클론을 확인하기 위해 96-well plate에 항원 100 ng을 4 ℃에서 하룻밤 코팅하였다. 항원을 코팅한 plate는 PBS-T로 3회 세척하였다. 이후 3% BSA로 상온에서 plate를 1시간 동안 blocking하였다. Blocking 이후 plate를 PBS-T로 3회 세척한 다음 파지 용액을 가하여 상온에서 1시간 동안 반응시켰다. Plate를 PBS-T로 3회 세척하고, 파지 특이적 항체를 Blocking를 사용하여 1 : 2000으로 희석한 다음, 상온에서 1시간 동안 반응시켰다. Plate를 PBS-T로 3회 세척하고, HRP가 붙은 항체를 1 : 5000으로 희석하여 상온에서 1시간 반응시킨 다음, 5회 세척 후, TMB 기질을 첨가한 후 stop solution을 각각 well에 첨가하였다. 흡광도 평판 판독기를 이용하여 450 nm의 흡광도를 측정하여 평가하였다.To identify phage clones binding to the antigen, 100 ng of the antigen was coated on a 96-well plate overnight at 4°C. The antigen-coated plate was washed three times with PBS-T. Thereafter, the plate was blocked for 1 hour at room temperature with 3% BSA. After blocking, the plate was washed three times with PBS-T, and then phage solution was added and reacted at room temperature for 1 hour. The plate was washed three times with PBS-T, phage-specific antibody was diluted 1:2000 using Blocking, and reacted at room temperature for 1 hour. The plate was washed three times with PBS-T, the HRP-attached antibody was diluted 1:5000 and reacted at room temperature for 1 hour. After washing five times, a TMB substrate was added and a stop solution was added to each well. The absorbance was evaluated by measuring the absorbance at 450 nm using a plate reader.
실험예 5: 각 항원에 대한 단일 도메인 VHH 항체 확인Experimental Example 5: Identification of single domain VHH antibodies for each antigen
실험예 4에서 선별된 비브리오 파라해모라이티쿠스의 외막에 존재하는 외막 단백질(V1) 26개 및 흰반점 바이러스(WSSV) 12개의 파지 클론 중 V1 1개 및 WSSV 1개의 파지 클론을 선택하여 양성 파지 클론의 VHH 유전자 서열을 BI 프리즘 3100 유전자 분석기 (Applied Biosystems0)를 이용하여 아미노산 염기서열을 분석하였다. V1 양성 파지 클론의 VHH 유전자에 대한 아미노산 염기서열은 서열번호 1에, WSSV 양성 파지 클론의 VHH 유전자에 대한 아미노산 염기서열은 서열번호 2에 나타내었다.Among the 26 outer membrane protein (V1) and 12 white spot virus (WSSV) phage clones present in the outer membrane of Vibrio parahaemolyticus selected in Experimental Example 4, one V1 and one WSSV phage clone was selected, and the VHH gene sequence of the positive phage clone was analyzed by BI Prism 3100 Genetic Analyzer (Applied Biosystems0) to analyze the amino acid sequence. The amino acid sequence of the VHH gene of the V1-positive phage clone is shown in SEQ ID NO: 1, and the amino acid sequence of the VHH gene of the WSSV-positive phage clone is shown in SEQ ID NO: 2.
실험예 6: 단일 도메인 VHH 항체의 특이성 확인Experimental Example 6: Confirmation of specificity of single domain VHH antibody
선발된 VHH 항체 각각의 비브리오 파라해모라이티쿠스 및 흰반점 바이러스에 대한 반응성을 확인하였다.The reactivity of each of the selected VHH antibodies against Vibrio parahaemolyticus and white spot virus was confirmed.
구체적으로, 먼저 특정 항원 (V1 혹은 VP28)을 coating buffer를 이용해서 희석한 뒤, 100 ng/well이 되도록 분주한 후 4℃에서 Overnight 한다. 그리고 다음 날 washing buffer (PBS/T(0.05% Tween-20))로 각 well을 3회 세척한 후, Blocking buffer (0.5% skim milk in PBS)를 200㎕/well 씩 각 웰에 분주한 다음 상온에서 1hr 반응시킨다. Washing buffer (PBS/T(0.05% Tween-20))로 각 well을 3회 세척하고 나서 dilution buffer (0.5% skim milk in PBS/T(0.05% Tween-20))로 측정하고자 하는 VHH 항체를 희석하여 well당 100㎕ 처리한 다음 상온에서 1hr 반응시켰다. 그 다음 3회 washing한 후에 Biotin anti-HA를 dilution buffer에 1:5,000으로 희석하여 100 ㎕/well 분주한 다음 상온에서 1hr 반응시켰다. 반응 종료 후에 3회 washing 후 Streptavidin HRP를 dilution buffer에 1:10,000으로 희석하여 100㎕/well 분주하고 상온에서 30min 반응시킨다. 최종적으로 3회 washing 후에 TMB solution을 100 ㎕/well 분주하여 발색 반응시킨다. 반응 완료 후에 stop solution (1.6N H2SO4)을 100 ㎕/well 분주하여 발색을 중단하고 Microplate reader를 이용하여 450nm에서 흡광도를 측정한다. 이 결과를 Graphpad prism 프로그램을 이용하여 그린 다음에 감염 최대값과 감염 최소값의 평균값인 EC50은 VHH 항체가 특정 항원 (V1 혹은 VP28)에 50% 결합하는 값을 결정하여 VHH 항체의 결합 정도를 파악하였으며, 그 결과를 도 5 및 도 6에 나타내었다.Specifically, first, after diluting a specific antigen (V1 or VP28) using a coating buffer, dispensing to 100 ng/well, and overnight at 4°C. The next day, after washing each well three times with washing buffer (PBS/T (0.05% Tween-20)), 200 μl/well of Blocking buffer (0.5% skim milk in PBS) was dispensed into each well, followed by reaction at room temperature for 1 hr. After washing each well three times with washing buffer (PBS/T (0.05% Tween-20)), the VHH antibody to be measured was diluted with dilution buffer (0.5% skim milk in PBS/T (0.05% Tween-20)), treated with 100 μl per well, and reacted at room temperature for 1 hr. Then, after washing three times, Biotin anti-HA was diluted 1:5,000 in dilution buffer, and 100 μl/well was dispensed, followed by reaction at room temperature for 1 hr. After completion of the reaction, after washing three times, Streptavidin HRP was diluted 1:10,000 in dilution buffer, dispensed at 100 μl/well, and reacted at room temperature for 30 min. Finally, after washing 3 times, 100 μl/well of TMB solution was dispensed for color development. After completion of the reaction, 100 μl/well of stop solution (1.6N H2SO4) is dispensed to stop color development, and absorbance is measured at 450 nm using a microplate reader. After drawing this result using the Graphpad prism program, the EC 50 , which is the average value of the maximum infection value and the minimum infection value, determines the value at which the VHH antibody binds to a specific antigen (V1 or VP28) by 50% to determine the degree of binding of the VHH antibody. The results are shown in FIGS. 5 and 6.
도 5에서 확인할 수 있듯이, 본 발명에 따른 V1 VHH 항체의 비브리오 파라해모라이티쿠스(V1)(도 5a)와 비브리오 하베이(Vb)(도 5b)에 대한 특이적 결합 EC50 값(effective concentration of drug that causes 50% of the maximum response)이 각각 0.975 ng/ml 및 1.720 ng/ml로, 비브리오 파라해모라이티쿠스 뿐만 아니라 비브리오 하베이에 대한 반응성도 우수하였다.As can be seen in FIG. 5, the specific binding EC 50 values (effective concentration of drug that causes 50% of the maximum response) of the V1 VHH antibody according to the present invention to Vibrio parahaemolyticus (V1) (FIG. 5a) and Vibrio harvey (Vb) (FIG. 5b) were 0.975 ng/ml and 1.720 ng/ml, respectively, as well as Vibrio parahaemolyticus. Reactivity to bay was also excellent.
또한, 도 6에서 확인할 수 있듯이, 본 발명에 따른 WSSV VHH 항체의 흰반점 바이러스의 VP28 항원에 대한 특이적 결합 EC50값(effective concentration of drug that causes 50% of the maximum response)이 2.055 μg/ml로, 흰반점 바이러스에 대한 반응성이 우수하였다.In addition, as can be seen in FIG. 6, the specific binding EC 50 value (effective concentration of drug that causes 50% of the maximum response) of the WSSV VHH antibody according to the present invention to the VP28 antigen of the white spot virus was 2.055 μg/ml, showing excellent reactivity to the white spot virus.
실험예 7: 새우 조기 폐사 증후군에 대한 억제 효과 확인Experimental Example 7: Confirmation of inhibitory effect on shrimp premature mortality syndrome
본 발명에 따라 제조된 VHH 항체의 새우 조기 폐사 증후군 유발균 또는 바이러스의 억제 효과를 확인하였다.The inhibitory effect of the VHH antibody prepared according to the present invention against bacteria or viruses causing premature shrimp death syndrome was confirmed.
새우 조기 폐사 증후군 유발균 또는 바이러스의 억제 효과인 IC50 는 다음과 같이 측정한다. 구체적으로 폐사 증후군 유발균인 V1을 각각 TSB agar plate 균주도말 한 후 24시간 동안 28℃ incubation 배양 한다. 배양 후 단일 Colony를 취하여 TSB broth 배지에 접종하여 24시간 동안 28℃, Shaking incubation 배양 한다. 균수 농도 약 1 X 109 cfu /mL의 전종 균액을 준비 한다. 희석액 (배지)으로 10,000배 희석하여 약 1 X 105 cfu/mL 의 전종 균액을 준비 한다. 준비된 균액을 각 Well당 100 ㎕/㎖ 씩 분주 한다(최종 접종 균수는 5 X 104 cfu/mL). 각 Well 마다 VHH 항체 샘플을 넣어 최종 200㎕에 들어가도록 한 다음에 세균 생장율을 측정한다. 28℃ incubation, 150 rpm 24시간 배양하고 나서 ELISA Reader (OD 600nm)로 값을 측정한다. 생장율을 세균 접종 직후와 24시간 배양 후의 OD 600nm 참고하여 Graphpad prism 프로그램을 이용하여 그래프을 그린 다음에 세균 생장을 50% 억제하는 농도에서 IC50 (IC50, inhibitory concentration of drug that causes 50% of the maxium inhibition) 값을 결정한다. 결과를 하기 표 3 및 도 7에 나타내었다.IC 50 , which is the inhibitory effect of bacteria or viruses causing premature shrimp death syndrome, is measured as follows. Specifically, V1, the cause of death syndrome, was smeared on each TSB agar plate, and then incubated at 28 ° C for 24 hours. After culturing, take a single colony, inoculate it into TSB broth medium, and incubate at 28℃ for 24 hours by Shaking incubation. Prepare all strains with a bacterial count of about 1 X 10 9 cfu/mL. Dilute 10,000 times with diluent (medium) to prepare about 1 X 10 5 cfu/mL of all strains. 100 μl/ml of the prepared bacterial solution is dispensed per well (the final number of inoculated bacteria is 5 X 10 4 cfu/mL). Put a VHH antibody sample into each well so that it enters the final 200 μl, and then measure the bacterial growth rate. After incubation at 28℃, 150 rpm for 24 hours, the values are measured with an ELISA Reader (OD 600nm). After drawing a graph using the Graphpad prism program by referring to the OD 600nm immediately after bacterial inoculation and after 24 hours of incubation for the growth rate, determine the IC 50 (IC 50 , inhibitory concentration of drug that causes 50% of the maxium inhibition) value at a concentration that inhibits bacterial growth by 50%. The results are shown in Table 3 and FIG. 7 below.
농도density
(μg/ml)(μg/ml)
(균생장율)(bacterial growth rate)
표 3 및 도 7에서 확인할 수 있듯이, 비브리오 파라해모라이티쿠스(V1) 균의 생장을 1/2 정도 저해하는 농도 (IC50, inhibitory concentration of drug that causes 50% of the maxium inhibition)는 V1 VHH 항체 농도 2.5 μg/ml에서 비브리오 파라해모라이티쿠스(V1) 균의 생장을 50% 억제하였다.As can be seen in Table 3 and Figure 7, the concentration that inhibits the growth of Vibrio parahaemolyticus (V1) by about half (IC 50 , inhibitory concentration of drug that causes 50% of the maxium inhibition) is the V1 VHH antibody concentration of 2.5 μg / ml. The growth of Vibrio parahaemolyticus (V1) was inhibited by 50%.
또한, VP28 VHH 항체의 WSSV 바이러스의 감염 억제 능력은 WSSV의 메인 세포막 단백질인 VP28 expressing pseudo-virus를 감염시킨 동물세포에서 VP28 VHH를 농도별로 희석하여 첨가한 후에 VP28 expressing pseudo-virus가 동물세포에 감염한 정도를 GFP 형광 세포 수로 파악하고, pseudo-virus의 감염을 1/2 정도 저해하는 농도를 IC50 (IC50, inhibitory concentration of drug that causes 50% of the maxium inhibition) 농도라고 칭한다. 결과 값을 Graphpad prism 프로그램을 이용하여 그래프를 그린 다음에 바이러스 감염을 50% 억제하는 농도에서 IC50 값을 결정한다.In addition, the ability of the VP28 VHH antibody to inhibit WSSV virus infection was determined by adding VP28 VHH diluted by concentration to animal cells infected with VP28 expressing pseudo-virus, the main cell membrane protein of WSSV, and the degree of infection of animal cells by the VP28 expressing pseudo-virus was determined by the number of GFP fluorescent cells, and the concentration that inhibited pseudo-virus infection by half was IC 50 (IC 50 , inhibitory concentration of drug that causes 50% of The maxium inhibition is called the concentration. The resulting values are graphed using the Graphpad prism program, and then the IC 50 value is determined at a concentration that inhibits viral infection by 50%.
도 8 및 도 9에서 확인할 수 있듯이, WSSV (White Spot Syndrome Virus)의 감염 정도를 VP28 expressing pseudo-virus system으로 분석하였더니, 감염을 1/2 정도 저해하는 농도 (IC50, inhibitory concentration of drug that causes 50% of the maxium inhibition)는 WSSV VHH 항체 농도 약 10.3 μg/ml에서 VP28 expressing pseudo-virus 감염을 50% 억제하였다.As can be seen in Figures 8 and 9, the degree of infection of WSSV (White Spot Syndrome Virus) was analyzed by the VP28 expressing pseudo-virus system, and the concentration that inhibits infection by about half (IC 50 , inhibitory concentration of drug that causes 50% of the maxium inhibition) inhibited VP28 expressing pseudo-virus infection by 50% at a WSSV VHH antibody concentration of about 10.3 μg / ml.
<110> ADbiotech Co., Ltd. <120> MANUFACTURING METHOD OF SINGLE DOMAIN VHH ANTIBODY FOR PREVENTING OR TREATING EARLY MORTALITY SYNDROME <130> P20-0450 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> V1_L5_IgG3_long hinge type <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Val Ile Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met His Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys Ser 85 90 95 Leu Gly Ser Pro Pro Thr Asp Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln 115 120 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> WSSV_S95_IgG2_short hinge type <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Val Phe Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Lys Thr Leu Trp Gly Ala Asn Phe Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser Ala His His Ser Glu Asp Pro 115 120 <210> 3 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> V1_L5_IgG3 <400> 3 caggtgcagc tcgtggagtc tgggggaggc ttggcgcagc ctggggggtc tctgagactc 60 tcctgtgcag tatctggaag agtcatcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtacaac aagctatgca 180 gactccgtga agggccgttt caccgtctcc agagacaaca ccaagaacac ggtgtatctg 240 caaatgcaca gcctgaaacc tgaggattcg gccgtctatt actgtagcct tggatcgccc 300 ccaaccgact ggggccaggg gacccaggtc accgtctcct cagaacccaa gacaccaaaa 360 ccacaa 366 <210> 4 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> WSSV_S95_IgG2 <400> 4 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggaag agtcttcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtactac gaactatgca 180 gactccgtga agggccgatt caccatctcc agaaacaacg ccaagaacac ggtgtatcta 240 caaatgaaca gcctgaaacc tgacgacacg gccgtctatt actgcaacgt gaaaaccctc 300 tggggcgcta acttctgggg ccagggaacc caggtcaccg tctcctcggc gcaccacagc 360 gaagacccc 369 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IgG2, IgG3 Forward primer <400> 5 agktgcagct cgtggagtcn ggngg 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IgG2 Reverse primer <400> 6 ggggtcttcg ctgtggtgcg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IgG3 Reverse primer <400> 7 ttgtggtttt ggtgtcttgg g 21 <210> 8 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> VHH forward primer <400> 8 tgctcctcgc ggcccagccg gccatggctc aggtgcagct cgtggagtct gg 52 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> VHH Reverse primer(IgG3) <400> 9 atgatgatgt gcactagttt gtggttttgg tgtcttggg 39 <210> 10 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VHH Reverse primer(IgG2) <400> 10 atgatgatgt gcactagtgg ggtcttcgct gtggtgcg 38 <110> ADbiotech Co., Ltd. <120> MANUFACTURING METHOD OF SINGLE DOMAIN VHH ANTIBODY FOR PREVENTING OR TREATING EARLY MORTALITY SYNDROME <130> P20-0450 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 122 <212> PRT <213> artificial sequence <220> <223> V1_L5_IgG3_long hinge type <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Val Ile Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met His Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys Ser 85 90 95 Leu Gly Ser Pro Pro Thr Asp Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln 115 120 <210> 2 <211> 123 <212> PRT <213> artificial sequence <220> <223> WSSV_S95_IgG2_short hinge type <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Val Phe Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Lys Thr Leu Trp Gly Ala Asn Phe Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser Ala His His Ser Glu Asp Pro 115 120 <210> 3 <211> 366 <212> DNA <213> artificial sequence <220> <223> V1_L5_IgG3 <400> 3 caggtgcagc tcgtggagtc tgggggaggc ttggcgcagc ctggggggtc tctgagactc 60 tcctgtgcag tatctggaag agtcatcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtacaac aagctatgca 180 gactccgtga agggccgttt caccgtctcc agagacaaca ccaagaacac ggtgtatctg 240 caaatgcaca gcctgaaacc tgaggattcg gccgtctatt actgtagcct tggatcgccc 300 ccaaccgact ggggccaggg gacccaggtc accgtctcct cagaacccaa gacaccaaaa 360 ccacaa 366 <210> 4 <211> 369 <212> DNA <213> artificial sequence <220> <223> WSSV_S95_IgG2 <400> 4 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggaag agtcttcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtactac gaactatgca 180 gactccgtga agggccgatt caccatctcc agaaacaacg ccaagaacac ggtgtatcta 240 caaatgaaca gcctgaaacc tgacgacacg gccgtctatt actgcaacgt gaaaaccctc 300 tggggcgcta acttctgggg ccagggaacc caggtcaccg tctcctcggc gcaccacagc 360 gaagacccc 369 <210> 5 <211> 25 <212> DNA <213> artificial sequence <220> <223> IgG2, IgG3 Forward primer <400> 5 agktgcagct cgtggagtcn ggngg 25 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> IgG2 reverse primer <400> 6 ggggtcttcg ctgtggtgcg 20 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> IgG3 reverse primer <400> 7 ttgtggtttt ggtgtcttgg g 21 <210> 8 <211> 52 <212> DNA <213> artificial sequence <220> <223> VHH forward primer <400> 8 tgctcctcgc ggcccagccg gccatggctc aggtgcagct cgtggagtct gg 52 <210> 9 <211> 39 <212> DNA <213> artificial sequence <220> <223> VHH reverse primer (IgG3) <400> 9 atgatgatgt gcactagttt gtggttttgg tgtcttggg 39 <210> 10 <211> 38 <212> DNA <213> artificial sequence <220> <223> VHH reverse primer (IgG2) <400> 10 atgatgatgt gcactagtgg ggtcttcgct gtggtgcg 38
Claims (14)
새우 조기 폐사 증후군의 원인균의 외막 단백질 또는 바이러스 항원과 플라젤린(flagellin)의 혼합물을 낙타과 동물에 주입하여 면역화 시키는 단계를 포함하는 것을 특징으로 하는 단일 도메인 VHH 항체의 제조 방법.
A method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome,
A method for producing a single domain VHH antibody comprising the step of injecting a mixture of an outer membrane protein or viral antigen and flagellin of a causative bacterium of shrimp premature death syndrome into a camelid animal to immunize it.
상기 새우 조기 폐사 증후군의 원인균은 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)로 이루어진 군에서 선택되는 하나 이상의 균이고, 바이러스는 흰반점 바이러스(white spot syndrome virus, WSSV)인 것인, 단일 도메인 VHH 항체의 제조방법.
According to claim 1,
The causative agent of the shrimp premature death syndrome is one or more bacteria selected from the group consisting of Vibrio parahaemolyticus , Vibrio harveyi and Vibrio anguillarum , and the virus is a white spot virus (white spot syndrome virus, WSSV).
상기 플라젤린은 살모넬라균(Salmonella typhimurium), 비브리오 패혈증균(Vibrio vulnficus) 및 리스테리아균(Listeria monocytogenes)으로 이루어진 군에서 선택된 세균에서 유래된 것인, 단일 도메인 VHH 항체의 제조방법.
According to claim 1,
The flagellin is derived from a bacterium selected from the group consisting of Salmonella typhimurium , Vibrio vulnficus and Listeria monocytogenes .
상기 외막 단백질 또는 항원과 플라젤린의 혼합 비율은 5~20 : 1인 것인, 단일 도메인 VHH 항체의 제조방법.
According to claim 1,
The method of producing a single domain VHH antibody, wherein the mixing ratio of the outer membrane protein or antigen and flagellin is 5 to 20: 1.
A single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 1 prepared by the method of claim 1.
A nucleic acid molecule represented by SEQ ID NO: 3 encoding the single domain VHH antibody according to claim 5.
A recombinant vector comprising the nucleic acid molecule according to claim 6 .
A host cell transformed with the recombinant vector of claim 7.
A single domain VHH antibody for preventing or treating shrimp premature mortality syndrome represented by the amino acid sequence of SEQ ID NO: 2 prepared by the method of claim 1.
A nucleic acid molecule represented by SEQ ID NO: 4 encoding the single domain VHH antibody according to claim 9.
A recombinant vector comprising the nucleic acid molecule according to claim 10 .
A host cell transformed with the recombinant vector of claim 11.
A pharmaceutical composition for preventing or treating premature shrimp mortality syndrome comprising at least one of the single domain VHH antibodies according to claims 5 and 9 as an active ingredient.
A feed additive composition for the prevention or treatment of premature shrimp mortality syndrome comprising at least one of the single domain VHH antibodies according to claims 5 and 9 as an active ingredient.
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PCT/KR2022/001099 WO2023140401A1 (en) | 2022-01-19 | 2022-01-21 | Method for producing single domain vhh antibody for prevention or treatment of early mortality syndrome in shrimp |
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