KR102049976B1 - Antibody for shrimp’s early mortality syndrome and white spot syndrome virus, and applications thereof - Google Patents
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Abstract
Description
본 발명은 새우 조기 폐사 증후군 및 흰반점 바이러스에 대한 항체 및 이의 응용에 관한 것으로, 더욱 상세하게는 산란계에 새우 조기 폐사 증후군과 흰반점 바이러스에 대한 항원을 접종하고, 산란계가 낳은 알의 난황에서 분리한 항체와 이의 응용 기술에 관한 것이다.The present invention relates to antibodies against shrimp early mortality syndrome and white spot virus and its application, and more particularly, to inoculate antigens against shrimp early mortality syndrome and white spot virus, and to isolate the eggs from egg yolks laid. It relates to an antibody and its application technology.
최근 전세계적으로 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, 양식어류의 질병발생은 경제적 발전에 커다란 영향을 미치고 있는 실정이다.Recently, as the interest in improving the diet for the prevention of geriatric diseases has increased rapidly around the world, the demand for seafood is continuously increasing and the production of fish farming is increasing rapidly. Therefore, aquaculture has become a major economic tool in many countries, and disease outbreaks in farmed fish have a great impact on economic development.
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~72시간 이내에 폐사하게 된다. Among the farmed fish, vibrio and white spot viruses are known as the main causes of disease in shrimp. Vibrio genus is a Gram-negative bacillus, with one or more hair follicles on one end and two or more hairs depending on the species, and is a representative pathogen. Vibrio cholera ( Vibrio genus) causes cholera. colera ) The causes of shrimp Vibrio illness called Vibrio para itty Syracuse to Mora (Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi ) and Vibrio anguillarum , which are killed within 24 to 72 hours when shrimp are infected with these bacteria.
흰반점 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. White spot syndrome virus (WSSV) has a tail-like attachment and has rod-shaped capsids and envelopes. When shrimp are infected with the white spot virus, white spots appear on the carapace, appendage and cuticle of the shrimp, and the hepatopancreas becomes red, and 3 to 5 days after infection It will die.
따라서 새우의 질병을 예방 및 치료하기 위한 방안 중 하나로 난황항체(IgY)가 떠오르고 있다. 난황항체는 조류의 면역체계를 이용한 수동면역의 한 형태로, 어미 닭이 능동면역으로 획득한 면역항체는 난황으로 이행되어 자손에게 면역능이 전해진다. 난황황체는 이미 여러 연구에 의해 질병의 예방 및 치료효과가 증명되어 왔으며, 고농도로 사용하는 경우, 항생제와 거의 같은 수준의 치료효과를 기대할 수 있는 것으로 보고되어 있다. 또한, 완전식품인 계란에서 유래하므로 안전성에 대한 염려가 전혀 없는 매우 유용한 물질이라 할 수 있다.Therefore, yolk antibody (IgY) is emerging as one of the measures to prevent and treat shrimp diseases. Egg yolk antibodies are a form of passive immunization using the bird's immune system. The immune antibodies obtained by active chickens from mother chickens are transferred to egg yolk and the immune function is transmitted to the offspring. Egg yolk has already been proved by various studies to prevent and cure the disease, and when used in high concentrations, it is reported that it can be expected to have almost the same therapeutic effect as antibiotics. In addition, since it is derived from eggs, which are complete foods, it can be said to be a very useful substance without any safety concerns.
본 발명의 목적은, 새우 조기 폐사 증후군과 흰반점 증후군에 대한 항원을 산란계에 접종하여 면역화시키고, 면역화된 산란계로부터 얻은 알의 난황에서 분리한 항체와 이를 이용한 새우 폐사방지용 조성물, 사료 또는 사료첨가제, 질병 예방방법 및 새우 사육방법을 제공하는 것이다.An object of the present invention is to immunize the laying hens with antigens against shrimp early mortality syndrome and white spot syndrome, and the antibody isolated from egg yolk of the egg obtained from the immunized laying hens and the composition for preventing shrimp mortality using the same, feed or feed additives, To provide disease prevention and shrimp breeding methods.
본 발명은, (a) 새우 조기 폐사 증후군의 원인균인 비브리오 균(Vibrio genus) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종하여 산란계를 면역화시키는 단계; (b) 면역화된 산란계로부터 알을 산란시키는 단계; 및 (c) 산란된 알의 난황으로부터 항체를 분리하는 단계를 포함하는, 새우 폐사방지용 항체 제조방법을 제공한다.The present invention comprises: (a) immunizing a laying hen by inoculating an laying hen with an antigen consisting of Vibrio genus and white spot syndrome virus (WSSV) recombinant protein, which are the causative agents of early shrimp mortality syndrome; (b) laying eggs from the immunized laying hens; And (c) separating the antibody from the egg yolk of the laid eggs, providing an antibody for preventing shrimp mortality.
또한 본 발명은, (a) 새우 조기 폐사 증후군의 원인균인 비브리오 균(Vibrio genus), 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질 및 비브리오 균 외막단백질의 항원결정기 부위의 재조합 단백질로 이루어진 항원을 산란계에 접종하여 산란계를 면역화시키는 단계; (b) 면역화 된 산란계로부터 알을 산란시키는 단계; 및 (c) 산란된 알의 난황으로부터 항체를 분리하는 단계를 포함하는, 새우 폐사방지용 항체 제조방법을 제공한다.In another aspect, the present invention, (a) shrimp early pathogen of Vibrio of mortality syndrome bacteria (Vibrio genus), white spot virus (white spot syndrome virus, WSSV) recombinant proteins and vibrio antigen consisting of the recombinant proteins of the antigenic determinant sites of bacterial outer membrane protein Immunizing the laying hens by inoculating the laying hens; (b) laying eggs from the immunized laying hens; And (c) separating the antibody from the egg yolk of the laid eggs, providing an antibody for preventing shrimp mortality.
상기 비브리오 균은 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)으로 이루어질 수 있다.The Vibrio bacterium is Vibrio parahamamoraiticus ( Vibrio parahaemolyticus ), Vibrio harveyi and Vibrio anguillarum .
상기 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)은 0.5~1.5:0.5~1.5:0.3~1.0의 비로 혼합될 수 있고, 바람직하게는 0.7~1.3:0.7~1.3:0.4~0.7의 비로 혼합될 수 있으며, 더 바람직하게는 1.0:1.0:0.5의 비로 혼합될 수 있다.Vibrio parahaemoriticus ( Vibrio parahaemolyticus ), Vibrio harveyi ) and Vibrio anguillarum may be mixed in a ratio of 0.5-1.5: 0.5-1.5: 0.3-1.0, preferably in a ratio of 0.7-1.3: 0.7-1.3: 0.4-0.7, More preferably 1.0: 1.0: 0.5.
상기 흰반점 바이러스 재조합 단백질은 VP28일 수 있다.The white spot virus recombinant protein may be VP28.
상기 비브리오 균 외막단백질의 항원결정기 부위의 재조합 단백질은 OmpW(비브리오 파라해모라이티쿠스 외막단백질(Outer Membrane Protein) W) 및 OmpK(비브리오 하베이 외막단백질(Outer Membrane Protein) K)일 수 있다.Recombinant proteins of the epitope region of the Vibrio bacteria outer membrane protein may be OmpW (Vibrio para-Haemolyticus outer membrane protein (W) and OmpK (Vibrio Havey outer membrane protein (Outer Membrane Protein K)).
본 발명에서, OmpW 및 OmpK는 각각 V1W 및 VbK로 표현할 수 있다.In the present invention, OmpW and OmpK can be expressed as V1W and VbK, respectively.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 새우 폐사방지용 항체를 제공한다. In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided are antigens consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein inoculated into a laying hen, and there is provided an antibody for preventing shrimp mortality isolated from the egg yolk of the laying hen.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 항체를 포함하는 새우 조기 폐사 증후군 예방 또는 치료용 조성물을 제공한다.In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided is a composition for preventing or treating shrimp early mortality syndrome comprising inoculating an antigen consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein into a laying hen, and then separating the egg from the egg yolk of the laying hen. do.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 항체를 포함하는 새우 흰반점 증후군 예방 또는 치료용 조성물을 제공한다.In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided is a composition for the prevention or treatment of shrimp white spot syndrome, comprising an antibody isolated from egg yolk of eggs laid by the laying hen after inoculating an antigen consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein into the laying hen. do.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 항체를 포함하는 새우 사료 또는 사료 첨가제를 제공한다.In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio An antigen consisting of anguillarum ) and a white spot syndrome virus (WSSV) recombinant protein is inoculated into a laying hen, and there is provided a shrimp feed or feed additive comprising an antibody isolated from egg yolk of the laying hen.
상기 새우 사료 또는 사료 첨가제는, 새우 조기 폐사 증후군 및 새우 흰반점 증후군 예방 또는 치료용으로 사용할 수 있다.The shrimp feed or feed additive can be used for preventing or treating shrimp early mortality syndrome and shrimp white spot syndrome.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 항체를 포함하는 새우 조기 폐사 증후군 예방 또는 치료용 조성물을 새우에 처리하는 새우의 질병 예방 방법을 제공한다.In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio After inoculating an egg consisting of anguillarum ) and a white spot syndrome virus (WSSV) recombinant protein into a laying hen, a composition for preventing or treating early mortality syndrome comprising a shrimp isolated from the egg yolk of the laying hen Provides a method for preventing shrimp disease in processing.
또한 본 발명은, 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi), 비브리오 앵길라룸(Vibrio anguillarum) 및 흰반점 바이러스(white spot syndrome virus, WSSV) 재조합 단백질로 이루어진 항원을 산란계에 접종한 후, 산란계가 낳은 알의 난황으로부터 분리한 항체를 포함하는 새우 사료 또는 사료 첨가제를 새우에 급여하는 새우의 사육방법을 제공한다.In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Shrimp which inoculate an egg consisting of anguillarum and white spot syndrome virus (WSSV) recombinant protein into a laying hen, and then feed it with a shrimp feed or feed additive containing an antibody isolated from the egg yolk of the laying hen. It provides a breeding method of.
본 발명에 따르면 산란계에 새우 조기 폐사 증후군 및 흰반점 증후군에 대한 항원을 접종하여 면역화시키고, 면역화된 산란계로부터 얻은 알의 난황에서 항체를 분리함으로써 새우 폐사방지용 조성물, 사료 또는 사료첨가제로 유용하게 사용할 수 있다. According to the present invention, by inoculating immunizing eggs with antigens against shrimp early mortality syndrome and white spot syndrome, and separating the antibody from egg yolk of eggs obtained from the immunized laying hens, it can be usefully used as a composition for preventing shrimp mortality, feed or feed additives. have.
또한, 본 발명에 따른 항체는 새우 조기 폐사 증후군의 원인균인 비브리오 균과 흰반점 바이러스 항원의 구성이 시너지(상승) 효과를 일으켜 비브리오 균이나 흰반점 바이러스를 단독으로 사용한 항체보다 우수한 항체 역가를 보이고, 이에 따라 새우 조기 폐사 증후군 및 흰반점 증후군에 대한 예방 또는 치료효과가 우수하며, 산란계로부터 항체를 생산하기 때문에 안정성이 뛰어나고, 항체를 비교적 쉽게 생산할 수 있다는 특징이 있다.In addition, the antibody according to the present invention exhibits a synergistic (synergistic) effect of the composition of Vibrio bacteria and white spot virus antigens, which causes shrimp early mortality syndrome, and shows better antibody titers than antibodies using Vibrio or white spot virus alone. Accordingly, it is excellent in preventing or treating shrimp early mortality syndrome and white spot syndrome, and is excellent in stability because the antibody is produced from the laying hen, and the antibody can be produced relatively easily.
도 1은 흰반점 증후군 바이러스(WSSV)의 재조합 단백질 VP28의 발현을 SDS-PAGE 및 웨스턴 블로팅을 통해 확인한 결과로, A : SDS-PAGE 결과, B : 웨스턴 블로팅 결과, Lane 1 : IPTG 유도(induction) 전, Lane 2 : IPTG 유도 후, M : 단백질 마커(marker)이다.
도 2는 pET-32a(+) OmpW 플라스미드의 모식도이다.
도 3은 pET-32a(+) OmpK 플라스미드의 모식도이다.
도 4는 비브리오 파라해모라이티쿠스 외막단백질의 항원결정기 부위의 재조합 단백질 OmpW(V1W)의 발현을 SDS-PAGE 및 웨스턴 블로팅을 통해 확인한 결과로, A : SDS-PAGE 결과, B : 웨스턴 블로팅 결과, Lane 1 : pET-32a(+) 오리지널 벡터_BL21(DE3)을 IPTG로 유도(induction) 전, Lane 2 : pET-32a(+) 오리지널 벡터_BL21(DE3)을 1.0 mM IPTG로 유도 후, Lane 3 : OmpW + pET-32a(+) 벡터_BL21(DE3)을 IPTG로 유도 전, Lane 4 : OmpW + pET-32a(+) 벡터_OmpW_BL21(DE3)을 1.0 mM IPTG로 유도 후, M : 단백질 마커(marker)이다.
도 5는 비브리오 하베이 외막단백질의 항원결정기 부위의 재조합 단백질 OmpK(VbK)의 발현을 SDS-PAGE 및 웨스턴 블로팅을 통해 확인한 결과로, A : SDS-PAGE 결과, B : 웨스턴 블로팅 결과, Lane 1 : pET-32a(+) 벡터_BL21(DE3)을 1.0 mM IPTG로 유도전, Lane 2 : pET-32a(+) 벡터_OmpW_BL21(DE3)을 1.0 mM IPTG로 유도후, M : 단백질 마커(marker)이다.
도 6은 각 그룹별 백신 접종 후 V1(비브리오 파라해모라이티쿠스)에 대해 생성되는 난황항체 역가를 확인한 결과이다. 가로축은 접종주차(예: 1-1은 1차 1주), 세로축은 난황항체 역가를 나타낸다.
도 7은 각 그룹별 백신 접종 후 Vb(비브리오 하베이)에 대해 생성되는 난황항체 역가를 확인한 결과이다.
도 8은 각 그룹별 백신 접종 후 Va(비브리오 앵길라룸)에 대해 생성되는 난황항체 역가를 확인한 결과이다.
도 9는 각 그룹별 백신 접종 후 WSSV VP28(흰반점 증후군 바이러스 재조합 단백질)에 대해 생성되는 난황항체 역가를 확인한 결과이다.
도 10은 IgY를 SDS-PAGE 및 웨스턴 블로팅을 통해 확인한 결과이다.
도 11은 항원에 대한 결합 친화성을 SDS-PAGE 및 웨스턴 블로팅을 통해 확인한 결과로, 1: 비브리오 앵길라룸(Vibrio anguillarum), 2: 비브리오 하베이(Vibrio harveyi), 3: 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 4: WSSV VP28, M: 단백질 마커이다.
도 12는 각 그룹별 백신 접종 후 V1(비브리오 파라해모라이티쿠스)에 대한 성장억제 효과를 확인한 결과이다. 가로축은 접종 후 시간, 세로축은 O.D 값을 나타낸다.
도 13은 각 그룹별 백신 접종 후 Vb(비브리오 하베이)에 대한 성장억제 효과를 확인한 결과이다.
도 14는 각 그룹별 백신 접종 후 Va(비브리오 앵길라룸)에 대한 성장억제 효과를 확인한 결과이다. 1 is a result of confirming the expression of recombinant protein VP28 of white spot syndrome virus (WSSV) through SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting results, Lane 1: IPTG induction ( before induction, Lane 2: after IPTG induction, M: protein marker.
2 is a schematic diagram of the pET-32a (+) OmpW plasmid.
3 is a schematic diagram of the pET-32a (+) OmpK plasmid.
Figure 4 shows the expression of recombinant protein OmpW (V1W) of the epitope region of the Vibrio para-Haemolyticus outer membrane protein by SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting Results: Lane 1: before induction of pET-32a (+) original vector_BL21 (DE3) with IPTG, Lane 2: after induction of pET-32a (+) original vector_BL21 (DE3) with 1.0 mM IPTG Lane 3: Before inducing OmpW + pET-32a (+) vector_BL21 (DE3) to IPTG, Lane 4: Inducing OmpW + pET-32a (+) vector_OmpW_BL21 (DE3) to 1.0 mM IPTG, M : Protein marker.
Figure 5 shows the expression of recombinant protein OmpK (VbK) of the epitope region of the Vibrio Habei outer membrane protein by SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting results, Lane 1 : Before inducing pET-32a (+) vector_BL21 (DE3) with 1.0 mM IPTG, Lane 2: After inducing pET-32a (+) vector_OmpW_BL21 (DE3) with 1.0 mM IPTG, M: Protein marker )to be.
Figure 6 shows the results of confirming the yolk antibody titers generated for V1 (Vibrio parahaemoriaticus) after vaccination of each group. The horizontal axis shows the inoculation week (eg 1-1 is the first week), and the vertical axis shows the yolk antibody titer.
Figure 7 shows the results of confirming the yolk antibody titer generated for Vb (Vibrio Habei) after vaccination of each group.
Figure 8 is the result of confirming the yolk antibody titers generated for Va (Vibrio Anguilla Room) after vaccination of each group.
Figure 9 shows the result of confirming the yolk antibody titer generated for WSSV VP28 (white spot syndrome virus recombinant protein) after vaccination of each group.
10 shows the results of confirming IgY through SDS-PAGE and western blotting.
Figure 11 shows the binding affinity to the antigen through SDS-PAGE and Western blotting, 1: Vibrio anguillarum, 2: Vibrio harveyi, 3: Vibrio parahaemoriti Vibrio parahaemolyticus, 4: WSSV VP28, M: protein marker.
12 is a result of confirming the growth inhibitory effect on V1 (Vibrio parahaemoriaticus) after vaccination of each group. The horizontal axis represents time after inoculation and the vertical axis represents OD value.
Figure 13 shows the results of confirming the growth inhibitory effect on Vb (Vibrio Habei) after each group vaccination.
Figure 14 shows the results of confirming the growth inhibitory effect on Va (Vibrio Anguilla Room) after vaccination of each group.
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 본 발명의 목적, 특징, 장점은 이하의 실시예를 통하여 쉽게 이해될 것이다. 본 발명은 여기서 설명하는 실시예에 한정되지 않고, 다른 형태로 구체화될 수도 있다. 여기서 소개되는 실시예는 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다. 따라서 이하의 실시예에 의해 본 발명이 제한되어서는 안 된다.Hereinafter, the present invention will be described in more detail with reference to Examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to sufficiently convey the spirit of the present invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.
실험예 1: 새우 폐사방지용 항체 제조Experimental Example 1: Preparation of Anti-Shrimp Antibodies
1-1. 1-1. 조기폐사증후군Early mortality syndrome (early mortality syndrome, EMS) 항원 제조(early mortality syndrome, EMS) antigen production
항원 준비를 위하여 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus) KCTC 2471, 비브리오 하베이(Vibrio harveyi) KCTC 12724 및 비브리오 앵길라룸(Vibrio anguillarum) KCTC 2711BP를 분양받아, 3% NaCl이 첨가된 트립틱 소이 배지(tryptic soy broth, TSB)를 사용, 37℃에서 120 rpm으로 진탕 배양기(shaking incubator)에서 20시간동안 배양하였다. 배양된 배양액은 6,000 rpm에서 원심분리(10 min, 4℃)후 펠렛(pellet)을 수거하여 PBS로 2회 세척하고, 부유액 50 ㎖를 만들었다. 분광광도계(spectrophotometer)를 사용하여 O.D 600 nm에서 흡광도 값이 0.3이 되도록 맞추어 고정시켰다. 총 부피 대비 37% 포름알데히드(formaldehyde)를 넣고, 24시간동안 실온에서 불활화하여 면역을 위한 항원으로 사용하였다.Vibrio para to the antigen preparation to Mora ET kusu (Vibrio parahaemolyticus) KCTC 2471, Harvey V. (Vibrio harveyi) KCTC 12724 and Vibrio Anguilla Anguilla room (Vibrio anguillarum ) KCTC 2711BP was distributed and incubated for 20 hours in a shaking incubator at 37 ° C. at 120 rpm using tryptic soy broth (TSB) added with 3% NaCl. The culture broth was centrifuged at 6,000 rpm (10 min, 4 ° C), pellets were collected, washed twice with PBS, and 50 ml of suspension was prepared. A spectrophotometer was used to fix the absorbance value at 0.3 at OD 600 nm. 37% formaldehyde (formaldehyde) relative to the total volume was added and inactivated at room temperature for 24 hours to use as an antigen for immunity.
1-2. 흰반점 바이러스 재조합 단백질 제조1-2. White spot virus recombinant protein production
흰반점 바이러스의 재조합 단백질인 VP28을 항원으로 사용하기 위하여 LB(lysogeny broth) 배지를 제조하고 멸균한 뒤 암피실린(ampicillin)을 50 ug/ml의 농도로 첨가하여 준비하였다. 하루 전, LB 아가 플레이트(agar plate)에 접종-도말시켜 배양한 흰반점 바이러스의 VP28 단백질(GenBank: DQ007315.1)을 발현하는 재조합 대장균 콜로니를 LB 배지 양의 1%를 준비하고 접종하였다. 흰반점 바이러스(White Spot Syndrome Virus, WSSV)의 캡시드(capsid) 단백질인 VP28 재조합 단백질을 생산하기 위하여 대장균 BL21(DE3)을 사용하였다. T7 프로모터를 가지고 있는 pET30a(+) 벡터(vector)와 VP28 유전자의 라이게이션(ligation)을 통해 발현벡터를 제조하였으며 이를 대장균의 형질전환에 이용하였다. 대장균을 암피실린(ampicillin)(50 ㎍/㎖)이 첨가된 LB 평판배지(Luria-Bertani agar plate)에 도말하여 형질 전환된 콜로니를 확보하였고, 이를 암피실린이 첨가된 LB 액체배지 15 ㎖에 접종하여 37℃에서 16시간동안 진탕 배양하였다. 암피실린이 첨가된 새로운 LB 액체배지 1.5 L에 배양액 15 ㎖을 계대배양하였으며, 분광광도계를 사용하여 600 nm 파장에서 광학밀도(O.D)를 측정하였다. 광학밀도(O.D)가 0.6일 때 Isopropyl β-D-1-thiogalactopyranoside(IPTG)을 최종 1.0 mM 농도로 첨가함으로써 유전자 발현을 유도(induction)하였다.In order to use VP28, a recombinant protein of white spot virus, as an antigen, LB (lysogeny broth) medium was prepared, sterilized, and prepared by adding ampicillin at a concentration of 50 ug / ml. One day ago, 1% of LB medium was prepared and inoculated with recombinant E. coli colonies expressing VP28 protein (GenBank: DQ007315.1) of white spot virus cultured by inoculation-staining on an LB agar plate. E. coli BL21 (DE3) was used to produce VP28 recombinant protein, which is the capsid protein of White Spot Syndrome Virus (WSSV). An expression vector was prepared by ligation of the pET30a (+) vector with the T7 promoter and VP28 gene, which was used for transformation of E. coli. Escherichia coli was plated on Luria-Bertani agar plate to which ampicillin (50 μg / ml) was added to secure transformed colonies, which were inoculated into 15 ml of LB liquid medium to which ampicillin was added. Shake incubation for 16 hours at ℃. 15 ml of the culture medium was passaged in 1.5 L of fresh LB liquid medium to which ampicillin was added, and optical density (O.D) was measured at a wavelength of 600 nm using a spectrophotometer. Gene expression was induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final 1.0 mM concentration when the optical density (O.D) was 0.6.
IPTG 첨가 후 6시간동안 추가 배양한 뒤, 6,000 rpm에서 30분간 원심분리하여 셀을 수거하였다. IPTG에 의해 과발현된 셀을 세포 파쇄 완충액(cell lysis buffer) (50 mM Tris-HCI(pH 8.0), 1 mM EDTA, 100 mM NaCl)에 재부유시키고 1 mg/ml의 라이소자임(lysozyme)과 10 ug/ml의 DNase를 넣고 진탕 배양기(200 rpm, 37℃)에서 1시간동안 교반하였다. 1시간 후, 소니케이터(sonicator)를 이용하여 셀을 파쇄하고, 원심분리하여(6,000 rpm, 30분) 펠렛을 수거하였다. 라이소자임을 이용하여 상기 과정을 1회 반복하였다. 라이소자임으로 수득한 펠렛을 우레아 버퍼(urea buffer) (100 mM Tris-HCl(pH 8.0), 5 mM EDTA, 2 M Urea, 2% Triton X-100)를 이용하여 소니케이터로 한 번 더 파쇄하여 단백질을 수거하고, PBS(phosphate buffer saline)를 이용하여 투석과정을 거쳐 우레아를 제거하고, 수거된 단백질은 SDS-PAGE상에서 최종적으로 확인하였다. SDS-PAGE는 10%의 SDS-PAGE 겔을 사용하였다. Cells were harvested by further incubation for 6 hours after IPTG addition, followed by centrifugation at 6,000 rpm for 30 minutes. Cells overexpressed by IPTG were resuspended in cell lysis buffer (50 mM Tris-HCI (pH 8.0), 1 mM EDTA, 100 mM NaCl) and 1 mg / ml of lysozyme and 10 ug. DNase of / ml was added and stirred for 1 hour in a shake incubator (200 rpm, 37 ℃). After 1 hour, the cells were crushed using a sonicator and centrifuged (6,000 rpm, 30 minutes) to collect the pellets. The process was repeated once using lysozyme. Pellets obtained as lysozyme were further crushed with a sonicator using urea buffer (100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 2 M Urea, 2% Triton X-100) Proteins were collected, urea was removed by dialysis using PBS (phosphate buffer saline), and the collected proteins were finally identified on SDS-PAGE. SDS-PAGE used a 10% SDS-PAGE gel.
단백질 발현여부는 웨스턴 블로팅(western-blot)을 통해 추가로 확인하였다. 웨스턴 블로팅은 공지된 방법으로 수행하였으며, 실험에 사용한 첫 번째 항체로는 1:1,000으로 희석된 Anti-His-probe-IgG를 사용하였고, 두 번째 항체로는 1:5,000으로 희석된 Anti-Mouse-IgG-HRP을 사용하였다.Protein expression was further confirmed by western blotting. Western blotting was performed by a known method, and the first antibody used in the experiment was Anti-His-probe-IgG diluted 1: 1,000, and the second antibody was Anti-Mouse diluted 1: 5,000. -IgG-HRP was used.
단백질 발현여부를 확인한 결과, 12 kda에서의 단백질의 과발현이 관찰되었으며, 웨스턴 블로팅을 통해 재조합 단백질 타겟에 붙어있는 6x-His에 대해서 단백질 발현여부를 확인한 결과, 예상 단백질 사이즈로 발현된 것을 확인할 수 있었다(도 1). As a result of confirming the protein expression, overexpression of the protein was observed at 12 kda, and the protein expression of the 6x-His attached to the recombinant protein target by Western blotting was confirmed. (FIG. 1).
1-3. 비브리오 균 외막단백질의 재조합 단백질 제조1-3. Preparation of Recombinant Protein of Vibrio Outer Membrane
조기폐사증후군의 주요 항원인 비브리오 파라해모라이티쿠스와 비브리오 하베이에 대한 바깥쪽 외막단백질 부분 중 항원 결정기인 에피토프(epitope) 단백질의 재조합 단백질인 OmpW와 OmpK를 각각 YUAN Ye, WANG Xiuli, GUO Sheping, QIU Xuemei et al., Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus , Chinese Journal of Oceanology and Limnology, Vol. 29, No. 5, P. 952-957, 2011 및 Li Ningqiu, Bai Junjie, Wu Shuqin, Fu Xiaozhe, Lao Haihua, Ye Xing, Shi Cunbin et al., An outer membrane protein, OmpK , is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper ( Epinephelus coioides ), Fish & Shellfish Immunology, 25, 829-833, 2008에 공지된 방법으로 생산하고자 하였다. Among the outer membrane proteins for Vibrio para-Haamoraiticus and Vibrio Habei, the main antigens of early mortality syndrome, OmpW and OmpK, the recombinant proteins of epitope proteins, which are antigenic determinants, were YUAN Ye, WANG Xiuli, GUO Sheping, QIU Xuemei et al., Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus , Chinese Journal of Oceanology and Limnology, Vol. 29, No. 5, P. 952-957, 2011 and Li Ningqiu, Bai Junjie, Wu Shuqin, Fu Xiaozhe, Lao Haihua, Ye Xing, Shi Cunbin et al., An outer membrane protein, OmpK , is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper ( Epinephelus coioides ) , Fish & Shellfish Immunology, 25, 829-833, 2008.
OmpW 및 OmpK 재조합 단백질을 발현시키기 위해, 클로닝된 TA-Clone에 제한효소 BamHⅠ과 XhoⅠ을 반응시켜 삽입된 유전자만을 획득하였고, Novagen사의 pET32a(+) 벡터(vector)(#69015-3CN)와 결합시켜 재조합 클론을 생산하였다(도 2, 도 3). pET32a(+) 벡터는 N말단에 T7 프로모터(promoter) 유전자를 가지고 있어 삽입된 외부유전자에 해당하는 단백질을 과발현할 수 있으며 T7 프로모터 유전자 뒤에 6개의 히스티딘 잔기가 반복된 히스택(His-tag)이 존재하여 재조합단백질을 검출하는데 사용할 수 있다. 생산한 재조합 클론을 BL21(DE3) 컴피턴트 세포(Competent Cell)(#69450-3CN, Novagen)에 형질전환하여 단일 콜로니를 획득하였다. 3 ml의 LB 액체배지에 단일 콜로니를 접종하여 37℃에서 16시간동안 진탕 배양하였다. 배양액을 50 ml의 LB 액체배지에 재접종한 후 37℃에서 진탕 배양하였다. 분광광도계를 이용하여 1시간마다 흡광도(A600)를 측정하여 0.5~0.6에 도달할 때까지 배양하였다. 발현 시스템의 T7 프로모터를 이용하기 위해서 isopropyl-β-D-thio-galactopyranoside(IPTG; Sigma, USA)를 최종농도 1 mM가 되도록 첨가하고 배양하였다. 이후 배양액 10 ml을 획득하여 12000 rpm, 10 min 원심분리를 통해 균체만 획득하였다. 획득한 균체에 라이시스 버퍼(lysis buffer)(50 mM NaH2PO4, 300 mM NaCl)를 1 ml 처리하여 재부유시킨 후 100 μg/ml 라이소자임(lysozyme)을 첨가하여 25℃에서 30분간 반응시켰다. 이후, 소니케이터(sonicator)를 사용하여 용해(Lysis)시켰다. 다음으로 18000 rpm, 30 min 원심분리를 통해 용해성 부분과 불용성 부분을 분리하였고, 불용성 부분은 SDS-PAGE를 통해 단백질 발현 유무를 확인하였다(도 4, 도 5).In order to express OmpW and OmpK recombinant proteins, the cloned TA-Clone was reacted with restriction enzymes BamHI and XhoI to obtain only the inserted gene, and then combined with Novagen's pET32a (+) vector (# 69015-3CN). Recombinant clones were produced (FIG. 2, FIG. 3). The pET32a (+) vector has a T7 promoter gene at the N-terminus, which can overexpress the protein corresponding to the inserted external gene, and a his-tag whose six histidine residues are repeated after the T7 promoter gene. Present and can be used to detect recombinant proteins. The recombinant clones produced were transformed into BL21 (DE3) Competent Cells (# 69450-3CN, Novagen) to obtain a single colony. Single colonies were inoculated into 3 ml of LB broth and shaken at 37 ° C. for 16 hours. The culture was reinoculated into 50 ml of LB broth and shaken at 37 ° C. The absorbance (A600) was measured every hour using a spectrophotometer and cultured until it reached 0.5 to 0.6. In order to use the T7 promoter of the expression system, isopropyl-β-D-thio-galactopyranoside (IPTG; Sigma, USA) was added to a final concentration of 1 mM and incubated. Since 10 ml of the culture was obtained, only cells were obtained by centrifugation at 12000 rpm and 10 min. The obtained cells were resuspended with 1 ml of lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl), and then reacted at 25 ° C. for 30 minutes by adding 100 μg / ml lysozyme. . Then, the solution was dissolved using a sonicator. Next, the soluble part and the insoluble part were separated by centrifugation at 18000 rpm and 30 min, and the insoluble part was confirmed whether the protein was expressed through SDS-PAGE (FIGS. 4 and 5).
비브리오 파라해모라이티쿠스 외막단백질의 항원결정기 부위의 재조합 단백질 OmpW(Outer membrane protein W)의 유전자를 증폭하기 위하여 GenBank에 등록된 OmpW(strain zj2003; GenBank accession No: DQ425109.1)를 참조하여 프라이머(primer)를 합성하였으며, 이를 이용해 만든 재조합 클론과 염기서열을 비교한 결과 99%의 상동성을 보였다. 따라서 재조합 유전자 pET-32a OmpW로 형질전환된 BL21(DE3) 균주를 1.0 mM의 IPTG를 처리하여 4시간동안 배양한 결과, OmpW 재조합 단백질 생산에 적합하다고 확인하였다(도 4).In order to amplify the gene of the recombinant protein OmpW (Outer membrane protein W) at the epitope region of the Vibrio para-Haemolyticus outer membrane protein, the primer (see strain zj2003; GenBank accession No: DQ425109.1) was referred to. primers) were synthesized, and showed a 99% homology when compared with the recombinant clones made from them. Therefore, the BL21 (DE3) strain transformed with the recombinant gene pET-32a OmpW was incubated for 4 hours by treatment with 1.0 mM IPTG, which was confirmed to be suitable for OmpW recombinant protein production (FIG. 4).
아울러, 비브리오 하베이 외막단백질의 항원결정기 부위의 재조합 단백질 OmpK(Outer membrane protein K)의 유전자를 증폭하기 위해 GenBank에 등록된 OmpK(GenBank accession No: AY332563.1)를 참고하여 프라이머를 제작하였으며, 이를 PCR로 증폭하여 염기서열을 확인하였다. GenBank에서 참고한 염기서열과 비교하였을 때 98%의 염기서열 상동성을 보였으며, 재조합 클론 pET-32a OmpK을 사용하여 BL21(DE3)균주를 형질전환하였을 때, 1.0 mM IPTG를 처리한 후 4시간동안 배양한 결과, 효율적으로 OmpK 재조합 단백질을 생산할 수 있음을 확인하였다(도 5).In addition, primers were prepared by referring to OmpK (GenBank accession No .: AY332563.1) registered in GenBank to amplify the gene of the recombinant protein OmpK (Outer membrane protein K) at the epitope region of the Vibrio Havey outer membrane protein. By amplification to confirm the base sequence. Compared with the nucleotide sequence referenced by GenBank, the sequence homology was 98%. When recombinant BL21 (DE3) strain was transformed using recombinant clone pET-32a OmpK, 4 hours after treatment with 1.0 mM IPTG As a result of incubation, it was confirmed that OmpK recombinant protein can be efficiently produced (FIG. 5).
1-4. 난황항체(IgY) 생산을 위한 산란계 접종용 백신 제조1-4. Manufacture of Vaccine for Laying Hens for Production of Egg Yolk Antibody (IgY)
산란계 접종을 위해 백신용기, 고무전 캡, PBS(1×), 체, 집게, 주사기, 50 ml 튜브, 20 ul 피펫, 20 ul 팁, 플라스틱 비이커(500 mL, 1000 mL)를 준비하고, 실험예 1-1 내지 실험예 1-3에 따라 제조한 항원을 혼합한 백신을 다음과 같이 제조하였다. Vaccine containers, rubber caps, PBS (1 ×), sieves, forceps, syringes, 50 ml tubes, 20 ul pipettes, 20 ul tips, plastic beakers (500 mL, 1000 mL) were prepared for laying hens. The vaccine mixed with the antigen prepared according to 1-1 to Experimental Example 1-3 was prepared as follows.
37±1℃로 데워진 수조에 각각의 아쥬반트 오일(adjuvant oil)을 넣어 15~20분간 데웠다. 균질기(homogenizer)는 멸균증류수에 37% 포름알데히드 용액 0.1%를 첨가하여 소독하고 물기를 제거한 후, 70% 에탄올을 뿌려 건조시켰다. 냉장보관된 실험예 1-1 내지 실험예 1-3에 따라 제조된 항원들은 실온에 꺼내놓고, 하기 표 1의 구성에 따라 혼합 항원을 제조하였다. 혼합된 항원은 70% 에탄올을 분무하여 불로 달궈준 체에 무균상태로 걸러내었다. 항원 혼합 시, 항원의 최종 부피가 부족할 경우 멸균 PBS를 추가하였다. 혼합 항원과 아쥬반트 오일을 섞어주고, 균질기를 25분간 돌려서 산란계 접종용 백신을 제조하였다. 표 1의 백신에 포함된 항원 구성에서, 흰반점 바이러스 재조합 단백질만으로 이루어진 백신은 비교예 1, 새우 조기 폐사 증후군의 원인균인 비브리오 균으로만 이루어진 백신은 비교예 2, 동일한 비율로 혼합된 비브리오 균과 흰반점 바이러스 재조합 단백질의 혼합 항원으로 이루어진 백신은 실시예 1, 1:1:0.5:1:0.5:0.5의 비율로 혼합된 비브리오 균, 흰반점 바이러스 재조합 단백질 및 비브리오 균 외막단백질의 항원결정기 부위의 재조합 단백질인 OmpW(V1W)와 OmpK(VbK)의 혼합 항원으로 이루어진 백신은 실시예 2로 지정하였다.Each adjuvant oil was added to a water bath warmed at 37 ± 1 ° C. and warmed for 15 to 20 minutes. The homogenizer was sterilized by adding 0.1% of 37% formaldehyde solution to sterile distilled water, removing water, and then dried by spraying 70% ethanol. Antigens prepared according to refrigerated Experimental Example 1-1 to Experimental Example 1-3 were taken out at room temperature, and mixed antigens were prepared according to the configuration of Table 1 below. The mixed antigen was filtered aseptically on a sieve sprayed with 70% ethanol. At antigen mixing, sterile PBS was added when the final volume of antigen was insufficient. The mixed antigen and the adjuvant oil were mixed, and a homogenizer was turned for 25 minutes to prepare a vaccine for laying hens. In the antigen composition included in the vaccine of Table 1, the vaccine consisting of only the white spot virus recombinant protein is Comparative Example 1, the vaccine consisting only of Vibrio bacteria, the causative agent of shrimp early mortality syndrome, is compared with Comparative Example 2, Vibrio bacteria mixed in the same ratio. The vaccine consisting of a mixed antigen of the white spot virus recombinant protein was prepared in Example 1, in a ratio of 1: 1: 0.5: 1: 0.5: 0.5 of the epitope region of the Vibrio bacteria, the white spot virus recombinant protein and the Vibrio bacteria outer membrane protein. A vaccine consisting of a mixed antigen of recombinant proteins OmpW (V1W) and OmpK (VbK) was designated as Example 2.
- V1: Vibrio parahaemolyticus, Vb : Vibrio harveyi, Va : Vibrio anguillarum , WSSV : White Spot Syndrome Virus, V1W : Vibrio parahaemolyticus Outer Membrane Protein’s recombinant protein(OmpW), VbK : Vibrio harveyi Outer Membrane Protein’s recombinant protein(OmpK)V1: Vibrio parahaemolyticus , Vb: Vibrio harveyi , Va: Vibrio anguillarum , WSSV: White Spot Syndrome Virus, V1W: Vibrio parahaemolyticus Outer Membrane Protein's recombinant protein (OmpW), VbK: Vibrio harveyi Outer Membrane Protein's recombinant protein (OmpK)
1-5. 산란계 면역1-5. Laying Hens
실험예 1-4에 따라 제조된 백신은 22주령의 Hyline-brown 계열의 산란계에 1 ml씩 가슴근육에 접종하였다. The vaccine prepared according to Experimental Example 1-4 was inoculated into the chest muscle by 1 ml in a 22-week-old Hyline-brown family laying hen.
1-6. 난황 샘플 수거1-6. Egg yolk sample collection
난황샘플은 매일 면역화 된 산란계가 낳은 계란을 수거하는 것부터 시작하였다. 수거한 계란은 날짜별로 기입하여 7일 간격으로 수집하였고, 주차별로 역가 검사를 시행하였다. 각 주차별 역가확인용 샘플 채취를 위해 각 주차별 난황 5개를 무작위 수거하여 난황을 분리하였고, 중량 대비 10배에 해당되는 멸균 DW(deionized water)를 혼합하여 50 ml 튜브에 담아 24시간 냉동 보관하였다. 24시간 후 37℃에서 중탕시켜 녹인 후 4℃에서 3,500 rpm으로 30분간 원심분리하여 상등액을 취해 역가분석용 샘플로 보관하였다.The yolk sample began with the collection of eggs laid by the immunized laying hens daily. Collected eggs were collected by date and collected at 7-day intervals. In order to collect the potency sample for each parking lot, five egg yolks were randomly collected to separate egg yolks, and sterile DW (deionized water) equivalent to 10 times the weight was mixed and stored in a 50 ml tube for 24 hours. It was. After 24 hours, the mixture was dissolved in a hot water at 37 ° C., centrifuged at 3,500 rpm at 4 ° C. for 30 minutes, and the supernatant was taken and stored as a sample for potency analysis.
실험예 2: 난황항체 역가 측정Experimental Example 2: Measurement of yolk antibody titer
2-1. EMS 항원의 OMP(outer membrane protein) 분리2-1. OMP separation of EMS antigens
조기 폐사 증후군(EMS)의 항원인 비브리오 균의 배양이 끝나면 균 배양액을 원심관에 옮겨 4℃에서 9,000 rpm으로 30분간 원심분리하여 상층액을 버리고, PBS로 원심관을 2회 세척하고 10 mM HEPES 버퍼(GibcoBRL, USA) 500 ml로 균을 부유시킨 뒤, 다시 원심분리를 하고 1회 세척하였다. 원심분리 후 상층액을 버리고 펠렛을 10 mM HEPES 버퍼 300 ml로 부유한 후 30 ml씩 분주하여 초음파처리하여 항원을 용해(lysis)시켰다. 용해시킨 상층액을 4℃에서 8,000 rpm으로 30분간 원심분리하여 상층액을 분리하고 펠렛은 버렸다. 분리한 상층액에 1% N-라우로일 사르코신(N-Lauroly sarcosine) (SIGMA USA)을 첨가한 후 실온에서 10분간 정치시키고 4℃에서 15,000rpm으로 50분간 원심 분리하였다. 상층액을 버리고 세정제(detergent)를 제거하기 위해 펠렛을 10 mM HEPES 버퍼 50 ml로 다시 부유시켜 4℃에서 15,000 rpm으로 50분간 원심 분리하였다. 원심분리 후 상층액을 버리고 펠렛(OMP)을 10 mM HEPES 버퍼 25 ml로 부유시켰다. 펠렛을 부유한 후 초음파처리를 하고, 0.45 um 사이즈로 걸러낸 후 포르말린(formalin) 및 티메로살(thimerosal)을 첨가하였다. 단백질을 정량하고, OMP 항원의 항원성을 확인하기 위해 표준혈청을 사용하여 ELISA로 검사를 수행하였다.After cultivation of Vibrio bacteria, the antigen of early mortality syndrome (EMS), transfer the bacterial culture to a centrifuge tube, centrifuge at 9,000 rpm for 30 minutes at 4 ° C, discard the supernatant, wash the centrifuge tube twice with PBS, and wash 10 mM HEPES. The cells were suspended in 500 ml of buffer (GibcoBRL, USA), centrifuged again and washed once. After centrifugation, the supernatant was discarded and the pellet was suspended in 300 ml of 10 mM HEPES buffer, and then 30 ml aliquots were sonicated to dissolve the antigen. The dissolved supernatant was centrifuged at 4 ° C. at 8,000 rpm for 30 minutes to separate the supernatant, and the pellet was discarded. 1% N-Lauroly sarcosine (SIGMA USA) was added to the separated supernatant, and then allowed to stand at room temperature for 10 minutes and centrifuged at 15,000 rpm for 50 minutes at 4 ° C. The pellet was resuspended in 50 ml of 10 mM HEPES buffer and centrifuged at 15,000 rpm for 50 minutes to discard the supernatant and remove the detergent. After centrifugation the supernatant was discarded and the pellet (OMP) was suspended in 25 ml of 10 mM HEPES buffer. The pellet was suspended and sonicated, filtered to 0.45 um, and formalin and thimerosal were added. Proteins were quantified and tested by ELISA using standard serum to confirm the antigenicity of the OMP antigen.
2-2. ELISA(enzyme-linked immunosorbent assay)를 통한 난황항체 역가 확인2-2. Confirmation of Yolk Antibody Titer by ELISA (enzyme-linked immunosorbent assay)
난황 중의 특이 난황항체의 역가는 Indirect ELISA method 방법을 응용하여 측정하였다. 먼저 각 항원별 설정된 농도로 카보네이트 코팅 버퍼(carbonate coating buffer)에 희석하여 각 웰(well) 당 100 ul씩 96 웰 폴리스티렌 플레이트(polystyrene plate)에 코팅하여 4℃에서 오버나이트(over night)시키거나, 37℃에서 1시간동안 방치시켰다. PBS-T(phosphate buffer saline, 0.05% Tween 20, pH 7.4)로 세척 후, 2% BSA(bovine serum albumin)가 함유된 PBS 버퍼로 1시간동안 37℃에서 블로킹(blocking)하며 상기와 같은 방법으로 세척하였다. 음성대조군, 양성대조군 및 난황샘플을 2배씩 희석하여 웰 당 100 ㎕씩 넣고 37℃에서 1시간동안 정치시켰다. 1시간 후 3번 세척하고 2차 항체(anti-chicken: Sigma, U.S.A)를 PBS에 적정량 희석하여 각 웰에 100 ㎕씩 분주한 후 37℃에서 1시간동안 반응시켰다. 그 후 3번 세척을 하고 기질(substrate)로 TMB(3,3’,5,5’-Tetramethylbenzidine) 용액을 각 웰에 100 ul씩 분주한 다음 실온에서 약 10분간 반응시키고 정지용액(stop solution) 1.6 N H2SO4 100 ul를 분주하여 반응을 중지시켜 450 nm의 파장에서 ELISA 리더기로 각 웰의 흡광도를 측정하여 ELISA 값으로 나타내었다. 측정된 결과에 음성샘플의 평균값을 2배수하여 처리된 난황의 측정값에 대입하여 분석 데이터의 역가를 나타내었다.The titer of specific yolk antibody in egg yolk was measured by applying the Indirect ELISA method. First, dilute in a carbonate coating buffer at a predetermined concentration for each antigen, and coat 100 ul of each well on a 96 well polystyrene plate to overnight at 4 ° C., It was left at 37 ° C. for 1 hour. After washing with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH 7.4), blocking with PBS buffer containing 2% BSA (bovine serum albumin) at 37 ° C. for 1 hour. Washed. Negative control, positive control and egg yolk samples were diluted twice, and 100 μl per well was added and allowed to stand at 37 ° C. for 1 hour. After 1 hour, washed three times, and the secondary antibody (anti-chicken: Sigma, USA) diluted in an appropriate amount in PBS and 100 ㎕ divided into each well and reacted for 1 hour at 37 ℃. After washing three times, TMB (3,3 ', 5,5'-Tetramethylbenzidine) solution was dispensed into each well for 100 ul with a substrate and reacted for about 10 minutes at room temperature. The reaction was stopped by dispensing 100 ul of 1.6 NH 2 SO 4, and the absorbance of each well was measured with an ELISA reader at a wavelength of 450 nm. The average value of the negative sample was doubled in the measured result, and the titer of the analyzed data was represented by substituting the measured value of the processed yolk.
실험 결과, 도 6~도 9에 나타난 바와 같이 비브리오 균 3종과 흰반점 증후군 바이러스 재조합 단백질(WSSV VP28)에 대한 IgY 역가를 각 그룹별로 확인할 수 있었다. 비교예 1의 경우, WSSV VP28 항원을 제외하고는 다른 항원에 대해서는 항체역가가 형성되지 않았다. 비교예 2의 경우, WSSV VP28항원을 제외하고는 각 항원에 대하여 주차별로 서서히 역가가 상승하여 3차 접종 이후의 역가가 6,400까지 상승하는 것으로 나타났다. 실시예 1의 경우, 대상 항원에 대해서 접종 주차별로 역가가 안정적으로 상승하는 것으로 나타났으나, 역가 상승의 한계치가 평균적으로 6,400 이상의 역가가 올라가지 않는 것으로 나타났다. 실시예 2의 경우, 주차별로 역가가 상승하여 모든 항원에 대해서 평균 12,800 이상의 역가가 올라가는 것으로 나타났으며, 상승된 역가를 유지하는 기간도 다른 군에 비하여 길게 나타나는 결과로 나타났다. 상기 결과는, 비브리오 균 항원 및 흰반점 바이러스 재조합 단백질 항원을 단독으로 포함하는 비교예 1 및 비교예 2에 비해, 실시예 2에 의한 항원의 역가가 월등하게 우수하게 나타남을 보여준다. As a result, as shown in FIGS. 6 to 9, IgY titers of three Vibrio strains and the white spot syndrome virus recombinant protein (WSSV VP28) were confirmed for each group. In Comparative Example 1, antibody titers were not formed for other antigens except for the WSSV VP28 antigen. In Comparative Example 2, except for WSSV VP28 antigen, the titer gradually increased by parking for each antigen, and the titer after the third inoculation increased to 6,400. In the case of Example 1, the titer was increased stably according to the inoculation parking for the target antigen, but the threshold value of the titer increase was not higher than 6,400 on average. In the case of Example 2, the titer was increased by parking, and the average titer was increased to 12,800 or more for all antigens, and the period of maintaining the elevated titer was also shown to be longer than other groups. The results show that the titer of the antigen according to Example 2 is superior to that of Comparative Example 1 and Comparative Example 2 containing Vibrio bacteria antigen and white spot virus recombinant protein antigen alone.
실험예 3: 암모늄 설페이트(Ammonium sulfate) 처리에 따른 난황항체 분리Experimental Example 3: Isolation of Egg Yolk Antibodies Treated with Ammonium Sulfate
생산된 특이 난황항체 단백질을 순수하게 분리하기 위해 암모늄 설페이트 처리법을 이용하여 분리정제를 진행하였다. 고역가의 계란을 수거하고 난황 분리기를 이용하여 난백과 알끈을 제거하고, Whatman No.1 필터 페이퍼(filter paper)로 난막을 제거한 뒤, 비커에 난황만을 분리하였다. 분리한 난황의 양을 측정하여 1차 증류수를 중량 대비 4배의 양을 넣어 1시간 동안 교반한 뒤, 1 M HCl을 사용하여 pH 5로 조정하였다. 이 난황액을 -70℃ 초저온냉장고에서 하루 동안 보관하였다. 난황액을 실온에 꺼내 하루정도 녹인 뒤, 10분 동안 원심분리(9,000 rpm, 4℃)하여 상등액을 수득하여 진공 펌프(vaccum pump)를 사용하여 필터 페이퍼에 여과하여 준비하였다. 여과된 상등액의 부피를 측정하여 암모늄 설페이트를 얼음 조건에서 조금씩 첨가시켜 충분히 녹여주었다. 암모늄 설페이트 처리된 용액을 4℃에서 오버나이트 정치시킨 뒤, 30분 동안 원심분리(9,000 rpm, 4℃)하여 펠렛을 수거하여 1×PBS 버퍼에 재부유시켜 샘플을 수거하였다. 상등액은 부피를 측정하고 다시 암모늄 설페이트를 첨가하여 펠렛과 상등액을 수거하고 SDS-PAGE와 웨스턴 블로팅을 통해 특이 난황항체의 분리 정제 순도를 확인하였다. SDS-PAGE와 웨스턴 블로팅은 실험예 1-2와 동일한 조건으로 수행하였다.In order to purely separate the produced specific yolk antibody protein, separation and purification were performed using ammonium sulfate treatment. Eggs of high titer were collected, egg whites and strips were removed using an egg yolk separator, egg yolk was removed with Whatman No. 1 filter paper, and only egg yolks were separated in a beaker. The amount of separated egg yolk was measured, and the primary distilled water was added to 4 times the amount by weight, stirred for 1 hour, and then adjusted to pH 5 using 1 M HCl. This yolk solution was stored for one day in a -70 ° C cryogenic refrigerator. The yolk solution was taken out at room temperature, dissolved for about one day, and centrifuged (9,000 rpm, 4 ° C.) for 10 minutes to obtain a supernatant, which was prepared by filtration on filter paper using a vacuum pump. The volume of the filtered supernatant was measured, and ammonium sulfate was added little by little under ice conditions to sufficiently dissolve. The ammonium sulfate treated solution was left overnight at 4 ° C., then centrifuged (9,000 rpm, 4 ° C.) for 30 minutes to collect pellets and resuspended in 1 × PBS buffer to collect samples. The supernatant was measured by volume and again added ammonium sulphate to collect the pellet and the supernatant and confirmed the purity of the isolated purified yolk antibody through SDS-PAGE and Western blotting. SDS-PAGE and western blotting were performed under the same conditions as in Experimental Example 1-2.
시험 결과, IgY 항체의 특징인 중쇄(heavy chain)(70kda)와 경쇄(light chain)(21kda) 부분이 잘 나타나있으며, 웨스턴 블로팅으로 확인한 결과, 난황항체 단백질임을 확인할 수 있었다(도 10).As a result, the heavy chain (70kda) and light chain (21kda) portions of the IgY antibody are well represented and confirmed by Western blotting, it was confirmed that the yolk antibody protein (Fig. 10).
실험예 4: 항원에 대한 결합 친화성(binding affinity) 확인Experimental Example 4: Confirming the binding affinity to the antigen
생산된 난황항체의 항원에 대한 결합 친화성을 확인하기 위해, 항원을 PBS에 일정량 부유시키고, 5×샘플 염료(sample dye)를 첨가한 뒤 전기영동을 실시하고, 멤브레인에 트랜스퍼(transfer)를 진행하고 5% 탈지유(skim milk)를 이용하여 블로킹한 뒤, PBS-T로 세척하고 IgY 항체를 2 ug/ml 농도로 처리하여 4℃에서 오버나이트하고 PBS-T로 세척하였다. Anti-Chicken-IgG-HRP로 1:20,000 희석하여 처리한 뒤, HRP(horseradish peroxidase) 기질을 이용하여 결과를 확인하였다. To confirm the binding affinity of the produced egg yolk antibody to the antigen, the antigen was suspended in PBS in a certain amount, 5 × sample dye was added, followed by electrophoresis, and transfer to the membrane. After blocking with 5% skim milk, it was washed with PBS-T and treated with IgY antibody at a concentration of 2 ug / ml at 4 ° C. and washed with PBS-T. After diluting with 1: 20,000 with Anti-Chicken-IgG-HRP, the results were confirmed using a horseradish peroxidase (HRP) substrate.
실험 결과, 난황항체를 사용한 각 항원에 특이적인 부분에 난황항체가 결합하는 것을 확인할 수 있었다(도 11). As a result, it was confirmed that the yolk antibody binds to the specific portion of each antigen using the yolk antibody (Fig. 11).
실험예 5: EMS 항원균에 대한 성장억제 확인Experimental Example 5: Confirmation of growth inhibition for EMS antigen
난황항체를 EMS 항원균에 처리할 경우, 성장억제 효능을 보이는지 확인하기 위해, 1×105 cfu/ml 농도의 항원균에 동일하게 10 mg/ml 농도의 난황항체를 처리하고 0, 3, 6, 9, 12, 24, 36시간까지 O.D 600 nm에서 흡광도를 측정하여, 항원의 조성에 따른 효과를 확인하였다. EMS 항원균에 대해 아무것도 처리하지 않은 것을 대조군으로 지정하여 실험을 진행하였다. When yolk antibodies are treated with EMS antigens, the same treatment with 1 × 10 5 cfu / ml antigen bacteria to 10 mg / ml yolk antibodies is performed to determine whether they exhibit growth inhibition effects. Absorbance was measured at OD 600 nm until 9, 12, 24, 36 hours, and the effect of the composition of the antigen was confirmed. The experiment was conducted by designating that the control group was nothing treated with EMS antigen.
실험 결과, 비교예 1의 경우, EMS에 대한 항원이 없는 관계로 대조군(control)과 동일하게 성장곡선이 나타났으며, 비교예 2의 경우, 각 항원균에 대해서 배양 후 3~9시간 사이에 항원균의 성장이 다소 감소하였으나 이후에는 대조군과 동일한 패턴으로 성장곡선이 나타났다. 실시예 1의 경우, 3~9시간까지의 항원균의 성장을 억제하는 효능이 있는 것으로 나타났다. 실시예 2의 경우, 모든 항원균에 대해서 3~9시간 사이의 성장곡선이 다른 모든 실험군에 비해 낮은 O.D 값과 함께 낮은 성장곡선을 나타냈으며, 최종 36시간까지의 O.D 값도 다른 군에 비하여 낮은 것으로 보아, 앞서 항체역가 실험에서 확인한 바와 같이, 높은 항체 역가가 유지되어 항원에 대한 결합력이 향상되는 것으로 보여진다(도 12~도 14). As a result of the experiment, in the case of Comparative Example 1, there was no antigen for EMS, and thus the growth curve appeared in the same manner as the control. In Comparative Example 2, each of the antigens was cultured for 3 to 9 hours after incubation. Although the growth of the antigen was slightly reduced, the growth curve appeared in the same pattern as the control. In the case of Example 1, it was shown to have the effect of inhibiting the growth of antigen bacteria up to 3-9 hours. In Example 2, the growth curve between 3-9 hours for all antigens showed a low growth curve with a lower OD value than all other experimental groups, and the OD value up to the last 36 hours was also lower than that of the other groups. As shown in the above antibody titer experiments, it is shown that high antibody titers are maintained to improve the binding ability to the antigen (FIGS. 12 to 14).
실험예 6: 흰반점 바이러스에 대한 난황항체 효과 확인Experimental Example 6: Confirmation of the effect of yolk antibody on white spot virus
흰반점 바이러스(WSSV)에 대한 난황항체의 효과를 확인하기 위해, 공격접종 실험을 진행하였다. 40일된 흰다리 새우 유생을 25℃의 통기성 조건의 여과된 해수(염도 30‰)에서 14일 동안 IgY를 섭취시켰다. 새우의 구강 경로로 WSSV를 감염시켰다. 예비 실험에 따르면 4일째에 새우 폐사율이 50-70%에 이르기 때문에 이를 통제하기 위해, WSSV에 감염된 새우 1마리에 대해 집단 당 약 체중의 10%에 달하는 먹이를 먹였다. 축적된 폐사율은 96시간에 걸쳐 매일 기록하였으며, 결과는 폐사율(%)로 표현하였다. 아울러, WSSV를 감염시키지 않은 새우실험군을 정상 대조군, WSSV를 감염시킨 새우에 실험군을 양성 대조군으로 지정하여 실험을 진행하였다. To confirm the effect of yolk antibody on white spot virus (WSSV), challenge experiments were conducted. Forty days old white larvae larvae were fed IgY for 14 days in filtered seawater (salt 30 ‰) at 25 ° C. WSSV was infected by the oral route of shrimp. In preliminary experiments, shrimp mortality rates ranged from 50-70% on
실험 결과, WSSV에 대한 단독 항원 구성 시험군인 비교예 1의 경우, 양성 대조군(positive control) 대비 약 11%정도 폐사율을 감소시켜 것으로 나타났다. WSSV에 대한 항원이 없는 비교예 2의 경우, 폐사율이 68.75%인 것으로 나타났다. 복합항원구성인 실시예 1, 실시예 2의 경우 폐사율이 양성 대조군 대비 15~28.75%정도 감소한 것으로 나타났다. 항원 그룹 중, 실시예 2가 41.25%의 폐사율로 모든 실험군에서 가장 폐사율이 낮은 것으로 나타나, 실시예 2의 항원 조합이 새우 폐사방지용 백신으로 가장 적합한 것을 확인하였다(표 2). As a result, in the case of Comparative Example 1, the sole antigen composition test group against WSSV, the mortality was reduced by about 11% compared to the positive control. For Comparative Example 2 without the antigen against WSSV, mortality was found to be 68.75%. In Examples 1 and 2, which are complex antigens, mortality was reduced by 15 to 28.75% compared to the positive control. Among the antigen groups, Example 2 showed the lowest mortality in all experimental groups with a mortality of 41.25%, confirming that the antigen combination of Example 2 was most suitable as a vaccine for preventing shrimp mortality (Table 2).
후 폐사 개체수24h
After our population
후 폐사 개체수48h
After our population
후 폐사 개체수72h
After our population
후 폐사 개체수96h
After our population
대조군normal
Control
대조군positivity
Control
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (13)
(b) 면역화된 산란계로부터 알을 산란시키는 단계; 및
(c) 산란된 알의 난황으로부터 항체를 분리하는 단계를 포함하는, 새우 폐사 방지용 난황항체 제조방법에 관한 것으로,
상기 단계 (a)에서 비브리오 균은 비브리오 파라해모라이티쿠스(Vibrio parahaemolyticus), 비브리오 하베이(Vibrio harveyi) 및 비브리오 앵길라룸(Vibrio anguillarum)로서, 0.7~1.3:0.7~1.3:0.4~0.7의 비로 혼합되고, 상기 단계 (a)에서 흰반점 바이러스 재조합 단백질은 VP28이고, 상기 단계 (a)에서 비브리오 균 외막단백질의 항원결정기 부위의 재조합 단백질은 비브리오 파라해모라이티쿠스 유래 외막단백질 OmpW 및 비브리오 하베이 유래 외막단백질 OmpK인 것을 특징으로 하는, 새우 폐사방지용 난황항체 제조방법.
(a) Mixtures of Vibrio genus, white spot syndrome virus (WSSV) recombinant protein, and Vibrio bacterial outer membrane protein, which are the causative agents of early mortality of shrimp, in a ratio of 2.5: 1: 1 Inoculating the laying hens with the antigen to immunize the hens;
(b) laying eggs from the immunized laying hens; And
(C) relates to a method for producing egg yolk antibody for preventing shrimp mortality, comprising the step of separating the antibody from the egg yolk of the laid egg,
Vibrio bacteria in step (a) are Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum, with a ratio of 0.7-1.3: 0.7-1.3: 0.4-0.7 Mixed, the white spot virus recombinant protein in step (a) is VP28, and in step (a) the recombinant protein of the epitope site of Vibrio fungal envelope protein is derived from Vibrio parahaemoriticus derived envelope membrane OmpW and Vibrio Havey The outer membrane protein OmpK, characterized in that the egg yolk antibody for preventing shrimp mortality.
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CN113317249A (en) * | 2021-06-30 | 2021-08-31 | 渤海水产育种(海南)有限公司 | Breeding method for reducing diseases of seed shrimps |
CN114146071A (en) * | 2022-02-08 | 2022-03-08 | 西北农林科技大学深圳研究院 | Application of paeonol and its derivatives in preventing and treating leukoplakia syndrome |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20030069506A (en) * | 2002-02-21 | 2003-08-27 | 주식회사 리앤조바이오텍 | Anti-white spot syndrome virus igy |
KR101242821B1 (en) | 2011-01-31 | 2013-03-12 | 씨제이제일제당 (주) | Probiotics Agent Against Vibrio sp. |
Family Cites Families (5)
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CN100500849C (en) * | 2003-12-08 | 2009-06-17 | 中国水产科学研究院珠江水产研究所 | Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacteria |
CN102836428B (en) * | 2012-09-12 | 2014-01-08 | 淮海工学院 | Inactivated vaccine preparation method for egg yolk antibody for resisting litopenaeus vannamei red body disease |
CN105198988B (en) * | 2015-09-30 | 2018-07-13 | 大连理工大学 | Anti- Vibrio splindidus Yolk antibody and preparation method thereof |
CN108003240B (en) * | 2017-12-04 | 2020-06-05 | 西安德轩驰生物科技有限公司 | Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20030069506A (en) * | 2002-02-21 | 2003-08-27 | 주식회사 리앤조바이오텍 | Anti-white spot syndrome virus igy |
KR101242821B1 (en) | 2011-01-31 | 2013-03-12 | 씨제이제일제당 (주) | Probiotics Agent Against Vibrio sp. |
Non-Patent Citations (2)
Title |
---|
Cochin University of Science & technology. M. Manjusha, White spot syndrome virus in penaeids: hisoipathology, development of polyclonal antisera and a cocktail vaccine(2003)* * |
J Biol Sci. 2016, 16(1-2):1-11.* * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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KR102154172B1 (en) * | 2019-03-28 | 2020-09-10 | 전한택 | Probiotics for inhibiting white spot syndrome virus and Vibrio bacterium infecting crustacean and use thereof |
CN113317249A (en) * | 2021-06-30 | 2021-08-31 | 渤海水产育种(海南)有限公司 | Breeding method for reducing diseases of seed shrimps |
CN114146071A (en) * | 2022-02-08 | 2022-03-08 | 西北农林科技大学深圳研究院 | Application of paeonol and its derivatives in preventing and treating leukoplakia syndrome |
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