KR100600837B1 - Fusion bio-insecticide of baculoviral polyhedrin and toxic protein - Google Patents
Fusion bio-insecticide of baculoviral polyhedrin and toxic protein Download PDFInfo
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Abstract
본 발명은 배큘로바이러스의 다각체 단백질과 바실러스 투린지엔시스 (Bacillus thuringiensis, Bt)의 독성단백질이 융합된 융합단백질에 관한 것으로, 보다 상세하게는, 배큘로바이러스의 다각체 단백질과 바실러스 투린지엔시스의 독성단백질이 융합된 융합단백질을 유효성분으로 함유하는 해충 방제용 살충제에 관한 것이다. 본 발명의 다각체-Bt 독소 융합단백질은 대장균에서 응집체의 형태로 발현되므로 대량생산이 가능하고 분리정제가 간단하며 독성활성을 그대로 유지하여 저렴하고 효과적인 생물살충제의 생산에 널리 이용될 수 있다. The present invention relates to a fusion protein in which a polyhedral protein of baculovirus and a toxic protein of Bacillus thuringiensis ( Bt) are fused. More specifically, the polyhedral protein of Baculovirus and Bacillus thuringiensis It relates to an insecticide for controlling pests containing a fusion protein fused with toxic protein as an active ingredient. Polygon-Bt toxin fusion protein of the present invention is expressed in the form of aggregates in E. coli, so that mass production is possible, simple purification and separation of toxic activity can be widely used in the production of cheap and effective biopesticides.
배큘로바이러스, 다각체 단백질, 바실러스 투린지엔시스, 독성단백질, 생물살충제Baculovirus, Polyhedral Protein, Bacillus thuringiensis, Toxic Protein, Biopesticide
Description
도 1은 배큘로바이러스의 다각체 단백질(polyhedrin)과 바실러스 투린지엔시스(Bacillus thuringiensis, Bt)의 독성단백질이 융합된 융합단백질을 생산하기 위한 pTH-pol:Cry1Ac 벡터를 제작하는 과정을 나타내는 모식도이고, 1 is a schematic diagram showing a process for preparing a pTH-pol: Cry1Ac vector for producing a fusion protein in which a polyhedrin of Baculovirus and a toxic protein of Bacillus thuringiensis (Bt) are fused. ,
도 2는 상기 융합단백질의 응집체(inclusion body)를 생산하는 BL21 대장균 세포의 성장을 배양시간에 따라 측정한 그래프이고, Figure 2 is a graph measuring the growth of the BL21 Escherichia coli cells producing the inclusion body of the fusion protein according to the culture time,
도 3은 IPTG로 발현 유도 후의 상기 융합단백질의 응집체의 발현을 시간에 따라 웨스턴 블롯(Western blot)으로 분석한 사진이고, 3 is a photograph of Western blot analysis of the expression of the aggregates of the fusion protein after expression induction with IPTG,
도 4는 도 3의 웨스턴 블롯 결과로부터 상기 융합단백질 발현량을 상대적으로 비교한 그래프이고, 4 is a graph comparing the expression level of the fusion protein relative to the Western blot result of FIG.
도 5는 상기 융합단백질이 응집체 형태로 발현되는지 여부를 확인하기 위하여, 상기 융합단백질을 발현하는 대장균 전세포(whole cell), 세포파쇄물 및 세포상등액 시료를 웨스턴 블롯한 결과를 나타내는 사진이고, 5 is a photograph showing Western blot results of E. coli whole cell, cell lysate and supernatant samples expressing the fusion protein in order to confirm whether the fusion protein is expressed in aggregate form.
도 6은 상기 융합단백질을 pH 12의 알칼리 용액에서 용해시킨 후 키모트립신을 처리하여 다각체 부분을 제거하고, SDS-PAGE로 분석한 결과를 나타내는 사진이 고, 6 is a photograph showing the result of dissolving the fusion protein in an alkaline solution of
도 7은 상기 융합단백질을 pH 12의 알칼리 용액에서 용해시킨 후 트립신을 처리하여 다각체 부분을 제거하고, SDS-PAGE로 분석한 결과를 나타내는 사진이고, 7 is a photograph showing the result of dissolving the fusion protein in an alkaline solution of
도 8은 도 6의 SDS-PAGE 후 Cry1Ac 항체를 이용하여 웨스턴 블롯한 결과이고, 8 is a result of Western blot using Cry1Ac antibody after SDS-PAGE of FIG. 6,
도 9는 도 7의 SDS-PAGE 후 Cry1Ac 항체를 이용하여 웨스턴 블롯한 결과이고, 9 is a result of Western blot using Cry1Ac antibody after SDS-PAGE of FIG. 7,
도 10은 도 6의 SDS-PAGE 후 히스티딘 항체를 이용하여 웨스턴 블롯한 결과이고, 10 is a result of Western blot using histidine antibody after SDS-PAGE of FIG. 6,
도 11은 도 7의 SDS-PAGE 후 히스티딘 항체를 이용하여 웨스턴 블롯한 결과이고, 11 is a result of Western blot using histidine antibody after SDS-PAGE of FIG. 7,
도 12는 본 발명의 융합단백질의 해충에 대한 작용기작을 나타낸다. Figure 12 shows the mechanism of action against pests of the fusion protein of the present invention.
본 발명은 배큘로바이러스의 다각체 단백질과 바실러스 투린지엔시스 (Bacillus thuringiensis, Bt)의 독성단백질이 융합된 융합단백질(이하, 다각체-Bt 독소 융합단백질이라 칭함)에 관한 것으로, 보다 상세하게는, 배큘로바이러스의 다각체 단백질과 바실러스 투린지엔시스의 독성단백질이 융합된 융합단백질을 유효성 분으로 함유하는 해충 방제용 살충제에 관한 것이다. The present invention relates to a fusion protein (hereinafter, referred to as a polyhedron-Bt toxin fusion protein) in which a polyhedral protein of Baculovirus and a toxic protein of Bacillus thuringiensis ( Bt) are fused. In addition, the present invention relates to an insecticide for pest control containing a fusion protein in which a polyhedral protein of baculovirus and a toxic protein of Bacillus thuringiensis are fused.
곤충은 환경에 대한 적응력이 뛰어나며 현재 지구상의 생물 중에서 가장 많은 종을 차지하고 있다. 이러한 곤충들 중 일부는 상업적으로 중요한 다양한 농경 작물에 많은 손실을 일으키고 있으며, 이러한 해충을 방제하기 위하여 종래 화학살충제가 많이 사용되어 왔다. 그러나 화학살충제는 작용 생물에 대한 광범위한 스펙트럼으로 해충만이 아니라 익충 및 해충에 있는 기생충과 같은 비목적 생물도 박멸시키는 문제점 및 이를 자주 사용할 경우 이에 반복적으로 노출된 해충이 내성을 가질 수 있으며, 그 성분이 인체에 해롭다는 문제점이 있다.Insects are adaptable to the environment and are now the largest species of life on Earth. Some of these insects are causing a lot of losses to a variety of commercially important agricultural crops, and conventional chemical pesticides have been used to control these pests. However, chemical pesticides have a broad spectrum of working organisms that destroy not only pests but also untargeted organisms such as insects and parasites in pests and, if used frequently, pests repeatedly exposed to them may be resistant, There is a problem that this is harmful to the human body.
상기와 같은 이유로 화학살충제의 사용이 점차 제한되고 있고, 국내에서도 현재 사용중인 살충제의 상당량이 감축될 예정에 있으므로 농산물의 생산성을 증가시키기 위한 수단으로서 새로운 살충제의 개발이 시급한 실정이다. The use of chemical pesticides is increasingly limited because of the above reasons, and a significant amount of pesticides currently in use are expected to be reduced in Korea. Therefore, it is urgent to develop new insecticides as a means for increasing productivity of agricultural products.
곤충세포가 배큘로바이러스에 감염되면, 약 1g/L의 다각체 단백질이 발현되는데, 이 양은 전체 단백질의 약 50%를 차지하는 많은 양이다. 세포 내에 발현된 다각체 단백질은 결정을 이루는 구조로 생성되는데 이를 다각체 입자라고 부르며, 이 결정화된 다각체 입자는 아직 밝혀지지 않은 기작에 의하여 배큘로바이러스 입자를 포함하게 되어 곤충세포가 분해되어 세포 밖 환경에 노출시 바이러스를 자연환경으로부터 보호하는 역할을 하게 된다. 다각체 단백질에 의하여 보호되는 배큘로바이러스 입자는 나뭇잎 등에 붙어 있다가 곤충이 나뭇잎을 먹을 때, 곤충의 중 장으로 들어가 중장의 알칼리 조건과 알칼리성 단백질 분해효소에 의하여 다각체 단백질이 용해될 때 빠져나와 곤충세포를 감염하게 된다(T. Suzuki et al., 1997, Journal of Virology 78, 3073-3080; E. Kotani 등, 1999, Insect Molecular biology 8, 299-304). When insect cells are infected with baculovirus, about 1 g / L of the polyhedral protein is expressed, which is about 50% of the total protein. Polyhedral proteins expressed in cells are produced in a crystalline structure, which is called polyhedral particles, and these crystallized polyhedral particles contain baculovirus particles by a mechanism that has not yet been identified. Exposure to the outside environment protects the virus from the natural environment. Baculovirus particles protected by polyhedral proteins are attached to leaves, but when insects eat leaves, they enter the insect's middle intestine and escape when the polyhedral protein is dissolved by the alkaline intestine and alkaline protease. Insect cells are infected (T. Suzuki et al., 1997, Journal of Virology 78, 3073-3080; E. Kotani et al., 1999, Insect Molecular biology 8, 299-304).
종래 미국특허 제4,870,023호에는 외래 아미노산 서열과 융합되어 결정화에 참여하는 다각체 단백질의 일부분을 포함하고, 다른 다각체 단백질과 결정화되어 재조합 다각체(inclusion body)를 형성할 수 있는 다각체 융합단백질이 개시되어 있었다. 그러나, 이러한 다각체 융합단백질은 다각체를 형성할 수 있는 성질을 가진 것에 한정되며, 다각체의 형성에 참여하는 것에 관한 것이었다. 또한, 상기 다각체 단백질의 발현은 배큘로바이러스의 비필수 영역에서 다각체 유전자를 치환시켜, 배큘로바이러스 내에 삽입된 외래 아미노산 서열을 코드하는 DNA를 포함하는 재조합 배큘로바이러스에 의하여 이루어졌다. 또한, 상기 재조합 배큘로바이러스를 통하여 융합 단백질을 생산하기 위하여는 곤충세포를 배양하고, 바이러스를 접종하는 과정이 필요하였으므로 본 발명의 대장균 발현 벡터를 이용한 대장균 세포에서의 융합단백질 생산과는 차이가 있다.Conventional US Patent No. 4,870,023 includes a polyhedral fusion protein comprising a portion of a polyhedral protein that is fused with a foreign amino acid sequence to participate in crystallization and crystallized with other polyhedral proteins to form a recombinant polyhedron. Was initiated. However, such polyhedral fusion proteins are limited to those having the property of forming a polyhedron, and related to participating in the formation of the polyhedron. In addition, the expression of the polyhedral protein was performed by a recombinant baculovirus comprising DNA encoding a foreign amino acid sequence inserted into the baculovirus by substituting the polyhedral gene in the non-essential region of the baculovirus. In addition, in order to produce a fusion protein through the recombinant baculovirus, a process of culturing insect cells and inoculating a virus is required, which is different from the production of a fusion protein in E. coli cells using the E. coli expression vector of the present invention. .
현재 미생물 살충제로 사용되고 있는 것으로 곰팡이, 세균, 바이러스 등을 이용한 것이 있으며, 세균 중 바실러스 투린지엔시스가 배출하는 독소(BT)를 이용하여 미생물 살충제로 개발, 사용하고 있는 것이 세계적인 추세이다. 우리나라에서도 이러한 미생물 살충제인 BT제를 스위스로부터 수입하여 흰불나방과 배추흰나 비 방제용으로 판매, 보급하고 있으나 이 BT제는 다른 화학살충제보다 가격이 높다는 문제가 있다.Currently, it is used as a microbial insecticide, and there are molds, bacteria, viruses, etc., and microorganisms are being developed and used as microbial insecticides by using toxins (BT) discharged from Bacillus thuringiensis. In Korea, the microbial insecticide BT is imported from Switzerland and sold and distributed for white fire moth and cabbage white or non-control. However, there is a problem that the BT is higher in price than other chemical insecticides.
한편, 대장균 발현시스템은 외래 단백질의 발현을 위한 다양한 종류의 균주, 프로모터 및 발현벡터에 관한 연구가 수행되어 왔으며, 외래단백질을 낮은 비용으로 높은 수율로 발현시킬 수 있는 장점이 있는 발현시스템이다.On the other hand, the E. coli expression system has been studied on various kinds of strains, promoters and expression vectors for the expression of foreign proteins, it is an expression system that has the advantage of expressing the foreign protein in high yield at a low cost.
대장균 시스템에서 발현되는 재조합 단백질은 단백질의 물리화학적 특성에 따라 수용성 형태 또는 불용성의 응집체(inclusion body) 형태로 발현되며 수용성 형태로 발현되는 경우, 일반적으로 생물학적 활성을 가진다는 장점이 있으나, 분리정제가 쉽지 않고 단백질 분해효소에 의한 공격을 쉽게 받을 수 있다는 단점을 가진다. 이에 반하여, 응집체 형태로 발현되는 경우에는 생물학적 활성을 가지지 못하므로 분리정제 후 재접힘(refolding) 공정을 수행하여야 한다는 단점이 있으나 일반적으로 단백질 분해효소에 강하고 분리정제가 쉽다는 잇점이 있다(H. Lilie et al., 1998, Current Opinion in Biotechnology 9, 497-501). Recombinant protein expressed in E. coli system is expressed in the form of water-soluble or insoluble inclusion (inclusion body) according to the physicochemical properties of the protein, and when expressed in water-soluble form, generally has the advantage of biological activity, It is not easy and can be easily attacked by protease. On the contrary, when expressed in aggregate form, since it does not have biological activity, it has a disadvantage in that a refolding process must be performed after separation and purification, but it is generally resistant to proteolytic enzymes and easy to be separated and purified (H). Lilie et al., 1998, Current Opinion in Biotechnology 9, 497-501).
이에, 본 발명자들은 본 발명의 배큘로바이러스 유래의 다각체 단백질과 바실러스의 독성단백질을 융합한 융합단백질이 대장균에서 발현되는 경우, 응집체의 형태로 발현되어 대량으로 생산할 수 있음과 상기 융합단백질이 바실러스에서 생산된 독성단백질과 마찬가지로 표적해충에 대한 독성활성을 유지함을 발견하고 생물살충제로 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.Thus, the present inventors, when the fusion protein fusion of the baculovirus-derived polyhedral protein of the present invention and the toxic protein of Bacillus is expressed in E. coli, it can be expressed in the form of aggregates and produced in large quantities, and the fusion protein is Bacillus The present invention was completed by finding that it maintains toxic activity against a target pest as well as the toxic protein produced in the present invention and confirmed that it can be used as a biopesticide.
본 발명의 목적은 대장균으로부터 대량생산이 가능하고 분리정제가 용이한 생물살충제를 제공하는 것이다.
An object of the present invention is to provide a biopesticide that can be mass-produced and easily purified from E. coli.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명은 배큘로바이러스의 다각체 단백질과 독성단백질이 융합된 융합단백질 및 상기 융합단백질을 코딩하는 유전자 컨스트럭트를 제공한다.The present invention provides a fusion protein in which a polyhedral protein of a baculovirus and a toxic protein are fused and a gene construct encoding the fusion protein.
또한, 본 발명은 배큘로바이러스 다각체 단백질과 독성단백질이 융합된 융합단백질 발현벡터 및 상기 발현벡터로 형질전환된 형질전환체를 제공한다.The present invention also provides a fusion protein expression vector fused to a baculovirus polyhedral protein and a toxic protein and a transformant transformed with the expression vector.
또한, 본 발명은 배큘로바이러스 다각체 단백질과 독성단백질이 융합된 융합단백질 또는 상기 융합단백질을 발현하는 대장균 세포를 유효성분으로 함유하는 해충 방제용 살충제를 제공한다.In another aspect, the present invention provides a pesticide for insect control containing a fusion protein fused to a baculovirus polyhedral protein and a toxic protein or E. coli cells expressing the fusion protein as an active ingredient.
아울러, 본 발명은 상기 대장균 형질전환체를 배양하여 융합단백질을 분리하는 단계를 포함하는, 배큘로바이러스 다각체 단백질과 독성단백질이 융합된 융합단백질의 생산방법을 제공한다. In addition, the present invention provides a method for producing a fusion protein fused to a baculovirus polyhedral protein and a toxic protein comprising culturing the E. coli transformant to separate the fusion protein.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 배큘로바이러스의 다각체 단백질과 독성단백질이 융합된 융합단백질을 제공한다. The present invention provides a fusion protein in which a polyhedral protein of a baculovirus and a toxic protein are fused.
상기 배큘로바이러스 유래의 다각체 단백질은 특별히 제한되는 것은 아니며, AcNPV(Autographa california nuclear polyhedrosis virus), BmNPV(Bombyx mori nuclear polyhedrosis virus), HzNPV(Heliothis zea nuclear polyhedrosis virus) 등 여러 배큘로바이러스에서 유래하는 여러 종류의 다각체 단백질이 모두 포함될 수 있다. 독성단백질도 특별히 제한되지 아니하며, 바실러스 균이나 전갈의 독성단백질 등 여러 생물에서 유래하는 여러 종류의 독성단백질이 모두 포함될 수 있다. The polyhedral protein derived from baculovirus is not particularly limited, and may be derived from various baculoviruses such as Autographa california nuclear polyhedrosis virus (AcNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), Heliothis zea nuclear polyhedrosis virus (HzNPV), and the like. Several polyhedral proteins can all be included. Toxic proteins are not particularly limited, and may include all kinds of toxic proteins derived from various organisms such as Bacillus bacteria or scorpion toxic proteins.
상기 융합단백질의 다각체 단백질 부위는 바람직하게는, AcNPV 유래의 서열번호 4로 기재되는 아미노산 서열을 가질 수 있으며, 독성단백질 부위는 바람직하게는, 바실러스 투린지엔시스 유래의 서열번호 6으로 기재되는 아미노산 서열을 가질 수 있다. The polyhedral protein site of the fusion protein may preferably have an amino acid sequence as set forth in SEQ ID NO: 4 from AcNPV, and the toxic protein site preferably comprises an amino acid as described in SEQ ID NO: 6 from Bacillus thuringiensis. May have a sequence.
또한, 본 발명의 융합단백질의 다각체 단백질 부위의 유전자는 바람직하게는, AcNPV 유래의 서열번호 3의 DNA 서열을 가질 수 있으며, 독성단백질 부위의 유전자는 바람직하게는, 바실러스 투린지엔시스 유래의 서열번호 5의 DNA 서열을 가질 수 있다.Further, the gene of the polyhedral protein portion of the fusion protein of the present invention may preferably have a DNA sequence of SEQ ID NO: 3 from AcNPV, and the gene of the toxic protein portion is preferably a sequence derived from Bacillus thuringiensis. It may have a DNA sequence of the
또한, 본 발명은 배큘로바이러스의 다각체 단백질과 독성단백질이 융합된 융합단백질의 발현벡터 및 상기 발현벡터로 형질전환된 형질전환체를 제공한다.The present invention also provides an expression vector of a fusion protein in which a polyhedral protein of a baculovirus and a toxic protein are fused, and a transformant transformed with the expression vector.
본 발명의 융합단백질 발현벡터는, 상기 융합단백질을 코딩하는 유전자 컨스트럭트를 포함하는 DNA 서열을 가지는 것을 특징으로 하며 바람직하게는, 서열번호 7의 융합단백질 유전자를 포함하는 DNA 서열을 가질 수 있다. The fusion protein expression vector of the present invention is characterized by having a DNA sequence comprising the gene construct encoding the fusion protein, preferably, may have a DNA sequence comprising the fusion protein gene of SEQ ID NO: 7 . .
상기 발현벡터는 본 발명의 융합단백질을 발현할 수 있는 것이면, 세균, 효모, 동물세포, 곤충세포 및 식물세포용 발현벡터 어느 것이라도 포함될 수 있다. 대장균용 발현벡터로서, pET3, pET11(이상 Stratagene사), pBAD, pThioHis, pTrcHis(이상, Invitrogen사), pKK223-3, pGEX2T(이상 Amersham Pharmacia Biotech사) 이외에 pBTrp2, pBTac1, pBTac2(이상 Boehringer Mannheim사), pSE280(Invitrogen사), pGEMEX-1(Promega사), pQE-8(QIAGEN사), pKYPl0[일본 공개특허공보 제(소)58-110600호], pKYP200[Agric. Biol. Chem., 48, 669(1984)], pLSA1[Agric. Biol, Chem., 53, 277(1989)], pGEL1[Proc.Natl. Acad. Sci. USA, 82, 4306(1985)], pBluescript Ⅱ SK(-)(Stratagene사), pTrs30[에스케리치아 콜라이 JM109/pTrS30(FERM BP-5407)로부터 조제], pTrs32[에스케리치아 콜라이 JM109/pTrS32(FERM BP-5408)로부터 조제], pGHA2[에스케리치아 콜라이 IGHA2(FERM BP-400)로부터 조제, 일본 공개특허공보 제(소)60-221091호], pGKA2[에스케리치아 콜라이IGKA2(PERM BP-6798)로부터 조제, 일본 공개특허공보 제(소)60-221091호], pTerm2(US 4686191, US 4939094, US 5160735), pSupex, pUB110, pTP5, pCl94, pEG400[J. Bacteriol., 172, 2392(1990)], pGEX(Pharmacia사) 및 pET 시스템(Novagen사) 등을 들 수 있다. The expression vector may be any expression vector for bacteria, yeast, animal cells, insect cells and plant cells, as long as it can express the fusion protein of the present invention. As expression vectors for Escherichia coli, pBTrp2, pBTac1, pBTac2 (above Boehringer Mannheim) in addition to pET3, pET11 (above Stratagene), pBAD, pThioHis, pTrcHis (above Invitrogen), pKK223-3, pGEX2T (above Amersham Pharmacia Biotech) ), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol, Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (Stratagene), pTrs30 [Prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [Escarchia coli JM109 / pTrS32 ( Prepared from FERM BP-5408], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Laid-Open Patent Publication (Sho) 60-221091], pGKA2 [esquelizia coli IGKA2 (PERM BP- 6798), Japanese Laid-Open Patent Publication No. 60-221091], pTerm2 (US 4686191, US 4939094, US 5160735), pSupex, pUB110, pTP5, pCl94, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia) and pET system (Novagen).
또한, 형질전환 숙주세포는 본 발명의 융합단백질을 발현할 수 있는 것이면 세균, 효모, 동물세포, 곤충세포, 식물세포 어느 것이나 사용할 수 있다. In addition, as long as the transformed host cell can express the fusion protein of the present invention, any one of bacteria, yeast, animal cells, insect cells and plant cells can be used.
본 발명의 바람직한 실시예에서는 상기 융합단백질을 코딩하는 서열번호 7의 DNA 서열을 포함하는 대장균 발현벡터 pTH-pol:Cry1Ac을 제조하고, 상기 벡터로 형질전환된 대장균 형질전환체를 2004년 1월 7일 한국생명공학연구원 유전자은행에 기탁하였다(기탁번호 KCTC 10577BP). In a preferred embodiment of the present invention, the E. coli expression vector pTH-pol: Cry1Ac comprising the DNA sequence of SEQ ID NO: 7 encoding the fusion protein was prepared, and the E. coli transformant transformed with the vector 7 January 2004 It was deposited at the Korea Biotechnology Research Institute Gene Bank (Accession No. KCTC 10577BP).
또한, 본 발명은 배큘로바이러스의 다각체 단백질과 독성단백질이 융합된 융합단백질 또는 상기 융합단백질을 발현하는 형질전환 대장균 세포를 유효성분으로 함유하는 해충 방제용 살충제를 제공한다. In addition, the present invention provides a pesticide for insect control containing a fusion protein fusion protein or a toxic protein of the baculovirus or transformed E. coli cells expressing the fusion protein as an active ingredient.
본 발명의 융합단백질 및 형질전환 대장균 전세포를 이용하여 표적해충을 대상으로 독성활성 실험을 수행한 결과, 바실러스에서 생산된 독성단백질 또는 시판되고 있는 생물살충제와 동등한 독성활성을 나타내었다(표 1 참조). As a result of conducting toxic activity experiments on target pests using the fusion protein and transformed E. coli whole cells of the present invention, it showed toxic activity equivalent to that of toxic proteins produced by Bacillus or commercial biopesticides (see Table 1 ). ).
본 발명의 살충제의 표적해충에는 본 발명의 살충제가 독성활성을 나타내는어떠한 해충도 포함될 수 있으며, 바람직하게는 배추좀나방, 파밤나방, 담배거세미나방, 양배추은무늬 밤나방 및 배추흰나비가 포함될 수 있다.The target pests of the insecticide of the present invention may include any pests of which the insecticide of the present invention exhibits toxic activity, and may preferably include Chinese cabbage moth, parchment moth, tobacco germweed moth, cabbage silver pattern moth and cabbage butterfly.
아울러, 본 발명은 상기 대장균 형질전환체를 배양하여 융합단백질을 분리하는 단계를 포함하는, 배큘로바이러스 다각체 단백질과 독성단백질이 융합된 융합단백질의 생산방법을 제공한다. In addition, the present invention provides a method for producing a fusion protein fused to a baculovirus polyhedral protein and a toxic protein comprising culturing the E. coli transformant to separate the fusion protein.
상기 융합단백질의 분리에는 단백질의 분리에 사용되는 통상적인 분리방법, 예를 들면, 원심분리, 여과, 염석 및 크로마토그래피법 등이 사용될 수 있으며 본 발명의 다각체 단백질과 독성단백질이 융합된 융합단백질은 세포내 응집체 형태로 발현되므로 수용성 단백질로부터 원심분리를 이용하여 쉽게 분리하여 생산하는 것이 바람직하다.The separation of the fusion protein may be a conventional separation method used for separation of proteins, for example, centrifugation, filtration, salting and chromatographic methods may be used and the fusion protein fusion of the polyhedral protein and the toxic protein of the present invention. Since it is expressed in the form of intracellular aggregates, it is preferable to easily separate and produce from water-soluble proteins by centrifugation.
이하, 본 발명을 실시예를 통하여 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention by way of example, the scope of the present invention is not limited to these examples.
<실시예 1> 다각체 단백질과 Bt 독성단백질이 융합된 융합단백질의 제조Example 1 Preparation of Fusion Protein Converging Polygonal Protein and Bt Toxic Protein
<1-1> 융합단백질의 발현벡터 제조<1-1> Expression Vector Preparation of Fusion Protein
pTrcHisC 벡터(Invitrogen, 미국)의 NheI과 BglII 제한효소 자리에 다각체 단백질과 Bt 독성단백질이 융합된 융합단백질을 생산하는 유전자를 삽입하기 위하여, 다각체 단백질을 코딩하는 735 bp 유전자 polh(Autographa californica nuclear polyhedrosis virus(AcNPV), Genebank No. L22858)와 Bt 독성단백질을 코딩하는 1836 bp 유전자 Cry1Ac(Bacillus thuringiensis subsp. HD-73, Genebank No. M11068)를, 상기 두 유전자를 포함하고 있는 pTen21-Pol1Ac 벡터를 주형으로 하고, 서열번호 1(정방향 프라이머)과 서열번호 2(역방향 프라이머, UAA 정지코돈 서열을 포함한다)의 프라이머를 이용한 PCR 반응을 통하여 동시에 대량 증폭시켰다. 상기 PCR 산물을 pTrcHisC 벡터의 NheI과 BglII 제한효소 자리에 삽입하여 만들어진 벡터를 pTH-pol:Cry1Ac(7033 bp)이라 명명하고(도 1), 대장균 TOP10 (Invitrogen, 미국)에 도입하여 제조된 대장균 형질전환체를 2004년 1월 7일자로 한국생명공학연구원 유전자은행에 기탁하였다(기탁번호 KCTC 10577BP). The 735 bp gene polh ( Autographa), which encodes a polyhedral protein, is inserted into the Nhe I and Bgl II restriction enzyme sites of the pTrcHisC vector (Invitrogen, USA) to insert a gene that produces a fusion protein fused with a polyhedral protein and Bt toxic protein. californica nuclear polyhedrosis virus (AcNPV), Genebank No. L22858) and the 1836 bp gene Cry1Ac ( Bacillus thuringiensis subsp. The vector was used as a template, and mass amplification was carried out simultaneously by PCR using primers of SEQ ID NO: 1 (forward primer) and SEQ ID NO: 2 (reverse primer, including UAA stop codon sequence). A vector produced by inserting the PCR product into Nhe I and Bgl II restriction sites of the pTrcHisC vector was named pTH-pol: Cry1Ac (7033 bp) ( FIG. 1 ) and was prepared by introducing into E. coli TOP10 (Invitrogen, USA). E. coli transformants were deposited with the Korea Biotechnology Research Institute Gene Bank on January 7, 2004 (Accession No. KCTC 10577BP).
<1-2> 형질전환체의 배양 및 융합단백질의 발현 유도<1-2> Culture of transformants and expression of fusion proteins
상기 실시예 <1-1>에서 얻어진 발현벡터 pTH-pol:Cry1Ac를 대장균 BL21(Invitrogen, 미국)에 CaCl2 버퍼를 이용한 열충격 방법으로 도입시키고 앰피실린으로 선별된 형질전환체를 M9(6 g/L Na2HPO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 1% 포도당, 2 mM MgSO4, 0.1 mM CaCl2) 배지를 이용하여 배양하였다. 배양액의 흡광도가 600 nm에서 0.5가 되었을 때, IPTG를 첨가(1 mM)하여 재조합 융합단백질의 발현을 유도하였다. The expression vector pTH-pol: Cry1Ac obtained in Example <1-1> was introduced into E. coli BL21 (Invitrogen, USA) by a thermal shock method using CaCl 2 buffer, and the transformants selected with ampicillin were M9 (6 g / Culture was performed using L Na 2 HPO 4 , 0.5 g / L NaCl, 1 g / L NH 4 Cl, 1% glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 ) medium. When the absorbance of the culture was 0.5 at 600 nm, IPTG was added (1 mM) to induce the expression of the recombinant fusion protein.
<1-3> 융합단백질의 발현량 측정 <1-3> Expression of fusion proteins
융합단백질의 발현량 측정은 웨스턴 블롯(Western blot) 방법을 이용하여 수행하였다(도 3 및 도 4 참조). 매 시간 배양액으로부터 얻은 1 ml의 시료를 4℃, 5,000 rpm에서 10 분간 원심분리 하여 침전물(pellet)을 -80℃에 보관하여 두고 전세포(whole cell) 시료로 사용하였다. 상기 전세포 시료를 600 nm에서의 흡광도를 1이 되게 희석한 후, SDS-PAGE용 버퍼(0.5M Tris-HCl(pH 6.8), 10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.25% bromophenol blue) 100 ㎕를 첨가하고 100℃에서 5 분간 끓여 변성시켰다. 상기와 같이 준비된 시료를 15% SDS-폴리 아크릴아마이드 젤 상에서 전기영동을 수행한 뒤, 15V 전압 하에서 나이트로셀룰로즈 막 에 이동시키고 히스티딘 단백질 항체(Santa Cruz, CA, 미국)를 이용하여 발색반응 후 영상분석소프트웨어(Gel-Pro Analyzer, Media Cybernetics, 미국)를 이용하여 정량하였다(도 4). The expression level of the fusion protein was measured using a Western blot method (see FIGS . 3 and 4 ). 1 ml of the sample obtained from the culture medium was centrifuged at 4 ° C. and 5,000 rpm for 10 minutes to store the pellet at −80 ° C. and used as a whole cell sample. After diluting the absorbance at 600 nm to 1, the whole cell sample was prepared using SDS-PAGE buffer (0.5M Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.25%). 100 μl of bromophenol blue) was added thereto, and the mixture was boiled at 100 ° C. for 5 minutes for denaturation. After the electrophoresis on the 15% SDS-polyacrylamide gel, the sample prepared as described above was transferred to the nitrocellulose membrane under 15V voltage and image after color reaction using histidine protein antibody (Santa Cruz, CA, USA). Quantification was performed using analysis software (Gel-Pro Analyzer, Media Cybernetics, USA) ( FIG. 4 ).
그 결과, 본 발명의 융합단백질의 발현은 발현 유도 2 시간 이후, 급격하게 증가하여 10시간 이후까지 점차적으로 증가됨을 알 수 있다. As a result, it can be seen that the expression of the fusion protein of the present invention increases rapidly after 2 hours of expression induction and gradually increases after 10 hours.
<1-4> 웨스턴 블롯에 의한 융합단백질의 발현 형태 확인<1-4> Confirmation of the expression form of the fusion protein by Western blot
실제로 본 발명의 융합단백질이 대장균에서 응집체(inclusion body) 형태로 발현되는지 확인하기 위하여 전세포(whole cell) 시료, 세포 파쇄물 시료 및 상등액 시료를 각각 히스티딘 단백질 항체를 이용하여 웨스턴 블롯을 수행한 결과, 도 5에서 볼 수 있는 바와 같이, 세포 파쇄물 시료에서는 본 발명의 융합단백질 밴드가 검출되었으나, 상등액 시료의 경우에는 검출되지 않았다. 따라서, 본 발명의 융합단백질은 대장균에서 응집체의 형태로 발현됨을 알 수 있다.In fact, in order to confirm that the fusion protein of the present invention is expressed in the form of an inclusion body in Escherichia coli, a whole cell sample, a cell lysate sample and a supernatant sample were each subjected to Western blot using histidine protein antibody. As can be seen in Figure 5 , the cell lysate sample was detected in the fusion protein band of the present invention, but not in the supernatant sample. Therefore, it can be seen that the fusion protein of the present invention is expressed in the form of aggregates in E. coli.
<실시예 2> 융합단백질의 분리 Example 2 Isolation of Fusion Protein
본 발명의 다각체-Bt 독소 융합단백질은 실시예 <1-4>에서 확인된 바와 같이, 대장균 내에서 응집체 형태로 발현되어 원심분리기로 상등액과 세포 파쇄물을 분리하는 경우 파쇄물 부분에 존재하기 때문에 응집체의 분리방법을 이용하여 분리해 낼 수 있다. Polygon-Bt toxin fusion protein of the present invention, as confirmed in Example <1-4>, is expressed in the form of aggregates in Escherichia coli and aggregates because they are present in the fraction of the supernatant and cell lysates when centrifuge separates them. It can be separated using the separation method of.
구체적으로, 4℃, 5500 rpm의 조건에서 배양액을 15 분간 원심분리 하여 대 장균 세포를 회수하고 세포무게 당 3 ml의 용해 버퍼(50 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 8.0; 100 mM NaCl)를 넣고 부드럽게 세포조각을 소생시켰다. 그 후 4℃에서 세포무게 당 4 ㎕의 100 mM PMSF(단백질분해효소 활성 저해제; Sigma)와 10 mg/ml의 리소자임(lysozyme)을 80 ㎕ 가하여 약 20 분 동안 부드럽게 저어주고 (일반적으로 이 과정에서 PMSF를 넣었지만, 단백질 분해효소 절단 실험의 경우에는 단백질 분해효소 활성 저해제의 영향이 없어야 하기 때문에 PMSF를 첨가하지 않았다) 세포무게 당 4 mg의 데옥시콜릭산(deoxycholic acid)을 가하여 더 이상 끈적거리지 않을 때까지 37℃ 배양기에 놓아두었다(점도가 커지는 경우는 1 mg/ml 의 DNase I을 20 ㎍ 첨가하였다). 상기 세포 용액을 14,000 rpm에서 원심분리 하여 얻어진 침전물(pellet)을 1 ml의 물에 현탁시키고 이를 다시 14,000 rpm로 원심분리 하여 얻어진 침전물(pellet)을 0.1 M Tris-Cl(pH 8.5) 버퍼로 세척하였다. 위 과정을 여러 번 반복하여 본 발명의 융합단백질 응집체만이 높은 순도로 분리되도록 하였다. Specifically, E. coli cells were recovered by centrifuging the culture solution at 4 ° C and 5500 rpm for 15 minutes, and 3 ml of lysis buffer (50 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 8.0; 100) per cell weight was collected. mM NaCl) was added to gently resuspend the cells. Then at 4 ° C. add 4 μl of 100 mM PMSF (Protease Activity Inhibitor; Sigma) and 80 μl of 10 mg / ml lysozyme per cell weight and stir gently for about 20 minutes (generally in this process PMSF was added, but proteolytic cleavage experiments were not added because PMSF should not be affected by protease activity inhibitors.) 4 mg of deoxycholic acid per cell weight was no longer sticky. It was left in the incubator at 37 ° C until not used (20 μg of 1 mg / ml of DNase I was added when the viscosity increased). The pellet obtained by centrifuging the cell solution at 14,000 rpm was suspended in 1 ml of water and again centrifuged at 14,000 rpm to wash the pellet obtained with 0.1 M Tris-Cl (pH 8.5) buffer. . The above procedure was repeated several times so that only the fusion protein aggregates of the present invention were separated with high purity.
<실시예 3> 특이 단백질 분해효소를 이용한 다각체-Bt 독소 융합단백질의 절단Example 3 Cleavage of Polyhedral-Bt Toxin Fusion Protein Using Specific Protease
상기 융합단백질의 다각체 단백질 부분이 배큘로바이러스 유래의 다각체 단백질과 마찬가지로 특이 알칼리성 단백질 분해효소에 의하여 분해되는지 알아보기 위하여, α-키모트립신 및 TPCK(treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone) 처리된 트립신을 이용한 다각체-Bt 독소 융합단백질의 절단 실험을 수행하였다. In order to determine whether the polyhedral protein portion of the fusion protein is degraded by a specific alkaline protease like the polyhedral protein derived from baculovirus, α-chymotrypsin and TPCK (treated with L- (tosylamido-2-phenyl) The cleavage experiment of the polyhedral-Bt toxin fusion protein using ethyl chloromethyl ketone) treated trypsin was performed.
상기 α-키모트립신(C. Novillo 등, 1997, Archives of Insect Biochemistry and Physiology 36, 181-201; M.J. Lee 등, 1995, Insect Biochem. Molec. Biol. 25, 49-61; K.A. Johnston 등, 1995, Insect Biochem. Molec. Biol. 25, 375-383) 및 트립신(R. Milne & H Kaplan, 1993, Insect Biochem. Molec. Bio. 6, 663-673)은 곤충 중장의 특이 알칼리성 단백질 분해효소의 성분이 상기 효소들과 비슷한 물성을 가지고 있다는 연구 결과를 토대로 선택되었다. 키모트립신은 페닐알라닌, 트립토판 및 타이로신과 같은 아미노산이 존재할 경우 펩타이드의 결합을 절단하는 단백질 분해효소이고, 트립신은 라이신 및 아르기닌과 같은 아미노산이 존재할 경우 펩타이드 결합을 절단하는 단백질 분해효소이다. Α-chymotrypsin (C. Novillo et al., 1997, Archives of Insect Biochemistry and Physiology 36, 181-201; MJ Lee et al. , 1995, Insect Biochem. Molec. Biol. 25, 49-61; KA Johnston et al. , 1995, Insect Biochem.Molec . Biol. 25, 375-383) and trypsin (R. Milne & H Kaplan, 1993, Insect Biochem.Molec . Bio. 6, 663-673) It was selected based on the results of research showing similar physical properties with the enzymes. Chymotrypsin is a protease that cleaves peptide bonds when amino acids such as phenylalanine, tryptophan and tyrosine are present, and trypsin is a protease that cleaves peptide bonds when amino acids such as lysine and arginine are present.
절단실험을 위하여, <실시예 2>에서 분리한 응집체 형태의 다각체-Bt 독소 융합단백질을 강 알칼리 상태(pH 12)에서 약 3 시간 동안 용해시킨 3 ml 시료를 pH 가 다른 PBS(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 , pH 8.5, pH 12)로 각각 1/8 희석을 시킨 다음, 상기 단백질 분해효소를 2:1(질량/부피, 단백질 분해효소/용해된 다각체-Bt 독소 융합단백질) 비율로 가하여 반응시켰다. 반응 5 분 후 단백질 분해효소 활성 저해제인 100 mM PMSF를 5 ㎕ 가하여 반응을 정지시키고 반응 산물을 50 mM Tris-HCl(pH 8.5)에서 약 2 일 동안 4℃에서 투석하였다. 상기 시료를 15% SDS-폴리 아크릴아마이드 젤에 전기영동한 뒤 쿠마시블루(Coomasie blue) 염색 또는 실버 염색(Bio-Rad, 미국)을 이용하여 단백질 밴드를 검출하였다.For the cleavage experiment, a 3 ml sample obtained by dissolving the aggregate-polygon-Bt toxin fusion protein isolated in Example 2 in a strong alkaline state (pH 12) for about 3 hours was subjected to PBS (137 mM NaCl) having a different pH. , 1/8 dilution with 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 8.5, pH 12), and then the protease was 2: 1 (mass / volume, proteolytic). Enzyme / dissolved polymer-Bt toxin fusion protein) ratio and reacted. After 5 minutes, 5 µl of 100 mM PMSF, a protease activity inhibitor, was added to stop the reaction, and the reaction product was dialyzed at 4 ° C. for about 2 days in 50 mM Tris-HCl (pH 8.5). The samples were electrophoresed on 15% SDS-polyacrylamide gel and protein bands were detected using Coomasie blue staining or silver staining (Bio-Rad, USA).
도 6 및 도 7에서 나타난 바와 같이, 단백질 분해효소 처리 5 분 후의 시료를 실버 염색을 이용한 SDS-PAGE 분석을 하였을 경우, 다각체-Bt 독소 융합단백질 밴드(98 kDa)가 사라짐을 알 수 있다. 절단된 아미노산 절편들은 밴드 아랫부분의 진한 부분으로 나타나고 동시에 65 kDa 위치에서 진한 밴드가 나타나는 것을 볼 수 있었다. As shown in FIG . 6 and FIG. 7 , when the SDS-PAGE analysis using silver staining of the
보다 정확한 결과 확인을 위하여, 웨스턴 블롯 분석(도 8 내지 도 11)을 수행한 결과, 다각체-Bt 독소 융합단백질 밴드는 절단 반응 후 분해되어 사라지고 다각체 단백질에 비하여 어느 정도 단백질 분해효소에 저항할 수 있는 Bt 독소 단백질 부분(65 kDa)만이 밴드로 나타나는 것을 확인할 수 있었다. 이 65 kDa의 단백질 밴드는 융합단백질의 앞부분이 절단되어 활성을 가지는 크기와 일치한다(도 12). In order to confirm the results more accurately, Western blot analysis ( FIGS . 8-11 ) showed that the polyhedral-Bt toxin fusion protein bands were degraded and disappeared after cleavage reaction, and to some extent proteolytic enzyme resistance compared to the polyhedral protein. Only Bt toxin protein part (65 kDa) could be seen as a band. This 65 kDa protein band matches the size at which the front of the fusion protein is cleaved and active ( FIG. 12 ).
또한, Cry1Ac 항체(서울대학교, 한국)를 이용하여 분석하였을 경우에는 약 65 kDa과 30 kDa에서 진한 밴드가 검출되었으나(트립신의 경우, 보다 확실하게 검출되었음) 히스티딘 단백질 항체(Santa Cruz Biotechnology, 미국)를 이용하여 분석하였을 경우에는 상기 두 밴드가 검출되지 않았다. 상기 결과로부터 곤충의 중장에 있는 단백질 분해효소와 유사한 특이 단백질 분해효소에 의하여 다각체 단백질이 분해됨으로 해서 다각체 단백질의 앞 부분에 위치한 히스티딘 마커가 사라졌음을 알 수 있다. In addition, when analyzed using Cry1Ac antibody (Seoul National University, Korea), a dark band was detected at about 65 kDa and 30 kDa (in the case of trypsin, more reliably detected) histidine protein antibody (Santa Cruz Biotechnology, USA) The two bands were not detected when analyzed using. From the above results, it can be seen that the histidine marker located at the front of the polyhedral protein disappeared by degrading the polyhedral protein by a specific protease similar to the proteolytic enzyme in the middle of the insect.
<실시예 4> 다각체-Bt 독소 융합단백질의 독성활성 실험Example 4 Toxicity Activity Test of Polygon-Bt Toxin Fusion Protein
본 발명의 다각체-Bt 독소 융합단백질의 독성 활성을 확인하기 위하여 전세포(whole cell) 시료, 분리·정제된 Bt 독소 단백질 시료 및 다각체-Bt 독소 융합단백질 시료를 준비하고 표적해충으로 알려진 배추좀나방에 대한 독성활성 실험을 수행하였다(표 1 참조). 대조 시료로는 바실러스에서 생산 및 분리된 Bt 독소 단백질(표 1의 Standard)(동부한농화학, 한국)과 생물살충제 제품으로 시판되고 있는 투리싸이드(표 1의 Thuricide)(BONIDE, 미국)가 사용되었다. In order to confirm the toxic activity of the polyhedron-Bt toxin fusion protein of the present invention, a whole cell sample, an isolated and purified Bt toxin protein sample, and a polyhedral-Bt toxin fusion protein sample were prepared and known as target pests. Toxic activity experiments were performed on the moths (see Table 1 ). As control samples, Bt toxin protein produced and isolated from Bacillus (Standard of Table 1 ) (Eastern Agrochemical, Korea) and Turiicide ( Table 1 ) (BONIDE, USA) sold as a biopesticide product were used. .
각각의 시료는 분말상태로 건조시킨 후 희석용액(NaCl 8.5g, K2HPO4 6.0g, KH2PO4 3.0g, Tween 80 1%)에 적절한 농도로 희석시키고 배추잎을 약 2 분 동안 각각의 시료 희석용액에 침지하여 시료가 붙도록 한 다음 배추잎이 잘 마른 후 페트리 디쉬에 배추잎을 넣고 각각의 배추잎에 약 12 마리의 배추좀나방을 올려놓았다. 상온에서 적절한 습도와 함께 배양하고 3 일 동안 표적생물의 치사율을 측정하였다.Each sample was dried in powder form, diluted to an appropriate concentration in dilute solution (NaCl 8.5g, K 2 HPO 4 6.0g, KH 2 PO 4 3.0g, Tween 80 1%), and cabbage leaves were kept for about 2 minutes. After dipping in the dilution solution of the sample so that the sample was attached to the cabbage leaves are well dried, put the cabbage leaves in a petri dish and put about 12 cabbage moths on each cabbage leaf. Incubation with appropriate humidity at room temperature and mortality of the target organisms were measured for 3 days.
그 결과 표 1에 나타난 바와 같이, 본 발명의 다각체-Bt 독소 융합단백질(표 1의 Pol-Bt(2))은 시판되고 있는 생물살충제 제품이나 바실러스에서 생산된 Bt 독소 단백질과 유사한 독성활성을 가지고 있음을 알 수 있다. 구체적으로, 각 시료 처리 1일, 2일, 3일 후의 배추좀나방의 생충율과 방제가를 비교한 결과 본 발명의 다각체-Bt 독소 융합단백질 및 전세포 시료 모두 상기 대조시료를 처리한 경우와 비슷한 생충율 및 방제가를 나타내었다.As a result, as shown in Table 1 , the polyhedral-Bt toxin fusion protein of the present invention (Pol-Bt (2) of Table 1 ) has similar toxic activity to that of commercially available biopesticide products or Bt toxin proteins produced by Bacillus It can be seen that Specifically, as a result of comparing the virulence rate and control value of
따라서, 상기 독성활성실험 결과로부터 대장균에서 생산된 본 발명의 다각체 -Bt 독소 융합단백질 및 상기 융합단백질을 발현하고 있는 대장균 전세포는 바실러스에서 생산되는 독성단백질의 독성을 그대로 유지하여 생물살충제로 이용할 수 있음이 판명되었다. Therefore, the polyhedral -Bt toxin fusion protein of the present invention produced by E. coli and the E. coli whole cells expressing the fusion protein can be used as biopesticides by maintaining the toxicity of the toxic protein produced in Bacillus. It turned out to be possible.
본 발명의 배큘로바이러스의 다각체 단백질과 바실러스의 독성단백질이 융합된 다각체-Bt 독소 융합단백질은 대장균에서 응집체의 형태로 발현되므로 숙주세포의 부담을 덜어 대량생산이 가능하고 분리정제가 간단하며 독성활성을 그대로 유지하여 저렴하고 효과적인 생물살충제의 생산에 널리 이용될 수 있다. The polyhedral-Bt toxin fusion protein in which the polyhedral protein of baculovirus and the toxic protein of Bacillus of the present invention is fused is expressed in the form of aggregates in Escherichia coli, so as to reduce the burden of the host cell, mass production is simple, and purification is easy. It can be widely used for the production of cheap and effective biopesticides by maintaining toxic activity as it is.
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| US4870023A (en) * | 1987-03-16 | 1989-09-26 | American Biogenetic Sciences, Inc. | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
| US5770192A (en) * | 1991-03-22 | 1998-06-23 | Roussel-Uclaf | Biological control agents |
| US5885569A (en) * | 1994-03-25 | 1999-03-23 | Zeneca Limited | Biological insect control agent |
| KR19990079361A (en) * | 1998-04-03 | 1999-11-05 | 강석권 | Recombinant baculovirus, a method for producing the same, and microbial pesticides containing the same |
| KR20040001008A (en) * | 2002-06-26 | 2004-01-07 | 학교법인 포항공과대학교 | A fusion protein of baculoviral polyhedrin protein-target protein and method for producing the target protein using the same |
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| US4870023A (en) * | 1987-03-16 | 1989-09-26 | American Biogenetic Sciences, Inc. | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
| US5770192A (en) * | 1991-03-22 | 1998-06-23 | Roussel-Uclaf | Biological control agents |
| US5885569A (en) * | 1994-03-25 | 1999-03-23 | Zeneca Limited | Biological insect control agent |
| KR19990079361A (en) * | 1998-04-03 | 1999-11-05 | 강석권 | Recombinant baculovirus, a method for producing the same, and microbial pesticides containing the same |
| KR20040001008A (en) * | 2002-06-26 | 2004-01-07 | 학교법인 포항공과대학교 | A fusion protein of baculoviral polyhedrin protein-target protein and method for producing the target protein using the same |
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