KR100437889B1 - Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof - Google Patents
Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof Download PDFInfo
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- KR100437889B1 KR100437889B1 KR1020030059324A KR20030059324A KR100437889B1 KR 100437889 B1 KR100437889 B1 KR 100437889B1 KR 1020030059324 A KR1020030059324 A KR 1020030059324A KR 20030059324 A KR20030059324 A KR 20030059324A KR 100437889 B1 KR100437889 B1 KR 100437889B1
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- South Korea
- Prior art keywords
- reverse transcriptase
- virus
- leu
- ala
- seq
- Prior art date
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Abstract
Description
본 발명은 세망내피증 바이러스(Reticuloendotheliosis Virus)의 역전사 효소 및 이를 생산하는 방법에 관한 것으로서, 보다 상세하게는 서열 번호 2로 기재되는 아미노산 서열을 가지는 세망내피증 바이러스의 역전사 효소와 서열번호 1로 기재되는 염기서열을 가지는 세망내피증 바이러스의 역전사 효소 및 이를 생산하는 방법에 관한 것이다.The present invention relates to a reverse transcriptase of Reticuloendotheliosis Virus and a method for producing the same, more specifically, the reverse transcriptase of reticuloendothelial virus having an amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: The present invention relates to a reverse transcriptase enzyme and a method of producing the same.
일반적으로, 바이러스는 코어라고 불리는 중심 부분에 DNA 또는 RNA를 유전 물질로 가지고 있으며, 그 둘레가 단백질 껍데기로 덮인 간단한 구조로 되어 있다. 동물과 식물 세포에 감염되면 감염된 세포(숙주)가 갖고 있는 효소를 이용하여 유전 물질의 복제와 단백질 합성을 하고 증식한다. 자기 자신만으로는 증식할 수 없기 때문에 엄밀한 의미에서는 생물은 아니지만 미생물로 분류되는 일이 많다. 바이러스에는 DNA 바이러스와 RNA 바이러스가 있는데, 이 중 RNA 바이러스는 RNA를 유전 물질로서 가지고 있는 바이러스로, 숙주에 감염하면 RNA에서 DNA를 만들고 그 DNA를 숙주 염색체에 조합시켜 복제가 만들어지게 된다. 이와 같이, RNA 바이러스는 본래의 DNA →RNA라는 방향과는 거꾸로 RNA →DNA라는 역방향으로 전사를 일으키는 효소를 가지기 때문에 '레트로 바이러스(retrovirus)'라고 불린다.Generally, a virus has DNA or RNA as a genetic material in its central part, called the core, and has a simple structure surrounded by protein shells. When infected with animal and plant cells, the enzymes contained in the infected cells (hosts) are used to replicate and multiply genetic material and synthesize proteins. Because they cannot proliferate on their own, they are often not microorganisms, but are classified as microorganisms. Viruses include DNA viruses and RNA viruses, among which RNA viruses contain RNA as a genetic material, and when infected with a host, DNA is produced from RNA and combined with the DNA on a host chromosome to create a replica. As such, the RNA virus is called a "retrovirus" because it has an enzyme that causes transcription in the reverse direction of RNA → DNA in the reverse direction of the original DNA → RNA.
레트로 바이러스는 먼저 바이러스의 RNA가 숙주 세포 내로 들어가고 바이러스의 RNA는 상보적 DNA가닥의 합성을 위한 주형으로 쓰이며, 이로부터 합성된 DNA 가닥은 DNA 합성효소 활성을 갖는 효소의 작용을 통해 이중가닥으로 되는 과정을 거쳐 숙주세포 내에서 복제된다. 즉, 레트로 바이러스는 유전자 내에 역전사 효소를 암호화하는 유전자를 가지고 있어, 스스로 바이러스의 RNA 유전자를 DNA로 역전사 시켜 숙주의 염색체 내부로 들어가게 된다. 숙주의 내부 염색체에 접합된 DNA는 바이러스의 단백질 합성을 시작한다. 상기와 같은 특징으로, 레트로 바이러스들은 DNA 바이러스에 비해 더 많은 유전물질을 수용할 수 있고, 다양한 생물 종의 여러 세포에 침입할 수 있다. 이러한 레트로 바이러스는 동물뿐만 아니라 사람에 있어서도 HIV, HTLV-I (사람 T 세포 백혈병 바이러스)등, 면역부전이나 종양을 일으키는 바이러스로서 알려져 있으며, 동물과 식물 세포에 유전자를 운반하는 '벡터'로서 이용되는 일도 있다.Retroviruses first enter the viral RNA into the host cell and the viral RNA is used as a template for the synthesis of complementary DNA strands, from which the DNA strands are double-stranded through the action of enzymes with DNA synthase activity. The process is replicated in the host cell. In other words, retroviruses have genes encoding reverse transcriptase in their genes, which themselves reverse-transcribe the RNA genes of the virus into DNA and enter the chromosome of the host. DNA conjugated to the host's internal chromosome starts the protein synthesis of the virus. As such, retroviruses can accommodate more genetic material than DNA viruses and can invade various cells of various species. Such retroviruses are known as viruses that cause immunodeficiency or tumors, such as HIV and HTLV-I (human T-cell leukemia virus) in humans as well as animals, and are used as 'vectors' that carry genes to animal and plant cells. There is work.
이러한 레트로 바이러스의 역전사 효소는 RNA를 주형으로 하여 상보적인 염기서열의 cDNA를 합성하는 효소로서 RT-PCR 및 DNA 칩 등 RNA를 검출하는 실험에서 없어서는 안 되는 중요한 효소이며, RNA를 주형으로 cDNA를 합성하여 질병을 진단하거나 유전자 클로닝을 하는데 많이 사용된다(Mocharla et al.,Gene, 1990, 99:271-275). 따라서 이러한 산업적 이용가치가 높은 역전사 효소를 클로닝하여 재조합 단백질로 대장균에서 대량 발현하여 생산하게 되면 질병의 진단 또는 유전자 클로닝 등에 유용하게 사용할 수 있다.The reverse transcriptase of the retrovirus is an enzyme that synthesizes cDNA of complementary nucleotide sequences using RNA as a template, and is an essential enzyme in experiments for detecting RNA such as RT-PCR and DNA chips. It is widely used to diagnose diseases or to clone genes (Mocharla et al., Gene , 1990, 99: 271-275). Therefore, the high-value reverse transcriptase of industrial use can be cloned to produce a large amount of recombinant E. coli as a recombinant protein, which can be useful for diagnosing diseases or gene cloning.
대장균에서 재조합 단백질을 생산함에 있어 강력하면서도 유도 가능한 프로모터를 갖춘 다양한 발현벡터가 개발되어 외래 단백질의 생산에 이용되어 왔다. 그러나 이러한 재조합 단백질이 대장균에서 과다 발현될 때 세포함유체(inclusion body)로 알려진 불용성 결합체를 형성하는 경우가 종종 보고되어 왔다. 이런 불용성 단백질은 수용성 단백질로부터 쉽게 분리되는 특성을 갖고, 비자연적이고 환원된 형태로 기능적 활성을 갖지 못한다. 세포함유체의 형성은 생물 공정 과정과 단백질의 특성에 따라 장점이 되기도 하고 단점이 될 수도 있다. 구체적으로, 재조합 단백질이 대장균에서 세포함유체를 형성할 경우 세포파쇄 후 단순한 원심 분리방법으로 순도 80% 이상으로 손쉽게 정제가능하다는 초기 분리단계에서의 용이성 및 세포내 프로테아제(protease)의 공격에 의한 단백질 파괴(proteolysis) 현상이 감소하는 장점을 가지는 반면, 비활성 상태의 세포함유체를 다시 활성이 있는 단백질로 전환하기 위하여 변성 및 구조재형성(denaturation & refolding) 과정이 필요하고 구조재형성 과정에서의 낮은 분리 효율을 보여 정제 과정에서 비용이 많이 든다는 단점이 있다. 이러한 세포함유체의 형성은 숙주-벡터 시스템, 단백질의 특성, 배양 및 발현조건에 따라 영향을 받을 수 있으나 아직까지 일반적인 규칙성을 발견할 수는 없다.In producing recombinant proteins in E. coli, a variety of expression vectors with powerful and inducible promoters have been developed and used in the production of foreign proteins. However, it has often been reported that when these recombinant proteins are overexpressed in Escherichia coli, they form insoluble conjugates known as inclusion bodies. These insoluble proteins have the property of being easily separated from water soluble proteins and have no functional activity in their unnatural and reduced form. The formation of cell media can be both an advantage and a disadvantage, depending on the biological process and the nature of the protein. Specifically, when the recombinant protein forms a cell-containing medium in Escherichia coli, it is easy to be purified to a purity of 80% or more by simple centrifugation after cell disruption, and the protein due to the attack of intracellular protease is easy. While proteolysis has the advantage of decreasing, in order to convert inactive cell-containing media back to active proteins, denaturation and refolding processes are required and low separation efficiency in the process of structural reformation There is a drawback in that the purification process is expensive. The formation of such cell media may be affected by the host-vector system, the characteristics of the protein, the culture and the expression conditions, but there is no general regularity yet found.
일반적으로 재조합 단백질을 세포질 내에 수용성 단백질의 형태로 생산할 경우 특히 이중 황화 결합(disulfide bond)의 형성이 필요한 단백질인 경우 발현시스템의 제작 및 배양 조건은 상당히 까다로워진다. 반면에 세포함유체 형태로 생산할 경우 벡터 제작 및 배양조건은 단순하다는 장점을 갖고 있으며 이는 세포고농도 배양(HCDC)과 같은 방법으로 대량생산이 가능하다.In general, when the recombinant protein is produced in the form of a water-soluble protein in the cytoplasm, especially in the case of a protein that requires the formation of a double sulfide bond (disulfide bond), the production and culture conditions of the expression system is quite difficult. On the other hand, when producing in cell-containing form, the vector production and culture conditions have the advantage of simplicity, which can be mass-produced by the same method as high cell concentration culture (HCDC).
원하는 단백질이 세포함유체 형태로 생산이 되었을 경우 다시 활성이 있는 단백질로 만들기 위한 변성 및 구조재형성 과정이 필요하며 이 과정에서 단백질 고유의 성질에 따라 정제 효율이 달라지기 때문에 단백질의 손실을 최소화하는 정제과정의 개발이 매우 중요하다.If the protein of interest is produced in cell-containing form, denaturation and structural remodeling process are required to make the protein active again. In this process, the purification efficiency varies depending on the intrinsic properties of the protein, thereby minimizing protein loss. The development of the process is very important.
한편, 세망내피증은 레트로바이러스(retrovirus)에 속하는 세망내피증 바이러스(reticuloendotheliosis virus; REV)가 원인체로 작용하며, 면역능력이 심하게 저하되고 발육부진과 급, 만성 종양이 발생되는 증상을 보이는 바이러스성 질병으로 닭, 칠면조, 오리 등에 주로 발병된다. 상기 세망내피증은 호주, 일본, 미국, 영국 등 많은 나라에서 발생이 보고되고 있으며 특히 호주와 일본에서는 상기 세망내피증 바이러스가 오염된 마렉병 백신을 사용함으로써 대대적으로 발생한 사례도 있었다. 국내에서는 1991년 피해사례가 최초로 확인되었으며 국내에서 분리된 세망내피증 바이러스는 심한 면역억제능과 증체율저하 및 만성종양을 일으키는 것으로 확인되어 병원성이 높은 것으로 알려져 있다. 이러한 세망내피증 바이러스의 pol 유전자에 대한 DNA염기 서열은 현재까지 pol 유전자의 3'말단 부위와 5'말단 부위만 각각 알려져 있을 뿐, 전체 염기서열 정보는 알려지지 않았다.Reticuloendothelial virus (REV), which belongs to the retrovirus, acts as a causative agent, and has a severely reduced immunity and develops acute development, acute and chronic tumors. The disease is mainly caused by chickens, turkeys and ducks. The retinopathy has been reported in many countries, such as Australia, Japan, the United States, the United Kingdom, and especially in Australia and Japan, the case of using the Marek's disease vaccine contaminated with the reticulosis endothelial virus. In 1991, damage cases were first identified in Korea, and isolated reticuloendothelial virus is known to cause severe immunosuppressive function, decrease in body weight gain rate, and chronic tumor. The DNA base sequence of the pol gene of the reticuloendothelial virus is known only to the 3 'end and 5' end of the pol gene, but the entire nucleotide sequence information is unknown.
이에, 본 발명자들은 산업적 이용가치가 높은 새로운 재조합 역전사효소를 제공하기 위하여, 상기 세망내피증 바이러스로부터 pol유전자를 전체 염기서열을 분석하였고, 역전사 효소의 유전자를 클로닝하여 염기서열을 밝혔고, 또한 클로닝한 역전사 효소 유전자를 대장균에서 대량 발현시켜 불용성 단백질 형태로 생산된 비활성 역전사 효소 단백질을 활성형으로 변화시키는 방법을 개발함으로써 본 발명을 완성하였다.In order to provide a novel recombinant reverse transcriptase of high industrial value, the present inventors analyzed the entire sequence of the pol gene from the reticuloendothelial virus, cloned the gene of the reverse transcriptase, and revealed the nucleotide sequence. The present invention has been completed by developing a method of mass-expressing reverse transcriptase genes in E. coli to convert an inactive reverse transcriptase protein produced in an insoluble protein form into an active form.
본 발명의 목적은 세망내피증 바이러스(Reticuloendotheliosis Virus)의 역전사 효소 및 이를 생산하는 방법을 제공하는 것이다.It is an object of the present invention to provide a reverse transcriptase of Reticuloendotheliosis Virus and a method of producing the same.
도 1은 역전사 효소를 클로닝하기 위하여 사용한 pET21bREV-RT 벡터의 모식도이다. 1 is a schematic diagram of the pET21bREV-RT vector used for cloning reverse transcriptase.
도 2는 세망내피증 바이러스의 역전사 효소 균주를 세포파쇄 후 세포함유체(inclusion body)층의 분리까지의 단계별 에스디에스-페이지 젤(SDS-PAGE gel) 사진이다. FIG. 2 is a step-by-step SDS-PAGE gel photograph of the reverse transcriptase strain of reticuloendothelial virus until cell disruption and separation of the inclusion body layer. FIG.
레인 1 : 크기 마커(size marker)Lane 1: size marker
레인 2 : BL21DE3Lane 2: BL21DE3
레인 3 : BL21DE3+pET21bREV-RT 유도(induction) 없음Lane 3: BL21DE3 + pET21bREV-RT no induction
레인 4 : BL21DE3+pET21bREV-RT 유도Lane 4: BL21DE3 + pET21bREV-RT induction
레인 5 : BL21DE3+pET21bREV-RT 유도 / 수용성 분획Lane 5: BL21DE3 + pET21bREV-RT induction / water soluble fraction
레인 6 : BL21DE3+pET21bREV-RT 유도 / 불용성 펠렛 세척 분획Lane 6: BL21DE3 + pET21bREV-RT Induced / Insoluble Pellet Wash Fraction
레인 7 : BL21DE3+pET21bREV-RT 유도 / 불용성 세포함유체 분획Lane 7: BL21DE3 + pET21bREV-RT induction / insoluble cell containing fraction
도 3은 재구조 형성(refolding) 조건에 따른 세망내피증 바이러스의 역전사효소 활성을 나타내는 cDNA의 전기영동 사진이다. 3 is an electrophoretic photograph of cDNA showing reverse transcriptase activity of reticuloendothelial virus according to refolding conditions.
레인 1 : 크기 마커(size marker)Lane 1: size marker
레인 2 : 50mM Tris + 1mMGSSG + 10mM GSHLane 2: 50mM Tris + 1mMGSSG + 10mM GSH
레인 3 : 50mM Tris + 1mM GSSG +10mM GSH +Tween20 0.02%Lane 3: 50mM Tris + 1mM GSSG + 10mM GSH + Tween20 0.02%
레인 4 : 50mM Tris + 1mM GSSG + 10mM GSH + 5% sucroseLane 4: 50mM Tris + 1mM GSSG + 10mM GSH + 5% sucrose
레인 5 : 50mM Tris + 1mM GSSG +10mM GSH +75 mM KCl, 3 mM MgCl2 Lane 5: 50 mM Tris + 1 mM GSSG +10 mM GSH +75 mM KCl, 3 mM MgCl 2
레인 6 : 50mM Tris + 1mM GSSG +10mM GSH + PEG8000 1mg/LLane 6: 50mM Tris + 1mM GSSG + 10mM GSH + PEG8000 1mg / L
레인 7 : 50mM Tris + 1mM GSSG +10mM GSH + 25mM CaCl2+ 25mM MgCl2 Lane 7: 50mM Tris + 1mM GSSG + 10mM GSH + 25mM CaCl 2 + 25mM MgCl 2
레인 8 : 50mM Tris + 1mM GSSG +10mM GSH + 0.1%BSALane 8: 50mM Tris + 1mM GSSG + 10mM GSH + 0.1% BSA
레인 9 : 50mM Tris + 2mM GSSG + 10mM GSHLane 9: 50mM Tris + 2mM GSSG + 10mM GSH
레인 10 : 50mM Tris + 2mM GSSG +10mM GSH +Tween20 0.02%Lane 10: 50mM Tris + 2mM GSSG + 10mM GSH + Tween20 0.02%
레인 11 : 50mM Tris + 2mM GSSG + 10mM GSH + 5% sucroseLane 11: 50mM Tris + 2mM GSSG + 10mM GSH + 5% sucrose
레인 12 : 50mM Tris + 2mM GSSG +10mM GSH +75 mM KCl, 3 mM MgCl2 Lane 12: 50 mM Tris + 2 mM GSSG + 10 mM GSH +75 mM KCl, 3 mM MgCl 2
레인 13 : 50mM Tris + 2mM GSSG +10mM GSH + PEG8000 1mg/LLane 13: 50mM Tris + 2mM GSSG + 10mM GSH + PEG8000 1mg / L
레인 14 : 50mM Tris + 2mM GSSG +10mM GSH + 25mM CaCl2+ 25mM MgCl2 Lane 14: 50mM Tris + 2mM GSSG + 10mM GSH + 25mM CaCl 2 + 25mM MgCl 2
레인 15 : 50mM Tris + 2mM GSSG +10mM GSH + 0.1%BSALane 15: 50mM Tris + 2mM GSSG + 10mM GSH + 0.1% BSA
레인 16 : 음성 대조군Lane 16: negative control
레인 17 : 양성 대조군Lane 17: positive control
상기 목적을 달성하기 위하여, 본 발명은 세망내피증 바이러스의 역전사 효소 및 이를 코딩하는 유전자를 제공한다.In order to achieve the above object, the present invention provides a reverse transcriptase of the retinopathy virus and a gene encoding the same.
또한, 본 발명은 상기 유전자를 포함하는 벡터 및 이를 포함하는 형질전환체를 제공한다.The present invention also provides a vector comprising the gene and a transformant comprising the same.
또한 본 발명은 숙주세포 내에서 대량 발현 시 불용성 단백질 형태로 생산되는 비활성 재조합 세망내피증 바이러스 역전사 효소를 수용성 활성형 단백질로 전환시키는 구조재형성 방법을 제공한다.The present invention also provides a method for restructuring the inactive recombinant retinopathy virus reverse transcriptase, which is produced in the form of an insoluble protein upon mass expression in a host cell, into a water-soluble active protein.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 1로 기재되는 세망내피증 바이러스의 역전사 효소 유전자, 그 상동형 유전자(homologs) 및 그 변이형 유전자(variants)를 제공한다. 또한, 본 발명은 서열번호 2로 기재되는 세망내피증 바이러스의 역전사 효소 또는 그 변이형 효소(variants)를 제공한다.The present invention provides a reverse transcriptase gene gene of homologous endothelial virus, homologs thereof and variants thereof described in SEQ ID NO: 1. The present invention also provides a reverse transcriptase or variant thereof of the reticuloendothelial virus described in SEQ ID NO: 2.
상기 세망내피증 바이러스의 역전사 효소 유전자의 "상동형 유전자(homologs)"는 서열번호 2로 기재되는 세망내피증 바이러스의 역전사 효소에 대한 코딩 서열을 포함하는 유전자를 뜻한다. 따라서, 상기 상동형 유전자의 범주에는 발현 시 서열번호 2로 기재되는 세망내피증 바이러스의 역전사 효소를 생산할 수 있는 모든 종류의 유전자가 포함된다.The "homologs" of the reverse transcriptase gene of the reticuloendothelial virus refer to a gene comprising a coding sequence for the reverse transcriptase of the reticuloendothelial virus described in SEQ ID NO: 2. Thus, the category of homologous genes includes all kinds of genes that are capable of producing reverse transcriptase of retinopathy virus described in SEQ ID NO: 2 when expressed.
상기 세망내피증 바이러스의 역전사 효소의 "변이형 효소(variants)"는 서열번호 2로 기재되는 세망내피증 바이러스의 역전사 효소 중 하나 이상의 아미노산 잔기를 결실, 부가, 삽입 또는 치환시킨 효소를 의미하며, 상기 세망내피증 바이러스의 역전사 효소 유전자의 "변이형 유전자(variants)"는 세망내피증 바이러스의 역전사 효소의 변이형 효소를 코딩하는 모든 유전자를 의미한다."Variants" of the reverse transcriptase of the reticulosis endothelial virus means an enzyme that deletes, adds, inserts or replaces at least one amino acid residue of the reverse transcriptase of the reticulosis endothelial virus of SEQ ID NO: 2, "Variants" of the reverse transcriptase gene of the retinopathy virus means all genes encoding the mutant enzyme of the reverse transcriptase of the retinopathy virus.
상기 변이형 효소는 서열번호 2로 기재되는 세망내피증 바이러스의 역전사 효소와 95% 이상의 상동성을 가지는 것이 바람직하며, 98% 이상의 상동성을 가지는 것이 더욱 바람직하다.The mutant enzyme preferably has at least 95% homology with the reverse transcriptase of reticuloendothelial virus described in SEQ ID NO: 2, and more preferably has at least 98% homology.
또한, 상기 변이형 효소 유전자는 서열번호 1로 기재되는 세망내피증 바이러스의 역전사 효소와 95% 이상의 상동성을 가지는 것이 바람직하며, 98% 이상의 상동성을 가지는 것이 더욱 바람직하다.In addition, the mutant enzyme gene preferably has at least 95% homology with the reverse transcriptase of reticuloendothelial virus described in SEQ ID NO: 1, and more preferably has at least 98% homology.
레트로 바이러스 유전자의 기본 구조는 프로모터인LTR(long terminalrepeat), 코어 단백질을 암호화하는gag, 역전사 효소를 암호화하는pol, 외피 단백질을 암호화하는env의LTR-gag-pol-env-LTR배열을 하고 있다. 모든 레트로 바이러스의 역전사 효소는pol유전자에 의해서 합성되며, RNA에서 직접 DNA를 합성하는 활성, DNA에서 DNA를 합성하는 활성 및 RNA를 분해하는 활성의 3가지 활성을 갖고 있다. pol 유전자는gag-pro-pol이라는 커다란 폴리단백질(polyprotein) 유전자의 일부분으로 전사되며,gag과pol은 그 사이의 정지 코돈(stop codon)을 인지하여 전사가 종결되거나 프레임 쉬프트(frame shift)가 일어나면서 새로 전사되는 방식 혹은 그대로 통과(stop codon readthrough)의해 한번에 폴리단백질로 전사된 후 바이러스의 단백질 분해 효소로 절단되는 방식 등 레트로 바이러스마다 다양하게 전사되어진다(Monica et al.,J.Biol. Chem, 1985, 260: 9326-9335; Luke et al.,Biochemistry, 1990, 29:1764-1769; Le Grice et al.,J. Virolo.,1991, 65:7004-7007).The basic structure of the retroviral gene is the LTR-gag-pol-env-LTR sequence of promoter LTR (long terminal repeat), gag encoding core protein, pol encoding reverse transcriptase, and env encoding envelope protein. The reverse transcriptase of all retroviruses is synthesized by the pol gene and has three activities: the activity of synthesizing DNA directly from RNA, the activity of synthesizing DNA from DNA, and the activity of digesting RNA. The pol gene is transcribed into a large polyprotein gene called gag-pro-pol , and gag and pol recognize stop codons between them, resulting in termination of transcription or frame shift. as is the new transfer method is or as it is passed through various methods such as transfer each retrovirus is cut by proteases of virus and then transferred to the (stop codon readthrough) at a time by the poly protein (Monica et al., J.Biol. Chem , 1985, 260: 9326-9335; Luke et al., Biochemistry , 1990, 29: 1764-1769; Le Grice et al., J. Virolo., 1991, 65: 7004-7007).
본 발명자들은 세망 내피증 바이러스를 숙주세포에 감염시켜 증식시킨 후 바이러스 게놈(genome)을 추출하여 모든 레트로 바이러스에서 역전사 효소를 코딩한다고 알려진 pol 유전자의 말단부위의 알려진 염기서열을 분석하여 제작한 공통 프라이머를 이용하여 pol 유전자의 전체 염기서열을 분석하였고 이를 토대로 추정된 세망내피증 바이러스의 역전사효소를 다른 균주들과 비교하였다. 또한, 상기의 분석된 pol 유전자 서열정보를 바탕으로 세망내피증 바이러스의 역전사 효소가 단량체일 것이라고 추정하고 이를 바탕으로 역전사 효소의 프라이머를 디자인하였다.또한 디자인된 서열을 갖는 역전사 효소를 대장균 발현벡터에 삽입하고 이를 대장균에 형질전환시킴으로써 상기 역전사 효소를 포함하는 벡터로 형질전환된 대장균 균주를 얻었다.The present inventors infect the host cells with the reticulum endothelial virus, and then propagate it, extract a viral genome, and analyze a known nucleotide sequence of the terminal region of the pol gene known to encode a reverse transcriptase in all retroviruses. The entire sequencing of the pol gene was analyzed by using and the reverse transcriptase of the reticuloendothelial virus estimated was compared with other strains. In addition, based on the analyzed pol gene sequence information, it was assumed that the reverse transcriptase of retinopathy virus was a monomer, and based on this, a primer of the reverse transcriptase was designed. A reverse transcriptase having the designed sequence was added to the E. coli expression vector. The E. coli strain transformed with the vector containing the reverse transcriptase was obtained by inserting and transforming E. coli.
그 결과, 본 발명의 신규한 세망내피증 바이러스 역전사 효소의 유전자 염기서열은 서열번호 1로 기재되고, 아미노산 서열은 서열번호 2로 기재됨을 확인하엿다. 또한 상기 유전자 염기서열을 다른 균주들과 BLAST SEARCH(National Center for Biotechnology Information ; NCBI)를 통해 비교ㆍ분석한 결과, 세망내피증 바이러스는 C형의 레트로 바이러스인 MMLV(moloney murine leukemia virus) 및 FelV(Feline leukemia virus)등과 각각 55%, 57%의 상동성을 가지는 레트로 바이러스인 것으로 나타났다.As a result, it was confirmed that the gene base sequence of the novel reticuloendothelial virus reverse transcriptase of the present invention is described by SEQ ID NO: 1, and the amino acid sequence is described by SEQ ID NO: 2. In addition, the gene sequence was compared with other strains through BLAST SEARCH (National Center for Biotechnology Information; NCBI). As a result, retinal endothelial viruses were MMLV (moloney murine leukemia virus) and FelV (type C retroviruses). Feline leukemia virus) and 55% and 57% homology to the retrovirus, respectively.
또한, 서열분석법을 통해 얻은 상기 서열 번호 2의 아미노산 서열을 전사 시 앞서 분석된 pol유전자 상에서 프레임 쉬프트를 통해 생산될 수 있는 서열과 비교하였을 때, 정지 코돈 그대로 통과(stop codon readthrough) 방식으로 생산되는 MMLV의 역전사 효소 아미노산 서열과 유사한 형태의 아미노산 서열을 보였다. 따라서, 상기 세망내피증 바이러스 역전사 효소는 C형 바이러스인 MMLV의 경우와 같이 정지 코돈 그대로 통과 방식으로 전사됨을 알 수 있었다.In addition, when the amino acid sequence of SEQ ID NO: 2 obtained through sequencing is compared with a sequence that can be produced through frame shift on the previously analyzed pol gene in transcription, it is produced in a stop codon readthrough manner. The amino acid sequence was similar in shape to the reverse transcriptase amino acid sequence of MMLV. Therefore, it was found that the reticuloendothelial virus reverse transcriptase is transcribed in a stop codon manner as it is in the case of MMLV, a type C virus.
또한, 3차 구조를 서열에 따라 예측할 수 있는 SWISS-MODEL(Protein modeling server made by Swiss Institute of Bioinformatics)을 사용하여 서열을 통해 3차 구조를 예측하고, 상기 예측된 구조를 알려져 있는 MMLV의 3차 구조와 비교하여 유사함을 판단하였다. 단백질의 3차 구조 형성에 있어 시스테인 잔기의 위치가 중요한데 MMLV의 역전사 효소와 와 본 발명의 세망내피증 바이러스 역전사 효소를 아미노산 서열에서 비교하였을 때 시스테인 잔기의 위치간격이 거의 일치하였다. 따라서, 상기 세망내피증 바이러스 역전사 효소 단백질의 3차 구조 또한 MMLV와 유사한 형태를 취함을 확인하였다.In addition, using a protein modeling server made by Swiss Institute of Bioinformatics (SWISS-MODEL), which can predict the tertiary structure according to the sequence, the tertiary structure is predicted through the sequence, and the predicted structure is the tertiary of MMLV. Similarity was determined by comparison with the structure. The position of cysteine residues is important in the formation of the tertiary structure of the protein. When the reverse transcriptase of MMLV and the reticuloendothelial virus reverse transcriptase of the present invention are compared in amino acid sequence, the positions of the cysteine residues are almost identical. Therefore, it was confirmed that the tertiary structure of the reticuloendothelial virus reverse transcriptase protein also took a form similar to that of MMLV.
또한, 본 발명은 세망내피증 바이러스의 역전사 효소 유전자를 포함하는 벡터 및 상기 벡터를 포함하는 형질전환체를 제공한다.The present invention also provides a vector comprising a reverse transcriptase gene of retinopathy virus and a transformant comprising the vector.
본 발명의 벡터의 제조에는 알려진 다양한 원핵세포용 또는 진핵세포용 벡터 중에서 발현 목적에 따라 선택된 임의의 벡터를 사용할 수 있으므로, 특정한 벡터에 국한되지 않음은 당업자에게 있어서는 자명하다. 또한, 벡터의 외래 유전자 삽입부위에 삽입되는 유전자의 크기, 염기서열 등도 공지의 기술에 의해 다양하게 변화시킬 수 있다. 또한, 상기 벡터를 포함하는 형질전환체는 숙주 세포에 상기 벡터를 도입하여 제조할 수 있으며, 그 도입 방법은 공지의 기술, 즉 열충격법, 전기천공법 등을 통해 도입할 수 있다. 또한, 형질전환체의 제조에 사용되는 숙주 세포는 특정한 세포에 국한되지 않음은 당업자에게 있어서 명백하며, 원핵생물 및 진핵생물의 모든 세포가 사용될 수 있다. 바람직하게는 상기 숙주세포는 대장균이다.In the production of the vector of the present invention, any vector selected according to the expression purpose from various known prokaryotic or eukaryotic vectors can be used, which is obvious to those skilled in the art. In addition, the size, nucleotide sequence, etc. of the gene inserted into the foreign gene insertion site of the vector can also be variously changed by known techniques. In addition, the transformant including the vector may be prepared by introducing the vector into a host cell, and the introduction method may be introduced through a known technique, that is, a thermal shock method or an electroporation method. In addition, it is apparent to those skilled in the art that the host cell used for the preparation of the transformant is not limited to a particular cell, and all cells of prokaryotes and eukaryotes can be used. Preferably the host cell is Escherichia coli.
본 발명자들은 본 발명의 세망내피증 바이러스의 역전사 효소 유전자를 포함하는 벡터로 형질전환된 대장균 형질전환체를 2003년 8월 20일자로 한국생명공학연구원 유전자은행에 기탁하였다(수탁번호: KCTC 10511BP).The present inventors deposited the E. coli transformants transformed with the vector containing the reverse transcriptase gene gene of the reticuloendothelial virus of the present invention to the Korea Biotechnology Research Institute Gene Bank (Accession No .: KCTC 10511BP). .
또한, 본 발명은In addition, the present invention
(1) 발현된 재조합 세망내피증 바이러스의 역전사 효소가 포함된 세포함유체를 분리하는 단계;(1) separating the cell-containing medium containing the reverse transcriptase of the expressed recombinant retinopathy virus;
(2) 분리된 세포함유체를 요소(urea) 또는 구아니딘 클로라이드(Guanidine chloride)를 포함하는 변성 완충액(denaturation buffer)에 용해시키는 단계;(2) dissolving the isolated cell-containing medium in a denaturation buffer containing urea or guanidine chloride;
(3) 용해된 단백질을 회복 완충액(renaturation buffer)에 반응시켜 구조재형성(refolding)하는 단계를 포함하는 불용성 단백질 형태로 생산된 비활성 재조합 세망내피증 바이러스의 역전사 효소를 수용성 활성형 단백질로 전환시키는 방법을 제공한다.(3) a method of converting the reverse transcriptase of an inactive recombinant retinopathy virus produced in an insoluble protein form into a water-soluble active protein, the method comprising the step of reacting the lysed protein with a recovery buffer to refold To provide.
상기 세망내피증 바이러스 역전사 효소를 포함하는 벡터로 형질전환된 대장균 균주를 배양하고, 발현된 역전사 효소 단백질을 상기 방법을 사용하여 수용성 활성형 단백질로 전환시킨 후 효소활성을 확인하고 종래의 역전사 효소와 비교하였다. 상기 실험에서 재조합 세망내피증 바이러스 역전사 효소를 생산하는 형질 전환체는 단량체로 생산을 하게 디자인되어 있으므로, 상기 수득된 역전사 효소가 활성을 보인다면 단량체로 판단할 수 있다.The E. coli strain transformed with the vector containing the reticulum endothelial virus reverse transcriptase was incubated, and the expressed reverse transcriptase protein was converted to a water-soluble active protein using the above method, followed by enzymatic activity and Compared. In the above experiment, the transformant that produces the recombinant retinopathy virus reverse transcriptase is designed to be produced as a monomer, and thus, if the obtained reverse transcriptase shows activity, it can be judged as a monomer.
그 결과, 상기 변성 및 구조 재형성 과정을 거쳐 수득된 재조합 세망내피증바이러스 역전사효소가 활성을 보임을 확인하였다. 따라서, HIV 또는 AMV의 역전사 효소 단백질은 이량체(dimer) 형태로 활성을 갖는 반면(Eisenman et al.,J. Virol, 1980, 36:62-78) 상기 재조합 세망내피증 바이러스 역전사효소는 단량체 형태로 역전사 효소의 활성을 갖고 있는 것을 알 수 있었다.As a result, it was confirmed that the recombinant reticuloendothelial virus reverse transcriptase obtained through the denaturation and structural remodeling process showed activity. Thus, the reverse transcriptase protein of HIV or AMV is active in dimer form (Eisenman et al., J. Virol , 1980, 36: 62-78) while the recombinant reticuloendothelial virus reverse transcriptase is in monomeric form. It can be seen that it has the activity of reverse transcriptase.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 분, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following Examples are not intended to illustrate the present invention, but the content of the present invention is not limited to the following Examples.
<실시예 1> 세망내피증 역전사 효소 유전자의 클로닝 및 염기서열 분석Example 1 Cloning and Sequence Analysis of Retinopathy Reverse Transcriptase Gene
세망내피증 바이러스를 계태아 섬유아세포(chien embryo fibroblast; CEF)에 감염시켜 증식한 후 바이러스 게놈을 추출하고, 상기 추출된 게놈으로부터 세망내피증 바이러스의 역전사 효소 유전자를 앞서 서열 분석된 pol 유전자를 바탕으로 합성된 프라이머를 중합효소 연쇄반응으로 증폭하여 세망내피증 바이러스 역전사 효소의 유전자를 얻었다. 구체적으로, G-DEX 게노믹 익스트렉션 키트(iNtRON Co., Korea)를 사용하여 제조사의 방침에 따라 게놈을 추출하고 여기서 추출된 게놈 1 ㎕를 주형으로 하여, 5 U/㎕의 Taq 중합효소(iNtRON co., Korea) 0.5 ㎕ 및 10X PCR 버퍼 2 ㎕, 10 pmole의 서열번호 3과 서열번호 4의 프라이머 각각 1 ㎕ 씩, 증류수 15.5 ㎕를 혼합하였다. 상기 반응액을 94℃에서 5분 동안 변성시킨 후, 94℃ 30초, 60℃ 30초, 72℃ 1분 10초로 30번 반복 실시하고 난 뒤 72℃에서 5분간 더반응시켰다. 상기 중합효소연쇄반응 산물은 1% 아가로스 젤에서 전기영동하여 EtBr(Ethidium Bromide)로 염색하여 관찰하였다.The reticulum endothelial virus is infected with chien embryo fibroblasts (CEFs) and propagated, followed by extraction of the viral genome, and the reverse transcriptase gene of reticulum endothelial virus from the extracted genome is based on the pol gene previously sequenced. Was synthesized by a polymerase chain reaction to obtain a gene for retinopathy of retinopathy virus. Specifically, using the G-DEX genomic extraction kit (iNtRON Co., Korea) to extract the genome according to the manufacturer's policy, using 1 μl extracted genome as a template, 5 U / μl Taq polymerase (iNtRON co., Korea) 0.5 µl, 2 µl of 10X PCR buffer, 1 µl of distilled water were mixed with 1 µl of primers of SEQ ID NO: 3 and SEQ ID NO: 4 of 10 pmole, respectively. The reaction solution was denatured at 94 ° C. for 5 minutes, then repeated 30 times at 94 ° C. 30 seconds, 60 ° C. 30 seconds, 72 ° C. 1 minute 10 seconds, and further reacted at 72 ° C. for 5 minutes. The polymerase chain reaction product was observed by electrophoresis on 1% agarose gel and stained with EtBr (Ethidium Bromide).
그 결과, 상기 프라이머를 사용하여 중합효소연쇄반응을 수행한 결과로 예측되는 크기의 증폭산물을 관찰하였다.As a result, the amplification product of the size predicted as a result of performing the polymerase chain reaction using the primer was observed.
상기 실험으로 얻은 역전사 효소의 유전자와 pET21b 벡터(Novagen co., US)를EcoRI과HindIII효소로 처리하여 반응시킨 후 정제하여 리가아제(ligase)로 연결시켰다. 구체적으로, 중합효소연쇄반응 산물 20 ㎕를 PCR 퀵스핀 PCR 프러덕트 퓨리피케이션 키트(iNtRON Co., Korea)를 사용하여 정제하고, pET21b 벡터와 함께 제한 효소EcoRI과HindIII를 처리한다. 상기 제한 효소 처리한 PCR 산물과 pET21b 벡터를 3 M 소듐 아세테이트(pH 5.2) 10 ㎕, 95% 에탄올 80 ㎕ 씩 각각 첨가하여 상온에서 5분간 방치한 후 13,000 rpm에서 20분간 원심분리하고 70% 에탄올로 세척한 후 침전물을 건조시키고 이를 3차 증류수 10 ㎕에 각각 용해시켰다. 상기 정제된 중합효소연쇄반응 산물 6 ㎕ 와 벡터 2 ㎕를 첨가하고 10X 연결 완충액(ligation buffer) 2 ㎕, 리가아제(ligase) 1 ㎕를 혼합하여 16℃에서 하루동안 놓아둔 후, 대장균에 칼슘 클로라이드 방법 등의 통상의 방법으로 형질전환 시켜 상기 역전사효소 유전자가 벡터에 삽입된 형질전환체를 확보하였다.The reverse transcriptase gene and the pET21b vector (Novagen co., US) obtained in the above experiments were treated with EcoRI and HindIII enzyme, and then purified and linked to ligase. Specifically, 20 μl of the polymerase chain reaction product is purified using a PCR quickspin PCR product purification kit (iNtRON Co., Korea) and treated with restriction enzymes EcoRI and HindIII together with the pET21b vector. 10 μl of 3 M sodium acetate (pH 5.2) and 80 μl of 95% ethanol were respectively added to the PCR product treated with the restriction enzyme and pET21b vector, and allowed to stand at room temperature for 5 minutes, followed by centrifugation at 13,000 rpm for 20 minutes, followed by 70% ethanol. After washing, the precipitate was dried and dissolved in 10 [mu] l of tertiary distilled water, respectively. 6 μl of the purified polymerase chain reaction product and 2 μl of the vector were added, 2 μl of 10 × ligation buffer and 1 μl of ligase were mixed and left at 16 ° C. for one day, followed by calcium chloride to E. coli. By transforming by a conventional method such as a method, a transformant having the reverse transcriptase gene inserted into a vector was obtained.
상기의 방법으로 얻은 역전사 효소 유전자가 삽입된 벡터는 효소의 절단 및 서열분석을 통해 분석되었다. 구체적으로, 상기 형질전환체로부터 얻은 벡터 DNA에 제한효소 EcoRI 과HindIII를 처리하여 전기영동한 후 예측되는 크기의 DNA 단편을 관찰하고, 상기 역전사 효소의 도입이 확인된 벡터를 ABI377 DNA 자동분석장치와 다이 종결 키트(dye terminator kit Perkin Elmer, Foster, CA)를 사용하여 염기서열을 분석하여 세망내피증 바이러스 역전사 효소의 염기서열을 결정하고, 이로부터 아미노산 서열을 추정하였다.The vector inserted with the reverse transcriptase gene obtained by the above method was analyzed through digestion and sequencing of the enzyme. Specifically, the vector DNA obtained from the transformant was treated with the restriction enzymes EcoRI and HindIII , followed by electrophoresis to observe a DNA fragment having a predicted size, and the vector was confirmed to be introduced with the ABI377 DNA automatic analyzer. The nucleotide sequence was analyzed using a die terminator kit Perkin Elmer, Foster, CA to determine the base sequence of the reticulovirus virus transcriptase, and the amino acid sequence was estimated therefrom.
그 결과, 상기 결정된 염기서열은 서열번호 1에 기재하였고, 상기 염기서열이 코딩하는 아미노산 서열을 서열번호 2에 기재하였다.As a result, the determined base sequence is described in SEQ ID NO: 1, and the amino acid sequence encoded by the base sequence is described in SEQ ID NO: 2.
또한, 본 발명자들은 본 발명의 세망내피증 바이러스의 역전사 효소 유전자를 포함하는 벡터로 형질전환된 대장균 형질전환체를 2003년 8월 20일자로 한국생명공학연구원 유전자은행에 기탁하였다(수탁번호: KCTC 10511BP)In addition, the present inventors deposited an E. coli transformant transformed with a vector containing the reverse transcriptase gene gene of the reticuloendothelial virus of the present invention to the Korea Biotechnology Research Institute Gene Bank (Acc. No .: KCTC). 10511BP)
〈실시예 2〉세망내피증 바이러스 역전사 효소의 발현<Example 2> Expression of reticuloendothelial virus reverse transcriptase
상기 실시예 1에서 획득한 세망내피증 바이러스 역전사효소의 삽입이 확인된 벡터를 가지는 형질전환된 균주를 표 1의 조성을 갖는 배지에서 배양한다.The transformed strain having the vector confirmed the insertion of the reticuloendothelial virus reverse transcriptase obtained in Example 1 is cultured in a medium having the composition of Table 1.
구체적으로, 표 1의 조성을 가지는 배지에서 pET21bREV-RT 벡터를 갖는BL21DE3대장균을 하루동안 배양하고, 이 배양액 2 ml을 본 배양에 접종하여 세망내피증 바이러스 역전사효소를 발현시킨다. 배양 도중, 세포배양액이 흡광도(O.D) 0.8에 이르면 IPTG(Isopropyl β-D- thiogalactopyranoside)을 최종농도가 1 mM이 되도록 처리하여 3시간 동안 더 배양한다. 상기 IPTG 처리과정을 단백질 발현의 유도(induction)라 한다. 배양 종료 후, 대장균을 4℃에서 6000 rpm으로 10분간 원심분리 하였다. 상기 방법으로 수득한 2 g의 대장균을 5 ml의 세포 융해 완충액 A(50 mM Tris-HCl(pH8.0), 1 mM EDTA(ethylendiamine tetraacetic acid), 25% 수크로즈, 2 mg/ml 라이소자임, 1 mM PMSF(phenylmethylsulfonyl fluoride))로 녹이고 4℃에서 30분간 진탕한 다음, 5 ml의 세포 융해 완충액 B(50 mM Tris-HCl (pH8.0), 1%(v/v) Triton X-100, 100 mM NaCl, 5 mM MgCl2)를 추가하여 다시 4℃에서 30 분간 진탕한 후, 상기 세포 융해물을 13,000 rpm에서 10분간 원심분리하여 수용성 단백질이 포함되어 있는 상층액과 불용성 단백질을 포함하는 세포함유체 펠렛층으로 나누고 상기 실험으로 얻은 단백질을 SDS-폴리아크릴아미드 젤에서 전기영동하였다.Specifically, BL21DE3 Escherichia coli with the pET21bREV-RT vector is incubated for one day in a medium having the composition shown in Table 1, and 2 ml of this culture is inoculated into the culture to express the reticuloendothelial virus reverse transcriptase. During the culture, when the cell culture solution reaches an absorbance (OD) of 0.8, IPTG (Isopropyl β-D-thiogalactopyranoside) is treated to a final concentration of 1 mM and further incubated for 3 hours. The IPTG process is called induction of protein expression. After incubation, E. coli was centrifuged at 6000 rpm for 10 minutes at 4 ℃. 2 g of Escherichia coli obtained by the above method was added to 5 ml of cell lysis buffer A (50 mM Tris-HCl, pH 8.0), 1 mM ethylendiamine tetraacetic acid (EDTA), 25% sucrose, 2 mg / ml lysozyme, 1 dissolved in mM PMSF (phenylmethylsulfonyl fluoride) and shaken at 4 ° C. for 30 minutes, then 5 ml of cell lysis buffer B (50 mM Tris-HCl (pH8.0), 1% (v / v) Triton X-100, 100 After shaking for another 30 minutes at 4 ° C. by adding mM NaCl and 5 mM MgCl 2 ), the cell lysate was centrifuged at 13,000 rpm for 10 minutes to carry out the supernatant containing the water-soluble protein and the cell containing the insoluble protein. The proteins obtained in the above experiments were divided into fluid pellet layers and electrophoresed on SDS-polyacrylamide gels.
그 결과, 세포함유체를 포함하는 불용성 단백질 펠렛층에서 세망내피증 바이러스의 역전사효소를 확인할 수 있었다(도 2). 이 결과로 세망내피증 바이러스의 역전사 효소는 불용성 단백질 형태로 세포함유체 내로 발현되며 이 세포함유체를 분리하는 과정을 통하여 비교적 간단하게 분리할 수 있음을 확인하였다.As a result, it was possible to confirm the reverse transcriptase of reticuloendothelial virus in the insoluble protein pellet layer containing the cell medium (FIG. 2). As a result, the reverse transcriptase of reticuloendothelial virus was expressed in the cell-containing body in the form of insoluble protein, and it was confirmed that the cell-containing body can be separated relatively simply by separating the cell-containing body.
〈실시예 3〉세망내피증 바이러스의 역전사 효소의 구조재형성 및 활성 확인Example 3 Structural Remodeling and Activity Confirmation of Reverse Transcriptase of Retinopathy Virus
상기 실시예 2의 세망내피증 바이러스 역전사 효소가 포함된 펠렛을 20 mg/ml의 농도로 pH 8.0의 변성 완충액(4 M GdnCl, 4 mM DTT, 50 mM Tris-HCl, and 1 mM EDTA (ethylenediaminetetraacetic acid))에 4℃에서 1 시간동안 녹인다. 상기 반응물을 다시 구조재형성(refolding) 시키기 위하여 20배 부피의 다양한 회복 완충액에 희석시켜 4℃에서 24시간 반응시킨다. 본 실험에서 사용한 다양한 회복 완충액의 조성을 표 2에 나타내었다.The pellet containing the reticuloendothelial virus reverse transcriptase of Example 2 was denatured in pH 8.0 at a concentration of 20 mg / ml (4 M GdnCl, 4 mM DTT, 50 mM Tris-HCl, and 1 mM EDTA (ethylenediaminetetraacetic acid). 1) at 4 ℃ for 1 hour. The reaction is diluted in 20-fold volume of various recovery buffers for refolding and reacted at 4 ° C. for 24 hours. The compositions of the various recovery buffers used in this experiment are shown in Table 2.
상기 각 단계의 반응물을 이용하여 역전사 중합효소연쇄반응(RT-PCR)을 통하여 구조 재형성된 세망내피증 바이러스의 역전사 효소의 활성을 확인하였다. 구체적으로, RNA 바이러스의 일종인 뉴캐슬병 바이러스(Newcastle Disease Virus; ND 바이러스)의 게놈을 주형으로하여 상기 각 단계로부터 얻은 세망내피증 바이러스 반응물을 이용하여 cDNA를 합성하고, 상기 합성된 cDNA를 중합효소 연쇄반응으로 증폭한 후 전기영동하였다. 도 3의 아가로스 젤의 상단부분은 4M 구아니딘 클로라이드를 사용하였고, 하단부분은 6M 구아니딘 클로라이드를 사용하여 불용성 REV-RT를 변성 용해시켜 상기 구조재형성 완충액 통해 구조 재형성 과정을 거쳐 활성을 측정한 것이다. 상기 실험에서 상기 각 단계의 반응물에 포함된 역전사 효소가 활성을 갖는다면 예측된 크기의 DNA 밴드를 확인할 수 있다.The reaction of each step was used to confirm the activity of the reverse transcriptase of the reshaped reticuloendothelial virus through reverse transcriptase polymerase chain reaction (RT-PCR). Specifically, cDNA is synthesized using the reticuloendothelial virus reactant obtained from each step using a genome of Newcastle Disease Virus (ND virus), which is a kind of RNA virus, and the synthesized cDNA is polymerase chained. After amplification by reaction and electrophoresis. The upper portion of the agarose gel of Figure 3 was used 4M guanidine chloride, the lower portion was 6M guanidine chloride was used to denature the insoluble REV-RT by dissolving the structure reforming buffer to measure the activity through the structure reforming process. . In the experiment, if the reverse transcriptase contained in the reaction of each step is active, it is possible to determine the DNA band of the predicted size.
도 3에 나타난 결과에서 보는 바와 같이, 상기 실험의 중합효소연쇄반응물을 전기영동한 아가로즈 젤 상의 여러 레인에서 예측된 크기의 DNA 밴드를 확인하였으며, 이로써 상기 변성 및 구조 재형성 과정을 거쳐 수득된 재조합 세망내피증 바이러스 역전사효소가 활성을 보임을 알 수 있었다. 그 중에서 특히 레인 4에서 가장 진한 DNA 밴드를 나타내므로, 표 2의 4번 회복완충액의 조성을 이용할 때, 재조합 세망내피증 바이러스 역전사 효소가 가장 잘 재구조 형성되어 활성이 큰 것을 알 수 있었다.As shown in the results shown in Figure 3, the DNA band of the expected size in the lanes on the agarose gel electrophoresis of the polymerase chain reaction of the experiment was confirmed, thereby obtained through the denaturation and structural reformation process Recombinant retinopathy virus reverse transcriptase showed activity. In particular, since the darkest DNA band is shown in lane 4, when using the composition of the recovery buffer No. 4 of Table 2, it was found that the recombinant reticuloendothelial virus reverse transcriptase was restructured best and had high activity.
상기에서 살펴본 바와 같이, 본 발명의 세망내피증 바이러스의 역전사 효소는 진단 및 유전자 클로닝에 유용하게 사용될 수 있다.As discussed above, the reverse transcriptase of the reticuloendothelial virus of the present invention can be usefully used for diagnosis and gene cloning.
<110> YOON, Seong Jun <120> Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof <130> 3p-07-11B <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2031 <212> DNA <213> Reticuloendotheliosis Virus <400> 1 acggctcccc tggaagagga ataccgtttg tttttagagg cgccgataca aaatgttacg 60 ctgctagagc agtggaaacg ggaaatcccg aaagtctggg ccgagataaa tcccccgggg 120 ttggcatcca cacaagcccc cattcatgtc cagctattaa gtaccgccct acctgtgagg 180 gtaagacagt atcctattac cctggaggca aaacgaagcc tgcgcgaaac tattcgcaaa 240 ttcagagcag cgggcattct gagacccgtc cactccccct ggaacactcc cctcctccct 300 gtgcgaaagt ccggcacgtc cgagtatcgg atggttcaag atttaaggga agtaaacaag 360 agagtagaga ctattcaccc aactgtccct aacccataca ccctcctcag cctattaccc 420 cctgaccgaa tatggtattc tgttttggac ctgaaagacg cctttttctg catccctctg 480 gcccctgagt cgcaattgat cttcgcattc gagtgggcag atgcggagga aggagaatca 540 gggcaactaa cctggactag gctacctcag ggttttaaga actcacccac cctttttgat 600 gaagccctta acagggatct gcagggattt cgacttgatc accctttcgt ttccctcctt 660 cagtatgtag gcgacttact tattgcggct gatacacaag ccgcatgcct gagtgccacc 720 cgagacttac tcatgacttt agcggagctg gggtaccggg tttcagggaa gaaggcccag 780 ctctgtcaag aggaagtcac atacttgggg ttcaagatcc ataagggatc acggacccta 840 tcaaatagcc gaactcaggc tattcttcag ataccagtac cgaaaaccaa aaggcaggtg 900 cgcgaatttt taggtacaat tgggtactgc aggctttgga tcccggggtt tgccgaactt 960 gcccagcccc tatatgcggc aacgcggggg ggaaatgatc ctttagtgtg gggcgaaaaa 1020 gaggaagaag cgttccagag cttgaaacta gccctcacac agccaccagc tttggccctg 1080 ccctccctag ataagccttt ccagctcttc gtagaagaaa caggcggggc ggccaaggga 1140 gtgctcacac aggccctcgg tccatggaaa ggacccgtag cctacttatc caaaaggctg 1200 gaccctgtgg cagctggctg gccccggtgc ctccgagcca ttgcggcagc cgctcttctc 1260 acaagagaag cctctaaact taccttcggg caggacatag agatcacttc gtcccataac 1320 ttggagagtt tgctgcgatc ccctcctgac gggtggctga ccaatgctcg catcacgcag 1380 taccaggtac tgctcctgga ccctccccga gtgcgcttta agcagactgc ggccctcaat 1440 ccggctaccc tgttacccga gacagacgat gcgctgccaa tacaccattg cctggacact 1500 ttagattctc tgacctccac ccgccccgac ctgacggatc agcccctggc acaagcggaa 1560 gcctccctgt tcactgacgg cggcagctac atacgagacg gaaaacgata caccggggca 1620 gcagtggtaa ctctggactc tgttatttgg gcagaacccc tcccaattgg aacatcagcc 1680 cagaaagcag aattgatcgc actaacaaag gccctggaat ggagcaagga catgagcgtc 1740 aacatctaca cggatagccg ttatgcattc gccactctac acgtccatgg gatgatctac 1800 agagaaaggg gactgttgac agcagggggg aaggctatca aaaacgcccc cgaaattcta 1860 gccttgctga ccgcggtctg gctacctaaa cgggtagctg tgatgcattg caagggacac 1920 cagaaagacg atgcacccac atcaactggt aaccgacggg cagatgaggt ggctcgagaa 1980 gtagctatac gtcctttgag cacccaggcc accatctccg atgcaccgga t 2031 <210> 2 <211> 677 <212> PRT <213> Reticuloendotheliosis Virus <400> 2 Thr Ala Pro Leu Glu Glu Glu Tyr Arg Leu Phe Leu Glu Ala Pro Ile 1 5 10 15 Gln Asn Val Thr Leu Leu Glu Gln Trp Lys Arg Glu Ile Pro Lys Val 20 25 30 Trp Ala Glu Ile Asn Pro Pro Gly Leu Ala Ser Thr Gln Ala Pro Ile 35 40 45 His Val Gln Leu Leu Ser Thr Ala Leu Pro Val Arg Val Arg Gln Tyr 50 55 60 Pro Ile Thr Leu Glu Ala Lys Arg Ser Leu Arg Glu Thr Ile Arg Lys 65 70 75 80 Phe Arg Ala Ala Gly Ile Leu Arg Pro Val His Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Arg Lys Ser Gly Thr Ser Glu Tyr Arg Met Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Thr Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Thr Leu Leu Ser Leu Leu Pro Pro Asp Arg Ile 130 135 140 Trp Tyr Ser Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Ile Pro Leu 145 150 155 160 Ala Pro Glu Ser Gln Leu Ile Phe Ala Phe Glu Trp Ala Asp Ala Glu 165 170 175 Glu Gly Glu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu Asn Arg Asp Leu Gln 195 200 205 Gly Phe Arg Leu Asp His Pro Phe Val Ser Leu Leu Gln Tyr Val Gly 210 215 220 Asp Leu Leu Ile Ala Ala Asp Thr Gln Ala Ala Cys Leu Ser Ala Thr 225 230 235 240 Arg Asp Leu Leu Met Thr Leu Ala Glu Leu Gly Tyr Arg Val Ser Gly 245 250 255 Lys Lys Ala Gln Leu Cys Gln Glu Glu Val Thr Tyr Leu Gly Phe Lys 260 265 270 Ile His Lys Gly Ser Arg Thr Leu Ser Asn Ser Arg Thr Gln Ala Ile 275 280 285 Leu Gln Ile Pro Val Pro Lys Thr Lys Arg Gln Val Arg Glu Phe Leu 290 295 300 Gly Thr Ile Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Leu 305 310 315 320 Ala Gln Pro Leu Tyr Ala Ala Thr Arg Gly Gly Asn Asp Pro Leu Val 325 330 335 Trp Gly Glu Lys Glu Glu Glu Ala Phe Gln Ser Leu Lys Leu Ala Leu 340 345 350 Thr Gln Pro Pro Ala Leu Ala Leu Pro Ser Leu Asp Lys Pro Phe Gln 355 360 365 Leu Phe Val Glu Glu Thr Gly Gly Ala Ala Lys Gly Val Leu Thr Gln 370 375 380 Ala Leu Gly Pro Trp Lys Gly Pro Val Ala Tyr Leu Ser Lys Arg Leu 385 390 395 400 Asp Pro Val Ala Ala Gly Trp Pro Arg Cys Leu Arg Ala Ile Ala Ala 405 410 415 Ala Ala Leu Leu Thr Arg Glu Ala Ser Lys Leu Thr Phe Gly Gln Asp 420 425 430 Ile Glu Ile Thr Ser Ser His Asn Leu Glu Ser Leu Leu Arg Ser Pro 435 440 445 Pro Asp Gly Trp Leu Thr Asn Ala Arg Ile Thr Gln Tyr Gln Val Leu 450 455 460 Leu Leu Asp Pro Pro Arg Val Arg Phe Lys Gln Thr Ala Ala Leu Asn 465 470 475 480 Pro Ala Thr Leu Leu Pro Glu Thr Asp Asp Ala Leu Pro Ile His His 485 490 495 Cys Leu Asp Thr Leu Asp Ser Leu Thr Ser Thr Arg Pro Asp Leu Thr 500 505 510 Asp Gln Pro Leu Ala Gln Ala Glu Ala Ser Leu Phe Thr Asp Gly Gly 515 520 525 Ser Tyr Ile Arg Asp Gly Lys Arg Tyr Thr Gly Ala Ala Val Val Thr 530 535 540 Leu Asp Ser Val Ile Trp Ala Glu Pro Leu Pro Ile Gly Thr Ser Ala 545 550 555 560 Gln Lys Ala Glu Leu Ile Ala Leu Thr Lys Ala Leu Glu Trp Ser Lys 565 570 575 Asp Met Ser Val Asn Ile Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr 580 585 590 Leu His Val His Gly Met Ile Tyr Arg Glu Arg Gly Leu Leu Thr Ala 595 600 605 Gly Gly Lys Ala Ile Lys Asn Ala Pro Glu Ile Leu Ala Leu Leu Thr 610 615 620 Ala Val Trp Leu Pro Lys Arg Val Ala Val Met His Cys Lys Gly His 625 630 635 640 Gln Lys Asp Asp Ala Pro Thr Ser Thr Gly Asn Arg Arg Ala Asp Glu 645 650 655 Val Ala Arg Glu Val Ala Ile Arg Pro Leu Ser Thr Gln Ala Thr Ile 660 665 670 Ser Asp Ala Pro Asp 675 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer1 for pol1 <400> 3 ggggaattcg acggctcccc tggaag 26 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer2 for pol1 <400> 4 gggaagctta tccggtgcat cggag 25<110> YOON, Seong Jun <120> Reverse transcriptase of Reticuloendotheliosis Virus and method for production according <130> 3p-07-11B <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2031 <212> DNA < 213> Reticuloendotheliosis Virus <400> 1 acggctcccc tggaagagga ataccgtttg tttttagagg cgccgataca aaatgttacg 60 ctgctagagc agtggaaacg ggaaatcccg aaagtctggg ccgagataaa tcccccgggg 120 ttggcatcca cacaagcccc cattcatgtc cagctattaa gtaccgccct acctgtgagg 180 gtaagacagt atcctattac cctggaggca aaacgaagcc tgcgcgaaac tattcgcaaa 240 ttcagagcag cgggcattct gagacccgtc cactccccct ggaacactcc cctcctccct 300 gtgcgaaagt ccggcacgtc cgagtatcgg atggttcaag atttaaggga agtaaacaag 360 agagtagaga ctattcaccc aactgtccct aacccataca ccctcctcag cctattaccc 420 cctgaccgaa tatggtattc tgttttggac ctgaaagacg cctttttctg catccctctg 480 gcccctgagt cgcaattgat cttcgcattc gagtgggtcg g agc tttaaga actcacccac cctttttgat 600 gaagccctta acagggatct gcagggattt cgacttgatc accctttcgt ttccctcctt 660 cagtatgtag gcgacttact tattgcggct gatacacaag ccgcatgcct gagtgccacc 720 cgagacttac tcatgacttt agcggagctg gggtaccggg tttcagggaa gaaggcccag 780 ctctgtcaag aggaagtcac atacttgggg ttcaagatcc ataagggatc acggacccta 840 tcaaatagcc gaactcaggc tattcttcag ataccagtac cgaaaaccaa aaggcaggtg 900 cgcgaatttt taggtacaat tgggtactgc aggctttgga tcccggggtt tgccgaactt 960 gcccagcccc tatatgcggc aacgcggggg ggaaatgatc ctttagtgtg gggcgaaaaa 1020 gaggaagaag cgttccagag cttgaaacta gccctcacac agccaccagc tttggccctg 1080 ccctccctag ataagccttt ccagctcttc gtagaagaaa caggcggggc ggccaaggga 1140 gtgctcacac aggccctcgg tccatggaaa ggacccgtag cctacttatc caaaaggctg 1200 gaccctgtgg cagctggctg gccccggtgc ctccgagcca ttgcggcagc cgctcttctc 1260 acaagagaag cctctaaact taccttcggg caggacatag agatcacttc gtcccataac 1320 ttggagagtt tgctgcgatc ccctcctgac gg gtggctga ccaatgctcg catcacgcag 1380 taccaggtac tgctcctgga ccctccccga gtgcgcttta agcagactgc ggccctcaat 1440 ccggctaccc tgttacccga gacagacgat gcgctgccaa tacaccattg cctggacact 1500 ttagattctc tgacctccac ccgccccgac ctgacggatc agcccctggc acaagcggaa 1560 gcctccctgt tcactgacgg cggcagctac atacgagacg gaaaacgata caccggggca 1620 gcagtggtaa ctctggactc tgttatttgg gcagaacccc tcccaattgg aacatcagcc 1680 cagaaagcag aattgatcgc actaacaaag gccctggaat ggagcaagga catgagcgtc 1740 aacatctaca cggatagccg ttatgcattc gccactctac acgtccatgg gatgatctac 1800 agagaaaggg gactgttgac agcagggggg aaggctatca aaaacgcccc cgaaattcta 1860 gccttgctga ccgcggtctg gctacctaaa cgggtagctg tgatgcattg caagggacac 1920 cagaaagacg atgcacccac atcaactggt aaccgacggg cagatgaggt ggctcgagaa 1980 gtagctatac gtcctttgag cacccaggcc accatctccg atgcaccgga t 2031 <210> 2 <211> 677 <212> PRT <213> Reticuloendotheliosis Virus <400 > 2 Thr Ala Pro Leu Glu Glu Gl u Tyr Arg Leu Phe Leu Glu Ala Pro Ile 1 5 10 15 Gln Asn Val Thr Leu Leu Glu Gln Trp Lys Arg Glu Ile Pro Lys Val 20 25 30 Trp Ala Glu Ile Asn Pro Pro Gly Leu Ala Ser Thr Gln Ala Pro Ile 35 40 45 His Val Gln Leu Leu Ser Thr Ala Leu Pro Val Arg Val Arg Gln Tyr 50 55 60 Pro Ile Thr Leu Glu Ala Lys Arg Ser Leu Arg Glu Thr Ile Arg Lys 65 70 75 80 Phe Arg Ala Ala Gly Ile Leu Arg Pro Val His Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Arg Lys Ser Gly Thr Ser Glu Tyr Arg Met Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Thr Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Thr Leu Leu Ser Leu Leu Pro Pro Asp Ar g Ile 130 135 140 Trp Tyr Ser Val Leu Asp Leu Lys Asp Ala Phe Cys Ile Pro Leu 145 150 155 160 Ala Pro Glu Ser Gln Leu Ile Phe Ala Phe Glu Trp Ala Asp Ala Glu 165 170 175 Glu Gly Glu Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu Asn Arg Asp Leu Gln 195 200 205 Gly Phe Arg Leu Asp His Pro Phe Val Ser Leu Leu Gln Tyr Val Gly 210 215 220 Asp Leu Leu Ile Ala Ala Asp Thr Gln Ala Ala Cys Leu Ser Ala Thr 225 230 235 240 Arg Asp Leu Leu Met Thr Leu Ala Glu Leu Gly Tyr Arg Val Ser Gly 245 250 255 Lys Lys Ala Gln Leu Cys Gln Glu Glu Val Thr Tyr Leu Gly Phe Lys 260 265 270 Ile His Lys Gly Ser Arg Thr Leu Ser Asn Ser Arg Thr Gln Ala Ile 275 280 285 Leu Gln Ile Pro Val Pro Lys Thr Lys Arg Gln Val Arg Glu Phe Leu 290 295 300 Gly Thr Ile Gly Tyr Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Leu 305 310 315 320 Ala Gln Pro Leu Tyr Ala Ala Thr Arg Gly Gly Asn Asp Pro Leu Val 325 330 335 Trp Gly Glu Lys Glu Glu Glu Ala Phe Gln Ser Leu Lys Leu Ala Leu 340 345 350 Thr Gln Pro Pro Ala Leu Ala Leu Pro Ser Leu Asp Lys Pro Phe Gln 355 360 365 Leu Phe Val Glu Glu Thr Gly Gly Ala Ala Lys Gly Val Leu Thr Gln 370 375 380 Ala Leu Gly Pro Trp Lys Gly Pro Val Ala Tyr Leu Ser Lys Arg Leu 385 390 395 400 A sp Pro Val Ala Ala Gly Trp Pro Arg Cys Leu Arg Ala Ile Ala Ala 405 410 415 Ala Ala Leu Leu Thr Arg Glu Ala Ser Lys Leu Thr Phe Gly Gln Asp 420 425 430 Ile Glu Ile Thr Ser Ser His Asn Leu Glu Ser Leu Leu Arg Ser Pro 435 440 445 Pro Asp Gly Trp Leu Thr Asn Ala Arg Ile Thr Gln Tyr Gln Val Leu 450 455 460 Leu Leu Asp Pro Pro Arg Val Arg Phe Lys Gln Thr Ala Ala Leu Asn 465 470 475 480 Pro Ala Thr Leu Leu Pro Glu Thr Asp Ala Leu Pro Ile His His 485 490 495 Cys Leu Asp Thr Leu Asp Ser Leu Thr Ser Thr Arg Pro Asp Leu Thr 500 505 510 Asp Gln Pro Leu Ala Gln Ala Glu Ala Ser Leu Phe Thr Asp Gly Gly 515 520 525 Ser Tyr Ile Arg Asp Gly Lys Arg T yr Thr Gly Ala Ala Val Val Thr 530 535 540 Leu Asp Ser Val Ile Trp Ala Glu Pro Leu Pro Ile Gly Thr Ser Ala 545 550 555 560 Gln Lys Ala Glu Leu Ile Ala Leu Thr Lys Ala Leu Glu Trp Ser Lys 565 570 575 Asp Met Ser Val Asn Ile Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr 580 585 590 Leu His Val His Gly Met Ile Tyr Arg Glu Arg Gly Leu Leu Thr Ala 595 600 605 Gly Gly Lys Ala Ile Lys Asn Ala Pro Glu Ile Leu Ala Leu Leu Thr 610 615 620 Ala Val Trp Leu Pro Lys Arg Val Ala Val Met His Cys Lys Gly His 625 630 635 640 Gln Lys Asp Asp Ala Pro Thr Ser Thr Gly Asn Arg Arg Ala Asp Glu 645 650 655 Val Ala Arg Glu Val Ala Ile Arg Pro Leu Ser Thr Gln Ala Thr I le 660 665 670 Ser Asp Ala Pro Asp 675 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer1 for pol1 <400> 3 ggggaattcg acggctcccc tggaag 26 <210> 4 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> primer2 for pol1 <400> 4 gggaagctta tccggtgcat cggag 25
Claims (8)
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020030059324A KR100437889B1 (en) | 2003-08-26 | 2003-08-26 | Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof |
| PCT/KR2003/002607 WO2005019445A1 (en) | 2003-08-26 | 2003-11-28 | Reverse transcriptase of reticuloendotheliosis virus and method for producing thereof |
| AU2003284784A AU2003284784A1 (en) | 2003-08-26 | 2003-11-28 | Reverse transcriptase of reticuloendotheliosis virus and method for producing thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020030059324A KR100437889B1 (en) | 2003-08-26 | 2003-08-26 | Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof |
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| Publication Number | Publication Date |
|---|---|
| KR100437889B1 true KR100437889B1 (en) | 2004-07-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020030059324A Expired - Lifetime KR100437889B1 (en) | 2003-08-26 | 2003-08-26 | Reverse transcriptase of Reticuloendotheliosis Virus and method for production thereof |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR100437889B1 (en) |
| AU (1) | AU2003284784A1 (en) |
| WO (1) | WO2005019445A1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995027047A2 (en) * | 1994-04-01 | 1995-10-12 | Gen Probe Inc | Highly-purified recombinant reverse transcriptase |
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2003
- 2003-08-26 KR KR1020030059324A patent/KR100437889B1/en not_active Expired - Lifetime
- 2003-11-28 WO PCT/KR2003/002607 patent/WO2005019445A1/en active Application Filing
- 2003-11-28 AU AU2003284784A patent/AU2003284784A1/en not_active Abandoned
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| Publication number | Publication date |
|---|---|
| WO2005019445A1 (en) | 2005-03-03 |
| AU2003284784A1 (en) | 2005-03-10 |
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