KR101885438B1 - Gene therapy vector system and prodrug genes - Google Patents

Gene therapy vector system and prodrug genes Download PDF

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KR101885438B1
KR101885438B1 KR1020160160087A KR20160160087A KR101885438B1 KR 101885438 B1 KR101885438 B1 KR 101885438B1 KR 1020160160087 A KR1020160160087 A KR 1020160160087A KR 20160160087 A KR20160160087 A KR 20160160087A KR 101885438 B1 KR101885438 B1 KR 101885438B1
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김연수
강문경
백민정
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충남대학교산학협력단
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Abstract

본 발명은 암세포 조직으로 효율적인 암의 치료를 위한 유전자의 전달을 가능하게 하는 티미딘 키나아제(HSV-TK) 및 시토신 디아미나아제(CD) 유전자를 포함하는 복제가능 레트로바이러스 벡터 시스템에 관한 것이다. 구체적으로 본 발명의 HSV-TK 및 CD 유전자를 모두 포함하며, 바이러스 감염 중 재조합이 일어나지 않아 치료 유전자의 소실이 없는, 안정성이 뛰어난 TK/CD 병용 자가복제 레트로바이러스 벡터 시스템은 전구약물인 GCV나 5-FC를 이용하여 암세포의 사멸을 유도할 수 있고, TK나 CD 중 어느 하나를 이용한 암치료에 내성을 나타내는 암에도 치료 유전자 및 그에 따른 전구 약물을 선택적으로 적용할 수 있으므로, 암의 치료를 위한 약학적 조성물로 유용하게 사용될 수 있다.
The present invention relates to a replicable retroviral vector system comprising a thymidine kinase (HSV-TK) and a cytosine deaminase (CD) gene that enables the delivery of a gene for efficient cancer therapy to cancer cell tissue. Specifically, the highly stable TK / CD duplicated retroviral vector system, which contains all of the HSV-TK and CD genes of the present invention and has no recombination during viral infection and does not lose the therapeutic gene, -FC can be used to induce the death of cancer cells, and the therapeutic gene and the prodrug thereof can be selectively applied to cancer that is resistant to cancer treatment using either TK or CD. Therefore, May be useful as pharmaceutical compositions.

Description

유전자치료 벡터시스템 및 전구약물 유전자{GENE THERAPY VECTOR SYSTEM AND PRODRUG GENES}GENE THERAPY VECTOR SYSTEM AND PRODRUG GENES BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

본 발명은 암세포 조직으로 효율적인 암의 치료를 위한 유전자의 전달을 가능하게 하는 티미딘 키나아제(thymidine kinase; HSV-TK) 및 시토신 디아미나아제(cytosine deaminase; CD) 유전자를 포함하는 복제가능 레트로바이러스 벡터 시스템에 관한 것이다.The present invention relates to a replication-competent retroviral vector comprising a thymidine kinase (HSV-TK) gene and a cytosine deaminase (CD) gene enabling the gene transfer for efficient cancer therapy to cancer cell tissue ≪ / RTI >

유전자 치료란, 환자의 세포 또는 조직에서 질환을 유발하는 비정상 유전자를 대체하거나 유전자나 질병을 치료하는데 도움이 되는 유전자를 삽입하여 질환을 치료하는 기술을 통칭한다. 유전자 치료제의 개발 초기에는 외부의 DNA를 표적 세포의 염색체 내에 삽입하여 특정 유전자를 발현하도록 만드는 것이 유전자 치료의 주된 개념이었다. 그러나, 최근에는 안티센스 올리고디옥시뉴클레오티드, 작은 리보핵산(siRNA) 등을 이용하여 특정 질환과 관련된 유전자의 발현을 억제하는 안티센스 요법도 범주에 포함된다.Gene therapy refers to a technique for treating a disease by replacing an abnormal gene that causes a disease in a patient's cell or tissue, or inserting a gene that is useful for treating a gene or disease. In the early stage of gene therapy, the main idea of gene therapy was to insert external DNA into the chromosome of the target cell to express a specific gene. However, recently, antisense therapies for inhibiting the expression of genes related to a specific disease using antisense oligodeoxynucleotides, small ribonucleic acid (siRNA) and the like are also included in the category.

이러한 유전자 치료는 지금까지의 치료법과는 전혀 다른 개념의 접근 방법으로 질환의 근본 원인을 분자 수준에서 규명하여 치료할 수 있다. 또한, 염기서열 특이적인 작용이기 때문에 주요 질환과 관련된 인자를 제거함으로써 다른 치료법에서 문제시되는 불필요한 부작용을 최소화할 수 있다. 또한, 이와 같이 유전자를 표적으로 하는 방법은 발현의 수준을 조절하고자 하는 유전자의 염기서열만 알면 치료제의 제작에서 별다른 최적화가 필요하지 않아, 항체나 화합물 치료제에 비해 제조과정이 매우 간단하다. 또한, 타 치료제가 표적화할 수 없는 표적도 질병의 원인이 되는 유전자만 알면 얼마든지 표적화할 수 있어, 차세대 치료제로서 잠재 능력이 충분하다. 이와 관련하여 기존의 의료기술로 치료가 어려운 난치병, 암, 에이즈, 유전질환, 신경계질환 등에 유전자 치료를 적용하여 치료 가능성을 높인 여러 연구 결과들이 있으며 실제 임상 실험도 진행되고 있다(YOUNG et al, 2006).This gene therapy can be treated at the molecular level by treating the root cause of the disease as a completely different approach from the conventional treatments. In addition, since it is a sequence-specific action, it is possible to minimize unnecessary side effects, which are problematic in other therapeutic methods, by removing factors related to major diseases. In addition, in the method of targeting a gene as described above, it is very simple to manufacture the gene as compared with the antibody or the compound therapeutic agent, since the nucleotide sequence of the gene to be regulated is not required to be optimized. In addition, the target gene can not be targeted by any other therapeutic agent, and only the gene that causes the disease can be targeted, so that it has sufficient potential as a next-generation therapeutic agent. In this regard, there are a number of studies that have improved the therapeutic potential by applying gene therapy to incurable diseases, cancers, AIDS, genetic diseases, and neurological diseases that are difficult to treat with conventional medical technology, and actual clinical trials are underway (YOUNG et al , 2006 ).

유전자 치료제는 유전자 전달체와 치료 유전자로 구성된다. 유전자를 생체 내로 전달하기 위한 도구인 유전자 전달체는 크게 바이러스성 전달체와 비바이러스성 전달체로 나눌 수 있다. 바이러스성 전달체는 바이러스 유전자의 대부분, 혹은 일부 필수 유전자를 없애서 스스로 복제할 수 없게 만들고 여기에 치료 유전자를 대신 삽입하여 제조된다(Lotze MT et al., Cancer Gene Therapy, 9:692-699, 2002). 바이러스성 전달체는 고효율로 유전자를 전달할 수 있으나, 바이러스의 종류에 따라 대량생산의 어려움, 면역반응 유발, 독성, 또는 복제가능 바이러스의 출현 등과 같은 문제점을 갖는다. 현재 유전자 치료제 개발에 활용되고 있는 주요 바이러스성 전달체로는 레트로바이러스, 렌티 바이러스, 아데노 바이러스, 아데노 부속 바이러스(AAV), 허피스 심플렉스 바이러스 및 폭스바이러스 등이 있다. 한편, 비바이러스성 전달체는 면역반응을 유도하지 않으며 독성이 낮고 대량생산이 용이하지만 유전자 전달효율이 낮고 그 발현이 일시적이다. Gene therapy consists of gene carriers and therapeutic genes. Gene transporters, which are tools for delivering genes in vivo, can be broadly divided into viral carriers and nonviral carriers. Viral transporters are produced by dislodging most or some essential genes of the viral gene, making them non-replicable by themselves and inserting therapeutic genes therein (Lotze MT et al ., Cancer Gene Therapy , 9: 692-699, 2002) . Viral transporters are able to deliver genes with high efficiency, but they have problems such as difficulty in mass production, induction of immune reaction, toxicity, or appearance of replicable virus depending on the kind of virus. Currently, the main viral carriers used in the development of gene therapy products are retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), herpes simplex viruses and poxviruses. On the other hand, nonviral carriers do not induce an immune response, are low in toxicity and easy to mass-produce, but have low gene transfer efficiency and their expression is transient.

임상에서 많이 쓰이는 바이러스성 전달체 중 하나인 레트로바이러스 벡터는 1990년 미국 국립보건원에서 실시된 최초의 유전자 치료의 임상시험에 사용된 벡터로서 치료 유전자를 안정하게 삽입하기에 가장 유용한 벡터로 여겨지고 있다. 현재 몰로니 설치류 백혈병 바이러스(MoMLV)에 기초한 레트로바이러스 벡터가 각종 유전자 치료를 위한 임상 시험에 사용되고 있다. Retroviral vectors, one of the most viral carriers in clinical practice, are considered to be the most useful vectors for stable insertion of therapeutic genes as vectors used in clinical trials of the first gene therapy in the US National Institutes of Health in 1990. Retroviral vectors based on Molloid rodential leukemia virus (MoMLV) are currently being used in clinical trials for various gene therapies.

자가복제가 제한된 복제불능의 레트로바이러스 벡터는 비교적 큰 유전자를 삽입할 수 있으며, 역가도 106~107 pfu/㎖ 정도로서 대상세포에의 감염에도 큰 문제가 없다. 또한, 포장세포주(packaging cell line)가 개발되어 있어 제조방법이 용이하다. 게다가, 레트로바이러스 벡터는 레트로바이러스의 플라스미드 내로 치료 유전자를 삽입하고 이를 포장세포에 감염시켜 재조합 바이러스를 생성시키고, 이를 대상 세포에 감염시키는 방식으로 규모확대(scale-up)가 가능하다. 그러나 염색체로의 삽입 과정에서 유전자 삽입에 따른 돌연변이가 일어날 수 있다. Non-replicable retroviral vectors with limited self-replication are capable of inserting relatively large genes and have an inverse degree of 10 6 to 10 7 pfu / ml. In addition, since a packaging cell line has been developed, a manufacturing method is easy. In addition, retroviral vectors can be scaled up by inserting therapeutic genes into retroviral plasmids, infecting the packaging cells to produce recombinant viruses, and infecting target cells. However, mutations may occur due to gene insertion during chromosomal integration.

한편, 암세포와 같은 증식성 세포에서 자가복제가 가능한 복제가능 레트로바이러스 벡터는 지놈(genome) 안정성에서 많은 논란이 있으며, 유전자 치료를 위한 자가복제 바이러스 벡터로서 개발시 외래도입이 가능한 유전자 크기가 약 1.3 kb로 제한되어 다양한 치료유전자의 도입이 어렵다(J. of virology, Vol.75, 6989-6998, 2001).On the other hand, replication-competent retroviral vectors capable of self-replication in proliferating cells such as cancer cells have many controversies in the genome stability, and self-replicating viral vectors for gene therapy have a gene size of about 1.3 kb, making it difficult to introduce various therapeutic genes ( J. of virology , Vol. 75, 6989-6998, 2001).

유전자 치료제에 사용되는 치료 유전자로서 허피스 심플렉스 바이러스 티미딘 키나아제 또는 시토신 디아미나아제와 같이 전구약물 투여에 의해 암세포의 자살을 유도하는 유전자, 인터류킨-12 또는 GM-CSF 등과 같이 면역반응을 촉진할 수 있는 사이토카인 유전자, CEA 또는 Her-2 등과 같이 종양특이적인 항원유전자가 많이 사용되고 있다(Gottesman MM, Cancer Gene Therapy, 10:501-508, 2003). 자살 유전자는 암세포로 전달된 후 암을 사멸시키고, 사이토카인 유전자 또는 종양특이적 항원 유전자는 암에 대한 면역반응을 활성화 시켜 암세포를 공격한다.The therapeutic gene used in the gene therapy agent may be a gene that induces suicide of cancer cells by administration of a prodrug such as herpes simplex virus thymidine kinase or cytosine deaminase and may induce an immune response such as interleukin-12 or GM-CSF (Gottesman MM, Cancer Gene Therapy , 10: 501-508, 2003) have been used in many cases, such as cytokine gene, CEA or Her-2. The suicide gene is transmitted to the cancer cells and kills the cancer, and the cytokine gene or the tumor-specific antigen gene activates the immune response to the cancer and attacks cancer cells.

최근, 악성종양에 대해 선택적으로 항종양효과를 나타내는 효소/전구약물 합성기법의 연구가 활발하게 진행되고 있다. 실제로, 자살 유전자를 암 조직에서 발현시키고, 이의 전구체를 생체에 전신적으로 투여하면 정상세포에서는 독성이 나타나지 않고 치료 유전자가 발현된 종양세포에서만 전구체가 독성물질로 전환되어 종양세포를 파괴한다. Recently, there have been active researches on the synthesis of enzyme / prodrug drugs selectively showing antitumor effect against malignant tumors. Indeed, when suicide genes are expressed in cancer tissues and their precursors are systemically administered to the body, toxicities do not appear in normal cells, and only the tumor cells expressing the therapeutic gene are converted to toxic substances to destroy the tumor cells.

가장 널리 사용되고 있는 자살 유전자 중 하나는 허피스 심플렉스 바이러스 티미딘 키나아제(HSV-TK)이다. 이는 세포에 무해한 간사이클로비르(GCV)라는 전구약물을 효소반응을 통해 세포독성을 가지는 물질로 바꿈으로써 자살유전자를 가지는 세포뿐만 아니라, 간극연접을 통해 인접한 세포의 사멸도 유도하는 방관자 효과(bystander effect)를 갖는다. 이는 임상 3상까지 수행되어 효능과 안정성이 입증되었다(human gene therapy, 4:725-731, 1993; molecular therapy, 1:195-203, 2000).One of the most widely used suicide genes is the herpes simplex virus thymidine kinase (HSV-TK). By transforming a prodrug called hepatocyclovir (GCV), which is innocuous to cells, into a cytotoxic substance through an enzymatic reaction, it can be used not only as a cell having a suicide gene but also as a bystander effect ). This was done to Phase 3 clinical trials, demonstrating efficacy and safety ( human gene therapy , 4: 725-731, 1993; molecular therapy , 1: 195-203, 2000).

또 다른 자살 유전자는 시토신 디아미나아제(CD)로, 5-플루오로사이토신(5-FC)을 강력한 항암제인 5-플루오로우라실(5-FU)로 탈아민화시킨다. 5-FU는 5-플루오로우리딘 삼인산(5-FUTP)과 5-플루오로데옥시우리딘 일인산(5-FdUMP)으로 대사된다. 리보핵산으로 융합된 5-FUTP는 리보솜 리보핵산과 전달자 리보핵산의 합성 과정을 방해하고, 5-FdUMP는 티미딘 합성효소를 비가역적으로 저해함으로써, DNA 합성을 억제한다. 따라서 TK 또는 CD가 발현되는 종양세포는 GCV와 5-FC와 같은 전구약물을 독성 대사물로 전환시켜 선택적으로 암을 사멸시킨다.Another suicide gene is cytosine deaminase (CD), which is 5-fluoro cytosine (5-FC), which is a strong anticancer drug, 5-fluorouracil (5-FU). 5-FU is metabolized by 5-fluorouridine triphosphate (5-FUTP) and 5-fluorodeoxyuridine phosphate (5-FdUMP). 5-FUTP fused to ribonucleic acid interferes with the synthesis of ribosomal ribonucleic acid and transporter ribonucleic acid, and 5-FdUMP inhibits DNA synthesis by irreversibly inhibiting thymidine synthase. Thus, tumor cells expressing TK or CD selectively convert prodrugs such as GCV and 5-FC to toxic metabolites to selectively kill cancer.

두 종류 이상의 치료 유전자를 유전자 치료에 동시에 적용하는 기술은 치료 효율 측면에서 뛰어나며, 특히 특정 유전자 치료에 내성을 나타내는 경우에 유용하다. 관련하여, 근래 TK 및 CD 투여에 의한 치료에 내성을 나타내는 암이 보고된 바, TK 및 CD를 동시에 암 조직에서 발현시킬 수 있는 유전자 치료 벡터 시스템은 큰 이점을 지닌다. 그러나 HSV-TK 및 CD를 모두 복제가능 레트로바이러스 벡터(Replicating- Retrovirus Vector; RRV)에 도입하면 지놈 크기가 약 10 kb 이상이 되어 단일 레트로바이러스 벡터에 삽입하는 것은 사실상 불가능하다. 또한, 유전자 치료를 위한 복제가능 레트로바이러스 벡터는 원래 레트로바이러스가 가진 지놈 RNA 외에 외래 유전자가 도입되기 때문에 지놈 RNA의 크기가 증가하고 비상동 염기서열이 추가되며 유전자 재조합이 일어나 치료 유전자가 소실될 가능성이 높아 벡터의 구축이 어렵다. Techniques that simultaneously apply two or more therapeutic genes to gene therapy are excellent in terms of therapeutic efficiency and are particularly useful when they are resistant to specific gene therapy. Regarding cancer which has recently been shown to be resistant to treatment with TK and CD administration, gene therapy vector systems capable of simultaneously expressing TK and CD in cancer tissues have great advantages. However, when HSV-TK and CD are introduced into a replicating-retrovirus vector (RRV), it is virtually impossible to insert HSV-TK and CD into a single retroviral vector with a size of about 10 kb or more. In addition, the replication-competent retroviral vector for gene therapy has the advantage of increasing the size of the genome RNA and adding the non-kinase sequence because the foreign gene is introduced in addition to the genome RNA originally possessed by the retrovirus, It is difficult to construct a vector.

이에, 본 발명자들은 재조합이 일어나지 않는 안전한 유전자 치료용 바이러스 벡터 시스템을 개발하던 중, HSV-TK 및 CD 유전자를 모두 포함하며, 바이러스 감염 중 재조합이 일어나지 않아 치료 유전자의 소실이 없는, 안정성이 뛰어난 TK/CD 병용 자가복제 레트로바이러스 벡터 시스템을 개발하였다. 상기 벡터는 전구약물인 GCV나 5-FC를 이용하여 암세포의 사멸을 유도할 수 있고, TK나 CD 중 어느 하나를 이용한 암치료에 내성을 나타내는 암에도 치료 유전자 및 그에 따른 전구 약물을 선택적으로 적용할 수 있어 본 발명의 벡터를 암 예방 또는 치료용 약학적 조성물로 사용할 수 있음을 확인함으로써 본 발명을 완성하였다. Accordingly, the inventors of the present invention have found that when developing a viral vector system for safe gene therapy that does not cause recombination, the present inventors have found that a recombinant vector containing all the HSV-TK and CD genes and the recombinant TK / CD combined self-replicating retrovirus vector system. The vector may induce the death of cancer cells using GCV or 5-FC, a prodrug, and selectively treat a therapeutic gene and its prodrug against cancer that is resistant to cancer treatment using either TK or CD And that the vector of the present invention can be used as a pharmaceutical composition for cancer prevention or treatment, thereby completing the present invention.

본 발명의 목적은, 티미딘 키나아제 및 시토신 디아미나아제 유전자를 포함하는 암의 치료를 위한 복제가능 레트로바이러스 벡터 시스템을 제공하는 것이다.It is an object of the present invention to provide a replicable retroviral vector system for the treatment of cancer comprising a thymidine kinase and a cytosine deaminase gene.

본 발명의 다른 목적은, 상기 레트로바이러스 벡터 시스템을 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스가 감염된 숙주세포를 제공하는 것이다.It is another object of the present invention to provide a recombinant retrovirus comprising the retroviral vector system and a host cell infected with the recombinant retrovirus.

본 발명의 또 다른 목적은, 상기 재조합 레트로바이러스를 포함하는 암 치료용 약학 조성물 및 유전자 전달 조성물을 제공하는 것이다.Still another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the recombinant retrovirus and a gene transfer composition.

본 발명의 또 다른 목적은, 상기 복제가능 레트로바이러스 시스템의 제조방법을 제공하는 것이다.It is yet another object of the present invention to provide a method for producing the replicable retroviral system.

상기 목적을 달성하기 위하여, 본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템을 제공한다.In order to achieve the above object, the present invention provides a recombinant expression vector comprising MuLV Gag-Pol gene, a promoter and a cytosine deaminase gene; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus.

또한, 본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템을 제공한다.The present invention also relates to a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the thymidine kinase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the cytosine deaminase gene of the virus.

또한, 본 발명은 상기 벡터 시스템을 포함하는 재조합 레트로바이러스를 제공한다.The present invention also provides a recombinant retrovirus comprising the vector system.

또한, 본 발명은 상기 재조합 레트로바이러스로 형질감염된 숙주 세포를 제공한다.The present invention also provides a host cell transfected with the recombinant retrovirus.

또한, 본 발명은 상기 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus as an active ingredient.

또한, 본 발명은 상기 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물을 제공한다.In addition, the present invention provides a gene transfer composition for treating cancer comprising the recombinant retrovirus.

또한, 본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법을 제공한다.The present invention also provides a method for producing a recombinant expression vector comprising the steps of: preparing a first recombinant expression vector comprising a Gag-Pol gene, a promoter and a cytosine deaminase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus.

아울러, 본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법을 제공한다.In addition, the present invention provides a method for producing a recombinant vector comprising the steps of: preparing a first recombinant expression vector comprising the Gag-Pol gene of MuLV, a promoter and a thymidine kinase gene; And producing a second recombinant expression vector comprising the Env gene, the promoter and the cytosine deaminase gene of the virus.

본 발명의 HSV-TK 및 CD 유전자를 모두 포함하며, 바이러스 감염 중 재조합이 일어나지 않아 치료 유전자의 소실이 없는, 안정성이 뛰어난 TK/CD 병용 자가복제 레트로바이러스 벡터 시스템은 전구약물인 GCV나 5-FC를 이용하여 암세포의 사멸을 유도할 수 있고, TK나 CD 중 어느 하나를 이용한 암치료에 내성을 나타내는 암에도 치료 유전자 및 그에 따른 전구 약물을 선택적으로 적용할 수 있으므로, 암의 치료를 위한 약학적 조성물로 유용하게 사용될 수 있다.The highly stable TK / CD duplicated retroviral vector system, which contains all of the HSV-TK and CD genes of the present invention and does not undergo recombination during viral infection and does not lose the therapeutic gene, can be used as the prodrug GCV or 5-FC Can be used to induce the death of cancer cells. Therapeutic genes and their corresponding prodrugs can also be selectively applied to cancer that is resistant to cancer treatment using either TK or CD. Therefore, May be useful as a composition.

도 1은 spRRVe-P1-TK(A), sRRVgp-P1-RFP(A), sRRVgp-P1-TK(B) 및 spRRVe-P1-GFP(B) 벡터의 구조를 나타낸 모식도이다.
도 2는 spRRVe-P1-TK/sRRVgp-P1-RFP 및 sRRVgp-P1-TK/spRRVe-P1-GFP 바이러스의 역가를 측정한 결과를 나타내는 도이다.
도 3은 spRRVe-P1-TK/sRRVgp-P1-RFP(A) 및 sRRVgp-P1-TK/spRRVe-P1-GFP(B) 바이러스 벡터의 재조합 여부를 확인한 도이다.
도 4는 spRRVe-P1-TK 벡터의 재조합 유형을 분석한 도이다.
도 5는 spRRVe-P1-yCD 및 sRRVgp-P1-RFP 벡터의 구조를 나타낸 모식도이다.
도 6은 spRRVe-P1-yCD 및 sRRVgp-P1-RFP 벡터의 재조합 여부를 확인한 도이다.
도 7은 spRRVe-P1-yCD 벡터의 재조합 유형을 분석한 도이다.
도 8은 spRRVe-P1-yCD/sRRVgp-P1-TK 벡터의 구조를 나타낸 모식도이다.
도 9는 spRRVe-P1-yCD/sRRVgp-P1-TK 벡터의 재조합 여부를 확인한 도이다.
도 10은 spRRVe-P1-yCD 벡터의 재조합 유형을 분석한 도이다.
도 11은 sRRVgp-P1-TK 벡터의 재조합 유형을 분석한 도이다.
도 12는 spRRVe-P1-yCD/sRRVgp-P1-TK 바이러스의 약물 감수성을 확인한 도이다.
도 13은 spRRVe-P1-CDa, spRRVe-P1-CDb 및 spRRVe-P1-CDc 벡터의 구조를 나타낸 모식도이다.
도 14는 spRRVe-P1-CDa, spRRVe-P1-CDb 및 spRRVe-P1-CDc 벡터의 재조합 여부를 확인한 도이다.
도 15는 spRRVe-P1-CDa 벡터의 재조합 유형을 분석한 도이다.
도 16은 spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 및 spRRVe-P1-CD9 벡터의 구조를 나타낸 모식도이다.
도 17a는 p1 및 p2 단계에서 spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 및 spRRVe-P1-CD9 바이러스의 약물 감수성을 확인한 도이다.
도 17b는 p3 및 p4 단계에서 spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 및 spRRVe-P1-CD9 바이러스의 약물 감수성을 확인한 도이다.
도 18은 spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 및 spRRVe-P1-CD9 벡터의 재조합 여부를 확인한 도이다.
도 19a는 spRRVe-P1-CD6 벡터의 재조합 유형을 분석한 도이다.
도 19b는 spRRVe-P1-CD6 벡터의 재조합 유형을 분석한 도의 이어지는 부분이다.
도 20a는 spRRVe-P1-CD8 벡터의 재조합 유형을 분석한 도이다.
도 20b는 spRRVe-P1-CD8 벡터의 재조합 유형을 분석한 도의 이어지는 부분이다.
도 21은 spRRVe-P2-yCD 및 spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8 및 spRRVe-P2-CD9 벡터의 구조를 나타낸 모식도이다.
도 22a는 p1 및 p3 단계에서 spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8 및 spRRVe-P2-CD9 바이러스의 약물 감수성을 확인한 도이다.
도 22b는 p5 및 p7 단계에서 spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8 및 spRRVe-P2-CD9 바이러스의 약물 감수성을 확인한 도이다.
도 22c는 p8 및 p9 단계에서 spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8 및 spRRVe-P2-CD9 바이러스의 약물 감수성을 확인한 도이다.
도 23은 spRRVe-P2-yCD, spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8, spRRVe-P2-CD9 및 sRRVgp-P1-TK 벡터의 재조합 여부를 확인한 도이다.
도 24는 sRRVgp-P1-TK 벡터의 재조합 유형을 분석한 도이다.
도 25는 sRRVgp-P2-TK 벡터의 구조를 나타낸 모식도이다.
도 26a는 p1 및 p3 단계에서 spRRVe-P2-CD6, spRRVe-P2-CD7 및 spRRVe-P2-CD8 바이러스의 약물 감수성을 확인한 도이다.
도 26b는 p5 및 p7 단계에서 spRRVe-P2-CD6, spRRVe-P2-CD7 및 spRRVe-P2-CD8 바이러스의 약물 감수성을 확인한 도이다.
도 26c는 p9 및 p11 단계에서 spRRVe-P2-CD6, spRRVe-P2-CD7 및 spRRVe-P2-CD8 바이러스의 약물 감수성을 확인한 도이다.
도 27a는 spRRVe-P2-CD6, spRRVe-P2-CD7 또는 spRRVe-P2-CD8/sRRVgp-P2-TK 벡터의 재조합 여부를 MuLV4194F 및 MFGSacⅠR 프라이머를 이용하여 확인한 도이다.
도 27b는 spRRVe-P2-CD6, spRRVe-P2-CD7 또는 spRRVe-P2-CD8/sRRVgp-P2-TK 벡터의 재조합 여부를 GaLV1624F 및 MFGSacⅠR 프라이머를 이용하여 확인한 도이다.
도 28a는 spRRVe-P2-CD6 벡터의 재조합 유형을 분석한 도이다.
도 28b는 spRRVe-P2-CD6 벡터의 재조합 유형을 분석한 도의 이어지는 부분이다.
도 29a는 spRRVe-P2-CD8 벡터의 재조합 유형을 분석한 도이다.
도 29b는 spRRVe-P2-CD8 벡터의 재조합 유형을 분석한 도의 이어지는 부분이다.
도 30은 spRRVe-P2-CD6, spRRVe-P2-CD10, spRRVe-P2-CD11, spRRVe-P2-CD12, spRRVe-P2-CD13, spRRVe-P2-CD14, spRRVe-P2-CD15 또는 spRRVe-P2-CD16 바이러스의 약물 감수성을 확인한 도이다.
도 31은 sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7, sRRVgp-P2-CD8 벡터의 구조를 나타낸 모식도이다.
도 32는 spRRVe-P2-TK 벡터의 구조를 나타낸 모식도이다.
도 33a는 spRRVe-P2-TK 벡터의 서열을 나타낸 도이다.
도 33b는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33c는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33d는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33e는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33f는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33g는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33h는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33i는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33j는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33k는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 33l는 spRRVe-P2-TK 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 34는 sRRVgp-P2-CD6 벡터의 구조를 나타낸 모식도이다.
도 35a는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도이다.
도 35b는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35c는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35d는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35e는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35f는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35g는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35h는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35i는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 35j는 sRRVgp-P2-CD6 벡터의 서열을 나타낸 도의 이어지는 부분이다.
도 36a는 spRRVe-P2-TK/sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 벡터의 재조합 여부를 GaLV124F 및 MFGSac1R 프라이머를 이용하여 확인한 도이다.
도 36b는 spRRVe-P2-TK/sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 벡터의 재조합 여부를 MuLV4194F 또는 MuLV7130F 및 MFGSac1R 프라이머를 이용하여 확인한 도이다.
도 37a는 p1 및 p2 단계에서 spRRVe-P2-TK/sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 바이러스의 약물 감수성을 확인한 도이다.
도 37b는 p3 및 p4 단계에서 spRRVe-P2-TK/sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 바이러스의 약물 감수성을 확인한 도이다.
도 37c는 p5, p6 및 p7 단계에서 spRRVe-P2-TK/sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 바이러스의 약물 감수성을 확인한 도이다. FIG. 1 is a schematic diagram showing the structures of spRRVe-P1-TK (A), sRRVgp-P1-RFP (A), sRRVgp-P1-TK (B) and spRRVe-P1-GFP (B)
FIG. 2 is a graph showing the results of measuring the activity of spRRVe-P1-TK / sRRVgp-P1-RFP and sRRVgp-P1-TK / spRRVe-P1-GFP virus.
FIG. 3 is a diagram for confirming recombination of spRRVe-P1-TK / sRRVgp-P1-RFP (A) and sRRVgp-P1-TK / spRRVe-P1-GFP (B) viral vectors.
Figure 4 is an analysis of the recombination type of the spRRVe-P1-TK vector.
FIG. 5 is a schematic diagram showing the structures of spRRVe-P1-yCD and sRRVgp-P1-RFP vectors.
FIG. 6 is a diagram for confirming whether recombination of spRRVe-P1-yCD and sRRVgp-P1-RFP vectors is performed.
Figure 7 is an analysis of the recombination type of the spRRVe-Pl-yCD vector.
8 is a schematic diagram showing the structure of the spRRVe-Pl-yCD / sRRVgp-P1-TK vector.
FIG. 9 is a diagram for confirming the recombination of the spRRVe-P1-yCD / sRRVgp-P1-TK vector.
10 is an analysis of the recombination type of the spRRVe-P1-yCD vector.
11 is an analysis of the recombinant type of the sRRVgp-P1-TK vector.
FIG. 12 shows the drug susceptibility of spRRVe-P1-yCD / sRRVgp-P1-TK virus.
13 is a schematic diagram showing the structures of spRRVe-P1-CDa, spRRVe-P1-CDb and spRRVe-P1-CDc vectors.
FIG. 14 is a diagram for confirming whether or not recombination of spRRVe-P1-CDa, spRRVe-P1-CDb and spRRVe-P1-CDc vectors.
15 is an analysis of the recombination type of the spRRVe-P1-CDa vector.
P1-CD8, spRRVe-P1-CD8, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe- 3 is a schematic diagram showing the structure of a vector.
Figure 17A is a graph showing the effect of the spRRVe-P1-CD8 and spRRVe-P1-CD8, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe- spRRVe-P1-CD9 < / RTI > virus.
P1-CD8, spRRVe-P1-CD8, spRRVe-P1-CD8, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe- spRRVe-P1-CD9 < / RTI > virus.
P1-CD8, spRRVe-P1-CD8, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe- And it is confirmed whether or not the vector is recombined.
19A is an analysis of the recombination type of the spRRVe-P1-CD6 vector.
Figure 19b is a continuation of the analysis of the recombinant type of the spRRVe-Pl-CD6 vector.
20A is an analysis of the recombination type of the spRRVe-P1-CD8 vector.
Figure 20b is a continuation of the analysis of the recombinant type of the spRRVe-P1-CD8 vector.
P2-CD8, spRRVe-P2-CD8, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe- 3 is a schematic diagram showing the structure of a vector.
Figure 22A is a graph showing the effect of the spRRVe-P2-CD8, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe- As shown in FIG.
Figure 22B is a graph showing the effect of the spRRVe-P2-CD8, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe- As shown in FIG.
Figure 22C is a graph showing the effect of the spRRVe-P2-CD8, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe- As shown in FIG.
P2-CD8, spRRVe-P2-CD8, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe- And the sRRVgp-P1-TK vector.
24 is an analysis of the recombination type of the sRRVgp-P1-TK vector.
25 is a schematic diagram showing the structure of sRRVgp-P2-TK vector.
FIG. 26A is a diagram showing the drug susceptibility of spRRVe-P2-CD6, spRRVe-P2-CD7 and spRRVe-P2-CD8 viruses at steps p1 and p3.
FIG. 26B shows the drug susceptibility of spRRVe-P2-CD6, spRRVe-P2-CD7 and spRRVe-P2-CD8 viruses at the p5 and p7 stages.
FIG. 26C shows the drug susceptibility of spRRVe-P2-CD6, spRRVe-P2-CD7 and spRRVe-P2-CD8 viruses at the p9 and p11 stages.
FIG. 27A is a view showing the recombination of spRRVe-P2-CD6, spRRVe-P2-CD7 or spRRVe-P2-CD8 / sRRVgp-P2-TK vector using MuLV4194F and MFGSacIR primers.
FIG. 27B is a view showing the recombination of spRRVe-P2-CD6, spRRVe-P2-CD7 or spRRVe-P2-CD8 / sRRVgp-P2-TK vector using GaLV1624F and MFGSacIR primers.
28A is an analysis of the recombination type of the spRRVe-P2-CD6 vector.
Figure 28b is a continuation of the analysis of the recombinant type of the spRRVe-P2-CD6 vector.
29A is an analysis of the recombination type of the spRRVe-P2-CD8 vector.
Figure 29b is a continuation of the analysis of the recombinant type of the spRRVe-P2-CD8 vector.
FIG. 30 is a graph showing the effect of the spRRVe-P2-CD16, spRRVe-P2-CD10, spRRVe-P2-CD11, spRRVe-P2-CD12, spRRVe- The drug susceptibility of the virus is confirmed.
31 is a schematic diagram showing the structures of sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 and sRRVgp-P2-CD8 vectors.
32 is a schematic diagram showing the structure of the spRRVe-P2-TK vector.
33A is a diagram showing the sequence of the spRRVe-P2-TK vector.
Figure 33B is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33C is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
33D is a continuation of the diagram showing the sequence of the spRRVe-P2-TK vector.
Figure 33E is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33F is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33g is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33h is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33i is a continuation of the diagram showing the sequence of the spRRVe-P2-TK vector.
Figure 33J is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 33K is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
Figure 331 is a continuation of the figure showing the sequence of the spRRVe-P2-TK vector.
34 is a schematic diagram showing the structure of sRRVgp-P2-CD6 vector.
35A is a diagram showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35B is a continuation of the diagram showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35c is a continuation of the figure showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35d is a continuation of the diagram showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35E is a continuation of the diagram showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35F is a continuation of the figure showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35g is a continuation of the diagram showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35h is a continuation of the figure showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35i is a continuation of the figure showing the sequence of the sRRVgp-P2-CD6 vector.
Figure 35J is a continuation of the figure showing the sequence of the sRRVgp-P2-CD6 vector.
FIG. 36A shows the recombination of spRRVe-P2-TK / sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 vectors using GaLV124F and MFGSac1R primers.
FIG. 36B is a diagram showing the recombination of spRRVe-P2-TK / sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 vectors using MuLV4194F or MuLV7130F and MFGSac1R primers.
FIG. 37A shows the drug susceptibility of spRRVe-P2-TK / sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 viruses at steps p1 and p2.
FIG. 37B shows the drug susceptibility of spRRVe-P2-TK / sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 viruses at steps p3 and p4.
FIG. 37C shows the drug susceptibility of spRRVe-P2-TK / sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 virus at the p5, p6 and p7 stages.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템을 제공한다.The present invention relates to a first recombinant expression vector comprising the Gag-Pol gene of MuLV, a promoter and a cytosine deaminase gene; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus.

상기 티미딘 키나아제 유전자는 서열번호 15의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 폴리뉴클레오티드는 티미딘 키나아제 단백질의 아미노산 서열을 코딩하는 폴리뉴클레오티드 서열뿐만 아니라, 상기 폴리뉴클레오티드와 실질적으로 동일한 염기서열을 갖는 폴리뉴클레오티드 및 이의 단편을 포함할 수 있다. 실질적으로 동일한 염기서열을 갖는 폴리뉴클레오티드는 본 발명의 폴리뉴클레오티드와 80％ 이상, 구체적으로는 90％ 이상, 더욱 구체적으로는 95％ 이상의 상동성을 가질 수 있다. 상술한 바와 같이, 본 발명의 폴리뉴클레오티드는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 염기서열이 치환, 결실 또는 삽입된 변이체를 포함할 수 있다.The thymidine kinase gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: The polynucleotide may comprise a polynucleotide sequence encoding the amino acid sequence of a thymidine kinase protein, as well as a polynucleotide having a base sequence substantially the same as the polynucleotide and a fragment thereof. A polynucleotide having substantially the same base sequence may have 80% or more, specifically 90% or more, more specifically 95% or more homology with the polynucleotide of the present invention. As described above, the polynucleotides of the present invention may include mutants in which one or more base sequences are substituted, deleted or inserted, so long as they encode a protein having equivalent activity.

상기 시토신 디아미나아제 유전자는 인간 코돈으로 최적화된 것일 수 있다.The cytosine deaminase gene may be one which has been optimized to the human codon.

본 명세서에서 사용된 용어, "인간 코돈으로 최적화된 것"은 숙주 세포 내에서 DNA가 단백질로 전사 및 번역될 때 아미노산을 지정하는 코돈 사이에 숙주에 따라 선호도가 높은 코돈이 존재하는데, 사람의 코돈으로 치환함으로써 그 핵산이 암호화하는 아미노산 또는 단백질의 발현 효율을 증가시키는 것을 의미한다.As used herein, the term "optimized to human codon" means that there are codons with a high preference according to the host among the codons designating amino acids when the DNA is transcribed and translated into protein in the host cell, Means that the expression efficiency of the amino acid or protein encoded by the nucleic acid is increased.

상기 시토신 디아미나아제 유전자는 서열번호 16, 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21, 서열번호 22, 서열번호 23, 서열번호 24, 서열번호 25, 서열번호 26, 서열번호 27, 서열번호 28, 서열번호 29, 서열번호 30 또는 서열번호 31의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 폴리뉴클레오티드는 상술한 바와 같은 특징을 갖는 변이체를 포함할 수 있다. The cytosine deaminase gene is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, , SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31. The polynucleotide may include variants having the above-mentioned characteristics.

상기 티미딘 키나아제 또는 시토신 디아미나아제 유전자는 전구체 약물을 활성화시킬 수 있다. 상기 전구체 약물은 간사이클로비르 및 5-플루오로사이토신으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. 본 발명의 일 실시예에서, 티미딘 키나아제 유전자는 간사이클로비르를, 시토신 디아미나아제 유전자는 5-플루오로사이토신을 활성화시킬 수 있다.The thymidine kinase or cytosine deaminase gene may activate the precursor drug. The precursor drug may be any one or more selected from the group consisting of hepcyclovir and 5-fluorocytosine. In one embodiment of the present invention, the thymidine kinase gene can activate hepcyclovir and the cytosine deaminase gene can activate 5-fluorocytosine.

상기 Gag 유전자는 레트로바이러스 코어를 구성하는 4종의 단백질을 암호화하는 폴리뉴클레오티드일 수 있다. 한편, 상기 Pol 유전자는 레트로바이러스의 역전사 효소를 암호화하는 폴리뉴클레오티드일 수 있고, 상기 Env 유전자는 레트로바이러스의 외피 당단백질을 암호화하는 폴리뉴클레오티드일 수 있다.The Gag gene may be a polynucleotide encoding four proteins constituting the retrovirus core. Meanwhile, the Pol gene may be a polynucleotide encoding a reverse transcriptase of a retrovirus, and the Env gene may be a polynucleotide encoding a retrovirus envelope glycoprotein.

상기 MuLV-Gag 유전자는 쥐 백혈병 바이러스(Murine Leukemia virus)의 Gag 유전자로서 서열번호 32의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 MuLV-Pol 유전자는 쥐 백혈병 바이러스의 Pol 유전자로서 서열번호 33의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 MuLV의 Gag-Pol 유전자는 서열번호 32 및 서열번호 33의 염기서열이 융합된 염기서열로 구성되는 폴리뉴클레오티드일 수 있다.The MuLV-Gag gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 32 as a Gag gene of Murine Leukemia virus. The MuLV-Pol gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 33 as Pol gene of murine leukemia virus. The Gag-Pol gene of MuLV may be a polynucleotide consisting of a nucleotide sequence fused with the nucleotide sequence of SEQ ID NO: 32 and SEQ ID NO: 33.

상기 프로모터는 MCMV 초기 프로모터(MCMV immediate-early promoter), EF1α 프로모터, HCMV 초기 프로모터, PGK 프로모터 및 hTERT 프로모터와 같은 암 특이적 프로모터로 이루어진 군으로부터 유래되는 어느 하나일 수 있다. 상기 EF1α 프로모터는 서열번호 34의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. The promoter may be any one derived from the group consisting of a cancer specific promoter such as MCMV immediate-early promoter, EF1? Promoter, HCMV early promoter, PGK promoter and hTERT promoter. The EF1? Promoter may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 34.

상기 바이러스의 Env 유전자는 긴팔원숭이 백혈병 바이러스(Gibbon ape Leukemia virus; GaLV), 암포트로픽 쥐 백혈병 바이러스(amphotropic MuLV), 친이종 쥐 백혈병 바이러스(xenotropic MuLV), 고양이 내재성 바이러스(RD114), 수포성 구내염바이러스(vesicular stomatitis virus; VSV) 및 홍역바이러스(measles virus; MV)의 Env 유전자로 이루어진 군으로부터 유래되는 어느 하나일 수 있다. 상기 긴팔원숭이 백혈병 바이러스의 Env 유전자는 서열번호 35의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 폴리뉴클레오티드는 상술한 바와 같은 특징을 갖는 변이체를 포함할 수 있다.The Env gene of the virus may be selected from the group consisting of Gibbon ape Leukemia virus (GaLV), amphotropic MuLV, xenotropic MuLV, cat endogenous virus (RD114), vesicular stomatitis The virus may be any one derived from the group consisting of Env genes of vesicular stomatitis virus (VSV) and measles virus (MV). The Env gene of the gibbon terminal leukemia virus may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 35. The polynucleotide may include variants having the above-mentioned characteristics.

본 명세서에서 사용된 용어, "복제가능"은 특정 유전자를 포함하는 바이러스 게놈(genome)이 형질 도입되거나 동물 세포나 특정 유전자가 포함된 바이러스 벡터가 감염된 세포에서 상기 바이러스 벡터가 스스로 복제할 수 있다는 것을 의미한다.As used herein, the term "replicable" means that the viral vector can self-replicate in a cell transfected with a viral genome containing a specific gene or infected with a viral vector containing an animal cell or a specific gene it means.

본 명세서에서 사용된 용어, "복제가능 레트로바이러스 벡터"는 비용균성 바이러스를 생산하는 벡터로서, 핵막이 깨지는 틈을 타 핵 안으로 들어가기 때문에 분열하는 세포, 즉 암세포에 특이적으로 감염될 수 있어 삽입된 유전자가 다른 정상세포에서 발현되는 것을 막을 수 있다. 따라서, 상기 벡터는 암세포로 안전하게 유전자를 전달할 수 있고, 바이러스의 복제가 가능하기 때문에 유전자의 전달 효율도 증대시킬 수 있다.As used herein, the term "replicable retrovirus vector" refers to a vector that produces cost-effective viruses. Since the nuclear envelope breaks through the cracks into other nuclei, it can specifically infect dividing cells, The gene can be prevented from being expressed in other normal cells. Therefore, the vector can safely transmit the gene to the cancer cells, and the replication efficiency of the gene can be increased because the virus can be replicated.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 시토신 디아미나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터가 재조합이 일어나지 않음을 확인하였다(도 36a 및 36b).In a specific embodiment of the present invention, the present inventors isolated the MuLV-Gag and MuLV-Pol genes and the GalV-EnV gene so as to express them in independent vectors, respectively. The P2 promoter and the cytosine diaminase gene were used as vectors having Gag and Pol genes , And the P2 promoter and the thymidine kinase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 31). Further, the present inventors confirmed that the vector did not undergo recombination (FIGS. 36A and 36B).

또한, 본 발명은 MuLV의 Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템을 제공한다.The present invention also relates to a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the thymidine kinase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the cytosine deaminase gene of the virus.

상기 벡터 시스템은 상술한 바와 같은 특징을 갖는다. 일례로, 상기 티미딘 키나아제 유전자는 전구체 약물인 간사이클로비르를, 상기 시토신 디아미나아제 유전자는 전구체 약물인 5-플루오로사이토신을 활성화 시킬 수 있다. The vector system has the features described above. For example, the thymidine kinase gene may activate a precursor drug, ganciclovir, and the cytosine diaminase gene may activate a precursor drug, 5-fluorocytosine.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 티미딘 키나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 시토신 디아미나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 25 참조). In a specific embodiment of the present invention, the present inventors isolated MuLV-Gag and MuLV-Pol genes and GalV-EnV genes so as to express them in independent vectors, respectively. The P2 promoter and thymidine kinase gene were inserted into a vector having Gag and Pol genes Cloning was performed, and the P2 promoter and cytosine deaminase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 25).

또한, 본 발명은 상기 벡터 시스템을 포함하는 재조합 레트로바이러스를 제공한다.The present invention also provides a recombinant retrovirus comprising the vector system.

상기 벡터 시스템은 상술한 바와 같은 특징을 가질 수 있다.The vector system may have the features described above.

한편, 상기 재조합 레트로바이러스는 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터로부터 생산될 수 있다. 이때, 상기 제 1 재조합 발현벡터 및 제 2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다.On the other hand, the recombinant retrovirus includes a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the cytosine deaminase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus. At this time, the first recombinant expression vector and the second recombinant expression vector may be included in the recombinant retrovirus or together.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 시토신 디아미나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터로 바이러스를 생산하였다.In a specific embodiment of the present invention, the present inventors isolated the MuLV-Gag and MuLV-Pol genes and the GalV-EnV gene so as to express them in independent vectors, respectively. The P2 promoter and the cytosine diaminase gene were used as vectors having Gag and Pol genes , And the P2 promoter and the thymidine kinase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 31). In addition, the present inventors produced viruses with the above vectors.

또한, 본 발명은 상기 재조합 레트로바이러스로 형질감염된 숙주 세포를 제공한다.The present invention also provides a host cell transfected with the recombinant retrovirus.

상기 벡터 시스템은 상술한 바와 같은 특징을 가질 수 있다.The vector system may have the features described above.

상기 숙주 세포는 NS/O 골수종 세포(NS/O myeloma cell), 인간 293T 세포, 중국 햄스터 난소 세포(CHO cell), HeLa 세포, CapT 세포(인간 양수 유래 세포) COS 세포, 케나인 D17 세포 또는 펠라인 PG4 세포일 수 있다. 본 발명의 일 실시예에서, 상기 숙주 세포는 인간 293T 세포일 수 있다.The host cell may be a NS / O myeloma cell, a human 293T cell, a Chinese hamster ovary cell (CHO cell), a HeLa cell, a CapT cell (human amniotic fluid cell) COS cell, a Kenan D17 cell, Gt; PG4 < / RTI > cells. In one embodiment of the invention, the host cell may be a human 293T cell.

상기 형질감염은 당업계에 공지된 방법에 따라 수행될 수 있다. 일례로, 리포펙타민 방법, 미세 주입법, 칼슘 포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법 및 유전자 밤바드먼트로 이루어진 군으로부터 선택되는 어느 하나 이상의 방법으로 수행될 수 있다. 본 발명의 일 실시예에서, 상기 형질감염은 리포펙타민 방법으로 수행될 수 있다.The transfection can be carried out according to methods known in the art. For example, the method may be carried out by any one or more methods selected from the group consisting of lipofectamine method, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, DEAE-dextran treatment method and gene bombardment . In one embodiment of the invention, the transfection can be carried out by the lipofectamine method.

상기 형질감염된 세포는 동물세포의 배양에 통상적으로 이용되는 배지를 사용하여 배양될 수 있다. 일례로, 상기 배지는 Eagles's MEM, a-MEM, Iscove'sMEM, 199 배지, CMRL 1066, RPMI 1640, F12, F10, DMEM, DMEM과 F12의 혼합배지, Way-mouth's MB752/1, McCoy's 5A 및 MCDB 시리즈 배지로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 본 발명의 일 실시예에서, 상기 배지는 DMEM일 수 있다.The transfected cells may be cultured using a medium commonly used for culturing animal cells. For example, the medium may be selected from the group consisting of Eagles's MEM, a-MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, mixed medium of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A and MCDB And a series medium. In one embodiment of the present invention, the medium may be DMEM.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 유전자 및 시토신 디아미나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 유전자 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터로 바이러스를 생산하여 재조합 유형 분석을 통해 재조합이 일어나지 않음을 확인하였다(도 36a 및 36b).In a specific embodiment of the present invention, the present inventors isolated MuLV-Gag and MuLV-Pol genes and GalV-EnV genes so as to express them in independent vectors, respectively. The P2 promoter gene and the cytosine deaminase gene were amplified with Gag and Pol gene The P2 promoter gene and the thymidine kinase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 31). In addition, the present inventors produced viruses from the above vectors and confirmed that recombination did not occur through analysis of recombinant type (FIGS. 36A and 36B).

또한, 본 발명은 상기 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus as an active ingredient.

한편, 상기 재조합 레트로바이러스는 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터로부터 생산될 수 있다. 이때, 상기 제 1 재조합 발현벡터 및 제 2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다. On the other hand, the recombinant retrovirus includes a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the cytosine deaminase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus. At this time, the first recombinant expression vector and the second recombinant expression vector may be included in the recombinant retrovirus or together.

상기 레트로바이러스는 분열 중인 모든 세포를 포적할 수 있으며, 구체적으로 상기 세포는 암세포일 수 있다. 상기 암세포는 점액상 세포 암종, 둥근 세포암종, 국소적 진행 종양, 전이성 암, 어윙 (Ewing) 육종, 암전이, 림프성 전이, 편평상피 세포 암종, 식도 편평 상피 세포 암종, 경구 암종, 다발성 골수종, 급성 림프구성 백혈병, 급성 비림프구성 백혈병, 만성 림프구 백혈병, 만성 골수구 백혈병, 모양 세포성 백혈병, 유출 림프종 (체강계 림프종), 흉선 림프종 폐암, 소 세포 폐암종, 피부 T 세포 림프종, 호지킨 림프종, 비-호지킨성 림프종, 부신 피질 암, ACTH-생성 종양, 비소세포 폐암, 유방암, 소 세포 암종, 도관 암종, 위암, 결장암, 결장직장암, 결장직장 종양 형성과 관련된 용종, 췌장암, 간암, 방광암, 1차 표면 방광 종양, 방광의 침습성 전이 세포 방광암종, 근 침윤성 방광암, 전립선암, 대장암, 신장암, 간암, 식도암, 난소 암종, 자궁경부암, 자궁내막암, 융모암, 난소암, 원발성 복막상피 신생물, 자궁 경관암종, 질암, 외음부암, 자궁암, 난포 중 고형 종양, 고환암, 음경암, 신장 세포 암종, 뇌암, 두경부암, 구경부암, 신경아세포종, 뇌간 신경교종, 신경교종, 중추신경계 중 전이성 종양 세포 침윤, 골종, 골육종, 악성 흑색종, 인간 피부 케라티노사이트의 종양 진행, 편평상피 세포 암종, 갑상선 암, 망막아종, 신경아세포종, 중피종, 빌름스 종양, 담낭암, 영양아세포 종양, 혈관주위세포종, 또는 카포시 육종과 같은 암으로부터 유래된 세포를 포함할 수 있다.The retrovirus may be capable of covering all the cells that are dividing, and specifically, the cells may be cancer cells. The cancer cells may be selected from the group consisting of point-like liquid cell carcinoma, round cell carcinoma, local advanced tumor, metastatic cancer, Ewing sarcoma, metastasis, lymphatic metastasis, squamous cell carcinoma, esophageal squamous cell carcinoma, Acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, shape cell leukemia, leukemic lymphoma (thrombosed lymphoma), thymoma lymphoma lung cancer, small cell lung carcinoma, skin T cell lymphoma, Hodgkin lymphoma , Non-Hodgkin's lymphoma, adrenocortical cancer, ACTH-producing tumor, non-small cell lung cancer, breast cancer, small cell carcinoma, ductal carcinoma, stomach cancer, colon cancer, colorectal cancer, colon associated with rectal tumorigenesis, pancreatic cancer, liver cancer, bladder cancer , Primary surface bladder tumor, invasive metastatic bladder carcinoma of bladder, muscular invasive bladder cancer, prostate cancer, colon cancer, renal cancer, liver cancer, esophageal cancer, ovarian carcinoma, cervical cancer, endometrial cancer, villus Cancer of the ovary, primary peritoneal epithelial neoplasm, cervical carcinoma, vaginal cancer, vulvar cancer, uterine cancer, solid tumors in the follicle, testicular cancer, penile cancer, renal cell carcinoma, brain cancer, head and neck cancer, Glioma, metastatic tumor cell invasion in central nervous system, osteoma, osteosarcoma, malignant melanoma, tumor progression of human skin keratinocyte, squamous cell carcinoma, thyroid cancer, retinoblastoma, neuroblastoma, mesothelioma, And may include cells derived from cancer, such as a malignant neoplasm, an adenocarcinoma tumor, an angioendocrine tumor, or Kaposi sarcoma.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 시토신 디아미나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터가 간사이클로비르 및 5-플루오로사이토신에 대한 약물감수성이 우수함을 확인하였다(도 37a, 37b 및 37c).In a specific embodiment of the present invention, the present inventors isolated the MuLV-Gag and MuLV-Pol genes and the GalV-EnV gene so as to express them in independent vectors, respectively. The P2 promoter and the cytosine diaminase gene were used as vectors having Gag and Pol genes , And the P2 promoter and the thymidine kinase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 31). Furthermore, the present inventors confirmed that the vector had excellent drug sensitivity to hepcyclovir and 5-fluoro cytosine (FIGS. 37A, 37B and 37C).

상기 약학 조성물은 조성물 전체 중량에 대하여 유효성분인 본 발명에 따른 조성물을 10 내지 95 중량%로 포함할 수 있다. 또한, 본 발명의 조성물은 상기 유효성분 외에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 추가로 함유할 수 있다.The pharmaceutical composition may contain 10 to 95% by weight of the composition according to the present invention, which is an active ingredient, based on the total weight of the composition. The composition of the present invention may further contain, in addition to the above-mentioned effective ingredients, one or more additional active ingredients having the same or similar functions.

본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약제학적으로 허용 가능한 담체는 조성물을 생체 내에 전달하는데 적합한 것이면 특별히 제한되지 않으며, 예로서, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 또는 이들 성분 중 1 성분 이상을 혼합한 것일 수 있다. 이때, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo. Examples include Merck Index, 13th ed., Merck & Inc. Sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of at least one of these components. At this time, other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added as necessary.

상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다.When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

본 발명의 조성물은 경구제제 또는 비경구제제로 제형화 될 수 있다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스, 락토오스 및 젤라틴 등을 섞어 조제될 수 있다. 또한, 마그네슘 스티레이트, 탈크 같은 윤활제들도 첨가될 수 있다. 한편, 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 여기에는 습윤제, 감미제, 방향제, 보존제 등과 같은 부형제가 포함될 수 있다.The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like. Lubricants such as magnesium stearate and talc may also be added. On the other hand, liquid formulations include suspensions, solutions, emulsions or syrups, which may contain excipients such as wetting agents, sweeteners, fragrances, preservatives and the like.

비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제 등의 주사제가 포함될 수 있다. Formulations for parenteral administration may include injections such as sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions.

비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여될수 있으며, 비경구 투여는 피부 외용 또는 복강내 주사, 직장내 주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식중 선택될 수 있다.The composition of the present invention can be administered orally or parenterally according to the desired method, and parenteral administration can be carried out by external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, Can be selected.

본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여된다. 이는 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물 등에 따라 달라질 수 있다. 본 발명의 조성물은 단독 또는 다른 치료제와 병용하여 투여될 수 있다. 병용 투여시, 투여는 순차적 또는 동시일 수 있다. The composition according to the invention is administered in a pharmaceutically effective amount. It may vary depending on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered concomitantly, the administration can be sequential or simultaneous.

그러나, 바람직한 효과를 위해서, 본 발명에 따른 조성물에 포함되는 유효성분의 양은 0.001 ~ 10,000 mg/㎏, 구체적으로는 0.1 g ~ 5 g/kg 일 수 있다. 상기 투여는 하루에 1회일 수 있고, 수회로 나뉠 수도 있다.However, for the desired effect, the amount of the active ingredient contained in the composition according to the present invention may be 0.001 to 10,000 mg / kg, specifically 0.1 g to 5 g / kg. The administration may be one time per day and may be divided into several times.

또한, 본 발명은 상기 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물을 제공한다.In addition, the present invention provides a gene transfer composition for treating cancer comprising the recombinant retrovirus.

한편, 상기 재조합 레트로바이러스는 MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터로부터 생산될 수 있다. 이때, 상기 제 1 재조합 발현벡터 및 제 2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다. On the other hand, the recombinant retrovirus includes a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the cytosine deaminase gene of MuLV; And a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus. At this time, the first recombinant expression vector and the second recombinant expression vector may be included in the recombinant retrovirus or together.

상기 암은 상술한 바와 같은 암을 포함할 수 있다.The arm may include an arm as described above.

본 명세서에서 사용된 용어, "유전자 전달 조성물"은 유전자를 표적 세포 내로 전달할 수 있는 조성물을 의미한다. As used herein, the term "gene delivery composition" refers to a composition capable of delivering a gene into a target cell.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 시토신 디아미나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터가 간사이클로비르 및 5-플루오로사이토신에 대한 약물감수성이 우수함을 확인하였다(도 37a, 37b 및 37c).In a specific embodiment of the present invention, the present inventors isolated the MuLV-Gag and MuLV-Pol genes and the GalV-EnV gene so as to express them in independent vectors, respectively. The P2 promoter and the cytosine diaminase gene were used as vectors having Gag and Pol genes , And the P2 promoter and the thymidine kinase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 31). Furthermore, the present inventors confirmed that the vector had excellent drug sensitivity to hepcyclovir and 5-fluoro cytosine (FIGS. 37A, 37B and 37C).

또한, 본 발명은 1) MuLV의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및 2) 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법을 제공한다.The present invention also provides a method for producing a recombinant expression vector, comprising the steps of: 1) preparing a first recombinant expression vector comprising the MuLV Gag-Pol gene, a promoter and a cytosine deaminase gene; And 2) preparing a second recombinant expression vector comprising the Env gene, the promoter and the thymidine kinase gene of the virus.

상기 벡터 시스템은 상술한 바와 같은 특징을 갖는다. 일례로, 상기 티미딘 키나아제 유전자는 전구체 약물인 간사이클로비르를, 상기 시토신 디아미나아제 유전자는 전구체 약물인 5-플루오로사이토신을 활성화시킬 수 있다. The vector system has the features described above. For example, the thymidine kinase gene may activate a precursor drug, ganciclovir, and the cytosine diaminase gene may activate a precursor drug, 5-fluorocytosine.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 시토신 디아미나아제 유전자 및 티미딘 키나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 31 참조). 또한, 본 발명자들은 상기 벡터가 재조합이 일어나지 않음을 확인하였다(도 36a 및 36b).In a specific example of the present invention, the present inventors isolated MuLV-Gag and MuLV-Pol genes and GalV-EnV genes so as to express them in independent vectors, respectively, and compared the P2 promoter and cytosine diaminase gene and the thymidine kinase gene with the Env gene To construct a replicable retrovirus vector (see Fig. 31). Further, the present inventors confirmed that the vector did not undergo recombination (FIGS. 36A and 36B).

아울러, 본 발명은 1) MuLV의 Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및 2) 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법을 제공한다.In addition, the present invention provides a method for producing a recombinant expression vector, comprising: 1) preparing a first recombinant expression vector comprising MuLV Gag-Pol gene, a promoter and a thymidine kinase gene; And 2) preparing a second recombinant expression vector comprising the Env gene, the promoter and the cytosine deaminase gene of the virus.

상기 벡터 시스템은 상술한 바와 같은 특징을 갖는다. 일례로, 상기 티미딘 키나아제 유전자는 전구체 약물인 간사이클로비르를, 상기 시토신 디아미나아제 유전자는 전구체 약물인 5-플루오로사이토신을 활성화 시킬 수 있다. The vector system has the features described above. For example, the thymidine kinase gene may activate a precursor drug, ganciclovir, and the cytosine diaminase gene may activate a precursor drug, 5-fluorocytosine.

본 발명의 구체적인 실시예에서, 본 발명자들은 MuLV-Gag 및 MuLV-Pol 유전자와 GalV-EnV 유전자가 각각 독립된 벡터에서 발현하도록 분리하였으며, P2 프로모터 및 티미딘 키나아제 유전자를 Gag 및 Pol 유전자가 있는 벡터에 클로닝하고, P2 프로모터 및 시토신 디아미나아제 유전자를 Env 유전자가 있는 벡터에 클로닝하여 복제가능 레트로바이러스 벡터를 구축하였다(도 25 참조). In a specific embodiment of the present invention, the present inventors isolated MuLV-Gag and MuLV-Pol genes and GalV-EnV genes so as to express them in independent vectors, respectively. The P2 promoter and thymidine kinase gene were inserted into a vector having Gag and Pol genes Cloning was performed, and the P2 promoter and cytosine deaminase gene were cloned into a vector having the Env gene to construct a replicable retrovirus vector (see FIG. 25).

이하, 본 발명을 하기 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by the following examples.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

허피스Herpice 심플렉스Simplex 바이러스의  Virus 티미딘Thymidine 키나아제 Kinase 유전자를 단독으로 포함하는 복제가능 레트로바이러스 벡터 시스템의 구축Construction of a replicable retroviral vector system containing the gene alone

<1-1> 바이러스 벡터의 제작<1-1> Production of virus vector

gag, pol 및 MuLV-env의 유전자가 하나의 지놈으로 합성되는 기존의 RRV 벡터를 gag-pol과 MuLV-env가 각각 독립된 벡터에서 발현하도록 분리하였으며, env는 영장류 감염에 친화적인 GaLV(Gibbon ape Leukemia virus)의 env 유전자로 치환하였다. HSV-TK 유전자를 gag-pol 벡터 또는 GaLV-env 벡터에 각각 클로닝 하였으며, 표적 마커로 형광유전자를 각 벡터에 교차 클로닝하여 벡터 시스템을 구축하였다.The existing RRV vector, in which gag, pol and MuLV-env genes were synthesized in a single genome, was isolated so that gag-pol and MuLV-env could be expressed in independent vectors, respectively. Env was obtained from Gibbon ape Leukemia virus) env gene. The HSV-TK gene was cloned into a gag-pol vector or a GaLV-env vector, respectively, and a vector system was constructed by cross-cloning the fluorescent gene into each vector with a target marker.

구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.Specifically, virus vectors were prepared by the following method.

1. spRRVe-P1-TK 벡터: 프로모터 1(P1; MCMV 초기 프로모터) 아래에 멀티 클로닝 부위(multi-cloning site)를 갖고 있는 spRRVe-P1-mcs 벡터를 PmeI 제한효소로 자른 후, CIAP(alkaline phosphatase, Calf intestinal)를 처리하였다. pSXLC-TK 벡터(Sugimoto et al., 1994, Bio/Technology 12, 694-698)를 NcoI 제한효소와 XhoI 제한효소로 자른 후, TK 유전자를 회수하였다. 회수한 TK 유전자를 T4 DNA 중합효소를 이용하여 평활 말단(blunt end)으로 변환시킨 후, spRRVe-P1-mcs 벡터를 BamHI 및 SalI 제한효소로 잘라 CIAP를 처리하여 회수한 뒤 클로닝하여 spRRVe-P1-TK 벡터를 제작하였다(도 1A).1. spRRVe-P1-TK vector: The spRRVe-P1-mcs vector having a multi-cloning site under the promoter 1 (P1; MCMV early promoter) was digested with PmeI restriction enzyme, and CIAP (alkaline phosphatase , Calf intestinal). After digesting the pSXLC-TK vector (Sugimoto et al. , 1994, Bio / Technology 12, 694-698) with NcoI restriction enzyme and XhoI restriction enzyme, TK gene was recovered. The recovered TK gene was transformed into a blunt end using T4 DNA polymerase. The spRRVe-P1-mcs vector was cut with BamHI and SalI restriction enzymes and recovered by treatment with CIAP and cloned into spRRVe-P1- TK vector (FIG. 1A).

2. sRRVgp-P1-RFP 벡터: pol과 3'-LTR 사이에 EcoRI 절단 부위를 포함하고 있는 sRRVgp 벡터(대한민국 공개특허 제10-1381064호)를 EcoRI 제한효소로 자른 후, CIAP를 처리하였다. 본 발명자들이 구축한 Lenti-P1-RFP(monomer) 벡터(충남대학교)를 PpuMI 제한효소와 NcoI 제한효소로 자른 후, P1-RFP 유전자를 회수하였다. 상기 P1-RFP 유전자를 T4 DNA 중합효소를 이용하여 sRRVgp 벡터에 클로닝하여 제작하였다(도 1A).2. sRRVgp-P1-RFP vector: The sRRVgp vector containing the EcoRI cleavage site between pol and 3'-LTR (Korean Patent Laid-Open No. 10-1381064) was digested with EcoRI restriction enzyme and treated with CIAP. The P1-RFP gene was recovered after the Lenti-P1-RFP (monomer) vector constructed by the present inventors (Chungnam National University) was digested with PpuMI restriction enzyme and NcoI restriction enzyme. The P1-RFP gene was cloned into the sRRVgp vector using T4 DNA polymerase (FIG. 1A).

3. sRRVgp-P1-TK 벡터: EcoRI으로 자른 후 CIAP 처리한 sRRVgp 벡터에 EcoRI, PmeI 제한효소 및 P1 프로모터를 포함하도록(EcoRI-P1-PmeI-EcoRI) PCR한 산물을 클로닝하여 완성한 sRRVgp-P1 벡터를 PmeI 제한효소로 자른 후, CIAP를 처리하였다. 본 발명자들이 구축한 spRRVe-P1-TK 벡터(충남대학교)를 주형으로 서열번호 1의 염기서열을 갖는 HSV-TK-EcoRI-F 프라이머(5'-CGGAATTCATGGCTTCGTACCCCGGCCA-3') 및 서열번호 2의 염기서열을 갖는 HSV-TK-EcoRI-R 프라이머(5'-GCGAATTCTCAGTAGCCTCCCCCATCTC-3')를 이용하여 EcoRI 절단 부위가 포함되도록 TK 유전자를 증폭하였다. 이를 회수하여 EcoRI 제한효소로 자른 후, sRRVgp-P1 벡터에 클로닝하여 제작하였다(도 1B).3. sRRVgp-P1-TK vector: An sRRVgp-P1 vector obtained by cloning the PCR product (EcoRI-P1-PmeI-EcoRI) so as to include EcoRI, PmeI restriction enzyme and P1 promoter in the sRRVgp vector cut with EcoRI and CIAP- Was digested with PmeI restriction enzyme and then treated with CIAP. The HSV-TK-EcoRI-F primer (5'-CGGAATTCATGGCTTCGTACCCCGGCCA-3 ') having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2 (5'-GCGAATTCTCAGTAGCCTCCCCCATCTC-3 ') containing the EcoRI cleavage site was used to amplify the TK gene. This was recovered and cut with EcoRI restriction enzyme and cloned into sRRVgp-P1 vector (FIG. 1B).

4. spRRVe-P1-GFP 벡터: GFP를 발현하는 레트로바이러스 벡터인 Retro-MCMV-GFP 벡터(MFG-mCMV-GFP, 대한민국 공개특허 제10-1381064호)의 gag 유전자와 GFP 유전자 사이에 GaLV-env 유전자를 클로닝 하기 위해 본 발명자들이 구축한 Retro-P1-GFP 벡터(충남대학교)를 PmeI 제한효소로 잘랐다. 본 발명자들이 구축한 MYK-GaLV 벡터(충남대학교)를 주형으로 서열번호 3의 염기서열을 갖는 GaLV-PmlI-F 프라이머(5'-CGGCACGTGATGGTATTGCTGCCTGGG-3') 및 서열번호 4의 염기서열을 갖는 GaLV-PmlI-R 프라이머(5'-GCCCACGTGTTAAAGGTTACCTTCGTT-3')를 이용하여 PmlI 절단 부위가 포함되도록 GaLV-env 유전자를 증폭하였다. 이를 회수하여 PmlI 제한효소로 자른 후, Retro-P1-GFP 벡터에 클로닝하여 제작하였다(도 1B).4. spRRVe-P1-GFP vector: A gag gene of the Retro-MCMV-GFP vector (MFG-mCMV-GFP, Korean Patent Publication No. 10-1381064), which is a retroviral vector expressing GFP, To clone the gene, Retro-P1-GFP vector (Chungnam National University) constructed by the present inventors was cut with PmeI restriction enzyme. (5'-CGGCACGTGATGGTATTGCTGCCTGGG-3 ') having the nucleotide sequence of SEQ ID NO: 3 and a GaLV-PmlI-F primer having the nucleotide sequence of SEQ ID NO: 4 as a template, using the MYK-GaLV vector (Chungnam National University) The GaLV-env gene was amplified using the PmlI-R primer (5'-GCCCACGTGTTAAAGGTTACCTTCGTT-3 ') to include the PmlI cleavage site. This was recovered, cut with PmlI restriction enzyme, and cloned into Retro-P1-GFP vector (FIG. 1B).

<1-2><1-2> 바이러스 생산 Virus production

실시예 <1-1>에서 제조한 벡터로 바이러스를 생산하기 위해 spRRVe-P1-TK 벡터와 sRRVgp-P1-RFP 벡터의 조합(spRRVe-P1-TK/sRRVgp-P1-RFP) 및 sRRVgp-P1-TK 벡터와 spRRVe-P1-GFP 벡터의 조합(sRRVgp-P1-TK/spRRVe-P1-GFP)으로 바이러스를 생산하였다.(SpRRVe-P1-TK / sRRVgp-P1-RFP) and sRRVgp-P1-RFP vector combination of the spRRVe-P1-TK vector and the sRRVgp-P1- RFP vector in order to produce the virus with the vector prepared in Example <1-1> (SRRVgp-P1-TK / spRRVe-P1-GFP) of the TK vector and the spRRVe-P1-GFP vector.

구체적으로, 형질감염 하루 전날 293T 세포를 6 웰 플레이트에 6×105 개/well의 농도로 접종하였다. 다음날 세포가 접종된 배지를 FBS 및 항생제가 없는 0.8 ㎖의 DMEM 배지로 교체한 후, 세포배양기에 플레이트를 다시 넣어주었다. 한편, 형질감염을 위한 용액을 준비하기 위해 2개의 1.5 ㎖ 튜브에 각각 DMEM을 100 ㎕씩 넣었다. 하나의 튜브에는 1 ㎍의 RRV(spRRVe-P1-TK/sRRVgp-P1-RFP 또는 sRRVgp-P1-TK/spRRVe-P1-GFP) DNA와 5 ㎕의 PLUS 시약(invitrogen)을, 다른 하나의 튜브에는 3 ㎕의 리포펙타민(invitrogen)을 넣고 10초간 섞어준 후 15분 동안 상온에 두었다. 그 다음 리포펙타민이 들어있는 튜브의 용액을 PLUS 시약이 들어있는 튜브에 넣고 10초간 섞어준 후, 20분 동안 상온에 두었다. 세포가 배양된 플레이트에 RRV DNA, PLUS 시약 및 리포펙타민이 들어있는 용액을 웰 당 208 ㎕씩 세포가 떨어지지 않게 방울방울 떨어뜨린 후, 4시간 동안 배양하였다. 4시간 후, 배지를 제거하고 3% FBS가 들어있는 DMEM을 세포가 떨어지지 않게 넣어주고, 48시간 동안 배양하였다. 48시간 후, 상층액을 얻어 0.45 ㎛ 주사기 필터로 여과한 후, 100 kDa의 단백질을 정제하는 15 ㎖ 센트리콘(centricon)으로 30 ㎖의 여과액을 정제하였다. 순도를 높이기 위해 15 ㎖의 D-PBS로 2번 여과하여 10배 농축한 후, 50 ㎕씩 분주하여 -80℃ 초저온냉동고에 보관하였다.Specifically, the day before transfection 293T cells were inoculated into 6 well plates at a concentration of 6 x 10 5 cells / well. The next day, the medium inoculated with the cells was replaced with 0.8 ml of DMEM medium free of FBS and antibiotics, and the plate was re-inserted into the cell incubator. Meanwhile, to prepare the solution for transfection, 100 μl of DMEM was added to each of two 1.5 ml tubes. One tube contained 1 μg of RRV (spRRVe-P1-TK / sRRVgp-P1-RFP or sRRVgp-P1-TK / spRRVe-P1-GFP) DNA and 5 μl of PLUS reagent (invitrogen) 3 μl of lipofectamine (Invitrogen) was added and mixed for 10 seconds and then kept at room temperature for 15 minutes. The solution containing the lipofectamine solution was then placed in a tube containing the PLUS reagent, mixed for 10 seconds, and placed at room temperature for 20 minutes. Cells were plated on a plate containing a solution containing RRV DNA, PLUS reagent and lipofectamine at a rate of 208 쨉 l per well so that the cells did not fall off and then cultured for 4 hours. After 4 hours, the medium was removed and DMEM containing 3% FBS was added to the cells so that the cells did not fall off and cultured for 48 hours. After 48 hours, the supernatant was collected, filtered through a 0.45 mu m syringe filter, and then 30 mL of the filtrate was purified with 15 mL centricon to purify 100 kDa protein. To increase the purity, the solution was filtered twice with 15 ml of D-PBS and concentrated 10 times. Then, 50 쨉 l of each solution was dispensed and stored at -80 캜 in a cryogenic freezer.

<1-3> 생산된 바이러스의 &Lt; 1-3 > 역가Potency 측정 Measure

실시예 <1-2>에서 생산된 바이러스의 역가를 유세포 분석기 및 qPCR을 통해 측정하였다. The activity of the virus produced in Example < 1-2 > was measured by flow cytometry and qPCR.

구체적으로, 바이러스 감염 하루 전날 신경교종세포(U87MG)를 6웰 플레이트에 접종하였다. 다음날 새로운 배지에 1X, 10X, 50X, 100X, 500X의 농도가 되도록 10배로 농축하여 분리한 바이러스를 순차 희석하였다. 희석된 바이러스 1 ㎖에 4 ㎍/㎖ 농도의 폴리브랜(polybrene)을 첨가한 후, 세포의 수를 측정하고, 신경교종세포에 바이러스를 감염시켰다. 48시간 후에 바이러스의 역전사 저해제인 50 uM의 아지도티미딘을 첨가하고 24시간 동안 반응시켜 바이러스의 증식을 억제하였다. 이후, GFP 발현을 유세포 분석기로 정량화하여 하기 수학식 1과 같이 역가를 계산하였다. Specifically, glioma cells (U87MG) were inoculated on 6-well plates the day before the virus infection. On the next day, the viruses were concentrated and diluted 10 times to a concentration of 1X, 10X, 50X, 100X, and 500X in a fresh medium. A polybrene at a concentration of 4 / / ml was added to 1 ml of the diluted virus, the number of cells was measured, and the glioma cells were infected with the virus. Forty-eight hours later, 50 袖 M azatrimidine, a reverse transcription inhibitor of the virus, was added and reacted for 24 hours to inhibit virus proliferation. Then, GFP expression was quantitated by a flow cytometer and the activity was calculated as shown in the following formula (1).

[수학식 1][Equation 1]

TU/㎖ = (감염 전 세포수 × GFP의 비율(%))/희석배율 TU / ml = (number of cells before infection × ratio of GFP (%)) / dilution factor

또한, 레트로바이러스 역가 세트(Cat. #6166, Takara, Japan)를 이용하여 제조사의 방법에 따라 real-time PCR을 수행하여 생산된 바이러스의 역가를 계산하였다.Real-time PCR was performed according to the manufacturer's method using a retrovirus titer set (Cat. # 6166, Takara, Japan) to calculate the titer of the virus produced.

그 결과, 표 1 및 도 2에 나타낸 바와 같이, 바이러스가 감염되기 직전의 신경교종세포의 개수는 1.533×105 개였으며, 신경교종세포에 대한 바이러스 역가는 약 6.13×107 TU/㎖이었다(표 1 및 도 2).As a result, as shown in Table 1 and FIG. 2, the number of glioma cells immediately before the virus infection was 1.533 × 10 5 , and the virus reverse ratio to glioma cells was about 6.13 × 10 7 TU / ml Table 1 and Fig. 2).

신경교종세표에 대한 바이러스의 역가The titer of the virus against the glioma grade
spRRVe-P1-TK/sRRVgp-P1-RFP에 의해 감염된 신경교종세포에서 GFP의 비율(%)(%) of GFP in glioma cells infected by spRRVe-P1-TK / sRRVgp-P1-RFP 10X 희석10X dilution 50X 희석50X dilution 100X 희석100X dilution 500X 희석 500X dilution 1회1 time 16.716.7 8.38.3 4.74.7 1.21.2 2회Episode 2 17.517.5 8.68.6 4.74.7 0.40.4 3회3rd time 17.917.9 8.28.2 4.34.3 0.80.8 평균Average 17.3617.36 8.368.36 4.564.56 0.80.8 TU/㎖TU / ml 2.66×107 2.66 x 10 7 6.41×107 6.41 x 10 7 7.00×107 7.00 x 10 7 6.13×107 6.13 × 10 7

<1-4> 복제가능 레트로바이러스 벡터의 재조합 여부 확인<1-4> Confirmation of the recombination of the replicable retrovirus vector

유전자치료용 복제가능 레트로바이러스 벡터는 도입된 외래 유전자에 의해 지놈 RNA의 크기가 증가하고 비상동 염기서열이 추가되어 재조합이 일어날 가능성이 높다. 따라서 효율적이고 안정적인 유전자 치료용 복제가능 레트로바이러스 벡터를 구축하기 위해서는 개발단계에서 재조합 여부 및 그 정도를 확인하는 것이 매우 중요하다. 실시예 <1-1>에서 구축한 벡터의 안정성을 확인하기 위해 하기와 같은 실험을 수행하였다.Replicable retroviral vectors for gene therapy are likely to increase the size of the genomic RNA by the introduced foreign gene and to add an exonucleotide sequence, resulting in recombination. Therefore, in order to construct an efficient and stable replication-competent retroviral vector for gene therapy, it is very important to confirm the degree of recombination and its degree at the development stage. In order to confirm the stability of the vector constructed in Example <1-1>, the following experiment was conducted.

구체적으로, 전날 계대한 신경교종세포(U-87MG)에 106 TU의 spRRVe-P1-TK/sRRVgp-P1-RFP 또는 sRRVgp-P1-TK/spRRVe-P1-GFP 바이러스를 3일 동안 감염시켰다. 상층액을 그 전날 새로 계대한 U-87MG 세포에 3일 간격으로 8번 순차 감염하여 이를 p1(passage1)~p8(passage8)이라 명명하였고, 형광현미경으로 각 단계의 세포에서 발현되는 RFP 또는 GFP를 관찰하였다. Specifically, 10 6 TU of spRRVe-P1-TK / sRRVgp-P1-RFP or sRRVgp-P1-TK / spRRVe-P1-GFP virus was infected for 3 days in the glioma glioma cell line (U-87MG) The supernatant was sequentially infected with U-87MG cells on the previous day at 8-day intervals at intervals of 3 days. The cells were named p1 (passage1) to p8 (passage8), and RFP or GFP Respectively.

또한, 같은 단계의 세포로부터 지놈 DNA를 분리하여, Env 벡터 및 gag-pol 벡터에 특이적 프라이머인 서열번호 5의 염기서열을 갖는 MuLV4194F 프라이머(5'-AGCAAGCTATTGGCCACTG-3'), 서열번호 6의 염기서열을 갖는 GaLV(1624)F 프라이머(5'-GACTCAGTCAGCAAGTTAGAG-3') 및 서열번호 7의 염기서열을 갖는 MFGSaclR 프라이머(5'-CAATCGGAGGACTGGCGCCCCGAGTGA-3')를 사용하여 PCR을 수행해 예상되는 크기로 유전자가 증폭되는지를 확인하였다. PCR 반응 튜브에 100 ng의 지놈 DNA, 1X 반응 버퍼, 0.25 mM dNTP, 0.2 pmol 정방향 프라이머, 0.2 pmol 역방향 프라이머, 0.2 unit Taq 중합효소를 넣고 최종 부피가 20 ㎕이 되도록 멸균된 증류수를 첨가하였다. 이후, 하기 표 1의 조건으로 PCR을 수행한 후, 1% 아가로오스 젤에 증폭된 DNA를 로딩하여 분석하였다. Further, the genomic DNA was isolated from the cells of the same step, and the MuLV4194F primer (5'-AGCAAGCTATTGGCCACTG-3 ') having the nucleotide sequence of SEQ ID NO: 5, which is a specific primer for Env vector and gag-pol vector, PCR was performed using the GaLV (1624) F primer (5'-GACTCAGTCAGCAAGTTAGAG-3 ') having the sequence of SEQ ID NO: 7 and the MFGSaclR primer (5'-CAATCGGAGGACTGGCGCCCCGAGTGA-3' Amplified. To the PCR reaction tube, 100 ng of genomic DNA, 1X reaction buffer, 0.25 mM dNTP, 0.2 pmol forward primer, 0.2 pmol reverse primer and 0.2 unit Taq polymerase were added and sterilized distilled water was added to a final volume of 20 μl. Thereafter, PCR was carried out under the conditions shown in Table 1, and DNA amplified in 1% agarose gel was loaded and analyzed.

PCR 조건PCR conditions 단계step 온도Temperature 시간time 사이클수Number of cycles 변성(denaturation)Denaturation 94℃94 ° C 3분3 minutes 1One
중합(polymerization)
Polymerization
94℃94 ° C 30초30 seconds
28
28
60℃60 ° C 30초30 seconds 72℃72 ℃ 2분 30초2 minutes 30 seconds 신장(extension)Extension 72℃72 ℃ 7분7 minutes 1One

그 결과, 도 3에 나타낸 바와 같이, spRRVe-P1-TK 벡터에서 발현된 TK 유전자는 p2 단계부터 밴드의 강도가 약해졌고 p3 단계부터는 그 크기가 급격히 작아졌다. 반면, sRRVgp-P1-RFP 벡터에서 발현된 RFP 유전자는 p8 단계까지 같은 크기의 PCR 산물이 지속적으로 증폭되었다(도 3A). 한편, sRRVgp-P1-TK 벡터 및 spRRVe-P1-GFP 벡터에서는 재조합 변이가 일어나지 않았다(도 3B). 상기 결과로부터 TK 유전자는 spRRVe-P1 벡터보다 sRRVgp-P1 벡터에 위치해 있을 때 더 안정함을 확인하였다.As a result, as shown in Fig. 3, the intensity of the band of the TK gene expressed in the spRRVe-P1-TK vector was weakened from the p2 stage and sharply decreased from the p3 stage. On the other hand, the RFP gene expressed in the sRRVgp-P1-RFP vector was continuously amplified with the same size PCR product until the p8 step (Fig. 3A). On the other hand, no recombination mutations occurred in the sRRVgp-P1-TK vector and the spRRVe-P1-GFP vector (Fig. 3B). From the above results, it was confirmed that the TK gene was more stable when located in the sRRVgp-P1 vector than the spRRVe-P1 vector.

<1-5> <1-5> spRRVespRRVe -P1-TK 벡터의 재조합 유형 분석Analysis of recombinant type of P1-TK vector

상기 실시예 <1-4>에서 spRRVe-P1-TK/sRRVgp-P1-RFP 조합의 지놈 DNA를 이용한 PCR 반응 후, 기대 크기보다 작아진 spRRVe-P1-TK 벡터의 p4 단계 재조합 밴드(*)의 PCR 산물을 회수하여 유전자 분석을 위해 pGEM-T 벡터에 클로닝하였다.After the PCR reaction using the genomic DNA of the combination of spRRVe-P1-TK / sRRVgp-P1-RFP in the above Example <1-4>, the p4 step recombination band (*) of the spRRVe-P1- The PCR product was recovered and cloned into pGEM-T vector for gene analysis.

그 결과, 도 4에 나타낸 바와 같이, 회수된 PCR 산물은 GaLV env와 P1 사이부터 3'-LTR 직전까지 약 2 kb 유전자가 소실되거나, GaLV env와 P1 사이부터 HSV-TK 말단까지 약 1.8 kb 유전자가 소실되는 경향을 보였으며, 이는 반복되는 염기서열인 AGAAAAAGGGGGGAAT 또는 ATGGGG 사이에 재조합이 원인임을 확인하였다(도 4). As a result, as shown in Fig. 4, the recovered PCR product showed that about 2 kb of the gene was lost from the position between GaLV env and P1 to just before the 3'-LTR, or about 1.8 kb from the position between GaLV env and P1 to the end of HSV- (Fig. 4), which was confirmed to be due to recombination between the repeated base sequence AGAAAAAAGGGGGGAAT or ATGGGG (Fig. 4).

시토신 Cytosine 디아미나아제Diaminase 유전자 단독 복제가능 레트로바이러스 벡터 시스템 구축 Construction of gene-only replicable retrovirus vector system

<2-1> 바이러스 벡터의 제작<2-1> Production of virus vector

실시예 <1-4>에서 TK 유전자가 sRRVgp-P1 벡터에 위치할 때 재조합이 일어나지 않아, 이미 임상에서 치료도구로 사용되고 있는 다른 자살유전자인 CD 유전자를 spRRVe-P1 벡터에 도입한 벡터 시스템을 구축하였다.In the example <1-4>, when the TK gene is located in the sRRVgp-P1 vector, no recombination occurs and a vector system is constructed in which the CD gene, another suicide gene already used as a therapeutic tool in clinical practice, is introduced into the spRRVe-P1 vector Respectively.

구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.Specifically, virus vectors were prepared by the following method.

1. spRRVe-P1-yCD 벡터: pcDNA-yCD 벡터(원자력병원, 이기호박사님 제공)를 XhoI 및 HindIII 제한효소로 자른 후, yCD(서열번호 16)를 회수하였다. 본 발명자들이 구축한 spRRVe-P1-MCS 벡터(충남대학교)를 PmeI 제한효소로 자른 후, yCD 유전자를 상기 위치에 클로닝하여 제작하였다(도 5).1. spRRVe-P1-yCD vector: yCD (SEQ ID NO: 16) was recovered after digesting pcDNA-yCD vector (Nuclear Hospital, provided by Dr. Kiho) with XhoI and HindIII restriction enzymes. The spRRVe-P1-MCS vector constructed by the present inventors (Chungnam National University) was digested with PmeI restriction enzyme, and the yCD gene was cloned into the above position (FIG. 5).

2. sRRVgp-P1-RFP 벡터: 상기 실시예 <1-1>과 같은 방법으로 제작하였다(도 5).2. sRRVgp-P1-RFP vector: Produced in the same manner as in Example <1-1> (FIG. 5).

<2-2><2-2> 바이러스 생산 Virus production

실시예 <2-1>에서 제조한 벡터로 바이러스를 생산하기 위해 spRRVe-P1-yCD 벡터와 sRRVgp-P1-RFP 벡터를 조합(spRRVe-P1-yCD/sRRVgp-P1-RFP)하여 상기 실시예 <1-2>의 방법으로 바이러스를 생산하였다.The spRRVe-P1-yCD vector and the sRRVgp-P1-RFP vector were combined (spRRVe-P1-yCD / sRRVgp-P1-RFP) to produce the virus from the vector prepared in Example <2-1> 1-2. &Lt; / RTI >

<2-3> 복제가능 레트로바이러스 벡터의 재조합 여부 확인<2-3> Confirmation of the recombination of replicable retrovirus vector

실시예 <2-1>에서 구축한 벡터의 안정성 확인은 spRRVe-P1-yCD/sRRVgp-P1-RFP 바이러스를 3일 간격으로 5번 순차 감염시킨 것과 PCR 수행시, 72℃ 온도에서 1분 30초 동안 중합시키는 것을 제외하고는, 실시예 <1-4>와 동일한 방법 및 조건으로 수행되었다.The stability of the vector constructed in Example <2-1> was confirmed by sequential infection of spRRVe-P1-yCD / sRRVgp-P1-RFP virus at intervals of 3 days and by PCR at 72 ° C for 1 minute 30 seconds Was carried out in the same manner and under the same conditions as in Example < 1-4 >.

그 결과, 도 6에 나타낸 바와 같이, spRRV-P1-yCD 벡터는 P1 단계부터 재조합이 크게 일어난 반면 sRRVgp-P1-RFP 벡터는 재조합이 일어나지 않았다(도 6).As a result, as shown in Fig. 6, the recombination was largely occurred in the spRRV-P1-yCD vector from the P1 stage, whereas the sRRVgp-P1-RFP vector did not recombine (Fig. 6).

<2-4> <2-4> spRRVespRRVe -P1--P1- yCDyCD 벡터의 재조합 유형 분석 Analysis of recombination type of vector

상기 실시예 <2-3>에서 spRRVe-P1-yCD/sRRVgp-P1-RFP 조합의 지놈 DNA를 이용한 PCR 반응 후, 기대 크기보다 작아진 spRRVe-P1-yCD 벡터의 p4 단계 재조합 밴드(*)의 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 하여 12개 클론에 대한 유전자 분석을 하였다.After the PCR reaction using the genomic DNA of the combination of spRRVe-P1-yCD / sRRVgp-P1-RFP in the above Example <2-3>, the p4 step recombination band (*) of the spRRVe-P1- The PCR product was recovered and cloned into pGEM-T vector to perform gene analysis on 12 clones.

그 결과, 도 7에 나타낸 바와 같이, 모두 2종류의 재조합 유형이 분석되었는데. 2종류 모두 GaLV Env와 P1 사이부터 yCD와 3'-LTR 사이에서 재조합이 일어나 유전자 소실이 일어남을 확인하였다(도 7).As a result, as shown in Fig. 7, two types of recombination types were analyzed. It was confirmed that recombination between yCD and 3'-LTR occurred between GaLV Env and P1 in both kinds, and gene loss occurred (Fig. 7).

시토신 Cytosine 디아미나아제Diaminase 유전자와  Gene and 티미딘Thymidine 키나아제 유전자를 모두 포함하는 복제가능 레트로바이러스 벡터 시스템의 구축 Construction of a replicable retroviral vector system including all kinase genes

<3-1> 바이러스 생산 <3-1> Virus production

spRRVe-P1-yCD 벡터와 sRRVgp-P1-TK 벡터의 조합(spRRVe-P1-yCD/sRRVgp-P1-TK)으로 상기 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다(도 8).The virus was produced in the same manner as in Example <1-2> with the combination of spRRVe-P1-yCD vector and sRRVgp-P1-TK vector (spRRVe-P1-yCD / sRRVgp-P1-TK) (FIG.

<3-2> 복제가능 레트로바이러스 벡터의 재조합 여부 확인<3-2> Confirmation of whether the replicable retrovirus vector is recombined

실시예 <3-1>에서 바이러스 생산에 이용한 벡터의 안정성 확인은 spRRVe-P1-yCD/sRRVgp-P1-TK 바이러스를 3일 간격으로 5번 순차 감염시킨 것을 제외하고는, 실시예 <1-4>와 동일한 방법 및 조건으로 수행되었다.The stability of the vector used for virus production in Example <3-1> was confirmed in Example <1-4> except that spRRVe-P1-yCD / sRRVgp-P1-TK virus was sequentially infected five times at intervals of 3 days &Gt;. &lt; / RTI &gt;

그 결과, 도 9에 나타낸 바와 같이, spRRVe-P1-yCD 벡터는 p1 단계부터 재조합이 발생했다(도 9).As a result, as shown in Fig. 9, recombination occurred in the spRRVe-P1-yCD vector from the p1 stage (Fig. 9).

<3-3> <3-3> spRRVespRRVe -P1--P1- yCDyCD 벡터의 재조합 유형 분석 Analysis of recombination type of vector

상기 실시예 <3-2>에서 spRRVe-P1-yCD/sRRVgp-P1-TK 조합의 지놈 DNA를 이용한 PCR 반응 후, 기대 크기보다 작아진 spRRVe-P1-yCD 벡터의 p4 단계 재조합 밴드(*)의 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 하여 10개 클론에 대한 유전자 분석을 하였다.After the PCR reaction using the genomic DNA of the combination of spRRVe-P1-yCD / sRRVgp-P1-TK in the above Example <3-2>, the p4 step recombination band (*) of the spRRVe-P1- The PCR product was recovered and cloned into pGEM-T vector to perform gene analysis for 10 clones.

그 결과, 도 10에 나타낸 바와 같이, 모두 3종류의 재조합 유형이 분석되었는데. 2종류는 GaLV Env와 P1 사이부터 yCD와 3'-LTR 사이에 재조합이 일어났으며, 1종류는 GaLV Env와 P1 사이부터 yCD 유전자 말단까지 재조합이 일어나 유전자 소실이 일어남을 확인하였다(도 10).As a result, as shown in Fig. 10, all three types of recombination types were analyzed. 2 recombination occurred between GaLV Env and P1 between yCD and 3'-LTR, and one type showed recombination between GaLV Env and P1 to the end of yCD gene (FIG. 10) .

<3-4> <3-4> sRRVgpsRRVgp -P1-TK 벡터의 재조합 유형 분석Analysis of recombinant type of P1-TK vector

상기 실시예 <3-2>에서 spRRVe-P1-yCD/sRRVgp-P1-TK 조합의 지놈 DNA를 이용한 PCR 반응 후 기대 크기보다 작아진 sRRVgp-P1-TK 벡터의 p1 단계 재조합 밴드(*)의 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 하여 20개 클론에 대한 유전자 분석을 하였다.PCR of the p1-step recombination band (*) of the sRRVgp-P1-TK vector which was smaller than the expected size after the PCR reaction using the genomic DNA of the combination of spRRVe-P1-yCD / sRRVgp-P1-TK in the above Example <3-2> The product was recovered and cloned into pGEM-T vector to perform gene analysis for 20 clones.

그 결과, 도 11에 나타낸 바와 같이, 모두 5종류의 재조합 유형이 분석되었는데, 주로 Pol 말단부터 HSV-TK 유전자 내 말단 또는 HSV-TK와 3'-LTR 사이에서 재조합이 일어나 유전자 소실이 일어남을 확인하였다(도 11).As a result, as shown in Fig. 11, all five types of recombinant types were analyzed, and it was confirmed that recombination occurred mainly between the Pol terminal and the end in the HSV-TK gene or HSV-TK and 3'-LTR, (Fig. 11).

<3-5> <3-5> spRRVespRRVe -P1--P1- yCDyCD // sRRVgpsRRVgp -P1-TK 바이러스의 약물 감수성 확인- Identification of drug susceptibility of P1-TK virus

상기 실시예 <3-1>에서 생산된 spRRVe-P1-yCD/sRRVgp-P1-TK 바이러스의 간사이클로비르(GCV) 및 5-플루오로사이토신(5-FC)에 대한 약물감수성을 확인하였다.The drug susceptibility of the spRRVe-P1-yCD / sRRVgp-P1-TK virus produced in Example <3-1> to hepcyclovir (GCV) and 5-fluorocytosine (5-FC) was confirmed.

구체적으로, spRRVe-P1-yCD/sRRVgp-P1-TK 바이러스를 PLUS 시약(invitrogen) 및 리포펙타민(invitrogen)을 이용하여 293T 세포주에 공형질주입(co-transfection)하였다. 2일 후 바이러스의 상층액을 회수하고, 전날 6웰에 1.5 X105개로 계대한 U-87MG 세포에 바이러스와 8 ㎕/㎖ 농도의 폴리브랜을 첨가하여 8시간 동안 감염시켰다. 감염 5일 후(postinfection 5d), 세포의 상층액을 취하여 전날 6웰에 1.5 X105개로 계대한 U-87MG 세포에 재감염 하였으며(p1), 이후 같은 방법으로 p4 까지 순차감염 하였다. 각 감염 단계의 세포에 트립신-EDTA를 처리하여 단일세포로 만들어, 12웰 플레이트에 1.5 X105개로 계대하였고, 계대한 다음날부터 30 ㎕/㎖ 농도의 GCV와 1 mM 농도의 5-FC를 각각 5일 또는 8일 동안 처리하여 세포 사멸을 확인하였다.Specifically, spRRVe-P1-yCD / sRRVgp-P1-TK virus was co-transfected into 293T cell line using PLUS reagent and lipofectamine. Recovering the virus supernatant after 2 days and to 1.5 X10 5 to open-circuit based on U-87MG cells in a 6-well the day before addition of virus and poly branch 8 ㎕ / ㎖ concentrations were infected for 8 hours. After 5 days of infection (postinfection 5d), the supernatant of the cells was taken and re-infected with U-87MG cells (p1) in 1.5 x 10 5 cells in 6 wells of the previous day. The cells of each infected stage were treated with trypsin-EDTA to form single cells. The cells were transferred to 12-well plates at 1.5 × 10 5 cells and 5 μl of GCV at 30 μl / ml and 5-FC at 1 mM concentration Lt; / RTI &gt; days or 8 days to confirm cell death.

그 결과, 도 12에 나타낸 바와 같이, spRRV-P1-yCD/sRRVgp-P1-TK 바이러스는 GCV에 대한 약물 감수성은 p3 단계까지 지속되는 반면, 5-FC에 대한 약물 감수성은 재조합이 발생하지 않은 post-infection 5d 단계에서조차도 나타나지 않았다(도 12).As a result, as shown in Fig. 12, the drug susceptibility to GCV of the spRRV-P1-yCD / sRRVgp-P1-TK virus continued to the p3 level, while the drug susceptibility to 5-FC -infection did not occur even in step 5d (FIG. 12).

spRRVespRRVe -P1--P1- CDaCDa , , spRRVespRRVe -P1--P1- CDbCDb 및  And spRRVespRRVe -P1--P1- CDcCDc 바이러스 벡터 시스템의 구축 Construction of virus vector system

<4-1> 바이러스 벡터의 제작&Lt; 4-1 > Production of virus vector

실시예 <3-3>에서 spRRVe-P1-yCD 벡터의 재조합이 주로 GaLV Env 유전자와 P1 프로모터 사이부터 3'-LTR과 yCD 유전자 사이에서 발생하였으므로, 바이러스 합성에 불필요한 염기서열을 제거하여 벡터 시스템을 구축하였다.Since the recombination of the spRRVe-P1-yCD vector occurred mainly between the 3'-LTR and the yCD gene between the GaLV Env gene and the P1 promoter in Example <3-3>, the vector system unnecessary for the viral synthesis was removed, Respectively.

구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.Specifically, virus vectors were prepared by the following method.

1. spRRVe-P1-CDa 벡터: spRRVe-P1-yCD 벡터를 주형으로 서열번호 8의 염기서열을 갖는 spRRVe-CDa-F 프라이머(5'-GAAGGTAACCTTTAATTCAATAACAGGAAAG-3') 및 서열번호 9의 염기서열을 갖는 spRRVe-CDa-R 프라이머(5'-CTTTCCTGTTATTGAATTAAAGGTTACCTTC-3')를 이용한 오버랩 PCR을 수행하여 제작하였다(도 13).1. a spRRVe-CDa-F primer (5'-GAAGGTAACCTTTAATTCAATAACAGGAAAG-3 ') having the nucleotide sequence of SEQ ID NO: 8 as a template and a nucleotide sequence of SEQ ID NO: (Fig. 13) using the spRRVe-CDa-R primer (5'-CTTTCCTGTTATTGAATTAAAGGTTACCTTC-3 ').

2. spRRVe-P1-CDb 벡터: spRRVe-P1-CD 벡터를 주형으로 서열번호 10의 염기서열을 갖는 spRRVe-CDb-F 프라이머(5'-GAAGATATTGGTGAGTAGCTATAAAATAAAAGATTTT-3') 및 서열번호 11의 염기서열을 갖는 spRRVe-CDb-R 프라이머(5'-AAAATCTTTTATTTTATAGCTACTCACCAATATCTTC-3')를 이용한 오버랩 PCR을 수행하여 제작하였다(도 13).(5'-GAAGATATTGGTGAGTAGCTATAAAATAAAAAGATTTT-3 ') having the nucleotide sequence of SEQ ID NO: 10 and a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 11 as a template, using the spRRVe-P1-CDb vector: spRRVe- (Fig. 13) using the spRRVe-CDb-R primer (5'-AAAATCTTTTATTTTATAGCTACTCACCAATATCTTC-3 ').

3. spRRVe-P1-CDc 벡터: spRRVe-P1-CD 벡터를 주형으로 서열번호 12의 염기서열을 갖는 spRRVe-CDc-F 프라이머(5'-ACCACCGTAGAACGCAATGGTGACAGGGGGAAT-3') 및 서열번호 13의 염기서열을 갖는 spRRVe-CDc-R 프라이머(5'-ATTCCCCCTGTCACCATTGCGTTCTACGGTGGT-3')를 이용한 오버랩 PCR을 수행하여 제작하였다(도 13).3. spRRVe-P1-CDc vector: spRRVe-CDc-F primer (5'-ACCACCGTAGAACGCAATGGTGACAGGGGGAAT-3 ') having the nucleotide sequence of SEQ ID NO: 12 as the template and the nucleotide sequence of SEQ ID NO: (Fig. 13) using an SPRRVe-CDc-R primer (5'-ATTCCCCCTGTCACCATTGCGTTCTACGGTGGT-3 ').

<4-2> 바이러스 생산 <4-2> Virus production

실시예 <4-1>에서 제조한 벡터를 이용한 바이러스의 생산은 spRRVe-P1-CDa, spRRVe-P1-CDb 또는 spRRVe-P1-CDc 벡터와 sRRVgp-P1-RFP 벡터의 조합 및 spRRVe-P1-CDa, spRRVe-P1-CDb 또는 spRRVe-P1-CDc 벡터와 sRRVgp-P1-TK 벡터의 조합으로 실시예 <1-2>와 동일한 방법으로 수행되었다.The production of virus using the vector prepared in Example <4-1> was carried out using a combination of spRRVe-P1-CDa, spRRVe-P1-CDb or spRRVe-P1-CDc vector and sRRVgp- , spRRVe-P1-CDb or spRRVe-P1-CDc vector and sRRVgp-P1-TK vector in the same manner as in Example <1-2>.

<4-3> <4-3> spRRVespRRVe -P1--P1- CDaCDa , , spRRVespRRVe -P1--P1- CDbCDb 및  And spRRVespRRVe -P1--P1- CDcCDc 벡터의 재조합 여부 확인 Check whether the vector is reassembled

실시예 <4-1>에서 구축한 벡터의 안정성 확인은 실시예 <4-2>에서 생산한 6종류의 바이러스를 감염시킨 것을 제외하고는, 실시예 <2-3>과 동일한 방법으로 벡터의 재조합 여부를 확인하였다.The stability of the vector constructed in Example <4-1> was confirmed in the same manner as in Example <2-3, except that the six kinds of viruses produced in Example <4-2> were infected. And confirmed the recombination.

그 결과, 도 14에 나타낸 바와 같이, spRRVe-P1-CDa 및 spRRVe-P1-CDc 벡터는 p2 단계부터 재조합이 크게 일어났고, spRRVe-P1-CDb 벡터는 p2부터 재조합이 발생하여 p3에서는 PCR 반응물을 확인할 수 없었다(도 14). As a result, as shown in FIG. 14, the spRRVe-P1-CDa and spRRVe-P1-CDc vectors exhibited a large recombination at the p2 stage and the recombination at the spRRVe-P1- (Fig. 14).

<4-4> <4-4> spRRVespRRVe -P1--P1- CDaCDa 벡터의 재조합 유형 분석 Analysis of recombination type of vector

실시예 <4-3>에서 spRRVe-P1-CDa/sRRVgp-P1-RFP 및 spRRVe-P1-CDa/sRRVgp-P1-TK 조합의 지놈 DNA를 이용한 PCR 반응 후, 기대 크기보다 작아진 spRRVe-P1-CDa 벡터의 p2 및 p3 단계 재조합 밴드(*)의 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 하여 24개 클론에 대한 유전자 분석을 하였다.P1-CDa / sRRVgp-P1-CDa / sRRVgp-P1-RFP and spRRVe-P1-CDa / sRRVgp-P1-TK combination in Example <4-3> The PCR products of the p2 and p3 step recombination bands (*) of the CDa vector were collected and cloned into the pGEM-T vector to analyze the gene for 24 clones.

그 결과, 도 15에 나타낸 바와 같이, P1을 중심으로 yCD 유전자 내부 또는 yCD 유전자와 3'-LTR 사이에서 여전히 재조합이 일어남을 확인하였다(도 15).As a result, as shown in Fig. 15, it was confirmed that still recombination occurred within the yCD gene or between the yCD gene and the 3'-LTR centering on P1 (Fig. 15).

인간 코돈으로 최적화된 CD 유전자 발굴CD gene discovery optimized with human codon

상기 <실시예 2> 내지 <실시예 4>에서 사용한 yCD 유전자는 감염시 재조합이 일어날 뿐만 아니라 5-FC에 대한 약물 감수성이 매우 낮으므로 이를 극복하기 위해 8종의 인간 코돈으로 최적화된 CD 유전자(CD2∼CD9)를 발굴하였다.Since the yCD gene used in Examples 2 to 4 is not only susceptible to recombination during infection but also has a low sensitivity to 5-FC, the CD gene optimized for 8 human codons CD2 to CD9) were excavated.

구체적으로, CD2는 토카젠(Tocagen)에서 코돈 최적화를 통해 개발한 CD 유전자로 양성대조군으로서 사용되었다. CD3는 CD2 유전자에서 인간 코돈으로 최적화가 안된 12개의 코돈을 최적화(optimization)한 유전자이며, CD4는 spRRVe-P1-yCD, spRRVe-P1-CDa, spRRVe-P1-CDb 및 spRRVe-P1-CDc 벡터의 재조합이 많이 일어난 32개 위치를 모두 돌연변이시킨 유전자이다. 또한, 5종의 CD5~CD9는 효모의 CD를 인간 코돈으로 최적화시킨 유전자이다. 상기 8종의 CD 유전자는 코스모젠에 의뢰하여 합성하였다. 상기 CD2∼CD9 유전자의 서열은 하기 표 3과 같다.Specifically, CD2 was used as a positive control for the CD gene developed through codon optimization in Tocagen. CD3 is a gene that optimizes twelve codons that have not been optimized as a human codon in the CD2 gene and CD4 is a gene encoding the spRRVe-P1-yCD, spRRVe-P1-CDa, spRRVe-P1-CDb and spRRVe- It is a gene that mutates all 32 positions where recombination occurs. In addition, the five CD5 to CD9 genes are genes in which yeast CD is optimized to the human codon. The 8 CD genes were synthesized by asking Cosmosen. The sequences of the CD2 to CD9 genes are shown in Table 3 below.

인간 코돈으로 최적화된 CD 유전자의 서열Sequence of CD gene optimized with human codon

인간 코돈으로 최적화된 유전자 서열

Optimized gene sequence with human codon yCD
뉴클레오티드
서열과
유사성(%) yCD
Nucleotides
Sequence and
Similarity (%) yCD
단백질
서열과유사성(%)yCD
protein
Sequence and similarity (%)

CD2
(서열번호 17)

CD2
(SEQ ID NO: 17) ATGGTGACCGGCGGCATGGCCTCCAAGTGGGATCAAAAGGGCATGGATATCGCTTACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTAAATGGTGACCGGCGGCATGGCCTCCAAGTGGGATCAAAAGGGCATGGATATCGCTTACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTAA


79


79



98


98

CD3
(서열번호 18)

CD3
(SEQ ID NO: 18) ATGGTGACCGGCGGCATGGCCTCCAAGTGGGACCAAAAGGGCATGGATATCGCTTACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCCATCGGCGGCTGCCTGATCAACAACAAGGACGGCAGCGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGCGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGCACCGGCGCTATCATCATGTACGGCATCCCTAGGTGCGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAGACCAGGGGCCACGAGGTGGTGGTGGTGGACGACGAGAGGTGCAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCCCAGGACTGGTTCGAGGACATCGGCGAGTAAATGGTGACCGGCGGCATGGCCTCCAAGTGGGACCAAAAGGGCATGGATATCGCTTACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCCATCGGCGGCTGCCTGATCAACAACAAGGACGGCAGCGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGCGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGCACCGGCGCTATCATCATGTACGGCATCCCTAGGTGCGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAGACCAGGGGCCACGAGGTGGTGGTGGTGGACGACGAGAGGTGCAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCCCAGGACTGGTTCGAGGACATCGGCGAGTAA


76


76


98


98

CD4
(서열번호 19)

CD4
(SEQ ID NO: 19) ATGGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAGAACTGCGGCAGGCTGGAAGGCAAGGTGTACAAAGATACCACTCTGTACACCACCCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATACTTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGACTGGTTCGAGGACATCGGCGAGTAAATGGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAGAACTGCGGCAGGCTGGAAGGCAAGGTGTACAAAGATACCACTCTGTACACCACCCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATACTTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGACTGGTTCGAGGACATCGGCGAGTAA


95


95


100


100

CD5
(서열번호 20)

CD5
(SEQ ID NO: 20) ATGGTGACTGGCGGCATGGCATCCAAGTGGGACCAGAAGGGGATGGACATAGCATATGAAGAGGCCGCGTTGGGATATAAGGAGGGCGGTGTACCAATCGGGGGCTGCCTCATTAACAATAAAGATGGCTCCGTTCTGGGTCGCGGCCACAACATGAGGTTTCAGAAGGGCAGTGCGACGCTCCACGGAGAAATCAGCACACTGGAAAATTGTGGGCGATTGGAGGGGAAAGTGTATAAGGATACAACTCTCTACACCACTCTCAGCCCCTGCGATATGTGCACAGGCGCAATCATAATGTACGGCATTCCCCGATGCGTGGTGGGGGAGAACGTGAACTTCAAGAGCAAAGGAGAGAAATATCTTCAGACCAGAGGACACGAAGTAGTGGTGGTGGATGATGAACGCTGCAAGAAAATCATGAAACAGTTTATAGATGAACGACCACAAGACTGGTTCGAGGATATCGGCGAATAGATGGTGACTGGCGGCATGGCATCCAAGTGGGACCAGAAGGGGATGGACATAGCATATGAAGAGGCCGCGTTGGGATATAAGGAGGGCGGTGTACCAATCGGGGGCTGCCTCATTAACAATAAAGATGGCTCCGTTCTGGGTCGCGGCCACAACATGAGGTTTCAGAAGGGCAGTGCGACGCTCCACGGAGAAATCAGCACACTGGAAAATTGTGGGCGATTGGAGGGGAAAGTGTATAAGGATACAACTCTCTACACCACTCTCAGCCCCTGCGATATGTGCACAGGCGCAATCATAATGTACGGCATTCCCCGATGCGTGGTGGGGGAGAACGTGAACTTCAAGAGCAAAGGAGAGAAATATCTTCAGACCAGAGGACACGAAGTAGTGGTGGTGGATGATGAACGCTGCAAGAAAATCATGAAACAGTTTATAGATGAACGACCACAAGACTGGTTCGAGGATATCGGCGAATAG


77


77


100


100

CD6
(서열번호 21)

CD6
(SEQ ID NO: 21) ATGGTTACTGGAGGGATGGCCAGTAAATGGGACCAGAAGGGTATGGATATTGCATACGAGGAGGCCGCTTTGGGATACAAGGAGGGGGGTGTCCCTATAGGCGGTTGCCTGATCAATAATAAAGACGGCTCTGTCTTGGGAAGAGGACACAATATGCGCTTTCAGAAGGGAAGCGCCACCCTGCATGGAGAGATCTCTACCCTCGAAAATTGCGGAAGGCTCGAAGGCAAAGTTTACAAAGATACCACCCTCTACACAACGCTGTCCCCCTGTGATATGTGCACCGGTGCCATTATCATGTATGGCATCCCACGCTGCGTTGTAGGAGAGAATGTAAACTTCAAATCCAAGGGAGAGAAGTATCTCCAGACCCGAGGGCACGAAGTTGTGGTGGTGGACGATGAAAGGTGTAAGAAGATCATGAAGCAGTTCATAGATGAGCGGCCTCAGGACTGGTTCGAGGATATTGGCGAATGAATGGTTACTGGAGGGATGGCCAGTAAATGGGACCAGAAGGGTATGGATATTGCATACGAGGAGGCCGCTTTGGGATACAAGGAGGGGGGTGTCCCTATAGGCGGTTGCCTGATCAATAATAAAGACGGCTCTGTCTTGGGAAGAGGACACAATATGCGCTTTCAGAAGGGAAGCGCCACCCTGCATGGAGAGATCTCTACCCTCGAAAATTGCGGAAGGCTCGAAGGCAAAGTTTACAAAGATACCACCCTCTACACAACGCTGTCCCCCTGTGATATGTGCACCGGTGCCATTATCATGTATGGCATCCCACGCTGCGTTGTAGGAGAGAATGTAAACTTCAAATCCAAGGGAGAGAAGTATCTCCAGACCCGAGGGCACGAAGTTGTGGTGGTGGACGATGAAAGGTGTAAGAAGATCATGAAGCAGTTCATAGATGAGCGGCCTCAGGACTGGTTCGAGGATATTGGCGAATGA


77


77


100


100

CD7
(서열번호 22)

CD7
(SEQ ID NO: 22) ATGGTAACTGGTGGCATGGCCTCAAAGTGGGATCAGAAAGGAATGGACATCGCTTACGAGGAGGCCGCACTGGGCTATAAGGAGGGCGGCGTCCCTATAGGCGGTTGCCTGATTAACAATAAAGACGGCTCAGTGCTGGGAAGGGGGCACAACATGAGATTTCAGAAAGGCAGCGCAACTCTGCACGGCGAAATCTCCACTCTGGAGAACTGCGGGCGGCTGGAGGGAAAGGTTTATAAAGATACTACCTTGTATACAACTCTGTCCCCCTGCGATATGTGCACCGGCGCCATCATAATGTACGGAATACCCAGGTGCGTGGTGGGAGAGAACGTGAATTTTAAGTCAAAAGGTGAGAAGTACCTGCAGACTCGCGGCCATGAGGTGGTTGTTGTTGACGATGAAAGGTGCAAGAAGATTATGAAGCAGTTCATTGATGAAAGACCCCAGGACTGGTTTGAGGATATCGGAGAGTAGATGGTAACTGGTGGCATGGCCTCAAAGTGGGATCAGAAAGGAATGGACATCGCTTACGAGGAGGCCGCACTGGGCTATAAGGAGGGCGGCGTCCCTATAGGCGGTTGCCTGATTAACAATAAAGACGGCTCAGTGCTGGGAAGGGGGCACAACATGAGATTTCAGAAAGGCAGCGCAACTCTGCACGGCGAAATCTCCACTCTGGAGAACTGCGGGCGGCTGGAGGGAAAGGTTTATAAAGATACTACCTTGTATACAACTCTGTCCCCCTGCGATATGTGCACCGGCGCCATCATAATGTACGGAATACCCAGGTGCGTGGTGGGAGAGAACGTGAATTTTAAGTCAAAAGGTGAGAAGTACCTGCAGACTCGCGGCCATGAGGTGGTTGTTGTTGACGATGAAAGGTGCAAGAAGATTATGAAGCAGTTCATTGATGAAAGACCCCAGGACTGGTTTGAGGATATCGGAGAGTAG


79


79


100


100

CD8
(서열번호 23)

CD8
(SEQ ID NO: 23) ATGGTTACTGGGGGAATGGCATCTAAGTGGGATCAGAAAGGTATGGACATCGCTTATGAAGAGGCTGCTCTCGGCTACAAAGAGGGTGGAGTGCCTATCGGAGGGTGCCTGATCAACAACAAGGACGGCAGTGTGCTGGGGAGGGGCCACAATATGAGGTTCCAAAAAGGCTCCGCCACTCTCCACGGGGAAATTAGTACCCTCGAGAATTGCGGACGATTGGAAGGGAAGGTGTACAAGGATACAACACTGTACACCACCCTGTCACCCTGTGATATGTGCACAGGCGCCATTATCATGTACGGAATCCCTAGATGTGTCGTGGGGGAGAATGTAAACTTCAAAAGTAAGGGGGAGAAATATCTCCAGACCCGGGGGCACGAAGTCGTCGTTGTGGACGATGAACGGTGTAAGAAGATCATGAAGCAGTTTATCGATGAGAGGCCCCAGGACTGGTTCGAAGACATCGGGGAATAAATGGTTACTGGGGGAATGGCATCTAAGTGGGATCAGAAAGGTATGGACATCGCTTATGAAGAGGCTGCTCTCGGCTACAAAGAGGGTGGAGTGCCTATCGGAGGGTGCCTGATCAACAACAAGGACGGCAGTGTGCTGGGGAGGGGCCACAATATGAGGTTCCAAAAAGGCTCCGCCACTCTCCACGGGGAAATTAGTACCCTCGAGAATTGCGGACGATTGGAAGGGAAGGTGTACAAGGATACAACACTGTACACCACCCTGTCACCCTGTGATATGTGCACAGGCGCCATTATCATGTACGGAATCCCTAGATGTGTCGTGGGGGAGAATGTAAACTTCAAAAGTAAGGGGGAGAAATATCTCCAGACCCGGGGGCACGAAGTCGTCGTTGTGGACGATGAACGGTGTAAGAAGATCATGAAGCAGTTTATCGATGAGAGGCCCCAGGACTGGTTCGAAGACATCGGGGAATAA


79


79


100


100

CD9
(서열번호 24)

CD9
(SEQ ID NO: 24) ATGGTTACAGGGGGAATGGCAAGTAAATGGGATCAAAAAGGGATGGATATAGCCTATGAGGAAGCGGCGCTGGGCTATAAAGAGGGAGGGGTGCCGATAGGTGGCTGTCTTATTAATAACAAAGACGGGAGTGTGTTGGGCAGAGGCCACAATATGCGATTTCAAAAAGGGTCCGCGACATTGCACGGAGAGATCAGCACCCTGGAGAATTGCGGAAGGTTGGAGGGAAAAGTGTATAAGGACACCACCCTCTATACCACACTGTCTCCATGTGATATGTGTACCGGTGCCATCATAATGTACGGGATTCCTCGCTGCGTAGTGGGAGAGAATGTTAACTTTAAAAGCAAGGGAGAGAAGTATTTGCAAACCCGGGGCCACGAAGTGGTGGTGGTGGACGACGAGCGATGTAAGAAAATCATGAAGCAATTTATCGATGAGCGGCCTCAAGATTGGTTCGAAGATATCGGCGAGTGAATGGTTACAGGGGGAATGGCAAGTAAATGGGATCAAAAAGGGATGGATATAGCCTATGAGGAAGCGGCGCTGGGCTATAAAGAGGGAGGGGTGCCGATAGGTGGCTGTCTTATTAATAACAAAGACGGGAGTGTGTTGGGCAGAGGCCACAATATGCGATTTCAAAAAGGGTCCGCGACATTGCACGGAGAGATCAGCACCCTGGAGAATTGCGGAAGGTTGGAGGGAAAAGTGTATAAGGACACCACCCTCTATACCACACTGTCTCCATGTGATATGTGTACCGGTGCCATCATAATGTACGGGATTCCTCGCTGCGTAGTGGGAGAGAATGTTAACTTTAAAAGCAAGGGAGAGAAGTATTTGCAAACCCGGGGCCACGAAGTGGTGGTGGTGGACGACGAGCGATGTAAGAAAATCATGAAGCAATTTATCGATGAGCGGCCTCAAGATTGGTTCGAAGATATCGGCGAGTGA


78


78


100


100

spRRVespRRVe -P1-CDs 바이러스 벡터 시스템의 구축-P1-CDs Construction of virus vector system

<6-1> 바이러스 벡터의 제작&Lt; 6-1 > Production of virus vector

<실시예 5>에서 발굴한 인간 코돈으로 최적화된 CD 유전자를 이용하여 벡터 시스템을 구축하였다.A vector system was constructed using the CD gene optimized for the human codon discovered in Example 5.

구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.Specifically, virus vectors were prepared by the following method.

1. spRRVe-P1-CD2 벡터: spRRVe-P1-RFP 벡터를 BamHI 및 ClaI 제한효소로 잘라 RFP를 제거한 자리에 CD2 유전자를 클로닝하여 제작하였다(도 16).1. spRRVe-P1-CD2 vector: The spRRVe-P1-RFP vector was cut with BamHI and ClaI restriction enzymes and the CD2 gene was cloned into the place where RFP was removed (FIG. 16).

2. spRRVe-P1-CD3 벡터: spRRVe-P1-RFP 벡터를 BamHI 및 ClaI 제한효소로 잘라 RFP를 제거한 자리에 CD3 유전자를 클로닝하여 제작하였다(도 16).2. spRRVe-P1-CD3 vector: The spRRVe-P1-RFP vector was cut with BamHI and ClaI restriction enzymes and the CD3 gene was cloned into the place where RFP was removed (FIG. 16).

3. spRRVe-P1-CD4 벡터: spRRVe-P1-RFP 벡터를 HpaI 제한효소로 잘라 RFP를 제거한 자리에 CD4 유전자를 클로닝하여 제작하였다(도 16).3. spRRVe-P1-CD4 vector: The spRRVe-P1-RFP vector was cut with HpaI restriction enzyme and the CD4 gene was cloned in place of RFP (FIG. 16).

4. spRRVe-P1-CD5 벡터: spRRVe-P1 벡터의 P1 아래에 다중 클로닝 자리(multi-cloning site)를 포함한 spRRVe-P1-mcs 벡터를 만들어, MluI 및 SalI 제한효소로 자른 후, CD5 유전자를 상기 위치에 클로닝하여 제작하였다(도 16).4. spRRVe-P1-CD5 vector: A spRRVe-P1-mcs vector containing a multi-cloning site under P1 of the spRRVe-P1 vector was prepared and cut with MluI and SalI restriction enzymes, (Fig. 16).

5. spRRVe-P1-CD6 벡터: spRRVe-P1 벡터의 P1 아래에 다중 클로닝 자리(multi-cloning site)를 포함한 spRRVe-P1-mcs 벡터를 만들어, MluI 및 SalI 제한효소로 자른 후, CD6 유전자를 상기 위치에 클로닝하여 제작하였다(도 16).5. spRRVe-P1-CD6 vector: An spRRVe-P1-mcs vector containing a multi-cloning site under P1 of the spRRVe-P1 vector was prepared and cut with MluI and SalI restriction enzymes, (Fig. 16).

6. spRRVe-P1-CD7 벡터: spRRVe-P1 벡터의 P1 아래에 다중 클로닝 자리(multi-cloning site)를 포함한 spRRVe-P1-mcs 벡터를 만들어, MluI 및 SalI 제한효소로 자른 후, CD7 유전자를 상기 위치에 클로닝하여 제작하였다(도 16).6. spRRVe-P1-CD7 vector: An spRRVe-P1-mcs vector containing a multi-cloning site under P1 of the spRRVe-P1 vector was prepared and cut with MluI and SalI restriction enzymes, (Fig. 16).

7. spRRVe-P1-CD8 벡터: spRRVe-P1 벡터의 P1 아래에 다중 클로닝 자리(multi-cloning site)를 포함한 spRRVe-P1-mcs 벡터를 만들어, MluI 및 SalI 제한효소로 자른 후, CD8 유전자를 상기 위치에 클로닝하여 제작하였다(도 16).7. spRRVe-P1-CD8 vector: A spRRVe-P1-mcs vector containing a multi-cloning site under P1 of the spRRVe-P1 vector was prepared and cut with MluI and SalI restriction enzymes, (Fig. 16).

8. spRRVe-P1-CD9 벡터: spRRVe-P1 벡터의 P1 아래에 다중 클로닝 자리(multi-cloning site)를 포함한 spRRVe-P1-mcs 벡터를 만들어, MluI 및 SalI 제한효소로 자른 후, CD9 유전자를 상기 위치에 클로닝하여 제작하였다(도 16).8. spRRVe-P1-CD9 vector: The spRRVe-P1-mcs vector containing a multi-cloning site under P1 of the spRRVe-P1 vector was prepared and cut with MluI and SalI restriction enzymes, (Fig. 16).

<6-2> 바이러스 생산 <6-2> Virus production

실시예 <6-1>에서 제조한 벡터로 바이러스를 생산하기 위해 spRRVe-P1-yCD, spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 또는 spRRVe-P1-CD9 벡터와 sRRVgp-P1-TK 벡터의 조합으로 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다.P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD3, spRRVe-P1-CD3, -CD6, spRRVe-P1-CD7, spRRVe-P1-CD8 or spRRVe-P1-CD9 vector and the sRRVgp-P1-TK vector in the same manner as in Example <1-2>.

<6-3> <6-3> spRRVespRRVe -P1-CDs/-P1-CDs / sRRVgpsRRVgp -P1-TK 바이러스의 약물 감수성 확인- Identification of drug susceptibility of P1-TK virus

실시예 <6-2>에서 생산한 바이러스의 GCV 및 5-FC에 대한 약물감수성 확인은 실시예 <6-2>에서 생산한 바이러스를 공형질주입했다는 것만 제외하고는 실시예 <3-5>와 동일한 방법으로 확인하였다.The susceptibility of the virus produced in Example <6-2> to GCV and 5-FC was confirmed in Example <3-5> except that the virus produced in Example <6-2> . &Lt; / RTI &gt;

그 결과, 도 17a 및 17b에 나타낸 바와 같이, spRRVe-P1-CD2/sRRVgp-P1-TK, spRRVe-P1-CD6/sRRVgp-P1-TK, spRRVe-P1-CD7/sRRVgp-P1-TK 및 spRRVe-P1-CD8/sRRVgp-P1-TK 바이러스는 p3 단계까지 GCV와 5-FC에 대해 약물 감수성을 보였다. 그러나 p4 단계부터는 모든 바이러스 벡터가 약물 감수성이 떨어짐을 확인하였다(도 17a 및 17b).As shown in FIGS. 17A and 17B, spRRVe-P1-CD2 / sRRVgp-P1-TK, spRRVe-P1-CD6 / sRRVgp-P1-TK, spRRVe- The P1-CD8 / sRRVgp-P1-TK virus showed drug susceptibility to GCV and 5-FC until p3. From the p4 level, however, it was confirmed that all the viral vectors were inferior in drug sensitivity (FIGS. 17A and 17B).

<6-4> <6-4> spRRVespRRVe -P1-CDs 벡터의 재조합 여부 확인Check whether P1-CDs vectors are recombined

실시예 <6-2>에서 바이러스 생산에 이용된 벡터의 안정성 확인은 실시예 <6-2>에서 생산한 바이러스를 3일 간격으로 4번 순차 감염시킨 것과 PCR 수행시, 72℃ 온도에서 3분 20초 동안 중합시키며, 서열번호 10의 염기서열을 갖는 GaLV(488)F 프라이머(5'-GGCTAGAATCCCTATATGTA-3')를 사용하는 것을 제외하고는, 실시예 <1-4>와 동일한 방법 및 조건으로 수행되었다.In order to confirm the stability of the vector used for virus production in Example <6-2>, the viruses produced in Example <6-2> were sequentially infected four times at intervals of 3 days, and at the time of PCR, Except that a GaLV (488) F primer (5'-GGCTAGAATCCCTATATGTA-3 ') having the nucleotide sequence of SEQ ID NO: 10 was used for 20 seconds, and the same procedure and conditions as in Example < .

그 결과, 도 18에 나타낸 바와 같이, spRRVe-P1-yCD, spRRVe-P1-CDs 및 sRRVgp-P1-TK 벡터 모두 p2 단계부터 재조합이 일어나 p3 또는 p4 단계부터 유전자 소실이 일어났다(도 18). 이는 p3 단계부터 약물 감수성이 떨어지는 실시예 <6-3>의 결과와도 유의하다.As a result, as shown in Fig. 18, both spRRVe-P1-yCD, spRRVe-P1-CDs and sRRVgp-P1-TK vectors underwent recombination at the p2 stage and gene loss occurred at the p3 or p4 stage (Fig. 18). This is also evident from the results of the Example <6-3> in which the drug sensitivity is poor from the p3 level.

<6-5> <6-5> spRRVespRRVe -P1-CDs 벡터의 재조합 유형 분석Analysis of recombinant types of P1-CDs vectors

상기 실시예 <6-3>에서 5-FC에 대한 약물 감수성이 우수하였던 spRRVe-P1-CD6 및 spRRVe-P1-CD8 벡터의 p2 및 p3 단계 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 한 후, 24개 클론에 대한 유전자 분석을 하였다.The p2 and p3 PCR products of the spRRVe-P1-CD6 and spRRVe-P1-CD8 vectors, which were excellent in drug sensitivity to 5-FC, were recovered and cloned into pGEM-T vector in Example <6-3> Genetic analysis was performed on 24 clones.

그 결과, 도 19a 내지 20b에 나타낸 바와 같이, 다양한 유형의 재조합 변이가 일어났는데, 주로 P1을 중심으로 CD 내부 또는 CD 부터 3'-LTR 사이에서 재조합이 일어났다(도 19a 내지 20b).As a result, as shown in Figs. 19A to 20B, various types of recombination mutations occurred, and recombination mainly occurred within the CD or between CD and 3'-LTR centered on P1 (Figs. 19A to 20B).

spRRVespRRVe -P2-CDs 바이러스 벡터 시스템 구축-P2-CDs virus vector system construction

<7-1> 바이러스 벡터의 제작<7-1> Construction of virus vector

<실시예 1> 내지 <실시예 6>의 결과, spRRV-P1 벡터의 재조합 변이는 주로 P1 프로모터 부위를 중심으로 일어났다. 이는 P1 프로모터의 반복적인 염기서열로 인한 것으로 예상되어 P1 대신 P2 유전자 조절부위로 교체한 벡터 시스템을 구축하였다. As a result of <Examples 1 to 6>, recombination mutations of the spRRV-P1 vector mainly occurred at the P1 promoter region. This was expected due to the repetitive nucleotide sequence of the P1 promoter and constructed a vector system that was replaced with a P2 gene control site instead of P1.

구체적으로, 각 spRRVe-P1-CDs 벡터의 P1 프로모터(MCMV 초기 프로모터) 부위를 제거하고 P1 프로모터 부위에서 반복적인 염기서열이 제거된 P2 프로모터(EF1α 프로모터) 부위를 클로닝하여 spRRVe-P2-yCD 및 spRRVe-P2-CDs 벡터를 구축하였다. 이때 상대적으로 약물 감수성이 약한 CD4 유전자는 제외하였다(도 21).Specifically, the P1 promoter (MCMV early promoter) site of each spRRVe-P1-CDs vector was removed and a P2 promoter (EF1? Promoter) region in which a repetitive base sequence was removed at the P1 promoter site was cloned to generate spRRVe-P2-yCD and spRRVe -P2-CDs vectors were constructed. At this time, the CD4 gene having a relatively weak drug sensitivity was excluded (FIG. 21).

<7-2> 바이러스 생산 <7-2> Virus production

실시예 <7-1>에서 제조한 벡터로 바이러스를 생산하기 위해 spRRVe-P2-yCD, spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, spRRVe-P2-CD8 또는 spRRVe-P2-CD9 벡터와 sRRVgp-P1-TK 벡터 조합으로 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다.The spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2 -CD7, spRRVe-P2-CD8 or spRRVe-P2-CD9 vector and sRRVgp-P1-TK vector in the same manner as in Example <1-2>.

<7-3> <7-3> spRRVespRRVe -P2--P2- yCDyCD // sRRVgpsRRVgp -P1-TK 및 -P1-TK and spRRVespRRVe -P2-CDs/-P2-CDs / sRRVgpsRRVgp -P1-TK 바이러스의 약물 감수성 확인- Identification of drug susceptibility of P1-TK virus

상기 실시예 <7-2>에서 생산한 바이러스의 GCV 및 5-FC에 대한 약물감수성 확인은 실시예 <7-2>에서 생산한 바이러스를 공형질주입하여, p1 에서 p9까지 순차감염시켰으며, GCV와 5-FC를 각각 9일 또는 12일 동안 처리하여 세포사멸을 관찰한 것만 제외하고는 실시예<3-5>와 동일한 방법으로 확인하였다.In order to confirm the drug susceptibility of GCV and 5-FC of the virus produced in Example <7-2>, the virus produced in Example <7-2> was co-infected and sequentially infected from p1 to p9, GCV and 5-FC were treated for 9 days or 12 days, respectively, to observe apoptosis.

그 결과, 도 22a 내지 22c에 나타낸 바와 같이, spRRVe-P1-CDs/sRRVgp-P1-TK와 달리 spRRVe-P2-CDs/sRRVgp-P1-TK는 p8 단계까지 세포 사멸이 관찰되었으며, p9 단계부터는 급격히 세포 사멸능이 감소하였다. 한편, spRRVe-P2-CD6, spRRVe-P2-CD7 및 spRRVe-P2-CD8은 양성 대조군인 spRRVe-P2-CD2 보다는 세포 사멸능이 떨어지나 spRRVe-P2-yCD 보다는 5-FC에 대한 약물 감수성이 많이 개선되었다(도 22a 내지 22c).As a result, as shown in FIGS. 22A to 22C, cell death was observed up to the p8 level in spRRVe-P2-CDs / sRRVgp-P1-TK unlike spRRVe-P1-CDs / sRRVgp-P1-TK, And decreased cell killing ability. On the other hand, spRRVe-P2-CD6, spRRVe-P2-CD7 and spRRVe-P2-CD8 showed less cytotoxic activity than positive control group spRRVe-P2-CD2 but significantly improved drug sensitivity to 5-FC than spRRVe-P2-yCD (Figs. 22A to 22C).

<7-4> <7-4> spRRVespRRVe -P2--P2- yCDyCD // sRRVgpsRRVgp -P1-TK 및 -P1-TK and spRRVespRRVe -P2-CDs/-P2-CDs / sRRVgpsRRVgp -P1-TK 벡터의 재조합 여부 확인- Confirmation of recombination of P1-TK vector

실시예 <7-1>에서 구축한 벡터의 안정성 확인은 실시예 <7-2>에서 생산한 8종류의 바이러스를 감염시킨 것을 제외하고는, 실시예 <6-4>와 동일한 방법 및 조건으로 수행되었다. The stability of the vector constructed in Example <7-1> was confirmed in the same manner and under the same conditions as in Example <6-4> except that eight kinds of viruses produced in Example <7-2> were infected. .

그 결과, 도 23에 나타낸 바와 같이, spRRVe-P2-CD5를 제외한 spRRVe-P2-CDs 벡터는 p4 단계까지 재조합이 일어나지 않은 반면, sRRVgp-P1-TK 벡터는 p1 단계 또는 p2 단계부터 재조합이 크게 일어났다(도 23). As a result, as shown in FIG. 23, the spRRVe-P2-CDs vector except for spRRVe-P2-CD5 did not recombine to the p4 stage, whereas the sRRVgp-P1-TK vector showed a large recombination at the p1 or p2 stage (Fig. 23).

<7-5> <7-5> sRRVgpsRRVgp -P1-TK 벡터의 재조합 유형 분석Analysis of recombinant type of P1-TK vector

상기 실시예 <7-4>에서 spRRVe-P2-yCD/sRRVgp-P1-TK 및 spRRVe-P2-CDs/sRRVgp-P1-TK 벡터 조합의 지놈 DNA를 이용한 PCR 반응 후, 기대 크기보다 작아진 sRRVgp-P1-TK 벡터의 p3 및 p4 단계 재조합 밴드의 PCR 산물을 회수하여 유전자 분석을 하였다. After the PCR reaction using the genomic DNA of the combination of spRRVe-P2-yCD / sRRVgp-P1-TK and spRRVe-P2-CDs / sRRVgp-P1-TK vector in Example <7-4>, sRRVgp- The PCR products of the p3 and p4 step recombination bands of the P1-TK vector were recovered and genetically analyzed.

그 결과, 도 24에 나타낸 바와 같이, P1과 TK 사이에서 결실이 되는 재조합이 일어남을 확인하였다(도 24).As a result, as shown in Fig. 24, it was confirmed that recombination that resulted in deletion between P1 and TK occurred (Fig. 24).

sRRVgpsRRVgp -P2-TK 바이러스 벡터 시스템 구축-P2-TK virus vector system construction

<8-1> 바이러스 벡터의 제작<8-1> Construction of virus vector

<실시예 7>의 결과 spRRVe-P2-CDs 벡터는 P1 조절부위를 사용한 벡터에 비해 재조합이 일어나는 빈도가 낮았으나, sRRVgp-P1-TK 벡터는 여전히 P1 조절부위를 중심으로 결실이 일어나 P1 프로모터 부위를 P2 프로모터 부위로 교체한 sRRVgp-P2-TK 벡터 시스템을 구축하였다.As a result of Example 7, the recombination frequency of the spRRVe-P2-CDs vector was lower than that of the vector using the P1 regulatory region, but the sRRVgp-P1-TK vector was still deleted around the P1 regulatory region, Was replaced with a P2 promoter site to construct the sRRVgp-P2-TK vector system.

구체적으로, P2 프로모터에 EcoRI, NotI 및 PmeI 제한효소 서열이 포함되도록(EcoRI-P2-NotI-PmeI-EcoRI) PCR 한 후, EcoRI으로 잘라 본 발명자들이 구축한 sRRVgp 벡터(충남대학교)의 Pol 유전자 위치 아래에 상기 PCR 산물을 삽입하여 sRRVgp-P2 벡터를 제작하였다. 이후, HSV-TK 유전자에 NotI과 PmeI 제한효소 서열이 포함되도록(NotI-HSV TK-PmeI) PCR 한 후, NotI 및 PmeI 제한효소로 잘라 P2 유전자 아래에 클로닝하여 sRRVgp-P2-TK 벡터를 완성하였다(도 25).Specifically, PCR was performed using EcoRI-P2-NotI-PmeI-EcoRI so as to include EcoRI, NotI and PmeI restriction enzyme sequences in the P2 promoter, followed by restriction of the Pol gene position of the sRRVgp vector (Chungnam National University) The PCR product was inserted below to prepare an sRRVgp-P2 vector. Thereafter, the HSV-TK gene was subjected to PCR (NotI-HSV TK-PmeI) so as to include NotI and PmeI restriction enzyme sequences, followed by cloning into the Not2 and PmeI restriction enzymes under the P2 gene to complete the sRRVgp-P2-TK vector (Fig. 25).

<8-2> 바이러스 생산 <8-2> Virus production

spRRVe-P2-CD2, 재조합을 극복한 spRRVe-P2-CD6, spRRVe-P2-CD7 또는 spRRVe-P2-CD8 벡터와 sRRVgp-P2-TK 벡터 조합으로 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다. The virus was obtained in the same manner as in Example <1-2> with the combination of spRRVe-P2-CD2, spRRVe-P2-CD6, spRRVe-P2-CD7 or spRRVe-P2-CD8 and sRRVgp- Respectively.

<8-3> <8-3> spRRVespRRVe -P2-CDs/-P2-CDs / sRRVgpsRRVgp -P2-TK 바이러스의 약물 감수성 확인Identification of drug susceptibility of P2-TK virus

상기 실시예 <8-2>에서 생산한 바이러스의 GCV 및 5-FC에 대한 약물감수성 확인은 실시예 <8-2>에서 생산한 바이러스를 공형질주입하여, p1 에서 p11까지 순차감염시켰으며, GCV와 5-FC를 각각 5일 또는 7일 동안 처리하여 세포사멸을 관찰한 것만 제외하고는 실시예<3-5>와 동일한 방법으로 확인하였다.In order to confirm the drug susceptibility of GCV and 5-FC of the virus produced in the above Example <8-2>, the virus produced in Example <8-2> was co-injected and sequentially infected from p1 to p11, GCV and 5-FC were treated for 5 days or 7 days, respectively, and cell death was observed.

그 결과, 도 26a 내지 26c에 나타낸 바와 같이, spRRVe-P2-yCD 벡터를 제외한 spRRVe-P2-CD6, spRRVe-P2-CD7 및 spRRVe-P2-CD8 벡터는 p11 단계까지 양성 대조군인 spRRVe-P2-CD2와 유의하게 5-FC에 대한 약물 감수성을 나타내었다(도 26a 내지 26c).As a result, as shown in Figs. 26A to 26C, spRRVe-P2-CD6, spRRVe-P2-CD7 and spRRVe-P2-CD8 vectors except the spRRVe-P2-yCD vector showed a positive control group spRRVe-P2- (Fig. 26 (a) to 26 (c)).

<8-4> <8-4> spRRVespRRVe -P2-CDs/-P2-CDs / sRRVgpsRRVgp -P2-TK 벡터의 재조합 여부 확인Confirmation of recombination of -P2-TK vector

실시예 <8-1>에서 구축한 벡터의 안정성 확인은 실시예 <8-2>에서 생산한 4종류의 바이러스를 3일 간격으로 7번 순차 감염시킨 것을 제외하고는, 실시예 <6-4>와 동일한 방법 및 조건으로 수행되었다.The stability of the vector constructed in Example < 8-1 > was confirmed in Example < 6-4 > except that four kinds of viruses produced in Example < &Gt;. &lt; / RTI &gt;

그 결과, 도 27a 및 27b에 나타낸 바와 같이, spRRVe-P2-yCD 벡터는 p4 단계부터 재조합이 일어났고, spRRVe-P2-CD2 벡터는 p7 단계부터, spRRVe-P2-CD6 벡터는 p6 단계부터, spRRVe-P2-CD7 및 spRRVe-P2-CD8 벡터는 p5 단계부터 재조합이 일어났다. 특히, sRRVgp-P2-TK 벡터와 spRRVe-P2-CD2 벡터의 조합에서는 유독 이른 단계에서 재조합이 일어났다(도 27a 및 27b). As a result, as shown in Figs. 27A and 27B, the spRRVe-P2-yCD vector underwent recombination at the p4 stage, the spRRVe-P2-CD2 vector at the p7 stage, the spRRVe- -P2-CD7 and spRRVe-P2-CD8 vectors had recombination at the p5 step. In particular, the combination of the sRRVgp-P2-TK vector and the spRRVe-P2-CD2 vector resulted in recombination at an early stage of toxicity (FIGS. 27A and 27B).

<8-5> <8-5> spRRVespRRVe -P2-CD6 및 -P2-CD6 and spRRVespRRVe -P2-CD8 벡터의 재조합 유형 분석Analysis of recombination type of P2-CD8 vector

상기 실시예 <8-4>에서 재조합이 일어난 spRRVe-P2-CD6 및 spRRVe-P2-CD8 벡터의 p7 단계 재조합 밴드의 PCR 산물을 회수하여 pGEM-T 벡터에 클로닝 한 후, 19개 클론에 대한 유전자 분석을 하였다.The PCR products of the p7-step recombination bands of the recombinant spRRVe-P2-CD6 and spRRVe-P2-CD8 vectors in Example <8-4> were recovered and cloned into the pGEM-T vector, Respectively.

그 결과, 도 28a 내지 29b에 나타낸 바와 같이, P2 부터 3'LTR 직전 부분에서 재조합이 일어남을 확인하였다(도 28a 내지 29b).As a result, as shown in Figs. 28A to 29B, it was confirmed that recombination occurred in the portion immediately before P2 to 3'LTR (Figs. 28A to 29B).

인간 코돈으로 최적화된 7종의 CD 유전자 추가 발굴Seven species of CD gene optimized by human codon

상기 실시예에서 yCD 유전자보다 약물 감수성이 뛰어난 CD6, CD7 및 CD8 유전자를 발굴하였으나, 약물에 대한 세포 사멸효과가 더 극대화된 CD 유전자를 확보하기 위해 7종의 인간 코돈으로 최적화된 CD 유전자(CD10∼CD16)를 추가로 발굴하였다.In this example, CD6, CD7 and CD8 genes having superior drug susceptibility than the yCD gene were excavated. However, in order to obtain a CD gene having a more highly apoptotic effect on the drug, CD genes optimized for seven human codons (CD10, CD16) was further excavated.

구체적으로, 7종의 CD10~CD16은 효모의 CD를 인간 코돈으로 최적화시킨 유전자이다. 상기 7종의 CD 유전자는 코스모젠에 의뢰하여 합성하였다. 상기 CD10∼CD16 유전자의 서열은 하기 표 4와 같다.Specifically, the seven kinds of CD10 to CD16 are genes in which yeast CD is optimized to the human codon. The seven CD genes were synthesized by asking Cosmogen. The sequence of the CD10-CD16 gene is shown in Table 4 below.

인간 코돈으로 최적화된 CD 유전자 서열CD gene sequence optimized with human codon

인간 코돈으로 최적화된 유전자 서열

Optimized gene sequence with human codon yCD
뉴클레오티드
서열과
유사성(%) yCD
Nucleotides
Sequence and
Similarity (%) yCD
단백질
서열과유사성(%)yCD
protein
Sequence and similarity (%)

CD10
(서열번호 25)

CD10
(SEQ ID NO: 25) ATGGTAACCGGAGGTATGGCATCCAAGTGGGACCAAAAAGGAATGGACATAGCATATGAAGAAGCAGCCCTGGGCTACAAGGAGGGAGGGGTTCCGATTGGCGGTTGTCTTATAAATAATAAAGACGGTAGTGTTCTTGGCAGGGGTCACAACATGAGATTCCAAAAGGGGAGTGCTACACTTCACGGCGAAATAAGCACCTTGGAAAACTGTGGTAGACTTGAGGGAAAAGTGTACAAGGACACGACCCTTTATACGACGCTGTCCCCTTGTGATATGTGCACCGGCGCTATCATCATGTATGGAATACCACGATGCGTAGTGGGAGAGAATGTTAATTTCAAGAGTAAGGGCGAGAAGTACCTTCAGACCAGGGGGCACGAGGTAGTAGTAGTTGACGATGAGCGATGCAAGAAGATTATGAAACAATTCATTGACGAGAGGCCGCAGGATTGGTTTGAAGACATCGGCGAATAGATGGTAACCGGAGGTATGGCATCCAAGTGGGACCAAAAAGGAATGGACATAGCATATGAAGAAGCAGCCCTGGGCTACAAGGAGGGAGGGGTTCCGATTGGCGGTTGTCTTATAAATAATAAAGACGGTAGTGTTCTTGGCAGGGGTCACAACATGAGATTCCAAAAGGGGAGTGCTACACTTCACGGCGAAATAAGCACCTTGGAAAACTGTGGTAGACTTGAGGGAAAAGTGTACAAGGACACGACCCTTTATACGACGCTGTCCCCTTGTGATATGTGCACCGGCGCTATCATCATGTATGGAATACCACGATGCGTAGTGGGAGAGAATGTTAATTTCAAGAGTAAGGGCGAGAAGTACCTTCAGACCAGGGGGCACGAGGTAGTAGTAGTTGACGATGAGCGATGCAAGAAGATTATGAAACAATTCATTGACGAGAGGCCGCAGGATTGGTTTGAAGACATCGGCGAATAG


80


80



100


100

CD11
(서열번호 26)

CD11
(SEQ ID NO: 26) ATGGTGACAGGGGGTATGGCAAGCAAATGGGATCAGAAGGGTATGGACATCGCATACGAGGAGGCGGCCTTGGGCTATAAGGAAGGCGGCGTACCTATAGGGGGGTGCCTTATTAACAATAAGGACGGGAGCGTCCTGGGCAGAGGTCACAACATGAGGTTCCAAAAGGGTTCAGCAACCCTGCATGGCGAAATAAGCACCCTTGAGAATTGTGGGAGGTTGGAGGGTAAGGTGTACAAGGATACCACGCTTTATACCACCTTGAGTCCTTGCGACATGTGCACAGGCGCTATAATCATGTATGGAATACCGCGCTGTGTTGTAGGAGAAAATGTAAACTTCAAGAGTAAAGGAGAAAAATACTTGCAAACGCGGGGACACGAAGTGGTAGTTGTCGATGATGAGCGGTGCAAAAAAATCATGAAGCAGTTCATTGACGAACGCCCCCAAGACTGGTTCGAAGACATTGGGGAGTAGATGGTGACAGGGGGTATGGCAAGCAAATGGGATCAGAAGGGTATGGACATCGCATACGAGGAGGCGGCCTTGGGCTATAAGGAAGGCGGCGTACCTATAGGGGGGTGCCTTATTAACAATAAGGACGGGAGCGTCCTGGGCAGAGGTCACAACATGAGGTTCCAAAAGGGTTCAGCAACCCTGCATGGCGAAATAAGCACCCTTGAGAATTGTGGGAGGTTGGAGGGTAAGGTGTACAAGGATACCACGCTTTATACCACCTTGAGTCCTTGCGACATGTGCACAGGCGCTATAATCATGTATGGAATACCGCGCTGTGTTGTAGGAGAAAATGTAAACTTCAAGAGTAAAGGAGAAAAATACTTGCAAACGCGGGGACACGAAGTGGTAGTTGTCGATGATGAGCGGTGCAAAAAAATCATGAAGCAGTTCATTGACGAACGCCCCCAAGACTGGTTCGAAGACATTGGGGAGTAG


78


78



100


100

CD12
(서열번호 27)

CD12
(SEQ ID NO: 27) ATGGTAACGGGTGGGATGGCTAGCAAGTGGGACCAGAAAGGCATGGATATAGCGTATGAAGAAGCGGCGTTGGGTTACAAAGAGGGCGGCGTTCCCATCGGTGGCTGCCTTATCAATAATAAAGACGGCTCCGTCCTTGGCCGGGGACACAATATGCGCTTCCAAAAGGGCAGCGCCACACTTCACGGTGAGATCTCCACGCTGGAGAATTGTGGGCGACTTGAGGGGAAAGTCTACAAGGACACAACTTTGTACACAACACTTAGCCCGTGCGATATGTGTACGGGAGCCATAATCATGTACGGCATCCCGCGCTGCGTGGTAGGAGAGAACGTAAATTTTAAGTCAAAAGGAGAAAAATATCTTCAGACCAGGGGCCACGAGGTGGTTGTCGTGGACGACGAGAGATGTAAAAAGATCATGAAACAGTTTATTGATGAAAGACCACAGGATTGGTTTGAGGACATCGGTGAGTAGATGGTAACGGGTGGGATGGCTAGCAAGTGGGACCAGAAAGGCATGGATATAGCGTATGAAGAAGCGGCGTTGGGTTACAAAGAGGGCGGCGTTCCCATCGGTGGCTGCCTTATCAATAATAAAGACGGCTCCGTCCTTGGCCGGGGACACAATATGCGCTTCCAAAAGGGCAGCGCCACACTTCACGGTGAGATCTCCACGCTGGAGAATTGTGGGCGACTTGAGGGGAAAGTCTACAAGGACACAACTTTGTACACAACACTTAGCCCGTGCGATATGTGTACGGGAGCCATAATCATGTACGGCATCCCGCGCTGCGTGGTAGGAGAGAACGTAAATTTTAAGTCAAAAGGAGAAAAATATCTTCAGACCAGGGGCCACGAGGTGGTTGTCGTGGACGACGAGAGATGTAAAAAGATCATGAAACAGTTTATTGATGAAAGACCACAGGATTGGTTTGAGGACATCGGTGAGTAG


79


79


100


100


CD13
(서열번호 28)

CD13
(SEQ ID NO: 28) ATGGTTACAGGAGGTATGGCTTCAAAGTGGGATCAAAAAGGGATGGACATCGCCTATGAAGAAGCAGCGTTGGGATACAAAGAAGGGGGGGTTCCCATAGGAGGTTGCCTTATCAACAATAAAGATGGAAGCGTTCTTGGGCGAGGGCACAATATGAGATTTCAAAAAGGTTCAGCCACTCTCCATGGAGAAATTTCAACTCTCGAAAACTGTGGTCGCCTTGAGGGCAAGGTTTATAAGGATACCACCCTCTACACTACCCTGTCACCCTGCGACATGTGTACAGGTGCAATTATAATGTACGGAATCCCTCGGTGTGTGGTGGGGGAGAACGTGAATTTTAAGTCCAAAGGTGAAAAATATCTCCAAACTCGCGGGCATGAAGTCGTCGTTGTTGATGATGAGAGGTGTAAAAAGATTATGAAACAATTCATAGACGAGAGGCCACAGGATTGGTTTGAGGACATAGGGGAGTAGATGGTTACAGGAGGTATGGCTTCAAAGTGGGATCAAAAAGGGATGGACATCGCCTATGAAGAAGCAGCGTTGGGATACAAAGAAGGGGGGGTTCCCATAGGAGGTTGCCTTATCAACAATAAAGATGGAAGCGTTCTTGGGCGAGGGCACAATATGAGATTTCAAAAAGGTTCAGCCACTCTCCATGGAGAAATTTCAACTCTCGAAAACTGTGGTCGCCTTGAGGGCAAGGTTTATAAGGATACCACCCTCTACACTACCCTGTCACCCTGCGACATGTGTACAGGTGCAATTATAATGTACGGAATCCCTCGGTGTGTGGTGGGGGAGAACGTGAATTTTAAGTCCAAAGGTGAAAAATATCTCCAAACTCGCGGGCATGAAGTCGTCGTTGTTGATGATGAGAGGTGTAAAAAGATTATGAAACAATTCATAGACGAGAGGCCACAGGATTGGTTTGAGGACATAGGGGAGTAG


78


78


100


100

CD14
(서열번호 29)

CD14
(SEQ ID NO: 29) ATGGTGACTGGGGGTATGGCTTCCAAATGGGATCAGAAAGGAATGGATATAGCATACGAAGAAGCAGCTCTCGGGTACAAAGAGGGTGGAGTACCCATTGGGGGATGCCTCATCAACAACAAGGATGGGAGTGTCCTTGGGCGAGGTCACAATATGCGATTCCAGAAGGGGAGCGCGACGCTCCACGGGGAGATAAGTACGCTGGAGAACTGCGGGAGGCTTGAAGGCAAGGTCTACAAAGATACCACACTCTACACGACCCTCAGCCCTTGCGACATGTGTACGGGTGCGATCATCATGTATGGAATACCGCGATGCGTAGTAGGAGAGAACGTGAACTTCAAGTCCAAAGGCGAAAAGTATCTCCAGACGCGCGGCCACGAAGTGGTAGTGGTAGACGACGAAAGGTGCAAGAAGATAATGAAGCAGTTTATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATTGGCGAGTAGATGGTGACTGGGGGTATGGCTTCCAAATGGGATCAGAAAGGAATGGATATAGCATACGAAGAAGCAGCTCTCGGGTACAAAGAGGGTGGAGTACCCATTGGGGGATGCCTCATCAACAACAAGGATGGGAGTGTCCTTGGGCGAGGTCACAATATGCGATTCCAGAAGGGGAGCGCGACGCTCCACGGGGAGATAAGTACGCTGGAGAACTGCGGGAGGCTTGAAGGCAAGGTCTACAAAGATACCACACTCTACACGACCCTCAGCCCTTGCGACATGTGTACGGGTGCGATCATCATGTATGGAATACCGCGATGCGTAGTAGGAGAGAACGTGAACTTCAAGTCCAAAGGCGAAAAGTATCTCCAGACGCGCGGCCACGAAGTGGTAGTGGTAGACGACGAAAGGTGCAAGAAGATAATGAAGCAGTTTATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATTGGCGAGTAG


76


76


100


100

CD15
(서열번호 30)

CD15
(SEQ ID NO: 30) ATGGTTACTGGCGGCATGGCTTCTAAGTGGGATCAGAAGGGCATGGATATAGCCTATGAAGAAGCAGCACTGGGATACAAAGAGGGAGGGGTACCAATTGGGGGATGTCTGATTAATAACAAAGACGGAAGTGTACTCGGTCGCGGGCATAATATGAGATTCCAAAAAGGCTCTGCAACGTTGCACGGCGAAATCAGCACGCTCGAAAATTGCGGGAGGCTGGAGGGAAAGGTTTACAAGGATACCACTCTCTATACCACACTGTCACCATGTGATATGTGTACGGGGGCTATAATAATGTATGGAATCCCCCGCTGCGTCGTGGGCGAAAACGTCAACTTTAAGTCTAAGGGGGAAAAGTATTTGCAAACGCGCGGTCATGAGGTCGTTGTAGTCGATGACGAGAGATGCAAAAAAATAATGAAGCAGTTTATTGACGAGAGACCTCAGGACTGGTTCGAAGACATCGGGGAGTAGATGGTTACTGGCGGCATGGCTTCTAAGTGGGATCAGAAGGGCATGGATATAGCCTATGAAGAAGCAGCACTGGGATACAAAGAGGGAGGGGTACCAATTGGGGGATGTCTGATTAATAACAAAGACGGAAGTGTACTCGGTCGCGGGCATAATATGAGATTCCAAAAAGGCTCTGCAACGTTGCACGGCGAAATCAGCACGCTCGAAAATTGCGGGAGGCTGGAGGGAAAGGTTTACAAGGATACCACTCTCTATACCACACTGTCACCATGTGATATGTGTACGGGGGCTATAATAATGTATGGAATCCCCCGCTGCGTCGTGGGCGAAAACGTCAACTTTAAGTCTAAGGGGGAAAAGTATTTGCAAACGCGCGGTCATGAGGTCGTTGTAGTCGATGACGAGAGATGCAAAAAAATAATGAAGCAGTTTATTGACGAGAGACCTCAGGACTGGTTCGAAGACATCGGGGAGTAG


77


77


100


100

CD16
(서열번호 31)

CD16
(SEQ ID NO: 31) ATGGTGACAGGAGGAATGGCCAGCAAGTGGGATCAGAAGGGAATGGATATTGCCTACGAGGAGGCCGCCCTGGGCTACAAGGAGGGGGGCGTGCCAATTGGCGGATGTCTGATTAACAACAAGGATGGGAGCGTGCTGGGAAGAGGACACAACATGAGATTTCAGAAGGGAAGCGCAACCTTGCACGGAGAAATTAGCACCCTTGAGAACTGCGGGCGGCTTGAAGGCAAGGTCTATAAAGACACTACACTTTATACTACCTTGTCTCCATGTGATATGTGTACAGGCGCCATTATTATGTACGGAATTCCTAGATGCGTCGTGGGAGAGAACGTGAACTTTAAGAGCAAGGGAGAGAAGTACCTGCAGACAAGAGGACACGAGGTGGTGGTGGTGGATGATGAGAGATGTAAGAAGATCATGAAGCAGTTCATCGATGAGAGGCCCCAGGATTGGTTTGAGGACATCGGCGAGTGAATGGTGACAGGAGGAATGGCCAGCAAGTGGGATCAGAAGGGAATGGATATTGCCTACGAGGAGGCCGCCCTGGGCTACAAGGAGGGGGGCGTGCCAATTGGCGGATGTCTGATTAACAACAAGGATGGGAGCGTGCTGGGAAGAGGACACAACATGAGATTTCAGAAGGGAAGCGCAACCTTGCACGGAGAAATTAGCACCCTTGAGAACTGCGGGCGGCTTGAAGGCAAGGTCTATAAAGACACTACACTTTATACTACCTTGTCTCCATGTGATATGTGTACAGGCGCCATTATTATGTACGGAATTCCTAGATGCGTCGTGGGAGAGAACGTGAACTTTAAGAGCAAGGGAGAGAAGTACCTGCAGACAAGAGGACACGAGGTGGTGGTGGTGGATGATGAGAGATGTAAGAAGATCATGAAGCAGTTCATCGATGAGAGGCCCCAGGATTGGTTTGAGGACATCGGCGAGTGA


78


78


100


100

spRRVespRRVe -P2-CDs 바이러스 벡터 시스템 구축-P2-CDs virus vector system construction

<10-1> 바이러스 벡터의 제작<10-1> Construction of virus vector

<실시예 9>에서 발굴한 인간 코돈으로 최적화된 CD 유전자를 spRRVe-P2 벡터에 클로닝하여 spRRVe-P2-CDs 벡터를 제작하였다.The spRRVe-P2-CDs vector was prepared by cloning the CD gene optimized with the human codon excised in Example 9 into the spRRVe-P2 vector.

<10-2> 바이러스 생산<10-2> Virus production

실시예 <10-1>에서 제조한 벡터로 바이러스를 생산하기 위해 실시예 <10-1>에서 제작한 7종류의 spRRVe-P2-CDs 벡터와 sRRVgp-P2-TK 벡터 조합으로 상기 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다. The spRRVe-P2-CDs vector and the sRRVgp-P2-TK vector prepared in Example <10-1> were used to produce viruses from the vector prepared in Example <10-1> -2 &gt;. &lt; / RTI &gt;

<10-3> <10-3> spRRVespRRVe -P2-CDs/-P2-CDs / sRRVgpsRRVgp -P2-TK 바이러스의 약물 감수성 확인Identification of drug susceptibility of P2-TK virus

상기 실시예 <10-2>에서 생산한 바이러스의 GCV 및 5-FC에 대한 약물감수성 확인은 실시예 <10-2>에서 생산한 바이러스를 공형질주입하여, p1 에서 p5까지 순차감염시켰으며, GCV와 5-FC를 각각 6일 또는 8일 동안 처리하여 세포사멸을 관찰한 것만 제외하고는 실시예<3-5>와 동일한 방법으로 확인하였다.In order to confirm the drug susceptibility of GCV and 5-FC of the virus produced in the above Example <10-2>, the virus produced in Example <10-2> was co-transfected and sequentially infected from p1 to p5, GCV and 5-FC were treated for 6 days or 8 days, respectively, to observe apoptosis.

그 결과, 도 30에 나타낸 바와 같이, CD6 유전자보다 더 우수한 CD 유전자가 없음을 확인하였다(도 30).As a result, as shown in Fig. 30, it was confirmed that there was no CD gene superior to the CD6 gene (Fig. 30).

spRRVespRRVe -P2-TK, -P2-TK, sRRVgpsRRVgp -P2-CD2, -P2-CD2, sRRVgpsRRVgp -P2-CD6, -P2-CD6, sRRVgpsRRVgp -P2-CD7 및 -P2-CD7 and sRRVgpsRRVgp -P2-CD8 바이러스 벡터 시스템 구축-P2-CD8 virus vector system construction

<11-1> 바이러스 벡터의 제작<11-1> Construction of virus vector

상기 실시예 <8-4>에서 순차감염단계가 늘어나면서 P2 프로모터를 포함하는 spRRVe-P2-CDs 벡터도 재조합이 발생하여 이를 개선하기 위해 spRRVe-P2-TK/sRRVgp-P2-CDs 조합으로 바이러스 벡터 시스템을 구축하였다.In order to improve the spRRVe-P2-CDs vector including the P2 promoter as the sequential infectious steps are increased in the above Example <8-4>, the recombination occurs in the spRRVe-P2-TK / sRRVgp-P2- System.

구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.Specifically, virus vectors were prepared by the following method.

1. spRRVe-P2-TK 벡터: TK 유전자를 주형으로 서열번호 11의 염기서열을 갖는 HSV-TK-BamHI-F 프라이머(5'-CGGGATCCATGGCTTCGTACCCCTGCCAT-3') 및 서열번호 12의 염기서열을 갖는 HSV-TK-SalI-R 프라이머(5'-CGGTCGACTCAGTTAGCCTCCCCCATCTC-3')를 이용하여 PCR 한 후, BamHI 및 SalI 제한효소로 잘랐다. 자른 산물을 spRRVe-P2-mcs 벡터의 BamHI-SalI 제한효소 자리에 클로닝하여 spRRVe-P2-TK 벡터(서열번호 13)를 제작하였다(도 31, 32 및 33a 내지 33ㅣ). 1. spRRVe-P2-TK vector: TK-BamHI-F primer (5'-CGGGATCCATGGCTTCGTACCCCTGCCAT-3 ') having the nucleotide sequence of SEQ ID NO: 11 and the HSV-TK-SalI-R primer having the nucleotide sequence of SEQ ID NO: -CGGTCGACTCAGTTAGCCTCCCCCATCTC-3 ') and then digested with BamHI and SalI restriction enzymes. The truncated product was cloned into the BamHI-SalI restriction enzyme site of the spRRVe-P2-mcs vector to construct the spRRVe-P2-TK vector (SEQ ID NO: 13) (FIGS. 31, 32 and 33a to 33).

2. sRRVgp-P2-yCD 벡터: yCD 유전자를 주형으로 서열번호 36의 염기서열을 갖는 CD-NotⅠ-F 프라이머(5'-CGGCGGCCGCATGGTGACAGGGGGAATGGC-3') 및 서열번호 37의 염기서열을 갖는 CD-PmeⅠ-R 프라이머(5'-CGGTTTAAACCTACTCACCAATATCTTCAAA-3')를 이용하여 PCR한 후, NotI 및 PmeI 제한효소로 잘랐다. 자른 산물을 sRRVgp-P2 벡터의 NotI-PmeI 제한효소 자리에 클로닝하여 sRRVgp-P2-yCD 벡터를 제작하였다.(5'-CGGCGGCCGCATGGTGACAGGGGGAATGGC-3 ') having the nucleotide sequence of SEQ ID NO: 36 and the CD-PmeI-Fc primer having the nucleotide sequence of SEQ ID NO: 37 as the template, using the sRRVgp- R primer (5'-CGGTTTAAACCTACTCACCAATATCTTCAAA-3 ') and then digested with NotI and PmeI restriction enzymes. The crop product was cloned into the NotI-PmeI restriction enzyme site of the sRRVgp-P2 vector to construct sRRVgp-P2-yCD vector.

3. sRRVgp-P2-CD2 벡터: CD2 유전자를 주형으로 서열번호 38의 염기서열을 갖는 CD2-NotⅠ-F 프라이머(5'-CGGCGGCCGCATGGTGACCGGCGGCATGGC-3') 및 서열번호 39의 염기서열을 갖는 CD2-PmeⅠ-R 프라이머(5'-CGGTTTAAACTTACTCGCCGATATCCTCGA-3')를 이용하여 PCR한 후, NotI 및 PmeI 제한효소로 잘랐다. 자른 산물을 sRRVgp-P2 벡터의 NotI-PmeI 제한효소 자리에 클로닝하여 sRRVgp-P2-CD2 벡터를 제작하였다.3. sRRVgp-P2-CD2 vector: CD2-NotI-F primer (5'-CGGCGGCCGCATGGTGACCGGCGGCATGGC-3 ') having the nucleotide sequence of SEQ ID NO: 38 as the template of CD2 gene and CD2- R primer (5'-CGGTTTAAACTTACTCGCCGATATCCTCGA-3 ') and then digested with NotI and PmeI restriction enzymes. The crop product was cloned into the NotI-PmeI restriction site of the sRRVgp-P2 vector to construct the sRRVgp-P2-CD2 vector.

4. sRRVgp-P2-CD6 벡터: CD6 유전자를 주형으로 서열번호 40의 염기서열을 갖는 CD6-NotⅠ-F 프라이머(5'-CGGCGGCCGCATGGTTACTGGAGGGATGGC-3') 및 서열번호 41의 염기서열을 갖는 CD6-PmeⅠ-R 프라이머(5'-CGGTTTAAACTCATTCGCCAATATCCTCGA-3')를 이용하여 PCR한 후, NotI 및 PmeI 제한효소로 잘랐다. 자른 산물을 sRRVgp-P2 벡터의 NotI-PmeI 제한효소 자리에 클로닝하여 sRRVgp-P2-CD6 벡터(서열번호 14)를 제작하였다(도 31, 34 및 35a 내지 35j). (5'-CGGCGGCCGCATGGTTACTGGAGGGATGGC-3 ') having the nucleotide sequence of SEQ ID NO: 40 and CD6-PmeI-F having the nucleotide sequence of SEQ ID NO: 41 as the template, the sRRVgp-P2- R primer (5'-CGGTTTAAACTCATTCGCCAATATCCTCGA-3 '), and then digested with NotI and PmeI restriction enzymes. The cleaved product was cloned into the NotI-PmeI restriction enzyme site of the sRRVgp-P2 vector to construct sRRVgp-P2-CD6 vector (SEQ ID NO: 14) (FIGS. 31, 34 and 35a to 35j).

5. sRRVgp-P2-CD7 벡터: CD7 유전자를 주형으로 서열번호 42의 염기서열을 갖는 CD7-NotⅠ-F 프라이머(5'-CGGCGGCCGCATGGTAACTGGTGGCATGGC-3') 및 서열번호 43의 염기서열을 갖는 CD7-PmeⅠ-R 프라이머(5'-CGGTTTAAACCTACTCTCCGATATCCTCAA-3')를 이용하여 PCR한 후, NotI 및 PmeI 제한효소로 잘랐다. 자른 산물을 sRRVgp-P2 벡터의 NotI-PmeI 제한효소 자리에 클로닝하여 sRRVgp-P2-CD7 벡터를 제작하였다.(5'-CGGCGGCCGCATGGTAACTGGTGGCATGGC-3 ') having the nucleotide sequence of SEQ ID NO: 42 and CD7-PmeI-F having the nucleotide sequence of SEQ ID NO: 43 as the template for the sRRVgp-P2- R primer (5'-CGGTTTAAACCTACTCTCCGATATCCTCAA-3 ') and then digested with NotI and PmeI restriction enzymes. The cleaved product was cloned into the NotI-PmeI restriction enzyme site of the sRRVgp-P2 vector to construct the sRRVgp-P2-CD7 vector.

6. sRRVgp-P2-CD8 벡터: CD8 유전자를 주형으로 서열번호 44의 염기서열을 갖는 CD8-NotⅠ-F 프라이머(5'-CGGCGGCCGCATGGTTACTGGGGGAATGG-3') 및 서열번호 45의 염기서열을 갖는 CD8-PmeⅠ-R 프라이머(5'-CGGTTTAAACTTATTCCCCGATGTCTTCGA-3')를 이용하여 PCR한 후, NotI 및 PmeI 제한효소로 잘랐다. 자른 산물을 sRRVgp-P2 벡터의 NotI-PmeI 제한효소 자리에 클로닝하여 sRRVgp-P2-CD8 벡터를 제작하였다.(5'-CGGCGGCCGCATGGTTACTGGGGGAATGG-3 ') having the nucleotide sequence of SEQ ID NO: 44 and CD8-PmeI-F having the nucleotide sequence of SEQ ID NO: 45 as the template for the sRRVgp-P2- R primer (5'-CGGTTTAAACTTATTCCCCGATGTCTTCGA-3 ') and then digested with NotI and PmeI restriction enzymes. The crop product was cloned into the NotI-PmeI restriction enzyme site of the sRRVgp-P2 vector to construct the sRRVgp-P2-CD8 vector.

<11-2> 바이러스 생산 <11-2> Virus production

실시예 <11-1>에서 제조한 sRRVgp-P2-yCD, sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 또는 sRRVgp-P2-CD8 벡터와 spRRVe-P2-TK 벡터를 조합하여 상기 실시예 <1-2>와 동일한 방법으로 바이러스를 생산하였다. The sRRVgp-P2-yCD, sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7 or sRRVgp-P2-CD8 vector prepared in Example <11-1> The virus was produced in the same manner as in Example <1-2>.

<11-3> <11-3> spRRVespRRVe -P2-TK/-P2-TK / sRRVgpsRRVgp -P2-CD(2,6,7,8)-P2-CD (2,6,7,8) 벡터의 재조합 여부 확인Check whether the vector is reassembled

실시예 <11-1>에서 구축한 벡터의 안정성 확인은 spRRVe-P2-TK/sRRVgp-P2-CD(2,6,7,8) 바이러스를 3일 간격으로 7번 순차 감염시킨 것과 PCR 수행시, 72℃ 온도에서 3분 동안 중합시키는 것을 제외하고는, 실시예 <1-4>와 동일한 방법 및 조건으로 수행되었다.The stability of the vector constructed in Example <11-1> was confirmed by sequential infection of spRRVe-P2-TK / sRRVgp-P2-CD (2,6,7,8) , And polymerization was carried out at a temperature of 72 DEG C for 3 minutes, in the same manner as in Example < 1-4 >.

그 결과, 도 36a 및 36b에 나타낸 바와 같이, sRRVgp-P2-CD6, sRRVgp-P2-CD7 및 sRRVgp-P2-CD8 벡터는 p7 단계까지 재조합이 일어나지 않았다(도 36a 및 36b). As a result, as shown in Figs. 36A and 36B, sRRVgp-P2-CD6, sRRVgp-P2-CD7 and sRRVgp-P2-CD8 vectors did not recombine up to the p7 step (Figs. 36A and 36B).

<11-4> <11-4> spRRVespRRVe -P2-TK/-P2-TK / sRRVgpsRRVgp -P2-CDs 벡터의 재조합 유형 분석Analysis of recombination types of -P2-CDs vectors

상기 실시예 <11-3>에서 spRRVe-P2-TK/sRRVgp-P2-yCD, spRRVe-P2-TK/sRRVgp-P2-CD2, spRRVe-P2-TK/sRRVgp-P2-CD6, spRRVe-P2-TK/sRRVgp-P2-CD7, spRRVe-P2-TK/sRRVgp-P2-CD8 벡터 조합의 지놈 DNA를 이용한 PCR 반응 후, spRRVe-P2-TK, sRRVgp-P2-yCD 및 sRRVgp-P2-CD(2,6,7,8) 벡터의 p6 및 p7 단계 PCR 산물을 회수하여 유전자 분석을 하였다. In the above Example 11-3, spRRVe-P2-TK / sRRVgp-P2-yCD, spRRVe-P2-TK / sRRVgp-P2-CD2, spRRVe-P2-TK / sRRVgp- P2-TK, sRRVgp-P2-yCD and sRRVgp-P2-CD (2,6 (SEQ ID NO: 2)) after the PCR reaction using the genomic DNA of the combination of sRRVgp-P2-CD7 and spRRVe- , 7,8) vector p6 and p7 step PCR products were collected and gene analysis was performed.

그 결과, 도 36a 및 36b에 나타낸 바와 같이, sRRVgp-P2-CD6, sRRVgp-P2-CD7 및 sRRVgp-P2-CD8 벡터는 재조합이 일어나지 않았으며, 이는 양성대조군으로 이용한 sRRVgp-P2-CD2 벡터보다도 더 안정성이 뛰어났다(도 36a 및 36b).As a result, as shown in FIGS. 36A and 36B, no recombination occurred in the sRRVgp-P2-CD6, sRRVgp-P2-CD7 and sRRVgp-P2-CD8 vectors, which was more than the sRRVgp-P2-CD2 vector used as a positive control The stability was excellent (Figs. 36A and 36B).

<11-5> <11-5> spRRVespRRVe -P2-TK/-P2-TK / sRRVgpsRRVgp -P2-CDs 바이러스의 약물 감수성 확인Identification of drug susceptibility of -P2-CDs virus

상기 실시예 <11-2>에서 생산한 바이러스의 GCV 및 5-FC에 대한 약물감수성 확인은 실시예 <11-2>에서 생산한 바이러스를 공형질주입하여, p1 에서 p7까지 순차감염시켰으며, GCV와 5-FC를 각각 8일 또는 12일 동안 처리하여 세포사멸을 관찰한 것만 제외하고는 실시예<3-5>와 동일한 방법으로 확인하였다.In order to confirm the drug susceptibility of GCV and 5-FC of the virus produced in Example <11-2>, the virus produced in Example <11-2> was co-infected and sequentially infected from p1 to p7, GCV and 5-FC were treated for 8 days or 12 days, respectively, to observe apoptosis.

그 결과, 도 37a 내지 37c에 나타낸 바와 같이, sRRVgp-P2-CD6, sRRVgp-P2-CD7, sRRVgp-P2-CD8 및 spRRVe-P2-TK 벡터는 p7 단계까지 세포 사멸이 잘 일어났다(도 37a 내지 37c).As a result, as shown in Figs. 37A to 37C, sRRVgp-P2-CD6, sRRVgp-P2-CD7, sRRVgp-P2-CD8 and spRRVe- Cell death was evident until p7 (Figs. 37A to 37C).

<110> The Industry & Academic Cooperation in Chungnam National University <120> GENE THERAPY VECTOR SYSTEM AND PRODRUG GENES <130> 2016P-11-010 <160> 45 <170> KoPatentIn 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-EcoRI-F primer <400> 1 cggaattcat ggcttcgtac cccggcca 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-EcoRI-R primer <400> 2 gcgaattctc agtagcctcc cccatctc 28 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaLV-PmlI-F primer <400> 3 cggcacgtga tggtattgct gcctggg 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaLV-PmlI-R primer <400> 4 gcccacgtgt taaaggttac cttcgtt 27 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MuLV4194F primer <400> 5 agcaagctat tggccactg 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GaLV1624 F primer <400> 6 gactcagtca gcaagttaga g 21 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MFGSaclR primer <400> 7 caatcggagg actggcgccc cgagtga 27 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-CDa-F primer <400> 8 gaaggtaacc tttaattcaa taacaggaaa g 31 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-CDa-R primer <400> 9 ctttcctgtt attgaattaa aggttacctt c 31 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GaLV488 F primer <400> 10 ggctagaatc cctatatgta 20 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-BamHI-F primer <400> 11 cgggatccat ggcttcgtac ccctgccat 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-SalI-R primer <400> 12 cggtcgactc agttagcctc ccccatctc 29 <210> 13 <211> 9735 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-P2-TK vector <400> 13 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat 4020 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg 4080 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac 4140 gttctttttc gcaacgggtt tgccgccaga acacagggat ccatggcttc gtacccctgc 4200 catcaacacg cgtctgcgtt cgaccaggct gcgcgttctc gcggccatag caaccgacgt 4260 acggcgttgc gccctcgccg gcagcaagaa gccacggaag tccgcctgga gcagaaaatg 4320 cccacgctac tgcgggttta tatagacggt cctcacggga tggggaaaac caccaccacg 4380 caactgctgg tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact 4440 tactggcagg tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc 4500 ctcgaccagg gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata 4560 acaatgggca tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag 4620 gctgggagct cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc 4680 gccgccctcc tgtgctaccc ggccgcgcga taccttatgg gcagcatgac cccccaggcc 4740 gtgctggcgt tcgtggccct catcccgccg accttgcccg gcacaaacat cgtgttgggg 4800 gcccttccgg aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg 4860 cttgacctgg ctatgctggc cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg 4920 cggtatctgc agggcggcgg gtcgtggcgg gaggattggg gacagctttc ggggacggcc 4980 gtgccgcccc agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac 5040 acgttattta ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtacaac 5100 gtgtttgcct gggccttgga cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc 5160 ctggattacg accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg 5220 atggtccaga cccacgtcac cacccccggc tccataccga cgatctgcga cctggcgcgc 5280 acgtttgccc gggagatggg ggaggctaac tgagtcgaca tcgatggtac cagatccgat 5340 aaaataaaag gttttattta gtctcctgaa aaagggggga atgaaagacc ccacctgtag 5400 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag 5460 aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag 5520 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga 5580 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 5640 agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc 5700 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 5760 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 5820 cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa 5880 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag 5940 tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa aattaatttg 6000 gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac attggcttcc 6060 ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt aagatagtat 6120 ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc catttgttgt 6180 tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta aagatcctac 6240 actatagttc aagctagact attagctact ctgtaaccca gggtgacctt gaagtcatgg 6300 gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta tcatttttgg 6360 tatatgattg attgattgat tgatgtgtgt gtgtgtgatt gtgtttgtgt gtgtgactgt 6420 gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat gtgtgtgtgt gagtgtgtgt 6480 gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt gtatgactgt gtgtgtgtgt 6540 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa aaaatattct atggtagtga 6600 gagccaacgc tccggctcag gtgtcaggtt ggtttttgag acagagtctt tcacttagct 6660 tggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 6720 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 6780 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 6840 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc 6900 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 6960 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 7020 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 7080 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 7140 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 7200 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 7260 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 7320 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 7380 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 7440 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 7500 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 7560 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 7620 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 7680 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 7740 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 7800 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 7860 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 7920 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 7980 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 8040 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 8100 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 8160 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 8220 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 8280 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 8340 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 8400 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 8460 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 8520 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 8580 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 8640 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 8700 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 8760 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 8820 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 8880 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 8940 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 9000 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 9060 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 9120 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 9180 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 9240 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 9300 ctttgctcct aggagtttcc taatacttcc caaactcaaa tatataaagc atttgacttg 9360 ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt taaaatgtta 9420 attccatttt aaatgcacag atgtttttat ttcataaggg tttcaatgtg catgaatgct 9480 gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg aaattactta 9540 gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct gctgagcaag 9600 ccagtttgca tctgtcagga tcaatttccc attatgccag tcatattaat tactagtcaa 9660 ttagttgatt tttatttttg acatatacat gtgaatgaaa gaccccacct gtaggtttgg 9720 caagctagct taagt 9735 <210> 14 <211> 10010 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-P2-CD6 vector <400> 14 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcgggcaga 8040 gcgcacatcg cccgcagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc 8100 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 8160 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 8220 caacgggttt gccgccagaa cacaggcggc cgccgatggt tactggaggg atggccagta 8280 aatgggacca gaagggtatg gatattgcat acgaggaggc cgctttggga tacaaggagg 8340 ggggtgtccc tataggcggt tgcctgatca ataataaaga cggctctgtc ttgggaagag 8400 gacacaatat gcgctttcag aagggaagcg ccaccctgca tggagagatc tctaccctcg 8460 aaaattgcgg aaggctcgaa ggcaaagttt acaaagatac caccctctac acaacgctgt 8520 ccccctgtga tatgtgcacc ggtgccatta tcatgtatgg catcccacgc tgcgttgtag 8580 gagagaatgt aaacttcaaa tccaagggag agaagtatct ccagacccga gggcacgaag 8640 ttgtggtggt ggacgatgaa aggtgtaaga agatcatgaa gcagttcata gatgagcggc 8700 ctcaggactg gttcgaggat attggcgaat gagtttaaac gaattcataa aataaaagat 8760 tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct 8820 agcttaagta acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt 8880 cagatcaagg tcaggaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt 8940 aagcagttcc tgccccggct cagggccaag aacagatgga acagctgaat atgggccaaa 9000 caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca 9060 gatgcggtcc agccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa 9120 ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg 9180 ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc 9240 cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt 9300 tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc 9360 cgtcagcggg ggtctttcat tgttacttaa agttacattg gcttccttga aataaacatg 9420 gagtattcag aatgtgtcat aaatatttct aattttaaga tagtatctcc attggctttc 9480 tactttttct tttatttttt tttgtcctct gtcttccatt tgttgttgtt gttgtttgtt 9540 tgtttgtttg ttggttggtt ggttaatttt tttttaaaga tcctacacta tagttcaagc 9600 tagactatta gctactctgt aacccagggt gaccttgaag tcatgggtag cctgctgttt 9660 tagccttccc acatctaaga ttacaggtat gagctatcat ttttggtata ttgattgatt 9720 gattgattga tgtgtgtgtg tgtgattgtg tttgtgtgtg tgactgtgaa aatgtgtgta 9780 tgggtgtgtg tgaatgtgtg tatgtatgtg tgtgtgtgag tgtgtgtgtg tgtgtgtgca 9840 tgtgtgtgtg tgtgactgtg tctatgtgta tgactgtgtg tgtgtgtgtg tgtgtgtgtg 9900 tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa atattctatg gtagtgagag ccaacgctcc 9960 ggctcaggtg tcaggttggt ttttgagaca gagtctttca cttagcttgg 10010 <210> 15 <211> 1131 <212> DNA <213> human herpesvirus 1 <400> 15 atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60 ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120 cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180 gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240 gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300 tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360 atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420 cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480 ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540 agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600 acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660 cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720 ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780 cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840 cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900 aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960 cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020 ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080 atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131 <210> 16 <211> 406 <212> DNA <213> Saccharomyces cerevisiae <400> 16 atggtgacag ggggaatggc aagcaagtgg gatcagaagg gtatggacat tgcctatgag 60 gaggcggcct taggttacaa agagggtggt gttcctattg gcggatgtct tatcaataac 120 aaagacggaa gtgttctcgg tcgtggtcac aacatgagat ttcaaaaggg atccgccaca 180 ctacatggtg agatctccac tttggaaaac tgtgggagat tagagggcaa agtgtacaaa 240 gataccactt tgtatacgac gctgtctcca tgcgacatgt gtacaggtgc catcatcatg 300 tatggtattc cacgctgtgt tgtcggtgag aacgttaatt tcaaaagtaa gggcgagaaa 360 tatttacaaa ctagaggtca cgaggttgtt gttgttgacg atgaga 406 <210> 17 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD2 <400> 17 atggtgaccg gcggcatggc ctccaagtgg gatcaaaagg gcatggatat cgcttacgag 60 gaggccctgc tgggctacaa ggagggcggc gtgcctatcg gcggctgtct gatcaacaac 120 aaggacggca gtgtgctggg caggggccac aacatgaggt tccagaaggg ctccgccacc 180 ctgcacggcg agatctccac cctggagaac tgtggcaggc tggagggcaa ggtgtacaag 240 gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gtaccggcgc tatcatcatg 300 tacggcatcc ctaggtgtgt gatcggcgag aacgtgaact tcaagtccaa gggcgagaag 360 tacctgcaaa ccaggggcca cgaggtggtg gttgttgacg atgagaggtg taagaagctg 420 atgaagcagt tcatcgacga gaggcctcag gactggttcg aggatatcgg cgagtaa 477 <210> 18 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD3 <400> 18 atggtgaccg gcggcatggc ctccaagtgg gaccaaaagg gcatggatat cgcttacgag 60 gaggccctgc tgggctacaa ggagggcggc gtgcccatcg gcggctgcct gatcaacaac 120 aaggacggca gcgtgctggg caggggccac aacatgaggt tccagaaggg ctccgccacc 180 ctgcacggcg agatctccac cctggagaac tgcggcaggc tggagggcaa ggtgtacaag 240 gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gcaccggcgc tatcatcatg 300 tacggcatcc ctaggtgcgt gatcggcgag aacgtgaact tcaagtccaa gggcgagaag 360 tacctgcaga ccaggggcca cgaggtggtg gtggtggacg acgagaggtg caagaagctg 420 atgaagcagt tcatcgacga gaggccccag gactggttcg aggacatcgg cgagtaa 477 <210> 19 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD4 <400> 19 atggtgacag ggggaatggc aagcaagtgg gatcagaagg gtatggacat tgcctatgag 60 gaggcggcct taggttacaa agagggtggt gttcctattg gcggatgtct tatcaataac 120 aaagacggaa gtgttctcgg tcgtggtcac aacatgagat ttcaaaaggg atccgccaca 180 ctacatggtg agatctccac tttggagaac tgcggcaggc tggaaggcaa ggtgtacaaa 240 gataccactc tgtacaccac cctgtctcca tgcgacatgt gtacaggtgc catcatcatg 300 tatggtattc cacgctgtgt tgtcggtgag aacgttaatt tcaaaagtaa gggcgagaaa 360 tacttgcaaa ccaggggcca cgaggtggtg gttgttgacg atgagaggtg taaaaagatc 420 atgaaacaat ttatcgatga aagacctcag gactggttcg aggacatcgg cgagtaa 477 <210> 20 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD5 <400> 20 atggtgactg gcggcatggc atccaagtgg gaccagaagg ggatggacat agcatatgaa 60 gaggccgcgt tgggatataa ggagggcggt gtaccaatcg ggggctgcct cattaacaat 120 aaagatggct ccgttctggg tcgcggccac aacatgaggt ttcagaaggg cagtgcgacg 180 ctccacggag aaatcagcac actggaaaat tgtgggcgat tggaggggaa agtgtataag 240 gatacaactc tctacaccac tctcagcccc tgcgatatgt gcacaggcgc aatcataatg 300 tacggcattc cccgatgcgt ggtgggggag aacgtgaact tcaagagcaa aggagagaaa 360 tatcttcaga ccagaggaca cgaagtagtg gtggtggatg atgaacgctg caagaaaatc 420 atgaaacagt ttatagatga acgaccacaa gactggttcg aggatatcgg cgaatag 477 <210> 21 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD6 <400> 21 atggttactg gagggatggc cagtaaatgg gaccagaagg gtatggatat tgcatacgag 60 gaggccgctt tgggatacaa ggaggggggt gtccctatag gcggttgcct gatcaataat 120 aaagacggct ctgtcttggg aagaggacac aatatgcgct ttcagaaggg aagcgccacc 180 ctgcatggag agatctctac cctcgaaaat tgcggaaggc tcgaaggcaa agtttacaaa 240 gataccaccc tctacacaac gctgtccccc tgtgatatgt gcaccggtgc cattatcatg 300 tatggcatcc cacgctgcgt tgtaggagag aatgtaaact tcaaatccaa gggagagaag 360 tatctccaga cccgagggca cgaagttgtg gtggtggacg atgaaaggtg taagaagatc 420 atgaagcagt tcatagatga gcggcctcag gactggttcg aggatattgg cgaatga 477 <210> 22 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD7 <400> 22 atggtaactg gtggcatggc ctcaaagtgg gatcagaaag gaatggacat cgcttacgag 60 gaggccgcac tgggctataa ggagggcggc gtccctatag gcggttgcct gattaacaat 120 aaagacggct cagtgctggg aagggggcac aacatgagat ttcagaaagg cagcgcaact 180 ctgcacggcg aaatctccac tctggagaac tgcgggcggc tggagggaaa ggtttataaa 240 gatactacct tgtatacaac tctgtccccc tgcgatatgt gcaccggcgc catcataatg 300 tacggaatac ccaggtgcgt ggtgggagag aacgtgaatt ttaagtcaaa aggtgagaag 360 tacctgcaga ctcgcggcca tgaggtggtt gttgttgacg atgaaaggtg caagaagatt 420 atgaagcagt tcattgatga aagaccccag gactggtttg aggatatcgg agagtag 477 <210> 23 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD8 <400> 23 atggttactg ggggaatggc atctaagtgg gatcagaaag gtatggacat cgcttatgaa 60 gaggctgctc tcggctacaa agagggtgga gtgcctatcg gagggtgcct gatcaacaac 120 aaggacggca gtgtgctggg gaggggccac aatatgaggt tccaaaaagg ctccgccact 180 ctccacgggg aaattagtac cctcgagaat tgcggacgat tggaagggaa ggtgtacaag 240 gatacaacac tgtacaccac cctgtcaccc tgtgatatgt gcacaggcgc cattatcatg 300 tacggaatcc ctagatgtgt cgtgggggag aatgtaaact tcaaaagtaa gggggagaaa 360 tatctccaga cccgggggca cgaagtcgtc gttgtggacg atgaacggtg taagaagatc 420 atgaagcagt ttatcgatga gaggccccag gactggttcg aagacatcgg ggaataa 477 <210> 24 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD9 <400> 24 atggttacag ggggaatggc aagtaaatgg gatcaaaaag ggatggatat agcctatgag 60 gaagcggcgc tgggctataa agagggaggg gtgccgatag gtggctgtct tattaataac 120 aaagacggga gtgtgttggg cagaggccac aatatgcgat ttcaaaaagg gtccgcgaca 180 ttgcacggag agatcagcac cctggagaat tgcggaaggt tggagggaaa agtgtataag 240 gacaccaccc tctataccac actgtctcca tgtgatatgt gtaccggtgc catcataatg 300 tacgggattc ctcgctgcgt agtgggagag aatgttaact ttaaaagcaa gggagagaag 360 tatttgcaaa cccggggcca cgaagtggtg gtggtggacg acgagcgatg taagaaaatc 420 atgaagcaat ttatcgatga gcggcctcaa gattggttcg aagatatcgg cgagtga 477 <210> 25 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD10 <400> 25 atggtaaccg gaggtatggc atccaagtgg gaccaaaaag gaatggacat agcatatgaa 60 gaagcagccc tgggctacaa ggagggaggg gttccgattg gcggttgtct tataaataat 120 aaagacggta gtgttcttgg caggggtcac aacatgagat tccaaaaggg gagtgctaca 180 cttcacggcg aaataagcac cttggaaaac tgtggtagac ttgagggaaa agtgtacaag 240 gacacgaccc tttatacgac gctgtcccct tgtgatatgt gcaccggcgc tatcatcatg 300 tatggaatac cacgatgcgt agtgggagag aatgttaatt tcaagagtaa gggcgagaag 360 taccttcaga ccagggggca cgaggtagta gtagttgacg atgagcgatg caagaagatt 420 atgaaacaat tcattgacga gaggccgcag gattggtttg aagacatcgg cgaatag 477 <210> 26 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD11 <400> 26 atggtgacag ggggtatggc aagcaaatgg gatcagaagg gtatggacat cgcatacgag 60 gaggcggcct tgggctataa ggaaggcggc gtacctatag gggggtgcct tattaacaat 120 aaggacggga gcgtcctggg cagaggtcac aacatgaggt tccaaaaggg ttcagcaacc 180 ctgcatggcg aaataagcac ccttgagaat tgtgggaggt tggagggtaa ggtgtacaag 240 gataccacgc tttataccac cttgagtcct tgcgacatgt gcacaggcgc tataatcatg 300 tatggaatac cgcgctgtgt tgtaggagaa aatgtaaact tcaagagtaa aggagaaaaa 360 tacttgcaaa cgcggggaca cgaagtggta gttgtcgatg atgagcggtg caaaaaaatc 420 atgaagcagt tcattgacga acgcccccaa gactggttcg aagacattgg ggagtag 477 <210> 27 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD12 <400> 27 atggtaacgg gtgggatggc tagcaagtgg gaccagaaag gcatggatat agcgtatgaa 60 gaagcggcgt tgggttacaa agagggcggc gttcccatcg gtggctgcct tatcaataat 120 aaagacggct ccgtccttgg ccggggacac aatatgcgct tccaaaaggg cagcgccaca 180 cttcacggtg agatctccac gctggagaat tgtgggcgac ttgaggggaa agtctacaag 240 gacacaactt tgtacacaac acttagcccg tgcgatatgt gtacgggagc cataatcatg 300 tacggcatcc cgcgctgcgt ggtaggagag aacgtaaatt ttaagtcaaa aggagaaaaa 360 tatcttcaga ccaggggcca cgaggtggtt gtcgtggacg acgagagatg taaaaagatc 420 atgaaacagt ttattgatga aagaccacag gattggtttg aggacatcgg tgagtag 477 <210> 28 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD13 <400> 28 atggttacag gaggtatggc ttcaaagtgg gatcaaaaag ggatggacat cgcctatgaa 60 gaagcagcgt tgggatacaa agaagggggg gttcccatag gaggttgcct tatcaacaat 120 aaagatggaa gcgttcttgg gcgagggcac aatatgagat ttcaaaaagg ttcagccact 180 ctccatggag aaatttcaac tctcgaaaac tgtggtcgcc ttgagggcaa ggtttataag 240 gataccaccc tctacactac cctgtcaccc tgcgacatgt gtacaggtgc aattataatg 300 tacggaatcc ctcggtgtgt ggtgggggag aacgtgaatt ttaagtccaa aggtgaaaaa 360 tatctccaaa ctcgcgggca tgaagtcgtc gttgttgatg atgagaggtg taaaaagatt 420 atgaaacaat tcatagacga gaggccacag gattggtttg aggacatagg ggagtag 477 <210> 29 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD14 <400> 29 atggtgactg ggggtatggc ttccaaatgg gatcagaaag gaatggatat agcatacgaa 60 gaagcagctc tcgggtacaa agagggtgga gtacccattg ggggatgcct catcaacaac 120 aaggatggga gtgtccttgg gcgaggtcac aatatgcgat tccagaaggg gagcgcgacg 180 ctccacgggg agataagtac gctggagaac tgcgggaggc ttgaaggcaa ggtctacaaa 240 gataccacac tctacacgac cctcagccct tgcgacatgt gtacgggtgc gatcatcatg 300 tatggaatac cgcgatgcgt agtaggagag aacgtgaact tcaagtccaa aggcgaaaag 360 tatctccaga cgcgcggcca cgaagtggta gtggtagacg acgaaaggtg caagaagata 420 atgaagcagt ttatcgacga gaggcctcag gactggttcg aggatattgg cgagtag 477 <210> 30 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD15 <400> 30 atggttactg gcggcatggc ttctaagtgg gatcagaagg gcatggatat agcctatgaa 60 gaagcagcac tgggatacaa agagggaggg gtaccaattg ggggatgtct gattaataac 120 aaagacggaa gtgtactcgg tcgcgggcat aatatgagat tccaaaaagg ctctgcaacg 180 ttgcacggcg aaatcagcac gctcgaaaat tgcgggaggc tggagggaaa ggtttacaag 240 gataccactc tctataccac actgtcacca tgtgatatgt gtacgggggc tataataatg 300 tatggaatcc cccgctgcgt cgtgggcgaa aacgtcaact ttaagtctaa gggggaaaag 360 tatttgcaaa cgcgcggtca tgaggtcgtt gtagtcgatg acgagagatg caaaaaaata 420 atgaagcagt ttattgacga gagacctcag gactggttcg aagacatcgg ggagtag 477 <210> 31 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD16 <400> 31 atggtgacag gaggaatggc cagcaagtgg gatcagaagg gaatggatat tgcctacgag 60 gaggccgccc tgggctacaa ggaggggggc gtgccaattg gcggatgtct gattaacaac 120 aaggatggga gcgtgctggg aagaggacac aacatgagat ttcagaaggg aagcgcaacc 180 ttgcacggag aaattagcac ccttgagaac tgcgggcggc ttgaaggcaa ggtctataaa 240 gacactacac tttatactac cttgtctcca tgtgatatgt gtacaggcgc cattattatg 300 tacggaattc ctagatgcgt cgtgggagag aacgtgaact ttaagagcaa gggagagaag 360 tacctgcaga caagaggaca cgaggtggtg gtggtggatg atgagagatg taagaagatc 420 atgaagcagt tcatcgatga gaggccccag gattggtttg aggacatcgg cgagtga 477 <210> 32 <211> 1617 <212> DNA <213> Murine leukemia virus <400> 32 atgggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag 60 cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct 120 gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc 180 atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc 240 ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt 300 gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct 360 cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc 420 aaacctaaac ctcaagttct ttctgacagt ggggggccgc tcatcgacct acttacagaa 480 gaccccccgc cttataggga cccaagacca cccccttccg acagggacgg aaatggtgga 540 gaagcgaccc ctgcgggaga ggcaccggac ccctccccaa tggcatctcg cctacgtggg 600 agacgggagc cccctgtggc cgactccact acctcgcagg cattccccct ccgcgcagga 660 ggaaacggac agcttcaata ctggccgttc tcctcttctg acctttacaa ctggaaaaat 720 aataaccctt ctttttctga agatccaggt aaactgacag ctctgatcga gtctgttctc 780 atcacccatc agcccacctg ggacgactgt cagcagctgt tggggactct gctgaccgga 840 gaagaaaaac aacgggtgct cttagaggct agaaaggcgg tgcggggcga tgatgggcgc 900 cccactcaac tgcccaatga agtcgatgcc gcttttcccc tcgagcgccc agactgggat 960 tacaccaccc aggcaggtag gaaccaccta gtccactatc gccagttgct cctagcgggt 1020 ctccaaaacg cgggcagaag ccccaccaat ttggccaagg taaaaggaat aacacaaggg 1080 cccaatgagt ctccctcggc cttcctagag agacttaagg aagcctatcg caggtacact 1140 ccttatgacc ctgaggaccc agggcaagaa actaatgtgt ctatgtcttt catttggcag 1200 tctgccccag acattgggag aaagttagag aggttagaag atttaaaaaa caagacgctt 1260 ggagatttgg ttagagaggc agaaaagatc tttaataaac gagaaacccc ggaagaaaga 1320 gaggaacgta tcaggagaga aacagaggaa aaagaagaac gccgtaggac agaggatgag 1380 cagaaagaga aagaaagaga tcgtaggaga catagagaga tgagcaagct attggccact 1440 gtcgttagtg gacagaaaca ggatagacag ggaggagaac gaaggaggtc ccaactcgat 1500 cgcgaccagt gtgcctactg caaagaaaag gggcactggg ctaaagattg tcccaagaaa 1560 ccacgaggac ctcggggacc aagaccccag acctccctcc tgaccctaga tgactag 1617 <210> 33 <211> 3608 <212> DNA <213> Murine leukemia virus <400> 33 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 60 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 120 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 180 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 240 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 300 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 360 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 420 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 480 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 540 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 600 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 660 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 720 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 780 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 840 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 900 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 960 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 1020 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1080 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1140 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1200 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1260 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1320 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1380 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1440 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1500 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1560 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1620 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1680 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1740 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1800 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1860 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1920 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1980 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 2040 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2100 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2160 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2220 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2280 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2340 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 2400 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 2460 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 2520 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 2580 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 2640 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 2700 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 2760 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 2820 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 2880 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 2940 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 3000 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 3060 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 3120 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 3180 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 3240 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 3300 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 3360 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 3420 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 3480 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 3540 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 3600 ccccctaa 3608 <210> 34 <211> 212 <212> DNA <213> Homo sapiens <400> 34 gggcagagcg cacatcgccc gcagtccccg agaagttggg gggaggggtc ggcaattgat 60 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac ag 212 <210> 35 <211> 2058 <212> DNA <213> Gibbon leukemia virus <400> 35 atggtattgc tgcctgggtc catgcttctc acctcaaacc tgcaccacct tcggcaccag 60 atgagtcctg ggagctggaa aagactgatc atcctcttaa gctgcgtatt cggcggcggc 120 gggacgagtc tgcaaaataa gaacccccac cagcccatga ccctcacttg gcaggtactg 180 tcccaaactg gagacgttgt ctgggataca aaggcagtcc agcccccttg gacttggtgg 240 cccacactta aacctgatgt atgtgccttg gcggctagtc ttgagtcctg ggatatcccg 300 ggaaccgatg tctcgtcctc taaacgagtc agacctccgg actcagacta tactgccgct 360 tataagcaaa tcacctgggg agccataggg tgcagctacc ctcgggctag gactagaatg 420 gcaagctcta ccttctacgt atgtccccgg gatggccgga ccctttcaga agctagaagg 480 tgcggggggc tagaatccct atactgtaaa gaatgggatt gtgagaccac ggggaccggt 540 tattggctat ctaaatcctc aaaagacctc ataactgtaa aatgggacca aaatagcgaa 600 tggactcaaa aatttcaaca gtgtcaccag accggctggt gtaaccccct taaaatagat 660 ttcacagaca aaggaaaatt atccaaggac tggataacgg gaaaaacctg gggattaaga 720 ttctatgtgt ctggacatcc aggcgtacag ttcaccattc gcttaaaaat caccaacatg 780 ccagctgtgg cagtaggtcc tgacctcgtc cttgtggaac aaggacctcc tagaacgtcc 840 ctcgctctcc cacctcctct tcccccaagg gaagcgccac cgccatctct ccccgactct 900 aactccacag ccctggcgac tagtgcacaa actcccacgg tgagaaaaac aattgttacc 960 ctaaacactc cgcctcccac cacaggcgac agactttttg atcttgtgca gggggccttc 1020 ctaaccttaa atgctaccaa cccaggggcc actgagtctt gctggctttg tttggccatg 1080 ggcccccctt attatgaagc aatagcctca tcaggagagg tcgcctactc caccgacctt 1140 gaccggtgcc gctgggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agactagaga tttctgtatc 1380 caggtccagc tgattcctcg catctattac tatcctgaag aagttttgtt acaggcctat 1440 gacaattctc accccaggac taaaagagag gctgtctcac ttaccctagc tgttttactg 1500 gggttgggaa tcacggcggg aataggtact ggttcaactg ccttaattaa aggacctata 1560 gacctccagc aaggcctgac aagcctccag atcgccatag atgctgacct ccgggccctc 1620 caagactcag tcagcaagtt agaggactca ctgacttccc tgtccgaggt agtgctccaa 1680 aataggagag gccttgactt gctgtttcta aaagaaggtg gcctctgtgc ggccctaaag 1740 gaagagtgct gtttttacat agaccactca ggtgcagtac gggactccat gaaaaaactc 1800 aaagaaaaac tggataaaag acagttagag cgccagaaaa gccaaaactg gtatgaagga 1860 tggttcaata actccccttg gttcactacc ctgctatcaa ccatcgctgg gcccctatta 1920 ctcctccttc tgttgctcat cctcgggcca tgcatcatca ataagttagt tcaattcatc 1980 aatgatagga taagtgcagt taaaattctg gtccttagac aaaaatatca ggccctagag 2040 aacgaaggta acctttaa 2058 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD-Not1-F primer <400> 36 cggcggccgc atggtgacag ggggaatggc 30 <210> 37 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CD-Pme1-R primer <400> 37 cggtttaaac ctactcacca atatcttcaa a 31 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD2-Not1-F primer <400> 38 cggcggccgc atggtgaccg gcggcatggc 30 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD2-Pme1-R primer <400> 39 cggtttaaac ttactcgccg atatcctcga 30 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD6-Not1-F primer <400> 40 cggcggccgc atggttactg gagggatggc 30 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD6-Pme1-R primer <400> 41 cggtttaaac tcattcgcca atatcctcga 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD7-Not1-F primer <400> 42 cggcggccgc atggtaactg gtggcatggc 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD7-Pme1-R primer <400> 43 cggtttaaac ctactctccg atatcctcaa 30 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CD8-Not1-F primer <400> 44 cggcggccgc atggttactg ggggaatgg 29 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD8-Pme1-R primer <400> 45 cggtttaaac ttattccccg atgtcttcga 30 <110> The Industry & Academic Cooperation in Chungnam National University <120> GENE THERAPY VECTOR SYSTEM AND PRODRUG GENES <130> 2016P-11-010 <160> 45 <170> KoPatentin 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-EcoRI-F primer <400> 1 cggaattcat ggcttcgtac cccggcca 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-EcoRI-R primer <400> 2 gcgaattctc agtagcctcc cccatctc 28 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaLV-PmlI-F primer <400> 3 cggcacgtga tggtattgct gcctggg 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaLV-PmlI-R primer <400> 4 gcccacgtgt taaaggttac cttcgtt 27 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MuLV4194F primer <400> 5 agcaagctat tggccactg 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GaLV1624F primer <400> 6 gactcagtca gcaagttaga g 21 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MFGSaclR primer <400> 7 caatcggagg actggcgccc cgagtga 27 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-CDa-F primer <400> 8 gaaggtaacc tttaattcaa taacaggaaa g 31 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-CDa-R primer <400> 9 ctttcctgtt attgaattaa aggttacctt c 31 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GaLV488 F primer <400> 10 ggctagaatc cctatatgta 20 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-BamHI-F primer <400> 11 cgggatccat ggcttcgtac ccctgccat 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HSV-TK-SalI-R primer <400> 12 cggtcgactc agttagcctc ccccatctc 29 <210> 13 <211> 9735 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-P2-TK vector <400> 13 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 ccccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat 4020 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg 4080 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac 4140 gttctttttc gcaacgggtt tgccgccaga acacagggat ccatggcttc gtacccctgc 4200 catcaacacg cgtctgcgtt cgaccaggct gcgcgttctc gcggccatag caaccgacgt 4260 acggcgttgc gccctcgccg gcagcaagaa gccacggaag tccgcctgga gcagaaaatg 4320 cccacgctac tgcgggttta tatagacggt cctcacggga tggggaaaac caccaccacg 4380 caactgctgg tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact 4440 tactggcagg tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc 4500 ctcgaccagg gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata 4560 acaatgggca tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag 4620 gctgggagct cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc 4680 gccgccctcc tgtgctaccc ggccgcgcga taccttatgg gcagcatgac cccccaggcc 4740 gtgctggcgt tcgtggccct catcccgccg accttgcccg gcacaaacat cgtgttgggg 4800 gcccttccgg aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg 4860 cttgacctgg ctatgctggc cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg 4920 cggtatctgc agggcggcgg gtcgtggcgg gaggattggg gacagctttc ggggacggcc 4980 gtgccgcccc agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac 5040 acgttattta ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtacaac 5100 gtgtttgcct gggccttgga cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc 5160 ctggattacg accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg 5220 atggtccaga cccacgtcac cacccccggc tccataccga cgatctgcga cctggcgcgc 5280 acgtttgccc gggagatggg ggaggctaac tgagtcgaca tcgatggtac cagatccgat 5340 aaaataaaag gttttattta gtctcctgaa aaagggggga atgaaagacc ccacctgtag 5400 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag 5460 aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag 5520 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga 5580 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 5640 agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc 5700 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 5760 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 5820 cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa 5880 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag 5940 tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa aattaatttg 6000 gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac attggcttcc 6060 ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt aagatagtat 6120 ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc catttgttgt 6180 tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta aagatcctac 6240 actatagttc aagctagact attagctact ctgtaaccca gggtgacctt gaagtcatgg 6300 gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta tcatttttgg 6360 tatatgattg attgattgat tgatgtgtgt gtgtgtgatt gtgtttgtgt gtgtgactgt 6420 gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat gtgtgtgtgt gagtgtgtgt 6480 gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt gtatgactgt gtgtgtgtgt 6540 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa aaaatattct atggtagtga 6600 gagccaacgc tccggctcag gtgtcaggtt ggtttttgag acagagtctt tcacttagct 6660 tggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 6720 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 6780 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 6840 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc 6900 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 6960 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 7020 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 7080 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 7140 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 7200 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 7260 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 7320 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 7380 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 7440 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 7500 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 7560 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 7620 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 7680 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 7740 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 7800 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 7860 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 7920 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 7980 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 8040 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 8100 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 8160 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 8220 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 8280 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 8340 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 8400 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 8460 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 8520 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 8580 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 8640 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 8700 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 8760 ggcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 8820 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 8880 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 8940 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 9000 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 9060 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 9120 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 9180 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 9240 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 9300 ctttgctcct aggagtttcc taatacttcc caaactcaaa tatataaagc atttgacttg 9360 ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt taaaatgtta 9420 attccatttt aaatgcacagat atgtttttat ttcataaggg tttcaatgtg catgaatgct 9480 gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg aaattactta 9540 gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct gctgagcaag 9600 ccagtttgca tctgtcagga tcaatttccc attatgccag tcatattaat tactagtcaa 9660 ttagttgatt tttatttttg acatatacat gtgaatgaaa gaccccacct gtaggtttgg 9720 caagctagct taagt 9735 <210> 14 <211> 10010 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-P2-CD6 vector <400> 14 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcgggcaga 8040 gcgcacatcg cccgcagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc 8100 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 8160 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 8220 caacgggttt gccgccagaa cacaggcggc cgccgatggt tactggaggg atggccagta 8280 aatgggacca gaagggtatg gatattgcat acgaggaggc cgctttggga tacaaggagg 8340 ggggtgtccc tataggcggt tgcctgatca ataataaaga cggctctgtc ttgggaagag 8400 gacacaatat gcgctttcag aagggaagcg ccaccctgca tggagagatc tctaccctcg 8460 aaaattgcgg aaggctcgaa ggcaaagttt acaaagatac caccctctac acaacgctgt 8520 ccccctgtga tatgtgcacc ggtgccatta tcatgtatgg catcccacgc tgcgttgtag 8580 gagagaatgt aaacttcaaa tccaagggag agaagtatct ccagacccga gggcacgaag 8640 ttgtggtggt ggacgatgaa aggtgtaaga agatcatgaa gcagttcata gatgagcggc 8700 ctcaggactg gttcgaggat attggcgaat gagtttaaac gaattcataa aataaaagat 8760 tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct 8820 agcttaagta acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt 8880 cagatcaagg tcaggaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt 8940 aagcagttcc tgccccggct cagggccaag aacagatgga acagctgaat atgggccaaa 9000 cggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca 9060 gatgcggtcc agccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa 9120 ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg 9180 ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc 9240 cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt 9300 tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc 9360 cgtcagcggg ggtctttcat tgttacttaa agttacattg gcttccttga aataaacatg 9420 gagtattcag aatgtgtcat aaatatttct aattttaaga tagtatctcc attggctttc 9480 tactttttct tttatttttt tttgtcctct gtcttccatt tgttgttgtt gttgtttgtt 9540 tgtttgtttg ttggttggtt ggttaatttt tttttaaaga tcctacacta tagttcaagc 9600 tagactatta gctactctgt aacccagggt gaccttgaag tcatgggtag cctgctgttt 9660 tagccttccc acatctaaga ttacaggtat gagctatcat ttttggtata ttgattgatt 9720 gattgattga tgtgtgtgtg tgtgattgtg tttgtgtgtg tgactgtgaa aatgtgtgta 9780 tgggtgtgtg tgaatgtgtg tatgtatgtg tgtgtgtgag tgtgtgtgtg tgtgtgtgca 9840 tgtgtgtgtg tgtgactgtg tctatgtgta tgactgtgtg tgtgtgtgtg tgtgtgtgtg 9900 tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa atattctatg gtagtgagag ccaacgctcc 9960 ggctcaggtg tcaggttggt ttttgagaca gagtctttca cttagcttgg 10010 <210> 15 <211> 1131 <212> DNA <213> human herpesvirus 1 <400> 15 atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60 ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120 cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180 gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240 gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300 tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360 atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420 cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480 ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540 agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600 acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660 cgcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720 ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780 cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840 gt; aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960 cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020 ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080 atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131 <210> 16 <211> 406 <212> DNA <213> Saccharomyces cerevisiae <400> 16 atggtgacag ggggaatggc aagcaagtgg gatcagaagg gtatggacat tgcctatgag 60 gaggcggcct taggttacaa agagggtggt gttcctattg gcggatgtct tatcaataac 120 aaagacggaa gtgttctcgg tcgtggtcac aacatgagat ttcaaaaggg atccgccaca 180 ctacatggtg agatctccac tttggaaaac tgtgggagat tagagggcaa agtgtacaaa 240 gataccactt tgtatacgac gctgtctcca tgcgacatgt gtacaggtgc catcatcatg 300 tatggtattc cacgctgtgt tgtcggtgag aacgttaatt tcaaaagtaa gggcgagaaa 360 tatttacaaa ctagaggtca cgaggttgtt gttgttgacg atgaga 406 <210> 17 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD2 <400> 17 atggtgaccg gcggcatggc ctccaagtgg gatcaaaagg gcatggatat cgcttacgag 60 gaggccctgc tgggctacaa ggagggcggc gtgcctatcg gcggctgtct gatcaacaac 120 aaggacggca gtgtgctggg caggggccac aacatgaggt tccagaaggg ctccgccacc 180 ctgcacggcg agatctccac cctggagaac tgtggcaggc tggagggcaa ggtgtacaag 240 gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gtaccggcgc tatcatcatg 300 tacggcatcc ctaggtgtgt gatcggcgag aacgtgaact tcaagtccaa gggcgagaag 360 tacctgcaaa ccaggggcca cgaggtggtg gttgttgacg atgagaggtg taagaagctg 420 atgaagcagt tcatcgacga gaggcctcag gactggttcg aggatatcgg cgagtaa 477 <210> 18 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD3 <400> 18 atggtgaccg gcggcatggc ctccaagtgg gaccaaaagg gcatggatat cgcttacgag 60 gaggccctgc tgggctacaa ggagggcggc gtgcccatcg gcggctgcct gatcaacaac 120 aaggacggca gcgtgctggg caggggccac aacatgaggt tccagaaggg ctccgccacc 180 ctgcacggcg agatctccac cctggagaac tgcggcaggc tggagggcaa ggtgtacaag 240 gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gcaccggcgc tatcatcatg 300 tacggcatcc ctaggtgcgt gatcggcgag aacgtgaact tcaagtccaa gggcgagaag 360 tacctgcaga ccaggggcca cgaggtggtg gtggtggacg acgagaggtg caagaagctg 420 atgaagcagt tcatcgacga gaggccccag gactggttcg aggacatcgg cgagtaa 477 <210> 19 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD4 <400> 19 atggtgacag ggggaatggc aagcaagtgg gatcagaagg gtatggacat tgcctatgag 60 gaggcggcct taggttacaa agagggtggt gttcctattg gcggatgtct tatcaataac 120 aaagacggaa gtgttctcgg tcgtggtcac aacatgagat ttcaaaaggg atccgccaca 180 ctacatggtg agatctccac tttggagaac tgcggcaggc tggaaggcaa ggtgtacaaa 240 gataccactc tgtacaccac cctgtctcca tgcgacatgt gtacaggtgc catcatcatg 300 tatggtattc cacgctgtgt tgtcggtgag aacgttaatt tcaaaagtaa gggcgagaaa 360 tacttgcaaa ccaggggcca cgaggtggtg gttgttgacg atgagaggtg taaaaagatc 420 atgaaacaat ttatcgatga aagacctcag gactggttcg aggacatcgg cgagtaa 477 <210> 20 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD5 <400> 20 atggtgactg gcggcatggc atccaagtgg gaccagaagg ggatggacat agcatatgaa 60 gaggccgcgt tgggatataa ggagggcggt gtaccaatcg ggggctgcct cattaacaat 120 aaagatggct ccgttctggg tcgcggccac aacatgaggt ttcagaaggg cagtgcgacg 180 ctccacggag aaatcagcac actggaaaat tgtgggcgat tggaggggaa agtgtataag 240 gatacaactc tctacaccac tctcagcccc tgcgatatgt gcacaggcgc aatcataatg 300 tacggcattc cccgatgcgt ggtgggggag aacgtgaact tcaagagcaa aggagagaaa 360 tatcttcaga ccagaggaca cgaagtagtg gtggtggatg atgaacgctg caagaaaatc 420 atgaaacagt ttatagatga acgaccacaa gactggttcg aggatatcgg cgaatag 477 <210> 21 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD6 <400> 21 atggttactg gagggatggc cagtaaatgg gaccagaagg gtatggatat tgcatacgag 60 gaggccgctt tgggatacaa ggaggggggt gtccctatag gcggttgcct gatcaataat 120 aaagacggct ctgtcttggg aagaggacac aatatgcgct ttcagaaggg aagcgccacc 180 ctgcatggag agatctctac cctcgaaaat tgcggaaggc tcgaaggcaa agtttacaaa 240 gataccaccc tctacacaac gctgtccccc tgtgatatgt gcaccggtgc cattatcatg 300 tatggcatcc cacgctgcgt tgtaggagag aatgtaaact tcaaatccaa gggagagaag 360 tatctccaga cccgagggca cgaagttgtg gtggtggacg atgaaaggtg taagaagatc 420 atgaagcagt tcatagatga gcggcctcag gactggttcg aggatattgg cgaatga 477 <210> 22 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD7 <400> 22 atggtaactg gtggcatggc ctcaaagtgg gatcagaaag gaatggacat cgcttacgag 60 gaggccgcac tgggctataa ggagggcggc gtccctatag gcggttgcct gattaacaat 120 aaagacggct cagtgctggg aagggggcac aacatgagat ttcagaaagg cagcgcaact 180 ctgcacggcg aaatctccac tctggagaac tgcgggcggc tggagggaaa ggtttataaa 240 gatactacct tgtatacaac tctgtccccc tgcgatatgt gcaccggcgc catcataatg 300 tacggaatac ccaggtgcgt ggtgggagag aacgtgaatt ttaagtcaaa aggtgagaag 360 tacctgcaga ctcgcggcca tgaggtggtt gttgttgacg atgaaaggtg caagaagatt 420 atgaagcagt tcattgatga aagaccccag gactggtttg aggatatcgg agagtag 477 <210> 23 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD8 <400> 23 atggttactg ggggaatggc atctaagtgg gatcagaaag gtatggacat cgcttatgaa 60 gaggctgctc tcggctacaa agagggtgga gtgcctatcg gagggtgcct gatcaacaac 120 aaggacggca gtgtgctggg gaggggccac aatatgaggt tccaaaaagg ctccgccact 180 ctccacgggg aaattagtac cctcgagaat tgcggacgat tggaagggaa ggtgtacaag 240 gatacaacac tgtacaccac cctgtcaccc tgtgatatgt gcacaggcgc cattatcatg 300 tacggaatcc ctagatgtgt cgtgggggag aatgtaaact tcaaaagtaa gggggagaaa 360 tatctccaga cccgggggca cgaagtcgtc gttgtggacg atgaacggtg taagaagatc 420 atgaagcagt ttatcgatga gaggccccag gactggttcg aagacatcgg ggaataa 477 <210> 24 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD9 <400> 24 atggttacag ggggaatggc aagtaaatgg gatcaaaaag ggatggatat agcctatgag 60 gaagcggcgc tgggctataa agagggaggg gtgccgatag gtggctgtct tattaataac 120 aaagacggga gtgtgttggg cagaggccac aatatgcgat ttcaaaaagg gtccgcgaca 180 ttgcacggag agatcagcac cctggagaat tgcggaaggt tggagggaaa agtgtataag 240 gacaccaccc tctataccac actgtctcca tgtgatatgt gtaccggtgc catcataatg 300 tacgggattc ctcgctgcgt agtgggagag aatgttaact ttaaaagcaa gggagagaag 360 tatttgcaaa cccggggcca cgaagtggtg gtggtggacg acgagcgatg taagaaaatc 420 atgaagcaat ttatcgatga gcggcctcaa gattggttcg aagatatcgg cgagtga 477 <210> 25 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD10 <400> 25 atggtaaccg gaggtatggc atccaagtgg gaccaaaaag gaatggacat agcatatgaa 60 gaagcagccc tgggctacaa ggagggaggg gttccgattg gcggttgtct tataaataat 120 aaagacggta gtgttcttgg caggggtcac aacatgagat tccaaaaggg gagtgctaca 180 cttcacggcg aaataagcac cttggaaaac tgtggtagac ttgagggaaa agtgtacaag 240 gacacgaccc tttatacgac gctgtcccct tgtgatatgt gcaccggcgc tatcatcatg 300 tatggaatac cacgatgcgt agtgggagag aatgttaatt tcaagagtaa gggcgagaag 360 taccttcaga ccagggggca cgaggtagta gtagttgacg atgagcgatg caagaagatt 420 atgaaacaat tcattgacga gaggccgcag gattggtttg aagacatcgg cgaatag 477 <210> 26 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD11 <400> 26 atggtgacag ggggtatggc aagcaaatgg gatcagaagg gtatggacat cgcatacgag 60 gaggcggcct tgggctataa ggaaggcggc gtacctatag gggggtgcct tattaacaat 120 aaggacggga gcgtcctggg cagaggtcac aacatgaggt tccaaaaggg ttcagcaacc 180 ctgcatggcg aaataagcac ccttgagaat tgtgggaggt tggagggtaa ggtgtacaag 240 gataccacgc tttataccac cttgagtcct tgcgacatgt gcacaggcgc tataatcatg 300 tatggaatac cgcgctgtgt tgtaggagaa aatgtaaact tcaagagtaa aggagaaaaa 360 tacttgcaaa cgcggggaca cgaagtggta gttgtcgatg atgagcggtg caaaaaaatc 420 atgaagcagt tcattgacga acgcccccaa gactggttcg aagacattgg ggagtag 477 <210> 27 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD12 <400> 27 atggtaacgg gtgggatggc tagcaagtgg gaccagaaag gcatggatat agcgtatgaa 60 gaagcggcgt tgggttacaa agagggcggc gttcccatcg gtggctgcct tatcaataat 120 aaagacggct ccgtccttgg ccggggacac aatatgcgct tccaaaaggg cagcgccaca 180 cttcacggtg agatctccac gctggagaat tgtgggcgac ttgaggggaa agtctacaag 240 gacacaactt tgtacacaac acttagcccg tgcgatatgt gtacgggagc cataatcatg 300 tacggcatcc cgcgctgcgt ggtaggagag aacgtaaatt ttaagtcaaa aggagaaaaa 360 tatcttcaga ccaggggcca cgaggtggtt gtcgtggacg acgagagatg taaaaagatc 420 atgaaacagt ttattgatga aagaccacag gattggtttg aggacatcgg tgagtag 477 <210> 28 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD13 <400> 28 atggttacag gaggtatggc ttcaaagtgg gatcaaaaag ggatggacat cgcctatgaa 60 gaagcagcgt tgggatacaa agaagggggg gttcccatag gaggttgcct tatcaacaat 120 aaagatggaa gcgttcttgg gcgagggcac aatatgagat ttcaaaaagg ttcagccact 180 ctccatggag aaatttcaac tctcgaaaac tgtggtcgcc ttgagggcaa ggtttataag 240 gataccaccc tctacactac cctgtcaccc tgcgacatgt gtacaggtgc aattataatg 300 tacggaatcc ctcggtgtgt ggtgggggag aacgtgaatt ttaagtccaa aggtgaaaaa 360 tatctccaaa ctcgcgggca tgaagtcgtc gttgttgatg atgagaggtg taaaaagatt 420 atgaaacaat tcatagacga gaggccacag gattggtttg aggacatagg ggagtag 477 <210> 29 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD14 <400> 29 atggtgactg ggggtatggc ttccaaatgg gatcagaaag gaatggatat agcatacgaa 60 gaagcagctc tcgggtacaa agagggtgga gtacccattg ggggatgcct catcaacaac 120 aaggatggga gtgtccttgg gcgaggtcac aatatgcgat tccagaaggg gagcgcgacg 180 ctccacgggg agataagtac gctggagaac tgcgggaggc ttgaaggcaa ggtctacaaa 240 gataccacac tctacacgac cctcagccct tgcgacatgt gtacgggtgc gatcatcatg 300 tatggaatac cgcgatgcgt agtaggagag aacgtgaact tcaagtccaa aggcgaaaag 360 tatctccaga cgcgcggcca cgaagtggta gtggtagacg acgaaaggtg caagaagata 420 atgaagcagt ttatcgacga gaggcctcag gactggttcg aggatattgg cgagtag 477 <210> 30 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD15 <400> 30 atggttactg gcggcatggc ttctaagtgg gatcagaagg gcatggatat agcctatgaa 60 gaagcagcac tgggatacaa agagggaggg gtaccaattg ggggatgtct gattaataac 120 aaagacggaa gtgtactcgg tcgcgggcat aatatgagat tccaaaaagg ctctgcaacg 180 ttgcacggcg aaatcagcac gctcgaaaat tgcgggaggc tggagggaaa ggtttacaag 240 gataccactc tctataccac actgtcacca tgtgatatgt gtacgggggc tataataatg 300 tatggaatcc cccgctgcgt cgtgggcgaa aacgtcaact ttaagtctaa gggggaaaag 360 tatttgcaaa cgcgcggtca tgaggtcgtt gtagtcgatg acgagagat ca caaaaaaata 420 atgaagcagt ttattgacga gagacctcag gactggttcg aagacatcgg ggagtag 477 <210> 31 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized CD16 <400> 31 atggtgacag gaggaatggc cagcaagtgg gatcagaagg gaatggatat tgcctacgag 60 gaggccgccc tgggctacaa ggaggggggc gtgccaattg gcggatgtct gattaacaac 120 aaggatggga gcgtgctggg aagaggacac aacatgagat ttcagaaggg aagcgcaacc 180 ttgcacggag aaattagcac ccttgagaac tgcgggcggc ttgaaggcaa ggtctataaa 240 gacactacac tttatactac cttgtctcca tgtgatatgt gtacaggcgc cattattatg 300 tacggaattc ctagatgcgt cgtgggagag aacgtgaact ttaagagcaa gggagagaag 360 tacctgcaga caagaggaca cgaggtggtg gtggtggatg atgagagatg taagaagatc 420 atgaagcagt tcatcgatga gaggccccag gattggtttg aggacatcgg cgagtga 477 <210> 32 <211> 1617 <212> DNA <213> Murine leukemia virus <400> 32 atgggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag 60 cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct 120 gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc 180 atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc 240 ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt 300 gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct 360 cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc 420 aaacctaaac ctcaagttct ttctgacagt ggggggccgc tcatcgacct acttacagaa 480 gaccccccgc cttataggga cccaagacca cccccttccg acagggacgg aaatggtgga 540 gaagcgaccc ctgcgggaga ggcaccggac ccctccccaa tggcatctcg cctacgtggg 600 agacgggagc cccctgtggc cgactccact acctcgcagg cattccccct ccgcgcagga 660 ggaaacggac agcttcaata ctggccgttc tcctcttctg acctttacaa ctggaaaaat 720 aataaccctt ctttttctga agatccaggt aaactgacag ctctgatcga gtctgttctc 780 atcacccatc agcccacctg ggacgactgt cagcagctgt tggggactct gctgaccgga 840 gaagaaaaac aacgggtgct cttagaggct agaaaggcgg tgcggggcga tgatgggcgc 900 cccactcaac tgcccaatga agtcgatgcc gcttttcccc tcgagcgccc agactgggat 960 tacaccaccc aggcaggtag gaaccaccta gtccactatc gccagttgct cctagcgggt 1020 ctccaaaacg cgggcagaag ccccaccaat ttggccaagg taaaaggaat aacacaaggg 1080 cccaatgagt ctccctcggc cttcctagag agacttaagg aagcctatcg caggtacact 1140 ccttatgacc ctgaggaccc agggcaagaa actaatgtgt ctatgtcttt catttggcag 1200 tctgccccag acattgggag aaagttagag aggttagaag atttaaaaaa caagacgctt 1260 ggagatttgg ttagagaggc agaaaagatc tttaataaac gagaaacccc ggaagaaaga 1320 gaggaacgta tcaggagaga aacagaggaa aaagaagaac gccgtaggac agaggatgag 1380 cagaaagaga aagaaagaga tcgtaggaga catagagaga tgagcaagct attggccact 1440 gtcgttagtg gacagaaaca ggatagacag ggaggagaac gaaggaggtc ccaactcgat 1500 cgcgaccagt gtgcctactg caaagaaaag gggcactggg ctaaagattg tcccaagaaa 1560 ccacgaggac ctcggggacc aagaccccag acctccctcc tgaccctaga tgactag 1617 <210> 33 <211> 3608 <212> DNA <213> Murine leukemia virus <400> 33 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 60 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 120 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 180 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 240 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 300 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 360 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 420 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 480 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 540 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 600 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 660 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 720 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 780 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 840 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 900 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 960 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 1020 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1080 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1140 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1200 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1260 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1320 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1380 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1440 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1500 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1560 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1620 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1680 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1740 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1800 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1860 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1920 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1980 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 2040 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2100 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2160 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2220 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2280 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2340 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 2400 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 2460 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 2520 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 2580 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 2640 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 2700 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 2760 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 2820 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 2880 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 2940 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 3000 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 3060 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 3120 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 3180 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 3240 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 3300 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 3360 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 3420 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 3480 caccttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 3540 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 3600 ccccctaa 3608 <210> 34 <211> 212 <212> DNA <213> Homo sapiens <400> 34 gggcagagcg cacatcgccc gcagtccccg agaagttggg gggaggggtc ggcaattgat 60 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac agn 212 <210> 35 <211> 2058 <212> DNA <213> Gibbon leukemia virus <400> 35 atggtattgc tgcctgggtc catgcttctc acctcaaacc tgcaccacct tcggcaccag 60 atgagtcctg ggagctggaa aagactgatc atcctcttaa gctgcgtatt cggcggcggc 120 gggacgagtc tgcaaaataa gaacccccac cagcccatga ccctcacttg gcaggtactg 180 tcccaaactg gagacgttgt ctgggataca aaggcagtcc agcccccttg gacttggtgg 240 cccacactta aacctgatgt atgtgccttg gcggctagtc ttgagtcctg ggatatcccg 300 ggaaccgatg tctcgtcctc taaacgagtc agacctccgg actcagacta tactgccgct 360 tataagcaaa tcacctgggg agccataggg tgcagctacc ctcgggctag gactagaatg 420 gcaagctcta ccttctacgt atgtccccgg gatggccgga ccctttcaga agctagaagg 480 tgcggggggc tagaatccct atactgtaaa gaatgggatt gtgagaccac ggggaccggt 540 tattggctat ctaaatcctc aaaagacctc ataactgtaa aatgggacca aaatagcgaa 600 tggactcaaa aatttcaaca gtgtcaccag accggctggt gtaaccccct taaaatagat 660 ttcacagaca aaggaaaatt atccaaggac tggataacgg gaaaaacctg gggattaaga 720 ttctatgtgt ctggacatcc aggcgtacag ttcaccattc gcttaaaaat caccaacatg 780 ccagctgtgg cagtaggtcc tgacctcgtc cttgtggaac aaggacctcc tagaacgtcc 840 ctcgctctcc cacctcctct tcccccaagg gaagcgccac cgccatctct ccccgactct 900 aactccacag ccctggcgac tagtgcacaa actcccacgg tgagaaaaac aattgttacc 960 ctaaacactc cgcctcccac cacaggcgac agactttttg atcttgtgca gggggccttc 1020 ctaaccttaa atgctaccaa cccaggggcc actgagtctt gctggctttg tttggccatg 1080 ggcccccctt attatgaagc aatagcctca tcaggagagg tcgcctactc caccgacctt 1140 gaccggtgcc gctgggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agactagaga tttctgtatc 1380 caggtccagc tgattcctcg catctattac tatcctgaag aagttttgtt acaggcctat 1440 gacaattctc accccaggac taaaagagag gctgtctcac ttaccctagc tgttttactg 1500 gggttgggaa tcacggcggg aataggtact ggttcaactg ccttaattaa aggacctata 1560 gacctccagc aaggcctgac aagcctccag atcgccatag atgctgacct ccgggccctc 1620 caagactcag tcagcaagtt agaggactca ctgacttccc tgtccgaggt agtgctccaa 1680 aataggagag gccttgactt gctgtttcta aaagaaggtg gcctctgtgc ggccctaaag 1740 gaagagtgct gtttttacat agaccactca ggtgcagtac gggactccat gaaaaaactc 1800 aaagaaaaac tggataaaag acagttagag cgccagaaaa gccaaaactg gtatgaagga 1860 tggttcaata actccccttg gttcactacc ctgctatcaa ccatcgctgg gcccctatta 1920 ctcctccttc tgttgctcat cctcgggcca tgcatcatca ataagttagt tcaattcatc 1980 aatgatagga taagtgcagt taaaattctg gtccttagac aaaaatatca ggccctagag 2040 aacgaaggta acctttaa 2058 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD-Not1-F primer <400> 36 cggcggccgc atggtgacag ggggaatggc 30 <210> 37 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CD-Pme1-R primer <400> 37 cggtttaaac ctactcacca atatcttcaa a 31 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD2-Not1-F primer <400> 38 cggcggccgc atggtgaccg gcggcatggc 30 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD2-Pme1-R primer <400> 39 cggtttaaac ttactcgccg atatcctcga 30 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD6-Not1-F primer <400> 40 cggcggccgc atggttactg gagggatggc 30 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD6-Pme1-R primer <400> 41 cggtttaaac tcattcgcca atatcctcga 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD7-Not1-F primer <400> 42 cggcggccgc atggtaactg gtggcatggc 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD7-Pme1-R primer <400> 43 cggtttaaac ctactctccg atatcctcaa 30 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CD8-Not1-F primer <400> 44 cggcggccgc atggttactg ggggaatgg 29 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CD8-Pme1-R primer <400> 45 cggtttaaac ttattccccg atgtcttcga 30

Claims (19)

MuLV(MurineLeukemia virus)의 Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제(cytosine deaminase) 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제(thymidine kinase) 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템으로서,
상기 프로모터는 서열번호 34의 염기서열을 포함하는 EF1α 프로모터이고,
상기 시토신 디아미나아제 유전자는 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21, 서열번호 22, 서열번호 23, 또는 서열번호 24의 염기서열을 포함하는 것인, 복제가능 레트로바이러스 벡터 시스템.
A first recombinant expression vector comprising the Gag-Pol gene, the promoter and the cytosine deaminase gene of MuLV (Murine Leukemia virus); And a second recombinant expression vector comprising an Env gene of the virus, a promoter and a thymidine kinase gene,
Wherein the promoter is an EFl [alpha] promoter comprising the nucleotide sequence of SEQ ID NO: 34,
Wherein the cytosine deaminase gene comprises the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23 or 24, Retroviral vector system.
MuLV의Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터; 및 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 포함하는 복제가능 레트로바이러스 벡터 시스템으로서,
상기 프로모터는 서열번호 34의 염기서열을 포함하는 EF1α 프로모터이고,
상기 시토신 디아미나아제 유전자는 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21, 서열번호 22, 서열번호 23, 또는 서열번호 24의 염기서열을 포함하는 것인, 복제가능 레트로바이러스 벡터 시스템.
A first recombinant expression vector comprising MuLV's Gag-Pol gene, a promoter and a thymidine kinase gene; And a second recombinant expression vector comprising an Env gene of the virus, a promoter and a cytosine deaminase gene,
Wherein the promoter is an EFl [alpha] promoter comprising the nucleotide sequence of SEQ ID NO: 34,
Wherein the cytosine deaminase gene comprises the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23 or 24, Retroviral vector system.
삭제delete 삭제delete 제 1항 또는 제 2항에 있어서, 상기 바이러스의 Env 유전자는 긴팔원숭이 백혈병 바이러스(Gibbon ape Leukemia virus; GaLV), 암포트로픽 쥐 백혈병 바이러스(amphotropic MuLV), 친이종 쥐 백혈병 바이러스(xenotropic MuLV), 고양이 내재성 바이러스(RD114), 수포성 구내염바이러스(vesicular stomatitis virus; VSV) 및 홍역바이러스(measles virus; MV)의 Env 유전자로 이루어진 군으로부터 유래되는 어느 하나인, 복제가능 레트로바이러스 벡터 시스템.
The method according to claim 1 or 2, wherein the Env gene of the virus is selected from the group consisting of Gibbon ape Leukemia virus (GaLV), amphotropic MuLV, xenotropic MuLV, Wherein the vector is derived from the group consisting of the Env gene of endogenous virus (RD114), vesicular stomatitis virus (VSV) and measles virus (MV).
제 5항에 있어서, GaLV의 Env 유전자가 서열번호 35의 염기서열을 갖는 폴리뉴클레오티드인, 복제가능 레트로바이러스 벡터 시스템.
6. The replicable retrovirus vector system according to claim 5, wherein the Env gene of GaLV is a polynucleotide having the nucleotide sequence of SEQ ID NO: 35.
제 1항 또는 제 2항에 있어서, 상기 티미딘 키나아제 유전자가 서열번호 15의 염기서열을 갖는 폴리뉴클레오티드인, 복제가능 레트로바이러스 벡터 시스템.
3. A replicable retroviral vector system according to claim 1 or 2, wherein the thymidine kinase gene is a polynucleotide having the nucleotide sequence of SEQ ID NO:
삭제delete 삭제delete 제 1항 또는 제 2항에 있어서, 상기 티미딘 키나아제 또는 시토신 디아미나아제 유전자가 전구체 약물을 활성화시키는, 복제가능 레트로바이러스 벡터 시스템.
3. A replicable retroviral vector system according to claim 1 or 2, wherein said thymidine kinase or cytosine deaminase gene activates a precursor drug.
제 10항에 있어서, 상기 전구체 약물이 간사이클로비르 및 5-플루오로사이토신으로 구성된 군으로부터 선택되는 어느 하나 이상인, 복제가능 레트로바이러스 벡터 시스템.
11. A replicable retroviral vector system according to claim 10, wherein the precursor drug is at least one selected from the group consisting of hepcyclovir and 5-fluorocytosine.
제 1항 또는 제 2항에 있어서, MuLV의 Gag-Pol 유전자가 서열번호 32 및 서열번호 33의 염기서열이 융합된 염기서열을 갖는 폴리뉴클레오티드인, 복제가능 레트로바이러스 벡터 시스템.
3. The replication-competent retrovirus vector system according to claim 1 or 2, wherein the Gag-Pol gene of MuLV is a polynucleotide having a nucleotide sequence fused with the nucleotide sequence of SEQ ID NO: 32 and SEQ ID NO: 33.
제 1항 또는 제 2항의 벡터 시스템을 포함하는 재조합 레트로바이러스.
A recombinant retrovirus comprising the vector system of any one of claims 1 or 2.
제 13항의 재조합 레트로바이러스로 형질감염된 숙주 세포.
15. A host cell transfected with the recombinant retrovirus of claim 13.
제 13항의 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물.
A pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus of claim 13 as an active ingredient.
제 15항에 있어서, 상기 암이 점액상 세포 암종, 둥근 세포 암종, 국소적 진행 종양, 전이성 암, 어윙(Ewing) 육종, 암전이, 림프성 전이, 편평상피 세포 암종, 식도 편평상피 세포 암종, 경구 암종, 다발성골수종, 급성 림프구성 백혈병, 급성 비림프구성 백혈병, 만성 림프구 백혈병, 만성 골수구 백혈병, 모양 세포성 백혈병, 유출 림프종 (체강계 림프종), 흉선 림프종 폐암, 소세포 폐암종, 피부 T 세포 림프종, 호지킨 림프종, 비-호지킨성 림프종, 부신 피질 암, ACTH-생성 종양, 비소세포 폐암, 유방암, 소세포 암종, 도관 암종, 위암, 결장암, 결장직장암, 결장직장 종양 형성과 관련된 용종, 췌장암, 간암, 방광암, 1차 표면 방광 종양, 방광의 침습성 전이 세포 방광암종, 근 침윤성 방광암, 전립선암, 대장암, 신장암, 간암, 식도암, 난소 암종, 자궁경부암, 자궁내막암, 융모암, 난소암, 원발성 복막상피 신생물, 자궁 경관 암종, 질암, 외음부암, 자궁암, 난포 중 고형 종양, 고환암, 음경암, 신장 세포 암종, 뇌암, 두경부암, 구경부암, 신경아세포종, 뇌간 신경교종, 신경교종, 중추신경계 중 전이성 종양 세포 침윤, 골종, 골육종, 악성 흑색종, 인간 피부 케라티노사이트의 종양 진행, 편평상피 세포 암종, 갑상선 암, 망막아종, 신경아세포종, 중피종, 빌름스 종양, 담낭암, 영양아세포 종양, 혈관주위세포종, 또는 카포시 육종인, 암의 예방 또는 치료용 약학 조성물.
16. The method of claim 15, wherein said cancer is selected from the group consisting of point cloud squamous cell carcinoma, round cell carcinoma, local advanced tumor, metastatic cancer, Ewing sarcoma, metastasis, lymphatic metastasis, squamous cell carcinoma, esophageal squamous cell carcinoma, The present invention relates to a method for treating a disease selected from the group consisting of acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, shape cell leukemia, leukemic lymphoma (thymic lymphoma), thymoma lymphoma lung cancer, Lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, adrenocortical cancer, ACTH-producing tumor, non-small cell lung cancer, breast cancer, small cell carcinoma, ductal carcinoma, stomach cancer, colon cancer, colorectal cancer, , Cancer of the liver, bladder cancer, primary surface bladder tumor, invasive metastatic bladder cancer of the bladder, muscular invasive bladder cancer, prostate cancer, colorectal cancer, kidney cancer, liver cancer, esophageal cancer, ovarian cancer, cervical cancer, uterine cancer The present invention relates to a method for treating a cancer selected from the group consisting of cancer, choriocarcinoma, ovarian cancer, primary peritoneal epithelial neoplasm, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, solid tumor in follicular cancer, testicular cancer, Neuroblastoma, metastatic tumor cell invasion in the central nervous system, osteoma, osteosarcoma, malignant melanoma, tumor progression of human skin keratinocyte, squamous cell carcinoma, thyroid cancer, retinoblastoma, neuroblastoma, mesothelioma, Tumor, gallbladder cancer, nasocomial tumor, angioplasty, or Kaposi's sarcoma.
제 13항의 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물.
14. A gene transfer composition for treating cancer comprising the recombinant retrovirus of claim 13.
1) MuLV의Gag-Pol 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및
2) 바이러스의 Env 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법으로서,
상기 프로모터는 서열번호 34의 염기서열을 포함하는 EF1α 프로모터이고,
상기 시토신 디아미나아제 유전자는 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21, 서열번호 22, 서열번호 23, 또는 서열번호 24의 염기서열을 포함하는 것인, 복제가능 레트로바이러스 벡터 시스템의 제조방법.
1) preparing a first recombinant expression vector comprising the Gag-Pol gene, the promoter and the cytosine deaminase gene of MuLV; And
2) preparing a second recombinant expression vector comprising an Env gene of the virus, a promoter and a thymidine kinase gene,
Wherein the promoter is an EFl [alpha] promoter comprising the nucleotide sequence of SEQ ID NO: 34,
Wherein the cytosine deaminase gene comprises the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23 or 24, A method for producing a retroviral vector system.
1) MuLV의Gag-Pol 유전자, 프로모터 및 티미딘 키나아제 유전자를 포함하는 제 1 재조합 발현벡터를 제조하는 단계; 및
2) 바이러스의 Env 유전자, 프로모터 및 시토신 디아미나아제 유전자를 포함하는 제 2 재조합 발현벡터를 제조하는 단계를 포함하는 복제가능 레트로바이러스 벡터 시스템의 제조방법으로서,
상기 프로모터는 서열번호 34의 염기서열을 포함하는 EF1α 프로모터이고,
상기 시토신 디아미나아제 유전자는 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21, 서열번호 22, 서열번호 23, 또는 서열번호 24의 염기서열을 포함하는 것인, 복제가능 레트로바이러스 벡터 시스템의 제조방법.








1) preparing a first recombinant expression vector comprising the Gag-Pol gene of MuLV, a promoter and a thymidine kinase gene; And
2) preparing a second recombinant expression vector comprising the Env gene of the virus, a promoter and a cytosine deaminase gene,
Wherein the promoter is an EFl [alpha] promoter comprising the nucleotide sequence of SEQ ID NO: 34,
Wherein the cytosine deaminase gene comprises the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23 or 24, A method for producing a retroviral vector system.








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