공개특허 (19) 대한민국특허청 (KR) (12) 공개특허공보 (A) (11) 공개번호 (43) 공개일자 2010년02월03일 (51) Int. Cl. C12N 7/02 ( ) C12N 7/00 (20

Transcription

1 (19) 대한민국특허청 (KR) (12) 공개특허공보 (A) (11) 공개번호 (43) 공개일자 2010년02월03일 (51) Int. Cl. C12N 7/02 ( ) C12N 7/00 ( ) C12N 15/38 ( ) (21) 출원번호 (22) 출원일자 2008 년 07 월 24 일 심사청구일자 전체청구항수 : 총 4 항 2008 년 07 월 24 일 (71) 출원인 차의과학대학교산학협력단 포천시동교동 차의과학대학교내 (72) 발명자 황동연 경기도성남시분당구서현동효자촌동아아파트 (74) 대리인 오국진 (54) 2 종이상의표적유전자를동시에함유하는바이러스의제조방법 (57) 요약 본발명은 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를, fhsv pac 및 pebhicp27 과함께수용세포에공동 - 형질도입시켜패키징 (packaging) 시키는단계를포함하는, 2 종이상의표적유전자를동시에함유하는바이러스의제조방법을제공한다. 대표도 - 도 3-1 -

2 이발명을지원한국가연구개발사업 과제고유번호 SC-5170 부처명 교육과학기술부 연구사업명 세포응용연구사업 연구과제명 안전하고효율적으로역분화, stemness 유지및분화시킬수있는방법들의개발및 GPCR 들을중심으로한기작연구 주관기관 세포응용연구사업단 연구기간 2008년 04월 01일 ~ 2009년 03월 31일 - 2 -

3 특허청구의범위청구항 1 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를, fhsv pac 및 pebhicp27과함께수용세포에공동-형질도입시켜패키징 (packaging) 시키는단계를포함하는, 2 종이상의표적유전자를동시에함유하는바이러스의제조방법. 청구항 2 제1항에있어서, 상기헤르페스심플렉스바이러스앰플리콘플라스미드가서열번호 1의염기서열을갖는것을특징으로하는제조방법. 청구항 3 제1항에있어서, 상기표적유전자가 2 종이상의역분화유도인자를코딩하는유전자인것을특징으로하는제조방법. 청구항 4 제3항에있어서, 상기표적유전자가서열번호 2 내지 7의유전자로부터 2 종이상선택되는것을특징으로하는제조방법. 명세서 발명의상세한설명 [0001] 기술분야본발명은 2 종이상의표적유전자를동시에함유하는바이러스의제조방법에관한것으로, 더욱상세하게는 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를수용세포에공동-형질도입시켜패키징시키는단계를포함하는 2 종이상의표적유전자를동시에함유하는바이러스의제조방법에관한것이다. [0002] [0003] 배경기술헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid, HSV amplicon plasmid) 는 DNA 복제 origin (oris) 과 virion DNA cleavage/packaging (pac) 에필요한최소한의바이러스 DNA 요소 (viral DNA element) 와박테리아에서증식할때필요한 DNA 요소 (bacterial DNA element) 만을가지고있다 (Spaete, R.R., and Frenkel, N. (1982). The herpes simplex virus amplicon: a new eukaryotic defective-virus cloning-amplifying vector. Cell 30, ). 크기제한이있는다른바이러스벡터와는달리 HSV amplicon 바이러스 vector는 150kb 의 DNA 까지도바이러스캡시드 (viral capsid) 안에패키징 (packaging) 시킬수있어 large transgene capacity를가지고있으며, 플라스미드내부에바이러스단백질을만들수있는유전자가없기때문에 ("gutted vector") 숙주세포에독성을나타내지않는장점이있다. 또한여러종류의세포에유전자를전달할수있는능력도있다. 최근에는헤르페스심플렉스바이러스제작시조금씩오염되어세포독성을유발할가능성이있었던 defective Herpes simplex 나 adeno helper-virus 없이 HSV amplicon plasmid vector를바이러스캡시드에패키징할수있는시스템즉, 헬퍼바이러스의오염가능성을없앤시스템이개발된바있다 (raefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, A.I. (1996). Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. Journal of virology 70, ; Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X.O., and Chiocca, E.A. (2001). Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome. Mol Ther 3, ; Saeki, Y., Ichikawa, T., Saeki, A., Chiocca, E.A., Tobler, K., Ackermann, M., Breakefield, X.O., and Fraefel, C. (1998). Herpes simplex virus type - 3 -

4 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replicationcompetent virus progeny and packaging of amplicon vectors. Human gene therapy 9, ; Stavropoulos, T.A., and Strathdee, C.A. (1998). An enhanced packaging system for helper-dependent herpes simplex virus vectors. Journal of virology 72, ). [0004] 한편, 종래의바이러스벡터시스템을이용하여두개이상의유전자발현유닛 (gene expression unit) 을한개의바이러스 [ 즉, 바이러스입자 (viral particle)] 내에도입하고자할경우, 하나의 HSV amplicon plasmid 안에두개이상의유전자들을발현하는발현유닛 ( 프로모터-1개유전자-polyA 의발현카세트가 2개이상혹은 IRES를이용하여한개의프러모터에의해두개의유전자들이발현되도록만든카세트 ) 을별도로제작하여바이러스에도입하여야한다. 그러나이경우, 플라스미드즉발현유닛의크기가커지면접합 (ligation) 이어려워져제작이곤란할뿐만아니라, 여러조합으로유전자를전달할때그유전자조합에따라각조합의유전자구조체를각각별도로제작하여야하는문제가있다. 발명의내용 [0005] [0006] 해결하고자하는과제본발명자들은 HSV amplicon plasmid vector를사용하여 2 이상의유전자를하나의세포에동시에전달할수있는방법을개발하고자다양한연구를수행하였다. 그결과, 각각의표적유전자를발현하는앰플리콘플라스미드를두개이상섞어이들을헬퍼 BAC DNA 와 pebhicp27 플라스미드와함께수용세포 (permissive cell line) 에동시-형질도입 (co-transfection) 시켜패키징시켰을때, 놀랍게도각각의다른앰플리콘플라스미드상에존재하는표적유전자들이동시에하나의바이러스내에패키징될수있다는것을발견하였다. 이는, 두개이상의앰플리콘플라스미드들을패키징하여바이러스를제조하더라도 rolling-circle mechanism에의해서한종류의앰플리콘플라스미드만이연쇄상으로 (concatenate) 도입되게된다는 ( 도 2 참조 ) 기존의학설만으로는설명할수없는새로운발견이다. 따라서, 본발명은각각의표적유전자를포함하는앰플리콘플라스미드들을수용세포내에동시-형질도입시켜패키징시키는단계를포함하는, 즉 2 종이상의표적유전자를동시에함유하는바이러스를쉽게제조하는방법을제공하는것을목적으로한다. [0007] [0008] 과제해결수단본발명의일태양에따라, 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를, fhsv pac 및 pebhicp27과함께수용세포에공동-형질도입시켜패키징 (packaging) 시키는단계를포함하는, 2 종이상의표적유전자를동시에함유하는바이러스를간편하게제조할수있는방법이제공된다. 상기헤르페스심플렉스바이러스앰플리콘플라스미드는서열번호 1의염기서열을가질수있으며, 상기표적유전자들은 2 종이상의역분화유도유전자들일수있고, 예를들어서열번호 2 내지 7의유전자로부터 2 종이상선택될수있다. [0009] 효과본발명에의해, 표적유전자가재조합된재조합플라스미드를 fhsv pac 및 pebhicp27와함께수용세포에공동-형질도입시켜패키징함에있어서, 상기재조합플라스미드가 2 종이상일경우즉 2 종이상의재조합플라스미드존재하에서패키징을수행할경우, 각각의재조합플라스미드에함유된표적유전자들이동시에바이러스에도입된다는것이발견되었다. 이는, 두개이상의앰플리콘플라스미드들을패키징하여바이러스를제조하더라도 rolling-circle mechanism 기작에의해서한종류의앰플리콘플라스미드만이연쇄상으로 (concatenate) 바이러스캡시드안에도입되게된다는기존의설 ( 도 2 참조 ) 을감안할때, 매우놀라운것이다. 따라서, 본발명의바이러스제조방법은 2 종이상의역분화유도인자를코딩하는유전자들이도입된바이러스제조에유용하게사용될수있다. [0010] 발명의실시를위한구체적인내용 본발명은 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를, fhsv pac

5 및 pebhicp27 과함께수용세포에공동 - 형질도입시켜패키징 (packaging) 시키는단계를포함하는, 2 종이상의 표적유전자를동시에함유하는헤르페스심플렉스바이러스앰플리콘바이러스의제조방법을제공한다. [0011] [0012] [0013] 본발명에따른제조방법은 2 종이상의표적유전자를동시에포함하는헤르페스심플렉스바이러스를제조하는데있어서, Saeki et al. 이개발한무-헬퍼바이러스시스템 (helper virus-free system) 을사용한다 ( 도 1 참조 ). 상기시스템에서는 pac (packaging) 시그날을제외한전 HSV-1 게놈이 BAC (bacterial artificial chromosome) 에넣어져있고 (fhsv pac 27 0+), 안전성을높이기위해 ICP27 유전자를 BAC DNA에서제거하여또다른플라스미드인 pebhicp27로서제공해준다. 이세종류의플라스미드를베로 (vero) 2-2세포등의수용세포 (permissive cell line) 에공동-형질도입 (co-transfection) 해주면 BAC DNA 와 pebhicp27 플라스미드로부터생산되는 HSV 입자생성에필요한유전자산물로인하여 HSV particle의구조가만들어지고, 그내부로앰플리콘 DNA 가 HSV 캡시드 (capsid) 의용량 (capacity) 인 150 kb 가될때까지 rolling circle mechanism에의해연쇄상으로 (concatenate) 이어져캡시드내부를채우게된다 ( 도 2 참조 ). 본발명의제조방법은상기 Saeki et al. 등의무-헬퍼바이러스시스템에있어서, 2 종이상의앰플리콘 DNA 즉, 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드 (Herpes simplex virus amplicon plasmid) 에재조합시켜얻어진 2 종이상의재조합플라스미드를공동-형질도입시킬경우, 2 종이상의표적유전자를동시에함유하는바이러스가제조된다는새로운발견에기초한다. 본발명의제조방법에있어서, 상기표적유전자는특별히제한된것이아니며, 바이러스를매개체로통하여숙주세포에도입하고자하는모든유전자를포함한다. 바람직하게는분화된세포의재프로그램화에관여하는역분화유도인자 (reprogramming-inducing factor) 를코딩하는유전자가포함된다. 상기역분화유도인자는기존에밝혀져있는역분화인자즉, Sox2, Oct3/4, Nanog, Klf4, Lin28, 또는 Myc를코딩하는유전자일수있으며, 이들은각각서열번호 2 내지 7의염기서열을갖는다. 당업자는상기한 Sox2, Oct3/4, Nanog, Klf4, Lin28, c-myc 뿐만아니라, 역분화유도에관여하는것으로알려지거나알려질다양한인자들이본발명에따른제조방법에제한없이적용될수있음을인식할것이다. 상기재조합플라스미드는 2 종이상의표적유전자를각각별도의헤르페스심플렉스바이러스앰플리콘플라스미드, 바람직하게는더서열번호 1의염기서열을갖는헤르페스심플렉스바이러스앰플리콘플라스미드에재조합시켜얻어질수있다. 상기재조합은 NheI, NotI, BamHI, BglII, XbaI 및 EcoRI 등의적절한제한효소를사용하여통상의유전공학적방법으로표적유전자를클로닝하여수행할수있다. [0014] [0015] 이하, 본발명을실시예를통하여더욱상세히설명한다. 그러나이들실시 으로, 본발명의범위가이들실시예에한정되는것은아니다. 실시예 예는본발명을예시하기위한것 [0016] [0017] [0018] [0019] 모델유전자로서 GFP(green fluorescence protein) 을코딩하는유전자 ( 서열번호 8) 및 DsRed2(red fluorescence protein) 를코딩하는유전자 ( 서열번호 9) 를선정하였다. 헤르페스심플레스앰플리콘플라스미드인 phgcx(saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X.O., and Chiocca, E.A. (2001). Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome. Mol Ther 3, ) 내에는 GFP를코딩하는유전자가헤르페스심플렉스바이러스 immediate early 프로모터 (IE4/5 promoter) 뒤에이미붙어있기때문에그자체로 GFP를발현하는재조합플라스미드로사용하였다. phgcx를 NruI과 NotI 제한효소의이중처리로 GFP를잘라낸뒤 CIP (calf intestinal alkaline phosphatase) 처리를하여벡터를제작하였다. 그다음 pdsred2 (Invitrogen) 상의 DsRed2 유전자를 SmaI 과 NotI 제한효소의이중처리로잘라내어만들어진벡터와라이게이션 (ligation) 시켜 DsRed2 를코딩하는유전자가헤르페스심플렉스바이러스 immediate early 프러모터 (IE4/5 promoter) 뒤에위치하는재조합플라스미드 (phsvamp-dsred2) 를제작하였다. 상기에서얻어진두개의플라스미드들을혼합하여 Saeki, Y. 등의방법 (Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X.O., and Chiocca, E.A. (2001). Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome. Mol Ther 3, ) 을변형하여, 베로 2-2 세포 (vero 2-2 cell: African green monkey kidney cell; from Dr. Rozanne Sandri-Goldin,. University of California, Irvine, CA, USA) 에공동-형질도입시켜패키징을수행하였다. 즉, 60 mm 세포배양용기에 phgcx(0.116 ug), phsvamp-dsred2(1.392 ug), pebhicp27(0.07 ug) 및 - 5 -

6 helper-bac DNA (fhsv PAC 27 0+; ug) 를섞어서 FugeneHD 8:2 로 Opti-MEM으로같이섞은후상온에서 15 분후베로 2-2 세포위에골고루떨어뜨려주었다. 37 에서 4시간동안세포배양한뒤 3 ml의 6% FBS로보충된 DMEM을넣어주고 60시간동안 HSV 앰플리콘바이러스가만들어지도록하였다. 세포 scraper로세포들을긁어모은뒤드라이아이스 / 에탄올수욕 (bath) 및 37 의수욕 (water bath) 에서 3번얼렸다녹였다하면서, 세포내에있는 HSV 앰플리콘바이러스들이배지로방출되도록하였다. 배양액을 2000 x g로원심분리하여세포침전물을제외한상층액을얻었다. 이렇게배지내에얻어진바이러스를인간 glioblastoma 세포주인 G16-9 세포 ( 인간글리오블라스토마, glioblastoma; Gli36 derived cell line tnat express HSV-1 VP 16 constitutuvely; widely available from many labs) 에감염시킨후 2-3일후에형광단백질들 (GFP 및 DsRed2) 을발현하는세포들의수를측정하였다. 그결과는도 3과같다. 도 3에서알수있는바와같이, 본발명에따른제조방법으로제조된바이러스로감염된세포의약 80% 정도가 GFP와 DsRed2 를하나의세포안에공통적으로발현함을알수있다. [0020] [0021] 비교예실시예 1에서얻어진 GFP를발현하는재조합플라스미드및 DsRed2를발현하는재조합플라스미드를각각별도로실시예 1과동일한방법으로패키징하고, 얻어진바이러스각각을혼합하여 G16-9 세포에감염시켜형광단백질들 (GFP 및 DsRed2) 의발현을측정하였다. 그결과는도 4와같다. 도 4에서알수있는바와같이, 약 2.6% 정도의세포들만이 GFP와 DsRed2를한세포안에공통적으로발현됨을알수있다. [0022] [0023] [0024] [0025] 도면의간단한설명도 1은 fhsv pac 및 pebhicp27 시스템을이용한무-헬퍼바이러스시스템을통해앰플리콘벡터를제작하는과정을나타내는모식도이다. 도 2는종래의패키징방법으로서앰플리콘플라스미드가 rolling circle mechanism 에의해연쇄상으로 (concatenate) 여러카피가이어져앰플리콘벡터가제작되는과정을나타내는모식도이다. 도 3 은각각 GFP와 DsRed2를발현하는유닛을가진두종류의다른앰플리콘플라스미드를 BAC DNA 와 pebhicp27 와함께공동-형질도입 (co-transfection) 하여얻어진바이러스를 G16-9 세포에감염시켜 GFP 및 DsRed2를공동발현하는세포가전체세포에서차지하는비율을측정한결과를나타낸다 (*P= ; #P= ). 도 4는 GFP와 DsRed2를함유하는앰플리콘플라스미드로부터각각별도로바이러스를제작하고, 얻어진 GFP를발현하는바이러스와 DsRed2를발현하는바이러스를함께섞어서 G16-9 세포에감염시켜 GFP 및 / 또는 DsRed2를발현하는세포의비율을측정한결과를나타낸다 (*P= ; #P= 0.02)

7 도면 도면 1 도면 2-7 -

8 도면 3 도면 4 서열목록 <110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Method for producing viruses co-packaged with two or more target genes - 8 -

9 <130> PN0181 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 6723 <212> DNA <213> herpes simplex virus 7 <400> 1 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 60 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 120 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 180 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata 240 cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg gtaccgagct 300 cggatccact agtccagtgt ggtggaattc tgcagatatc cagcacagtg gcggccgctc 360 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 420 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 480 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 540 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcgttaat 600 taaaattcca gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt caaaaataat 660 aataaccggg caggccatgt ctgcccgtat ttcgcgtaag gaaatccatt atgtactatt 720 taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt tttatcatgg

10 gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc agcaaaagtg 840 atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa ccgctgtttg 900 gtctgctttc tgacaaactc ggaacttgtt tattgcagct tataatggtt acaaataaag 960 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttg 1020 tccaaactca tcaatgtatc ttatcatgtc tggatctctg acctgagatt ggcggcactg 1080 aggtagagat gcccgaaccc ccccgaggga gcgcgggacg cgccggggag ggctggggcc 1140 ggggagggct ggggccgggg agggctgggg ccggggaggg ctggggccgg ggagggctgg 1200 ggccggggag ggctggggcc ggggagggct ggggctgggg agggctgggg ctggggaggg 1260 ggcggtggtg tgtagcagga gcggtgtgtt gcgccggggt acgtctggag gagcgggagg 1320 tgcgcggtga cgtgtggatg aggaacagga gttgttgcgc ggtgagttgt cgctgtgagt 1380 tgtgttgttg ggcaggtgtg gtggatgacg tgacgtgtga cgtgcggagt gcgccgtgct 1440 ctgttggttt cacctgtggc agcccgggcc ccccgcgggc gcgcgcgcgc gcaaaaaagg 1500 cgggcggcgg tccgggcggc gtgcgcgcgc gcggcgggcg tggggggcgg ggccgcggga 1560 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1620 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1680 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1740 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ccagacgccg aaaacgggcc 1800 ccccccaaaa cacacccccc gggggtcgcg cgcggccctt taaagcggtg gcggcgggca 1860 gcccgggccc cccgcggccg agactagcga gttagacagg caagcactac tcgcctctgc 1920 acgcacatgc ttgcctgtca aactctacca ccccggcacg ctctctgtct ccatggcccg

11 ccgccgccgc catcgcggcc cccgccgccc ccggccgccc gggcccacgg gcgccgtccc 2040 aaccgcacag tcccaggtaa cgagctcgaa ttaattcttg aagacgaaag ggcctcgtga 2100 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagggt cgacccaggt 2160 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 2220 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 2280 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 2340 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 2400 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 2460 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 2520 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 2580 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 2640 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 2700 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 2760 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 2820 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 2880 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 2940 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 3000 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 3060 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata

12 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 3180 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 3240 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 3300 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 3360 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 3420 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 3480 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 3540 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 3600 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 3660 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 3720 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 3780 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 3840 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 3900 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 3960 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 4020 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 4080 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 4140 ataggtcgac ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 4200 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 4260 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg

13 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 4380 cagccggcca ttatgcacga ccccgccccg acgccggcac gccgggggcc cgtggccgcg 4440 gcccgttggt cgaacccccg gccccgccca tccgcgccat ctgccatggg cggggcgcga 4500 gggcgggtgg gtccgcgccc cgccccgcat ggcatctcat taccgcccga tccggcggtt 4560 tccgcttccg ttccgcatgc taacgaggaa cgggcagggg gcggggcccg ggccccgact 4620 tcccggttcg gcggtaatga gatacgagcc ccgcgcgccc gttggccgtc cccgggcccc 4680 ccggtcccgc ccgccggacg ccgggaccaa cgggacggcg ggcggcccaa gggccgcccg 4740 ccttgccgcc cccccattgg ccggcgggcg ggaccgcccc aagggggcgg ggccgccggg 4800 taaaagaagt gagaacgcga agcgttcgca cttcgtccca atatatatat attattaggg 4860 cgaagtgcga gcactggcgc cgtgcccgac tccgcgccgg ccccgggggc gggcccgggc 4920 ggcggggggc gggtctctcc ggcgcacata aaggcccggc gcgaccgacg cccgcagacg 4980 gcgccggcca cgaacgacgg gagcggctgc ggagcacgcg gaccgggagc gggagtcgca 5040 gagggccgtc ggagcggacg gcgtcggcat cgcgacgccc cggctcggga tcgggatcgc 5100 atcggaaagg gacacgcgga cgcggggggg aaagacccgc ccaccccacc cacgaaacac 5160 aggggacgca ccccgggggc ctccgacgac agaaacccac cggtccgcct tttttgcacg 5220 ggtaagcacc ttgggtgggc ggaggagggg gggacgcggg ggtggaggag gggggacgcg 5280 ggggcggagg aggggggacg cgggggcgga ggagggggga cgcgggggcg gaggaggggg 5340 gacgcggggg cggaggaggg ggctcacccg cgttcgtgcc ttcccgcagg aggaacgtcc 5400 tcgtcgataa gctgatccat cgccaccatg gtgagcaagg gcgaggagct gttcaccggg

14 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 5520 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 5580 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 5640 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 5700 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 5760 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 5820 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 5880 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 5940 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 6000 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 6060 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 6120 ctcggcatgg acgagctgta caagtaaagc ggccaacttg tttattgcag cttataatgg 6180 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 6240 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg gtttgaagat 6300 cttccgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 6360 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 6420 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 6480 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggactatt 6540 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 6600 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg

15 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 6720 ttt 6723 <210> 2 <211> 954 <212> DNA <213> Homo sapiens <400> 2 atgtacaaca tgatggagac ggagctgaag ccgccgggcc cgcagcaaac ttcggggggc 60 ggcggcggca actccaccgc ggcggcggcc ggcggcaacc agaaaaacag cccggaccgc 120 gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc 180 caggagaacc ccaagatgca caactcggag atcagcaagc gcctgggcgc cgagtggaaa 240 cttttgtcgg agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg 300 cacatgaagg agcacccgga ttataaatac cggccccggc ggaaaaccaa gacgctcatg 360 aagaaggata agtacacgct gcccggcggg ctgctggccc ccggcggcaa tagcatggcg 420 agcggggtcg gggtgggcgc cggcctgggc gcgggcgtga accagcgcat ggacagttac 480 gcgcacatga acggctggag caacggcagc tacagcatga tgcaggacca gctgggctac 540 ccgcagcacc cgggcctcaa tgcgcacggc gcagcgcaga tgcagcccat gcaccgctac 600 gacgtgagcg ccctgcagta caactccatg accagctcgc agacctacat gaacggctcg 660 cccacctaca gcatgtccta ctcgcagcag ggcacccctg gcatggctct tggctccatg 720 ggttcggtgg tcaagtccga ggccagctcc agcccccctg tggttacctc ttcctcccac 780 tccagggcgc cctgccaggc cggggacctc cgggacatga tcagcatgta tctccccggc

16 gccgaggtgc cggaacccgc cgcccccagc agacttcaca tgtcccagca ctaccagagc 900 ggcccggtgc ccggcacggc cattaacggc acactgcccc tctcacacat gtga 954 <210> 3 <211> 1083 <212> DNA <213> Homo sapiens <400> 3 atggcgggac acctggcttc ggatttcgcc ttctcgcccc ctccaggtgg tggaggtgat 60 gggccagggg ggccggagcc gggctgggtt gatcctcgga cctggctaag cttccaaggc 120 cctcctggag ggccaggaat cgggccgggg gttgggccag gctctgaggt gtgggggatt 180 cccccatgcc ccccgccgta tgagttctgt ggggggatgg cgtactgtgg gccccaggtt 240 ggagtggggc tagtgcccca aggcggcttg gagacctctc agcctgaggg cgaagcagga 300 gtcggggtgg agagcaactc cgatggggcc tccccggagc cctgcaccgt cacccctggt 360 gccgtgaagc tggagaagga gaagctggag caaaacccgg aggagtccca ggacatcaaa 420 gctctgcaga aagaactcga gcaatttgcc aagctcctga agcagaagag gatcaccctg 480 ggatatacac aggccgatgt ggggctcacc ctgggggttc tatttgggaa ggtattcagc 540 caaacgacca tctgccgctt tgaggctctg cagcttagct tcaagaacat gtgtaagctg 600 cggcccttgc tgcagaagtg ggtggaggaa gctgacaaca atgaaaatct tcaggagata 660 tgcaaagcag aaaccctcgt gcaggcccga aagagaaagc gaaccagtat cgagaaccga 720 gtgagaggca acctggagaa tttgttcctg cagtgcccga aacccacact gcagcagatc 780 agccacatcg cccagcagct tgggctcgag aaggatgtgg tccgagtgtg gttctgtaac 840 cggcgccaga agggcaagcg atcaagcagc gactatgcac aacgagagga ttttgaggct

17 gctgggtctc ctttctcagg gggaccagtg tcctttcctc tggccccagg gccccatttt 960 ggtaccccag gctatgggag ccctcacttc actgcactgt actcctcggt ccctttccct 1020 gagggggaag cctttccccc tgtctccgtc accactctgg gctctcccat gcattcaaac 1080 tga 1083 <210> 4 <211> 918 <212> DNA <213> Homo sapiens <400> 4 atgagtgtgg atccagcttg tccccaaagc ttgccttgct ttgaagcatc cgactgtaaa 60 gaatcttcac ctatgcctgt gatttgtggg cctgaagaaa actatccatc cttgcaaatg 120 tcttctgctg agatgcctca cacggagact gtctctcctc ttccttcctc catggatctg 180 cttattcagg acagccctga ttcttccacc agtcccaaag gcaaacaacc cacttctgca 240 gagaagagtg tcgcaaaaaa ggaagacaag gtcccggtca agaaacagaa gaccagaact 300 gtgttctctt ccacccagct gtgtgtactc aatgatagat ttcagagaca gaaatacctc 360 agcctccagc agatgcaaga actctccaac atcctgaacc tcagctacaa acaggtgaag 420 acctggttcc agaaccagag aatgaaatct aagaggtggc agaaaaacaa ctggccgaag 480 aatagcaatg gtgtgacgca gaaggcctca gcacctacct accccagcct ttactcttcc 540 taccaccagg gatgcctggt gaacccgact gggaaccttc caatgtggag caaccagacc 600 tggaacaatt caacctggag caaccagacc cagaacatcc agtcctggag caaccactcc 660 tggaacactc agacctggtg cacccaatcc tggaacaatc aggcctggaa cagtcccttc

18 tataactgtg gagaggaatc tctgcagtcc tgcatgcagt tccagccaaa ttctcctgcc 780 agtgacttgg aggctgcctt ggaagctgct ggggaaggcc ttaatgtaat acagcagacc 840 actaggtatt ttagtactcc acaaaccatg gatttattcc taaactactc catgaacatg 900 caacctgaag acgtgtga 918 <210> 5 <211> 1440 <212> DNA <213> Homo sapiens <400> 5 atgaggcagc cacctggcga gtctgacatg gctgtcagcg acgcgctgct cccatctttc 60 tccacgttcg cgtctggccc ggcgggaagg gagaagacac tgcgtcaagc aggtgccccg 120 aataaccgct ggcgggagga gctctcccac atgaagcgac ttcccccagt gcttcccggc 180 cgcccctatg acctggcggc ggcgaccgtg gccacagacc tggagagcgg cggagccggt 240 gcggcttgcg gcggtagcaa cctggcgccc ctacctcgga gagagaccga ggagttcaac 300 gatctcctgg acctggactt tattctctcc aattcgctga cccatcctcc ggagtcagtg 360 gccgccaccg tgtcctcgtc agcgtcagcc tcctcttcgt cgtcgccgtc gagcagcggc 420 cctgccagcg cgccctccac ctgcagcttc acctatccga tccgggccgg gaacgacccg 480 ggcgtggcgc cgggcggcac gggcggaggc ctcctctatg gcagggagtc cgctccccct 540 ccgacggctc ccttcaacct ggcggacatc aacgacgtga gcccctcggg cggcttcgtg 600 gccgagctcc tgcggccaga attggacccg gtgtacattc cgccgcagca gccgcagccg 660 ccaggtggcg ggctgatggg caagttcgtg ctgaaggcgt cgctgagcgc ccctggcagc 720 gagtacggca gcccgtcggt catcagcgtc agcaaaggca gccctgacgg cagccacccg

19 gtggtggtgg cgccctacaa cggcgggccg ccgcgcacgt gccccaagat caagcaggag 840 gcggtctctt cgtgcaccca cttgggcgct ggaccccctc tcagcaatgg ccaccggccg 900 gctgcacacg acttccccct ggggcggcag ctccccagca ggactacccc gaccctgggt 960 cttgaggaag tgctgagcag cagggactgt caccctgccc tgccgcttcc tcccggcttc 1020 catccccacc cggggcccaa ttacccatcc ttcctgcccg atcagatgca gccgcaagtc 1080 ccgccgctcc attaccaaga gctcatgcca cccggttcct gcatgccaga ggagcccaag 1140 ccaaagaggg gaagacgatc gtggccccgg aaaaggaccg ccacccacac ttgtgattac 1200 gcgggctgcg gcaaaaccta cacaaagagt tcccatctca aggcacacct gcgaacccac 1260 acaggtgaga aaccttacca ctgtgactgg gacggctgtg gatggaaatt cgcccgctca 1320 gatgaactga ccaggcacta ccgtaaacac acggggcacc gcccgttcca gtgccaaaaa 1380 tgcgaccgag cattttccag gtcggaccac ctcgccttac acatgaagag gcatttttaa <210> 6 <211> 630 <212> DNA <213> Homo sapiens <400> 6 atgggctccg tgtccaacca gcagtttgca ggtggctgcg ccaaggcggc agaagaggcg 60 cccgaggagg cgccggagga cgcggcccgg gcggcggacg agcctcagct gctgcacggt 120 gcgggcatct gtaagtggtt caacgtgcgc atggggttcg gcttcctgtc catgaccgcc 180 cgcgccgggg tcgcgctcga ccccccagtg gatgtctttg tgcaccagag taagctgcac

20 atggaagggt tccggagctt gaaggagggt gaggcagtgg agttcacctt taagaagtca 300 gccaagggtc tggaatccat ccgtgtcacc ggacctggtg gagtattctg tattgggagt 360 gagaggcggc caaaaggaaa gagcatgcag aagcgcagat caaaaggaga caggtgctac 420 aactgtggag gtctagatca tcatgccaag gaatgcaagc tgccacccca gcccaagaag 480 tgccacttct gccagagcat cagccatatg gtagcctcat gtccgctgaa ggcccagcag 540 ggccctagtg cacagggaaa gccaacctac tttcgagagg aagaagaaga aatccacagc 600 cctaccctgc tcccggaggc acagaattga 630 <210> 7 <211> 1365 <212> DNA <213> Homo sapiens <400> 7 ctggattttt ttcgggtagt ggaaaaccag cagcctcccg cgacgatgcc cctcaacgtt 60 agcttcacca acaggaacta tgacctcgac tacgactcgg tgcagccgta tttctactgc 120 gacgaggagg agaacttcta ccagcagcag cagcagagcg agctgcagcc cccggcgccc 180 agcgaggata tctggaagaa attcgagctg ctgcccaccc cgcccctgtc ccctagccgc 240 cgctccgggc tctgctcgcc ctcctacgtt gcggtcacac ccttctccct tcggggagac 300 aacgacggcg gtggcgggag cttctccacg gccgaccagc tggagatggt gaccgagctg 360 ctgggaggag acatggtgaa ccagagtttc atctgcgacc cggacgacga gaccttcatc 420 aaaaacatca tcatccagga ctgtatgtgg agcggcttct cggccgccgc caagctcgtc 480 tcagagaagc tggcctccta ccaggctgcg cgcaaagaca gcggcagccc gaaccccgcc 540 cgcggccaca gcgtctgctc cacctccagc ttgtacctgc aggatctgag cgccgccgcc

21 tcagagtgca tcgacccctc ggtggtcttc ccctaccctc tcaacgacag cagctcgccc 660 aagtcctgcg cctcgcaaga ctccagcgcc ttctctccgt cctcggattc tctgctctcc 720 tcgacggagt cctccccgca gggcagcccc gagcccctgg tgctccatga ggagacaccg 780 cccaccacca gcagcgactc tgaggaggaa caagaagatg aggaagaaat cgatgttgtt 840 tctgtggaaa agaggcaggc tcctggcaaa aggtcagagt ctggatcacc ttctgctgga 900 ggccacagca aacctcctca cagcccactg gtcctcaaga ggtgccacgt ctccacacat 960 cagcacaact acgcagcgcc tccctccact cggaaggact atcctgctgc caagagggtc 1020 aagttggaca gtgtcagagt cctgagacag atcagcaaca accgaaaatg caccagcccc 1080 aggtcctcgg acaccgagga gaatgtcaag aggcgaacac acaacgtctt ggagcgccag 1140 aggaggaacg agctaaaacg gagctttttt gccctgcgtg accagatccc ggagttggaa 1200 aacaatgaaa aggcccccaa ggtagttatc cttaaaaaag ccacagcata catcctgtcc 1260 gtccaagcag aggagcaaaa gctcatttct gaagaggact tgttgcggaa acgacgagaa 1320 cagttgaaac acaaacttga acagctacgg aactcttgtg cgtaa 1365 <210> 8 <211> 749 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac

22 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtactaatga 720 ccccgtaatt gattactatt aataactaa 749 <210> 9 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 60 accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120 cacaacaccg tgaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc

23 ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240 gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300 gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360 aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480 acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540 tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600 atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660 caccacctgt tcctgtag