CN1833024A

CN1833024A - 含有衍生自基因型2a丙肝病毒(HCV)基因组的核酸的核酸构建体和具有转移入其中的这样的核酸构建体的细胞

Info

Publication number: CN1833024A
Application number: CN200380110406.5A
Authority: CN
Inventors: 胁田隆字; 加藤孝宣; 伊达朋子
Original assignee: Tokyo Metropolitan Organization Medical Research Institute; Bartenschlager Ralf; Toray Industries Inc
Current assignee: Bartenschlager Ralf; Tokyo Metropolitan Institute of Medical Science; Toray Industries Inc
Priority date: 2003-05-26
Filing date: 2003-11-25
Publication date: 2006-09-13
Anticipated expiration: 2023-11-25
Also published as: CN1833024B; US20080032323A1; WO2004104198A1; US20110092688A1; US20130183754A1; AU2003284689A1; CA2525698C; CA2525698A1; JP4694208B2; US8754061B2; DE60333042D1; US8383371B2; JPWO2004104198A1; EP1627917A1; US7935676B2; AU2003284689A8; EP1627917B1; ES2347237T3; EP1627917A4

Abstract

一种复制子RNA，其含有的碱基序列至少包含：基因型2a丙肝病毒基因组RNA的5′非翻译区，编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的碱基序列以及3′非翻译区。

Description

含有衍生自基因型2a丙肝病毒(HCV)基因组的核酸的核酸构建体和具有转移入其中的这样的核酸构建体的细胞

技术领域

本发明涉及基因型2a丙肝病毒的复制子RNA、其中导入了复制子RNA的复制子-复制细胞，和提高复制子RNA复制效率的方法。

背景技术

丙肝病毒(HCV)是一种属于黄病毒科(Flaviviridae)的病毒。其基因组是单链有义链RNA，且已知该病毒导致丙肝。近期的研究已经揭示，根据基因型或血清型，丙肝病毒被分成多种类型。根据Simmonds等人利用HCV株核苷酸序列所作的系统发生分析，该方法是目前主流的HCV基因型分类方法，将HCV分成六个基因型：基因型1a、基因型1b、基因型2a、基因型2b、基因型3a和基因型3b(参见Simmonds，P.等人，Hepatology，(1994)10，第1321-1324页)。这些类型的每一个进一步被分成若干亚型。迄今已经确定了许多HCV基因型的全长基因组核苷酸序列(参见JP专利公开(Kokai)No.2002-171978A；Choo等人，Science，(1989)244，第359-362页；Kato等人，J.Med.Virol.，(2001)64(3)，第334-339页；Okamoto，H等人，J.Gen.Virol.，(1992)73，第673-679页；和Mori，S.等人，Biochem.Biophis.Res.Commun.，(1992)183，第334-342页)。

HCV通过持续感染导致慢性肝炎。目前，全世界观察到的慢性肝炎的主要原因是持续性HCV感染。实际上，约50％的持续感染个体发展成了慢性肝炎。这些患者中约有20％在10至20年中转变成了肝硬化，而且其中有些患者还发展至比如肝癌这样的晚期致命病理学状况。

目前主要通过采用干扰素-α或干扰素-β的治疗方法、或者组合使用干扰素-α和三氮唑核苷(嘌呤-核苷衍生物)的治疗方法，来治疗丙肝。然而，甚至在进行这些治疗后，在所有接受治疗的患者中也只有约60％观察到了治疗效果。当在产生效果后停止治疗时，超过半数的患者疾病复发。已知干扰素的治疗效果与HCV基因型相关，而且据说对基因型1b更低且对基因型2a更高(参见Yoshioka等人，Hepatology，(1992)16(2)，第293-299页)。

开发有效针对丙肝的治疗剂或预防剂是一个重要目标，工业国家的丙肝发病率高，目前对丙肝还没有病因疗法，而且丙肝最终会导致严重的结果。因此，迫切需要开发HCV-特异性化疗法和疫苗疗法。开发抗-HCV剂的一个目的可能是抑制HCV复制或抑制HCV感染细胞。

直至最近，仍难以在细胞培养系统中增殖HCV以及用HCV感染培养的细胞。此外，黑猩猩是唯一可以被HCV感染而且可以在实验中使用的动物，因此难以开展对HCV复制机制和HCV感染机制的研究。然而，近来，已经制得了HCV-衍生的自主复制RNA这样的HCV亚基因组RNA复制子(参见JP专利申请(Kokai)No.2001-17187A；Lohmann等人，Science，(1999)285，第110-113页；Blight等人，Science，(2000)290，第1972-1974页；Friebe等人，J.Virol.，(2001)75(24)，第12047-12057页；Ikeda等人，J.Virol.，(2002)76(6)，第2997-3006页)，这使人们可以用培养的细胞来分析HCV的复制机制。这些HCV亚基因组RNA复制子各自通过用新霉素抗性基因和连接在抗性基因下游的EMCV IRES取代HCV基因型1b基因组RNA 5′非翻译区中HCV IRES下游的结构基因制得。已经证实，当将RNA复制子导入Huh7细胞并在新霉素存在下进行培养时，其可以在人肝癌细胞Huh7细胞中自主复制。

然而，关于这样的细胞内HCV的RNA复制系统，目前只有那些利用HCV基因型1b基因组RNA的系统已经制备出来。由于有报道说不同的HCV基因型编码的病毒蛋白也不同，所以仅仅通过分析从HCV基因型1b衍生而来的亚基因组RNA复制子难以充分阐明HCV的复制机制。此外，基于干扰素的治疗效果依据HCV基因型而不同这一事实，仅利用包含HCV基因型1b亚基因组RNA复制子的HCV复制系统尤其难于开发对各种HCV类型都有效的抗-HCV剂。

发明概述

将本申请要求了优先权的第2003-148242号和第2003-329115号日本专利申请的内容引入本文。

本发明的一个目的是提供还未曾制备出复制子RNA的HCV基因型的HCV-衍生的复制子RNA。

为实现上述目的所作的深入研究的结果是，我们已经成功地制备出了HCV基因型2a的复制子RNA。

也即，本发明如下。

[1]一种复制子RNA，其含有的核苷酸序列至少包含：基因型2a丙肝病毒基因组RNA的5′非翻译区，编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的核苷酸序列以及3′非翻译区。优选该复制子RNA还包含至少一个选择标记基因或报告基因、以及至少一种IRES序列。

[2]一种复制子RNA，其含有的核苷酸序列包含：包含由SEQ IDNO：9或10之中任一所示的核苷酸序列的5′非翻译区；至少一个选择标记基因或报告基因；IRES序列；编码基因型2a丙肝病毒基因组RNA的NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的核苷酸序列；和含有由SEQ ID NO：11或12之中任一所示的核苷酸序列的3′非翻译区。

[3]上述[1]或[2]的复制子RNA，其中所述基因型2a丙肝病毒基因组RNA是一种含有由SEQ ID NO：3或5所示核苷酸序列的RNA。

[4]一种复制子RNA，其含有以下RNA(a)或(b)：

(a)含有由SEQ ID NO：1或2所示核苷酸序列的RNA；和

(b)含有由缺失、取代或添加1至10个核苷酸而衍生自SEQ IDNO：1或2所示核苷酸序列、且能够自主复制的RNA。

[5]一种复制子-复制细胞，它是通过将上述[1]至[4]之中任一的复制子RNA导入细胞制得的。对于该复制子-复制细胞，优选复制子所导入的细胞是真核细胞，更优选是人肝-衍生细胞、人子宫颈-衍生细胞或人胎肾-衍生细胞，且更进一步优选是选自Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞和293细胞中的任何一种细胞。

[6]上述[1]至[4]的复制子RNA，其用于制备或评估针对丙肝病毒感染的治疗剂或诊断剂。

[7]上述[5]的复制子-复制细胞，其用于制备或评估针对丙肝病毒感染的治疗剂或诊断剂。

[8]上述[1]至[4]的复制子RNA，其用于制备针对丙肝病毒感染的疫苗。

[9]上述[5]的复制子-复制细胞，其用于制备针对丙肝病毒感染的疫苗。

[10]一种制备基因型2a丙肝病毒的复制子RNA的方法，其包括从上述[5]的复制子-复制细胞提取复制子RNA。

[11]一种制备基因型2a丙肝病毒的病毒蛋白的方法，其包括培养上述[5]的复制子-复制细胞，并从所得培养产物得到病毒蛋白。

[12]一种筛选促进或抑制丙肝病毒复制的物质的方法，其包括在测试物质存在下培养上述[5]的复制子-复制细胞，并检测所得培养产物中复制子RNA的复制。

[13]一种提高基因型2a丙肝病毒复制子RNA复制效率的方法，其包括进行一个或多个以下步骤：从上述[5]的复制子-复制细胞获得已复制的复制子RNA，并将由此获得的已复制复制子RNA导入与复制子-复制细胞不同的细胞，从而制成一种新的复制子-复制细胞。在该方法中，更优选与最初被导入复制子-复制细胞的复制子RNA相比复制效率提高优选至少2倍。

[14]一种制备复制效率提高的基因型2a丙肝病毒的复制子RNA的方法，其包括进行一个或多个以下步骤：从上述[5]的复制子-复制细胞获得已复制的复制子RNA，并将由此获得的已复制复制子RNA导入与复制子-复制细胞不同的细胞，从而制成一种新的复制子-复制细胞；和从最终获得的复制子-复制细胞得到已复制的复制子RNA。

[15]一种制备复制效率提高的基因型2a丙肝病毒的复制子RNA的方法，其包括检测通过上述[14]的方法为提高复制效率而制得的复制子RNA与最初导入复制子-复制细胞的复制子RNA间的核苷酸突变或氨基酸突变；和将由此所检测到的核苷酸突变或氨基酸突变导入复制效率欲提高的复制子RNA。

[16]一种复制子RNA，其含有衍生自SEQ ID NO：1所示核苷酸序列、具有至少一个选自以下(a)至(u)的突变的核苷酸序列：

(a)核苷酸位点7157处从A突变为G；

(b)核苷酸位点4955处从C突变为U；

(c)核苷酸位点4936处从A突变为G；

(d)核苷酸位点5000处从A突变为G；

(e)核苷酸位点7288处从A突变为G；

(f)核苷酸位点5901处从G突变为U；

(g)核苷酸位点6113处从A突变为U；

(h)核苷酸位点2890处从A突变为G；

(i)核苷酸位点6826处从C突变为A；

(j)核苷酸位点6887处从C突变为A；

(k)核苷酸位点6580处从U突变为A；

(l)核苷酸位点7159处从U突变为C；

(m)核苷酸位点7230处从U突变为A；

(n)核苷酸位点6943处从C突变为A；

(o)核苷酸位点5687处从G突变为A；

(p)核苷酸位点6110处从A突变为G；

(q)核苷酸位点5550处从U突变为C；

(r)核苷酸位点7217处从A突变为G；

(s)核苷酸位点3643处从A突变为G；

(t)核苷酸位点5851处从G突变为A；和

(u)核苷酸位点5914处从G突变为A。

根据本发明，首次提供了衍生自HCV基因型2a株的HCV-RNA复制子。根据本发明的复制子-复制细胞可用作连续生产基因型2a HCV衍生的RNA和HCV蛋白的培养系统。此外，根据本发明的复制子-复制细胞可用作筛选各种影响HCV复制和/或HCV蛋白翻译的物质的测试系统。

附图简述

附图1是构建用于制备根据本发明的HCV-RNA复制子的模板DNA的步骤的示意图。附图1上部显示其中插入了病毒基因组的pJFH1或pJCH1内部区域的结构。附图1下部显示通过用含有新霉素抗性基因和EMCV IRES的DNA片段取代pJFH1或pJCH1的一部分病毒基因组-插入区而构建的、其中插入了病毒基因组的质粒DNApSGREP-JFH1或pSGREP-JCH1内部区域的结构。附图1中的符号如下所述。T7，T7RNA启动子；G，插入到所插入的JFH-1-或JCH-1-衍生的DNA 5′末端上游、和T7RNA启动子序列3′末端下游的dGTP；5′NTR，5′非翻译区；核心，核心蛋白；和3′NTR，3′非翻译区。E1和E2表示包膜蛋白。NS2、NS3、NS4A、NS4B、NS5A和NS5B表示非结构蛋白。Age I、Cla I和Xba I分别表示限制酶Age I、Cla I和Xba I的切割位点。GDD，对应于NS5B蛋白活性中心的氨基酸基序GDD的位置；neo，新霉素抗性基因；和EMCV IRES，脑心肌炎病毒的内部核糖体进入位点(EMCV IRES)。

附图2A至F显示rSGREP-JFH1的核苷酸序列。

附图3A至F显示rSGREP-JCH1的核苷酸序列。

附图4给出了显示其中分别转染了rSGREP-JFH1、rSGREP-JFH1/GND和rSGREP-JFH1/dGDD的Huh7细胞的群体形成照片。上部的三个转染的RNA各自的量是100ng，且下部的三个转染的RNA各自的量是300ng。向各培养皿中添加了1.0mg/ml G418。

附图5给出了显示在培养基中G418浓度为0.5mg/ml时其中分别转染了rSGREP-JFH1和rSGREP-JCH1的Huh7细胞的群体形成照片。这些转染的RNA各自的量为100ng。

附图6给出了显示所作的绿豆核酸酶处理对转染的细胞群体形成能力的影响的照片。两种情况中所转染的rSGREP-JFH1 RNA的量均为100ng。培养基中的G418浓度均为1.0mg/ml。

附图7给出了显示当用来自经rSGREP-JFH1转染而确立的复制子-复制细胞克隆的总细胞RNA再转染另一Huh7细胞时的群体形成的照片。左侧照片显示，在采用来自第六号复制子-复制细胞克隆的总细胞RNA时观察到形成96个群体的结果。右侧照片显示，在采用来自合并克隆的总细胞RNA时观察到形成77个群体的结果。两种情况中，RNA均以包含1×10⁷拷贝复制子RNA的量进行再转染。

附图8给出了显示用RNA印迹法使用rSGREP-JFH1-特异性探针检测来自通过将总细胞RNA(源自通过rSGREP-JFH1转染确立的复制子-复制细胞)再转染进另一Huh7细胞而获得的细胞克隆的总RNA的结果照片。对各泳道说明如下。10⁸表示向提取自Huh7细胞的总RNA添加10⁸拷贝体外合成的复制子RNA而制得的样品。10⁷表示向提取自Huh7细胞的总RNA添加10⁷拷贝体外合成的复制子RNA而制得的样品。Huh7，提取自未转染的Huh7细胞的总RNA；合并克隆，提取自合并克隆的总RNA；和1-11，各提取自第1至11号细胞克隆的总RNA。“复制子RNA”表示指示rSGREP-JFH1大小的分子量标记的电泳位置，“28S”表示指示4.5kb大小分子量的核糖体RNA标记的电泳位置，和“18S”表示指示1.9kb大小分子量的核糖体RNA标记的电泳位置。

附图9给出了显示存在或不存在新霉素抗性基因向经rSGREP-JFH1-或rSGREP-JCH1-衍生的已复制复制子RNA再转染的细胞克隆中的宿主细胞基因组DNA中整合的照片。照片左侧泳道的说明如下。M，DNA分子量标记；1-8，第1至8号rSGREP-JFH1-衍生的细胞克隆；N，未转染的Huh7细胞；和P，阳性对照(新霉素抗性基因的PCR扩增产物)。此外，照片右侧泳道的说明如下。M，DNA分子量标记；和1-6，第1至6号rSGREP-JCH1-衍生的细胞克隆。

附图10给出了显示由经rSGREP-JFH1-或rSGREP-JCH1-衍生的已复制复制子RNA再转染的细胞克隆所表达的NS3蛋白的检测结果照片。左侧照片的第1至8泳道表示第1至8号rSGREP-JFH1-衍生的细胞克隆。右侧照片的第1至6泳道表示第1至6号rSGREP-JCH1-衍生的细胞克隆。右侧照片的泳道P表示NS3蛋白(阳性对照)，和N表示提取自未转染的Huh7细胞的蛋白(阴性对照)。

附图11显示，获自通过将rSGREP-JFH1-衍生已复制复制子RNA再转染进Huh7细胞而确立的21个细胞克隆的复制子RNA中的核苷酸突变位置。突变位置用标有表4所示核苷酸号的线条表示。粗线条表示非同义取代，而细线条表示同义取代。

附图12给出了显示用rSGREP-JFH1对A，HepG2细胞；B，IMY-N9细胞；C，293细胞；或D，HeLa细胞转染结果的照片。向各培养皿添加0.8mg/ml G418。

附图13给出了显示对复制子-复制细胞克隆RNA印迹结果的照片。泳道1，HepG2(阴性对照)；泳道2，10⁸拷贝的合成RNA；泳道3，10⁷拷贝的合成RNA；泳道4，Hep-IH-1(源自HepG2细胞)；泳道5，Hep-IH-3(源自HepG2细胞)；泳道6，Hep-IH-5(源自HepG2细胞)；泳道7，Hep-IH-11(源自HepG2细胞)；泳道8，Hep-IH-13(源自HepG2细胞)；泳道9，IMY-IH-3(源自IMY-N9细胞)；泳道10，IMY-IH-4(源自IMY-N9细胞)；泳道11，IMY-IH-7(源自IMY-N9细胞)；泳道12，IMY-IH-10(源自IMY-N9细胞)；泳道13，包含在其中转染的293-IH的细胞库(源自293细胞)；泳道14，HeLa-IH-9(源自HeLa细胞)；泳道15，HeLa-IH-12(源自HeLa细胞)；泳道16，HeLa-IH-13(源自HeLa细胞)；和泳道17，HeLa(阴性对照)。

附图14给出了显示为证实新霉素抗性基因整合进基因组DNA进行的电泳结果的照片。对HepG2复制子细胞(上部)和IMY-N9复制子细胞(下部)的各基因组DNA，用PCR分析检测了新霉素抗性基因。M，DNA大小标记；P，阳性对照；H，HepG2细胞；I，IMY-N9细胞；和，PCR产物。

附图15给出了显示用蛋白印迹法对源自复制子-复制细胞克隆的NS3和NS5a蛋白的分析结果的照片。

实施本发明的最佳方式

本发明详述如下。

1.根据本发明的HCV-衍生的复制子RNA

丙肝病毒基因组(HCV)是包含约9600个核苷酸的单链(+)链RNA。该基因组RNA含有5′非翻译区(也表示为5′NTR或5′UTR)，由结构区和非结构区组成的翻译区，和3′非翻译区(也表示为3′NTR或3′UTR)。结构区中编码HCV结构蛋白，且非结构区中编码多个非结构蛋白。

这样的HCV结构蛋白和非结构蛋白是如此产生的：从翻译区翻译成HCV结构蛋白和非结构蛋白的连续形式(多蛋白)，多蛋白经蛋白酶限制性降解，之后分别释放结构蛋白(核心，E1和E2)和非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)。在这些结构蛋白和非结构蛋白，也即，HCV病毒蛋白中，核心是核心蛋白，E1和E2是包膜蛋白，且非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)是参与病毒自身复制的蛋白。NS2已知具有金属蛋白酶活性，且NS3已知具有丝氨酸蛋白酶活性(N末端侧三分之一处)和解旋酶活性(C-末端侧三分之二处)。此外，NS4A是NS3蛋白酶活性的辅因子，且已报道NS5B具有RNA-依赖的RNA聚合酶活性。此外，已报道基因型2a HCV基因组具有类似的基因结构(参见JP专利公开(Kokai)No.2002-171978 A)。

我们已经用这样的基因型2a HCV基因组构建了能够自主复制的RNA。具体地说，本发明的HCV-衍生的复制子RNA是一种RNA构建体，其包含基因型2a HCV基因组的全部或部分RNA，并能够自主复制。

在本说明书中，将通过改变HCV病毒基因组制备的、且能自主复制的RNA称作“复制子RNA”或“RNA复制子”。将从基因型2a HCV人工制得的、且能够自主复制的RNA称作“衍生自基因型2a HCV的复制子RNA”。在本说明书中，HCV-衍生的复制子RNA也称作HCV-RNA复制子。

在本发明中，“基因型2a丙肝病毒”或“基因型2a HCV”意指根据Simmonds等人的国际分类被鉴定为基因型2a的丙肝病毒。本发明的“基因型2a丙肝病毒”或“基因型2a HCV”不仅包括具有天然存在的HCV基因组RNA的病毒，而且还包括具有通过人为改变天然存在的HCV基因组序列而制得的基因组RNA的病毒。基因型2a HCV的特定例子包括JFH-1株和JCH-1株病毒(参见JP专利公开(Kokai)No.2002-171978 A)。

此外，“基因型2a丙肝病毒基因组RNA”意指，含有基因型2a丙肝病毒单链(+)链有义RNA且具有其整个基因组区域的核苷酸序列的RNA。基因型2a丙肝病毒基因组RNA优选为含有SEQ ID NO：3或5所示核苷酸序列的RNA，但不限于此。

在本申请说明书中，“5′非翻译区”(5′NTR或5′UTR)、“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”、“编码核心蛋白的序列”(核心区或C区)、“编码E1蛋白的序列”(E1区)、“编码E2蛋白的序列”(E2区)、“编码N2蛋白的序列”(NS2区)、“编码NS3蛋白的序列”(NS3区)、“编码NS4A蛋白的序列”(NS4A区)、“编码NS4B蛋白的序列”(NS4B区)、“编码NS5A蛋白的序列”(NS5A区)、“编码NS5B蛋白的序列”(NS5B区)和“3′非翻译区”(3′NTR或3′UTR)、以及其它特定区域或位点根据编码JFH-1株基因组完整区域的全长cDNA(JFH-1克隆)核苷酸序列SEQ ID NO：3确定，所述JFH-1株是基因型2a HCV。SEQ IDNO：3的核苷酸序列可以参考登录号AB047639从国际DNA数据库(DDBJ/EMBL/GenBank)获得。具体地说，当将某一特定HCV RNA序列与SEQ ID NO：3所示核苷酸序列进行比对时，与SEQ ID NO：3所示核苷酸序列第1至340位核苷酸比对的序列是该RNA的“5′非翻译区”，与SEQ ID NO：3所示核苷酸序列第3431至9442位核苷酸比对的序列是编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列，与SEQ ID NO：3所示核苷酸序列第3431至5323位核苷酸比对的序列是“编码NS3蛋白的序列”，与SEQ ID NO：3所示核苷酸序列第5324至5485位核苷酸比对的序列是“编码NS4A蛋白的序列”，与SEQ ID NO：3所示核苷酸序列第5486至6268位核苷酸比对的序列是“编码NS4B蛋白的序列”，与SEQ ID NO：3所示核苷酸序列第6269至7666位核苷酸比对的序列是“编码NS5A蛋白的序列”，与SEQ ID NO：3所示核苷酸序列第7667至9442位核苷酸比对的序列是“编码NS5B蛋白的序列”，与SEQ ID NO：3所示核苷酸序列第9443至9678位核苷酸比对的序列是“3′非翻译区”。另外，此处，在“所比对的”序列中可能存在缺口、添加、缺失、取代等。而且，上述“特定HCV”不限于此，且包括JFH-1株或JCH-1株、或由其衍生的病毒株。

本发明HCV RNA-复制子的一种实施方案是含有的核苷酸序列至少包含基因型2a丙肝病毒基因组RNA 5′非翻译区，编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列和3′非翻译区的复制子RNA。该复制子RNA还可以至少包含一个选择标记基因或一个报告基因，和至少一个IRES序列。此外，该复制子RNA还可以包含编码基因型2a丙肝病毒基因组RNA上除NS3、NS4A、NS4B、NS5A和NS5B蛋白之外的病毒蛋白的序列。

本发明HCV RNA-复制子的另一优选实施方案是复制子RNA，其含有如下核苷酸序列，所述核苷酸序列包含含有SEQ ID NO：9或10所示核苷酸序列的5′非翻译区，至少一种选择标记基因或报告基因，IRES序列，编码基因型2a丙肝病毒基因组RNA的NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列，和含有SEQ ID NO：11或12所示核苷酸序列的3′非翻译区。此处，SEQ ID NO：9和10所示核苷酸序列分别是本发明复制子RNA rSGREP-JFH1(SEQ IDNO：1)和rSGREP-JCH1(SEQ ID NO：2)的5′非翻译区序列。此外，SEQ ID NO：11和12所示核苷酸序列分别是本发明复制子RNArSGREP-JFH1(SEQ ID NO：1)和rSGREP-JCH1(SEQ ID NO：2)的3′非翻译区序列。

本发明HCV RNA-复制子的更优选实施方案是由含有SEQ IDNO：1或2所示核苷酸序列的RNA组成的复制子RNA。此外，本发明范围内还包括含有衍生自SEQ ID NO：1或2所示核苷酸序列并缺失、取代或添加了1至50、1至30、1至10、1至6、或1至若干个(2至5个)核苷酸的核苷酸序列、且能够自主复制的复制子RNA作为一种优选实施方案。在本发明中，“能够自主复制”意指，在将该复制子RNA导入细胞时，该复制子RNA能够使其自身全长序列在细胞中复制。例如，该自主复制能力可以通过如下方法证实：将复制子RNA转染进Huh7细胞、培养Huh7细胞、从所得培养产物中的细胞提取RNA和利用能够特异性检测转染的复制子RNA的探针对所提取的RNA作RNA印迹杂交，从而检测复制子RNA的存在。然而，该方法的例子不限于此。用于证实自主复制能力的特定程序可以根据本说明书实施例中给出的说明来进行，比如那些测量群体形成能力、证实HCV蛋白表达或检测复制子RNA的实施例。

在本发明中，“选择标记基因”意指能够为细胞提供选择性的基因，由此仅选择出表达该基因的细胞。选择标记基因的一般例子是抗生素抗性基因。在本发明中，选择标记基因的优选例子包括新霉素抗性基因、胸苷激酶基因、卡那霉素抗性基因、吡啶硫胺素抗性基因、腺苷酰转移酶基因、Zeocin抗性基因和嘌呤霉素抗性基因。优选新霉素抗性基因和胸苷激酶基因，更优选新霉素抗性基因。然而，本发明的选择标记基因不限于这些基因。

此外在本发明中，“报告基因”意指编码作为该基因表达标记的基因产物的标记基因。报告基因的一般例子包括，催化发光反应或呈色发应的酶的结构基因。本发明中报告基因的优选例子包括转座子Tn9-衍生的氯霉素乙酰转移酶基因、大肠杆菌(Escherichia coli)-衍生的β葡糖醛酸糖苷酶或β半乳糖苷酶基因、萤光素酶基因、绿色荧光蛋白基因、来自水母的水母发光蛋白基因和分泌型胎盘碱性磷酸酶(SEAP)基因。然而，本发明的报告基因不限于这些基因。

复制子RNA中可以只包含上述选择标记基因和报告基因之一或者包含二者。

在本发明中，“IRES序列”意指可以通过在RNA内部结合核糖体而引发翻译的内部核糖体进入位点。本发明IRES序列的优选实例包括但不限于，EMCV IRES(脑心肌炎病毒内部核糖体进入位点)、FMDV IRES和HCV IRES。更优选EMCV IRES和HCV IRES，且EMCV IRES是最优选序列。

本发明的复制子RNA还可包含另一HCV株或另一基因型HCV的基因组RNA上的序列。例如，复制子RNA还可以包含基因型1b的HCV基因组片段。另一HCV株的例子株包括但不限于：HCV-1、HCV-H、HC-J1、HCT-18、H77、DK-7、US11、S14、HCT23、HCV-Th、DR1、DR4、HCT27、S18、SW1、DK9、H90、TD-6E1、S9、HCV-BK、T10、DK1、HC-J4、HCV-J、HK3、HK8、HK5、HCV-G3、IND5、IND8、P10、D1、D3、SW2、T3、S45、SA10、US6、HCV-JK1、HCV-JK4、HCV-JK3、HCV-JK2、HCV-JT、HC-J2、HCV-T、HK4、HC-G9、Z1、Bi、S.I.、Cho、J.M.、HCV-J6、T4、T9、US10、HC-J5、T2、HC-J7、DK11、SW3、DK8、T8、HC-J8、S83、HK2、HC-J6、HC-J8、BEBE1、HCV-J6、HCV-J8、HD10-2、BR36-9、S52、S54、S2、BR33-1、HK10、DK12、HCV-TR、BA-1、BA-2、DK13、Z1、Z4、Z6、Z7、HK2、SA1、SA4、SA5、SA7、SA13、SA6、NZL1、SA30、EG-13、HCV-K3a/650、ED43、EUH1480、EUHK2、Th580、VN235、VN405、VN004、JK049、JK046、JFH-1、JCH-1、JCH-2、JCH-3、JCH-4、JCH-5、JCH-6、J6CF和H77。

本发明的复制子RNA优选在5′-最远侧具有基因型2a HCV基因组RNA 5′非翻译区、和在3′-最远侧具有基因型2a HCV基因组RNA3′非翻译区。选择标记基因或报告基因可以连接在IRES序列上游、或“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”的上游或下游，或插入“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”的中间。

本发明的复制子RNA更优选在5′-最远侧具有基因型2a HCV基因组RNA 5′非翻译区、依次位于5′非翻译区下游的选择标记基因或报告基因、IRES序列和“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”，以及在3′-最远侧具有基因型2aHCV基因组RNA 3′非翻译区。

本发明复制子RNA的例子可以包括包含欲在导入了所述复制子RNA的细胞内表达的任何外源基因和上述序列的RNA。外源基因也可以连接在5′非翻译区下游、或连接在选择标记基因或报告基因的上游或下游、或连接在“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”的上游或下游、或可以插入“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和N S5B蛋白的序列”中间。当包含外源基因的复制子RNA在导入RNA的细胞中翻译时，它能够表达由该外源基因编码的蛋白。因此，包含外源基因的复制子RNA还适宜用于其目的是在细胞内生成特定基因产物的基因疗法等。

本发明的复制子RNA还可以包含核酶。将核酶插入以使复制子RNA 5′侧的选择标记基因、报告基因或外源基因与该复制子RNA 3′侧包括IRES序列和“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”的那些基因连接，由此该核酶能够通过其自-切割活性将二者切割并分离。

在本发明的复制子RNA中，将上述选择标记基因、报告基因、编码基因型2a丙肝病毒基因组RNA上的病毒蛋白的序列、编码除基因型2a外的基因型的HCV病毒蛋白的序列、外源基因等连接，由此它们能够以正确的读框从复制子RNA翻译。在这些序列中，蛋白-编码序列可以通过蛋白酶切割位点等彼此相连，由此在蛋白被表达为具有从基因型2a丙肝病毒的“编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的序列”翻译出的多蛋白的融合蛋白后，该融合蛋白被蛋白酶分离成各蛋白。

2.制备本发明的复制子RNA

可以用本领域技术人员已知的任何遗传工程技术制备本发明HCV RNA-复制子。例如，可以用以下方法制备HCV RNA-复制子，但制备方法不限于此。

首先，根据标准程序将对应于基因型2a丙肝病毒基因组RNA完整区域的DNA连接到RNA启动子下游以制备DNA克隆。如本文中所使用的，“对应于RNA的DNA”意指具有通过以T(胸腺嘧啶)取代U(尿嘧啶)而衍生自RNA核苷酸序列的核苷酸序列的DNA。上述RNA启动子优选为质粒克隆中所含的RNA启动子。RNA启动子的例子不限，但尤其优选T7 RNA启动子。

接着，对由此制得的DNA克隆，例如，用包含选择标记基因或报告基因以及其下游所连接的IRES序列的DNA片段取代位于5′非翻译区下游的结构区(核心序列、E1序列和E2序列)和编码NS2蛋白的序列。在该取代中，除结构区外的部分，比如位于5′非翻译区3′末端侧的片段或一部分编码NS3蛋白的序列，可以用源自另一基因型HCV的序列取代。

随后，以经过取代的DNA克隆为模板，用RNA聚合酶合成RNA。RNA合成可以通过标准程序从5′非翻译区和IRES序列引发。当模板DNA是质粒克隆时，则用限制酶将上述连接在RNA启动子下游的DNA区从质粒克隆切除，之后可以以DNA片段为模板合成RNA。此外，优选待合成的RNA的3′末端与病毒基因组RNA的3′非翻译区一致，且未添加或缺失其它序列。由此合成的RNA即为本发明的复制子RNA。

3.制备其中导入了基因型2a HCV复制子RNA的复制子-复制细胞

将如上所述制得的复制子RNA导入在其中该复制子RNA应当进行复制的细胞，由此能够获得复制子RNA在其中进行连续复制的细胞。在本说明书中，将复制子RNA在其中进行连续复制的细胞称作“复制子-复制细胞”。

可以使用任何细胞作为将向其中导入复制子RNA的细胞，只要它能够进行继代培养。这样的细胞优选为真核细胞，更优选为人肝-衍生细胞、人子宫颈-衍生细胞或人胎肾-衍生细胞，以及更优选为选自Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞和293细胞的的任何细胞。对于这些细胞，可以利用可商购的细胞，这些细胞可以获自细胞保藏单位，或者可以使用从任何细胞(例如，癌细胞或干细胞)确立的细胞系。

为大量生产HCV蛋白，比如为生产疫苗，优选使用可以大量培养的细胞作为上述细胞。从这一观点出发，优选除Huh7细胞外的细胞。

可以用本领域技术人员已知的任何技术将复制子RNA导入细胞。这样的导入方法的例子包括电穿孔、基因枪法、脂质转染法、磷酸钙法、微注射法、和DEAE琼脂糖凝胶法。优选使用电击法。

目的复制子RNA可以单独导入，或可以与其它核酸混合后导入。为在将待导入的RNA量维持在特定水平的同时改变复制子RNA的量，将目的复制子RNA与提取自其中导入了RNA的细胞的总细胞RNA混合，之后将该混合物用于导入细胞。用于导入细胞的复制子RNA的量可以依赖于所使用的导入方法来确定，且优选介于1皮克和100微克，更优选介于10皮克和10微克。

当用包含选择标记基因或报告基因的复制子RNA来导入细胞时，可以利用选择标记基因或报告基因的表达来选择其中导入了复制子RNA并且该复制子RNA在其中连续复制的细胞。具体地说，例如，这样的其中已经导入了复制子RNA的细胞可以在能够通过选择标记基因或报告基因的表达在其上选择细胞的培养基中进行培养。作为例子，当复制子RNA含有新霉素抗性基因作为选择标记基因时，将细胞内已经导入了复制子RNA的细胞接种在培养皿中。培养16至24小时后，向培养皿中添加浓度为0.05毫克/毫升至3.0毫克/毫升的G418(新霉素)。接种后优选连续培养细胞10天至40天，更优选14天至28天，同时每周更换两次培养液。接着，用结晶紫将存活细胞染色，由此可以选择其中导入了复制子RNA并且复制子RNA在其中连续复制的细胞作为所形成的群体。

用标准程序克隆存活细胞、然后继续培养该细胞可以从所形成的群体获得克隆细胞。本说明书中将这样所得的、目的复制子RNA在其中连续复制的细胞克隆称作“复制子-复制细胞克隆”。

关于所确立的细胞克隆，优选检测已经从细胞克隆中所导入的复制子RNA复制的复制子RNA、证实所导入的复制子RNA中的选择标记基因或报告基因向宿主基因组DNA中整合的存在或不存在、并证实HCV蛋白的表达，以证实目的复制子RNA确实连续复制这一事实。

可以根据本领域技术人员已知的任何RNA检测技术检测已经从细胞克隆中所导入的复制子RNA复制的复制子RNA(在本说明书下文中方便地称作“已复制复制子RNA”)。例如，可以用特异于所导入的复制子RNA的DNA片段为探针、对提取自细胞克隆的总RNA做RNA印迹杂交法来进行检测。

此外，所导入的复制子RNA中的选择标记基因或报告基因向宿主基因组DNA中整合的存在或不存在可以通过如下方法进行证实，例如，对提取自细胞克隆的宿主基因组DNA做PCR以扩增至少一部分选择标记基因或报告基因、之后证实扩增产物的存在或不存在。然而，相关方法的例子不限于此。对已经证实了扩增产物的细胞克隆，认为它带有整合进宿主基因组的选择标记基因或报告基因。因此，对于细胞克隆，复制子RNA自身不能在细胞内连续复制。这种情况下，如下所述，可以通过进行证实HCV蛋白表达的试验来证实复制子RNA是否连续复制。

HCV蛋白表达可以通过如下方法进行证实，例如，产生针对欲从所导入的复制子RNA表达的HCV蛋白的抗体、并与提取自细胞克隆的蛋白反应。该方法通过通过任何一种本领域技术人员已知的蛋白检测方法来进行。具体地说，例如，将提取自细胞克隆的蛋白样品印迹到硝酸纤维素膜上，使膜与抗-HCV蛋白抗体(例如，抗-NS3-特异性抗体或收集自丙肝患者的抗血清)反应，且之后检测抗-HCV蛋白抗体。如果从提取自细胞克隆的蛋白中检测到了HCV蛋白，则可推论该细胞克隆持续复制HCV-衍生的复制子RNA以表达HCV蛋白。

如上所述，可以获得已经证实持续复制目的复制子RNA的细胞克隆(复制子-复制细胞克隆)。此外，在本发明中，复制子RNA可以通过本领域技术人员已知的任何一种方法获得，例如，通过从复制子-复制细胞中提取RNA、然后用电泳法从RNA中分离复制子RNA。本发明还涉及这样一种制备复制子RNA的方法。此外，优选地，本发明的复制子-复制细胞可用于生产HCV蛋白。本领域技术人员可以根据任何标准方法从复制子-复制细胞获得HCV蛋白。具体地说，例如，基因型2a丙肝病毒的病毒蛋白可以通过如下方法制备：培养复制子-复制细胞、经标准程序从所得培养产物(包括培养细胞和培养基)收集蛋白、之后通过检测等方法用抗-HCV蛋白抗体从蛋白选择性获得病毒蛋白。

此外，当本发明的复制子-复制细胞持续复制含有外源基因的复制子RNA时，可通过用复制子-复制细胞表达外源基因得到外源基因编码的蛋白。具体地说，例如，通过培养复制子-复制细胞、经标准程序从所得培养产物(包括培养细胞和培养基)收集蛋白、之后用针对目的蛋白的抗体通过检测等从蛋白中选择性获得蛋白，可以获得外源基因编码的蛋白。

4.将提高复制效率的突变导入源自基因型2a HCV的复制子RNA

根据本发明，在复制子-复制细胞中复制或生成的复制子RNA(已复制的复制子RNA)中经常发生提高复制效率的突变。这样的突变可能是适应性突变。

利用这一事实，可以在本发明中促进向本发明的复制子RNA中导入提高复制效率的突变。

具体地说，重复一次或多次，优选1至10次，更优选1至5次，以及更优选1至2次的如下步骤，所述步骤包括通过提取等方法从第一复制子-复制细胞(优选，其中已经导入了本发明复制子RNA的复制子-复制细胞)获得第一已复制的复制子RNA、之后将第一已复制的复制子RNA再次导入另一细胞中以制备第二复制子-复制细胞，由此可以向复制子-复制细胞内的复制子RNA中高频率导入提高复制效率的突变。

可以使用任何细胞作为其中再导入了已复制的复制子RNA的细胞。这样的细胞优选源自与复制子RNA最初所导入的细胞相同的生物物种，更优选源自与复制子RNA最初所导入的细胞相同的生物物种的相同组织，且更优选与复制子RNA最初所导入的细胞的细胞系相同的细胞系。

因此在本发明中，用上述方法，可以制备其中导入了提高复制效率的突变的复制子RNA。具体地说，重复一次或多次，优选1至10次，更优选1至5次，以及更优选1至2次如下步骤，所述步骤包括通过提取等方法从第一复制子-复制细胞(优选，其中已经导入了本发明复制子RNA的复制子-复制细胞)获得第一已复制的复制子RNA、之后将第一已复制的复制子RNA再次导入另一细胞中以制备第二复制子-复制细胞。随后，通过提取等方法从重复步骤结束时最终获得的复制子-复制细胞中获得已复制的复制子RNA，由此产生复制效率提高了的复制子RNA。

本发明中，通过上述方法，复制子RNA的复制效率可以提高至少2倍，优选10至100倍，且更优选100至10000倍。

关于通过这样的方法制得从而具有提高的复制效率的复制子RNA，优选用已知方法确定核苷酸序列，例如，通过用反转录PCR获得cDNA并对这样的cDNA进行测序。此外，将由此确定的核苷酸序列或由该核苷酸序列编码的氨基酸序列与最初导入细胞的复制子RNA的核苷酸序列进行比较，由此可以鉴定适应性突变。尤其优选导致由复制子RNA编码的病毒蛋白中的氨基酸突变的非同义取代，作为提高复制效率的适应性突变。

本发明还提供一种方法，藉由该方法，通过标准程序将所鉴定的适应性突变导入复制效率待提高的复制子RNA，可以生产具有提高的复制效率的基因型2a丙肝病毒的复制子RNA。

如上述制备以具有提高的复制效率的复制子RNA可用于在已用于该方法的细胞中大量生产复制子RNA。

可以通过本领域技术人员已知的方法确定本发明复制子RNA的复制效率。例如，可以根据以下方法确定。分别以0.0001、0.0003、0.001、0.003、0.01、0.03、0.1、0.3和1.0微克的量将复制子RNA转染入Huh7细胞，以类似于上述试验技术的方法用G418选择性培养21天，之后对所形成的群体计数(群体数)。将所导入的RNA的量与所形成的群体数相比以确定所导入的复制子RNA的量的范围，在该范围内群体形成以量-依赖方式提高。用该范围内所形成的群体数以除所导入的RNA的量，并将所得值视为每微克的群体形成活性。该方程如下。

群体形成活性[(群体形成单位，或CFU)/微克]＝所形成的群体数[群体]/所导入的RNA的量[微克]

将由此计算出的群体形成活性视作表示所导入复制子RNA的复制效率的值。具体地说，群体形成活性越高，复制子RNA的复制效率越高。此外，由每个所形成的群体所导入的复制子RNA的拷贝数表示的复制子RNA的复制效率也可以通过群体-形成能力显示。也即，这种情况下，该能力可以根据以下方程计算。

群体形成能力＝所导入的复制子RNA拷贝数[拷贝]/所形成的群体数[群体]

5.本发明的其它实施方案

本发明的复制子RNA-复制细胞还可用作，例如，筛选促进或抑制丙肝病毒复制的物质的测试系统。具体地说，例如，在测试物质存在下培养复制子复制细胞，检测所得培养产物中的复制子RNA的复制，之后检测测试物质是否促进或抑制复制子RNA的复制，由此可以筛选出促进或抑制丙肝病毒复制的物质。在这种情况下，检测所得培养产物中复制子RNA的复制可通过如下方法进行：检测提取自复制子RNA-复制细胞的RNA中复制子RNA的量或存在或不存在复制子RNA、或检测培养产物或培养产物中所含的复制子RNA-复制细胞中的蛋白中所含的HCV蛋白的量或存在或不存在HCV蛋白。

这样的利用本发明的复制子RNA-复制细胞的测试细胞系统可以指向生产或评估用于治疗丙肝病毒感染的治疗剂或诊断剂。这样的目的的特定例子包括以下例子。

(1)寻找抑制基因型2a HCV增殖的物质

抑制基因型2a HCV增殖的物质的例子包括直接或间接影响基因型2a HCV增殖的有机化合物、和通过与基因型2a HCV基因组中的靶序列或其互补链杂交而直接或间接影响HCV增殖或HCV蛋白翻译的反义寡核苷酸。

(2)评估在细胞培养物中具有抗病毒作用的各种物质

各种物质的例子包括通过合理药物设计和高通量筛选获得的物质(例如，分离和纯化的酶)等。

(3)鉴定用于治疗基因型2a HCV感染患者的新攻击靶

为鉴定在HCV病毒增殖过程中起重要作用的宿主细胞蛋白，例如，可以使用本发明的复制子-复制细胞。

(4)评估HCV病毒获得对药物等的抗性的能力和鉴定与这种抗性相关的突变

(5)生产可作为抗原用于开发、生产和评估丙肝病毒感染的诊断剂或治疗剂的病毒蛋白

(6)用于生产可用于开发、生产和评估丙肝病毒感染的诊断剂或治疗剂的HCV病毒或病毒样颗粒的病毒基因组复制系统

(7)生产可用作针对基因型2a HCV的疫苗的疫苗抗原

(8)生产在向其中整合了外源基因后用于基因治疗的肝细胞-定向遗传载体

6.实施例

根据以下实施例和附图本发明将得到更具体地描述。然而，本发明的技术范围并不限于这些实施例。

实施例1：制备复制子RNA

(A)构建表达载体

从包含病毒株全长基因组cDNA的JFH-1克隆获得了对应于丙肝病毒JFH-1株(基因型2a)(分离自暴发性肝脏衰竭患者)病毒基因组完整区域的DNA。将该DNA插入到已被插入pUC19质粒中的T7 RNA启动子序列下游。由此构建的质粒DNA在下文中称作pJFH1。类似地，从包含病毒株全长基因组cDNA的JCH-1克隆获得了对应于丙肝病毒JCH-1株(基因型2a)(分离自慢性肝炎患者)病毒基因组完整区域的DNA。将该DNA插入到已被插入pUC19质粒中的T7 RNA启动子序列下游。由此构建的质粒DNA在下文中称作pJCH1。此外，上述JFH1克隆和JCH-1克隆的制备描述于JP专利公开(Kokai)No.2002-171978A和Kato等人，J.Med.Virol.，(2001)64(3)，第334-339页。而且，JFH-1克隆全长cDNA核苷酸序列已登记于国际DNA数据库(DDBJ/EMBL/GenBank)，登录号为AB047639，且JCH-1克隆全长cDNA核苷酸序列的登录号为AB047640。

由此所构建的质粒DNA pJFH1和pJCH1的结构示于附图1上部。“T7”表示T7 RNA启动子，而“G”表示插入到所插入的JFH-1-或JCH-1-衍生DNA的5′末端上游和T7 RNA启动子序列3′末端下游的dGTP。从“5′NTR”至“3′NTR”的区域是对应于丙肝病毒完整基因组区域的DNA。

接下来，用新霉素抗性基因(neo；也称作新霉素磷酸转移酶基因)和EMCV-IRES(脑心肌炎病毒内部核糖体进入位点)取代质粒DNA pJFH1和pJCH1的结构区和部分非结构区，从而分别构建出质粒DNA pSGREP-JFH1和pSGREP-JCH1(附图1下部)。该构建程序根据在先的报道(Lohmann等人，Science，(1999)285，第110-113页)来进行。具体地说，用限制酶Age I和Cla I切割质粒pJFH1和pJCH1，并在Age I和Cla I限制位点间插入以下片段以进行连接：通过PCR扩增将源自pJFH-1的5′NTR至核心区范围内的序列和源自pRSV5NEO的新霉素抗性基因结合以及之后用限制酶Age I和Pme I对其进行切割制得的片段；和，通过PCR扩增将EMCV IRES至NS3区域范围内的序列结合以及之后用限制酶Pme I和Cla I对其进行切割制得的片段。

此外，向pSGREP-JFH1的NS5B区域导入一个使氨基酸基序GDD(对应于NS5B区域所编码的RNA聚合酶的活性中心)突变为GND的突变，由此制得突变体质粒克隆pSGREP-JFH1/GND。

此外，向pSGREP-JFH1的NS5B区域导入一个使一段包含氨基酸基序GDD(对应于NS5B区域所编码的RNA聚合酶的活性中心)的10个连续氨基酸的序列缺失的突变，由此制得突变体质粒克隆pSGREP-JFH1/dGDD。

上述制备的突变体克隆pSGREP-JFH1/GND和pSGREP-JFH1/dGDD不能表达复制子RNA复制所需的活性NS5B蛋白，因为这些克隆所编码的NS5B蛋白的活性位点氨基酸序列已经突变了。

(B)制备复制子RNA

为制备用于合成复制子RNA的模板DNA，将上述构建的表达载体pSGREP-JFH1、pSGREP-JCH1、pSGREP-JFH1/GND 和pSGREP-JFH1/dGDD各用限制酶Xba I进行切割。

然后，50μl反应液中各含10至20μg这些Xba I-切割的片段，之后进一步于30℃用20U绿豆核酸酶温育30分钟进行处理。绿豆核酸酶是一种选择性催化双链DNA中的单链部分降解的酶。一般，当直接以上述Xba I-切割的片段为模板合成RNA时，会合成在3’末端过量添加有CUGA四核苷酸(Xba I识别序列的一部分)的复制子RNA。因此，在本实施例中，用绿豆核酸酶处理Xba I切割的片段，以从片段中除去CUGA四核苷酸。根据通用的方法处理已经用绿豆核酸酶处理过的含Xba I-切割片段的溶液以除去蛋白，由此纯化已经除去了CUGA四核苷酸的Xba I-切割片段并将其用作模板DNA。

接着，用T7 RNA聚合酶从模板DNA体外合成RNA。对这一RNA合成，使用了Ambion，Inc.的MEGAscript。根据生产商的说明，用20μl含0.5至1.0微克模板DNA的反应液进行反应。

RNA合成完成之后，向反应液中加入DNA酶(2U)，于37℃反应15分钟。用酸性苯酚提取RNA以进一步除去模板DNA。将以这种方式从上述源自pSGREP-JFH1、pSGREP-JCH1、pSGREP-JFH1/GND和pSGREP-JFH1/dGDD的模板DNA合成的RNA(复制子RNA)分别命名为rSGREP-JFH1、rSGREP-JCH1、rSGREP-JFH1/GND和rSGREP-JFH1/dGDD。关于这些复制子RNA的核苷酸序列，rSGREP-JFH1的核苷酸序列示于SEQ ID NO：1和附图2A～F，rSGREP-JCH1的核苷酸序列示于SEQ ID NO：2和附图3A～F，rSGREP-JFH1/GND的核苷酸序列示于SEQ ID NO：7，和rSGREP-JFH1/dGDD的核苷酸序列示于SEQ ID NO：8。

实施例2：确立复制子-复制细胞克隆

(C)复制子RNA的转染，确定转染细胞的群体形成能力和确立细胞克隆

将各以上合成的复制子RNA(rSGREP-JFH1，rSGREP-JCH1，rSGREP-JFH1/GND和rSGREP-JFH1/dGDD)以不同的量与提取自Huh7细胞的总细胞RNA混合，从而使总RNA量为10μg。然后，用电穿孔将混合RNA导入Huh7细胞。将经过电穿孔处理的Huh7细胞接种于培养皿，之后培养16至24小时。向培养皿中加入不同浓度的G418(新霉素)。其后，继续培养，其中每周更换两次培养液。接种培养21天后，用结晶紫对存活细胞染色。对被染色的群体计数，然后计算每μg转染的复制子RNA的被染色群体数。

对已经观察到了群体形成的rSGREP-JFH1或rSGREP-JCH1-转染的细胞，从上述培养21天后的培养皿进一步克隆存活细胞群体，并继续培养。通过这样对群体进行克隆，可以确立几个细胞克隆株。

对已确立的细胞克隆，如以下实施例4所述检测已复制的复制子RNA、证实新霉素抗性基因向宿主基因组DNA中整合的存在或不存在、并证实HCV蛋白的表达。将已经证实了复制子在细胞中复制的细胞克隆视为复制子-复制细胞克隆。

(D)各转染细胞的群体形成能力

上述转染的结果是，对于rSGREP-JFH1-转染的Huh7细胞，当G418浓度为1.0mg/ml时，每μg转染的复制子RNA的群体形成能力是94700CFU(群体形成单位)/μg·RNA(附图4左栏)。相反，在其中各转染了rSGREP-JFH1/dGDD和rSGREP-JFH1/GND的Huh7细胞中未观察到群体形成(附图4中栏和右栏)。这暗示，其中已转染了rSGREP-JFH1复制子RNA的Huh7细胞的群体形成能力依赖于rSGREP-JFH1所表达的NS5B(RNA聚合酶)活性。具体地说，认为在已形成群体的细胞中，rSGREP-JFH1复制子RNA由于由rSGREP-JFH1所表达的NS5B的作用而自主复制，且新霉素抗性基因持续表达以维持G418抗性，由此使得细胞能够生长。

另一方面，在其中已转染了rSGREP-JCH1的Huh7细胞中，倘若G418浓度为1至0.5mg/ml，则未观察到群体形成(附图5)。当G418浓度被降至0.25mg/ml时，在其中已转染了rSGREP-JCH1的Huh7细胞中观察到了群体形成。

此外，将上述(B)中所获的表达载体pSGREP-JFH1的Xba I切割片段不经用绿豆核酸酶处理而用作合成RNA的模板DNA，以合成复制子RNA。用该复制子RNA以与上述(C)类似的方式转染Huh7细胞。不经绿豆核酸酶处理而制得的复制子RNA在3’末端过量加有CUGA四核苷酸。

结果，其中已转染了不经绿豆核酸酶处理制得的复制子RNA的Huh7细胞的群体形成能力降至512CFU/μg·RNA(附图6左侧)。该结果揭示，复制子RNA的3’末端序列影响转染细胞的群体形成能力。

实施例3

(E)来自复制子-复制细胞的已复制的复制子RNA的再转染

从根据实施例2的说明将rSGREP-JFH1转染进Huh7细胞所确立的复制子-复制细胞克隆，经标准程序提取了总RNA。通过RNA印迹分析和定量RT-PCR法测定细胞RNA中所含的已复制的复制子RNA的拷贝数。

根据Molecular Cloning，A laboratory Manual，第二版，J.Sambrook，E.F.Fritsch，T.Maniatis，Cold Spring HarborLaboratory Press(1989)中的说明进行RNA印迹分析。具体地说，对提取自细胞的RNA做变性凝胶电泳。电泳后，将RNA转移到带正电荷的尼龙膜上。将从pSGREP-JFH1制得的³²P-标记的DNA或RNA探针与转移至上述膜上的RNA杂交。接下来洗膜，然后对胶片曝光，以检测复制子-特异的RNA带。

根据Takeuchi T，Katsume A，Tanaka T，Abe A，Inoue K，Tsukiyama-Kohara K，Kawaguchi R，Tanaka S和Kohara M.，Real-time detection system for quantification of Hepatitis C virusgenome，Gastroenterology 116：636-642(1999)，利用定量RT-PCR通过检测HCV RNA内的5′非翻译区RNA来进行对复制子RNA的检测。具体地说，用合成引物通过PCR扩增从细胞提取的RNA中的复制子RNA：R6-130-S17，5′-CGGGAGAGCCATAGTGG-3′(SEQ IDNO：13)和R6-290-R19，5′-AGTACCACAAGGCCTTTCG-3′(SEQID NO：14)；TaqMan探针；R6-148-S21FT，5′-CTGCGGAACCGGTGAGTACAC-3′(SEQ ID NO：15)和EZ rTthRNA PCR试剂盒，之后用ABI Prism 7700序列检测系统进行检测。

接着，将提取自克隆6(上述复制子-复制细胞克隆中的)和合并克隆(通过收集一整皿已形成群体的复制子-复制细胞并对其进行培养而制得)的总细胞RNA等分试样通过再转染各自导入另外的Huh7细胞。根据以上测定的复制子RNA的拷贝数，将转染所用的总细胞RNA制成包含1×10⁷拷贝复制子RNA。如上述(C)所述进行转染，之后在1mg/ml G418的浓度条件下进行选择培养。从而，观察到了复制子-复制细胞的群形成能力(附图7)。这种情况下，当从所得群体数计算时，每1×10⁶拷贝用于转染的复制子RNA的群体形成能力是一个群体或更多。

另一方面，当根据RNA的重量和长度来计算时，以pSGREP-JFH1为模板且用T7RNA聚合酶体外合成的体外合成RNA的拷贝数约为2×10¹¹拷贝/μg·RNA。对于用体外合成的RNA以与上述方法类似的方式进行转染的情况，群体形成能力是每5×10⁷拷贝1个群体。这些结果揭示，当以相同的拷贝数将提取自复制子-复制细胞的细胞RNA和体外合成的RNA作为复制子RNA各自转染Huh7细胞时，使用Huh7细胞内复制的复制子RNA使群体形成能力约为体外合成的RNA群体形成能力的50倍。

实施例4

(F)检测复制子RNA

根据上述(E)，通过将获自用rSGREP-JFH1转染Huh7细胞所确立的复制子-复制细胞克隆的总RNA再转染进另一Huh7细胞来确立细胞克隆[克隆1至11]。利用酸性苯酚提取法，分别从所确立的细胞克隆和合并克隆(通过收集一整皿已形成群体的细胞克隆并对其进行培养而制得)提取了总RNA。随后利用RNA印迹法以pSGREP-JFH1-特异性探针为探针分析总RNA。使用了：自未转染的Huh7细胞类似提取的总RNA(附图8，示为“Huh7”)、通过向提取自Huh7细胞的总RNA添加10⁷拷贝的体外合成的复制子RNA制得的样品(附图8，示为“10⁷”)、通过向提取自Huh7细胞的总RNA添加10⁸拷贝的体外合成的复制子RNA制得的样品(附图8，示为“10⁸”)，作为对照。附图8中，1至11表示细胞克隆编号。

结果，用pSGREP-JFH1-特异性探针检测到了约与rSGREP-JFH1大小相同的RNA(附图8)。由此证实，最初转染的源自rSGREP-JFH1的复制子RNA在细胞克隆中进行了复制和增殖。此外还显示，细胞克隆彼此间已复制的复制子RNA的量不相同。在附图8中，例如，克隆2、6、9和10含有高量的已复制的复制子RNA，克隆4、8和11含有低量的已复制的复制子RNA。

(G)证实新霉素抗性基因向基因组DNA中整合的存在或不存在

对于如实施例3所述通过复制子RNA再转染所得的细胞克隆，为证实各细胞克隆对G418的抗性不是由于新霉素抗性基因整合进基因组而产生的，用新霉素抗性基因-特异性引物：有义引物，NEO-S3：5′-AACAAGATGGATTGCACGCA-3′(SEQ ID NO：16)和反义引物，NEO-R：5′-CGTCAAGAAGGCGATAGAAG-3′(SEQ ID NO：17)，并以提取自各细胞克隆的宿主细胞基因组DNA作为模板，进行了PCR扩增。此处所使用的细胞克隆是通过再转染rSGREP-JFH1-衍生的已复制复制子RNA所得的第1至8号细胞克隆(rSGREP-JFH1-衍生的细胞克隆No.1至8)，和通过再转染rSGREP-JCH1-衍生的已复制复制子RNA所得的第1至6号细胞克隆(rSGREP-JCH1-衍生的细胞克隆No.1至6)。结果如附图9所示，在所检查的八个rSGREP-JFH1-衍生的细胞克隆中，未观察到显示新霉素抗性基因扩增的阳性克隆。对于rSGREP-JCH1-衍生细胞克隆，所检查的六个克隆中只有一个是阳性克隆(附图9，右图中的泳道3)。该阳性克隆被认为已经通过rSGREP-JCH1-衍生的已复制复制子RNA中的新霉素抗性基因整合进宿主细胞基因组DNA，获得了G418。由此认为在阳性克隆中，不同于其它克隆，复制子RNA本身未在细胞内自主复制。这得到了以下(H)所示的从阳性克隆未检测到HCV蛋白的试验结果的证实。

(H)检测HCV蛋白

用标准程序从rSGREP-JFH1-和rSGREP-JCH1-转染的细胞克隆提取蛋白，并用SDS-PAGE和蛋白印迹法进行分析(附图10)。所检查的细胞克隆与上述(G)中所使用的相同：rSGREP-JFH1-衍生的细胞克隆No.1至8和rSGREP-JCH1-衍生的细胞克隆No.1至6。此外，把通过将包含NS3基因的表达质粒DNA瞬间转染进Huh7细胞所得细胞的细胞提取物视为阳性对照(NS3蛋白)。而且，以提取自未转染的Huh7细胞的蛋白作为阴性对照。将提取自各细胞克隆的蛋白样品印迹至PVDF膜(Immobilon-P，Millipore)，之后用抗-NS3-特异性抗体(由Dr.Moradpour；Wolk B，等人，J.Virology.2000，74：2293-2304提供)检测由已复制的复制子RNA编码的NS3蛋白。如附图10所示，在rSGREP-JFH1-衍生的细胞克隆No.1至8和rSGREP-JCHI-衍生的细胞克隆No.1、2和4至6中，检测到了与阳性对照大小相同的蛋白。在rSGREP-JCH1-衍生的细胞克隆No.3(上述(G)中检测为阳性克隆的克隆)中，未检测到NS3蛋白的表达。也即，在rSGREP-JCH1-衍生的细胞克隆No.3中，证实没有复制子RNA的复制。在未转染的Huh7细胞中未检测到NS3蛋白，这揭示，在检测到NS3蛋白的细胞克隆中，转染的复制子RNA自主复制由此NS3蛋白得以表达。

此外，通过以丙肝患者血清作为抗体，在上述已证实了NS3蛋白表达的各细胞克隆中，还证实了NS5a蛋白从复制子RNA的表达。

基于上述(G)和(H)的结果，证实了复制子RNA在通过复制子RNA转染所确立的细胞克隆中发生了复制。

实施例5

(I)适应性突变分析

根据实施例3的说明，将获自通过用rSGREP-JFH1转染Huh7细胞所确立的复制子-复制细胞克隆的总RNA再转染进另一Huh7细胞，由此确立了21个细胞克隆。用标准程序各从这些细胞克隆提取总RNA。利用作为模板的总RNA、反转录酶Superscript II(Invitrogen)和引物9641R-IH(5′-GCACTCTCTGCAGTCATGCGGCTCACGGAC-3′(SEQ ID NO：18))合成了对应于复制子RNA的cDNA。经反转录反应合成cDNA的反应液组成如下所示。

反应液组成	流体体积(μl)
反应液组成	流体体积(μl)	5×第一链缓冲液	4
2mM dNTP	5	5×第一链缓冲液	4
2mM dNTP	5	0.1M DTT	1
9651R-IH引物(100μM)	1	0.1M DTT	1
9651R-IH引物(100μM)	1	DW(蒸馏水)	6.5
样品RNA(2mg/mL)	1	DW(蒸馏水)	6.5
样品RNA(2mg/mL)	1	RNA酶抑制剂(Promega)(40U/μL)	0.5
Superscript II RT(Invitrogen)	1	RNA酶抑制剂(Promega)(40U/μL)	0.5
Superscript II RT(Invitrogen)	1	总计	20μl

在cDNA合成反应中，将上述除RNA酶抑制剂和Superscript II外的试剂混合制成第一反应液。将该溶液于90℃加热3分钟，之后在冰上冷却。随后，向该反应液中加入RNA酶抑制剂和SuperscriptII，之后使溶液于42℃反应1小时，接着再于70℃反应15分钟。

此外，用所得cDNA和五个引物组根据以下程序进行PCR扩增，由此获得了覆盖几乎整个复制子RNA区域的DNA扩增片段。所使用的引物组和由各引物组扩增出的区域示于下表1和表2。

表1

扩增片段的名称	引物组		扩增的区域
	引物组			引物1	引物2
	A/	42S-IH		引物1	引物2	433R-neo	41-470
B/	A/	42S-IH	C/S17ssp	4680R-IH	28-3026	433R-neo	41-470
B/	C/	4534S-IH	C/S17ssp	4680R-IH	28-3026	7279R-IH	2880-5625
D/	C/	4534S-IH	7198S-IH	9367R-IH	5544-7713	7279R-IH	2880-5625
D/	E/	9247S-NF	7198S-IH	9367R-IH	5544-7713	9576R-NF	7597-7960

表1中，扩增区域用其在rSGREP-JFH1(SEQ ID NO：1)中所对应的核苷酸号表示。

表2

引物名称	核苷酸序列(5′→3′)	SEQ ID NO：
引物名称	核苷酸序列(5′→3′)	SEQ ID NO：	42S-IH	CCCCTGTGAGGAACTACTGTCTTCACGC	SEQ ID NO：19
C/S17ssp	CCGGGAGAGCCATAGTGGTCTGCG	SEQ ID NO：20	42S-IH	CCCCTGTGAGGAACTACTGTCTTCACGC	SEQ ID NO：19
C/S17ssp	CCGGGAGAGCCATAGTGGTCTGCG	SEQ ID NO：20	4534S-IH	CCACTCAAAGAAAAAGTGTGACGAGCTCGC	SEQ ID NO：21
7198S-IH	GGCTTGGGCACGGCCTGA	SEQ ID NO：22	4534S-IH	CCACTCAAAGAAAAAGTGTGACGAGCTCGC	SEQ ID NO：21
7198S-IH	GGCTTGGGCACGGCCTGA	SEQ ID NO：22	9247S-NF	GCGGTGAAGACCAAGCTCAAACTCACTCCA	SEQ ID NO：23
433R-neo	AGAACCTGCGTGCAATCCATC	SEQ ID NO：24	9247S-NF	GCGGTGAAGACCAAGCTCAAACTCACTCCA	SEQ ID NO：23
433R-neo	AGAACCTGCGTGCAATCCATC	SEQ ID NO：24	4680R-IH	CCCGTCATGAGGGCGTCGGTGGC	SEQ ID NO：25
7279R-IH	ACCAGCAACGGTGGGCGGTTGGTAATC	SEQ ID NO：26	4680R-IH	CCCGTCATGAGGGCGTCGGTGGC	SEQ ID NO：25
7279R-IH	ACCAGCAACGGTGGGCGGTTGGTAATC	SEQ ID NO：26	9367R-RI	GGCACGCGACACGCTGTG	SEQ ID NO：27
9576R-NF	AGCTAGCCGTGACTAGGGCTAAGATGGAGC	SEQ ID NO：28	9367R-RI	GGCACGCGACACGCTGTG	SEQ ID NO：27

该PCR反应中的反应液组成如下所示。

反应液组成	流体体积(μl)
反应液组成	流体体积(μl)	引物1(10μM)	1.0
引物2(10μM)	1.0	引物1(10μM)	1.0
引物2(10μM)	1.0	2.5mM dNTPs	5.0
10×LA缓冲液	5.0	2.5mM dNTPs	5.0
10×LA缓冲液	5.0	MgCl₂(25mM)	5.0
LA Taq(TAKARA)(5U/μl)	0.3	MgCl₂(25mM)	5.0
LA Taq(TAKARA)(5U/μl)	0.3	DW(蒸馏水)	30.7
模板cDNA	2.0	DW(蒸馏水)	30.7
模板cDNA	2.0	总计	50μl

此外，PCR反应条件如下：95℃ 2分钟；98℃ 10秒，之后68℃ 8分钟，进行35个循环；和72℃ 7分钟；之后将温度保持在4℃。

确定如上述所得各PCR产物的核苷酸序列，之后将对应于DNA序列的RNA序列与rSGREP-JFH1的序列进行比较。结果示于表3。

表3

区域	同义取代	非同义取代	总突变数
区域	同义取代	非同义取代	总突变数	NS3	0	5	5
NS4A	0	2	2	NS3	0	5	5
NS4A	0	2	2	NS4B	0	3	3
NS5A	0	7	7	NS4B	0	3	3
NS5A	0	7	7	NS5B	3	5	8
总计	3	22	25	NS5B	3	5	8

如表3所示，在21个细胞克隆中观察到的核苷酸突变总数为25。这些突变中有22个是导致氨基酸突变的非同义取代。这些突变的类型如表4所示。此外，这些突变在非结构区中的位置示于附图11。

表4

克隆名称	突变位点
	突变位点				核苷酸号	核苷酸突变	氨基酸突变	氨基酸号
	C1	7098	AG	无	核苷酸号	核苷酸突变	氨基酸突变	氨基酸号
7157		7098	AG	无	AG	YC	2824
7157		C2	4955	CU	AG	YC	2824	AV	2090
C3	4936	C2	4955	CU	AG	TA	2084	AV	2090
	4936	5000	AG	YC	AG	TA	2084	2105
	7287	5000	AG	YC	AG	无		2105
	7287	7288	AG	MV	AG	无		2868
	C4	7288	AG	MV	5901	GU	ED	2868	2405
6113		AU	HL	2476	5901	GU	ED		2405
6113		AU	HL	2476	C5	2890	AG	KE	1402
C6	7209	AG	无		C5	2890	AG	KE	1402

在表4和附图11中，“C1至C6”表示带有已发现具有突变的复制子RNA的复制子-复制细胞克隆C1至C6。“核苷酸号”表示复制子RNA rSGREP-JFH1核苷酸序列(SEQ ID NO：1)中相应的核苷酸号。“氨基酸号”表示由JFH-1克隆编码的氨基酸序列(SEQ IDNO：4)中对应的氨基酸号。突变位点的核苷酸和氨基酸类型根据其通用符号表示。如表4所示，在克隆C2中，复制子RNA上对应于SEQ ID NO：1第4955位核苷酸的核苷酸从C(胞嘧啶)突变为U(尿嘧啶)，其导致对应于SEQ ID NO：4第2090位氨基酸的氨基酸从A(丙氨酸)突变为V(缬氨酸)。

此外，附图11所示的突变位置用表4中所示的核苷酸号和线条表示。粗线条表示非同义取代，而细线条表示同义取代。

有两个克隆根本不含有导致氨基酸突变的核苷酸突变。当对这两个克隆做RNA印迹分析，据显示，在这两个克隆中，所复制的复制子RNA量低于那些含有带有导致氨基酸突变的核苷酸突变的已复制复制子RNA的细胞克隆。由此认为，复制子RNA内导致氨基酸突变的核苷酸突变是一种提高复制子RNA在Huh7细胞中的复制效率的适应性突变。

实施例6

(J)用除Huh7细胞外的细胞确立复制子-复制细胞克隆

根据实施例1所述方法，将rSGREP-JFH1转染进除Huh7细胞之外的一些肝癌细胞和非-肝-衍生细胞。将所转染的细胞接种在培养皿上并然后培养。观察群体形成并对群体计数。用于转染的细胞如下：

(1)HepG2细胞(代表性肝癌细胞和Huh7细胞)

(2)IMY-N9细胞(由Ito等人确立；HepG2细胞和人原代培养肝细胞的融合细胞(Hepatology 2001，34：566-572))

(3)HeLa细胞(人子宫颈癌-衍生细胞(Can Cer Res.1952，12：264-265))

(4)293细胞(人胎肾衍生细胞(Gen.Virol.1977，36：59-72))

分别用HepG2细胞、IMY-N9细胞、HeLa细胞或293细胞转染的结果示于附图12A至D。如附图12A至D所示，对于rSGREP-JFH1-转染的细胞，所有HepG2细胞、IMY-N9细胞、HeLa细胞和293细胞都显示群体形成。

对于所确立的细胞克隆，如下面(L)和(M)所述，检测已复制的复制子RNA，证实新霉素抗性基因向宿主基因组DNA中整合的存在或不存在，和证实HCV蛋白的表达。将已证实了复制子在细胞中复制的细胞克隆视作复制子-复制细胞克隆。具体地说，据证实，用rSGREP-JFH1还使人们能够利用除Huh7细胞之外的肝癌细胞和以前未能成功用于制备HCV复制子-复制细胞的非肝细胞制备HCV复制子-复制细胞(Blight等人，Science，(2000)290；1972-1974)。

(K)用除Huh7细胞之外的细胞检测复制子-复制细胞中的复制子RNA

根据Molecular Cloning，A laboratory Manual，第二版，J.Sambrook，E.F.Fritsch，T.Maniatis，Cold Spring HarborLaboratory Press(1989)的说明做RNA印迹分析。根据上述(J)部分的说明，用酸性苯酚提取法从将rSGREP-JFH1分别转染进HepG2、IMY-N9或HeLa细胞而确立的各个复制子-复制细胞克隆、以及从将rSGREP-JFH1转染进239细胞而确立的复制子-复制细胞的合并克隆(通过收集一整皿已形成群体的细胞克隆并对其进行培养而制得)，提取总RNA。接着，用RNA印迹法以pSGREP-JFH1-特异性探针为探针分析总RNA。使用了：从未经转染的Huh7细胞和HepG2细胞中类似提取的总RNA(附图13中的泳道1和17)、通过向提取自Huh7细胞的总RNA中添加10⁷或10⁸拷贝的体外合成的复制子RNA制得的RNA(附图13中的泳道2和3)，作为对照。结果，用pSGREP-JFH1-特异性探针检测到了约与rSGREP-JFH1相同大小的RNA(附图13)。因此，证实了，最初转染的源自rSGREP-JFH1的复制子RNA在细胞克隆中进行了复制和增殖。此外还揭示，已复制的复制子RNA的量依赖于细胞类型而不同，并发现IMY-N9细胞尤其有效地复制复制子RNA。此外还揭示，克隆彼此间的已复制的复制子RNA的量各不相同。

(L)证实新霉素抗性基因向基因组DNA中整合的存在或不存在

对于由此确立的复制子RNA-复制细胞克隆，为证实各细胞克隆对G418的抗性不是由于新霉素抗性基因整合进基因组而产生的，用新霉素抗性基因-特异性引物(有义引物，NEO-S3：5′-AACAAGATGGATTGCACGCA-3′(SEQ ID NO：29)，反义引物，NEO-R：5′-CGTCAAGAAGGCGATAGAAG-3′(SEQ ID NO：30))，并以提取自各细胞克隆的宿主细胞基因组DNA作为模板，进行了PCR扩增。此处所使用的细胞克隆是通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进HepG2细胞所得的第1、5、7、8、9、10、11、12和13号细胞克隆和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进IMY-N9细胞所得的第3、4、5、6、7、8、9、10和11号细胞克隆。结果如附图14所示，在所检查的九个通过将rSGREP-JFH1导入HepG2细胞获得的细胞克隆中，未观察到显示新霉素抗性基因扩增的阳性克隆。在所检查的九个通过将rSGREP-JFH1导入IMY-N9细胞获得的细胞克隆中，未观察到显示新霉素抗性基因扩增的阳性克隆。

对于用通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进HeLa细胞所得的细胞克隆，和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进293细胞所得的细胞克隆，也做了类似的检查。然而，未观察到显示新霉素抗性基因扩增的阳性克隆。

(M)HCV蛋白的检测

用标准程序从所确立的细胞克隆提取蛋白，之后用SDS-PAGE和蛋白印迹法对其进行分析(附图15)。这种情况中所检查的细胞克隆与上述部分中所使用的细胞克隆相同：通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进HepG2细胞所得的第1、5、7、8、9、10、11、12和13号细胞克隆和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进IMY-N9细胞所得的第3、4、5、6、7、8、9、10和11号细胞克隆。此外，根据以前的报道(Lehmann等人，Science，(1999))，把通过将rSGREP-JFH1导入Huh7细胞所制得的HCV RNA复制子-复制细胞克隆视为阳性对照(附图15，泳道4-1，C6)。此外，将提取自未转染的细胞的蛋白用作阴性对照(附图15，泳道N)。将提取自各细胞克隆的蛋白样品印迹到PVDF膜(Immobilon-P，Millipore)上，之后用抗-NS3-特异性抗体(由Dr.Moradpour；Wolk B，等人，J.Virology.2000，74：2293-2304提供)检测由已复制的复制子RNA编码的NS3蛋白。如附图15上部所示，在通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染所得的第1、5、7、8、9、10、11、12和13号细胞克隆和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进IMY-N9细胞所得的第3、4、5、6、7、8、9、10和11号细胞克隆中检测到了与阳性对照大小相同的蛋白。

此外，通过以丙肝患者血清作为抗体，在上述已证实表达NS3蛋白的各细胞克隆中，证实了NS5a蛋白从复制子RNA的表达。本试验中，除用抗-NS5a抗体替代患者血清外，以类似于NS3蛋白表达情况中的方式做了检查。结果，如附图15下部所示，在通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染所得的第1、5、7、8、9、10、11、12和13号细胞克隆、和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进IMY-N9细胞所得的第3、4、5、6、7、8、9、10和11号细胞克隆中检测到了与阳性对照大小相同的蛋白。

当对通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进HeLa细胞所得的细胞克隆和通过将rSGREP-JFH1-衍生的已复制复制子RNA再转染进293细胞所得的细胞克隆做类似的检查时，可以证实NS3和NS5a蛋白的表达。

如上所述，证实了复制子RNA在通过复制子RNA的转染而确立的细胞克隆中发生了复制。

实施例7

(N)适应性突变分析

根据实施例3的说明，将获自通过将rSGREP-JFH1转染进HepG2和HeLa细胞所确立的复制子-复制细胞克隆的总RNA分别再转染进各细胞系的另一细胞，由此为HepG2细胞确立了14个细胞克隆，为HeLa细胞确立了8个细胞克隆。对这些细胞克隆的每一个，通过标准程序提取了总RNA。使用作为模板的总RNA，反转录酶Superscript II(Invitrogen)和引物9641R-IH(5′-GCACTCTCTGCAGTCATGCGGCTCACGGAC-3′(SEQ ID NO：31))合成了对应于复制子RNA的cDNA。经反转录反应合成cDNA的反应液组成如下所示。

反应液组成	流体体积(μl)
反应液组成	流体体积(μl)	5×第一链缓冲液	4
2mM dNTP	5	5×第一链缓冲液	4

0.1M DTT	1
0.1M DTT	1	9651R-IH引物(100μM)	1
DW(蒸馏水)	6.5	9651R-IH引物(100μM)	1
DW(蒸馏水)	6.5	样品RNA(2mg/mL)	1
RNA酶抑制剂(Promega)(40U/μL)	0.5	样品RNA(2mg/mL)	1
RNA酶抑制剂(Promega)(40U/μL)	0.5	Superscript II RT(Invitrogen)	1
总计	20μl	Superscript II RT(Invitrogen)	1

在cDNA合成反应中，将上述除RNA酶抑制剂和Superscript II外的试剂混合制成第一反应液。将该第一反应液于90℃加热3分钟，之后在冰上冷却。随后，向该反应液中加入RNA酶抑制剂和Superscript II，之后使溶液于42℃反应1小时，接着再于70℃反应15分钟。

此外，用所得cDNA和五个引物组根据以下程序进行PCR扩增，由此获得了覆盖几乎整个复制子RNA区域的DNA扩增片段。所使用的引物组和由各引物组扩增出的区域示于下表5和表6。

表5

扩增片段的名称	引物组		扩增的区域
	引物组			引物1	引物2
	A	42S-IH		引物1	引物2	433R-neo	41-470
B	A	42S-IH	C/S17ssp	4680R-IH	28-3026	433R-neo	41-470
B	C	4534S-IH	C/S17ssp	4680R-IH	28-3026	7279R-IH	2280-5625
D	C	4534S-IH	7198S-IH	9367R-IH	5544-7713	7279R-IH	2280-5625
D	E	9247S-NF	7198S-IH	9367R-IH	5544-7713	9576R-NF	7597-7966

在该表中，扩增区域用其在rSGREP-JFH1(SEQ ID NO：1)中所对应的核苷酸号表示。

表6

引物名称	核苷酸序列(5′→3′)	SEQ ID NO：
引物名称	核苷酸序列(5′→3′)	SEQ ID NO：	43S-IH	CCCCTGTGAGGAACTACTGTCTTCACGC	SEQ ID NO：32
C/S17ssp	CCGGGAGAGCCATAGTGGTCTGCG	SEQ ID NO：33	43S-IH	CCCCTGTGAGGAACTACTGTCTTCACGC	SEQ ID NO：32
C/S17ssp	CCGGGAGAGCCATAGTGGTCTGCG	SEQ ID NO：33	4534S-IH	CCACTCAAAGAAAAAGTGTGACGAGCTCGC	SEQ ID NO：34
7198S-IH	GGCTTGGGCACGGCCTGA	SEQ ID NO：35	4534S-IH	CCACTCAAAGAAAAAGTGTGACGAGCTCGC	SEQ ID NO：34
7198S-IH	GGCTTGGGCACGGCCTGA	SEQ ID NO：35	9247S-NF	GCGGTGAAGACCAAGCTCAAACTCACTCCA	SEQ ID NO：36
433R-neo	AGAACCTGCGTGCAATCCATC	SEQ ID NO：37	9247S-NF	GCGGTGAAGACCAAGCTCAAACTCACTCCA	SEQ ID NO：36
433R-neo	AGAACCTGCGTGCAATCCATC	SEQ ID NO：37	4680R-IH	CCCGTCATGAGGGCGTCGGTGGC	SEQ ID NO：38
7279R-IH	ACCAGCAACGGTGGGCGGTTGGTAATC	SEQ ID NO：39	4680R-IH	CCCGTCATGAGGGCGTCGGTGGC	SEQ ID NO：38
7279R-IH	ACCAGCAACGGTGGGCGGTTGGTAATC	SEQ ID NO：39	9367R-IH	GGAACGCGACACGCTGTG	SEQ ID NO：40
9576R-NF	AGCTAGCCGTGACTAGGGCTAAGATGGAGC	SEQ ID NO：41	9367R-IH	GGAACGCGACACGCTGTG	SEQ ID NO：40

该PCR反应中的反应液组成如下所示。

此外，PCR反应条件如下：95℃ 2分钟；98℃ 10秒，之后68℃ 8分钟，进行35个循环；随后72℃ 7分钟；之后将温度保持在4℃。

确定如上述所得各PCR产物的核苷酸序列，之后将对应于DNA序列的RNA序列与rSGREP-JFH1的序列进行比较。结果示于表7和表8。

表7：HepG2细胞中的JFH-1复制子适应性突变的分析

克隆	突变位点		突变
	突变位点		突变		核苷酸号	氨基酸号	核苷酸	氨基酸
	HepIH1	6826	2714	CA	核苷酸号	氨基酸号	核苷酸	氨基酸	QK
HepIH3	HepIH1	6826	2714	CA	6887	2734	CA	TN	QK
HepIH3	HepIH5	6887		UA	6887	2734	CA	TN	无
HepIH8	HepIH5	6887		UA	6580	2632	UA	ST	无
	7159	2825	UC	YH	6580	2632	UA	ST
	7159	2825	UC	YH	HepIH9	3342		AG	无
3594		CA	无			3342		AG	无
3594		CA	无	7230		2848	UA	NK
HepIH10	5052		UC	7230		2848	UA	NK	无
	5052		UC	6943	2753	CA	PT		无
	HepIH12	无		6943	2753	CA	PT
HepIH13	HepIH12	无		4302		CU	无
	5687	2334	GA	4302		CU	无	GD
	5687	2334	GA	6110	2475	AG	YC	GD

如表7所示，对于HepG2细胞，在8个细胞克隆中共观察到了13个核苷酸突变，这些突变中有8个是导致氨基酸突变的非同义取代。这些突变的类型示于表8。另一方面，对于HeLa细胞，在3个细胞克隆中总共观察到了7个核苷酸突变，这些突变中有5个是导致氨基酸突变的非同义取代。这些突变的类型示于表8。

表8：HeLa细胞中的JFH-1复制子适应性突变的分析

克隆	突变位点		突变
	突变位点		突变		核苷酸号	氨基酸号	核苷酸	氨基酸
	HeLaH1	无			核苷酸号	氨基酸号	核苷酸	氨基酸
HeLaIH2	HeLaH1	无			5550	2272	UC	SP
	6252		AG	无	5550	2272	UC	SP
	6252		AG	无	7182		UC	无
	7217	2844	AG	HR	7182		UC	无
	7217	2844	AG	HR	HeLaIH5	3643	1653	AG	MV
5851	2389	GA	AT			3643	1653	AG	MV
5851	2389	GA	AT	5914		2410	GA	EK

在表7中，“HepIH号”表示含有复制子RNA、且已经用HepG2细胞克隆了的复制子-复制细胞克隆的克隆号。“核苷酸号”表示复制子RNA rSGREP-JFH1核苷酸序列(SEQ ID NO：1)中相应的核苷酸号。“氨基酸号”表示由JFH-1克隆编码的氨基酸序列(SEQ IDNO：4)中对应的氨基酸号。突变位点的核苷酸和氨基酸类型根据其通用符号表示。如表7所示，例如，在克隆HepIH1中，复制子RNA上对应于SEQ ID NO：第6826位的核苷酸从C突变为A，由此导致对应于SEQ ID NO：第2714位氨基酸的氨基酸从Q突变为E。类似地，在表8中，“HeLaIH号”表示含有复制子RNA、且已经用HeLa细胞克隆了的复制子-复制细胞克隆的克隆号。

此外，当对在根本不含有导致氨基酸突变的核苷酸突变的克隆做RNA印迹分析时，据显示，由该克隆所复制的复制子RNA的量低于那些使带有导致氨基酸突变的核苷酸突变的复制子RNA复制的细胞克隆。由此得出结论，复制子RNA内诱导氨基酸突变的核苷酸突变是一种提高复制子RNA在细胞中的复制效率的适应性突变。

工业实用性

本发明的复制子-复制细胞可用作连续生产HCV基因型2a-衍生的RNA和HCV蛋白的培养系统。此外，本发明的复制子-复制细胞可用作筛选各种影响HCV复制和/或HCV蛋白翻译的物质的测试系统。

本文所引用的所有公开文本、专利和专利申请都全文引入本文作为参考。

无文本序列表

SEQ ID NO：1.人工序列说明：复制子

SEQ ID NO：2.人工序列说明：复制子

SEQ ID NO：7.人工序列说明：复制子

SEQ ID NOS：8至12.人工序列说明：合成的RNA

SEQ ID NOS：13至41.人工序列说明：合成的DNA

序列表

<110>Toray Industries Inc.

Tokyo Metropolitan Organization for Medical Research

Johannes Gutenberg-Universitaet Mainz

<120>含有衍生自基因型2a丙肝病毒(HCV)基因组的核酸的核酸构建体和具有转移入其中的这样的核酸构建体的细胞

<130>PH-1953-PCT

<140>PCT/JP2003/015038

<141>2003-11-25

<150>JP 2003-148242

<151>2003-05-26

<150>JP 2003-329115

<151>2003-09-19

<160>41

<170>PatentIn Ver.2.1

<210>1

<211>8024

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：复制子

<220>

<223>Inventor：Wakita，Takaji

Inventor：Kato，Takanobu

Inventor：Date，Tomoko

<400>1

accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360

ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420

cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480

aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540

ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600

guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660

aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720

uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780

ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840

ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900

cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960

uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020

cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080

ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140

ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200

ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260

cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320

aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380

ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440

caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500

gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560

uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620

ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680

caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740

acgugguuuu ccuuugaaaa acacgaugau accauggcuc ccaucacugc uuaugcccag 1800

caaacacgag gccuccuggg cgccauagug gugaguauga cggggcguga caggacagaa 1860

caggccgggg aaguccaaau ccuguccaca gucucucagu ccuuccucgg aacaaccauc 1920

ucggggguuu uguggacugu uuaccacgga gcuggcaaca agacucuagc cggcuuacgg 1980

gguccgguca cgcagaugua cucgagugcu gagggggacu ugguaggcug gcccagcccc 2040

ccugggacca agucuuugga gccgugcaag uguggagccg ucgaccuaua ucuggucacg 2100

cggaacgcug augucauccc ggcucggaga cgcggggaca agcggggagc auugcucucc 2160

ccgagaccca uuucgaccuu gaaggggucc ucgggggggc cggugcucug cccuaggggc 2220

cacgucguug ggcucuuccg agcagcugug ugcucucggg gcguggccaa auccaucgau 2280

uucauccccg uugagacacu cgacguuguu acaaggucuc ccacuuucag ugacaacagc 2340

acgccaccgg cugugcccca gaccuaucag gucggguacu ugcaugcucc aacuggcagu 2400

ggaaagagca ccaagguccc ugucgcguau gccgcccagg gguacaaagu acuagugcuu 2460

aaccccucgg uagcugccac ccugggguuu ggggcguacc uauccaaggc acauggcauc 2520

aaucccaaca uuaggacugg agucaggacc gugaugaccg gggaggccau cacguacucc 2580

acauauggca aauuucucgc cgaugggggc ugcgcuagcg gcgccuauga caucaucaua 2640

ugcgaugaau gccacgcugu ggaugcuacc uccauucucg gcaucggaac gguccuugau 2700

caagcagaga cagccggggu cagacuaacu gugcuggcua cggccacacc ccccggguca 2760

gugacaaccc cccaucccga uauagaagag guaggccucg ggcgggaggg ugagaucccc 2820

uucuauggga gggcgauucc ccuauccugc aucaagggag ggagacaccu gauuuucugc 2880

cacucaaaga aaaaguguga cgagcucgcg gcggcccuuc ggggcauggg cuugaaugcc 2940

guggcauacu auagaggguu ggacgucucc auaauaccag cucagggaga uguggugguc 3000

gucgccaccg acgcccucau gacgggguac acuggagacu uugacuccgu gaucgacugc 3060

aauguagcgg ucacccaagc ugucgacuuc agccuggacc ccaccuucac uauaaccaca 3120

cagacugucc cacaagacgc ugucucacgc agucagcgcc gcgggcgcac agguagagga 3180

agacagggca cuuauaggua uguuuccacu ggugaacgag ccucaggaau guuugacagu 3240

guagugcuuu gugagugcua cgacgcaggg gcugcguggu acgaucucac accagcggag 3300

accaccguca ggcuuagagc guauuucaac acgcccggcc uacccgugug ucaagaccau 3360

cuugaauuuu gggaggcagu uuucaccggc cucacacaca uagacgccca cuuccucucc 3420

caaacaaagc aagcggggga gaacuucgcg uaccuaguag ccuaccaagc uacggugugc 3480

gccagagcca aggccccucc cccguccugg gacgccaugu ggaagugccu ggcccgacuc 3540

aagccuacgc uugcgggccc cacaccucuc cuguaccguu ugggcccuau uaccaaugag 3600

gucacccuca cacacccugg gacgaaguac aucgccacau gcaugcaagc ugaccuugag 3660

gucaugacca gcacgugggu ccuagcugga ggaguccugg cagccgucgc cgcauauugc 3720

cuggcgacug gaugcguuuc caucaucggc cgcuugcacg ucaaccagcg agucgucguu 3780

gcgccggaua aggagguccu guaugaggcu uuugaugaga uggaggaaug cgccucuagg 3840

gcggcucuca ucgaagaggg gcagcggaua gccgagaugu ugaaguccaa gauccaaggc 3900

uugcugcagc aggccucuaa gcaggcccag gacauacaac ccgcuaugca ggcuucaugg 3960

cccaaagugg aacaauuuug ggccagacac auguggaacu ucauuagcgg cauccaauac 4020

cucgcaggau ugucaacacu gccagggaac cccgcggugg cuuccaugau ggcauucagu 4080

gccgcccuca ccaguccguu gucgaccagu accaccaucc uucucaacau caugggaggc 4140

ugguuagcgu cccagaucgc accacccgcg ggggccaccg gcuuugucgu caguggccug 4200

gugggggcug ccgugggcag cauaggccug gguaaggugc ugguggacau ccuggcagga 4260

uauggugcgg gcauuucggg ggcccucguc gcauucaaga ucaugucugg cgagaagccc 4320

ucuauggaag augucaucaa ucuacugccu gggauccugu cuccgggagc ccugguggug 4380

ggggucaucu gcgcggccau ucugcgccgc cacgugggac cgggggaggg cgcgguccaa 4440

uggaugaaca ggcuuauugc cuuugcuucc agaggaaacc acgucgcccc uacucacuac 4500

gugacggagu cggaugcguc gcagcgugug acccaacuac uuggcucucu uacuauaacc 4560

agccuacuca gaagacucca caauuggaua acugaggacu gccccauccc augcuccgga 4620

uccuggcucc gcgacgugug ggacuggguu ugcaccaucu ugacagacuu caaaaauugg 4680

cugaccucua aauuguuccc caagcugccc ggccuccccu ucaucucuug ucaaaagggg 4740

uacaagggug ugugggccgg cacuggcauc augaccacgc gcugcccuug cggcgccaac 4800

aucucuggca auguccgccu gggcucuaug aggaucacag ggccuaaaac cugcaugaac 4860

accuggcagg ggaccuuucc uaucaauugc uacacggagg gccagugcgc gccgaaaccc 4920

cccacgaacu acaagaccgc caucuggagg guggcggccu cggaguacgc ggaggugacg 4980

cagcaugggu cguacuccua uguaacagga cugaccacug acaaucugaa aauuccuugc 5040

caacuaccuu cuccagaguu uuucuccugg guggacggug ugcagaucca uagguuugca 5100

cccacaccaa agccguuuuu ccgggaugag gucucguucu gcguugggcu uaauuccuau 5160

gcugucgggu cccagcuucc cugugaaccu gagcccgacg cagacguauu gagguccaug 5220

cuaacagauc cgccccacau cacggcggag acugcggcgc ggcgcuuggc acggggauca 5280

ccuccaucug aggcgagcuc cucagugagc cagcuaucag caccgucgcu gcgggccacc 5340

ugcaccaccc acagcaacac cuaugacgug gacauggucg augccaaccu gcucauggag 5400

ggcggugugg cucagacaga gccugagucc agggugcccg uucuggacuu ucucgagcca 5460

auggccgagg aagagagcga ccuugagccc ucaauaccau cggagugcau gcuccccagg 5520

agcggguuuc cacgggccuu accggcuugg gcacggccug acuacaaccc gccgcucgug 5580

gaaucgugga ggaggccaga uuaccaaccg cccaccguug cugguugugc ucuccccccc 5640

cccaagaagg ccccgacgcc ucccccaagg agacgccgga cagugggucu gagcgagagc 5700

accauaucag aagcccucca gcaacuggcc aucaagaccu uuggccagcc ccccucgagc 5760

ggugaugcag gcucguccac gggggcgggc gccgccgaau ccggcggucc gacguccccu 5820

ggugagccgg cccccucaga gacagguucc gccuccucua ugcccccccu cgagggggag 5880

ccuggagauc cggaccugga gucugaucag guagagcuuc aaccuccccc ccaggggggg 5940

gggguagcuc ccgguucggg cucggggucu uggucuacuu gcuccgagga ggacgauacc 6000

accgugugcu gcuccauguc auacuccugg accggggcuc uaauaacucc cuguagcccc 6060

gaagaggaaa aguugccaau caacccuuug aguaacucgc uguugcgaua ccauaacaag 6120

guguacugua caacaucaaa gagcgccuca cagagggcua aaaagguaac uuuugacagg 6180

acgcaagugc ucgacgccca uuaugacuca gucuuaaagg acaucaagcu agcggcuucc 6240

aaggucagcg caaggcuccu caccuuggag gaggcgugcc aguugacucc accccauucu 6300

gcaagaucca aguauggauu cggggccaag gagguccgca gcuuguccgg gagggccguu 6360

aaccacauca aguccgugug gaaggaccuc cuggaagacc cacaaacacc aauucccaca 6420

accaucaugg ccaaaaauga gguguucugc guggaccccg ccaagggggg uaagaaacca 6480

gcucgccuca ucguuuaccc ugaccucggc guccgggucu gcgagaaaau ggcccucuau 6540

gacauuacac aaaagcuucc ucaggcggua augggagcuu ccuauggcuu ccaguacucc 6600

ccugcccaac ggguggagua ucucuugaaa gcaugggcgg aaaagaagga ccccaugggu 6660

uuuucguaug auacccgaug cuucgacuca accgucacug agagagacau caggaccgag 6720

gaguccauau accaggccug cucccugccc gaggaggccc gcacugccau acacucgcug 6780

acugagagac uuuacguagg agggcccaug uucaacagca agggucaaac cugcgguuac 6840

agacguugcc gcgccagcgg ggugcuaacc acuagcaugg guaacaccau cacaugcuau 6900

gugaaagccc uagcggccug caaggcugcg gggauaguug cgcccacaau gcugguaugc 6960

ggcgaugacc uaguagucau cucagaaagc caggggacug aggaggacga gcggaaccug 7020

agagccuuca cggaggccau gaccagguac ucugccccuc cuggugaucc ccccagaccg 7080

gaauaugacc uggagcuaau aacauccugu uccucaaaug ugucuguggc guugggcccg 7140

cggggccgcc gcagauacua ccugaccaga gacccaacca cuccacucgc ccgggcugcc 7200

ugggaaacag uuagacacuc cccuaucaau ucauggcugg gaaacaucau ccaguaugcu 7260

ccaaccauau ggguucgcau gguccuaaug acacacuucu ucuccauucu caugguccaa 7320

gacacccugg accagaaccu caacuuugag auguauggau caguauacuc cgugaauccu 7380

uuggaccuuc cagccauaau ugagagguua cacgggcuug acgccuuuuc uaugcacaca 7440

uacucucacc acgaacugac gcggguggcu ucagcccuca gaaaacuugg ggcgccaccc 7500

cucagggugu ggaagagucg ggcucgcgca gucagggcgu cccucaucuc ccguggaggg 7560

aaagcggccg uuugcggccg auaucucuuc aauugggcgg ugaagaccaa gcucaaacuc 7620

acuccauugc cggaggcgcg ccuacuggac uuauccaguu gguucaccgu cggcgccggc 7680

gggggcgaca uuuuucacag cgugucgcgc gcccgacccc gcucauuacu cuucggccua 7740

cuccuacuuu ucguaggggu aggccucuuc cuacuccccg cucgguagag cggcacacac 7800

uagguacacu ccauagcuaa cuguuccuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 7860

uuuuuuuuuu cuuuuuuuuu uuuuucccuc uuucuucccu ucucaucuua uucuacuuuc 7920

uuucuuggug gcuccaucuu agcccuaguc acggcuagcu gugaaagguc cgugagccgc 7980

augacugcag agagugccgu aacuggucuc ucugcagauc augu 8024

<210>2

<211>8024

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：复制子

<400>2

acccgccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgucua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

ugcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa aucccaaacc 360

ucaaagaaaa accaaaagaa acacuaaccg ucgcccaaug auugaacaag auggauugca 420

cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480

aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540

ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600

guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660

aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720

uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780

ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840

ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900

cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960

uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020

cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080

ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140

ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200

ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260

cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320

aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380

ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440

caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500

gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560

uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620

ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680

caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740

acgugguuuu ccuuugaaaa acacgauaau accauggccc ccaucaccgc uuacgcccag 1800

cagacacgag gucucuuggg cucuauagug gugagcauga cggggcguga caagacagaa 1860

caggccgggg agguccaagu ccuguccaca gucacucagu ccuuccucgg aacauccauu 1920

ucgggggucu uauggacugu uuaccacgga gcuggcaaca agacacuagc cggcucgcgg 1980

ggcccgguca cgcagaugua cucgagcgcc gagggggacu uggucgggug gcccagcccu 2040

ccugggacca aaucuuugga gccguguacg uguggagcgg ucgaccugua uuuggucacg 2100

cggaacgcug augucauccc ggcucgaaga cgcggggaca agcggggagc gcugcucucc 2160

ccgagacccc uuucgaccuu gaaggggucc ucggggggac cugugcuuug cccuaggggc 2220

cacgcugucg gaaucuuccg ggcagcugug ugcucucggg guguggcuaa guccauagau 2280

uucauccccg uugagacgcu cgacaucguc acgcggucuc ccaccuuuag ugacaacagc 2340

acaccaccag cugugcccca gaccuaucag gugggguacu ugcacgcccc cacuggcagu 2400

ggaaaaagca ccaagguccc cgucgcguac gccgcccagg gguauaaagu gcuggugcuc 2460

aaucccucgg uggcugccac ccugggauuu ggggcguacu uguccaaggc acauggcauc 2520

aaccccaaca uuaggacugg agucagaacu gugacgaccg gggagcccau uacauacucc 2580

acguauggua aauuccucgc cgaugggggc ugcgcaggcg gcgccuauga caucaucaua 2640

ugcgaugaau gccacucugu ggaugcuacc acuauucucg gcaucgggac aguccuugac 2700

caagcagaga cagccggggu caggcuaacu guacuggcca cggccacgcc ccccgggucg 2760

gugacaaccc cccaucccaa uauagaggag guagcccucg gacaggaggg ugagaucccc 2820

uucuauggga gggcguuucc ccugucuuac aucaagggag ggaggcacuu gauuuucugc 2880

cacucaaaga aaaaguguga cgagcucgca acggcccuuc ggggcauggg cuugaacgcu 2940

guggcauauu acagaggguu ggacgucucc auaauaccaa cucaaggaga uguggugguc 3000

guugccaccg acgcccucau gacgggguau acuggagacu uugacuccgu gaucgacugc 3060

aacguagcgg ucacccaggc cguagacuuc agccuggacc ccaccuucac uauaaccaca 3120

cagacugucc cgcaagacgc ugucucacgu agucagcgcc gagggcgcac ggguagagga 3180

agacugggca uuuauaggua uguuuccacu ggugagcgag ccucaggaau guuugacagu 3240

guaguacucu gugagugcua cgacgcagga gcugcuuggu augagcucuc accaguggag 3300

acgaccguca ggcucagggc guauuucaac acgccuggcu ugccugugug ccaggaccac 3360

cuugaguuuu gggaggcagu uuucaccggc cucacacaca uagacgcuca uuuccuuucc 3420

cagacaaagc agucggggga aaauuucgca uacuuaguag ccuaucaggc cacagugugc 3480

gccagggcca aagcgccccc cccguccugg gacgucaugu ggaagugcuu gacucgacuc 3540

aagcccacgc uugugggccc uacaccucuc cuguaccguu ugggcucugu uaccaacgag 3600

gucacccuua cacaccccgu gacaaaauac aucgccacau gcaugcaagc ugaccucgag 3660

gucaugacca gcacgugggu ccuggcuggg ggagucuuag cagccgucgc cgcguauugc 3720

uuagcgaccg gguguguuuc caucauuggc cguuuacaca ucaaccagcg agcugucguc 3780

gcuccggaca aggagguccu cuaugaggcu uuugaugaga uggaggaaug ugccuccaga 3840

gcggcucucc uugaagaggg gcagcggaua gccgagaugc ugaaguccaa gauccaaggc 3900

uuauugcagc aagccucuaa acaggcccag gacauacaac ccgcugugca agcuucgugg 3960

cccaagaugg agcaauucug ggccaaacau auguggaacu ucauaagcgg cauucaguac 4020

cucgcaggac ugucaacacu gccagggaac ccugcugugg cuuccaugau ggcauucagc 4080

gccgcccuca ccaguccguu gucaacuagc accaccaucc uucuuaacau ucuggggggc 4140

uggcuggcgu cccaaauugc gccacccgcg ggggccacug gcuuuguugu caguggccug 4200

gugggagcug cuguuggcag cauaggcuug gguaaagugc ugguggacau ccuggcaggg 4260

uauggugcgg gcauuucggg ggcccucguc gcguuuaaga ucaugucugg cgagaagccc 4320

uccauggagg augucaucaa cuugcugccu gggauucugu cuccaggugc ucugguggug 4380

ggagucaucu gcgcggccau ucugcgccgc caugugggac cgggggaagg cgcgguccaa 4440

uggaugaaca ggcuuaucgc cuucgcuucc agaggaaacc acgucgcccc uacucacuac 4500

gugacggagu cggaugcguc gcagcguguc acccaacugc uuggcucucu cacuauaacu 4560

agucuacuca ggagacuuca caacuggauc acugaggauu gccccauccc augcgccggc 4620

ucguggcucc gcgaugugug ggacuggguc uguaccaucc uaacagacuu uaagaacugg 4680

cugaccucca agcuguuccc aaagaugccu ggccuccccu uuaucucuug ccaaaagggg 4740

uacaagggcg ugugggccgg cacuggcauc augaccacac gaugccccug cggcgccaac 4800

aucucuggca acguccgcuu gggcucuaug agaaucacag gacccaaaac cugcaugaac 4860

accuggcagg ggaccuuucc uaucaauugu uauacagaag gccagugcuu gccgaaaccc 4920

gcguuaaacu ucaagaccgc caucuggaga guggcggccu cagaguacgc ggaagugacg 4980

cagcacggau cauaugccua uauaacaggg cugaccacug acaacuuaaa agucccuugc 5040

caacuccccu cuccagaguu uuucucuugg guggacggag uacaaaucca uagguccgcc 5100

cccacaccaa agccguuuuu ccgggaugag gucucguuca gcguugggcu caauucauuu 5160

gucgucgggu cucagcuucc cugugacccu gagcccgaca cugagguagu gauguccaug 5220

cuaacagacc caucccauau cacggcggag gcugcagcgc ggcguuuagc gcggggguca 5280

cccccaucug aggcaagcuc cucagcgagc cagcugucgg cgccaucgcu gcgagccacc 5340

ugcaccaccc acgguaggac cuaugaugug gacauggugg augccaaccu guucaugggg 5400

ggcggcguga uucggauaga gucugagucc aaaguggucg uucuggacuc ccucgacuca 5460

augaccgagg aagagggcga ccuugagccu ucaguaccau cggaguauau gcuccccagg 5520

aagagguucc caccggccuu accggcuugg gcgcggccug auuacaaccc accgcuugug 5580

gaaucgugga agaggccaga uuaccaacca cccacuguug cgggcugugc ucuccccccc 5640

cccaaaaaga ccccgacgcc uccuccaagg agacgccgga cagugggucu gagcgagagc 5700

accauaggag augcccucca acagcuggcc aucaaguccu uuggccagcc ccccccaagc 5760

ggcgauucag gccuuuccac gggggcggac gccgccgacu ccggcgaucg gacacccccu 5820

gacgaguugg cucuuucgga gacagguucu accuccucca ugcccccccu cgagggggag 5880

ccuggggacc cagaccugga gccugagcag guagagcuuc aaccuccucc ccaggggggg 5940

gaggcagcuc ccggcucgga cucggggucc uggucuacuu gcuccgagga ggaugacucc 6000

gucgugugcu gcuccauguc auauuccugg accggggcuc uaauaacucc uuguagcccc 6060

gaagaggaaa aguugccaau uaacuccuug agcaacucgc uguugcgaua ccauaacaag 6120

guauacugua cuacaucaaa gagugccuca cuaagggcua aaaagguaac uuuugauagg 6180

augcaagugc ucgacgccua uuaugauuca gucuuaaagg acaucaagcu agcggccucc 6240

aaggucagcg caaggcuccu caccuuagag gaggcgugcc aauugacccc accccacucu 6300

gcaagaucca aguauggguu uggggcuaag gagguccgca gcuuguccgg gagggccguc 6360

aaccacauca aguccgugug gaaggaccuc uuggaagacu cacaaacacc aauuccuaca 6420

accaucaugg ccaaaaauga gguguucugc guggaccccg ccaagggggg uaaaaaacca 6480

gcucgccuua ucguuuaccc ugaccucggc gucagggucu gcgagaagau ggcccuuuau 6540

gaugucacac aaaagcuucc ucaggcggug augggggcuu cuuauggcuu ccaguacucc 6600

cccgcucagc ggguggaguu ucucuugaag gcaugggcgg aaaagagaga cccuaugggu 6660

uuuucguaug auacccgaug cuuugacuca accgucacug agagagacau caggacugag 6720

gaguccauau accaggccug cuccuuaccc gaggaggccc gaacugccau acacucgcug 6780

acugagagac ucuauguggg agggcccaug uucaacagca agggccaguc cugcggguac 6840

aggcguugcc gcgccagcgg ggugcuuacc acuaguaugg ggaacaccau cacaugcuau 6900

guaaaagccc uagcggcuug caaggcugcg gggauaauug cgcccacgau gcugguaugc 6960

ggcgacgacu uggucgucau cucagaaagc caggggacug aggaggacga gcggaaccug 7020

agagccuuca cggaggcuau gaccagguau ucugccccuc cuggugaccc ccccagaccg 7080

gaauaugacc uggagcuaau aacaucuugu uccucaaacg ugucuguggc acuuggccca 7140

cagggccgcc gcagauacua ccugaccaga gaccccacca cuucaauugc ccgggcugcc 7200

ugggaaacag uuagacacuc cccugucaau ucauggcugg gaaacaucau ccaguacgcu 7260

ccaaccauau ggguucgcau gguccugaug acacacuucu ucuccauucu cauggcccag 7320

gacacccuag accagaaccu uaacuuugaa auguacggau cgguguacuc cgugaguccu 7380

cuggaccucc cagccauaau ugaaagguua cacgggcuug acgccuucuc ucugcacaca 7440

uacacucccc acgaacugac gcggguggcu ucagcccuca gaaaacuugg ggcgccaccc 7500

cucagagcgu ggaagagucg ggcgcgugca guuagggcgu cccucaucuc ccgugggggg 7560

agggcggccg uuugcggucg guaccucuuc aacugggcgg ugaagaccaa gcucaaacuc 7620

acuccuuugc cggaggcacg ccuccuggau uuguccaguu gguuuaccgu cggcgccggc 7680

gggggcgaca uuuaucacag cgugucgcgu gcccgacccc gccuauuacu ccuuagccua 7740

cuccuacuuu cuguaggggu aggccucuuc cuacuccccg cucgauagag cggcacacau 7800

uagcuacacu ccauagcuaa cuguuccuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 7860

uuuuuuuuuu cuuuuuuuuu uuuuucccuc uuucuucccu ucucaucuua uucuacuuuc 7920

uuucuuggug gcuccaucuu agcccuaguc acggcuagcu gugaaagguc cgugagccgc 7980

augacugcag agagugccgu aacuggucuc ucugcagauc augu 8024

<210>3

<211>9678

<212>DNA

<213>丙肝病毒

<220>

<221>CDS

<222>(341).(9442)

<400>3

acctgcccct aataggggcg acactccgcc atgaatcact cccctgtgag gaactactgt 60

cttcacgcag aaagcgccta gccatggcgt tagtatgagt gtcgtacagc ctccaggccc 120

ccccctcccg ggagagccat agtggtctgc ggaaccggtg agtacaccgg aattgccggg 180

aagactgggt cctttcttgg ataaacccac tctatgcccg gccatttggg cgtgcccccg 240

caagactgct agccgagtag cgttgggttg cgaaaggcct tgtggtactg cctgataggg 300

cgcttgcgag tgccccggga ggtctcgtag accgtgcacc atg agc aca aat cct 355

Met Ser Thr Asn Pro

1 5

aaa cct caa aga aaa acc aaa aga aac acc aac cgt cgc cca gaa gac 403

Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Glu Asp

10 15 20

gtt aag ttc ccg ggc ggc ggc cag atc gtt ggc gga gta tac ttg ttg 451

Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu

25 30 35

ccg cgc agg ggc ccc agg ttg ggt gtg cgc acg aca agg aaa act tcg 499

Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Thr Thr Arg Lys Thr Ser

40 45 50

gag cgg tcc cag cca cgt ggg aga cgc cag ccc atc ccc aaa gat cgg 547

Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Asp Arg

55 60 65

cgc tcc act ggc aag gcc tgg gga aaa cca ggt cgc ccc tgg ccc cta 595

Arg Ser Thr Gly Lys Ala Trp Gly Lys Pro Gly Arg Pro Trp Pro Leu

70 75 80 85

tat ggg aat gag gga ctc ggc tgg gca gga tgg ctc ctg tcc cce cga 643

Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg

90 95 100

ggc tct cgc ccc tcc tgg ggc ccc act gac ccc cgg cat agg tcg cgc 691

Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg

105 110 115

aac gtg ggt aaa gtc atc gac acc cta acg tgt ggc ttt gcc gac ctc 739

Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu

120 125 130

atg ggg tac atc ccc gtc gta ggc gcc ccg ctt agt ggc gcc gcc aga 787

Met Gly Tyr Ile Pro Val Val Gly Ala Pro Leu Ser Gly Ala Ala Arg

135 140 145

gct gtc gcg cac ggc gtg aga gtc ctg gag gac ggg gtt aat tat gca 835

Ala Val Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala

150 155 160 165

aca ggg aac cta ccc ggt ttc ccc ttt tct atc ttc ttg ctg gcc ctg 883

Thr Gly Asn Leu Pro Gly Phe Pro Phe Ser Ile Phe Leu Leu Ala Leu

170 175 180

ttg tcc tgc atc acc gtt ccg gtc tct gct gcc cag gtg aag aat acc 931

Leu Ser Cys Ile Thr Val Pro Val Ser Ala Ala Gln Val Lys Asn Thr

185 190 195

agt agc agc tac atg gtg acc aat gac tgc tcc aat gac agc atc act 979

Ser Ser Ser Tyr Met Val Thr Asn Asp Cys Ser Asn Asp Ser Ile Thr

200 205 210

tgg cag ctc gag gct gcg gtt ctc cac gtc ccc ggg tgc gtc ccg tgc 1027

Trp Gln Leu Glu Ala Ala Val Leu His Val Pro Gly Cys Val Pro Cys

215 220 225

gag aga gtg ggg aat acg tca cgg tgt tgg gtg cca gtc tcg cca aac 1075

Glu Arg Val Gly Asn Thr Ser Arg Cys Trp Val Pro Val Ser Pro Asn

230 235 240 245

atg gct gtg cgg cag ccc ggt gcc ctc acg cag ggt ctg cgg acg cac 1123

Met Ala Val Arg Gln Pro Gly Ala Leu Thr Gln Gly Leu Arg Thr His

250 255 260

atc gat atg gtt gtg atg tcc gcc acc ttc tgc tct gct ctc tac gtg 1171

Ile Asp Met Val Val Met Ser Ala Thr Phe Cys Ser Ala Leu Tyr Val

265 270 275

ggg gac ctc tgt ggc ggg gtg atg ctc gcg gcc cag gtg ttc atc gtc 1219

Gly Asp Leu Cys Gly Gly Val Met Leu Ala Ala Gln Val Phe Ile Val

280 285 290

tcg ccg cag tac cac tgg ttt gtg caa gaa tgc aat tgc tcc atc tac 1267

Ser Pro Gln Tyr His Trp Phe Val Gln Glu Cys Asn Cys Ser Ile Tyr

295 300 305

cct ggc acc atc act gga cac cgc atg gca tgg gac atg atg atg aac 1315

Pro Gly Thr Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn

310 315 320 325

tgg tcg ccc acg gcc acc atg atc ctg gcg tac gtg atg cgc gtc ccc 1363

Trp Ser Pro Thr Ala Thr Met Ile Leu Ala Tyr Val Met Arg Val Pro

330 335 340

gag gtc atc ata gac atc gtt agc ggg gct cac tgg ggc gtc atg ttc 1411

Glu Val Ile Ile Asp Ile Val Ser Gly Ala His Trp Gly Val Met Phe

345 350 355

ggc ttg gcc tac ttc tct atg cag gga gcg tgg gcg aag gtc att gtc 1459

Gly Leu Ala Tyr Phe Ser Met Gln Gly Ala Trp Ala Lys Val Ile Val

360 365 370

atc ctt ctg ctg gcc gct ggg gtg gac gcg ggc acc acc acc gtt gga 1507

Ile Leu Leu Leu Ala Ala Gly Val Asp Ala Gly Thr Thr Thr Val Gly

375 380 385

ggc gct gtt gca cgt tcc acc aac gtg att gcc ggc gtg ttc agc cat 1555

Gly Ala Val Ala Arg Ser Thr Asn Val Ile Ala Gly Val Phe Ser His

390 395 400 405

ggc cct cag cag aac att cag ctc att aac acc aac ggc agt tgg cac 1603

Gly Pro Gln Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly Ser Trp His

410 415 420

atc aac cgt act gcc ttg aat tgc aat gac tcc ttg aac acc ggc ttt 1651

Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly Phe

425 430 435

ctc gcg gcc ttg ttc tac acc aac cgc ttt aac tcg tca ggg tgt cca 1699

Leu Ala Ala Leu Phe Tyr Thr Asn Arg Phe Asn Ser Ser Gly Cys Pro

440 445 450

ggg cgc ctg tcc gcc tgc cgc aac atc gag gct ttc cgg ata ggg tgg 1747

Gly Arg Leu Ser Ala Cys Arg Asn Ile Glu Ala Phe Arg Ile Gly Trp

455 460 465

ggc acc cta cag tac gag gat aat gtc acc aat cca gag gat atg agg 1795

Gly Thr Leu Gln Tyr Glu Asp Asn Val Thr Asn Pro Glu Asp Met Arg

470 475 480 485

ccg tac tgc tgg cac tac ccc cca aag ccg tgt ggc gta gtc ccc gcg 1843

Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Val Val Pro Ala

490 495 500

agg tct gtg tgt ggc cca gtg tac tgt ttc acc ccc agc ccg gta gta 1891

Arg Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val

505 510 515

gtg ggc acg acc gac aga cgt gga gtg ccc acc tac aca tgg gga gag 1939

Val Gly Thr Thr Asp Arg Arg Gly Val Pro Thr Tyr Thr Trp Gly Glu

520 525 530

aat gag aca gat gtc ttc cta ctg aac agc acc cga ccg ccg cag ggc 1987

Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr Arg Pro Pro Gln Gly

535 540 545

tca tgg ttc ggc tgc acg tgg atg aac tcc act ggt ttc acc aag act 2035

Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Thr

550 555 560 565

tgt ggc gcg cca cct tgc cgc acc aga gct gac ttc aac gcc agc acg 2083

Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp Phe Asn Ala Ser Thr

570 575 580

gac ttg ttg tgc cct acg gat tgt ttt agg aag cat cct gat gcc act 2131

Asp Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala Thr

585 590 595

tat att aag tgt ggt tct ggg ccc tgg ctc aca cca aag tgc ctg gtc 2179

Tyr Ile Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Lys Cys Leu Val

600 605 610

cac tac cct tac aga ctc tgg cat tac ccc tgc aca gtc aat ttt acc 2227

His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr

615 620 625

atc ttc aag ata aga atg tat gta ggg ggg gtt gag cac agg ctc acg 2275

Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Thr

630 635 640 645

gcc gca tgc aac ttc act cgt ggg gat cgc tgc gac ttg gag gac agg 2323

Ala Ala Cys Asn Phe Thr Arg Gly Asp Arg Cys Asp Leu Glu Asp Arg

650 655 660

gac agg agt cag ctg tct cct ctg ttg cac tct acc acg gaa tgg gcc 2371

Asp Arg Ser Gln Leu Ser Pro Leu Leu His Ser Thr Thr Glu Trp Ala

665 670 675

atc ctg ccc tgc acc tac tca gac tta ccc gct ttg tca act ggt ctt 2419

Ile Leu Pro Cys Thr Tyr Ser Asp Leu Pro Ala Leu Ser Thr Gly Leu

680 685 690

ctc cac ctt cac cag aac atc gtg gac gta caa tac atg tat ggc ctc 2467

Leu His Leu His Gln Asn Ile Val Asp Val Gln Tyr Met Tyr Gly Leu

695 700 705

tca cct gct atc aca aaa tac gtc gtt cga tgg gag tgg gtg gta ctc 2515

Ser Pro Ala Ile Thr Lys Tyr Val Val Arg Trp Glu Trp Val Val Leu

710 715 720 725

tta ttc ctg ctc tta gcg gac gcc aga gtc tgc gcc tgc ttg tgg atg 2563

Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp Met

730 735 740

ctc atc ttg ttg ggc cag gcc gaa gca gca ttg gag aag ttg gtc gtc 2611

Leu Ile Leu Leu Gly Gln Ala Glu Ala Ala Leu Glu Lys Leu Val Val

745 750 755

ttg cac gct gcg agt gcg gct aac tgc cat ggc ctc cta tat ttt gcc 2659

Leu His Ala Ala Ser Ala Ala Asn Cys His Gly Leu Leu Tyr Phe Ala

760 765 770

atc ttc ttc gtg gca gct tgg cac atc agg ggt cgg gtg gtc ccc ttg 2707

Ile Phe Phe Val Ala Ala Trp His Ile Arg Gly Arg Val Val Pro Leu

775 780 785

acc acc tat tgc ctc act ggc cta tgg ccc ttc tgc cta ctg ctc atg 2755

Thr Thr Tyr Cys Leu Thr Gly Leu Trp Pro Phe Cys Leu Leu Leu Met

790 795 800 805

gca ctg ccc cgg cag gct tat gcc tat gac gca cct gtg cac gga cag 2803

Ala Leu Pro Arg Gln Ala Tyr Ala Tyr Asp Ala Pro Val His Gly Gln

810 815 820

ata ggc gtg ggt ttg ttg ata ttg atc acc ctc ttc aca ctc acc ccg 2851

Ile Gly Val Gly Leu Leu Ile Leu Ile Thr Leu Phe Thr Leu Thr Pro

825 830 835

ggg tat aag acc ctc ctc ggc cag tgt ctg tgg tgg ttg tgc tat ctc 2899

Gly Tyr Lys Thr Leu Leu Gly Gln Cys Leu Trp Trp Leu Cys Tyr Leu

840 845 850

ctg acc ctg ggg gaa gcc atg att cag gag tgg gta cca ccc atg cag 2947

Leu Thr Leu Gly Glu Ala Met Ile Gln Glu Trp Val Pro Pro Met Gln

855 860 865

gtg cgc ggc ggc cgc gat ggc atc gcg tgg gcc gtc act ata ttc tgc 2995

Val Arg Gly Gly Arg Asp Gly Ile Ala Trp Ala Val Thr Ile Phe Cys

870 875 880 885

ccg ggt gtg gtg ttt gac att acc aaa tgg ctt ttg gcg ttg ctt ggg 3043

Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu Leu Ala Leu Leu Gly

890 895 900

cct gct tac ctc tta agg gcc gct ttg aca cat gtg ccg tac ttc gtc 3091

Pro Ala Tyr Leu Leu Arg Ala Ala Leu Thr His Val Pro Tyr Phe Val

905 910 915

aga gct cac gct ctg ata agg gta tgc gct ttg gtg aag cag ctc gcg 3139

Arg Ala His Ala Leu Ile Arg Val Cys Ala Leu Val Lys Gln Leu Ala

920 925 930

ggg ggt agg tat gtt cag gtg gcg cta ttg gcc ctt ggc agg tgg act 3187

Gly Gly Arg Tyr Val Gln Val Ala Leu Leu Ala Leu Gly Arg Trp Thr

935 940 945

ggc acc tac atc tat gac cac ctc aca cct atg tcg gac tgg gcc gct 3235

Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met Ser Asp Trp Ala Ala

950 955 960 965

agc ggc ctg cgc gac tta gcg gtc gcc gtg gaa ccc atc atc ttc agt 3283

Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Ile Ile Phe Ser

970 975 980

ccg atg gag aag aag gtc atc gtc tgg gga gcg gag acg gct gca tgt 3331

Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala Glu Thr Ala Ala Cys

985 990 995

ggg gac att cta cat gga ctt ccc gtg tcc gcc cga ctc ggc cag gag 3379

Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala Arg Leu Gly Gln Glu

1000 1005 1010

atc ctc ctc ggc cca gct gat ggc tac acc tcc aag ggg tgg aag ctc 3427

Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser Lys Gly Trp Lys Leu

1015 1020 1025

ctt gct ccc atc act gct tat gcc cag caa aca cga ggc ctc ctg ggc 3475

Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly

1030 1035 1040 1045

gcc ata gtg gtg agt atg acg ggg cgt gac agg aca gaa cag gcc ggg 3523

Ala Ile Val Val Ser Met Thr Gly Arg Asp Arg Thr Glu Gln Ala Gly

1050 1055 1060

gaa gtc caa atc ctg tcc aca gtc tct cag tcc ttc ctc gga aca acc 3571

Glu Val Gln Ile Leu Ser Thr Val Ser Gln Ser Phe Leu Gly Thr Thr

1065 1070 1075

atc tcg ggg gtt ttg tgg act gtt tac cac gga gct ggc aac aag act 3619

Ile Ser Gly Val Leu Trp Thr Val Tyr His Gly Ala Gly Asn Lys Thr

1080 1085 1090

cta gcc ggc tta cgg ggt ccg gtc acg cag atg tac tcg agt gct gag 3667

Leu Ala Gly Leu Arg Gly Pro Val Thr Gln Met Tyr Ser Ser Ala Glu

1095 1100 1105

ggg gac ttg gta ggc tgg ccc agc ccc cct ggg acc aag tct ttg gag 3715

Gly Asp Leu Val Gly Trp Pro Ser Pro Pro Gly Thr Lys Ser Leu Glu

1110 1115 1120 1125

ccg tgc aag tgt gga gcc gtc gac cta tat ctg gtc acg cgg aac gct 3763

Pro Cys Lys Cys Gly Ala Val Asp Leu Tyr Leu Val Thr Arg Asn Ala

1130 1135 1140

gat gtc atc ccg gct cgg aga cgc ggg gac aag cgg gga gca ttg ctc 3811

Asp Val Ile Pro Ala Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu

1145 1150 1155

tcc ccg aga ccc att tcg acc ttg aag ggg tcc tcg ggg ggg ccg gtg 3859

Ser Pro Arg Pro Ile Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val

1160 1165 1170

ctc tgc cct agg ggc cac gtc gtt ggg ctc ttc cga gca gct gtg tgc 3907

Leu Cys Pro Arg Gly His Val Val Gly Leu Phe Arg Ala Ala Val Cys

1175 1180 1185

tct cgg ggc gtg gcc aaa tcc atc gat ttc atc ccc gtt gag aca ctc 3955

Ser Arg Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu Thr Leu

1190 1195 1200 1205

gac gtt gtt aca agg tct ccc act ttc agt gac aac agc acg cca ccg 4003

Asp Val Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser Thr Pro Pro

1210 1215 1220

gct gtg ccc cag acc tat cag gtc ggg tac ttg cat gct cca act ggc 4051

Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His Ala Pro Thr Gly

1225 1230 1235

agt gga aag agc acc aag gtc cct gtc gcg tat gcc gcc cag ggg tac 4099

Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr Ala Ala Gln Gly Tyr

1240 1245 1250

aaa gta cta gtg ctt aac ccc tcg gta gct gcc acc ctg ggg ttt ggg 4147

Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly

1255 1260 1265

gcg tac cta tcc aag gca cat ggc atc aat ccc aac att agg act gga 4195

Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro Asn Ile Arg Thr Gly

1270 1275 1280 1285

gtc agg acc gtg atg acc ggg gag gcc atc acg tac tcc aca tat ggc 4243

Val Arg Thr Val Met Thr Gly Glu Ala Ile Thr Tyr Ser Thr Tyr Gly

1290 1295 1300

aaa ttt ctc gcc gat ggg ggc tgc gct agc ggc gcc tat gac atc atc 4291

Lys Phe Leu Ala Asp Gly Gly Cys Ala Ser Gly Ala Tyr Asp Ile Ile

1305 1310 1315

ata tgc gat gaa tgc cac gct gtg gat gct acc tcc att ctc ggc atc 4339

Ile Cys Asp Glu Cys His Ala Val Asp Ala Thr Ser Ile Leu Gly Ile

1320 1325 1330

gga acg gtc ctt gat caa gca gag aca gcc ggg gtc aga cta act gtg 4387

Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Val Arg Leu Thr Val

1335 1340 1345

ctg gct acg gcc aca ccc ccc ggg tca gtg aca acc ccc cat ccc gat 4435

Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Thr Pro His Pro Asp

1350 1355 1360 1365

ata gaa gag gta ggc ctc ggg cgg gag ggt gag atc ccc ttc tat ggg 4483

Ile Glu Glu Val Gly Leu Gly Arg Glu Gly Glu Ile Pro Phe Tyr Gly

1370 1375 1380

agg gcg att ccc cta tcc tgc atc aag gga ggg aga cac ctg att ttc 4531

Arg Ala Ile Pro Leu Ser Cys Ile Lys Gly Gly Arg His Leu Ile Phe

1385 1390 1395

tgc cac tca aag aaa aag tgt gac gag ctc gcg gcg gcc ctt cgg ggc 4579

Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly

1400 1405 1410

atg ggc ttg aat gcc gtg gca tac tat aga ggg ttg gac gtc tcc ata 4627

Met Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Ile

1415 1420 1425

ata cca gct cag gga gat gtg gtg gtc gtc gcc acc gac gcc ctc atg 4675

Ile Pro Ala Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met

1430 1435 1440 1445

acg ggg tac act gga gac ttt gac tcc gtg atc gac tgc aat gta gcg 4723

Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Val Ala

1450 1455 1460

gtc acc caa gct gtc gac ttc agc ctg gac ccc acc ttc act ata acc 4771

Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Thr

1465 1470 1475

aca cag act gtc cca caa gac gct gtc tca cgc agt cag cgc cgc ggg 4819

Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly

1480 1485 1490

cgc aca ggt aga gga aga cag ggc act tat agg tat gtt tcc act ggt 4867

Arg Thr Gly Arg Gly Arg Gln Gly Thr Tyr Arg Tyr Val Ser Thr Gly

1495 1500 1505

gaa cga gcc tca gga atg ttt gac agt gta gtg ctt tgt gag tgc tac 4915

Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu Cys Glu Cys Tyr

1510 1515 1520 1525

gac gca ggg gct gcg tgg tac gat ctc aca cca gcg gag acc acc gtc 4963

Asp Ala Gly Ala Ala Trp Tyr Asp Leu Thr Pro Ala Glu Thr Thr Val

1530 1535 1540

agg ctt aga gcg tat ttc aac acg ccc ggc cta ccc gtg tgt caa gac 5011

Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu Pro Val Cys Gln Asp

1545 1550 1555

cat ctt gaa ttt tgg gag gca gtt ttc acc ggc ctc aca cac ata gac 5059

His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His Ile Asp

1560 1565 1570

gcc cac ttc ctc tcc caa aca aag caa gcg ggg gag aac ttc gcg tac 5107

Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Glu Asn Phe Ala Tyr

1575 1580 1585

cta gta gcc tac caa gct acg gtg tgc gcc aga gcc aag gcc cct ccc 5155

Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro

1590 1595 1600 1605

ccg tcc tgg gac gcc atg tgg aag tgc ctg gcc cga ctc aag cct acg 5203

Pro Ser Trp Asp Ala Met Trp Lys Cys Leu Ala Arg Leu Lys Pro Thr

1610 1615 1620

ctt gcg ggc ccc aca cct ctc ctg tac cgt ttg ggc cct att acc aat 5251

Leu Ala Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Pro Ile Thr Asn

1625 1630 1635

gag gtc acc ctc aca cac cct ggg acg aag tac atc gcc aca tgc atg 5299

Glu Val Thr Leu Thr His Pro Gly Thr Lys Tyr Ile Ala Thr Cys Met

1640 1645 1650

caa gct gac ctt gag gtc atg acc agc acg tgg gtc cta gct gga gga 5347

Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly Gly

1655 1660 1665

gtc ctg gca gcc gtc gcc gca tat tgc ctg gcg act gga tgc gtt tcc 5395

Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val Ser

1670 1675 1680 1685

atc atc ggc cgc ttg cac gtc aac cag cga gtc gtc gtt gcg ccg gat 5443

Ile Ile Gly Arg Leu His Val Asn Gln Arg Val Val Val Ala Pro Asp

1690 1695 1700

aag gag gtc ctg tat gag gct ttt gat gag atg gag gaa tgc gcc tct 5491

Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser

1705 1710 1715

agg gcg gct ctc atc gaa gag ggg cag cgg ata gcc gag atg ttg aag 5539

Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met Leu Lys

1720 1725 1730

tcc aag atc caa ggc ttg ctg cag cag gcc tct aag cag gcc cag gac 5587

Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln Ala Gln Asp

1735 1740 1745

ata caa ccc gct atg cag gct tca tgg ccc aaa gtg gaa caa ttt tgg 5635

Ile Gln Pro Ala Met Gln Ala Ser Trp Pro Lys Val Glu Gln Phe Trp

1750 1755 1760 1765

gcc aga cac atg tgg aac ttc att agc ggc atc caa tac ctc gca gga 5683

Ala Arg His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly

1770 1775 1780

ttg tca aca ctg cca ggg aac ccc gcg gtg gct tcc atg atg gca ttc 5731

Leu Ser Thr Leu Pro Gly Asn Pro Ala Val Ala Ser Met Met Ala Phe

1785 1790 1795

agt gcc gcc ctc acc agt ccg ttg tcg acc agt aec acc atc ctt ctc 5779

Ser Ala Ala Leu Thr Ser Pro Leu Ser Thr Ser Thr Thr Ile Leu Leu

1800 1805 1810

aac atc atg gga ggc tgg tta gcg tcc cag atc gca cca ccc gcg ggg 5827

Asn Ile Met Gly Gly Trp Leu Ala Ser Gln Ile Ala Pro Pro Ala Gly

1815 1820 1825

gcc acc ggc ttt gtc gtc agt ggc ctg gtg ggg gct gcc gtg ggc agc 5875

Ala Thr Gly Phe Val Val Ser Gly Leu Val Gly Ala Ala Val Gly Ser

1830 1835 1840 1845

ata ggc ctg ggt aag gtg ctg gtg gac atc ctg gca gga tat ggt gcg 5923

Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala

1850 1855 1860

ggc att tcg ggg gcc ctc gtc gca ttc aag atc atg tct ggc gag aag 5971

Gly Ile Ser Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys

1865 1870 1875

ccc tct atg gaa gat gtc atc aat cta ctg cct ggg atc ctg tct ccg 6019

Pro Ser Met Glu Asp Val Ile Asn Leu Leu Pro Gly lle Leu Ser Pro

1880 1885 1890

gga gcc ctg gtg gtg ggg gtc atc tgc gcg gcc att ctg cgc cgc cac 6067

Gly Ala Leu Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg His

1895 1900 1905

gtg gga ccg ggg gag ggc gcg gtc caa tgg atg aac agg ctt att gcc 6115

Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala

1910 1915 1920 1925

ttt gct tcc aga gga aac cac gtc gcc cct act cac tac gtg acg gag 6163

Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Thr Glu

1930 1935 1940

tcg gat gcg tcg cag cgt gtg acc caa cta ctt ggc tct ctt act ata 6211

Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly Ser Leu Thr Ile

1945 1950 1955

acc agc cta ctc aga aga ctc cac aat tgg ata act gag gac tgc ccc 6259

Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile Thr Glu Asp Cys Pro

1960 1965 1970

atc cca tgc tcc gga tcc tgg ctc cgc gac gtg tgg gac tgg gtt tgc 6307

Ile Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Val Cys

1975 1980 1985

acc atc ttg aca gac ttc aaa aat tgg ctg acc tct aaa ttg ttc ccc 6355

Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr Ser Lys Leu Phe Pro

1990 1995 2000 2005

aag ctg ccc ggc ctc ccc ttc atc tct tgt caa aag ggg tac aag ggt 6403

Lys Leu Pro Gly Leu Pro Phe Ile Ser Cys Gln Lys Gly Tyr Lys Gly

2010 2015 2020

gtg tgg gcc ggc act ggc atc atg acc acg cgc tgc cct tgc ggc gcc 6451

Val Trp Ala Gly Thr Gly Ile Met Thr Thr Arg Cys Pro Cys Gly Ala

2025 2030 2035

aac atc tct ggc aat gtc cgc ctg ggc tct atg agg atc aca ggg cct 6499

Asn Ile Ser Gly Asn Val Arg Leu Gly Ser Met Arg Ile Thr Gly Pro

2040 2045 2050

aaa acc tgc atg aac acc tgg cag ggg acc ttt cct atc aat tgc tac 6547

Lys Thr Cys Met Asn Thr Trp Gln Gly Thr Phe Pro Ile Asn Cys Tyr

2055 2060 2065

acg gag ggc cag tgc gcg ccg aaa ccc ccc acg aac tac aag acc gcc 6595

Thr Glu Gly Gln Cys Ala Pro Lys Pro Pro Thr Asn Tyr Lys Thr Ala

2070 2075 2080 2085

atc tgg agg gtg gcg gcc tcg gag tac gcg gag gtg acg cag cat ggg 6643

Ile Trp Arg Val Ala Ala Ser Glu Tyr Ala Glu Val Thr Gln His Gly

2090 2095 2100

tcg tac tcc tat gta aca gga ctg acc act gac aat ctg aaa att cct 6691

Ser Tyr Ser Tyr Val Thr Gly Leu Thr Thr Asp Asn Leu Lys Ile Pro

2105 2110 2115

tgc caa cta cct tct cca gag ttt ttc tcc tgg gtg gac ggt gtg cag 6739

Cys Gln Leu Pro Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln

2120 2125 2130

atc cat agg ttt gca ccc aca cca aag ccg ttt ttc cgg gat gag gtc 6787

Ile His Arg Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val

2135 2140 2145

tcg ttc tgc gtt ggg ctt aat tcc tat gct gtc ggg tcc cag ctt ccc 6835

Ser Phe Cys Val Gly Leu Asn Ser Tyr Ala Val Gly Ser Gln Leu Pro

2150 2155 2160 2165

tgt gaa cct gag ccc gac gca gac gta ttg agg tcc atg cta aca gat 6883

Cys Glu Pro Glu Pro Asp Ala Asp Val Leu Arg Ser Met Leu Thr Asp

2170 2175 2180

ccg ccc cac atc acg gcg gag act gcg gcg cgg cgc ttg gca cgg gga 6931

Pro Pro His Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Ala Arg Gly

2185 2190 2195

tca cct cca tct gag gcg agc tcc tca gtg agc cag cta tca gca ccg 6979

Ser Pro Pro Ser Glu Ala Ser Ser Ser Val Ser Gln Leu Ser Ala Pro

2200 2205 2210

tcg ctg cgg gcc acc tgc acc acc cac agc aac acc tat gac gtg gac 7027

Ser Leu Arg Ala Thr Cys Thr Thr His Ser Asn Thr Tyr Asp Val Asp

2215 2220 2225

atg gtc gat gcc aac ctg ctc atg gag ggc ggt gtg gct cag aca gag 7075

Met Val Asp Ala Asn Leu Leu Met Glu Gly Gly Val Ala Gln Thr Glu

2230 2235 2240 2245

cct gag tcc agg gtg ccc gtt ctg gac ttt ctc gag cca atg gcc gag 7123

Pro Glu Ser Arg Val Pro Val Leu Asp Phe Leu Glu Pro Met Ala Glu

2250 2255 2260

gaa gag agc gac ctt gag ccc tca ata cca tcg gag tgc atg ctc ccc 7171

Glu Glu Ser Asp Leu Glu Pro Ser Ile Pro Ser Glu Cys Met Leu Pro

2265 2270 2275

agg agc ggg ttt cca cgg gcc tta ccg gct tgg gca cgg cct gac tac 7219

Arg Ser Gly Phe Pro Arg Ala Leu Pro Ala Trp Ala Arg Pro Asp Tyr

2280 2285 2290

aac ccg ccg ctc gtg gaa tcg tgg agg agg cca gat tac caa ccg ccc 7267

Asn Pro Pro Leu Val Glu Ser Trp Arg Arg Pro Asp Tyr Gln Pro Pro

2295 2300 2305

acc gtt gct ggt tgt gct ctc ccc ccc ccc aag aag gcc ccg acg cct 7315

Thr Val Ala Gly Cys Ala Leu Pro Pro Pro Lys Lys Ala Pro Thr Pro

2310 2315 2320 2325

ccc cca agg aga cgc cgg aca gtg ggt ctg agc gag agc acc ata tca 7363

Pro Pro Arg Arg Arg Arg Thr Val Gly Leu Ser Glu Ser Thr Ile Ser

2330 2335 2340

gaa gcc ctc cag caa ctg gcc atc aag acc ttt ggc cag ccc ccc tcg 7411

Glu Ala Leu Gln Gln Leu Ala Ile Lys Thr Phe Gly Gln Pro Pro Ser

2345 2350 2355

agc ggt gat gca ggc tcg tcc acg ggg gcg ggc gcc gcc gaa tcc ggc 7459

Ser Gly Asp Ala Gly Ser Ser Thr Gly Ala Gly Ala Ala Glu Ser Gly

2360 2365 2370

ggt ccg acg tcc cct ggt gag ccg gcc ccc tca gag aca ggt tcc gcc 7507

Gly Pro Thr Ser Pro Gly Glu Pro Ala Pro Ser Glu Thr Gly Ser Ala

2375 2380 2385

tcc tct atg ccc ccc ctc gag ggg gag cct gga gat ccg gac ctg gag 7555

Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu

2390 2395 2400 2405

tct gat cag gta gag ctt caa cct ccc ccc cag ggg ggg ggg gta gct 7603

Ser Asp Gln Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Gly Val Ala

2410 2415 2420

ccc ggt tcg ggc tcg ggg tct tgg tct act tgc tcc gag gag gac gat 7651

Pro Gly Ser Gly Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp Asp

2425 2430 2435

acc acc gtg tgc tgc tcc atg tca tac tcc tgg acc ggg gct cta ata 7699

Thr Thr Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Ile

2440 2445 2450

act ccc tgt agc ccc gaa gag gaa aag ttg cca atc aac cct ttg agt 7747

Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro Ile Asn Pro Leu Ser

2455 2460 2465

aac tcg ctg ttg cga tac cat aac aag gtg tac tgt aca aca tca aag 7795

Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr Cys Thr Thr Ser Lys

2470 2475 2480 2485

agc gcc tca cag agg gct aaa aag gta act ttt gac agg acg caa gtg 7843

Ser Ala Ser Gln Arg Ala Lys Lys Val Thr Phe Asp Arg Thr Gln Val

2490 2495 2500

ctc gac gcc cat tat gac tca gtc tta aag gac atc aag cta gcg gct 7891

Leu Asp Ala His Tyr Asp Ser Val Leu Lys Asp Ile Lys Leu Ala Ala

2505 2510 2515

tcc aag gtc agc gca agg ctc ctc acc ttg gag gag gcg tgc cag ttg 7939

Ser Lys Val Ser Ala Arg Leu Leu Thr Leu Glu Glu Ala Cys Gln Leu

2520 2525 2530

act cca ccc cat tct gca aga tcc aag tat gga ttc ggg gcc aag gag 7987

Thr Pro Pro His Ser Ala Arg Ser Lys Tyr Gly Phe Gly Ala Lys Glu

2535 2540 2545

gtc cgc agc ttg tcc ggg agg gcc gtt aac cac atc aag tcc gtg tgg 8035

Val Arg Ser Leu Ser Gly Arg Ala Val Asn His Ile Lys Ser Val Trp

2550 2555 2560 2565

aag gac ctc ctg gaa gac cca caa aca cca att ccc aca acc atc atg 8083

Lys Asp Leu Leu Glu Asp Pro Gln Thr Pro Ile Pro Thr Thr Ile Met

2570 2575 2580

gcc aaa aat gag gtg ttc tgc gtg gac ccc gcc aag ggg ggt aag aaa 8131

Ala Lys Asn Glu Val Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys

2585 2590 2595

cca gct cgc ctc atc gtt tac cct gac ctc ggc gtc cgg gtc tgc gag 8179

Pro Ala Arg Leu Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu

2600 2605 2610

aaa atg gcc ctc tat gac att aca caa aag ctt cct cag gcg gta atg 8227

Lys Met Ala Leu Tyr Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met

2615 2620 2625

gga gct tcc tat ggc ttc cag tac tcc cct gcc caa cgg gtg gag tat 8275

Gly Ala Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr

2630 2635 2640 2645

ctc ttg aaa gca tgg gcg gaa aag aag gac ccc atg ggt ttt tcg tat 8323

Leu Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr

2650 2655 2660

gat acc cga tgc ttc gac tca acc gtc act gag aga gac atc agg acc 8371

Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp Ile Arg Thr

2665 2670 2675

gag gag tcc ata tac cag gcc tgc tcc ctg ccc gag gag gcc cgc act 8419

Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro Glu Glu Ala Arg Thr

2680 2685 2690

gcc ata cac tcg ctg act gag aga ctt tac gta gga ggg ccc atg ttc 8467

Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Phe

2695 2700 2705

aac agc aag ggt caa acc tgc ggt tac aga cgt tgc cgc gcc agc ggg 8515

Asn Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly

2710 2715 2720 2725

gtg cta acc act agc atg ggt aac acc atc aca tgc tat gtg aaa gcc 8563

Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr Cys Tyr Val Lys Ala

2730 2735 2740

cta gcg gcc tgc aag gct gcg ggg ata gtt gcg ccc aca atg ctg gta 8611

Leu Ala Ala Cys Lys Ala Ala Gly Ile Val Ala Pro Thr Met Leu Val

2745 2750 2755

tgc ggc gat gac cta gta gtc atc tca gaa agc cag ggg act gag gag 8659

Cys Gly Asp Asp Leu Val Val Ile Ser Glu Ser Gln Gly Thr Glu Glu

2760 2765 2770

gac gag cgg aac ctg aga gcc ttc acg gag gcc atg acc agg tac tct 8707

Asp Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser

2775 2780 2785

gcc cct cct ggt gat ccc ccc aga ccg gaa tat gac ctg gag cta ata 8755

Ala Pro Pro Gly Asp Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile

2790 2795 2800 2805

aca tcc tgt tcc tca aat gtg tct gtg gcg ttg ggc ccg cgg ggc cgc 8803

Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu Gly Pro Arg Gly Arg

2810 2815 2820

cgc aga tac tac ctg acc aga gac cca acc act cca ctc gcc cgg gct 8851

Arg Arg Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala

2825 2830 2835

gcc tgg gaa aca gtt aga cac tcc cct atc aat tca tgg ctg gga aac 8899

Ala Trp Glu Thr Val Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn

2840 2845 2850

atc atc cag tat gct cca acc ata tgg gtt cgc atg gtc cta atg aca 8947

Ile Ile Gln Tyr Ala Pro Thr Ile Trp Val Arg Met Val Leu Met Thr

2855 2860 2865

cac ttc ttc tcc att ctc atg gtc caa gac acc ctg gac cag aac ctc 8995

His Phe Phe Ser Ile Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu

2870 2875 2880 2885

aac ttt gag atg tat gga tca gta tac tcc gtg aat cct ttg gac ctt 9043

Asn Phe Glu Met Tyr Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu

2890 2895 2900

cca gcc ata att gag agg tta cac ggg ctt gac gcc ttt tct atg cac 9091

Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala Phe Ser Met His

2905 2910 2915

aca tac tct cac cac gaa ctg acg cgg gtg gct tca gcc ctc aga aaa 9139

Thr Tyr Ser His His Glu Leu Thr Arg Val Ala Ser Ala Leu Arg Lys

2920 2925 2930

ctt ggg gcg cca ccc ctc agg gtg tgg aag agt cgg gct cgc gca gtc 9187

Leu Gly Ala Pro Pro Leu Arg Val Trp Lys Ser Arg Ala Arg Ala Val

2935 2940 2945

agg gcg tcc ctc atc tcc cgt gga ggg aaa gcg gcc gtt tgc ggc cga 9235

Arg Ala Ser Leu Ile Ser Arg Gly Gly Lys Ala Ala Val Cys Gly Arg

2950 2955 2960 2965

tat ctc ttc aat tgg gcg gtg aag acc aag ctc aaa ctc act cca ttg 9283

Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu

2970 2975 2980

ccg gag gcg cgc cta ctg gac tta tcc agt tgg ttc acc gtc ggc gcc 9331

Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser Trp Phe Thr Val Gly Ala

2985 2990 2995

ggc ggg ggc gac att ttt cac agc gtg tcg cgc gcc cga ccc cgc tca 9379

Gly Gly Gly Asp Ile Phe His Ser Val Ser Arg Ala Arg Pro Arg Ser

3000 3005 3010

tta ctc ttc ggc cta ctc cta ctt ttc gta ggg gta ggc ctc ttc cta 9427

Leu Leu Phe Gly Leu Leu Leu Leu Phe Val Gly Val Gly Leu Phe Leu

3015 3020 3025

ctc ccc gct cgg tag agcggcacac actaggtaca ctccatagct aactgttcct 9482

Leu Pro Ala Arg

3030

tttttttttt tttttttttt tttttttttt tttttttttt ttcttttttt tttttttccc 9542

tctttcttcc cttctcatct tattctactt tctttcttgg tggctccatc ttagccctag 9602

tcacggctag ctgtgaaagg tccgtgagcc gcatgactgc agagagtgcc gtaactggtc 9662

tctctgcaga tcatgt 9678

<210>4

<211>3033

<212>PRT

<213>丙肝病毒

<400>4

Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn

1 5 10 15

Arg Arg Pro Glu Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly

20 25 30

Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Thr

35 40 45

Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro

50 55 60

Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys Ala Trp Gly Lys Pro Gly

65 70 75 80

Arg Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp

85 90 95

Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro

100 105 110

Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys

115 120 125

Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Val Val Gly Ala Pro Leu

130 135 140

Ser Gly Ala Ala Arg Ala Val Ala His Gly Val Arg Val Leu Glu Asp

145 150 155 160

Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Phe Pro Phe Ser Ile

165 170 175

Phe Leu Leu Ala Leu Leu Ser Cys Ile Thr Val Pro Val Ser Ala Ala

180 185 190

Gln Val Lys Asn Thr Ser Ser Ser Tyr Met Val Thr Asn Asp Cys Ser

195 200 205

Asn Asp Ser Ile Thr Trp Gln Leu Glu Ala Ala Val Leu His Val Pro

210 215 220

Gly Cys Val Pro Cys Glu Arg Val Gly Asn Thr Ser Arg Cys Trp Val

225 230 235 240

Pro Val Ser Pro Asn Met Ala Val Arg Gln Pro Gly Ala Leu Thr Gln

245 250 255

Gly Leu Arg Thr His Ile Asp Met Val Val Met Ser Ala Thr Phe Cys

260 265 270

Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Gly Val Met Leu Ala Ala

275 280 285

Gln Val Phe Ile Val Ser Pro Gln Tyr His Trp Phe Val Gln Glu Cys

290 295 300

Asn Cys Ser Ile Tyr Pro Gly Thr Ile Thr Gly His Arg Met Ala Trp

305 310 315 320

Asp Met Met Met Asn Trp Ser Pro Thr Ala Thr Met Ile Leu Ala Tyr

325 330 335

Val Met Arg Val Pro Glu Val Ile Ile Asp Ile Val Ser Gly Ala His

340 345 350

Trp Gly Val Met Phe Gly Leu Ala Tyr Phe Ser Met Gln Gly Ala Trp

355 360 365

Ala Lys Val Ile Val Ile Leu Leu Leu Ala Ala Gly Val Asp Ala Gly

370 375 380

Thr Thr Thr Val Gly Gly Ala Val Ala Arg Ser Thr Asn Val Ile Ala

385 390 395 400

Gly Val Phe Ser His Gly Pro Gln Gln Asn Ile Gln Leu Ile Asn Thr

405 410 415

Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser

420 425 430

Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr Asn Arg Phe Asn

435 440 445

Ser Ser Gly Cys Pro Gly Arg Leu Ser Ala Cys Arg Asn Ile Glu Ala

450 455 460

Phe Arg Ile Gly Trp Gly Thr Leu Gln Tyr Glu Asp Asn Val Thr Asn

465 470 475 480

Pro Glu Asp Met Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys

485 490 495

Gly Val Val Pro Ala Arg Ser Val Cys Gly Pro Val Tyr Cys Phe Thr

500 505 510

Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Arg Gly Val Pro Thr

515 520 525

Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr

530 535 540

Arg Pro Pro Gln Gly Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Thr

545 550 555 560

Gly Phe Thr Lys Thr Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp

565 570 575

Phe Asn Ala Ser Thr Asp Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys

580 585 590

His Pro Asp Ala Thr Tyr Ile Lys Cys Gly Ser Gly Pro Trp Leu Thr

595 600 605

Pro Lys Cys Leu Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys

610 615 620

Thr Val Asn Phe Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val

625 630 635 640

Glu His Arg Leu Thr Ala Ala Cys Asn Phe Thr Arg Gly Asp Arg Cys

645 650 655

Asp Leu Glu Asp Arg Asp Arg Ser Gln Leu Ser Pro Leu Leu His Ser

660 665 670

Thr Thr Glu Trp Ala Ile Leu Pro Cys Thr Tyr Ser Asp Leu Pro Ala

675 680 685

Leu Ser Thr Gly Leu Leu His Leu His Gln Asn Ile Val Asp Val Gln

690 695 700

Tyr Met Tyr Gly Leu Ser Pro Ala Ile Thr Lys Tyr Val Val Arg Trp

705 710 715 720

Glu Trp Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys

725 730 735

Ala Cys Leu Trp Met Leu Ile Leu Leu Gly Gln Ala Glu Ala Ala Leu

740 745 750

Glu Lys Leu Val Val Leu His Ala Ala Ser Ala Ala Asn Cys His Gly

755 760 765

Leu Leu Tyr Phe Ala Ile Phe Phe Val Ala Ala Trp His Ile Arg Gly

770 775 780

Arg Val Val Pro Leu Thr Thr Tyr Cys Leu Thr Gly Leu Trp Pro Phe

785 790 795 800

Cys Leu Leu Leu Met Ala Leu Pro Arg Gln Ala Tyr Ala Tyr Asp Ala

805 810 815

Pro Val His Gly Gln Ile Gly Val Gly Leu Leu Ile Leu Ile Thr Leu

820 825 830

Phe Thr Leu Thr Pro Gly Tyr Lys Thr Leu Leu Gly Gln Cys Leu Trp

835 840 845

Trp Leu Cys Tyr Leu Leu Thr Leu Gly Glu Ala Met Ile Gln Glu Trp

850 855 860

Val Pro Pro Met Gln Val Arg Gly Gly Arg Asp Gly Ile Ala Trp Ala

865 870 875 880

Val Thr Ile Phe Cys Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu

885 890 895

Leu Ala Leu Leu Gly Pro Ala Tyr Leu Leu Arg Ala Ala Leu Thr His

900 905 910

Val Pro Tyr Phe Val Arg Ala His Ala Leu Ile Arg Val Cys Ala Leu

915 920 925

Val Lys Gln Leu Ala Gly Gly Arg Tyr Val Gln Val Ala Leu Leu Ala

930 935 940

Leu Gly Arg Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met

945 950 955 960

Ser Asp Trp Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu

965 970 975

Pro Ile Ile Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala

980 985 990

Glu Thr Ala Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala

995 1000 1005

Arg Leu Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser

1010 1015 1020

Lys Gly Trp Lys Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr

1025 1030 1035 1040

Arg Gly Leu Leu Gly Ala Ile Val Val Ser Met Thr Gly Arg Asp Arg

1045 1050 1055

Thr Glu Gln Ala Gly Glu Val Gln Ile Leu Ser Thr Val Ser Gln Ser

1060 1065 1070

Phe Leu Gly Thr Thr Ile Ser Gly Val Leu Trp Thr Val Tyr His Gly

1075 1080 1085

Ala Gly Asn Lys Thr Leu Ala Gly Leu Arg Gly Pro Val Thr Gln Met

1090 1095 1100

Tyr Ser Ser Ala Glu Gly Asp Leu Val Gly Trp Pro Ser Pro Pro Gly

1105 1110 1115 1120

Thr Lys Ser Leu Glu Pro Cys Lys Cys Gly Ala Val Asp Leu Tyr Leu

1125 1130 1135

Val Thr Arg Asn Ala Asp Val Ile Pro Ala Arg Arg Arg Gly Asp Lys

1140 1145 1150

Arg Gly Ala Leu Leu Ser Pro Arg Pro Ile Ser Thr Leu Lys Gly Ser

1155 1160 1165

Ser Gly Gly Pro Val Leu Cys Pro Arg Gly His Val Val Gly Leu Phe

1170 1175 1180

Arg Ala Ala Val Cys Ser Arg Gly Val Ala Lys Ser Ile Asp Phe Ile

1185 1190 1195 1200

Pro Val Glu Thr Leu Asp Val Val Thr Arg Ser Pro Thr Phe Ser Asp

1205 1210 1215

Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu

1220 1225 1230

His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr

1235 1240 1245

Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala

1250 1255 1260

Thr Leu Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro

1265 1270 1275 1280

Asn Ile Arg Thr Gly Val Arg Thr Val Met Thr Gly Glu Ala Ile Thr

1285 1290 1295

Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Ser Gly

1300 1305 1310

Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ala Val Asp Ala Thr

1315 1320 1325

Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly

1330 1335 1340

Val Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr

1345 1350 1355 1360

Thr Pro His Pro Asp Ile Glu Glu Val Gly Leu Gly Arg Glu Gly Glu

1365 1370 1375

Ile Pro Phe Tyr Gly Arg Ala Ile Pro Leu Ser Cys Ile Lys Gly Gly

1380 1385 1390

Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala

1395 1400 1405

Ala Ala Leu Arg Gly Met Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly

1410 1415 1420

Leu Asp Val Ser Ile Ile Pro Ala Gln Gly Asp Val Val Val Val Ala

1425 1430 1435 1440

Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile

1445 1450 1455

Asp Cys Asn Val Ala Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro

1460 1465 1470

Thr Phe Thr Ile Thr Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg

1475 1480 1485

Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Gln Gly Thr Tyr Arg

1490 1495 1500

Tyr Val Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val

1505 1510 1515 1520

Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Asp Leu Thr Pro

1525 1530 1535

Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu

1540 1545 1550

Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly

1555 1560 1565

Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly

1570 1575 1580

Glu Asn Phe Ala Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg

1585 1590 1595 1600

Ala Lys Ala Pro Pro Pro Ser Trp Asp Ala Met Trp Lys Cys Leu Ala

1605 1610 1615

Arg Leu Lys Pro Thr Leu Ala Gly Pro Thr Pro Leu Leu Tyr Arg Leu

1620 1625 1630

Gly Pro Ile Thr Asn Glu Val Thr Leu Thr His Pro Gly Thr Lys Tyr

1635 1640 1645

Ile Ala Thr Cys Met Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp

1650 1655 1660

Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala

1665 1670 1675 1680

Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Val Asn Gln Arg Val

1685 1690 1695

Val Val Ala Pro Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met

1700 1705 1710

Glu Glu Cys Ala Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile

1715 1720 1725

Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser

1730 1735 1740

Lys Gln Ala Gln Asp Ile Gln Pro Ala Met Gln Ala Ser Trp Pro Lys

1745 1750 1755 1760

Val Glu Gln Phe Trp Ala Arg His Met Trp Asn Phe Ile Ser Gly Ile

1765 1770 1775

Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val Ala

1780 1785 1790

Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Ser Thr Ser

1795 1800 1805

Thr Thr Ile Leu Leu Asn Ile Met Gly Gly Trp Leu Ala Ser Gln Ile

1810 1815 1820

Ala Pro Pro Ala Gly Ala Thr Gly Phe Val Val Ser Gly Leu Val Gly

1825 1830 1835 1840

Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu

1845 1850 1855

Ala Gly Tyr Gly Ala Gly Ile Ser Gly Ala Leu Val Ala Phe Lys Ile

1860 1865 1870

Met Ser Gly Glu Lys Pro Ser Met Glu Asp Val Ile Asn Leu Leu Pro

1875 1880 1885

Gly Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Ile Cys Ala Ala

1890 1895 1900

Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met

1905 1910 1915 1920

Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr

1925 1930 1935

His Tyr Val Thr Glu Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu

1940 1945 1950

Gly Ser Leu Thr Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile

1955 1960 1965

Thr Glu Asp Cys Pro Ile Pro Cys Ser Gly Ser Trp Leu Arg Asp Val

1970 1975 1980

Trp Asp Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr

1985 1990 1995 2000

Ser Lys Leu Phe Pro Lys Leu Pro Gly Leu Pro Phe Ile Ser Cys Gln

2005 2010 2015

Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr GlyIle Met Thr Thr Arg

2020 2025 2030

Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val Arg Leu Gly Ser Met

2035 2040 2045

Arg Ile Thr Gly Pro Lys Thr Cys Met Asn Thr Trp Gln Gly Thr Phe

2050 2055 2060

Pro Ile Asn Cys Tyr Thr Glu Gly Gln Cys Ala Pro Lys Pro Pro Thr

2065 2070 2075 2080

Asn Tyr Lys Thr Ala Ile Trp Arg Val Ala Ala Ser Glu Tyr Ala Glu

2085 2090 2095

Val Thr Gln His Gly Ser Tyr Ser Tyr Val Thr Gly Leu Thr Thr Asp

2100 2105 2110

Asn Leu Lys Ile Pro Cys Gln Leu Pro Ser Pro Glu Phe Phe Ser Trp

2115 2120 2125

Val Asp Gly Val Gln Ile His Arg Phe Ala Pro Thr Pro Lys Pro Phe

2130 2135 2140

Phe Arg Asp Glu Val Ser Phe Cys Val Gly Leu Asn Ser Tyr Ala Val

2145 2150 2155 2160

Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Ala Asp Val Leu Arg

2165 2170 2175

Ser Met Leu Thr Asp Pro Pro His Ile Thr Ala Glu Thr Ala Ala Arg

2180 2185 2190

Arg Leu Ala Arg Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Val Ser

2195 2200 2205

Gln Leu Ser Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Ser Asn

2210 2215 2220

Thr Tyr Asp Val Asp Met Val Asp Ala Asn Leu Leu Met Glu Gly Gly

2225 2230 2235 2240

Val Ala Gln Thr Glu Pro Glu Ser Arg Val Pro Val Leu Asp Phe Leu

2245 2250 2255

Glu Pro Met Ala Glu Glu Glu Ser Asp Leu Glu Pro Ser Ile Pro Ser

2260 2265 2270

Glu Cys Met Leu Pro Arg Ser Gly Phe Pro Arg Ala Leu Pro Ala Trp

2275 2280 2285

Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp Arg Arg Pro

2290 2295 2300

Asp Tyr Gln Pro Pro Thr Val Ala Gly Cys Ala Leu Pro Pro Pro Lys

2305 2310 2315 2320

Lys Ala Pro Thr Pro Pro Pro Arg Arg Arg Arg Thr Val Gly Leu Ser

2325 2330 2335

Glu Ser Thr Ile Ser Glu Ala Leu Gln Gln Leu Ala Ile Lys Thr Phe

2340 2345 2350

Gly Gln Pro Pro Ser Ser Gly Asp Ala Gly Ser Ser Thr Gly Ala Gly

2355 2360 2365

Ala Ala Glu Ser Gly Gly Pro Thr Ser Pro Gly Glu Pro Ala Pro Ser

2370 2375 2380

Glu Thr Gly Ser Ala Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly

2385 2390 2395 2400

Asp Pro Asp Leu Glu Ser Asp Gln Val Glu Leu Gln Pro Pro Pro Gln

2405 2410 2415

Gly Gly Gly Val Ala Pro Gly Ser Gly Ser Gly Ser Trp Ser Thr Cys

2420 2425 2430

Ser Glu Glu Asp Asp Thr Thr Val Cys Cys Ser Met Ser Tyr Ser Trp

2435 2440 2445

Thr Gly Ala Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro

2450 2455 2460

Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr

2465 2470 2475 2480

Cys Thr Thr Ser Lys Ser Ala Ser Gln Arg Ala Lys Lys Val Thr Phe

2485 2490 2495

Asp Arg Thr Gln Val Leu Asp Ala His Tyr Asp Ser Val Leu Lys Asp

2500 2505 2510

Ile Lys Leu Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Thr Leu Glu

2515 2520 2525

Glu Ala Cys Gln Leu Thr Pro Pro His Ser Ala Arg Ser Lys Tyr Gly

2530 2535 2540

Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Gly Arg Ala Val Asn His

2545 2550 2555 2560

Ile Lys Ser Val Trp Lys Asp Leu Leu Glu Asp Pro Gln Thr Pro Ile

2565 2570 2575

Pro Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Asp Pro Ala

2580 2585 2590

Lys Gly Gly Lys Lys Pro Ala Arg Leu Ile Val Tyr Pro Asp Leu Gly

2595 2600 2605

Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Ile Thr Gln Lys Leu

2610 2615 2620

Pro Gln Ala Val Met Gly Ala Ser Tyr Gly Phe Gln Tyr Ser Pro Ala

2625 2630 2635 2640

Gln Arg Val Glu Tyr Leu Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro

2645 2650 2655

Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu

2660 2665 2670

Arg Asp Ile Arg Thr Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro

2675 2680 2685

Glu Glu Ala Arg Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val

2690 2695 2700

Gly Gly Pro Met Phe Asn Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg

2705 2710 2715 2720

Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr

2725 2730 2735

Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly Ile Val Ala

2740 2745 2750

Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Ser Glu Ser

2755 2760 2765

Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala

2770 2775 2780

Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Arg Pro Glu Tyr

2785 2790 2795 2800

Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu

2805 2810 2815

Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu Thr Arg Asp Pro Thr Thr

2820 2825 2830

Pro Leu Ala Arg Ala Ala Trp Glu Thr Val Arg His Ser Pro Ile Asn

2835 2840 2845

Ser Trp Leu Gly Asn Ile Ile Gln Tyr Ala Pro Thr Ile Trp Val Arg

2850 2855 2860

Met Val Leu Met Thr His Phe Phe Ser Ile Leu Met Val Gln Asp Thr

2865 2870 2875 2880

Leu Asp Gln Asn Leu Asn Phe Glu Met Tyr Gly Ser Val Tyr Ser Val

2885 2890 2895

Asn Pro Leu Asp Leu Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp

2900 2905 2910

Ala Phe Ser Met His Thr Tyr Ser His His Glu Leu Thr Arg Val Ala

2915 2920 2925

Ser Ala Leu Arg Lys Leu Gly Ala Pro Pro Leu Arg Val Trp Lys Ser

2930 2935 2940

Arg Ala Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly Lys Ala

2945 2950 2955 2960

Ala Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu

2965 2970 2975

Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser Trp

2980 2985 2990

Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Phe His Ser Val Ser Arg

2995 3000 3005

Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu Leu Leu Leu Phe Val Gly

3010 3015 3020

Val Gly Leu Phc Leu Leu Pro Ala Arg

3025 3030

<210>5

<211>9674

<212>DNA

<213>丙肝病毒

<220>

<221>CDS

<222>(341)..(9442)

<400>5

acccgcccct aataggggcg acactccgcc atgaatcact cccctgtgag gaactactgt 60

cttcacgcag aaagcgtcta gccatggcgt tagtatgagt gtcgtacagc ctccaggccc 120

ccccctcccg ggagagccat agtggtctgc ggaaccggtg agtacaccgg aattgccggg 180

aagactgggt cctttcttgg ataaacccac tctatgcccg gccatttggg cgtgcccccg 240

caagactgct agccgagtag cgttgggttg cgaaaggcct tgtggtactg cctgataggg 300

tgcttgcgag tgccccggga ggtctcgtag accgtgcacc atg agc aca aat ccc 355

Met Ser Thr Asn Pro

1 5

aaa cct caa aga aaa acc aaa aga aac act aac cgt cgc cca caa gac 403

Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp

10 15 20

gtt aag ttt ccg ggc ggc ggc cag atc gtt ggc gga gta tac ttg ttg 451

Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu

25 30 35

ccg cgc agg ggc ccc agg ttg ggt gtg cgc gcg aca agg aag gct tcg 499

Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Ala Ser

40 45 50

gag cgg tcc cag cca cgt ggg agg cgc cag ccc atc ccc aaa cat cgg 547

Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys His Arg

55 60 65

cgc tcc act ggc aag tcc tgg ggg aag cca gga tac ccc tgg ccc ctg 595

Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pro Leu

70 75 80 85

tat ggg aat gag ggg ctc ggt tgg gca gga tgg ctc ctg tcc cct cga 643

Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg

90 95 100

ggt tcc cgt ccc tca tgg ggc ccc aat gac ccc cgg cat agg tcg cgc 691

Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro Arg His Arg Ser Arg

105 110 115

aat gtg ggt aag gtc atc gat acc cta acg tgc ggc ttt gcc gac ctc 739

Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu

120 125 130

ttg ggg tac gtc ccc gtc gta ggc gcc ccg ctt agt ggc gtt gcc agt 787

Leu Gly Tyr Val Pro Val Val Gly Ala Pro Leu Ser Gly Val Ala Ser

135 140 145

gct ctc gcg cac ggc gtg aga gtc ctg gag gac ggg gtt aat ttt gca 835

Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Phe Ala

150 155 160 165

aca ggg aac tta cct ggt tgc tcc ttt tct atc ttc ttg ctg gcc cta 883

Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu

170 175 180

ctg tcc tgc atc act act ccg gtc tct gct gtc caa gtg aag aac acc 931

Leu Ser Cys Ile Thr Thr Pro Val Ser Ala Val Gln Val Lys Asn Thr

185 190 195

age aac gcc tat atg gcg act aac gac tgt tcc aat gac agc atc act 979

Ser Asn Ala Tyr Met Ala Thr Asn Asp Cys Ser Asn Asp Ser Ile Thr

200 205 210

tgg cag ctt gag gcc gca gtc ctc cat gtc ccc ggg tgc gtc ccg tgc 1027

Trp Gln Leu Glu Ala Ala Val Leu His Val Pro Gly Cys Val Pro Cys

215 220 225

gag aaa atg ggg aac aca tca cgg tgc tgg ata cca gtc tca cca aac 1075

Glu Lys Met Gly Asn Thr Ser Arg Cys Trp Ile Pro Val Ser Pro Asn

230 235 240 245

gtg gct gtg cgg cag cct ggc gcc ctc acg cgg ggc ttg cgg acg cac 1123

Val Ala Val Arg Gln Pro Gly Ala Leu Thr Arg Gly Leu Arg Thr His

250 255 260

atc gac atg gtc gtg ttg tcc gce acg ctc tgc tcc gct ctc tac gtg 1171

Ile Asp Met Val Val Leu Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val

265 270 275

ggg gac ctc tgt ggc ggg gtg atg ctc gcg tcc cag atg ttc att gtc 1219

Gly Asp Leu Cys Gly Gly Val Met Leu Ala Ser Gln Met Phe Ile Val

280 285 290

tcg ccg cag cac cac tgg ttc gtg cag gaa tgc aat tgc tcc atc tac 1267

Ser Pro Gln His His Trp Phe Val Gln Glu Cys Asn Cys Ser Ile Tyr

295 300 305

cct ggc gcc atc act ggg cac cgt atg gca tgg gac atg atg atg aac 1315

Pro Gly Ala Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn

310 315 320 325

tgg tcg ccc acg acc acc atg atc ctg gcg tac gtg atg cgc gtt ccc 1363

Trp Ser Pro Thr Thr Thr Met Ile Leu Ala Tyr Val Met Arg Val Pro

330 335 340

gag gtc atc ata gac atc att agc gga gct cac tgg ggc gtc atg ttt 1411

Glu Val Ile Ile Asp Ile Ile Ser Gly Ala His Trp Gly Val Met Phe

345 350 355

ggc ctg gcc tac ttc tct atg cag gga gcg tgg gcg aag gtc gtt gtc 1459

Gly Leu Ala Tyr Phe Ser Met Gln Gly Ala Trp Ala Lys Val Val Val

360 365 370

atc ctc ctg ctg gcc tct ggg gtg gac gcg tac acc acc acg act ggg 1507

Ile Leu Leu Leu Ala Ser Gly Val Asp Ala Tyr Thr Thr Thr Thr Gly

375 380 385

agc gct gct ggg cgc act acc agt agc ctg gcc agc gcc ttc tcc cct 1555

Ser Ala Ala Gly Arg Thr Thr Ser Ser Leu Ala Ser Ala Phe Ser Pro

390 395 400 405

ggc gct cgg cag aac att cag ctc att aat acc aat ggt agc tgg cac 1603

Gly Ala Arg Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly Ser Trp His

410 415 420

atc aac cgc acc gcc ctg aat tgc aac gat tcc ttg cac acc ggc ttc 1651

Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu His Thr Gly Phe

425 430 435

ttc acg gcc ctg ttc tac atc cat aag ttc aac tcg tcg gga tgt ccc 1699

Phe Thr Ala Leu Phe Tyr Ile His Lys Phe Asn Ser Ser Gly Cys Pro

440 445 450

gag cgc ctg tcc gcc tgt cgc aac atc gag gac ttc cgg ata gga tgg 1747

Glu Arg Leu Ser Ala Cys Arg Asn Ile Glu Asp Phe Arg Ile Gly Trp

455 460 465

ggc gcc ctg caa tac gac gac aat gtc acc aat cca gaa gat atg agg 1795

Gly Ala Leu Gln Tyr Asp Asp Asn Val Thr Asn Pro Glu Asp Met Arg

470 475 480 485

cca tat tgc tgg cac tac cca cca aaa cag tgt ggc gta gtc ccc gca 1843

Pro Tyr Cys Trp His Tyr Pro Pro Lys Gln Cys Gly Val Val Pro Ala

490 495 500

ggg acc gtg tgc ggc cca gtg tac tgt ttc acc cct agc ccg gtg gta 1891

Gly Thr Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val

505 510 515

gtg ggc acg acc gat aga ctt gga gtg cct act tac acg tgg gga gag 1939

Val Gly Thr Thr Asp Arg Leu Gly Val Pro Thr Tyr Thr Trp Gly Glu

520 525 530

aat gag aca gat gtc ttc cta ttg aac agc acc cga cca ccg tcg ggg 1987

Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr Arg Pro Pro Ser Gly

535 540 545

tca tgg ttt ggc tgc acg tgg atg aac tcc act ggc ttc acc aag acc 2035

Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Thr

550 555 560 565

tgc ggc gca cca ccc tgc cgc act aga gct gac ttc aat acc agc aca 2083

Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp Phe Asn Thr Ser Thr

570 575 580

gat ctg ttg tgc ccc acg gac tgt ttt aga aaa cat cct gaa gcc act 2131

Asp Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr

585 590 595

tac atc aaa tgt ggt tcc ggg cct tgg ctc acg cca aag tgt ctg gtt 2179

Tyr Ile Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Lys Cys Leu Val

600 605 610

gac tac ccc tac agg ctc tgg cat tac cct tgc aca gtc aat tac tcc 2227

Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Tyr Ser

615 620 625

acc ttc aag atc agg atg tat gtg ggg gga gtt gag cac agg ctc atg 2275

Thr Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Met

630 635 640 645

gcc gcg tgc aat ttc act cgt ggg gat cgc tgc aac ttg gag gat agg 2323

Ala Ala Cys Asn Phe Thr Arg Gly Asp Arg Cys Asn Leu Glu Asp Arg

650 655 660

gac aga agt caa cag act cct ctg ttg cac tcc acc acg gaa tgg gcc 2371

Asp Arg Ser Gln Gln Thr Pro Leu Leu His Ser Thr Thr Glu Trp Ala

665 670 675

att ttg ccc tgc tct ttc tca gac ttg ccc gct ttg tcg act ggt ctt 2419

Ile Leu Pro Cys Ser Phe Ser Asp Leu Pro Ala Leu Ser Thr Gly Leu

680 685 690

ctc cac ctc cac caa aat atc gtg gac gta caa tat atg tat ggc ctg 2467

Leu His Leu His Gln Asn Ile Val Asp Val Gln Tyr Met Tyr Gly Leu

695 700 705

tca cct gcc ctc aca caa tat atc gtt cga tgg gag tgg gta gta ctc 2515

Ser Pro Ala Leu Thr Gln Tyr Ile Val Arg Trp Glu Trp Val Val Leu

710 715 720 725

tta ttc ctg ctc cta gcg gac gcc agg gtc tgc gcc tgc ttg tgg atg 2563

Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp Met

730 735 740

ctc atc ttg ctg ggc caa gcc gaa gca gca ctg gag aag ctg gtc gtc 2611

Leu Ile Leu Leu Gly Gln Ala Glu Ala Ala Leu Glu Lys Leu Val Val

745 750 755

ttg cac gct gcg agc gca gct agc tgc aat ggc ttc ctg tat ttt gtc 2659

Leu His Ala Ala Ser Ala Ala Ser Cys Asn Gly Phe Leu Tyr Phe Val

760 765 770

atc ttt ctc gtg gct gct tgg cac atc aag ggt agg gtg gtc ccc ttg 2707

Ile Phe Leu Val Ala Ala Trp His Ile Lys Gly Arg Val Val Pro Leu

775 780 785

gct gct tat tcc ctt act ggc ctg tgg ccg ttc tgc cta ctg ctc cta 2755

Ala Ala Tyr Ser Leu Thr Gly Leu Trp Pro Phe Cys Leu Leu Leu Leu

790 795 800 805

gca ctg ccc cag cag gct tac gcc tat gat gca tct gtg cac gga cag 2803

Ala Leu Pro Gln Gln Ala Tyr Ala Tyr Asp Ala Ser Val His Gly Gln

810 815 820

gtg ggc gcg gct ttg cta gta ctg att acc ctc ttt aca ctc acc ccg 2851

Val Gly Ala Ala Leu Leu Val Leu Ile Thr Leu Phe Thr Leu Thr Pro

825 830 835

ggg tat aag acc ctt ctc agc cag tcc ctg tgg tgg ttg tgc tat ctc 2899

Gly Tyr Lys Thr Leu Leu Ser Gln Ser Leu Trp Trp Leu Cys Tyr Leu

840 845 850

ctg acc ctg gcg gaa acc atg gtc cag gag tgg gca cca tcc atg cag 2947

Leu Thr Leu Ala Glu Thr Met Val Gln Glu Trp Ala Pro Ser Met Gln

855 860 865

gcg cgc ggc ggc cgt gat ggc atc ata tgg gcc gcc acc ata ttt tgc 2995

Ala Arg Gly Gly Arg Asp Gly Ile Ile Trp Ala Ala Thr Ile Phe Cys

870 875 880 885

ccg ggc gta gtg ttt gac ata acc aag tgg ctc tta gcg gtg ctt ggg 3043

Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu Leu Ala Val Leu Gly

890 895 900

cct ggt tac ctc cta aga ggt gct ttg acg cgc gtg cca tat ttc gtc 3091

Pro Gly Tyr Leu Leu Arg Gly Ala Leu Thr Arg Val Pro Tyr Phe Val

905 910 915

aga gcc cac gct ctg ctg aga atg tgc act atg gtg agg cac ctc gcg 3139

Arg Ala His Ala Leu Leu Arg Met Cys Thr Met Val Arg His Leu Ala

920 925 930

ggg ggt agg tac gtc cag atg gcg cta tta gcc ctt ggc agg tgg act 3187

Gly Gly Arg Tyr Val Gln Met Ala Leu Leu Ala Leu Gly Arg Trp Thr

935 940 945

ggc act tac atc tat gac cac ctc acc cct atg tcg gat tgg gct gct 3235

Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met Ser Asp Trp Ala Ala

950 955 960 965

agc ggc ctg cgg gac ttg gcg gtc gct gtg gag cct atc atc ttc agt 3283

Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Ile Ile Phe Ser

970 975 980

ccg atg gag aag aaa gtc atc gtt tgg gga gcg gag acg gct gcg tgc 3331

Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala Glu Thr Ala Ala Cys

985 990 995

ggg gac atc ttg cac gga ctt ccc gtg tcc gcc cga ctc ggt cgg gag 3379

Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala Arg Leu Gly Arg Glu

1000 1005 1010

atc ctc ctt ggc cca gct gat ggc tac acc tcc aag ggg tgg aag ctt 3427

Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser Lys Gly Trp Lys Leu

1015 1020 1025

ctc gcc ccc atc acc gct tac gcc cag cag aca cga ggt ctc ttg ggc 3475

Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly

1030 1035 1040 1045

tct ata gtg gtg agc atg acg ggg cgt gac aag aca gaa cag gcc ggg 3523

Ser Ile Val Val Ser Met Thr Gly Arg Asp Lys Thr Glu Gln Ala Gly

1050 1055 1060

gag gtc caa gtc ctg tcc aca gtc act cag tcc ttc ctc gga aca tcc 3571

Glu Val Gln Val Leu Ser Thr Val Thr Gln Ser Phe Leu Gly Thr Ser

1065 1070 1075

att tcg ggg gtc tta tgg act gtt tac cac gga gct ggc aac aag aca 3619

Ile Ser Gly Val Leu Trp Thr Val Tyr His Gly Ala Gly Asn Lys Thr

1080 1085 1090

cta gcc ggc tcg cgg ggc ccg gtc acg cag atg tac tcg agc gcc gag 3667

Leu Ala Gly Ser Arg Gly Pro Val Thr Gln Met Tyr Ser Ser Ala Glu

1095 1100 1105

ggg gac ttg gtc ggg tgg ccc agc cct cct ggg acc aaa tct ttg gag 3715

Gly Asp Leu Val Gly Trp Pro Ser Pro Pro Gly Thr Lys Ser Leu Glu

1110 1115 1120 1125

ccg tgt acg tgt gga gcg gtc gac ctg tat ttg gtc acg cgg aac gct 3763

Pro Cys Thr Cys Gly Ala Val Asp Leu Tyr Leu Val Thr Arg Asn Ala

1130 1135 1140

gat gtc atc ccg gct cga aga cgc ggg gac aag cgg gga gcg ctg ctc 3811

Asp Val Ile Pro Ala Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu

1145 1150 1155

tcc ccg aga ccc ctt tcg acc ttg aag ggg tcc tcg ggg gga cct gtg 3859

Ser Pro Arg Pro Leu Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val

1160 1165 1170

ctt tgc cct agg ggc cac gct gtc gga atc ttc cgg gca gct gtg tgc 3907

Leu Cys Pro Arg Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys

1175 1180 1185

tct cgg ggt gtg gct aag tcc ata gat ttc atc ccc gtt gag acg ctc 3955

Ser Arg Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu Thr Leu

1190 1195 1200 1205

gac atc gtc acg cgg tct ccc acc ttt agt gac aac agc aca cca cca 4003

Asp Ile Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser Thr Pro Pro

1210 1215 1220

gct gtg ccc cag acc tat cag gtg ggg tac ttg cac gcc ccc act ggc 4051

Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His Ala Pro Thr Gly

1225 1230 1235

agt gga aaa agc acc aag gtc ccc gtc gcg tac gcc gcc cag ggg tat 4099

Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr Ala Ala Gln Gly Tyr

1240 1245 1250

aaa gtg ctg gtg ctc aat ccc tcg gtg gct gcc acc ctg gga ttt ggg 4147

Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly

1255 1260 1265

gcg tac ttg tcc aag gca cat ggc atc aac ccc aac att agg act gga 4195

Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro Asn Ile Arg Thr Gly

1270 1275 1280 1285

gtc aga act gtg acg acc ggg gag ccc att aca tac tcc acg tat ggt 4243

Val Arg Thr Val Thr Thr Gly Glu Pro Ile Thr Tyr Ser Thr Tyr Gly

1290 1295 1300

aaa ttc ctc gcc gat ggg ggc tgc gca ggc ggc gcc tat gac atc atc 4291

Lys Phe Leu Ala Asp Gly Gly Cys Ala Gly Gly Ala Tyr Asp Ile Ile

1305 1310 1315

ata tgc gat gaa tgc cac tct gtg gat gct acc act att ctc ggc atc 4339

Ile Cys Asp Glu Cys His Ser Val Asp Ala Thr Thr Ile Leu Gly Ile

1320 1325 1330

ggg aca gtc ctt gac caa gca gag aca gcc ggg gtc agg cta act gta 4387

Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Val Arg Leu Thr Val

1335 1340 1345

ctg gcc acg gcc acg ccc ccc ggg tcg gtg aca acc ccc cat ccc aat 4435

Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Thr Pro His Pro Asn

1350 1355 1360 1365

ata gag gag gta gcc ctc gga cag gag ggt gag atc ccc ttc tat ggg 4483

Ile Glu Glu Val Ala Leu Gly Gln Glu Gly Glu Ile Pro Phe Tyr Gly

1370 1375 1380

agg gcg ttt ccc ctg tct tac atc aag gga ggg agg cac ttg att ttc 4531

Arg Ala Phe Pro Leu Ser Tyr Ile Lys Gly Gly Arg His Leu Ile Phe

1385 1390 1395

tgc cac tca aag aaa aag tgt gac gag ctc gca acg gcc ctt cgg ggc 4579

Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Thr Ala Leu Arg Gly

1400 1405 1410

atg ggc ttg aac gct gtg gca tat tac aga ggg ttg gac gtc tcc ata 4627

Met Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Ile

1415 1420 1425

ata cca act caa gga gat gtg gtg gtc gtt gcc acc gac gcc ctc atg 4675

Ile Pro Thr Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met

1430 1435 1440 1445

acg ggg tat act gga gac ttt gac tcc gtg atc gac tgc aac gta gcg 4723

Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Val Ala

1450 1455 1460

gtc acc cag gcc gta gac ttc agc ctg gac ccc acc ttc act ata acc 4771

Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Thr

1465 1470 1475

aca cag act gtc ccg caa gac gct gtc tca cgt agt cag cgc cga ggg 4819

Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly

1480 1485 1490

cgc acg ggt aga gga aga ctg ggc att tat agg tat gtt tcc act ggt 4867

Arg Thr Gly Arg Gly Arg Leu Gly Ile Tyr Arg Tyr Val Ser Thr Gly

1495 1500 1505

gag cga gcc tca gga atg ttt gac agt gta gta ctc tgt gag tgc tac 4915

Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu Cys Glu Cys Tyr

1510 1515 1520 1525

gac gca gga gct gct tgg tat gag ctc tca cca gtg gag acg acc gtc 4963

Asp Ala Gly Ala Ala Trp Tyr Glu Leu Ser Pro Val Glu Thr Thr Val

1530 1535 1540

agg ctc agg gcg tat ttc aac acg cct ggc ttg cct gtg tgc cag gac 5011

Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu Pro Val Cys Gln Asp

1545 1550 1555

cac ctt gag ttt tgg gag gca gtt ttc acc ggc ctc aca cac ata gac 5059

His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His Ile Asp

1560 1565 1570

gct cat ttc ctt tcc cag aca aag cag tcg ggg gaa aat ttc gca tac 5107

Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Phe Ala Tyr

1575 1580 1585

tta gta gcc tat cag gcc aca gtg tgc gcc agg gcc aaa gcg ccc ccc 5155

Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro

1590 1595 1600 1605

ccg tcc tgg gac gtc atg tgg aag tgc ttg act cga ctc aag ccc acg 5203

Pro Ser Trp Asp Val Met Trp Lys Cys Leu Thr Arg Leu Lys Pro Thr

1610 1615 1620

ctt gtg ggc cct aca cct ctc ctg tac cgt ttg ggc tct gtt acc aac 5251

Leu Val Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ser Val Thr Asn

1625 1630 1635

gag gtc acc ctt aca cac ccc gtg aca aaa tac atc gcc aca tgc atg 5299

Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr Cys Met

1640 1645 1650

caa gct gac ctc gag gtc atg acc agc acg tgg gtc ctg gct ggg gga 5347

Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly Gly

1655 1660 1665

gtc tta gca gcc gtc gcc gcg tat tgc tta gcg acc ggg tgt gtt tcc 5395

Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val Ser

1670 1675 1680 1685

atc att ggc cgt tta cac atc aac cag cga gct gtc gtc gct ccg gac 5443

Ile Ile Gly Arg Leu His Ile Asn Gln Arg Ala Val Val Ala Pro Asp

1690 1695 1700

aag gag gtc ctc tat gag gct ttt gat gag atg gag gaa tgt gcc tcc 5491

Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser

1705 1710 1715

aga gcg gct ctc ctt gaa gag ggg cag cgg ata gcc gag atg ctg aag 5539

Arg Ala Ala Leu Leu Glu Glu Gly Gln Arg Ile Ala Glu Met Leu Lys

1720 1725 1730

tcc aag atc caa ggc tta ttg cag caa gcc tct aaa cag gcc cag gac 5587

Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln Ala Gln Asp

1735 1740 1745

ata caa ccc gct gtg caa gct tcg tgg ccc aag atg gag caa ttc tgg 5635

Ile Gln Pro Ala Val Gln Ala Ser Trp Pro Lys Met Glu Gln Phe Trp

1750 1755 1760 1765

gcc aaa cat atg tgg aac ttc ata agc ggc att cag tac ctc gca gga 5683

Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly

1770 1775 1780

ctg tca aca ctg cca ggg aac cct gct gtg gct tcc atg atg gca ttc 5731

Leu Ser Thr Leu Pro Gly Asn Pro Ala Val Ala Ser Met Met Ala Phe

1785 1790 1795

agc gcc gcc ctc acc agt ccg ttg tca act agc acc acc atc ctt ctt 5779

Ser Ala Ala Leu Thr Ser Pro Leu Ser Thr Ser Thr Thr Ile Leu Leu

1800 1805 1810

aac att ctg ggg ggc tgg ctg gcg tcc caa att gcg cca ccc gcg ggg 5827

Asn Ile Leu Gly Gly Trp Leu Ala Ser Gln Ile Ala Pro Pro Ala Gly

1815 1820 1825

gcc act ggc ttt gtt gtc agt ggc ctg gtg gga gct gct gtt ggc agc 5875

Ala Thr Gly Phe Val Val Ser Gly Leu Val Gly Ala Ala Val Gly Ser

1830 1835 1840 1845

ata ggc ttg ggt aaa gtg ctg gtg gac atc ctg gca ggg tat ggt gcg 5923

Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala

1850 1855 1860

ggc att tcg ggg gcc ctc gtc gcg ttt aag atc atg tct ggc gag aag 5971

Gly Ile Ser Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys

1865 1870 1875

ccc tcc atg gag gat gtc atc aac ttg ctg cct ggg att ctg tct cca 6019

Pro Ser Met Glu Asp Val Ile Asn Leu Leu Pro Gly Ile Leu Ser Pro

1880 1885 1890

ggt gct ctg gtg gtg gga gtc atc tgc gcg gcc att ctg cgc cgc cat 6067

Gly Ala Leu Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg His

1895 1900 1905

gtg gga ccg ggg gaa ggc gcg gtc caa tgg atg aac agg ctt atc gcc 6115

Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala

1910 1915 1920 1925

ttc gct tcc aga gga aac cac gtc gcc cct act cac tac gtg acg gag 6163

Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Thr Glu

1930 1935 1940

tcg gat gcg tcg cag cgt gtc acc caa ctg ctt ggc tct ctc act ata 6211

Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly Ser Leu Thr Ile

1945 1950 1955

act agt cta ctc agg aga ctt cac aac tgg atc act gag gat tgc ccc 6259

Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile Thr Glu Asp Cys Pro

1960 1965 1970

atc cca tgc gcc ggc tcg tgg ctc cgc gat gtg tgg gac tgg gtc tgt 6307

Ile Pro Cys Ala Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Val Cys

1975 1980 1985

acc atc cta aca gac ttt aag aac tgg ctg acc tcc aag ctg ttc cca 6355

Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr Ser Lys Leu Phe Pro

1990 1995 2000 2005

aag atg cct ggc ctc ccc ttt atc tct tgc caa aag ggg tac aag ggc 6403

Lys Met Pro Gly Leu Pro Phe Ile Ser Cys Gln Lys Gly Tyr Lys Gly

2010 2015 2020

gtg tgg gcc ggc act ggc atc atg acc aca cga tgc ccc tgc ggc gcc 6451

Val Trp Ala Gly Thr Gly Ile Met Thr Thr Arg Cys Pro Cys Gly Ala

2025 2030 2035

aac atc tct ggc aac gtc cgc ttg ggc tct atg aga atc aca gga ccc 6499

Asn Ile Ser Gly Asn Val Arg Leu Gly Ser Met Arg Ile Thr Gly Pro

2040 2045 2050

aaa acc tgc atg aac acc tgg cag ggg acc ttt cct atc aat tgt tat 6547

Lys Thr Cys Met Asn Thr Trp Gln Gly Thr Phe Pro Ile Asn Cys Tyr

2055 2060 2065

aca gaa ggc cag tgc ttg ccg aaa ccc gcg tta aac ttc aag acc gcc 6595

Thr Glu Gly Gln Cys Leu Pro Lys Pro Ala Leu Asn Phe Lys Thr Ala

2070 2075 2080 2085

atc tgg aga gtg gcg gcc tca gag tac gcg gaa gtg acg cag cac gga 6643

Ile Trp Arg Val Ala Ala Ser Glu Tyr Ala Glu Val Thr Gln His Gly

2090 2095 2100

tca tat gcc tat ata aca ggg ctg acc act gac aac tta aaa gtc cct 6691

Ser Tyr Ala Tyr Ile Thr Gly Leu Thr Thr Asp Asn Leu Lys Val Pro

2105 2110 2115

tgc caa ctc ccc tct cca gag ttt ttc tct tgg gtg gac gga gta caa 6739

Cys Gln Leu Pro Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln

2120 2125 2130

atc cat agg tcc gcc ccc aca cca aag ccg ttt ttc cgg gat gag gtc 6787

Ile His Arg Ser Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val

2135 2140 2145

tcg ttc agc gtt ggg ctc aat tca ttt gtc gtc ggg tct cag ctt ccc 6835

Ser Phe Ser Val Gly Leu Asn Ser Phe Val Val Gly Ser Gln Leu Pro

2150 2155 2160 2165

tgt gac cct gag ccc gac act gag gta gtg atg tcc atg cta aca gac 6883

Cys Asp Pro Glu Pro Asp Thr Glu Val Val Met Ser Met Leu Thr Asp

2170 2175 2180

cca tcc cat atc acg gcg gag gct gca gcg cgg cgt tta gcg cgg ggg 6931

Pro Ser His Ile Thr Ala Glu Ala Ala Ala Arg Arg Leu Ala Arg Gly

2185 2190 2195

tca ccc cca tct gag gca agc tcc tca gcg agc cag ctg tcg gcg cca 6979

Ser Pro Pro Ser Glu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro

2200 2205 2210

tcg ctg cga gcc acc tgc acc acc cac ggt agg acc tat gat gtg gac 7027

Ser Leu Arg Ala Thr Cys Thr Thr His Gly Arg Thr Tyr Asp Val Asp

2215 2220 2225

atg gtg gat gcc aac ctg ttc atg ggg ggc ggc gtg att cgg ata gag 7075

Met Val Asp Ala Asn Leu Phe Met Gly Gly Gly Val Ile Arg Ile Glu

2230 2235 2240 2245

tct gag tcc aaa gtg gtc gtt ctg gac tcc ctc gac tca atg acc gag 7123

Ser Glu Ser Lys Val Val Val Leu Asp Ser Leu Asp Ser Met Thr Glu

2250 2255 2260

gaa gag ggc gac ctt gag cct tca gta cca tcg gag tat atg ctc ccc 7171

Glu Glu Gly Asp Leu Glu Pro Ser Val Pro Ser Glu Tyr Met Leu Pro

2265 2270 2275

agg aag agg ttc cca ccg gcc tta ccg gct tgg gcg cgg cct gat tac 7219

Arg Lys Arg Phe Pro Pro Ala Leu Pro Ala Trp Ala Arg Pro Asp Tyr

2280 2285 2290

aac cca ccg ctt gtg gaa tcg tgg aag agg cca gat tac caa cca ccc 7267

Asn Pro Pro Leu Val Glu Ser Trp Lys Arg Pro Asp Tyr Gln Pro Pro

2295 2300 2305

act gtt gcg ggc tgt gct ctc ccc ccc ccc aaa aag acc ccg acg cct 7315

Thr Val Ala Gly Cys Ala Leu Pro Pro Pro Lys Lys Thr Pro Thr Pro

2310 2315 2320 2325

cct cca agg aga cgc cgg aca gtg ggt ctg agc gag agc acc ata gga 7363

Pro Pro Arg Arg Arg Arg Thr Val Gly Leu Ser Glu Ser Thr Ile Gly

2330 2335 2340

gat gcc ctc caa cag ctg gcc atc aag tcc ttt ggc cag ccc ccc cca 7411

Asp Ala Leu Gln Gln Leu Ala Ile Lys Ser Phe Gly Gln Pro Pro Pro

2345 2350 2355

agc ggc gat tca ggc ctt tcc acg ggg gcg gac gcc gcc gac tcc ggc 7459

Ser Gly Asp Ser Gly Leu Ser Thr Gly Ala Asp Ala Ala Asp Ser Gly

2360 2365 2370

gat cgg aca ccc cct gac gag ttg gct ctt tcg gag aca ggt tct acc 7507

Asp Arg Thr Pro Pro Asp Glu Leu Ala Leu Ser Glu Thr Gly Ser Thr

2375 2380 2385

tcc tcc atg ccc ccc ctc gag ggg gag cct ggg gac cca gac ctg gag 7555

Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu

2390 2395 2400 2405

cct gag cag gta gag ctt caa cct cct ccc cag ggg ggg gag gca gct 7603

Pro Glu Gln Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Glu Ala Ala

2410 2415 2420

ccc ggc tcg gac tcg ggg tcc tgg tct act tgc tcc gag gag gat gac 7651

Pro Gly Ser Asp Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp Asp

2425 2430 2435

tcc gtc gtg tgc tgc tcc atg tca tat tcc tgg acc ggg gct cta ata 7699

Ser Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Ile

2440 2445 2450

act cct tgt agc ccc gaa gag gaa aag ttg cca att aac tcc ttg agc 7747

Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro Ile Asn Ser Leu Ser

2455 2460 2465

aac tcg ctg ttg cga tac cat aac aag gta tac tgt act aca tca aag 7795

Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr Cys Thr Thr Ser Lys

2470 2475 2480 2485

agt gcc tca cta agg gct aaa aag gta act ttt gat agg atg caa gtg 7843

Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp Arg Met Gln Val

2490 2495 2500

ctc gac gcc tat tat gat tca gtc tta aag gac atc aag cta gcg gcc 7891

Leu Asp Ala Tyr Tyr Asp Ser Val Leu Lys Asp Ile Lys Leu Ala Ala

2505 25l0 2515

tcc aag gtc agc gca agg ctc ctc acc tta gag gag gcg tgc caa ttg 7939

Ser Lys Val Ser Ala Arg Leu Leu Thr Leu Glu Glu Ala Cys Gln Leu

2520 2525 2530

acc cca ccc cac tct gca aga tcc aag tat ggg ttt ggg gct aag gag 7987

Thr Pro Pro His Ser Ala Arg Ser Lys Tyr Gly Phe Gly Ala Lys Glu

2535 2540 2545

gtc cgc agc ttg tcc ggg agg gcc gtc aac cac atc aag tcc gtg tgg 8035

Val Arg Ser Leu Ser Gly Arg Ala Val Asn His Ile Lys Ser Val Trp

2550 2555 2560 2565

aag gac ctc ttg gaa gac tca caa aca cca att cct aca acc atc atg 8083

Lys Asp Leu Leu Glu Asp Ser Gln Thr Pro Ile Pro Thr Thr Ile Met

2570 2575 2580

gcc aaa aat gag gtg ttc tgc gtg gac ccc gcc aag ggg ggt aaa aaa 8131

Ala Lys Asn Glu Val Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys

2585 2590 2595

cca gct cgc ctt atc gtt tac cct gac ctc ggc gtc agg gtc tgc gag 8179

Pro Ala Arg Leu Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu

2600 2605 2610

aag atg gcc ctt tat gat gtc aca caa aag ctt cct cag gcg gtg atg 8227

Lys Met Ala Leu Tyr Asp Val Thr Gln Lys Leu Pro Gln Ala Val Met

2615 2620 2625

ggg gct tct tat ggc ttc cag tac tcc ccc gct cag cgg gtg gag ttt 8275

Gly Ala Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Phe

2630 2635 2640 2645

ctc ttg aag gca tgg gcg gaa aag aga gac cct atg ggt ttt tcg tat 8323

Leu Leu Lys Ala Trp Ala Glu Lys Arg Asp Pro Met Gly Phe Ser Tyr

2650 2655 2660

gat acc cga tgc ttt gac tca acc gtc act gag aga gac atc agg act 8371

Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp Ile Arg Thr

2665 2670 2675

gag gag tcc ata tac cag gcc tgc tcc tta ccc gag gag gcc cga act 8419

Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro Glu Glu Ala Arg Thr

2680 2685 2690

gcc ata cac tcg ctg act gag aga ctc tat gtg gga ggg ccc atg ttc 8467

Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Phe

2695 2700 2705

aac agc aag ggc cag tcc tgc ggg tac agg cgt tgc cgc gcc agc ggg 8515

Asn Ser Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly

2710 2715 2720 2725

gtg ctt acc act agt atg ggg aac acc atc aca tgc tat gta aaa gcc 8563

Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr Cys Tyr Val Lys Ala

2730 2735 2740

cta gcg gct tgc aag gct gcg ggg ata att gcg ccc acg atg ctg gta 8611

Leu Ala Ala Cys Lys Ala Ala Gly Ile Ile Ala Pro Thr Met Leu Val

2745 2750 2755

tgc ggc gac gac ttg gtc gtc atc tca gaa agc cag ggg act gag gag 8659

Cys Gly Asp Asp Leu Val Val Ile Ser Glu Ser Gln Gly Thr Glu Glu

2760 2765 2770

gac gag cgg aac ctg aga gcc ttc acg gag gct atg acc agg tat tct 8707

Asp Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser

2775 2780 2785

gcc cct cct ggt gac ccc ccc aga ccg gaa tat gac ctg gag cta ata 8755

Ala Pro Pro Gly Asp Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile

2790 2795 2800 2805

aca tct tgt tcc tca aac gtg tct gtg gca ctt ggc cca cag ggc cgc 8803

Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu Gly Pro Gln Gly Arg

2810 2815 2820

cgc aga tac tac ctg acc aga gac ccc acc act tca att gcc cgg gct 8851

Arg Arg Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Ser Ile Ala Arg Ala

2825 2830 2835

gcc tgg gaa aca gtt aga cac tcc cct gtc aat tca tgg ctg gga aac 8899

Ala Trp Glu Thr Val Arg His Ser Pro Val Asn Ser Trp Leu Gly Asn

2840 2845 2850

atc atc cag tac gct cca acc ata tgg gtt cgc atg gtc ctg atg aca 8947

Ile Ile Gln Tyr Ala Pro Thr Ile Trp Val Arg Met Val Leu Met Thr

2855 2860 2865

cac ttc ttc tcc att ctc atg gcc cag gac acc cta gac cag aac ctt 8995

His Phe Phe Ser Ile Leu Met Ala Gln Asp Thr Leu Asp Gln Asn Leu

2870 2875 2880 2885

aac ttt gaa atg tac gga tcg gtg tac tcc gtg agt cct ctg gac ctc 9043

Asn Phe Glu Met Tyr Gly Ser Val Tyr Ser Val Ser Pro Leu Asp Leu

2890 2895 2900

cca gcc ata att gaa agg tta cac ggg ctt gac gcc ttc tct ctg cac 9091

Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala Phe Ser Leu His

2905 2910 2915

aca tac act ccc cac gaa ctg acg cgg gtg gct tca gcc ctc aga aaa 9139

Thr Tyr Thr Pro His Glu Leu Thr Arg Val Ala Ser Ala Leu Arg Lys

2920 2925 2930

ctt ggg gcg cca ccc ctc aga gcg tgg aag agt cgg gcg cgt gca gtt 9187

Leu Gly Ala Pro Pro Leu Arg Ala Trp Lys Ser Arg Ala Arg Ala Val

2935 2940 2945

agg gcg tcc ctc atc tcc cgt ggg ggg agg gcg gcc gtt tgc ggt cgg 9235

Arg Ala Ser Leu Ile Ser Arg Gly Gly Arg Ala Ala Val Cys Gly Arg

2950 2955 2960 2965

tac ctc ttc aac tgg gcg gtg aag acc aag ctc aaa ctc act cct ttg 9283

Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu

2970 2975 2980

ccg gag gca cgc ctc ctg gat ttg tcc agt tgg ttt acc gtc ggc gcc 9331

Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser Trp Phe Thr Val Gly Ala

2985 2990 2995

ggc ggg ggc gac att tat cac agc gtg tcg cgt gcc cga ccc cgc cta 9379

Gly Gly Gly Asp Ile Tyr His Ser Val Ser Arg Ala Arg Pro Arg Leu

3000 3005 3010

tta ctc ctt agc cta ctc cta ctt tct gta ggg gta ggc ctc ttc cta 9427

Leu Leu Leu Ser Leu Leu Leu Leu Ser Val Gly Val Gly Leu Phe Leu

3015 3020 3025

ctc ccc gct cga tag agcggcacac attagctaca ctccatagct aactgttcct 9482

Leu Pro Ala Arg

3030

tttttttttt tttttttttt tttttttttt tttttttctt tttttttttt tttccctctt 9542

tcttcccttc tcatcttatt ctactttctt tcttggtggc tccatcttag ccctggtcac 9602

ggctagctgt gaaaggtccg tgagccgcat gactgcagag agtgccgtaa ctggtctctc 9662

tgcagatcat gt 9674

<210>6

<211>3033

<212>PRT

<213>丙肝病毒

<400>6

Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn

1 5 10 15

Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly

20 25 30

Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala

35 40 45

Thr Arg Lys Ala Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro

50 55 60

Ile Pro Lys His Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly

65 70 75 80

Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp

85 90 95

Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro

100 105 110

Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys

115 120 125

Gly Phe Ala Asp Leu Leu Gly Tyr Val Pro Val Val Gly Ala Pro Leu

130 135 140

Ser Gly Val Ala Ser Ala Leu Ala His Gly Val Arg Val Leu Glu Asp

145 150 155 160

Gly Val Asn Phe Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile

165 170 175

Phe Leu Leu Ala Leu Leu Ser Cys Ile Thr Thr Pro Val Ser Ala Val

180 185 190

Gln Val Lys Asn Thr Ser Asn Ala Tyr Met Ala Thr Asn Asp Cys Ser

195 200 205

Asn Asp Ser Ile Thr Trp Gln Leu Glu Ala Ala Val Leu His Val Pro

210 215 220

Gly Cys Val Pro Cys Glu Lys Met Gly Asn Thr Ser Arg Cys Trp Ile

225 230 235 240

Pro Val Ser Pro Asn Val Ala Val Arg Gln Pro Gly Ala Leu Thr Arg

245 250 255

Gly Leu Arg Thr His Ile Asp Met Val Val Leu Ser Ala Thr Leu Cys

260 265 270

Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Gly Val Met Leu Ala Ser

275 280 285

Gln Met Phe Ile Val Ser Pro Gln His His Trp Phe Val Gln Glu Cys

290 295 300

Asn Cys Ser Ile Tyr Pro Gly Ala Ile Thr Gly His Arg Met Ala Trp

305 310 315 320

Asp Met Met Met Asn Trp Ser Pro Thr Thr Thr Met Ile Leu Ala Tyr

325 330 335

Val Met Arg Val Pro Glu Val Ile Ile Asp Ile Ile Ser Gly Ala His

340 345 350

Trp Gly Val Met Phe Gly Leu Ala Tyr Phe Ser Met Gln Gly Ala Trp

355 360 365

Ala Lys Val Val Val Ile Leu Leu Leu Ala Ser Gly Val Asp Ala Tyr

370 375 380

Thr Thr Thr Thr Gly Ser Ala Ala Gly Arg Thr Thr Ser Ser Leu Ala

385 390 395 400

Ser Ala Phe Ser Pro Gly Ala Arg Gln Asn Ile Gln Leu Ile Asn Thr

405 410 415

Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser

420 425 430

Leu His Thr Gly Phe Phe Thr Ala Leu Phe Tyr Ile His Lys Phe Asn

435 440 445

Ser Ser Gly Cys Pro Glu Arg Leu Ser Ala Cys Arg Asn Ile Glu Asp

450 455 460

Phe Arg Ile Gly Trp Gly Ala Leu Gln Tyr Asp Asp Asn Val Thr Asn

465 470 475 480

Pro Glu Asp Met Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Gln Cys

485 490 495

Gly Val Val Pro Ala Gly Thr Val Cys Gly Pro Val Tyr Cys Phe Thr

500 505 510

Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Leu Gly Val Pro Thr

515 520 525

Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe Leu Leu Asn Ser Thr

530 535 540

Arg Pro Pro Ser Gly Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Thr

545 550 555 560

Gly Phe Thr Lys Thr Cys Gly Ala Pro Pro Cys Arg Thr Arg Ala Asp

565 570 575

Phe Asn Thr Ser Thr Asp Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys

580 585 590

His Pro Glu Ala Thr Tyr Ile Lys Cys Gly Ser Gly Pro Trp Leu Thr

595 600 605

Pro Lys Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys

610 615 620

Thr Val Asn Tyr Ser Thr Phe Lys Ile Arg Met Tyr Val Gly Gly Val

625 630 635 640

Glu His Arg Leu Met Ala Ala Cys Asn Phe Thr Arg Gly Asp Arg Cys

645 650 655

Asn Leu Glu Asp Arg Asp Arg Ser Gln Gln Thr Pro Leu Leu His Ser

660 665 670

Thr Thr Glu Trp Ala Ile Leu Pro Cys Ser Phe Ser Asp Leu Pro Ala

675 680 685

Leu Ser Thr Gly Leu Leu His Leu His Gln Asn Ile Val Asp Val Gln

690 695 700

Tyr Met Tyr Gly Leu Ser Pro Ala Leu Thr Gln Tyr Ile Val Arg Trp

705 710 715 720

Glu Trp Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys

725 730 735

Ala Cys Leu Trp Met Leu Ile Leu Leu Gly Gln Ala Glu Ala Ala Leu

740 745 750

Glu Lys Leu Val Val Leu His Ala Ala Ser Ala Ala Ser Cys Asn Gly

755 760 765

Phe Leu Tyr Phe Val Ile Phe Leu Val Ala Ala Trp His Ile Lys Gly

770 775 780

Arg Val Val Pro Leu Ala Ala Tyr Ser Leu Thr Gly Leu Trp Pro Phe

785 790 795 800

Cys Leu Leu Leu Leu Ala Leu Pro Gln Gln Ala Tyr Ala Tyr Asp Ala

805 810 815

Ser Val His Gly Gln Val Gly Ala Ala Leu Leu Val Leu Ile Thr Leu

820 825 830

Phe Thr Leu Thr Pro Gly Tyr Lys Thr Leu Leu Ser Gln Ser Leu Trp

835 840 845

Trp Leu Cys Tyr Leu Leu Thr Leu Ala Glu Thr Met Val Gln Glu Trp

850 855 860

Ala Pro Ser Met Gln Ala Arg Gly Gly Arg Asp Gly Ile Ile Trp Ala

865 870 875 880

Ala Thr Ile Phe Cys Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu

885 890 895

Leu Ala Val Leu Gly Pro Gly Tyr Leu Leu Arg Gly Ala Leu Thr Arg

900 905 910

Val Pro Tyr Phe Val Arg Ala His Ala Leu Leu Arg Met Cys Thr Met

915 920 925

Val Arg His Leu Ala Gly Gly Arg Tyr Val Gln Met Ala Leu Leu Ala

930 935 940

Leu Gly Arg Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met

945 950 955 960

Ser Asp Trp Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu

965 970 975

Pro Ile Ile Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala

980 985 990

Glu Thr Ala Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala

995 1000 1005

Arg Leu Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser

1010 1015 1020

Lys Gly Trp Lys Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr

1025 1030 1035 1040

Arg Gly Leu Leu Gly Ser Ile Val Val Ser Met Thr Gly Arg Asp Lys

1045 1050 1055

Thr Glu Gln Ala Gly Glu Val Gln Val Leu Ser Thr Val Thr Gln Ser

1060 1065 1070

Phe Leu Gly Thr Ser Ile Ser Gly Val Leu Trp Thr Val Tyr His Gly

1075 1080 1085

Ala Gly Asn Lys Thr Leu Ala Gly Ser Arg Gly Pro Val Thr Gln Met

1090 1095 1100

Tyr Ser Ser Ala Glu Gly Asp Leu Val Gly Trp Pro Ser Pro Pro Gly

1105 1110 1115 1120

Thr Lys Ser Leu Glu Pro Cys Thr Cys Gly Ala Val Asp Leu Tyr Leu

1125 1130 1135

Val Thr Arg Asn Ala Asp Val Ile Pro Ala Arg Arg Arg Gly Asp Lys

1140 1145 1150

Arg Gly Ala Leu Leu Ser Pro Arg Pro Leu Ser Thr Leu Lys Gly Ser

1155 1160 1165

Ser Gly Gly Pro Val Leu Cys Pro Arg Gly His Ala Val Gly Ile Phe

1170 1175 1180

Arg Ala Ala Val Cys Ser Arg Gly Val Ala Lys Ser Ile Asp Phe Ile

1185 1190 1195 1200

Pro Val Glu Thr Leu Asp Ile Val Thr Arg Ser Pro Thr Phe Ser Asp

1205 1210 1215

Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu

1220 1225 1230

His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr

1235 1240 1245

Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala

1250 1255 1260

Thr Leu Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro

1265 1270 1275 1280

Asn Ile Arg Thr Gly Val Arg Thr Val Thr Thr Gly Glu Pro Ile Thr

1285 1290 1295

Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Gly Gly

1300 1305 1310

Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Val Asp Ala Thr

1315 1320 1325

Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly

1330 1335 1340

Val Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr

1345 1350 1355 1360

Thr Pro His Pro Asn Ile Glu Glu Val Ala Leu Gly Gln Glu Gly Glu

1365 1370 1375

Ile Pro Phe Tyr Gly Arg Ala Phe Pro Leu Ser Tyr Ile Lys Gly Gly

1380 1385 1390

Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala

1395 1400 1405

Thr Ala Leu Arg Gly Met Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly

1410 1415 1420

Leu Asp Val Ser Ile Ile Pro Thr Gln Gly Asp Val Val Val Val Ala

1425 1430 1435 1440

Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile

1445 1450 1455

Asp Cys Asn Val Ala Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro

1460 1465 1470

Thr Phe Thr Ile Thr Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg

1475 1480 1485

Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Leu Gly Ile Tyr Arg

1490 1495 1500

Tyr Val Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val

1505 1510 1515 1520

Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Ser Pro

1525 1530 1535

Val Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu

1540 1545 1550

Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly

1555 1560 1565

Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly

1570 1575 1580

Glu Asn Phe Ala Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg

1585 1590 1595 1600

Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu Thr

1605 1610 1615

Arg Leu Lys Pro Thr Leu Val Gly Pro Thr Pro Leu Leu Tyr Arg Leu

1620 1625 1630

Gly Ser Val Thr Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr

1635 1640 1645

Ile Ala Thr Cys Met Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp

1650 1655 1660

Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala

1665 1670 1675 1680

Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Ile Asn Gln Arg Ala

1685 1690 1695

Val Val Ala Pro Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met

1700 1705 1710

Glu Glu Cys Ala Ser Arg Ala Ala Leu Leu Glu Glu Gly Gln Arg Ile

1715 1720 1725

Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser

1730 1735 1740

Lys Gln Ala Gln Asp Ile Gln Pro Ala Val Gln Ala Ser Trp Pro Lys

1745 1750 1755 1760

Met Glu Gln Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile

1765 1770 1775

Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val Ala

1780 1785 1790

Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Ser Thr Ser

1795 1800 1805

Thr Thr Ile Leu Leu Asn Ile Leu Gly Gly Trp Leu Ala Ser Gln Ile

1810 1815 1820

Ala Pro Pro Ala Gly Ala Thr Gly Phe Val Val Ser Gly Leu Val Gly

1825 1830 1835 1840

Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu

1845 1850 1855

Ala Gly Tyr Gly Ala Gly Ile Ser Gly Ala Leu Val Ala Phe Lys Ile

1860 1865 1870

Met Ser Gly Glu Lys Pro Ser Met Glu Asp Val Ile Asn Leu Leu Pro

1875 1880 1885

Gly Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Ile Cys Ala Ala

1890 1895 1900

Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met

1905 1910 1915 1920

Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr

1925 1930 1935

His Tyr Val Thr Glu Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu

1940 1945 1950

Gly Ser Leu Thr Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile

1955 1960 1965

Thr Glu Asp Cys Pro Ile Pro Cys Ala Gly Ser Trp Leu Arg Asp Val

1970 1975 1980

Trp Asp Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr

1985 1990 1995 2000

Ser Lys Leu Phe Pro Lys Met Pro Gly Leu Pro Phe Ile Ser Cys Gln

2005 2010 2015

Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Ile Met Thr Thr Arg

2020 2025 2030

Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val Arg Leu Gly Ser Met

2035 2040 2045

Arg Ile Thr Gly Pro Lys Thr Cys Met Asn Thr Trp Gln Gly Thr Phe

2050 2055 2060

Pro Ile Asn Cys Tyr Thr Glu Gly Gln Cys Leu Pro Lys Pro Ala Leu

2065 2070 2075 2080

Asn Phe Lys Thr Ala Ile Trp Arg Val Ala Ala Ser Glu Tyr Ala Glu

2085 2090 2095

Val Thr Gln His Gly Ser Tyr Ala Tyr Ile Thr Gly Leu Thr Thr Asp

2100 2105 2110

Asn Leu Lys Val Pro Cys Gln Leu Pro Ser Pro Glu Phe Phe Ser Trp

2115 2120 2125

Val Asp Gly Val Gln Ile His Arg Ser Ala Pro Thr Pro Lys Pro Phe

2130 2135 2140

Phe Arg Asp Glu Val Ser Phe Ser Val Gly Leu Asn Ser Phe Val Val

2145 2150 2155 2160

Gly Ser Gln Leu Pro Cys Asp Pro Glu Pro Asp Thr Glu Val Val Met

2165 2170 2175

Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Ala Arg

2180 2185 2190

Arg Leu Ala Arg Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Ala Ser

2195 2200 2205

Gln Leu Ser Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Gly Arg

2210 2215 2220

Thr Tyr Asp Val Asp Met Val Asp Ala Asn Leu Phe Met Gly Gly Gly

2225 2230 2235 2240

Val Ile Arg Ile Glu Ser Glu Ser Lys Val Val Val Leu Asp Ser Leu

2245 2250 2255

Asp Ser Met Thr Glu Glu Glu Gly Asp Leu Glu Pro Ser Val Pro Ser

2260 2265 2270

Glu Tyr Met Leu Pro Arg Lys Arg Phe Pro Pro Ala Leu Pro Ala Trp

2275 2280 2285

Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp Lys Arg Pro

2290 2295 2300

Asp Tyr Gln Pro Pro Thr Val Ala Gly Cys Ala Leu Pro Pro Pro Lys

2305 2310 2315 2320

Lys Thr Pro Thr Pro Pro Pro Arg Arg Arg Arg Thr Val Gly Leu Ser

2325 2330 2335

Glu Ser Thr Ile Gly Asp Ala Leu Gln Gln Leu Ala Ile Lys Ser Phe

2340 2345 2350

Gly Gln Pro Pro Pro Ser Gly Asp Ser Gly Leu Ser Thr Gly Ala Asp

2355 2360 2365

Ala Ala Asp Ser Gly Asp Arg Thr Pro Pro Asp Glu Leu Ala Leu Ser

2370 2375 2380

Glu Thr Gly Ser Thr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly

2385 2390 2395 2400

Asp Pro Asp Leu Glu Pro Glu Gln Val Glu Leu Gln Pro Pro Pro Gln

2405 2410 2415

Gly Gly Glu Ala Ala Pro Gly Ser Asp Ser Gly Ser Trp Ser Thr Cys

2420 2425 2430

Ser Glu Glu Asp Asp Ser Val Val Cys Cys Ser Met Ser Tyr Ser Trp

2435 2440 2445

Thr Gly Ala Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro

2450 2455 2460

Ile Asn Ser Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr

2465 2470 2475 2480

Cys Thr Thr Ser Lys Ser Ala Ser Leu Arg Ala Lys Lys Val Thr Phe

2485 2490 2495

Asp Arg Met Gln Val Leu Asp Ala Tyr Tyr Asp Ser Val Leu Lys Asp

2500 2505 2510

Ile Lys Leu Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Thr Leu Glu

2515 2520 2525

Glu Ala Cys Gln Leu Thr Pro Pro His Ser Ala Arg Ser Lys Tyr Gly

2530 2535 2540

Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Gly Arg Ala Val Asn His

2545 2550 2555 2560

Ile Lys Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Gln Thr Pro Ile

2565 2570 2575

Pro Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Asp Pro Ala

2580 2585 2590

Lys Gly Gly Lys Lys Pro Ala Arg Leu Ile Val Tyr Pro Asp Leu Gly

2595 2600 2605

Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Thr Gln Lys Leu

2610 2615 2620

Pro Gln Ala Val Met Gly Ala Ser Tyr Gly Phe Gln Tyr Ser Pro Ala

2625 2630 2635 2640

Gln Arg Val Glu Phe Leu Leu Lys Ala Trp Ala Glu Lys Arg Asp Pro

2645 2650 2655

Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu

2660 2665 2670

Arg Asp Ile Arg Thr Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro

2675 2680 2685

Glu Glu Ala Arg Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val

2690 2695 2700

Gly Gly Pro Met Phe Asn Ser Lys Gly Gln Ser Cys Gly Tyr Arg Arg

2705 2710 2715 2720

Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr

2725 2730 2735

Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly Ile Ile Ala

2740 2745 2750

Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Ser Glu Ser

2755 2760 2765

Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala

2770 2775 2780

Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Arg Pro Glu Tyr

2785 2790 2795 2800

Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu

2805 2810 2815

Gly Pro Gln Gly Arg Arg Arg Tyr Tyr Leu Thr Arg Asp Pro Thr Thr

2820 2825 2830

Ser Ile Ala Arg Ala Ala Trp Glu Thr Val Arg His Ser Pro Val Asn

2835 2840 2845

Ser Trp Leu Gly Asn Ile Ile Gln Tyr Ala Pro Thr Ile Trp Val Arg

2850 2855 2860

Met Val Leu Met Thr His Phe Phe Ser Ile Leu Met Ala Gln Asp Thr

2865 2870 2875 2880

Leu Asp Gln Asn Leu Asn Phe Glu Met Tyr Gly Ser Val Tyr Ser Val

2885 2890 2895

Ser Pro Leu Asp Leu Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp

2900 2905 2910

Ala Phe Ser Leu His Thr Tyr Thr Pro His Glu Leu Thr Arg Val Ala

2915 2920 2925

Ser Ala Leu Arg Lys Leu Gly Ala Pro Pro Leu Arg Ala Trp Lys Ser

2930 2935 2940

Arg Ala Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly Arg Ala

2945 2950 2955 2960

Ala Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu

2965 2970 2975

Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser Trp

2980 2985 2990

Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Tyr His Ser Val Ser Arg

2995 3000 3005

Ala Arg Pro Arg Leu Leu Leu Leu Ser Leu Leu Leu Leu Ser Val Gly

3010 3015 3020

Val Gly Leu Phe Leu Leu Pro Ala Arg

3025 3030

<210>7

<211>8024

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：复制子

<400>7

accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360

ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420

cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480

aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540

ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600

guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660

aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720

uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780

ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840

ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900

cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960

uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020

cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080

ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140

ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200

ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260

cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320

aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380

ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440

caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500

gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560

uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620

ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680

caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740

acgugguuuu ccuuugaaaa acacgaugau accauggcuc ccaucacugc uuaugcccag 1800

caaacacgag gccuccuggg cgccauagug gugaguauga cggggcguga caggacagaa 1860

caggccgggg aaguccaaau ccuguccaca gucucucagu ccuuccucgg aacaaccauc 1920

ucggggguuu uguggacugu uuaccacgga gcuggcaaca agacucuagc cggcuuacgg 1980

gguccgguca cgcagaugua cucgagugcu gagggggacu ugguaggcug gcccagcccc 2040

ccugggacca agucuuugga gccgugcaag uguggagccg ucgaccuaua ucuggucacg 2100

cggaacgcug augucauccc ggcucggaga cgcggggaca agcggggagc auugcucucc 2160

ccgagaccca uuucgaccuu gaaggggucc ucgggggggc cggugcucug cccuaggggc 2220

cacgucguug ggcucuuccg agcagcugug ugcucucggg gcguggccaa auccaucgau 2280

uucauccccg uugagacacu cgacguuguu acaaggucuc ccacuuucag ugacaacagc 2340

acgccaccgg cugugcccca gaccuaucag gucggguacu ugcaugcucc aacuggcagu 2400

ggaaagagca ccaagguccc ugucgcguau gccgcccagg gguacaaagu acuagugcuu 2460

aaccccucgg uagcugccac ccugggguuu ggggcguacc uauccaaggc acauggcauc 2520

aaucccaaca uuaggacugg agucaggacc gugaugaccg gggaggccau cacguacucc 2580

acauauggca aauuucucgc cgaugggggc ugcgcuagcg gcgccuauga caucaucaua 2640

ugcgaugaau gccacgcugu ggaugcuacc uccauucucg gcaucggaac gguccuugau 2700

caagcagaga cagccggggu cagacuaacu gugcuggcua cggccacacc ccccggguca 2760

gugacaaccc cccaucccga uauagaaaag guaggccucg ggcgggaggg ugagaucccc 2820

uucuauggga gggcgauucc ccuauccugc aucaagggag ggagacaccu gauuuucugc 2880

cacucaaaga aaaaguguga cgagcucgcg gcggcccuuc ggggcauggg cuugaaugcc 2940

guggcauacu auagaggguu ggacgucucc auaauaccag cucagggaga uguggugguc 3000

gucgccaccg acgcccucau gacgggguac acuggagacu uugacuccgu gaucgacugc 3060

aauguagcgg ucacccaagc ugucgacuuc agccuggacc ccaccuucac uauaaccaca 3120

cagacugucc cacaagacgc ugucucacgc agucagcgcc gcgggcgcac agguagagga 3180

agacagggca cuuauaggua uguuuccacu ggugaacgag ccucaggaau guuugacagu 3240

guagugcuuu gugagugcua cgacgcaggg gcugcguggu acgaucucac accagcggag 3300

accaccguca ggcuuagagc guauuucaac acgcccggcc uacccgugug ucaagaccau 3360

cuugaauuuu gggaggcagu uuucaccggc cucacacaca uagacgccca cuuccucucc 3420

caaacaaagc aagcggggga gaacuucgcg uaccuaguag ccuaccaagc uacggugugc 3480

gccagagcca aggccccucc cccguccugg gacgccaugu ggaagugccu ggcccgacuc 3540

aagccuacgc uugcgggccc cacaccucuc cuguaccguu ugggcccuau uaccaaugag 3600

gucacccuca cacacccugg gacgaaguac aucgccacau gcaugcaagc ugaccuugag 3660

gucaugacca gcacgugggu ccuagcugga ggaguccugg cagccgucgc cgcauauugc 3720

cuggcgacug gaugcguuuc caucaucggc cgcuugcacg ucaaccagcg agucgucguu 3780

gcgccggaua aggagguccu guaugaggcu uuugaugaga uggaggaaug cgccucuagg 3840

gcggcucuca ucgaagaggg gcagcggaua gccgagaugu ugaaguccaa gauccaaggc 3900

uugcugcagc aggccucuaa gcaggcccag gacauacaac ccgcuaugca ggcuucaugg 3960

cccaaagugg aacaauuuug ggccagacac auguggaacu ucauuagcgg cauccaauac 4020

cucgcaggau ugucaacacu gccagggaac cccgcggugg cuuccaugau ggcauucagu 4080

gccgcccuca ccaguccguu gucgaccagu accaccaucc uucucaacau caugggaggc 4140

ugguuagcgu cccagaucgc accacccgcg ggggccaccg gcuuugucgu caguggccug 4200

gugggggcug ccgugggcag cauaggccug gguaaggugc ugguggacau ccuggcagga 4260

uauggugcgg gcauuucggg ggcccucguc gcauucaaga ucaugucugg cgagaagccc 4320

ucuauggaag augucaucaa ucuacugccu gggauccugu cuccgggagc ccugguggug 4380

ggggucaucu gcgcggccau ucugcgccgc cacgugggac cgggggaggg cgcgguccaa 4440

uggaugaaca ggcuuauugc cuuugcuucc agaggaaacc acgucgcccc uacucacuac 4500

gugacggagu cggaugcguc gcagcgugug acccaacuac uuggcucucu uacuauaacc 4560

agccuacuca gaagacucca caauuggaua acugaggacu gccccauccc augcuccgga 4620

uccuggcucc gcgacgugug ggacuggguu ugcaccaucu ugacagacuu caaaaauugg 4680

cugaccucua aauuguuccc caagcugccc ggccuccccu ucaucucuug ucaaaagggg 4740

uacaagggug ugugggccgg cacuggcauc augaccacgc gcugcccuug cggcgccaac 4800

aucucuggca auguccgccu gggcucuaug aggaucacag ggccuaaaac cugcaugaac 4860

accuggcagg ggaccuuucc uaucaauugc uacacggagg gccagugcgc gccgaaaccc 4920

cccacgaacu acaagaccgc caucuggagg guggcggccu cggaguacgc ggaggugacg 4980

cagcaugggu cguacuccua uguaacagga cugaccacug acaaucugaa aauuccuugc 5040

caacuaccuu cuccagaguu uuucuccugg guggacggug ugcagaucca uagguuugca 5100

cccacaccaa agccguuuuu ccgggaugag gucucguucu gcguugggcu uaauuccuau 5160

gcugucgggu cccagcuucc cugugaaccu gagcccgacg cagacguauu gagguccaug 5220

cuaacagauc cgccccacau cacggcggag acugcggcgc ggcgcuuggc acggggauca 5280

ccuccaucug aggcgagcuc cucagugagc cagcuaucag caccgucgcu gcgggccacc 5340

ugcaccaccc acagcaacac cuaugacgug gacauggucg augccaaccu gcucauggag 5400

ggcggugugg cucagacaga gccugagucc agggugcccg uucuggacuu ucucgagcca 5460

auggccgagg aagagagcga ccuugagccc ucaauaccau cggagugcau gcuccccagg 5520

agcggguuuc cacgggccuu accggcuugg gcacggccug acuacaaccc gccgcucgug 5580

gaaucgugga ggaggccaga uuaccaaccg cccaccguug cugguugugc ucuccccccc 5640

cccaagaagg ccccgacgcc ucccccaagg agacgccgga cagugggucu gagcgagagc 5700

accauaucag aagcccucca gcaacuggcc aucaagaccu uuggccagcc ccccucgagc 5760

ggugaugcag gcucguccac gggggcgggc gccgccgaau ccggcggucc gacguccccu 5820

ggugagccgg cccccucaga gacagguucc gccuccucua ugcccccccu cgagggggag 5880

ccuggagauc cggaccugga gucugaucag guagagcuuc aaccuccccc ccaggggggg 5940

gggguagcuc ccgguucggg cucggggucu uggucuacuu gcuccgagga ggacgauacc 6000

accgugugcu gcuccauguc auacuccugg accggggcuc uaauaacucc cuguagcccc 6060

gaagaggaaa aguugccaau caacccuuug aguaacucgc uguugcgaua ccauaacaag 6120

guguacugua caacaucaaa gagcgccuca cagagggcua aaaagguaac uuuugacagg 6180

acgcaagugc ucgacgccca uuaugacuca gucuuaaagg acaucaagcu agcggcuucc 6240

aaggucagcg caaggcuccu caccuuggag gaggcgugcc aguugacucc accccauucu 6300

gcaagaucca aguauggauu cggggccaag gagguccgca gcuuguccgg gagggccguu 6360

aaccacauca aguccgugug gaaggaccuc cuggaagacc cacaaacacc aauucccaca 6420

accaucaugg ccaaaaauga gguguucugc guggaccccg ccaagggggg uaagaaacca 6480

gcucgccuca ucguuuaccc ugaccucggc guccgggucu gcgagaaaau ggcccucuau 6540

gacauuacac aaaagcuucc ucaggcggua augggagcuu ccuauggcuu ccaguacucc 6600

ccugcccaac ggguggagua ucucuugaaa gcaugggcgg aaaagaagga ccccaugggu 6660

uuuucguaug auacccgaug cuucgacuca accgucacug agagagacau caggaccgag 6720

gaguccauau accaggccug cucccugccc gaggaggccc gcacugccau acacucgcug 6780

acugagagac uuuacguagg agggcccaug uucaacagca agggucaaac cugcgguuac 6840

agacguugcc gcgccagcgg ggugcuaacc acuagcaugg guaacaccau cacaugcuau 6900

gugaaagccc uagcggccug caaggcugcg gggauaguug cgcccacaau gcugguaugc 6960

ggcaaugacc uaguagucau cucagaaagc caggggacug aggaggacga gcggaaccug 7020

agagccuuca cggaggccau gaccagguac ucugccccuc cuggugaucc ccccagaccg 7080

gaauaugacc uggagcuaau aacauccugu uccucaaaug ugucuguggc guugggcccg 7140

cggggccgcc gcagauacua ccugaccaga gacccaacca cuccacucgc ccgggcugcc 7200

ugggaaacag uuagacacuc cccuaucaau ucauggcugg gaaacaucau ccaguaugcu 7260

ccaaccauau ggguucgcau gguccuaaug acacacuucu ucuccauucu caugguccaa 7320

gacacccugg accagaaccu caacuuugag auguauggau caguauacuc cgugaauccu 7380

uuggaccuuc cagccauaau ugagagguua cacgggcuug acgccuuuuc uaugcacaca 7440

uacucucacc acgaacugac gcggguggcu ucagcccuca gaaaacuugg ggcgccaccc 7500

cucagggugu ggaagagucg ggcucgcgca gucagggcgu cccucaucuc ccguggaggg 7560

aaagcggccg uuugcggccg auaucucuuc aauugggcgg ugaagaccaa gcucaaacuc 7620

acuccauugc cggaggcgcg ccuacuggac uuauccaguu gguucaccgu cggcgccggc 7680

gggggcgaca uuuuucacag cgugucgcgc gcccgacccc gcucauuacu cuucggccua 7740

cuccuacuuu ucguaggggu aggccucuuc cuacuccccg cucgguagag cggcacacac 7800

uagguacacu ccauagcuaa cuguuccuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 7860

uuuuuuuuuu cuuuuuuuuu uuuuucccuc uuucuucccu ucucaucuua uucuacuuuc 7920

uuucuuggug gcuccaucuu agcccuaguc acggcuagcu gugaaagguc cgugagccgc 7980

augacugcag agagugccgu aacuggucuc ucugcagauc augu 8024

<210>8

<211>7994

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：复制子

<400>8

accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360

ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420

cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480

aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540

ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600

guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660

aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720

uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780

ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840

ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900

cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960

uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020

cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080

ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140

ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200

ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260

cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320

aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380

ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440

caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500

gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560

uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620

ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680

caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740

acgugguuuu ccuuugaaaa acacgaugau accauggcuc ccaucacugc uuaugcccag 1800

caaacacgag gccuccuggg cgccauagug gugaguauga cggggcguga caggacagaa 1860

caggccgggg aaguccaaau ccuguccaca gucucucagu ccuuccucgg aacaaccauc 1920

ucggggguuu uguggacugu uuaccacgga gcuggcaaca agacucuagc cggcuuacgg 1980

gguccgguca cgcagaugua cucgagugcu gagggggacu ugguaggcug gcccagcccc 2040

ccugggacca agucuuugga gccgugcaag uguggagccg ucgaccuaua ucuggucacg 2100

cggaacgcug augucauccc ggcucggaga cgcggggaca agcggggagc auugcucucc 2160

ccgagaccca uuucgaccuu gaaggggucc ucgggggggc cggugcucug cccuaggggc 2220

cacgucguug ggcucuuccg agcagcugug ugcucucggg gcguggccaa auccaucgau 2280

uucauccccg uugagacacu cgacguuguu acaaggucuc ccacuuucag ugacaacagc 2340

acgccaccgg cugugcccca gaccuaucag gucggguacu ugcaugcucc aacuggcagu 2400

ggaaagagca ccaagguccc ugucgcguau gccgcccagg gguacaaagu acuagugcuu 2460

aaccccucgg uagcugccac ccugggguuu ggggcguacc uauccaaggc acauggcauc 2520

aaucccaaca uuaggacugg agucaggacc gugaugaccg gggaggccau cacguacucc 2580

acauauggca aauuucucgc cgaugggggc ugcgcuagcg gcgccuauga caucaucaua 2640

ugcgaugaau gccacgcugu ggaugcuacc uccauucucg gcaucggaac gguccuugau 2700

caagcagaga cagccggggu cagacuaacu gugcuggcua cggccacacc ccccggguca 2760

gugacaaccc cccaucccga uauagaagag guaggccucg ggcgggaggg ugagaucccc 2820

uucuauggga gggcgauucc ccuauccugc aucaagggag ggagacaccu gauuuucugc 2880

cacucaaaga aaaaguguga cgagcucgcg gcggcccuuc ggggcauggg cuugaaugcc 2940

guggcauacu auagaggguu ggacgucucc auaauaccag cucagggaga uguggugguc 3000

gucgccaccg acgcccucau gacgggguac acuggagacu uugacuccgu gaucgacugc 3060

aauguagcgg ucacccaagc ugucgacuuc agccuggacc ccaccuucac uauaaccaca 3120

cagacugucc cacaagacgc ugucucacgc agucagcgcc gcgggcgcac agguagagga 3180

agacagggca cuuauaggua uguuuccacu ggugaacgag ccucaggaau guuugacagu 3240

guagugcuuu gugagugcua cgacgcaggg gcugcguggu acgaucucac accagcggag 3300

accaccguca ggcuuagagc guauuucaac acgcccggcc uacccgugug ucaagaccau 3360

cuugaauuuu gggaggcagu uuucaccggc cucacacaca uagacgccca cuuccucucc 3420

caaacaaagc aagcggggga gaacuucgcg uaccuaguag ccuaccaagc uacggugugc 3480

gccagagcca aggccccucc cccguccugg gacgccaugu ggaagugccu ggcccgacuc 3540

aagccuacgc uugcgggccc cacaccucuc cuguaccguu ugggcccuau uaccaaugag 3600

gucacccuca cacacccugg gacgaaguac aucgccacau gcaugcaagc ugaccuugag 3660

gucaugacca gcacgugggu ccuagcugga ggaguccugg cagccgucgc cgcauauugc 3720

cuggcgacug gaugcguuuc caucaucggc cgcuugcacg ucaaccagcg agucgucguu 3780

gcgccggaua aggagguccu guaugaggcu uuugaugaga uggaggaaug cgccucuagg 3840

gcggcucuca ucgaagaggg gcagcggaua gccgagaugu ugaaguccaa gauccaaggc 3900

uugcugcagc aggccucuaa gcaggcccag gacauacaac ccgcuaugca ggcuucaugg 3960

cccaaagugg aacaauuuug ggccagacac auguggaacu ucauuagcgg cauccaauac 4020

cucgcaggau ugucaacacu gccagggaac cccgcggugg cuuccaugau ggcauucagu 4080

gccgcccuca ccaguccguu gucgaccagu accaccaucc uucucaacau caugggaggc 4140

ugguuagcgu cccagaucgc accacccgcg ggggccaccg gcuuugucgu caguggccug 4200

gugggggcug ccgugggcag cauaggccug gguaaggugc ugguggacau ccuggcagga 4260

uauggugcgg gcauuucggg ggcccucguc gcauucaaga ucaugucugg cgagaagccc 4320

ucuauggaag augucaucaa ucuacugccu gggauccugu cuccgggagc ccugguggug 4380

ggggucaucu gcgcggccau ucugcgccgc cacgugggac cgggggaggg cgcgguccaa 4440

uggaugaaca ggcuuauugc cuuugcuucc agaggaaacc acgucgcccc uacucacuac 4500

gugacggagu cggaugcguc gcagcgugug acccaacuac uuggcucucu uacuauaacc 4560

agccuacuca gaagacucca caauuggaua acugaggacu gccccauccc augcuccgga 4620

uccuggcucc gcgacgugug ggacuggguu ugcaccaucu ugacagacuu caaaaauugg 4680

cugaccucua aauuguuccc caagcugccc ggccuccccu ucaucucuug ucaaaagggg 4740

uacaagggug ugugggccgg cacuggcauc augaccacgc gcugcccuug cggcgccaac 4800

aucucuggca auguccgccu gggcucuaug aggaucacag ggccuaaaac cugcaugaac 4860

accuggcagg ggaccuuucc uaucaauugc uacacggagg gccagugcgc gccgaaaccc 4920

cccacgaacu acaagaccgc caucuggagg guggcggccu cggaguacgc ggaggugacg 4980

cagcaugggu cguacuccua uguaacagga cugaccacug acaaucugaa aauuccuugc 5040

caacuaccuu cuccagaguu uuucuccugg guggacggug ugcagaucca uagguuugca 5100

cccacaccaa agccguuuuu ccgggaugag gucucguucu gcguugggcu uaauuccuau 5160

gcugucgggu cccagcuucc cugugaaccu gagcccgacg cagacguauu gagguccaug 5220

cuaacagauc cgccccacau cacggcggag acugcggcgc ggcgcuuggc acggggauca 5280

ccuccaucug aggcgagcuc cucagugagc cagcuaucag caccgucgcu gcgggccacc 5340

ugcaccaccc acagcaacac cuaugacgug gacauggucg augccaaccu gcucauggag 5400

ggcggugugg cucagacaga gccugagucc agggugcccg uucuggacuu ucucgagcca 5460

auggccgagg aagagagcga ccuugagccc ucaauaccau cggagugcau gcuccccagg 5520

agcggguuuc cacgggccuu accggcuugg gcacggccug acuacaaccc gccgcucgug 5580

gaaucgugga ggaggccaga uuaccaaccg cccaccguug cugguugugc ucuccccccc 5640

cccaagaagg ccccgacgcc ucccccaagg agacgccgga cagugggucu gagcgagagc 5700

accauaucag aagcccucca gcaacuggcc aucaagaccu uuggccagcc ccccucgagc 5760

ggugaugcag gcucguccac gggggcgggc gccgccgaau ccggcggucc gacguccccu 5820

ggugagccgg cccccucaga gacagguucc gccuccucua ugcccccccu cgagggggag 5880

ccuggagauc cggaccugga gucugaucag guagagcuuc aaccuccccc ccaggggggg 5940

gggguagcuc ccgguucggg cucggggucu uggucuacuu gcuccgagga ggacgauacc 6000

accgugugcu gcuccauguc auacuccugg accggggcuc uaauaacucc cuguagcccc 6060

gaagaggaaa aguugccaau caacccuuug aguaacucgc uguugcgaua ccauaacaag 6120

guguacugua caacaucaaa gagcgccuca cagagggcua aaaagguaac uuuugacagg 6180

acgcaagugc ucgacgccca uuaugacuca gucuuaaagg acaucaagcu agcggcuucc 6240

aaggucagcg caaggcuccu caccuuggag gaggcgugcc aguugacucc accccauucu 6300

gcaagaucca aguauggauu cggggccaag gagguccgca gcuuguccgg gagggccguu 6360

aaccacauca aguccgugug gaaggaccuc cuggaagacc cacaaacacc aauucccaca 6420

accaucaugg ccaaaaauga gguguucugc guggaccccg ccaagggggg uaagaaacca 6480

gcucgccuca ucguuuaccc ugaccucggc guccgggucu gcgagaaaau ggcccucuau 6540

gacauuacac aaaagcuucc ucaggcggua augggagcuu ccuauggcuu ccaguacucc 6600

ccugcccaac ggguggagua ucucuugaaa gcaugggcgg aaaagaagga ccccaugggu 6660

uuuucguaug auacccgaug cuucgacuca accgucacug agagagacau caggaccgag 6720

gaguccauau accaggccug cucccugccc gaggaggccc gcacugccau acacucgcug 6780

acugagagac uuuacguagg agggcccaug uucaacagca agggucaaac cugcgguuac 6840

agacguugcc gcgccagcgg ggugcuaacc acuagcaugg guaacaccau cacaugcuau 6900

gugaaagccc uagcggccug caaggcugcg gggauaguug cgcccacaau cucagaaagc 6960

caggggacug aggaggacga gcggaaccug agagccuuca cggaggccau gaccagguac 7020

ucugccccuc cuggugaucc ccccagaccg gaauaugacc uggagcuaau aacauccugu 7080

uccucaaaug ugucuguggc guugggcccg cggggccgcc gcagauacua ccugaccaga 7140

gacccaacca cuccacucgc ccgggcugcc ugggaaacag uuagacacuc cccuaucaau 7200

ucauggcugg gaaacaucau ccaguaugcu ccaaccauau ggguucgcau gguccuaaug 7260

acacacuucu ucuccauucu caugguccaa gacacccugg accagaaccu caacuuugag 7320

auguauggau caguauacuc cgugaauccu uuggaccuuc cagccauaau ugagagguua 7380

cacgggcuug acgccuuuuc uaugcacaca uacucucacc acgaacugac gcggguggcu 7440

ucagcccuca gaaaacuugg ggcgccaccc cucagggugu ggaagagucg ggcucgcgca 7500

gucagggcgu cccucaucuc ccguggaggg aaagcggccg uuugcggccg augucucuuc 7560

aauugggcgg ugaagaccaa gcucaaacuc acuccauugc cggaggcgcg ccuacuggac 7620

uuauccaguu gguucaccgu cggcgccggc gggggcgaca uuuuucacag cgugucgcgc 7680

gcccgacccc gcucauuacu cuucggccua cuccuacuuu ucguaggggu aggccucuuc 7740

cuacuccccg cucgguagag cggcacacac uagguacacu ccauagcuaa cuguuccuuu 7800

uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu cuuuuuuuuu uuuuucccuc 7860

uuucuucccu ucucaucuua uucuacuuuc uuucuuggug gcuccaucuu agcccuaguc 7920

acggcuagcu gugaaagguc cgugagccgc augacugcag agagugccgu aacuggucuc 7980

ucugcagauc augu 7994

<210>9

<211>340

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：合成的RNA

<400>9

accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

cgcuugcgag ugccccggga ggucucguag accgugcacc 340

<210>10

<211>340

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：合成的RNA

<400>10

acccgccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgucua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

ugcuugcgag ugccccggga ggucucguag accgugcacc 340

<210>11

<211>236

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：合成的RNA

<400>11

agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 60

uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu 120

uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg 180

uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu 236

<210>12

<211>232

<212>RNA

<213>人工序列

<220>

<223>人工序列描述：合成的RNA

<400>12

agcggcacac auuagcuaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 60

uuuuuuuuuu uuuuuuucuu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu 120

cuacuuucuu ucuugguggc uccaucuuag cccuggucac ggcuagcugu gaaagguccg 180

ugagccgcau gacugcagag agugccguaa cuggucucuc ugcagaucau gu 232

<210>13

<211>17

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>13

cgggagagcc atagtgg 17

<210>14

<211>19

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>14

agtaccacaa ggcctttcg 19

<210>15

<211>21

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>15

ctgcggaacc ggtgagtaca c 21

<210>16

<211>20

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>16

aacaagatgg at tgcacgca 20

<210>17

<211>20

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>17

cgtcaagaag gcgatagaag 20

<210>18

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>18

gcactctctg cagtcatgcg gctcacggac 30

<210>19

<211>28

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>19

cccctgtgag gaactactgt cttcacgc 28

<210>20

<211>24

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>20

ccgggagagc catagtggtc tgcg 24

<210>21

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>21

ccactcaaag aaaaagtgtg acgagctcgc 30

<210>22

<211>18

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>22

ggcttgggca cggcctga 18

<210>23

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>23

gcggtgaaga ccaagctcaa actcactcca 30

<210>24

<211>21

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>24

agaacctgcg tgcaatccat c 21

<210>25

<211>23

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>25

cccgtcatga gggcgtcggt ggc 23

<210>26

<211>27

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>26

accagcaacg gtgggcggtt ggtaatc 27

<210>27

<211>18

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>27

ggcacgcgac acgctgtg 18

<210>28

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>28

agctagccgt gactagggct aagatggagc 30

<210>29

<211>20

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA(引物)

<400>29

aacaagatgg attgcacgca 20

<210>30

<211>20

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA(引物)

<400>30

cgtcaagaag gcgatagaag 20

<210>31

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>31

gcactctctg cagtcatgcg gctcacggac 30

<210>32

<211>28

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>32

cccctgtgag gaactactgt cttcacgc 28

<210>33

<211>24

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>33

ccgggagagc catagtggtc tgcg 24

<210>34

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>34

ccactcaaag aaaaagtgtg acgagctcgc 30

<210>35

<211>18

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA(引物)

<400>35

ggcttgggca cggcctga 18

<210>36

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>36

gcggtgaaga ccaagctcaa actcactcca 30

<210>37

<211>21

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>37

agaacctgcg tgcaatccat c 21

<210>38

<211>23

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>38

cccgtcatga gggcgtcggt ggc 23

<210>39

<211>27

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>39

accagcaacg gtgggcggtt ggtaatc 27

<210>40

<211>18

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>40

ggaacgcgac acgctgtg 18

<210>41

<211>30

<212>DNA

<213>人工序列

<220>

<223>人工序列描述：合成的DNA

<400>41

agctagccgt gactagggct aagatggagc 30

Claims

1.一种复制子RNA，其含有的核苷酸序列至少包含：基因型2a丙肝病毒基因组RNA的5′非翻译区，编码NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的核苷酸序列以及3′非翻译区。

2.权利要求1的复制子RNA，其包含至少一个选择标记基因或报告基因、以及至少一种IRES序列。

3.一种复制子RNA，其含有的核苷酸序列包含：包含由SEQ IDNO：9或10所示的核苷酸序列的5′非翻译区；至少一个选择标记基因或报告基因；IRES序列；编码基因型2a丙肝病毒基因组RNA的NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的核苷酸序列；和含有由SEQ ID NO：11或12所示的核苷酸序列的3′非翻译区。

4.权利要求1-3中任意一项的复制子RNA，其中所述基因型2a丙肝病毒基因组RNA是一种含有由SEQ ID NO：3或5所示核苷酸序列的RNA。

5.一种复制子RNA，其含有以下RNA(a)或(b)：

(a)含有由SEQ ID NO：1或2所示核苷酸序列的RNA；和

6.一种复制子-复制细胞，它是通过将权利要求1-5中任意一项的复制子RNA导入细胞制得的。

7.权利要求6的复制子-复制细胞，其中所述细胞是真核细胞。

8.权利要求7的复制子-复制细胞，其中所述真核细胞是人肝-衍生细胞、人子宫颈-衍生细胞或人胎肾-衍生细胞。

9.权利要求7的复制子-复制细胞，其中所述真核细胞是选自Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞和293细胞中的任何一种细胞。

10.权利要求1-5中任意一项的复制子RNA，其用于制备或评估针对丙肝病毒感染的治疗剂或诊断剂。

11.权利要求6-9中任意一项的复制子-复制细胞，其用于制备或评估针对丙肝病毒感染的治疗剂或诊断剂。

12.权利要求1-5中任意一项的复制子RNA，其用于制备针对丙肝病毒感染的疫苗。

13.权利要求6-9中任意一项的复制子-复制细胞，其用于制备针对丙肝病毒感染的疫苗。

14.一种制备基因型2a丙肝病毒的复制子RNA的方法，其包括从权利要求6-9中任意一项的复制子-复制细胞提取复制子RNA。

15.一种制备基因型2a丙肝病毒的病毒蛋白的方法，其包括培养权利要求6-9中任意一项的复制子-复制细胞，并从所得培养产物得到病毒蛋白。

16.一种筛选促进或抑制丙肝病毒复制的物质的方法，其包括在测试物质存在下培养权利要求6-9中任意一项的复制子-复制细胞，并检测所得培养产物中复制子RNA的复制。

17.一种提高基因型2a丙肝病毒复制子RNA复制效率的方法，其包括进行一个或多个以下步骤：从权利要求6-9中任意一项的复制子-复制细胞获得已复制的复制子RNA，并将由此获得的已复制复制子RNA导入与复制子-复制细胞不同的细胞，从而制成一种新的复制子-复制细胞。

18.权利要求17的方法，其中与最初被导入复制子-复制细胞的复制子RNA相比复制效率提高至少2倍。

19.一种制备复制效率提高的基因型2a丙肝病毒的复制子RNA的方法，其包括进行一个或多个以下步骤：从权利要求6-9中任意一项的复制子-复制细胞获得已复制的复制子RNA，并将由此获得的已复制复制子RNA导入与复制子-复制细胞不同的细胞，从而制成一种新的复制子-复制细胞；和从最终获得的复制子-复制细胞得到已复制的复制子RNA。

20.一种制备复制效率提高的基因型2a丙肝病毒的复制子RNA的方法，其包括检测通过权利要求19的方法为提高复制效率而制得的复制子RNA与最初导入复制子-复制细胞的复制子RNA间的核苷酸突变或氨基酸突变；和将由此所检测到的核苷酸突变或氨基酸突变导入复制效率欲提高的复制子RNA。

21.一种复制子RNA，其含有衍生自SEQ ID NO：1所示核苷酸序列、具有至少一个选自以下(a)至(u)的突变的核苷酸序列：

(a)核苷酸位点7157处从A突变为G；

(b)核苷酸位点4955处从C突变为U；

(c)核苷酸位点4936处从A突变为G；

(d)核苷酸位点5000处从A突变为G；

(e)核苷酸位点7288处从A突变为G；

(f)核苷酸位点5901处从G突变为U；

(g)核苷酸位点6113处从A突变为U；

(h)核苷酸位点2890处从A突变为G；

(i)核苷酸位点6826处从C突变为A；

(j)核苷酸位点6887处从C突变为A；

(k)核苷酸位点6580处从U突变为A；

(l)核苷酸位点7159处从U突变为C；

(m)核苷酸位点7230处从U突变为A；

(n)核苷酸位点6943处从C突变为A；

(o)核苷酸位点5687处从G突变为A；

(p)核苷酸位点6110处从A突变为G；

(q)核苷酸位点5550处从U突变为C；

(r)核苷酸位点7217处从A突变为G；

(s)核苷酸位点3643处从A突变为G；

(t)核苷酸位点5851处从G突变为A；和

(u)核苷酸位点5914处从G突变为A。