CN1720261A - 疫苗 - Google Patents
疫苗 Download PDFInfo
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- CN1720261A CN1720261A CNA2003801049852A CN200380104985A CN1720261A CN 1720261 A CN1720261 A CN 1720261A CN A2003801049852 A CNA2003801049852 A CN A2003801049852A CN 200380104985 A CN200380104985 A CN 200380104985A CN 1720261 A CN1720261 A CN 1720261A
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Abstract
本发明涉及用于治疗和预防人乳头状瘤病毒感染的方法及组合物。特别地，本发明还涉及编码E1和/或E2的核酸分子和适于DNA疫苗递送的载体，以及包含它们药物组合物。本发明也涉及制备所述分子、载体和组合物的方法。
Description
本发明涉及用于治疗和预防人乳头状瘤病毒感染的方法及组合物。特别地，本发明还涉及核酸分子，其通常编码基于来自不同HPV株早期抗原的多蛋白，并涉及适于DNA疫苗递送的载体，以及包含它们药物组合物。本发明也涉及制备所述分子、载体和组合物的方法，以及它们在医药中的应用。
发明背景
人乳头状瘤病毒具有高度组织和物种特异性。它感染上皮基底细胞并在细胞核内复制及完成其整个生命周期。病毒基因表达与上皮细胞分化紧密相连，而且衣壳组装和成熟仅发生在上皮细胞上层中完全分化了的上皮细胞中。
已知出现在生殖器疣中的感染性人乳头状瘤病毒的基因型是基因型6b或基因型11。大多数(～90％)生殖器疣是由HPV6b感染的，而约10％是由HPV11感染的。出现在与宫颈癌相关的感染中的主要感染基因型是HPV16和18。
人生殖器疣会在感染的位置上发展并且它们会演变成慢性疾病，持续长时间，或者，它们会自发地消退，消退过程中完全不留疤痕。引发该消退的因素尚不明确，但公认疾病恢复进程中可能涉及细胞应答。
乳头状瘤病毒并不是很具有天然免疫原性的，而且在自然感染过程中抗体仅在很晚才会产生(在消退期间或之后)，并且在一小部分患者中同时会有一些患者疾病消退而一点也不产生可检测的抗体。
在几种不同动物模型系统中已经广泛研究了用乳头状瘤病毒早期抗原进行的免疫接种。然而仅有一些报道研究了免疫接种治疗。例如，用包含牛乳头状瘤病毒(BPV)E1、E2、E4和E7蛋白的混合蛋白免疫治疗牛，相对于对照，在一定比例的动物中显示出降低的乳头状瘤病毒发病量。
在多种物种中已观察到了乳头状瘤病毒的感染，包括绵羊、狗、兔子、猴子、牛和人。基于DNA序列同源性的程度，人乳头状瘤病毒(HPV)已被分成80余种类型[Epidemiology and Biology of CervicalCancer Seminars in Surgical Oncology 1999 16：203-211.WolfgangMJ，Schoell MD，Janicek MF和Mirhashemi R.]，其中一些进一步划分为亚型(如6a和6b型)。乳头状瘤病毒一般感染上皮细胞，但不同类型的HPV会导致不同的疾病。例如，1-4、7、10和26-29型会造成良性疣，16、18、31、33、35、39、45、51、52、56、58、59和68与宫颈癌有关，而6和11型涉及生殖器疣(生殖道的非恶性湿疣)。
已证明HPV在组织培养中很难生长，所以没有传统的活的或减毒的病毒疫苗。由于缺乏在其中可研究人病毒的适当的动物模型，所以HPV疫苗的开发滞后。因为所述病毒是高度种属特异性的，所以用来自不同物种宿主的乳头状瘤病毒来感染动物是非常困难的，而这正是疫苗第一次在人体内试用之前进行安全测试所必需的。
乳头状瘤病毒具有DNA基因组，其编码了“早期”和“晚期”基因，称为E1到E7、L1和L2。所述早期基因序列已显示出具有如下功能：与病毒DNA复制和转录有关、与逃避宿主的免疫有关、与正常宿主细胞正常周期的改变以及与其他过程有关。例如E1蛋白是一种ATP依赖的DNA解旋酶，参与病毒DNA复制过程的起始，E2是一种调控蛋白，控制病毒基因表达和DNA复制。E2通过其既结合E1又结合病毒复制起点的能力，使E1在所述起点局部聚集，从而刺激病毒DNA复制的起始。E4蛋白具有多种未确定的功能，但结合宿主细胞骨架属于上述功能，E5似乎能延迟内体的酸化导致细胞表面EGF受体增加表达，已知E6和E7各自都能与细胞蛋白p53和pRB结合。E6和E7蛋白形成与宫颈癌有关的HPV类型，是已知的原癌基因。L1和L2编码两种病毒结构(衣壳)蛋白。
在历史上，疫苗一直被视为预防病原体感染的一种方式，如果发生感染，疫苗可激发免疫系统识别病原体并中和之。疫苗包含一种或多种来自病原体的抗原，通常是完整的生物体，可以是已灭活的或减弱(减毒)的形式，或是来自所述生物体的特定抗原肽。当免疫系统暴露于抗原时，产生使个体终生都保持免疫“记忆”的细胞。再次暴露于相同的抗原(如感染所述病原体时)会刺激特定的免疫应答，从而清除或失活感染的病原体。
有两种武器用于免疫应答：体液(抗体)应答和细胞介导的应答。衍生自细胞内复制的病原体(病毒和某些细菌)的蛋白抗原在受感染的宿主细胞中加工，释放小肽，然后这些小肽与I类主要组织相容性(MHC I)分子联合展示在受感染的细胞表面上。当该MHC I肽结合复合物与抗原特异性CD8+T细胞接触时，所述T细胞被激活，获得细胞毒性。这些细胞毒T细胞(CTLs)可溶解受感染的宿主细胞，从而限制感染病原体的复制和传播。免疫应答的另一重要武器受控于CD4+T细胞。当衍生自病原体的抗原释放进入细胞外环境时，它们可以被专门的抗原递呈细胞(APCs)摄取、并与MHC II类分子联合共同展示在这些细胞表面上。对该复合物中抗原的识别刺激CD4+T细胞分泌可溶性因子(细胞因子)，其能调节其他T细胞效应机制。B细胞产生抗体。抗原与分泌型抗体的结合可以中和病原体的感染，抗原与B细胞表面膜结合型抗体的结合可刺激B细胞的分裂，从而扩大B细胞的应答。一般来说，对控制细菌感染而言需要好的抗体应答，对控制病毒感染而言，需要抗体和细胞介导的免疫应答(CD8+和CD4+)二者。
据信，甚至在发生病原体感染后，通过免疫接种利用免疫系统灭活或清除病原体以控制和消除感染都是可能的。这种“治疗性”疫苗需要细胞介导的应答才能使之有效，并理想地可激发体液及细胞应答两者。
已证实(Benvenisty，N和Reshaf，L.PNAS 839551-9555)用磷酸钙沉淀的DNA对小鼠接种可引起该DNA编码的肽表达。随后，向小鼠肌肉注射未经沉淀的质粒DNA，发现所述DNA被摄入到所述肌肉细胞中，并表达编码的蛋白质。因为DNA的表达导致宿主细胞中所编码的病原体蛋白质的产生，如同天然感染的状况，这种机制能刺激治疗性免疫接种所需的细胞介导的免疫应答。DNA疫苗描述于W090/11092(Vical，INC.)中。
DNA疫苗除了肌肉内注射外，还可通过其他机制来递送。例如，向皮肤中递送，其利用了在诸如皮肤和粘膜的感染屏障组织中免疫机制高度活化的优势。向皮肤内递送可以通过注射、使用喷射式注射器(其使液体在压力下进入皮肤)或通过颗粒轰击来进行，其中DNA以足够密度包被在颗粒上以便穿透上皮(美国专利第5371015号)。这些颗粒射入皮肤导致直接转染上皮细胞和上皮郎罕氏细胞。郎罕氏细胞是抗原递呈细胞(APC)，其摄取DNA，表达编码的肽，并加工使抗原展示在细胞表面MHC蛋白上。转染的郎罕氏细胞迁移到淋巴结，在那儿它们将所展示的抗原片段递呈给淋巴细胞，引发免疫应答。通过向皮肤内进行颗粒递送，只需极少量的DNA(0.5-1μg)便能诱导免疫应答，这与已知在直接肌肉注射后产生免疫应答所需的毫克量的DNA形成对比。
已报道，例如在利用由L1和L2衣壳蛋白所形成的病毒样颗粒或单独利用这些蛋白质的研究(1)中，HPV免疫原性不佳。另外，已证实HPV基因在人或其他哺乳动物细胞中难以表达，造成了开发蛋白质亚单位疫苗的困难。已证实单胞质E1在哺乳动物细胞中在异源启动子控制下特别难以表达(J.Virology 199973，3062-3070.RemmM，Remm A和Mart Ustav.Human papilloma virus type 18 E1 istranslated from polycistronic MRNA by a discontinuous scanningmechanism)。E1的表达最经常通过使用包含质粒的HPV起点的体外DNA复制来检测，该质粒作为E1的替代物(Lu，JZJ，Sun等.J.VIROL 199367，7131-7139和Del Vecchio AM等.J.Virol 1992 66，5949-5958)。
国际专利申请WO 02/08435提供了HPV多核苷酸，其中序列优化为类似高表达的人基因的使用模式。具体公开了密码子优化的HPV6bE1和HPV 11 E2。
发明简述
本发明提供了新的核酸构建体，其用于预防并更具体地用于治疗人乳头状瘤病毒引起的生殖器疣、或其他HPV引起的后遗症。
根据本发明的第一个方面，提供了一种核酸构建体，其编码包含源自至少两个不同早期抗原表位的多蛋白。本发明优选提供一种核酸构建体，其编码包含源自至少三个不同早期抗原表位的多蛋白。本发明人所披露的这种构建体在动物模型中比单一蛋白质方法更有效。
发明详述
优选的构建体包括核酸，其编码来自两种不同的HPV基因型的E2，诸如HPV6b，和来自HPV-11的E2。另外，如果还具有E1编码序列，则也是优选的。E1优选来自于HPV 6或11。
优选的构建体包括具有如下排列的核酸分子：
1)HPV6bE1-HPV6bE2-HPV11E2
2)HPV6bE2-HPV6bE1-HPV11E2
3)HPV6bE2-HPV11E2-HPV6bE1
最优选地，上述多蛋白所有的核酸序列密码子被优化为类似于高表达人基因的密码子用法。E1和E2基因优选是基本上全长的或更优选是全长的。基本上全长的方式指具有至少85％，优选90％的E1和E2多肽得到编码。令人惊奇地，这种构建体，其表达具有与密码子优化的单个蛋白质相当的表达水平，并且编码多蛋白的单个质粒具有较三个单独质粒更便宜并更易于制备的优势。
优选的这些基因得到密码子优化，以便使其密码子使用模式类似于肌动蛋白，一种高表达的人基因产物。
多核酸序列可以是DNA序列，例如双链DNA序列。多核酸序列优选编码HPV 6、11、16、18、33或45型的HPV多肽，最优选编码11型、6a亚型或6b亚型。在某些实施方案中，所编码的氨基酸序列是野生型HPV氨基酸序列。在可选的实施方案中，所编码的氨基酸序列是突变的HPV氨基酸序列，其包含带有氨基酸变化的野生型序列，例如，氨基酸点突变，该变化足以使一个或多个多肽的天然生物功能减少或失活。所述突变的氨基酸序列仍期望保持野生型多肽的免疫原性。
由本发明多核苷酸编码的蛋白质也构成了本发明的一个方面。
就E1而言，其主要的生物学角色是在感染的细胞中启动病毒特异性DNA复制。优选将E1突变以失活其复制潜力。
优选的突变是：G 482 D
K 83 G
R 84 G
优选包括两个或多个突变。
最优选包括三个突变。
就E2而言，其是位点特异性结合核蛋白，作为主要复制起点识别蛋白的功能，并辅助启动前复制复合物的组装。优选E2蛋白是失活的。可达到该目的的优选突变为K111A。
根据本发明的一个方面，多核苷酸的密码子使用模式优选排除人高表达基因中RSCU值小于0.2的密码子。相关同义密码子使用(RSCU)值是密码子的观测数值除以期望以相同频率使用的该氨基酸所有密码子而得到的数值。本发明的多核苷酸一般具有大于0.3的高表达人基因的密码子使用系数，优选大于0.4，最优选大于0.5。根据本发明的第二个方面，提供了一种表达载体，其包含并能控制本发明所述的多核苷酸序列表达，所述多核苷酸编码具有来自两种或多种早期抗原表位的多肽。所述载体可适于控制异源DNA的表达，该表达可在细菌昆虫或哺乳动物细胞中进行，特别是在人细胞中。在一个实施方案中，所述表达载体为p7313PLc。
在另一方面，本发明提供了一种疫苗组合物，其包含本发明的蛋白质，或载体，或多核苷酸序列。疫苗组合物优选包含本发明所述的DNA载体。在优选的实施方案中，疫苗组合物包含众多用DNA包被的颗粒，优选金颗粒，该DNA包含含有多核苷酸序列的载体，该多核苷酸序列编码具有来自两种或多种早期抗原表位的多肽。在可选的实施方案中，所述疫苗组合物包含药学上可接受的赋形剂和本发明第二个方面所述的DNA载体。所述疫苗组合物也可包含佐剂。
在另一方面，本发明提供一种制备疫苗组合物的方法，包括构建编码具有来自两种或多种早期抗原表位的多肽的多核苷酸，并与药学上可接受的赋形剂配制。
还提供了本发明所述的多核苷酸或载体的应用，在治疗或预防HPV感染中的应用，优选HPV 6、11、16或18型的感染。本发明也提供了根据本发明的多核苷酸、载体的应用，在治疗或预防皮(皮肤)疣、生殖器疣、未确定意义的非典型鳞状细胞(ASCUS)、子宫颈发育异常、子宫颈上皮内瘤(CIN)或宫颈癌中的应用。因此，本发明也提供了根据本发明的多核苷酸或载体在制备治疗或预防HPV感染或其相关症状或疾病的疫苗中的应用。
本发明也提供了治疗或预防HPV感染或其相关症状或疾病的方法，包括施用有效剂量的根据本发明的蛋白质、多核苷酸或载体或疫苗。疫苗可以以单一剂量或多剂量的方式施用，例如以“基础-加强”(“prime-boost”)接种方案。在某些情况下，“基础”免疫接种可通过DNA疫苗递送，具体而言，通过颗粒介导的DNA递送根据本发明的多核苷酸，优选掺入到质粒衍生的载体，并通过施用包含相同多核苷酸序列的重组病毒载体来“加强”。可选地，用递送的DNA作为基础-加强接种方案的另一种武器(所述蛋白质与DNA所编码的蛋白质相同)，那么蛋白佐剂方法可承担基础或加强方法的部分作用。
在本发明整个说明书及所附权利要求中的词语“包含(comprise)”和“包括(include)”及诸如“包含(comprises)”、“包含(comprising)”、“包括(includes)”和“包括(including)”变体，都包含在所要进行的解释内。即，这些词意指可能包含上下文允许而没有明确表述的其他要素或整体。
术语“变体”指一种多核苷酸，其与本发明另一多核苷酸编码相同的氨基酸序列，但由于其遗传密码子的冗余性而具有不同的核苷酸序列，而同时保持相同的密码子使用模式，例如具有相同的密码子使用系数或密码子使用系数为所述另一多核苷酸的0.1之内，优选在0.05之内。
术语“密码子使用模式”指的是所述核苷酸序列、基因或一类基因(如高表达的哺乳动物基因)中所有密码子的平均频率。可在文献中找到包括人的哺乳动物密码子使用模式(参见如Nakamura等.Nucleic Acids Research 1996，24：214-215)。
在本发明的多核苷酸中，密码子使用模式从人乳头状瘤病毒典型的模式被改变为更接近于人所呈现的密码子偏好。“密码子使用系数”是衡量给定多核苷酸序列与目标物种相似程度的一个指标。对于许多物种的高表达基因而言，密码子频率可得自文献资源(参见例如Nakamura等.Nucleic Acids Research 1996，24：214-215)。对于二十种天然氨基酸中的每一种，标准化61种密码子中每种密码子的频率(表示为在所选基因种类的每1000个密码子中出现的次数)，从而将每种氨基酸最常用的密码子的值设定为1并将较不常用的密码子的频率设定在0和1之间。这样，对于靶物种高表达基因来说，61种密码子中的每一种都赋予了一个1或更小的值。为计算特定多核苷酸相对于该物种高表达基因的密码子使用系数，记录特定多核苷酸的每种密码子的设定值并算出所有这些值的几何平均值(通过把这些值的自然对数总和除以密码子总数，然后取反对数)。该系数具有介于0到1的值，而且系数越高，多核苷酸中会有越多的密码子是频繁使用的密码子。如果多核苷酸序列具有为1的密码子使用系数，则所有的密码子都是靶物种高表达基因“最常用的”的密码子。
较短的多核苷酸序列也包含在本发明的范围内。例如，本发明的多核苷酸可编码HPV蛋白的片段。只要多核苷酸能编码经证实具有HPV抗原性的多肽，则编码长度为至少8个，如1-10个，或至多20、50、60、70、80、100、150或200个氨基酸的片段的多核苷酸都被认为落入本发明的范围之内。具体而言，但不仅限于，本发明一个方面包括如下情况，即多核苷酸编码完整HPV蛋白序列的片段并代表该蛋白一种或多种不连续的表位。
如上所述，本发明包括包含本发明核苷酸序列的表达载体。上述表达载体按照分子生物学领域的常规方法来构建，例如，可包括使用质粒DNA以及合适的起始密码子、启动子、增强子和其它元件，如以正确的方向放置的必要聚腺苷酸化信号，从而使蛋白质表达。其它合适的载体对本领域技术人员来说是显而易见的。对于这方面的其它实例，我们可参考Sambrook等，Molecular Cloning：a LaborotoryManual.2nd Editon.CSH Laboratory Press.(1989)。
优选地，本发明的用于本发明载体中的多核苷酸可操作地连接于调控序列，所述调控序列能通过宿主细胞表达编码序列，即所述载体是一种表达载体。术语“可操作地连接”是指一种毗连关系，其中所述成分处于允许它们以预期的方式起作用的相互关系中。“可操作地连接”于编码序列的调控序列，如启动子，是以一种方式来放置，从而在与调控序列相容的条件下实现编码序列的表达。
载体可以是诸如质粒、人工染色体、病毒或噬菌体的载体，其具有复制起点，任选还具有用于表达所述多核苷酸的启动子以及任选具有启动子的调节子。载体可包含一种或多种可供选择的标记基因，例如在细菌质粒的中所用的氨苄青霉素或卡那霉素抗性基因或真菌载体的抗性基因。载体可以在体外应用，例如用于制备DNA或RNA或用于转染或转化宿主细胞，如哺乳动物宿主细胞。载体也可以经改造而在体内应用，例如用在DNA免疫接种或基因治疗方法中。
可选择启动子和其它表达调控信号以使其与设计要用来表达的宿主细胞相容。例如，哺乳动物启动子包括金属硫蛋白启动子，其能响应诸如镉的重金属从而被诱导，以及β肌动蛋白启动子。还可使用病毒启动子，如SV40大T抗原启动子、人巨细胞病毒(CMV)即刻早期(IE)启动子、劳斯肉瘤病毒LTR启动子、腺病毒启动子)、或HPV启动子，特别是HPV上游调控区(URR)。所有这些启动子都能在现有技术中很容易地得到。
合适的病毒载体的实例包括单纯疱疹病毒载体、包括慢病毒的牛痘病毒或α病毒载体和逆转录病毒、腺病毒和腺相关病毒。利用这些病毒进行的基因转移技术对本领域技术人员来说是已知的。例如可用逆转录病毒载体将本发明的多核苷酸稳定整合到宿主基因组中，尽管这种重组并不是优选的。复制缺陷型腺病毒载体仍保持游离体的形式，从而进行暂时性的表达。可以利用昆虫细胞中能驱动表达的载体(如杆状病毒载体)，或人细胞或细菌中的载体，以产生大量本发明多核苷酸编码的的HPV蛋白，比如可用作亚单位疫苗。优选的病毒载体是那些衍生自非人类的灵长类腺病毒，如C68黑猩猩腺病毒(US6,083,716)，也称为Pan 9。
当本发明的多核苷酸可供治疗剂使用时，如在DNA免疫接种中，可将核酸施用于要进行接种的哺乳动物，例如施用于人。以载体形式提供核酸，例如RNA或DNA，优选DNA，如上所述，其可在哺乳动物细胞中表达。可通过任何可获得的技术施用多核苷酸。例如，可以通过针注射将核酸导入，优选皮内、皮下或肌肉内注射。可选地，可利用核酸递送装置，如颗粒介导的DNA递送(PMDD)，通过皮肤直接递送核酸。在该方法中，惰性颗粒(如金珠)用核酸包被，并被加速至足以能使它们穿透受试者表面(如皮肤)的速度，例如通过发射装置的高压进行的发射方法。(包被了本发明核酸分子的颗粒包含在本发明的范围之内，载有该粒子的递送装置也在本发明的范围内)。
将裸露的多核苷酸或载体导入患者的合适技术包括用合适载体进行局部应用。例如通过鼻内、口腔、阴道内或直肠内施用，核酸可局部施用于皮肤，或粘膜表面。裸露的多核苷酸或载体可与药学上可接受的赋形剂一起存在，例如与磷酸盐缓冲液(PBS)一起。通过向组合物加入促进剂，如丁哌卡因，可进一步促进DNA的摄取。其他向受试者直接施用核酸的方法包括超声、电刺激、电穿孔以及US-5,697,901中所述的显微接种。
通过几种已知的转染技术可提高核酸构建体的摄取，例如那些包括应用转染制剂的那些技术。这些制剂的实例包括阳离子制剂，如磷酸钙和DEAE-Dextran以及脂质转染剂，如lipofectam和transfectam。要施用的核酸剂量可改变。通常，核酸以1pg到1mg范围内的剂量施用，对于颗粒介导的基因递送来说，优选为1pg到10μg，而其它的途径优选为10μg到1mg。
本发明的核酸序列也可通过用于基因治疗中的专用递送载体来进行施用。例如，基因治疗方法论述于Verme等，Nuture 1997，389：239-242中。病毒和非病毒系统都可以使用。基于病毒的系统包括基于逆转录病毒、慢病毒、腺病毒、腺相关病毒、疱疹病毒、金丝雀痘病毒和基于牛痘病毒的系统。基于非病毒的系统包括核酸的直接施用和基于脂质体的系统。
本发明的核酸序列也可以通过转化细胞的方式来进行施用。上述细胞包括从受试者收获的细胞。本发明的裸露多核苷酸或载体可在体外导入到上述细胞中，之后将转化的细胞输回受试者。本发明的多核苷酸可通过同源重组方式整合到已经存在于细胞中的核酸中。必要时，转化的细胞可以在体外培养，再将一个或多个所得的细胞用于本发明。通过已知的外科或显微外科技术(如，移植、显微注射等)能将细胞放置在患者适当的部位中。
本发明的疫苗组合物可包含佐剂化合物，其用来增加蛋白质本身或由质粒DNA编码的蛋白质所诱导的免疫应答。在此推荐改变密码子偏好以适合免疫接种的物种，将其作为增加表达从而强化免疫应答的方法，但是佐剂依旧是必不可少的，因为DNA疫苗倾向于在小鼠模型中效果很好，而在更多物种中，如非人类的灵长类中，其被认为是人可能的效果先兆，却出现了效果有点弱化的证据。
本发明的疫苗组合物也可包含佐剂，例如在一个实施方案中，为咪喹莫特、妥卡雷琐或明矾。
佐剂优选与本发明的疫苗组合物同时施用，并在一个优选的实施方案中把它们配制在一起。本发明预期的这种佐剂制剂包括，但该列表决不是毫无遗漏的因而并不排除其它制剂：合成的咪唑基喹啉，如咪喹莫特[S-26308，R-837]，(Harrison，等。‘Reduction of recurrentHSV disease using imiquimod alone or combined with a glycoproteinvaccine′，Vaccine 19：1820-1826，(2001))；和瑞西喹漠[S-28463，R-848](Vasilakos，等‘Adjuvant activates of immune responsemodifier R-848：Comparison with CpG ODN′，Cellular immunology204：64-74(2000).)，基本在抗原递呈细胞和T细胞表面表达的羰基席弗碱和胺，如妥卡雷琐(Rhodes，J.等‘Therapeutic potentiationof the immune system by costimulatory Schiff-base-forming drugs′，Nature 377：71-75(1995))，细胞因子，趋化因子和共刺激分子，Th1诱导子，如γ干扰素、IL-2、IL-12、IL-15和IL-18，Th2诱导子，如IL-4、IL-5、IL-6、IL-10和IL-13和其他趋化因子和共刺激基因，如MCP-1、MIP-1α、MIP-1β、RANTES、TCA-3、CD80，CD86和CD40L，其他免疫刺激靶向配体，如CTLA-4和L-选凝素，凋亡刺激蛋白和肽，如Fas，(49)，基于合成脂质的佐剂，如vaxfectin、(Reyes等，‘Vaxfectin enhances antigen specific antibody titres andmaintains TH1 type immune responses to plasmid DNAimmunization′，Vaccine 19：3778-3786)角鲨烯、α生育酚、聚山梨醇酯80、DOPC和胆固醇、内毒素，[LPS]，Butler，B.，‘Endotoxin，’‘Toll-like receptor 4，and the afferent limb of innate immunity′，Current Opinion in Microbiology 3：23-30(2000))；CpG寡和二核苷酸，Sato，Y.等，‘Immunostimulatory DNA sequences necessaryfor effective Intradermal gene immunization′，Science 273(5273)：352-354(1996).Hemmi，H.等.，‘A Toll-like receptor recognizesbacterial DNA’，Nature 408：740-745，(2000)和其他能激发Toll受体产生Th1-诱导的细胞因子的潜在配体，如合成的分枝杆菌脂蛋白、分枝杆菌蛋白P19、糖肽、磷壁酸和脂质A。
诱导以Th1型应答为主的某些优选佐剂组合物包括，例如，脂质A衍生物，如单磷酰基脂质A，或优选3-脱-氧-乙酰基单磷酰基脂质A.MPL佐剂，其可从Corixa公司(Seattle，WA；如参见，美国专利4,436,727；4,877,611；4,866,034和4,912,094)获得。包含CpG的寡核苷酸(其中CpG二核苷酸是非甲基化的)也诱导以Th1型为主的应答。这种寡核苷酸是公知的并如WO 96/02555、WO99/33488和美国专利6,008,200和5,856,462所述。免疫刺激性DNA序列也如Sato等，Science 273：352，1996所述。其他优选的佐剂包含皂苷，如Quil A，或其衍生物，包括QS21和QS7(AquilaBiopharmaceuticals Inc.，Framingham，MA)；七叶树皂角素；洋地黄皂苷；或丝石竹属(Gypsophila)或藜谷(Chenopodium quinoa)的皂苷。
在一个实施方案中，佐剂包含免疫刺激性的CpG寡核苷酸，如WO 96/02555所公开的。典型的免疫刺激性寡核苷酸长度在8-100个碱基之间并包含通式X1CpGX2，其中X1和X2是核苷酸碱基，而且C和G是非甲基化的。
优选的用于本发明佐剂或疫苗中的寡核苷酸优选包括两个或多个二核苷酸CpG模体，优选通过至少三个、更优选至少六个或更多核苷酸来分隔。本发明的寡核苷酸通常是脱氧核苷酸。在优选的实施方案中，寡核苷酸中核苷酸之间是磷酸硫酯键，尽管磷酸二酯键和其他核苷酸间的键也包含在本发明的范围内，包括具有核苷酸间混合连接键的寡核苷酸，如混合的磷酸硫酯/磷酸二酯。也可使用其他能稳定寡核苷酸的核苷酸间的键。生产磷酸硫酯寡核苷酸或磷酸二硫酯的方法如US5,666,153，US5,278,302和WO95/26204中所述。
优选的寡核苷酸实例具有以下序列。序列优选包含磷酸硫酯修饰的核苷酸间的连接键。
OLIGO 1：TCC ATG ACG TTC CTG ACG TT(CpG 1826)(SEQ ID NO 24)
OLIGO 2：TCT CCC AGC GTG CGC CAT(CpG 1758)(SEQ ID NO 25)
OLIGO 3：ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG(SEQ ID NO 26)
OLIGO 4：TCG TCG TTT TGT CGT TTT GTC GTT(CpG 2006)(SEQ ID NO 27)
OLIGO 5：TCC ATG ACG TTC CTG ATG CT(CpG 1668)(SEQ ID NO 28)
可选的CpG寡核苷酸可包含以上优选序列，其中它们另外还可具有非必要的缺失或添加。
本发明所用的CpG寡核苷酸可以通过现有技术中已知的任何方法来合成(如EP 468520)。利用自动合成仪可以方便地合成这种寡核苷酸。包含CpG寡核苷酸的佐剂配方可按照商品名称″ImmunEasy″从Qiagen处购买。
以下实施例和参考附图用以进一步描述本发明，其中：
图1是本发明的HPV免疫治疗性疫苗构建体的示意图。
图2是P70776be2的质粒图谱，该质粒编码经密码子优化并突变过的HPV 6b E2。
图3是p73p1c6be1的质粒图谱，该质粒编码经密码子优化并突变过的HPV 6b E1。
图4是p707711e2的质粒图谱，该质粒编码经密码子优化并突变过的HPV 11 E2。
图5是HPV 102的质粒图谱，该质粒编码p7313背景中的HPV11。
图6是HPV 104的质粒图谱，为来自HPV 6b的E2和来自p7313背景中的HPV 11的E2的融合体。
图7是HPV 105的质粒图谱，为经密码子优化、突变的HPV 6b的E2和来自HPV 11的E2的融合体。
图8是HPV 108的质粒图谱，为在p7313背景中经密码子优化、突变的HPV 6b E1。
图9是HPV 110的质粒图谱，为在p7313背景中经密码子优化、突变的HPV 6b E2。
图10是HPV 116的质粒图谱，为HPV 6b E1、HPV 6b E2、HPV11 E2。
图11是HPV 117的质粒图谱，为HPV 6b E2、HPV 11 E2、HPV6b E1。
图12是HPV 118的质粒图谱，为HPV 6b E2、HPV 11 E2、HPV6b E1。
图13为本发明三个多蛋白构建体在293T细胞中的蛋白质印迹分析。
图14显示体外CAT转录报告基因试验中KIIIA突变的E2丧失了功能。
图15显示小鼠中针对E1的细胞免疫应答。
图16显示针对E2的细胞免疫应答。
图17PMID之后用HPV 118进行的CTL试验的数据。
图18显示在COPV模型中施用E1/E2后疣减小了。
1.质粒：c/o突变的pWRG70776be2
感兴趣的基因：
HPV6be2基因大小约有1.1Kb，利用称为Syngene的可视化基础程序来创建(对于人的表达而言)密码子优化的序列。另外，所述序列在氨基酸位点第111位上包括了一个密码子变化，由此野生型中的赖氨酸残基(AAG)改变为丙氨酸残基(GCA)以产生突变基因。这个改变使得6be2的转录活性丧失。据此设计了可将整个基因和在5′和3′端所选择的限制性位点合并的重叠引物。
克隆：
用凝胶纯化1.1kb的PCR片段，并用限制性酶Not I和Bam HI消化，进而将其连接在载体pWRG7077(Powderject)中。该基因由全长即刻早期CMV启动子调控并具有牛生长激素聚A的尾部。
进行克隆测序，显示出许多碱基错误。鉴定出许多合适的克隆并利用限制性消化来构建正确的基因序列。从再次克隆中发现一个克隆C7在第497位上仅有一个碱基错误(T到C)。其他克隆在该区域中都是正确的，只需要简单的片段交换就能纠正该错误。最终的克隆C7a得到证实是密码子优化突变的6be2。(参见图2)
pWRG7077中的6be2序列(序列ID No.1)
ATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGA
GGAAAACAGCACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGT
CAGTGCTCCTGTACAAGGCCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTG
CCCCCGCTGAAGGTGAGCGAAGCCAAGGGCCACAACGCTATCGAGATGCAGATGCACCT
GGAGAGCCTGCTGCGGACCGAATACAGCATGGAGCCCTGGACTCTCCAGGAGACGTCCT
ACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCGCAAAGCGCGGCAAGACAGTTGAG
GTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGTGTGGACCGATGTCTA
CGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAGGGCATCT
ATTACACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAG
TATGGTTCCACCAAGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGC
CTCCGTGTCGTCCACCACCCAGGAAGTGAGCATTCCGGAGAGCACCACATACACCCCGG
CCCAAACGAGCACGCTCGTCAGCAGCAGCACCAAGGAGGACGCCGTCCAGACGCCCCCC
CGGAAGAGGGCCCGGGGGGTCCAGCAGTCTCCCTGCAATGCCCTGTGCGTTGCTCACAT
CGGCCCTGTCGATTCTGGGAACCACAATCTCATCACGAACAACCACGACCAGCACCAAA
GGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCCAGGGGGAGTCCAAC
TGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGACTTGAT
CAGTTCCACGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGG
TGACCTACGACTCCGAGGAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCG
ACAATCAGCCACAAGCTTGGCTTCATGTCCCTGCACCTGCTGTGA
氨基酸序列(Seq.ID No.2)
MEAIAKRLDA CQEQLLELYE ENSTDLHKHV LHWKCMRHES VLLYKAKQMG LSHIGMQVVP
PLKVSEAKGH NAIEMQMHLE SLLRTEYSME PWTLQETSYE MWQTPPKRCF AKRGKTVEVK
FDGCANNTMD YVVWTDVYVQ DNDTWVKVHS MVDAKGIYYT CGQFKTYYVN FVKEAEKYGS
TKHWEVCYGS TVICSPASVS STTQEVSIPE STTYTPAQTS TLVSSSTKED AVQTPPRKRA
RGVQQSPCNA LCVAHIGPVD SGNHNLITNN HDQHQRRNNS NSSATPIVQF QGESNCLKCF
RYRLNDRHRH LFDLTSSTWH WASSKAPHKH AIVTVTYDSE EQRQQFLDVV KIPPTISHKL
GFMSLHLL
2.质粒：p7313plc 6be1 c/o突变体
感兴趣的基因：
HPV6be1基因大小约有2Kb，利用被称为Syngene的统计可视化基础程序来创建(对于大肠杆菌和人的表达而言)密码子优化的野生型(wt)序列。据此设计了可将整个基因和在5′和3′端选择的限制性位点合并的重叠引物。然后用限制性酶Bam HI和Not I消化该合成的基因，进而将其连接在载体pCIN4中。从许多选择的克隆的测序数据中发现了大量碱基错误。通过把克隆#24中正确的Pst I-Bam HI片段与克隆#21中NotI-PST I片段合并在p7313-pic上，产生了正确的克隆。通过测序证实正确的克隆(#1)。为了产生突变，设计了引物从而改变以下氨基酸；在第83位赖氨酸(AAA)变为甘氨酸(GGA)，在第84位精氨酸(CGC)变为甘氨酸(GGC)以及在第482位甘氨酸(GGC)变为天冬酰胺(GAC)。
6bel的密码子优化突变的序列(Seq ID No.3)
ATGGCAGACGATTCCGGTACTGAGAACGAAGGTTCTGGTTGTACCGGTTGGTTCATGGT
TGAAGCAATCGTTCAGCATCCGACTGGTACCCAGATCTCCGATGACGAAGACGAAGAAG
TTGAAGATTCTGGTTACGACATGGTTGACTTCATCGATGACTCCAACATCACTCATAAC
TCTCTGGAAGCACAGGCTCTGTTTAACCGCCAGGAAGCTGATACCCATTACGCTACTGT
TCAGGACCTGGGAGGCAAATATCTGGGCTCTCCGTACGTTTCCCCGATCAACACTATCG
CAGAAGCAGTTGAGTCTGAAATCTCCCCGCGCCTGGACGCTATCAAACTGACTCGTCAG
CCGAAGAAGGTTAAACGTCGTCTGTTCCAGACTCGTGAACTGACCGACTCCGGTTACGG
TTATAGCGAAGTTGAGGCTGGCACCGGCACCCAGGTTGAAAAACACGGTGTACCGGAAA
ACGGCGGCGACGGTCAGGAAAAGGACACCGGCCGCGACATCGAGGGTGAGGAACACACC
GAAGCTGAAGCTCCGACTAACTCTGTTCGTGAACACGCAGGTACTGCGGGTATCCTGGA
ACTGCTGAAATGCAAAGACCTGCGCGCGGCTCTGCTGGGCAAATTCAAAGAATGCTTCG
GCCTGTCTTTCATTGACCTGATCCGTCCGTTTAAGTCTGACAAAACTACCTGTCTGGAC
TGGGTTGTAGCAGGCTTCGGCATCCACCACTCTATCTCTGAAGCATTCCAGAAACTGAT
CGAGCCGCTGTCTCTGTACGCGCACATCCAGTGGCTGACTAACGCTTGGGGTATGGTTC
TGCTGGTACTGCTGCGCTTTAAAGTAAACAAATCTCGTTCCACTGTTGCTCGTACTCTG
GCTACCCTGCTGAACATCCCGGAGAACCAGATGCTGATCGAACCGCCGAAAATCCAGTC
TGGTGTAGCTGCACTGTACTGGTTTCGTACTGGCATCTCTAACGCTAGCACTGTTATCG
GTGAAGCACCGGAATGGATCACTCGTCAGACCGTTATCGAACACGGTCTGGCAGATTCT
CAGTTCAAACTGACTGAAATGGTTCAGTGGGCATACGACAACGACATCTGCGAGGAATC
TGAAATTGCGTTCGAATACGCTCAGCGTGGCGACTTCGACTCCAACGCTCGTGCTTTCC
TGAACAGCAACATGCAGGCTAAATACGTAAAAGACTGCGCTACCATGTGCCGTCACTAC
AAACACGCGGAAATGCGTAAAATGTCTATCAAACAGTGGATCAAGCACCGCGGTTCTAA
AATCGAAGGTACCGGTAACTGGAAACCGATCGTTCAGTTCCTGCGCCATCAGAACATCG
AATTCATCCCGTTCCTGACCAAATTCAAGCTGTGGCTGCACGGTACCCCGAAAAAAAAC
TGCATCGCTATCGTAGGTCCACCGGACACTGACAAGTCTTACTTCTGTATGTCCCTGAT
CTCTTTCCTGGGCGGCACTGTAATCTCTCACGTTAACTCTTCCTCCCATTTCTGGCTGC
AGCCACTGGTAGACGCGAAAGTAGCTCTGCTGGACGACGCGACCCAGCCGTGCTGGATC
TACATGGATACTTACATGCGCAACCTGCTGGACGGTAACCCGATGTCTATCGACCGTAA
ACACAAAGCGCTGACTCTGATCAAGTGCCCGCCGCTGCTGGTAACTTCTAACATCGACA
TCACCAAGGAAGATAAATACAAGTACCTGCATACCCGTGTTACTACCTTTACTTTCCCG
AACCCGTTCCCGTTTGATCGTAACGGTAACGCTGTTTACGAACTGTCCAACACTAACTG
GAAATGCTTCTTCGAGCGTCTGTCTTCCTCCCTGGACATCCAGGACTCTGAAGATGAAG
AAGATGGTTCTAACTCTCAGGCTTTCCGTTGTGTTCCGGGTACTGTTGTTCGTACTCTG
TGA
氨基酸序列(Seq.ID No.4)
MADDSGTENE GSGCTGWFMV EAIVQHPTGT QISDDEDEEV EDSGYDMVDF
IDDSNITHNS LEAQALFNRQ EADTHYATVQ DLGGKYLGSP YVSPINTIAE
AVESEISPRL DAIKLTRQPK KVKRRLFQTR ELTDSGYGYS EVEAGTGTQV
EKHGVPENGG DGQEKDTGRD IEGEEHTEAE APTNSVREHA GTAGILELLK
CKDLRAALLG KFKECFGLSF IDLIRPFKSD KTTCLDWVVA GFGIHHSISE
AFQKLIEPLS LYAHIQWLTN AWGMVLLVLL RFKVNKSRST VARTLATLLN
IPENQMLIEP PKIQSGVAAL YWFRTGISNA STVIGEAPEW ITRQTVIEHG
LADSQFKLTE MVQWAYDNDI CEESEIAFEY AQRGDFDSNA RAFLNSNMQA
KYVKDCATMC RHYKHAEMRK MSIKQWIKHR GSKIEGTGNW KPIVQFLRHQ
NIEFIPFLTK FKLWLHGTPK KNCIAIVGPP DTDKSYFCMS LISFLGGTVI
SHVNSSSHFW LQPLVDAKVA LLDDATQPCW IYMDTYMRNL LDGNPMSIDR
KHKALTLIKC PPLLVTSNID ITKEDKYKYL HTRVTTFTFP NPFPFDRNGN
AVYELSNTNW KCFFERLSSS LDIQDSEDEE DGSNSQAFRC VPGTVVRTL
3.质粒：WRG7077 11e2c/o突变体
感兴趣的基因：
HPV11e2基因大小约有1.1Kb，利用被称为Syngene的可视化基础程序来创建(对于人的表达而言)密码子优化的序列。另外，所述序列在氨基酸位点第111位上包括了一个密码子变化，由此野生型中的赖氨酸残基(AAG)改变为丙氨酸残基(GCC)以产生突变基因。这个改变在文献中就已显示其使得E2蛋白的转录活性丧失了。据此设计了可将整个基因和在5′和3′端选择的限制性位点合并的重叠引物，所述引物用于装配合成的密码子优化的突变体11e2。
克隆：
用凝胶纯化1.2kb的PCR片段，并用限制性酶Not I和Bam HI消化，进而将其连接在载体pWRG7077(Powderject)中。该基因由全长即刻早期CMV启动子调控并具有牛生长激素聚A的尾部。
进行克隆测序，显示出许多碱基错误，接着纠正这些错误。最终的克隆F1发现是密码子优化突变的11E2。
pWRG7077中的11e2序列(Seq.ID No.5)
ATGGAAGCCATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGCTGTACGA
GGAGAACAGCATTGACATCCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGA
GCGTGCTGTTGCACAAGGCCAAGCAGATGGGCCTGTCCCACATAGGCCTTCAGGTGGTC
CCCCCTCTGACCGTGTCAGAGACAAAGGGCCATAACGCAATCGAGATGCAGATGCACCT
CGAGTCGCTGGCGAAAACACAGTACGGCGTGGAGCCATGGACCCTGCAGGACACCTCGT
ACGAAATGTGGCTGACCCCACCTAAGCGATGCTTCGCCAAACAGGGCAACACAGTGGAG
GTGAAGTTCGACGGCTGTGAGGATAACGTTATGGAGTATGTCGTGTGGACGCACATCTA
TCTGCAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACGCGAAGGGCATCT
ACTATACCTGTGGGCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAAAAG
TATGGCTCCACCAACCACTGGGAGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGC
CAGCGTGTCTAGCACTGTGCGCGAGGTGAGCATTGCCGAGCCGACCACGTACACCCCTG
CCCAGACGACCGCTCCGACCGTGTCTGCTTGTACTACCGAGGACGGCGTGAGCGCTCCA
CCCAGGAAGCGTGCGAGGGGCCCAAGCACCAACAACACCCTCTGTGTGGCGAACATTCG
CAGCGTCGACAGTACCATCAATAACATCGTGACGGATAACTATAACAAGCACCAGAGGC
GTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCCAGGGAGACAGCAATTGC
CTTAAGTGCTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAGCTCGCCTC
GTCGACGTGGCACTGGGCCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCA
CTTACTCCAGTGAGGAGCAGAGACAGCAGTTTCTGAACAGCGTGAAGATCCCACCGACG
ATCCGTCATAAGGTCGGCTTCATGTCACTGCATCTCCTGTGA
氨基酸序列(Seq.ID No.6)
MEAIAKRLDA CQDQLLELYE ENSIDIHKHI MHWKCIRLES VLLHKAKQMG LSHIGLQVVP
PLTVSETKGH NAIEMQMHLE SLAKTQYGVE PWTLQDTSYE MWLTPPKRCF AKQGNTVEVK
FDGCEDNVME YVVWTHIYLQ DNDSWVKVTS SVDAKGIYYT CGQFKTYYVN FNKEAQKYGS
TNHWEVCYGS TVICSPASVS STVREVSIAE PTTYTPAQTT APTVSACTTE DGVSAPPRKR
ARGPSTNNTL CVANIRSVDS TINNIVTDNY NKHQRRNNCH SAATPIVQLQ GDSNCLKCFR
YRLNDKYKHL FELASSTWHW ASPEAPHKNA IVTLTYSSEE QRQQFLNSVK IPPTIRHKVG
FMSLHLL
4.质粒：HPV102(p7313me 11e2c/o突变体)
感兴趣的基因：
将密码子优化突变的11e2从pWRG7077 11e2c/o突变体转移到另一个表达载体p7313me中。
克隆：
用限制性酶Bam HI和NotI将11e2c/o突变体片段从pWRG707711e2载体上切下。然后利用这些位点把该片段连接到p7313me载体中。
HPV102中的11e2序列(Seq.ID No.7)
ATGGAAGCCATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGCTGTACGA
GGAGAACAGCATTGACATCCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGA
GCGTGCTGTTGCACAAGGCCAAGCAGATGGGCCTGTCCCACATAGGCCTTCAGGTGGTC
CCCCCTCTGACCGTGTCAGAGACAAAGGGCCATAACGCAATCGAGATGCAGATGCACCT
CGAGTCGCTGGCGAAAACACAGTACGGCGTGGAGCCATGGACCCTGCAGGACACCTCGT
ACGAAATGTGGCTGACCCCACCTAAGCGATGCTTCGCCAAACAGGGCAACACAGTGGAG
GTGAAGTTCGACGGCTGTGAGGATAACGTTATGGAGTATGTCGTGTGGACGCACATCTA
TCTGCAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACGCGAAGGGCATCT
ACTATACCTGTGGGCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAAAAG
TATGGCTCCACCAACCACTGGGAGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGC
CAGCGTGTCTAGCACTGTGCGCGAGGTGAGCATTGCCGAGCCGACCACGTACACCCCTG
CCCAGACGACCGCTCCGACCGTGTCTGCTTGTACTACCGAGGACGGCGTGAGCGCTCCA
CCCAGGAAGCGTGCGAGGGGCCCAAGCACCAACAACACCCTCTGTGTGGCGAACATTCG
CAGCGTCGACAGTACCATCAATAACATCGTGACGGATAACTATAACAAGCACCAGAGGC
GTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCCAGGGAGACAGCAATTGC
CTTAAGTGCTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAGCTCGCCTC
GTCGACGTGGCACTGGGCCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCA
CTTACTCCAGTGAGGAGCAGAGACAGCAGTTTCTGAACAGCGTGAAGATCCCACCGACG
ATCCGTCATAAGGTCGGCTTCATGTCACTGCATCTCCTGTGA
氨基酸序列(Seq.ID No.8)
MEAIAKRLDA CQDQLLELYE ENSIDIHKHI MHWKCIRLES VLLHKAKQMG LSHIGLQVVP
PLTVSETKGH NAIEMQMHLE SLAKTQYGVE PWTLQDTSYE MWLTPPKRCF AKQGNTVEVK
FDGCEDNVME YVVWTHIYLQ DNDSWVKVTS SVDAKGIYYT CGQFKTYYVN FNKEAQKYGS
TNHWEVCYGS TVICSPASVS STVREVSIAE PTTYTPAQTT APTVSACTTE DGVSAPPRKR
ARGPSTNNTL CVANIRSVDS TINNIVTDNY NKHQRRNNCH SAATPIVQLQ GDSNCLKCFR
YRLNDKYKHL FELASSTWHW ASPEAPHKNA IVTLTYSSEE QRQQFLNSVK IPPTIRHKVG
FMSLHLL
5.质粒：HPV104(p7313me 6b/11e2c/o突变体)
感兴趣的基因：
将HPV102和HPV110用作模板，并用适当设计的引物利用2xPCR来构建6be2和11e2的融合蛋白。将～2.2kb的融合片段与融合蛋白起点的6be2一起克隆到p7313me表达载体中。
克隆：
用限制性酶Bam HI和Not I消化该2.2kb的融合片段，并将其连接到p7313me表达载体中。通过测序检查分离的克隆，显示没有错误带入。
HPV104中的6b/11e2融合序列(Seq.ID No.9)
ATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGAGGAAAACAG
CACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTGCTCCTGTACAAGG
CCAAGCAGATGGGGCTGTCCCACATCGGATGCAGGTCGTGCCCCCCGCTGAAGGTGAGCGAAGCCAAG
GGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAATACAGCATGGAGCC
CTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCGCAAAGCGCG
GCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGTGTGGACCGAT
GTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAGGGCATCTATTA
CACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAGTATGGTTCCACCA
AGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTCGTCCACCACCCAG
GAAGTGAGCATTCCGGAGAGCACCACATACACCCCGGCCCAAACGAGCACGCTCGTCAGCAGCAGCAC
CAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCAGTCTCCCTGCAATG
CCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATCACGAACAACCACGAC
CAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCCAGGGGGAGTCCAA
CTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGACTTGATCAGTTCCA
CGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGGTGACCTACGACTCCGAG
GAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCAGCCACAAGCTTGGCTTCAT
GTCCCTGCACCTGCTGATGGAAGCCATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGC
TGTACGAGGAGAACAGCATTGACATCCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGAGC
GTGCTGTTGCACAAGGCCAAGCAGATGGGCCTGTCCCACATAGGCCTTCAGGTGGTCCCCCCTCTGAC
CGTGTCAGAGACAAAGGGCCATAACGCAATCGAGATGCAGATGCACCTCGAGTCGCTGGCGAAAACAC
AGTACGGCGTGGAGCCATGGACCCTGCAGGACACCTCGTACGAAATGTGGCTGACCCCACCTAAGCGA
TGCTTCGCCAAACAGGGCAACACAGTGGAGGTGAAGTTCGACGGCTGTGAGGATAACGTTATGGAGTA
TGTCGTGTGGACGCACATCTATCTGCAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACG
CGAAGGGCATCTACTATACCTGTGGGCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAA
AAGTATGGCTCCACCAACCACTGGGAGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGCCAGCGT
GTCTAGCACTGTGCGCGAGGTGAGCATTGCCGAGCCGACCACGTACACCCCTGCCCAGACGACCGCTC
CGACCGTGTCTGCTTGTACTACCGAGGACGGCGTGAGCGCTCCACCCAGGAAGCGTGCGAGGGGCCCA
AGCACCAACAACACCCTCTGTGTGGCGAACATTCGCAGCGTCGACAGTACCATCAATAACATCGTGAC
GGATAACTATAACAAGCACCAGAGGCGTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCC
AGGGAGACAGCAATTGCCTTAAGTGCTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAG
CTCGCCTCGTCGACGTGGCACTGGGCCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCAC
TTACTCCAGTGAGGAGCAGAGACAGCAGTTTCTGAACAGCGTGAAGATCCCACCGACGATCCGTCATA
AGGTCGGCTTCATGTCACTGCATCTCCTGA
氨基酸序列(Seq.ID No.10)
MEAIAKRLDA CQEQLLELYE ENSTDLHKHV LHWKCMRHES VLLYKAKQMG LSHIGMQVVP
PLKVSEAKGH NAIEMQMHLE SLLRTEYSME PWTLQETSYE MWQTPPKRCF AKRGKTVEVK
FDGCANNTMD YVVWTDVYVQ DNDTWVKVHS MVDAKGIYYT CGQFKTYYVN FVKEAEKYGS
TKHWEVCYGS TVICSPASVS STTQEVSIPE STTYTPAQTS TLVSSSTKED AVQTPPRKRA
RGVQQSPCNA LCVAHIGPVD SGNHNLITNN HDQHQRRNNS NSSATPIVQF QGESNCLKCF
RYRLNDRHRH LFDLISSTWH WASSKAPHKH AIVTVTYDSE EQRQQFLDVV KIPPTISHKL
GFMSLHLLME AIAKRLDACQ DQLLELYEEN SIDIHKHIMH WKCIRLESVL LHKAKQMGLS
HIGLQVVPPL TVSETKGHNA IEMQMHLESL AKTQYGVEPW TLQDTSYEMW LTPPKRCFAK
QGNTVEVKFD GCEDNVMEYV VWTHIYLQDN DSWVKVTSSV DAKGIYYTCG QFKTYYVNFN
KEAQKYGSTN HWEVCYGSTV ICSPASVSST VREVSIAEPT TYTPAQTTAP TVSACTTEDG
VSAPPRKRAR GPSTNNTLCV ANIRSVDSTI NNIVTDNYNK HQRRNNCHSA ATPIVQLQGD
SNCLKCFRYR LNDKYKHLFE LASSTWHWAS PEAPHKNAIV TLTYSSEEQR QQFLNSVKIP
PTIRHKVGFM SLHLL
6.质粒：HPV105(p7313me 11/6be2c/o突变体)
感兴趣的基因：
将HPV102和HPV110用作模板，并用适当设计的引物利用2xPCR来构建6be2和11e2的融合蛋白。将～2.2kb的融合片段与融合蛋白起点的11e2一起克隆到p7313me表达载体中。
克隆：
用限制性酶Bam HI和Not I消化该2.2kb的融合片段，并将其连接到p7313me表达载体中。通过测序检查分离的克隆，显示没有错误带入。
HPV105中的11/6bE2融合序列(Seq.ID No.11)
ATGGAAGCCATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGCTGTACGAGGAGAACAG
CATTGACATCCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGAGCGTGCTGTTGCACAAGG
CCAAGCAGATGGGCCTGTCCCACATAGGCCTTCAGGTGGTCCCCCCTCTGACCGTGTCAGAGACAAAG
GGCCATAACGCAATCGAGATGCAGATGCACCTCGAGTCGCTGGCGAAAACACAGTACGGCGTGGAGCC
ATGGACCCTGCAGGACACCTCGTACGAAATGTGGCTGACCCCACCTAAGCGATGCTTCGCCAAACAGG
GCAACACAGTGGAGGTGAAAGTTCGACGGCTGTGAGGATAACGTATGGAGTATGTCGTGTGGACGCAC
ATCTATCTGCAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACGCGAAGGGCATCTACTA
TACCTGTGGGCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAAAAGTATGGCTCCACCA
ACCACTGGGAGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGCCAGCGTGTCTAGCACTGTGCGC
GAGGTGAGCATTGCCGAGCCGACCACGTACACCCCTGCCCAGACGACCGCTCCGACCGTGTCTGCTTG
TACTACCGAGGACGGCGTGAGCGCTCCACCCAGGAAGCGTGCGAGGGGCCCAAGCACCAACAACACCC
TCTGTGTGGCGAACATTCGCAGCGTCGACAGTACCATCAATAACATCGTGACGGATAACTATAACAAG
CACCAGAGGCGTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCCAGGGAGACAGCAATTG
CCTTAAGTGCTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAGCTCGCCTCGTCGACGT
GGCACTGGGCCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCACTTACTCCAGTGAGGAG
CAGAGACAGCAGTTTCTGAACAGCGTGAAGATCCCACCGACGATCCGTCATAAGGTCGGCTTCATGTC
ACTGCATCTCCTGATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGT
ACGAGGAAAACAGCACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTG
CTCCTGTACAAGGCCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTGCCCCCGCTGAAGGT
GAGCGAAGCCAAGGGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAAT
ACAGCATGGAGCCCTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGT
TTCGCAAAGCGCGGCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGT
GGTGTGGACCGATGTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCA
AGGGCATCTATTACACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAG
TATGGTTCCACCAAGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTC
GTCCACCACCCAGGAAGTGAGCATTCCGGAGAGCACCACATACACCCCGGCCCAAACGAGCACGCTCG
TCAGCAGCAGCACCAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCAG
TCTCCCTGCAATGCCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATCAC
GAACAACCACGACCAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCC
AGGGGGAGTCCAACTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGAC
TTGATCAGTTCCACGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGGTGAC
CTACGACTCCGAGGAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCAGCCACA
AGCTTGGCTTCATGTCCCTGCACCTGCTGA
氨基酸序列(Seq.ID No.12)
MEAIAKRLDA CQDQLLELYE ENSIDIHKHI MHWKCIRLES VLLHKAKQMG LSHIGLQVVP
PLTVSETKGH NAIEMQMHLE SLAKTQYGVE PWTLQDTSYE MWLTPPKRCF AKQGNTVEVK
FDGCEDNVME YVVWTHIYLQ DNDSWVKVTS SVDAKGIYYT CGQFKTYYVN FNKEAQKYGS
TNHWEVCYGS TVICSPASVS STVREVSIAE PTTYTPAQTT APTVSACTTE DGVSAPPRKR
ARGPSTNNTL CVANIRSVDS TINNIVTDNY NKHQRRNNCH SAATPIVQLQ GDSNCLKCFR
YRLNDKYKHL FELASSTWHW ASPEAPHKNA IVTLTYSSEE QRQQFLNSVK IPPTIRHKVG
PMSLHLLMEA IAKRLDACQE QLLELYEENS TDLHKHVLHW KCMRHESVLL YKAKQMGLSH
IGMQVVPPLK VSEAKGHNAI EMQMHLESLL RTEYSMEPWT LQETSYEMWQ TPPKRCFAKR
GKTVEVKFDG CANNTMDYVV WTDVYVQDND TWVKVHSMVD AKGIYYTCGQ FKTYYVNFVK
EAEKYGSTKH WEVCYGSTVI CSPASVSSTT QEVSIPESTT YTPAQTSTLV SSSTKEDAVQ
TPPRKRARGV QQSPCNALCV AHIGPVDSGN HNLITNNHDQ HQRRNNSNSS ATPIVQFQGE
SNCLKCFRYR LNDRHRHLFD LISSTWHWAS SKAPHKHAIV TVTYDSEEQR QQFLDVVKIP
PTISHKLGFM SLHLL
7.质粒：HPV108(p7313ie 6be1c/o突变体)
感兴趣的基因：
将密码子优化突变的6be1从p7313plc 6be1c/o突变体克隆N转移到载体p7313ie中。
克隆：
通过用Not I和Bam HI限制性酶消化将6be1c/o突变体片段从p7313plc 6be2克隆上切下。然后利用这些位点把该片段连接到p7313ie载体中。该基因由ie启动子(即刻早期cmv+外显子1)调控，且其后连接有兔b-球蛋白的聚腺苷酸化信号。
p7313ie中的6be1序列(Seq.ID No.13)
ATGGCAGACGATTCCGGTACTGAGAACGAAGGTTCTGGTTGTACCGGTTGGTTCATGGTTGAAGCAAT
CGTTCAGCATCCGACTGGTACCCAGATCTCCGATGACGAAGACGAAGAAGTTGAAGATTCTGGTTACG
ACATGGTTGACTTCATCGATGACTCCAACATCACTCATAACTCTCTGGAAGCACAGGCTCTGTTTAAC
CGCCAGGAAGCTGATACCCATTACGCTACTGTTCAGGACCTGGGAGGCAAATATCTGGGCTCTCCGTA
CGTTTCCCCGATCAACACTATCGCAGAAGCAGTTGAGTCTGAAATCTCCCCGCGCCTGGACGCTATCA
AACTGACTCGTCAGCCGAAGAAGGTTAAACGTCGTCTGTTCCAGACTCGTGAACTGACCGACTCCGGT
TACGGTTATAGCGAAGTTGAGGCTGGCACCGGCACCCAGGTTGAAAAACACGGTGTACCGGAAAACGG
CGGCGACGGTCAGGAAAAGGACACCGGCCGCGACATCGAGGGTGAGGAACACACCGAAGCTGAAGCTC
CGACTAACTCTGTTCGTGAACACGCAGGTACTGCGGGTATCCTGGAACTGCTGAAATGCAAAGACCTG
CGCGCGGCTCTGCTGGGCAAATTCAAAGAATGCTTCGGCCTGTCTTTCATTGACCTGATCCGTCCGTT
TAAGTCTGACAAAACTACCTGTCTGGACTGGGTTGTAGCAGGCTTCGGCATCCACCACTCTATCTCTG
AAGCATTCCAGAAACTGATCGAGCCGCTGTCTCTGTACGCGCACATCCAGTGGCTGACTAACGCTTGG
GGTATGGTTCTGCTGGTACTGCTGCGCTTTAAAGTAAACAAATCTCGTTCCACTGTTGCTCGTACTCT
GGCTACCCTGCTGAACATCCCGGAGAACCAGATGCTGATCGAACCGCCGAAAATCCAGTCTGGTGTAG
CTGCACTGTACTGGTTTCGTACTGGCATCTCTAACGCTAGCACTGTTATCGGTGAAGCACCGGAATGG
ATCACTCGTCAGACCGTTATCGAACACGGTCTGGCAGMTCTCAGTTCMAAACTGACTGAAATGGTTCA
GTGGGCATACGACAACGACATCTGCGAGGAATCTGAAATTGCGTTCGAATACGCTCAGCGTGGCGACT
TCGACTCCAACGCTCGTGCTTTCCTGAACAGCAACATGCAGGCTAAATACGTAAAAGACTGCGCTACC
ATGTGCCGTCACTACAAACACGCGGAAATGCGTAAAATGTCTATCAAACAGTGGATCAAGCACCGCGG
TTCTAAAATCGAAGGTACCGGTAACTGGAAACCGATCGTTCAGTTCCTGCGCCATCAGAACATCGAAT
TCATCCCGTTCCTGACCAAATTCAAGCTGTGGCTGCACGGTACCCCGAAAAAAAACTGCATCGCTATC
GTAGGTCCACCGGACACTGACAAGTCTTACTTCTGTATGTCCCTGATCTCTTTCCTGGGCGGCACTGT
AATCTCTCACGTTAACTCTTCCTCCCATTTCTGGCTGCAGCCACTGGTAGACGCGAAAGTAGCTCTGC
TGGACGACGCGACCCAGCCGTGCTGGATCTACATGGATACTTACATGCGCAACCTGCTGGACGGTAAC
CCGATGTCTATCGACCGTAAACACAAAGCGCTGACTCTGATCAAGTGCCCGCCGCTGCTGGTAACTTC
TAACATCGACATCACCAAGGAAGATAAATACAAGTACCTGCATACCCGTGTTACTACCTTTACTTTCC
CGAACCCGTTCCCGTTTGATCGTAACGGTAACGCTGTTTACGAACTGTCCAACACTAACTGGAAATGC
TTCTTCGAGCGTCTGTCTTCCTCCCTGGACATCCAGGACTCTGAAGATGAAGAAGATGGTTCTAACTC
TCAGGCTTTCCGTTGTGTTCCGGGTACTGTTGTTCGTACTCTGTGA
氨基酸序列(Seq.ID No.14)
MADDSGTENE GSGCTGWFMV EAIVQHPTGT QISDDEDEEV EDSGYDMVDF IDDSNITHNS
LEAQALFNRQ EADTHYATVQ DLGGKYLGSP YVSPINTIAE AVESEISPRL DAIKLTRQPK
KVKRRLFQTR ELTDSGYGYS EVEAGTGTQV EKHGVPENGG DGQEKDTGRD IEGEEHTEAE
APTNSVREHA GTAGILELLK CKDLRAALLG KFKECFGLSF IDLIRPFKSD KTTCLDWVVA
GFGIHHSISE AFQKLIEPLS LYAHIQWLTN AWGMVLLVLL RFKVNKSRST VARTLATLLN
IPENQMLIEP PKIQSGVAAL YWFRTGISNA STVIGEAPEW ITRQTVIEHG LADSQFKLTE
MVQWAYDNDI CEESEIAFEY AQRGDFDSNA RAFLNSNMQA KYVKDCATMC RHYKHAEMRK
MSIKQWIKHR GSKIEGTGNW KPIVQFLRHQ NIEFIPFLTK FKLWLHGTPK KNCIAIVGPP
DTDKSYFCMS LISFLGGTVI SHVNSSSHFW LQPLVDAKVA LLDDATQPCW IYMDTYMRNL
LDGNPMSIDR KHKALTLIKC PPLLVTSNID ITKEDKYKYL HTRVTTFTFP NPFPFDRNGN
AVYELSNTNW KCFFERLSSS LDIQDSEDEE DGSNSQAFRC VPGTVVRTL
8.质粒：HPV110(p7313ie 6be2c/o突变体)
感兴趣的基因：
将密码子优化突变的6be2从pWRG7077 6be2转移到载体p7313ie中。
克隆：
通过用Not I和Bam HI限制性消化将6be2c/o突变体片段从pWRG7077 6be2克隆上切下。然后利用这些位点把该片段连接到p7313ie载体中。该基因由ie启动子(即刻早期cmv+外显子1)调控，且其后连接有兔b-球蛋白的聚腺苷酸化信号。
p7313ie中的6be2序列(Seq.ID No.15)
ATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGAGGAAAACAG
CACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTGCTCCTGTACAAGG
CCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTGCCCCCGCTGAAGGTGAGCGAAGCCAAG
GGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAATACAGCATGGAGCC
CTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCGCAAAGCGCG
GCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGTGTGGACCGAT
GTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAGGGCATCTATTA
CACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAGTATGGTTCCACCA
AGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTCGTCCACCACCCAG
GAAGTGAGCATTCCGGAGAGCACCACATACACCCCGGCCCAAACGAGCACGCTCGTCAGCAGCAGCAC
CAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCAGTCTCCCTGCAATG
CCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATCACGAACAACCACGAC
CAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCCAGGGGGAGTCCAA
CTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGACTTGATCAGTTCCA
CGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGGTGACCTACGACTCCGAG
GAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCAGCCACAAGCTTGGCTTCAT
GTCCCTGCACCTGCTGTGA
氨基酸序列(Seq.ID No.16)
MEAIAKRLDA CQEQLLELYE ENSTDLHKHV LHWKCMRHES VLLYKAKQMG LSHIGMQVVP
PLKVSEAKGH NAIEMQMHLE SLLRTEYSME PWTLQETSYE MWQTPPKRCF AKRGKTVEVK
FDGCANNTMD YVVWTDVYVQ DNDTWVKVHS MVDAKGIYYT CGQFKTYYVN FVKEAEKYGS
TKHWEVCYGS TVICSPASVS STTQEVSIPE STTYTPAQTS TLVSSSTKED AVQTPPRKRA
RGVQQSPCNA LCVAHIGPVD SGNHNLITNN HDQHQRRNNS NSSATPIVQF QGESNCLKCF
RYRLNDRHRH LFDLISSTWH WASSKAPHKH AIVTVTYDSE EQRQQFLDVV KIPPTISHKL
GFMSLHLL
9.质粒：HPV116(p7313ie 6be1.6be211e2)
感兴趣的基因：
HPV116构建体中的多蛋白基因是三联融合蛋白，依次包含所有密码子都优化并突变的6be1、6be2、11e2。使用2个前述PCR片段，即6be1和6b/11e2，通过PCR来装配该多蛋白基因。通过PAGE和蛋白质印迹观察，基因大小为～4.1kb，产生了～170kD的多蛋白。
克隆：
用Bam HI+NotI限制性酶消化多蛋白基因，并将其连接到p7313ie载体中。对所选择克隆序列的分析显示出‘不寻常的’碱基改变，但通过不同片段交换即可克服。发现所得的克隆hpv116#1没有错误。
HPV116中的多蛋白序列(SEQ.ID No.17)
ATGGCAGACGATTCCGGTACTGAGAACGAAGGTTCTGGTTGTACCGGTTGGTTCATGGTTGAAGCAA
TCGTTCAGCATCCGACTGGTACCCAGATCTCCGATGACGAAGACGAAGAAGTTGAAGATTCTGGTTA
CGACATGGTTGACTTCATCGATGACTCCAACATCACTCATAACTCTCTGGAAGCACAGGCTCTGTTT
AACCGCCAGGAAGCTGATACCCATTACGCTACTGTTCAGGACCTGGGAGGCAAATATCTGGGCTCTC
CGTACGTTTCCCCGATCAACACTATCGCAGAAGCAGTTGAGTCTGAAATCTCCCCGCGCCTGGACGC
TATCAAACTGACTCGTCAGCCGAAGAAGGTTAAACGTCGTCTGTTCCAGACTCGTGAACTGACCGAC
TCCGGTTACGGTTATAGCGAAGTTGAGGCTGGCACCGGCACCCAGGTTGAAAAACACGGTGTACCGG
AAAACGGCGGCGACGGTCAGGAAAAGGACACCGGCCGCGACATCGAGGGTGAGGAACACACCGAAGC
TGAAGCTCCGACTAACTCTGTTCGTGAACACGCAGGTACTGCGGGTATCCTGGAACTGCTGAAATGC
AAAGACCTGCGCGCGGCTCTGCTGGGCAAATTCAAAGAATGCTTCGGCCTGTCTTTCATTGACCTGA
TCCGTCCGTTTAAGTCTGACAAAACTACCTGTCTGGACTGGGTTGTAGCAGGCTTCGGCATCCACCA
CTCTATCTCTGAAGCATTCCAGAAACTGATCGAGCCGCTGTCTCTGTACGCGCACATCCAGTGGCTG
ACTAACGCTTGGGGTATGGTTCTGCTGGTACTGCTGCGCTTTAAAGTAAACAAATCTCGTTCCACTG
TTGCTCGTACTCTGGCTACCCTGCTGAACATCCCGGAGAACCAGATGCTGATCGAACCGCCGAAAAT
CCAGTCTGGTGTAGCTGCACTGTACTGGTTTCGTACTGGCATCTCTAACGCTAGCACTGTTATCGGT
GAAGCACCGGAATGGATCACTCGTCAGACCGTTATCGAACACGGTCTGGCAGATTCTCAGTTCAAAC
TGACTGAAATGGTTCAGTGGGCATACGACAACGACATCTGCGAGGAATCTGAAATTGCGTTCGAATA
CGCTCAGCGTGGCGACTTCGACTCCAACGCTCGTGCTTTCCTGAACAGCAACATGCAGGCTAAATAC
GTAAAAGACTGCGCTACCATGTGCCGTCACTACAAACACGCGGAAATGCGTAAAATGTCTATCAAAC
AGTGGATCAAGCACCGCGGTTCTAAAATCGAAGGTACCGGTAACTGGAAACCGATCGTTCAGTTCCT
GCGCCATCAGAACATCGAATTCATCCCGTTCCTGACCAAATTCAAGCTGTGGCTGCACGGTACCCCG
AAAAAAAACTGCATCGCTATCGTAGGTCCACCGGACACTGACAAGTCTTACTTCTGTATGTCCCTGA
TCTCTTTCCTGGGCGGCACTGTAATCTCTCACGTTAACTCTTCCTCCCATTTCTGGCTGCAGCCACT
GGTAGACGCGAAAGTAGCTCTGCTGGACGACGCGACCCAGCCGTGCTGGATCTACATGGATACTTAC
ATGCGCAACCTGCTGGACGGTAACCCGATGTCTATCGACCGTAAACACAAAGCGCTGACTCTGATCA
AGTGCCCGCCGCTGCTGGTAACTTCTAACATCGACATCACCAAGGAAGATAAATACAAGTACCTGCA
TACCCGTGTTACTACCTTTACTTTCCCGAACCCGTTCCCGTTTGATCGTAACGGTAACGCTGTTTAC
GAACTGTCCAACACTAACTGGAAATGCTTCTTCGAGCGTCTGTCTTCCTCCCTGGACATCCAGGACT
CTGAAGATGAAGAAGATGGTTCTAACTCTCAGGCTTTCCGTTGTGTTCCGGGTACTGTTGTTCGTAC
TCTGATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGAGGAA
AACAGCACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTGCTCCTGT
ACAAGGCCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTGCCCCCGCTGAAGGTGAGCGA
AGCCAAGGGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAATACAGC
ATGGAGCCCTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCG
CAAAGCGCGGCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGT
GTGGACCGATGTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAG
GGCATCTATTACACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAGT
ATGGTTCCACCAAGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTC
GTCCACCACCCAGGAAGTGAGCATTCCGGAGAGACCACATACACCCCGGCCCAAACGAGCACGCTCG
TCAGCAGCAGCACCAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCA
GTCTCCCTGCAATGCCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATC
ACGAACAACCACGACCAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGT
TCCAGGGGGAGTCCAACTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTT
CGACTTGATCAGTTCCACGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACG
GTGACCTACGACTCCGAGGAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCA
GCCACAAGCTTGGCTTCATGTCCCTGCACCTGCTGATGGAAGCCATCGCGAAGAGGCTCGACGCCTG
CCAGGACCAGCTGCTCGAGCTGTACGAGGAGAACAGCATTGACATCCATAAGCACATCATGCACTGG
AAGTGCATTCGCCTGGAGAGCGTGCTGTTGCACAAGGCCAAGCAGATGGGCCTGTCCCACATAGGCC
TTCAGGTGGTCCCCCCTCTGACCGTGTCAGAGACAAAGGGCCATAACGCAATCGAGATGCAGATGCA
CCTCGAGTCGCTGGCGAAAACACAGTACGGCGTGGAGCCATGGACCCTGCAGGACACCTCGTACGAA
ATGTGGCTGACCCCACCTAAGCGATGCTTCGCCAAACAGGGCAACACAGTGGAGGTGAAGTTCGACG
GCTGTGAGGATAACGTTATGGAGTATGTCGTGTGGACGCACATCTATCTGCAGGACAACGACAGTTG
GGTGAAGGTGACCAGCTCCGTGGACGCGAAGGGCATCTACTATACCTGTGGGCAGTTTAAAACCTAC
TATGTGAACTTCAACAAAGAGGCCCAAAAGTATGGCTCCACCAACCACTGGGAGGTCTGCTATGGGA
GCACGGTGATTTGCTCTCCCGCCAGCGTGTCTAGCACTGTGCGCGAGGTGAGCATTGCCGAGCCGAC
CACGTACACCCCTGCCCAGACGACCGCTCCGACCGTGTCTGCTTGTACTACCGAGGACGGCGTGAGC
GCTCCACCCAGGAAGCGTGCGAGGGGCCCAAGCACCAACAACACCCTCTGTGTGGCGAACATTCGCA
GCGTCGACAGTACCATCAATAACATCGTGACGGATAACTATAACAAGCACCAGAGGCGTAACAACTG
TCACTCTGCCGCAACCCCCATCGTGCAGCTCCAGGGAGACAGCAATTGCCTTAAGTGCTTCCGCTAT
CGCCTCAACGACAAGTACAAGCACCTCTTTGAGCTCGCCTCGTCGACGTGGCACTGGGCCTCACCCG
AGGCACCTCACAAGAACGCCATCGTCACTCTCACTTACTCCAGTGAGGAGCAGAGACAGCAGTTTCT
GAACAGCGTGAAGATCCCACCGACGATCCGTCATAAGGTCGGCTTCATGTCACTGCATCTCCTGTGA
氨基酸序列(Seq.ID No.18)
MADDSGTENE GSGCTGWFMV EAIVQHPTGT QISDDEDEEV EDSGYDMVDF IDDSNITHNS
LEAQALFNRQ EADTHYATVQ DLGGKYLGSP YVSPINTIAE AVESEISPRL DAIKLTRQPK
KVKRRLFQTR ELTDSGYGYS EVEAGTGTQV EKHGVPENGG DGQEKDTGRD IEGEEHTEAE
APTNSVREHA GTAGILELLK CKDLRAALLG KFKECFGLSF IDLIRPFKSD KTTCLDWVVA
GFGIHHSISE AFQKLIEPLS LYAHIQWLTN AWGMVLLVLL RFKVNKSRST VARTLATLLN
IPENQMLIEP PKIQSGVAAL YWFRTGISNA STVIGEAPEW ITRQTVIEHG LADSQFKLTE
MVQWAYDNDI CEESEIAFEY AQRGDFDSNA RAFLNSNMQA KYVKDCATMC RHYKHAEMRK
MSIKQWIKHR GSKIEGTGNW KPIVQFLRHQ NIEFIPFLTK FKLWLHGTPK KNCIAIVGPP
DTDKSYFCMS LISFLGGTVI SHVNSSSHFW LQPLVDAKVA LLDDATQPCW IYMDTYMRNL
LDGNPMSIDR KHKALTLIKC PPLLVTSNID ITKEDKYKYL HTRVTTFTFP NPFPFDRNGN
AVYELSNTNW KCFFERLSSS LDIQDSEDEE DGSNSQAFRC VPGTVVRTLM EAIAKRLDAC
QEQLLELYEE NSTDLHKHVL HWKCMRHESV LLYKAKQMGL SHIGMQVVPP LKVSEAKGHN
AIEMQMHLES LLRTEYSMEP WTLQETSYEM WQTPPKRCFA KRGKTVEVKF DGCANNTMDY
VVWTDVYVQD NDTWVKVHSM VDAKGIYYTC GQFKTYYVNF VKEAEKYGST KHWEVCYGST
VICSPASVSS TTQEVSIPES TTYTPAQTST LVSSSTKEDA VQTPPRKRAR GVQQSPCNAL
CVAHIGPVDS GNHNLITNNH DCHQRRNNSN SSATPIVQFQ GESNCLKCFR YRLNDRHRHL
FDLISSTWHW ASSKAPHKHA IVTVTYDSEE QRQQFLDVVK IPPTISHKLG FMSLHLLMEA
IAKRLDACQD QLLELYEENS IDIHKHIMHW KCIRLESVLL HKAKQMGLSH IGLQVVPPLT
VSETKGHNAI EMQMHLESLA KTQYGVEPWT LQDTSYEMWL TPPKRCFAKQ GNTVEVKFDG
CEDNVMEYVV WTHIYLQDND SWVKVTSSVD AKGIYYTCGQ FKTYYVNFNK EAQKYGSTNH
WEVCYGSTVI CSPASVSSTV REVSIAEPTT YTPAQTTAPT VSACTTEDGV SAPPRKRARG
PSTNNTLCVA NIRSVDSTIN NIVTDNYNKH QRRNNCHSAA TPIVQLQGDS NCLKCFRYRL
NDKYKHLFEL ASSTWHWASP EAPHKNAIVT LTYSSEEQRQ QFLNSVKIPP TIRHKVGFMS
LHLL
10.质粒：HPV117(p7313ie 6be2.6be1.11e2)
感兴趣的基因：
HPV117构建体中的多蛋白基因是三联融合蛋白，依次包含所有密码子都优化并突变的6be2、6be1、11e2。使用3个前述PCR片段，即6be1和6b2和11e2，通过PCR来装配该多蛋白基因。通过PAGE和蛋白质印迹观察，基因大小为～4.1kb，产生了～170kD的多蛋白。
克隆：
用Bam HI+NotI限制性酶消化多蛋白基因，并将其连接到p7313ie载体中。对所选择克隆序列的分析显示出‘不寻常的’碱基改变，但通过不同片段交换即可克服。发现所得的克隆hpV117#6没有错误。HPV117中的多蛋白序列(SEQ.ID No.19)
ATGGAAGCTATTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGAGGAAAACAG
CACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTGCTCCTGTACAAGG
CCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTGCCCCCGCTGAAGGTGAGCGAAGCCAAG
GGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAATACAGCATGGAGCC
CTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCGCAAAGCGCG
GCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGTGTGGACCGAT
GTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAGGGCATCTATTA
CACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAGTATGGTTCCACCA
AGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTCGTCCACCACCCAG
GAAGTGAGCATTCCGGAGAGCACCACATACACCCCGGCCCAAACGAGCACGCTCGTCAGCAGCAGCAC
CAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCAGTCTCCCTGCAATG
CCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATCACGAACAACCACGAC
CAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCCAGGGGGAGTCCAA
CTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGACTTGATCAGTTCCA
CGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGGTGACCTACGACTCCGAG
GAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCAGCCACAAGCTTGGCTTCAT
GTCCCTGCACCTGCTGATGGCAGACGATTCCGGTACTGAGAACGAAGGTTCTGGTTGTACCGGTTGGT
TCATGGTTGAAGCAATCGTTCAGCATCCGACTGGTACCCAGATCTCCGATGACGAAGACGAAGAAGTT
GAAGATTCTGGTTACGACATGGTTGACTTCATCGATGACTCCAACATCACTCATAACTCTCTGGAAGC
ACAGGCTCTGTTTAACCGCCAGGAAGCTGATACCCATTACGCTACTGTTCAGGACCTGGGAGGCAAAT
ATCTGGGCTCTCCGTACGTTTCCCCGATCAACACTATCGCAGAAGCAGTTGAGTCTGAAATCTCCCCG
CGCCTGGACGCTATCAAACTGACTCGTCAGCCGAAGAAGGTTAAACGTCGTCTGTTCCAGACTCGTGA
ACTGACCGACTCCGGTTACGGTTATAGCGAAGTTGAGGCTGGCACCGGCACCCAGGTTGAAAAACACG
GTGTACCGGAAAACGGCGGCGACGGTCAGGAAAAGGACACCGGCCGCGACATCGAGGGTGAGGAACAC
ACCGAAGCTGAAGCTCCGACTAACTCTGTTCGTGAACACGCAGGTACTGCGGGTATCCTGGAACTGCT
GAAATGCAAAGACCTGCGCGCGGCTCTGCTGGGCAAATTCAAAGAATGCTTCGGCCTGTCTTTCATTG
ACCTGATCCGTCCGTTTAAGTCTGACAAAACTACCTGTCTGGACTGGGTTGTAGCAGGCTTCGGGATC
CACCACTCTATCTCTGAAGCATTCCAGAAACTGATCGAGCCGCTGTCTCTGTACGCGCACATCCAGTG
GCTGACTAACGCTTGGGGTATGGTTCTGCTGGTACTGCTGCGCTTTAAAGTAAACAAATCTCGTTCCA
CTGTTGCTCGTACTCTGGCTACCCTGCTGAACATCCCGGAGAACCAGATGCTGATCGAACCGCCGAAA
ATCCAGTCTGGTGTAGCTGCACTGTACTGGTTTCGTACTGGCATCTCTAACGCTAGCACTGTTATCGG
TGAAGCACCGGAATGGATCACTCGTCAGACCGTTATCGAACACGGTCTGGCAGATTCTCAGTTCAAAC
TGACTGAAATGGTTCAGTGGGCATACGACAACGACATCTGCGAGGAATCTGAAATTGCGTTCGAATAC
GCTCAGCGTGGCGACTTCGACTCCAACGCTCGTGCTTTCCTGAACAGCAACATGCAGGCTAAATACGT
AAAAGACTGCGCTACCATGTGCCGTCACTACAAACACGCGGAAATGCGTAAAATGTCTATCAAACAGT
GGATCAAGCACCGCGGTTCTAAAATCGAAGGTACCGGTAACTGGAAACCGATCGTTCAGTTCCTGCGC
CATCAGAACATCGAATTCATCCCGTTCCTGACCAAATTCAAGCTGTGGCTGCACGGTACCCCGAAAAA
AAACTGCATCGCTATCGTAGGTCCACCGGACACTGACAAGTCTTACTTCTGTATGTCCCTGATCTCTT
TCCTGGGCGGCACTGTAATCTCTCACGTTAACTCTTCCTCCCATTTCTGGCTGCAGCCACTGGTAGAC
GCGAAAGTAGCTCTGCTGGACGACGCGACCCAGCCGTGCTGGATCTACATGGATACTTACATGCGCAA
CCTGCTGGACGGTAACCCGATGTCTATCGACCGTAAACACAAAGCGCTGACTCTGATCAAGTGCCCGC
CGCTGCTGGTAACTTCTAACATCGACATCACCAAGGAAGATAAATACAAGTACCTGCATACCCGTGTT
ACTACCTTTACTTTCCCGAACCCGTTCCCGTTTGATCGTAACGGTAACGCTGTTTACGAACTGTCCAA
CACTAACTGGAAATGCTTCTTCGAGCGTCTGTCTTCCTCCCTGGACATCCAGGACTCTGAAGATGAAG
AAGATGGTTCTAACTCTCAGGCTTTCCGTTGTGTTCCGGGTACTGTTGTTCGTACTCTGATGGAAGCC
ATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGCTGTACGAGGAGAACAGCATTGACAT
CCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGAGCGTGCTGTTGCACAAGGCCAAGCAGA
TGGGCCTGTCCCACATAGGCCTTCAGGTGGTCCCCCCTCTGACCGTGTCAGAGACAAAGGGCCATAAC
GCAATCGAGATGCAGATGCACCTCGAGTCGCTGGCGAAAACACAGTACGGCGTGGAGCCATGGACCCT
GCAGGACACCTCGTACGAAATGTGGCTGACCCCACCTAAGCGATGCTTCGCCAAACAGGGCAACACAG
TGGAGGTGAAGTTCGACGGCTGTGAGGATAACGTTATGGAGTATGTCGTGTGGACGCACATCTATCTG
CAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACGCGAAGGGCATCTACTATACCTGTGG
GCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAAAAGTATGGCTCCACCAACCACTGGG
AGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGCCAGCGTGTCTAGCACTGTGCGCGAGGTGAGC
ATTGCCGAGCCGACCACGTACACCCCTGCCCAGACGACCGCTCCGACCGTGTCTGCTTGTACTACCGA
GGACGGCGTGAGCGCTCCACCCAGGAAGCGTGCGAGGGGCCCAAGCACCAACAACACCCTCTGTGTGG
CGAACATTCGCAGCGTCGACAGTACCATCAATAACATCGTGACGGATAACTATAACAAGCACCAGAGG
CGTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCCAGGGAGACAGCAATTGCCTTAAGTG
CTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAGCTCGCCTCGTCGACGTGGCACTGGG
CCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCACTTACTCCAGTGAGGAGCAGAGACAG
CAGTTTCTGAACAGCGTGAAGATCCCACCGACGATCCGTCATAAGGTCGGCTTCATGTCACTGCATCT
CCTGTGA
氨基酸序列(Seq.ID No.20)
MEAIAKRLDA CQEQLLELYE ENSTDLHKHV LHWKCMRHES VLLYKAKQMG LSHIGMQVVP
PLKVSEAKGH NAIEMQMHLE SLLRTEYSME PWTLQETSYE MWQTPPKRCF AKRGKTVEVK
FDGCANNTMD YVVWTDVYVQ DNDTWVKVHS MVDAKGIYYT CGQFKTYYVN FVKEAEKYGS
TJGWEVCYGS TVICSPASVS STTQEVSIPE STTYTPAQTS TTVSSSTKED AVQTPPRKRA
RGVQQSPCNA LCVAHIGPVD SGNHNLITNN HDQHQRRNNS NSSATPIVQF QGESNCLKCF
RYRLNDRHRH LFDLISSTWH WASSKAPHKH AIVTVTYDSE EQRQQFLDVV KIPPTISHKL
GFMSLHLLMA DDSGTENEGS GCTGWFMVEA IVQHPTGTQI SDDEDEEVED SGYDMVDFID
DSNITHNSLE AQALFNRQEA DTHYATVQDL GGKYLGSPYV SPINTIAEAV ESEISPRLDA
IKLTRQPKKV KRRLFQTREL TDSGYGYSEV EAGTGTQVEK HGVPENGGDG QEKDTGRDIE
GEEHTEAEAP TNSVREHAGT AGILELLKCK DLRAALLGKF KECFGLSFLD LIRPFKSDKT
TCLDWVVAGF GIHHSISEAF QKLIEPLSLY AHIQWLTNAW GMVLLVLLRF KVNKSRSTVA
RTLATLLNIP ENQMLIEPPK IQSGVAALYW FRTGISNAST VIGEAPEWIT RQTVIEHGLA
DSQFKLTEMV QWAYDNDICE ESEIAFEYAQ RGDFDSNARA FINSNMQAMY VKDCATMCRH
YKHAEMRKMS IKQWIKHRGS KIEGTGNWKP IVQFLRHQNI EFIPFLTKFK LWLHGTPKKN
CIAIVGPPDT DKSYFCMSLI SFLGGTVISH VNSSSHFWLQ PLVDAKVALL DDATQPCWIY
MDTYMRNLLD GNPMSIDRKH KALTLIKCPP LLVTSNIDIT KEDKYKYLHT RVTTFTFPNP
FPFDRNGNAV YELSNTNWKC FFERLSSSLD IQDSEDEEDG SNSQAFRCVP GTVVRTLMEA
IAKRLDACQD QLLELYEENS IDIHKHIMHW KCIRLESVLL HKAKQMGLSH IGLQVVPPLT
VSETKGHNAI EMQMHLESLA KTQYGVEPWT LQDTSYEMWL TPPKRCFAKQ GNTVEVKFDG
CEDNVMEYVV WTHIYLQDND SWVKVTSSVD AKGIYYTCGQ FKTYYVNFNK EAQKYGSTNH
WEVCYGSTVI CSPASVSSTV REVSIAEPTT YTPAQTTAPT VSACTTEDGV SAPPRKRARG
PSTNNTLCVA NIRSVDSTIN NIVTDNYNKH QRRNNCESAA TPIVQLQGDS NCLKCFRYRL
NDKYKHLFEL ASSTWHWASP EAPHKNAIVT LTYSSEEQRQ QFLNSVKIPP TIRHKVGFMS
LHLL
11.质粒：HPV118(p7313ie 6be2.11e2.6be1)
感兴趣的基因：
HPV118构建体中的多蛋白基因是三联融合蛋白，依次包含所有密码子都优化并突变的6be2、11e2、6be1。使用2个前述PCR片段，即6be1和11/6be2，通过PCR来装配该多蛋白基因。通过PAGE和蛋白质印迹观察，基因大小为～4.1kb，产生了～170kD的多蛋白。
克隆：
用Bam HI+NotI限制性酶消化多蛋白基因，并将其连接到p7313ie载体中。对所选择克隆序列的分析显示出‘不寻常的’碱基改变，但通过不同片段交换即可克服。发现所得的克隆hpv118#3没有错误。
HPV118中的多蛋白序列(SEQ.ID No.21)
ATGGAAGCTMTTGCCAAGCGACTGGACGCCTGCCAGGAGCAGCTGCTGGAGCTGTACGAGGAAAACAG
CACAGACCTCCACAAGCACGTGCTGCACTGGAAGTGCATGCGCCACGAGTCAGTGCTCCTGTACAAGG
CCAAGCAGATGGGGCTGTCCCACATCGGGATGCAGGTCGTGCCCCCGCTGAAGGTGAGCGAAGCCAAG
GGCCACAACGCTATCGAGATGCAGATGCACCTGGAGAGCCTGCTGCGGACCGAATACAGCATGGAGCC
CTGGACTCTCCAGGAGACGTCCTACGAAATGTGGCAGACTCCTCCGAAGCGCTGTTTCGCAAAGCGCG
GCAAGACAGTTGAGGTGAAATTCGATGGGTGCGCAAACAACACGATGGACTACGTGGTGTGGACCGAT
GTCTACGTGCAGGACAATGACACCTGGGTGAAGGTACATAGTATGGTGGATGCCAAGGGCATCTATTA
CACCTGCGGGCAGTTCAAGACGTACTACGTCAACTTCGTCAAGGAAGCCGAAAAGTATGGTTCCACCA
AGCACTGGGAGGTGTGTTACGGGAGTACTGTGATCTGCAGCCCCGCCTCCGTGTCGTCCACCACCCAG
GAAGTGAGCATTCCGGAGAGCACCACATACACCCCGGCCCAAACGAGCACGCTCGTCAGCAGCAGCAC
CAAGGAGGACGCCGTCCAGACGCCCCCCCGGAAGAGGGCCCGGGGGGTCCAGCAGTCTCCCTGCAATG
CCCTGTGCGTTGCTCACATCGGCCCTGTCGATTCTGGGAACCACAATCTCATCACGAACAACCACGAC
CAGCACCAAAGGCGCAACAACTCTAACAGCTCCGCAACTCCAATAGTGCAGTTCCAGGGGGAGTCCAA
CTGCCTCAAGTGTTTCCGCTACCGCCTCAACGACCGCCACCGCCACCTGTTCGACTTGATCAGTTCCA
CGTGGCACTGGGCCAGCAGCAAGGCGCCCCACAAACACGCTATCGTGACGGTGACCTACGACTCCGAG
GAGCAGAGGCAGCAGTTCCTGGACGTCGTGAAGATTCCTCCGACAATCAGCCACAAGCTTGGCTTCAT
GTCCCTGCACCTGCTGATGGAAGCCATCGCGAAGAGGCTCGACGCCTGCCAGGACCAGCTGCTCGAGC
TGTACGAGGAGAACAGCATTGACATCCATAAGCACATCATGCACTGGAAGTGCATTCGCCTGGAGAGC
GTGCTGTTGCACAAGGCCAAGCAGATGGGCCTGTCCCACATAGGCCTTCAGGTGGTCCCCCCTCTGAC
CGTGTCAGAGACAAAGGGCCATAACGCAATCGAGATGCAGATGCACCTCGAGTCGCTGGCGAAAACAC
AGTACGGCGTGGAGCCATGGACCCTGCAGGACACCTCGTACGAAATGTGGCTGACCCCACCTAAGCGA
TGCTTCGCCAAACAGGGCAACACAGTGGAGGTGAAGTTCGACGGCTGTGAGGATAACGTTATGGAGTA
TGTCGTGTGGACGCACATCTATCTGCAGGACAACGACAGTTGGGTGAAGGTGACCAGCTCCGTGGACG
CGAAGGGCATCTACTATACCTGTGGGCAGTTTAAAACCTACTATGTGAACTTCAACAAAGAGGCCCAA
AAGTATGGCTCCACCAACCACTGGGAGGTCTGCTATGGGAGCACGGTGATTTGCTCTCCCGCCAGCGT
GTCTAGCACTGTGCGCGAGGTGAGCATTGCCGAGCCGACCACGTACACCCCTGCCCAGACGACCGCTC
CGACCGTGTCTGCTTGTACTACCGAGGACGGCGTGAGCGCTCCACCCAGGAAGCGTGCGAGGGGCCCA
AGCACCAACAACACCCTCTGTGTGGCGAACATTCGCAGCGTCGACAGTACCATCAATAACATCGTGAC
GGATAACTATAACAAGCACCAGAGGCGTAACAACTGTCACTCTGCCGCAACCCCCATCGTGCAGCTCC
AGGGAGACAGCAATTGCCTTAAGTGCTTCCGCTATCGCCTCAACGACAAGTACAAGCACCTCTTTGAG
CTCGCCTCGTCGACGTGGCACTGGGCCTCACCCGAGGCACCTCACAAGAACGCCATCGTCACTCTCAC
TTACTCCAGTGAGGAGCAGAGACAGCAGTTTCTGAACAGCGTGAAGATCCCACCGACGATCCGTCATA
AGGTCGGCTTCATGTCACTGCATCTCCTGATGGCAGACGATTCCGGTACTGAGAACGAAGGTTCTGGT
TGTACCGGTTGGTTCATGCTTGAAGCAATCGTTCAGCATCCGACTGGTACCCAGATCTCCGATGACGA
AGACGAAGAAGTTGAAGATTCTGGTTACGACATGGTTGACTTCATCGATGACTCCAACATCACTCATA
ACTCTCTGGAAGCACAGGCTCTGTTTAACCGCCAGGAAGCTGATACCCATTACGCTACTGTTCAGGAC
CTGGGAGGCAAATATCTGGGCTCTCCGTACGTTTCCCCGATCAACACTATCGCAGAAGCAGTTGAGTC
TGAAATCTCCCCGCGCCTGGACGCTATCAAACTGACTCGTCAGCCGAAGAAGGTTAAACGTCGTCTGT
TCCAGACTCGTGAACTGACCGACTCCGGTTACGGTTATAGCGAAGTTGAGGCTGGCACCGGCACCCAG
GTTGAAAAACACGGTGTACCGGAAAACGGCGGCGACGGTCAGGAAAAGGACACCGGCCGCGACATCGA
GGGTGAGGAACACACCGAAGCTGAAGCTCCGACTAACTCTGTTCGTGAACACGCAGGTACTGCGGGTA
TCCTGGAACTGCTGAAATGCAAAGACCTGCGCGCGGCTCTGCTGGGCAAATTCAAAGAATGCTTCGGC
CTGTCTTTCATTGACCTGATCCGTCCGTTTAAGTCTGACAAAACTACCTGTCTGGACTGGGTTGTAGC
AGGCTTCGGCATCCACCACTCTATCTCTGAAGCATTCCAGAAACTGATCGAGCCGCTGTCTCTGTACG
CGCACATCCAGTGGCTGACTAACGCTTGGGGTATGGTTCTGCTGGTACTGCTGCGCTTTAAAGTAAAC
AAATCTCGTTCCACTGTTGCTCGTACTCTGGCTACCCTGCTGAACATCCCGGAGAACCAGATGCTGAT
CGAACCGCCGAAAATCCAGTCTGGTGTAGCTGCACTGTACTGGTTTCGTACTGGCATCTCTAACGCTA
GCACTGTTATCGGTGAAGCACCGGAATGGATCACTCGTCAGACCGTTATCGAACACGGTCTGGCAGAT
TCTCAGTTCAAACTGACTGAAATGGTTCAGTGGGCATACGACAACGACATCTGCGAGGAATCTGAAAT
TGCGTTCGAATACGCTCAGCGTGGCGACTTCGACTCCAACGCTCGTGCTTTCCTGAACAGCAACATGC
AGGCTAAATACGTAAAAGACTGCGCTACCATGTGCCGTCACTACAAACACGCGGAAATGCGTAAAATG
TCTATCAAACAGTGGATCAAGCACCGCGGTTCTAAAATCGAAGGTACCGGTAACTGGAAACCGATCGT
TCAGTTCCTGCGCCATCAGAACATCGAATTCATCCCGTTCCTGACCAAATTCAAGCTGTGGCTGCACG
GTACCCCGAAAAAAAACTGCATCGCTATCGTAGGTCCACCGGACACTGACAAGTCTTACTTCTGTATG
TCCCTGATCTCTTTCCTGGGCGGCACTGTAATCTCTCACGTTAACTCTTCCTCCCATTTCTGGCTGCA
GCCACTGGTAGACGCGAAAGTAGCTCTGCTGGACGACGCGACCCAGCCGTGCTGGATCTACATGGATA
CTTACATGCGCAACCTGCTGGACGGTAACCCGATGTCTATCGACCGTAAACACAAAGCGCTGACTCTG
ATCAAGTGCCCGCCGCTGCTGGTAACTTCTAACATCGACATCACCAAGGAAGATAAATACAAGTACCT
GCATACCCGTGTTACTACCTTTACTTTCCCGAACCCGTTCCCGTTTGATCGTAACGGTAACGCTGTTT
ACGAACTGTCCAACACTAACTGGAAATGCTTCTTCGAGCGTCTGTCTTCCTCCCTGGACATCCAGGAC
TCTGAAGATGAAGAAGATGGTTCTAACTCTCAGGCTTTCCGTTGTGTTCCGGGTACTGTTGTTCGTAC
TCTGTGA
氨基酸序列(Seq.ID No.22)
MEAIAKRLDA CQEQLLELYE ENSTDLHKHV LHWKCMRHES VLLYKAKQMG LSHIGMQVVP
PLKVSEAKGH NAIEMQMHLE SLLRTEYSME PWTLQETSYE MWWTPPKRCF AKRGKTVEVK
FDGCANNTMD YVVWTDVYVQ DNDTWVKVHS MVDAKGIYYT CGQFKTYYVN FVKEAEKYGS
TKHWEVCYGS TVICSPASVS STTQEVSIPE STTYTPAQTS TLVSSSTKED AVQTPPRKRA
RGVQQSPCNA LCVAHIGPVD SGNHNLITNN HDQHQRRNNS NSSATPIVQF QGESNCLKCF
RYRLNDRHRH LFDLISSTWH WASSKAPHKH AIVTVTYDSE EQRQQFLDVV KIPPTISHKL
GFMSLHLLME AIAKRLDACQ DQLLELYEEN SIDIHKHIMH WKCIRLESVL LHKAKQMGLS
HIGLQVVPPL TVSETKGHNA IEMQMHLESL AKTQYGVEPW TLQDTSYEMW LTPPKRCFAK
QGNTVEVKFD GCEDNVMEYV VWTHIYLQDN DSWVKVTSSV DADGIYYTCG QFKTYYVNFN
KEAQKYGSTN HWEVCYGSTV ICSPASVSST VREVSIAEPT TYTPAQTTAP TVSACTTEDG
VSAPPRKRAR GPSTNNTLCV ANIRSVDSTI NNIVTDNYNK HQRRNNCHSA ATPIVQLQGD
SNCLKCFRYR LNDKYKHLFE LASSTWHWAX PEAPHKNAIV TLTYSSEEQR QQFLNSVKIP
PTIRHKVGFM SLHLLMADDS GTENEGSGCT GWFMVEAIVQ HPTGTQISDD EDEEVEDSGY
DMVDFIDDSN ITHNSLEAQA LFNRQEADTH YATVQDLGGK YLGSPYVSPI NTIAEAVESE
ISPRLDAIKL TRQPKKVKRR LFQTRELTDS GYGYSEVEAG TGTQVEKHGV PENGGDGQEK
DTGRDIEGEE HTEAEAPTNS VREHAGTAGI LELLKCKDLR AALLGKFKEC FGLSFIDLIR
PFKSDKTTCL DWVVAGFGIH HSISEAFQKL IEPLSLYAHI QWLTNAWGMV LLVLLRFKVN
KSRSTVARTL ATLLNIPENQ MLIEPPKIQS GVAALYWFRT GISNASTVIG EAPEWITRQT
VIEHGLADSQ FKLTEWVQWA YDNDICEESE IAFEYAQRGD FDSNARAFLN SNMQAKYVKD
CATMCRHYKH AEMRKMSIKQ WIKHRGSKIE GTGNWKPIVQ FLRHQMIEFI PFLTKFKLWL
HGTPKKNCIA IVGPPDTDKS YFCMSLISFL GGTVISHVNS SSHFWLQPLV DAKVALLDDA
TQPCWIYMDT YMRNLLDGNP MSIDRKHKAL TLIKCPPLLV TSNIDITKED KYKYLHTRVT
TFTFPNPFPF DRNGNAVYEL SNTNWKCFFE RLSSSLDIQD SEDEEDGSNS QAFRCVPGTV
VRTL
从得自DaVid Hodgeson(Warwick大学)的质粒pDAH212的亚克隆中获得了ColE1尾部序列，并利用引物通过PCR扩增从而将EcoRI限制性位点安置在序列的末端。然后把尾部序列插入到p7313-PL的EcoRl位点中以产生质粒p7313-PLc。扩增的尾部序列用Genbank条目M11411来核对。
实施例2-在哺乳动物293T细胞中表达
哺乳动物293T细胞于37℃在5％CO2中生长过夜，对数生长期生长的终浓度为2X105个细胞/每个6孔Corning CostarTM(CorningScience Products，10The ValleyCentre，Gordon Road，HighWycombe，Bucks，UK)组织培养板。制备以下转染混合物并混合25分钟：
感兴趣的DNA 2μg
2μg
补足用的无菌双蒸水 16μl
OPTI-memTM(Gibco BRL，Paisley，Scotland) 8μl
LipofectamineTM(GibcoBRL) 6μl。
用OPTI-memTM将孔中的每一单细胞层仔细地洗两次。向每个孔中加入800μl OPTI-memTM。向转染混合物中加入200μl OPTI-memTM，混匀并轻轻地加到单细胞层上。在5％CO2中于37℃温育培养板5小时，然后弃置转染混合物及OPTI-memTM。用细胞生长培养基轻轻地洗单细胞层两次，并在5％CO2中于37C将最终转染的细胞在Dulbecco′s改良的Eagle培养基中温育24小时，其中培养基包含10％胎牛血清和29.2mg/ml L-谷氨酰胺。把细胞刮入微试管中，用PBS洗涤两次，离心沉淀并将细胞小球重新悬浮在SDS PageLaemmli染色液中。煮沸细胞小球并置于10％SDS Page凝胶上，在1X Tris甘氨酸SDS缓冲液中电泳。电泳后，将凝胶转移到硝化纤维膜(Amersham)上并进行蛋白质印迹。硝化纤维膜用含5％MarvelTM(Premier Beverages，Knighton，Adbaston，STAFFORD，UK)的PBS于室温封闭30分钟，并用PBS和0.1％Tween 20洗涤两次。用含5％MarvelTM的PBS稀释兔的多克隆抗体，所述抗体抗HPV6bE1的C末端蛋白序列(蛋白序列：CSSS LDIQD SEDEEDG SNSQAFRSeq.ID No.23)，并将其加到硝化纤维膜上。将其在室温中温育1小时并轻微振荡。也可用抗HPV11E1的多克隆抗体检查交叉反应性。除去稀释的抗体并用PBS和0.1％Tween 20洗膜三次。二级偶联物，即猪抗兔辣根过氧化酶(HRP)(DAKO)，在PBS和0.1％Tween 20中稀释到1∶20000。将其加到洗过的膜上并在室温下温育1小时并轻微振荡。然后用PBS和0.1％Tween 20彻底地洗膜。用化学发光HRP试剂盒(Amersham)检测转移到膜上的蛋白质。
结果：
结果(图13)显示了由HPV 116、117、118表达的正确蛋白质大小，HPV 116、117、118包含密码子优化的HPV多蛋白。
分别用各种构建体的～0.5ug DNA转柒HEK293T细胞并在24小时后收获该细胞。然后通过聚丙烯酰胺电泳分析这些样品，接着进行蛋白质印迹。用两个肽的抗体检测多蛋白表达(～180kd)：抗6BE1(编号.1097)和抗6bE2(编号.1101)。
实施例3
E1抗原失活和实验确证
HPV E1蛋白是非常保守的核蛋白，具有非特异性DNA结合、ATP酶和解pri旋酶活性。E1也可结合宿主细胞DNA聚合酶-α引发酶，亦结合HPV E2蛋白，然后HPV E2蛋白把E1‘补充’到起始前病毒DNA复制复合物中。E1的主要作用是启动感染细胞中的病毒DNA复制。
E1(和E2)的DNA复制功能是相对非特异的，现在已有许多研究表明来自一个基因型的E1和E2蛋白可启动带有来自不同基因型复制起始序列的质粒的特异性DNA复制。研究也表明，向已经包含低拷贝数HPV质粒的细胞中导入高表达的E1和E2能够造成该质粒显著扩增。这种混乱使其带有少量的安全危险隐患，本方案正寻求消除该隐患。因此，找到了使他们复制潜力失活的E1(和E2)突变。
E1突变G482D发生在高度保守的ATP结合一致性序列上，带有该突变的E1蛋白显示出具有多种功能缺陷。其他接近蛋白质N-末端的突变(K83G，R84G)显示出使E1丧失核定位性质。如果有额外水平的功能丧失和安全性，则不能定位到核中间，也可用来将E1从宿主复制蛋白和病毒DNA中分离。选择这些突变(G428D，K83G，R84G)并将其引入E1作为HPV DNA免疫治疗E1载体的一部分。
将HPV DNA的体外复制分析用于证实E1 DNA复制功能的丧失(由此推论，增加E2活性的复制突变失活也可在相同的试验中得到证实)。简而言之，E1和E2一起激活HPV复制起点，并且已知来自HPV 6b的E1和E2蛋白激活并使得DNA从HPV-11起点开始重新复制。编码我们密码子优化的E1和E2序列的质粒同带有HPV-11复制起点的质粒(ori质粒)一起共转染到293细胞中。通过收获共转染后48小时的细胞中的DNA可以测定所导入的ori质粒的E1和E2依赖性复制(Hirt溶胞)。提取的DNA首先用Hind III进行限制酶消化，然后用Dpn I消化未甲基化的、未复制的DNA。接着进行DNA的DNA印迹分析，ori质粒DNA用作为探针进行杂交。Dpn I消化后同ori质粒具有相同大小的条带就是表示体外重新复制的质粒DNA的标记物。
野生型的E1和E2(HPV119+HPV120)显示出明显的条带，表明有复制了的加入的质粒DNA。三个前导构建体中的每一个都呈阴性，(HPV116、HPV117和HPV118)显示了结果：没有复制。结论：前导构建体HPV116、HPV117和HPV118不具有DNA复制活性。
实施例4
乳头状瘤病毒的E2蛋白是位点特异性DNA结合核蛋白，其功能是主要复制起点识别蛋白的功能，并辅助启动前复制复合物的装配。根据(相对于其他转录因子位点的)位置和其同类结合位点的蛋白质亲核性，全长的E2蛋白也能用作病毒转录的抑制子和活化子。已知E2也影响几种宿主细胞启动子的转录。已经广泛研究了E2的突变失活，特别是一个点突变Lys 111→Ala(K111A)已显示出使得E2的转录和复制功能都失活。该突变也具有防止蛋白质核易位的额外好处。该突变(K111A)加入到每个E2抗原以作为免疫治疗性HPV DNA的一部分。
我们着手在体外CAT转录报告基因试验中验证K111A突变的E2和每个多蛋白构建体的功能丧失。我们使用两个阳性对照(源于有活性的E2蛋白)。有一个构建体表达未突变(有活性)的HPV-11E2蛋白，并有第二个载体表达BPV E2蛋白(一种强转录转活化子)。这些数据如图14所示。
结论：这些数据显示表达自天然(未突变)HPV 6b E2载体的蛋白质具有转录活性，而突变(K111A)的E2没有活性，多蛋白载体HPV116、HPV117和HPV118中的每一个也都没有活性。
实施例5
不同基因构建体HPV116、HPV117和HPV118的表达和比较
比较前导构建体HPV116、HPV117和HPV118的基因表达的研究无法鉴定出任何清楚的体外基因表达的差异。另外，多蛋白的表达与在单个质粒(HPV110)中各自(未融合)抗原的表达相等。同样重要的是，导入点突变并没有对基因表达产生影响(HPV108和HPV110)。
实施例6
小鼠体内的免疫原性研究
为比较三种不同构建体HPV116、HPV117和HPV118在体内的免疫原性，利用PMID免疫小鼠。
每次免疫过程包括对Balb/c(H-2Kd)或C57BL6(H-2Kb)小鼠剃去毛的腹部皮下注射0.5μg DNA，注射两次。用1μg DNA初次免疫动物，21天后用相同剂量进行强化免疫，并在强化5-7天后筛选。提取血清和脾脏，用于进行PMID后所产生的体液和细胞免疫应答的分析。
体液试验
利用标准ELISA方法并用重组E1和E2蛋白作为捕获抗原来评估PMID免疫的小鼠中所得到的抗体。在E2免疫的小鼠广泛免疫之后，并不能可靠地检测出抗体应答。我们无法确证小鼠中E1抗原的抗体的检测结果。这些弱的/未检测到的抗体应答与公开的文献内容一致。
细胞试验
用ELISPOT试验研究小鼠中的细胞免疫应答。该技术适于评估在已知密度培养基中的细胞频率，这些细胞能分泌特异性地应答上文免疫相容的MHC分子中出现的抗原的细胞因子。
简而言之，在合适的靶细胞出现抗原之前，将免疫动物中分离的脾细胞的单细胞悬液加入到专门的、包被有抗细胞因子捕获抗体的微滴定培养板中，并温育过夜。用结合在培养板上的抗体将细胞因子直接捕获在细胞周围的区域中，当细胞溶解并洗去时，其仍结合在那里。利用生物素化的抗细胞因子二抗与抗生物素蛋白链菌素碱性磷酸酶偶联物来进行检测。该酶对显色底物的作用允许目测细胞因子产生细胞的频率。
牛痘ELISPOT试验及数据
由于缺少明确的鼠T细胞表位，以能工程化表达靶抗原的重组牛痘病毒的形式提供了抗原。将这种病毒用于感染合适的靶细胞以产生针对ELISPOT试验中效应细胞的抗原。
在三个候选构建体对C57BL/6小鼠PMID后，检测对HPV 6bE1的应答。利用统计学分析2次单独的实验结果。代表性的实验结果如图15和16所示。
在小鼠中使用前导构建体和PMID所得到的解释性的免疫原性数据：
CTL试验和数据
活化的CD8+T细胞应答上文免疫相容MHC I分子中出现的特定肽，从而能够溶解细胞。根据Eu3+释放生物试验，即传统的铬释放试验的非放射性改良方法，可检测出该功能。
要应用该试验达到这些目的，则需要鉴定来源于HPV 6bE1蛋白主要序列的CD8+T细胞表位。可使用细胞因子ELISPOT通过对重叠了11个氨基酸的15肽所组成的肽库进行筛选可达到目的。通过标准流式细胞技术可将应答细胞群分为CD4+或CD8+T细胞。
该技术的基础涉及溶解Eu3+标记的靶细胞，该细胞用同类多肽脉冲刺激。在两小时温育过程中，通过细胞溶解性T细胞溶解靶细胞，从而把Eu3+释放到培养基上清液中。这可用时间解析荧光测定法来检测。当靶细胞用化学方法溶解时，特定的溶解被表示为检测到的溶解总量的百分比。
细胞免疫学数据的评估
HPV116、HPV117和HPV118的免疫学评估，包括在小鼠中将牛痘ELISPOT和CTL试验分析作为免疫学结果以重复PMID免疫研究。所有候选构建体都产生了对每个抗原的免疫应答。
总的来说，牛痘ELISPOT数据显示，通过突变或通过融合到E2抗原成分上并不能阻止对E1的应答。当在HPV-108(单6b E1构建体)、HPV116、HPV117和HPV118之间比较E1应答时，应答不具有统计学上的差异。可是牛痘ELISPOT数据揭示了对HPV-11E2抗原成分应答中的差异。用HPV118免疫的小鼠中的E2抗原特异性应答显著地大于用HPV116或HPV117免疫的小鼠。在此基础上HPV118似乎比HPV116或HPV117具有更好的免疫原性。
对E1抗原特异性CTL溶解的分析也可揭示出效果的趋势。用HPV118免疫的小鼠的T细胞所产生的特异性溶解的百分比高于HPV116或HPV117。该观察结果是可重复的。
合起来看，并在牛痘ELISPOT和CTL溶解数据的基础上来看，HPV118有更强的免疫原性。
结论：以纯免疫学标准来看，构建体HPV118具有最强的多蛋白免疫原性。
实施例7
对于抗犬口腔乳头状瘤病毒疾病而言，密码子优化的COPV E1/E2融合蛋白的PMID递送较密码子优化的E1或密码子优化的E2更有效
介绍
犬口腔乳头状瘤病毒(COPV)的动物模型是良好的人粘膜乳头状瘤病毒疾病的模拟体。在狗中由COPV所引起疾病的特点与发生在人中的非常相似(Nicholls等Virology 2001，283(1)31-39)。其是重要的粘膜乳头状瘤病毒疾病模型。COPV病毒感染犬粘膜上皮细胞，并在几周以后出现疣，然后又过了几周时间疣便自发消退了。COPV病毒编码了每种人乳头状瘤病毒基因的同源体(E1，E2，E4，E6，E7，L1和L2)。
已将狗COPV粘膜疾病模型用作为开发人病毒样颗粒(VLP)乳头状瘤病毒疫苗的理论基础中的关键模型(Ghim等，Vaccines 199525，375-379，Suzich等，PNAS 1995，9211553-11557)。人乳头状瘤病毒VLP疫苗现正在开发中，最近已完成了在人体中的早期临床试验。
我们指出当通过PMID施用时，编码密码子优化的E1和E2基因融合蛋白的质粒DNA能比单独编码密码子优化的E1或密码子优化的E2都更为有效地减轻疾病负担。
方法
密码子优化的E1和E2融合载体的构建
利用前述方法产生编码密码子优化的COPV E2序列的人工合成基因。将其融合到从克隆pCOPVE1c/o中收获的人工合成的密码子优化的COPV E1基因中，并插入载体WRG7077中以产生称为pCOPVE2/E1c/o的新克隆。该克隆表达包含COPV E2(N末端)和COPV E1(C末端)融合的多蛋白。通过蛋白质印迹确定多蛋白具有预期的大小。
用pCOPVE1c/o、pCOPVE2c/o和pCOPVE2/E1c/o免疫猎兔犬
用三种纯化的质粒pCOPVE1c/o、pCOPVE2c/o和pCOPVE2/E1c/o通过PMID来免疫猎兔犬。在12个皮肤位点免疫动物，腹部中线每侧具有6个非重叠位点。在全身麻醉条件下进行所有的免疫接种。每组五只动物。初次接种六周后，以同样方式使用相同步骤进行强化接种。
在最终强化免疫2周后，用感染性COPV病毒刺激免疫的动物。轻轻擦拭每只动物上唇的粘膜。在十个位点的每个(上唇每侧各五个)上使用10μl纯化的COPV病毒制品，并使之吸收几分钟。感染性COPV病毒的分离和纯化已有报道(Virology 1999，265(2)365-374)。
在用COPV病毒刺激后，每周检查粘膜刺激的位点。测定疣(乳头状瘤)出现的时间(刺激后)和疣的大小(mm)。
在用pCOPVE1c/o免疫的动物中，在刺激后的第7周，乳头状瘤在粘膜刺激的位置上开始产生。乳头状瘤继续生长，在第11周其大小达到＞3.5mm的平均大小。在用pCOPV E2c/o免疫的动物中，乳头状瘤首先在第8周出现，但是乳头状瘤平均大小在第11周才达到1.5mm。在用pCOPVE2/E1c/o免疫的动物中，疾病的初次征兆与其它组同时发生，而整个病情却显著地减轻了。pCOPVE2/E1c/o组中的(五只中的)一只动物完全阻止了疾病的发展，而该组中其他所有动物都仅产生非常小的乳头状瘤，该乳头状瘤在短期内就消退了(1-2周)。
在乳头状瘤病毒感染的动物模型中，编码COPV E1和COPV E2融合蛋白的质粒DNA对于防止疾病发展较COPV E1或COPV E2更为有效(图18)。
序列表
<110>Glaxo Group
<120>疫苗
<130>PG4961
<160>28
<170>用于Windows的FastSEQ 4.0版
<210>1
<211>1107
<212>DNA
<213>HPV
<400>1
atggaagcta ttgccaagcg actggacgcc tgccaggagc agctgctgga gctgtacgag 60
gaaaacagca cagacctcca caagcacgtg ctgcactgga agtgcatgcg ccacgagtca 120
gtgctcctgt acaaggccaa gcagatgggg ctgtcccaca tcgggatgca ggtcgtgccc 180
ccgctgaagg tgagcgaagc caagggccac aacgctatcg agatgcagat gcacctggag 240
agcctgctgc ggaccgaata cagcatggag ccctggactc tccaggagac gtcctacgaa 300
atgtggcaga ctcctccgaa gcgctgtttc gcaaagcgcg gcaagacagt tgaggtgaaa 360
ttcgatgggt gcgcaaacaa cacgatggac tacgtggtgt ggaccgatgt ctacgtgcag 420
gacaatgaca cctgggtgaa ggtacatagt atggtggatg ccaagggcat ctattacacc 480
tgcgggcagt tcaagacgta ctacgtcaac ttcgtcaagg aagccgaaaa gtatggttcc 540
accaagcact gggaggtgtg ttacgggagt actgtgatct gcagccccgc ctccgtgtcg 600
tccaccaccc aggaagtgag cattccggag agcaccacat acaccccggc ccaaacgagc 660
acgctcgtca gcagcagcac caaggaggac gccgtccaga cgcccccccg gaagagggcc 720
cggggggtcc agcagtctcc ctgcaatgcc ctgtgcgttg ctcacatcgg ccctgtcgat 780
tctgggaacc acaatctcat cacgaacaac cacgaccagc accaaaggcg caacaactct 840
aacagctccg caactccaat agtgcagttc cagggggagt ccaactgcct caagtgtttc 900
cgctaccgcc tcaacgaccg ccaccgccac ctgttcgact tgatcagttc cacgtggcac 960
tgggccagca gcaaggcgcc ccacaaacac gctatcgtga cggtgaccta cgactccgag 1020
gagcagaggc agcagttcct ggacgtcgtg aagattcctc cgacaatcag ccacaagctt 1080
ggcttcatgt ccctgcacct gctgtga 1107
<210>2
<211>368
<212>PRT
<213>HPV
<400>2
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His
20 25 30
Trp Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val
50 55 60
Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu
85 90 95
Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr
115 120 125
Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr
130 135 140
Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu
165 170 175
Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile
195 200 205
Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser
210 215 220
Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala
225 230 235 240
Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile
245 250 255
Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp
260 265 270
Gln His Gln Arg Arg Asn Asn Ser Asn Ser Sar Ala Thr Pro Ile Val
275 280 285
Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu
290 295 300
Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His
305 310 315 320
Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr
325 330 335
Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile
340 345 350
Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
355 360 365
<210>3
<211>1950
<212>DNA
<213>HPV
<400>3
atggcagacg attccggtac tgagaacgaa ggttctggtt gtaccggttg gttcatggtt 60
gaagcaatcg ttcagcatcc gactggtacc cagatctccg atgacgaaga cgaagaagtt 120
gaagattctg gttacgacat ggttgacttc atcgatgact ccaacatcac tcataactct 180
ctggaagcac aggctctgtt taaccgccag gaagctgata cccattacgc tactgttcag 240
gacctgggag gcaaatatct gggctctccg tacgtttccc cgatcaacac tatcgcagaa 300
gcagttgagt ctgaaatctc cccgcgcctg gacgctatca aactgactcg tcagccgaag 360
aaggttaaac gtcgtctgtt ccagactcgt gaactgaccg actccggtta cggttatagc 420
gaagttgagg ctggcaccgg cacccaggtt gaaaaacacg gtgtaccgga aaacggcggc 480
gacggtcagg aaaaggacac cggccgcgac atcgagggtg aggaacacac cgaagctgaa 540
gctccgacta actctgttcg tgaacacgca ggtactgcgg gtatcctgga actgctgaaa 600
tgcaaagacc tgcgcgcggc tctgctgggc aaattcaaag aatgcttcgg cctgtctttc 660
attgacctga tccgtccgtt taagtctgac aaaactacct gtctggactg ggttgtagca 720
ggcttcggca tccaccactc tatctctgaa gcattccaga aactgatcga gccgctgtct 780
ctgtacgcgc acatccagtg gctgactaac gcttggggta tggttctgct ggtactgctg 840
cgctttaaag taaacaaatc tcgttccact gttgctcgta ctctggctac cctgctgaac 900
atcccggaga accagatgct gatcgaaccg ccgaaaatcc agtctggtgt agctgcactg 960
tactggtttc gtactggcat ctctaacgct agcactgtta tcggtgaagc accggaatgg 1020
atcactcgtc agaccgttat cgaacacggt ctggcagatt ctcagttcaa actgactgaa 1080
atggttcagt gggcatacga caacgacatc tgcgaggaat ctgaaattgc gttcgaatac 1140
gctcagcgtg gcgacttcga ctccaacgct cgtgctttcc tgaacagcaa catgcaggct 1200
aaatacgtaa aagactgcgc taccatgtgc cgtcactaca aacacgcgga aatgcgtaaa 1260
atgtctatca aacagtggat caagcaccgc ggttctaaaa tcgaaggtac cggtaactgg 1320
aaaccgatcg ttcagttcct gcgccatcag aacatcgaat tcatcccgtt cctgaccaaa 1380
ttcaagctgt ggctgcacgg taccccgaaa aaaaactgca tcgctatcgt aggtccaccg 1440
gacactgaca agtcttactt ctgtatgtcc ctgatctctt tcctgggcgg cactgtaatc 1500
tctcacgtta actcttcctc ccatttctgg ctgcagccac tggtagacgc gaaagtagct 1560
ctgctggacg acgcgaccca gccgtgctgg atctacatgg atacttacat gcgcaacctg 1620
ctggacggta acccgatgtc tatcgaccgt aaacacaaag cgctgactct gatcaagtgc 1680
ccgccgctgc tggtaacttc taacatcgac atcaccaagg aagataaata caagtacctg 1740
catacccgtg ttactacctt tactttcccg aacccgttcc cgtttgatcg taacggtaac 1800
gctgtttacg aactgtccaa cactaactgg aaatgcttct tcgagcgtct gtcttcctcc 1860
ctggacatcc aggactctga agatgaagaa gatggttcta actctcaggc tttccgttgt 1920
gttccgggta ctgttgtt tactctgtga
<210>4
<211>649
<212>PRT
<213>HPV
<400>4
Met Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly
1 5 10 15
Trp Phe Met Val Glu Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile
20 25 30
Ser Asp Asp Glu Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val
35 40 45
Asp Phe Ile Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln
50 55 60
Ala Leu Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln
65 70 75 80
Asp Leu Gly Gly Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn
85 90 95
Thr Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala
100 105 110
Ile Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln
115 120 125
Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala
130 135 140
Gly Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu Asn Gly Gly
145 150 155 160
Asp Gly Gln Glu Lys Asp Thr Gly Arg Asp Ile Glu Gly Glu Glu His
165 170 175
Thr Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr
180 185 190
Ala Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu
195 200 205
Leu Gly Lys Phe Lys Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile
210 215 220
Arg Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu Asp Trp Val Val Ala
225 230 235 240
Gly Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile
245 250 255
Glu Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr Asn Ala Trp
260 265 270
Gly Met Val Leu Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg
275 280 285
Ser Thr Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn
290 295 300
Gln Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala Leu
305 310 315 320
Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr Va1 Ile Gly Glu
325 330 335
Ala Pro Glu Trp Ile Thr Arg Gln Thr Va1 Ile Glu His Gly Leu Ala
340 345 350
Asp Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp Asn
355 360 365
Asp Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly
370 375 380
Asp Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala
385 390 395 400
Lys Tyr Val Lys Asp Cys Ala Thr Met Cys Arg His Tyr Lys His Ala
405 410 415
Glu Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly Ser
420 425 430
Lys Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg
435 440 445
His Gln Asn Ile Glu Phe Ile Pro Phs Leu Thr Lys Phe Lys Leu Trp
450 455 460
Leu His Gly Thr Pro Cys Lys Asn Cys Ile Ala Ile Val Gly Pro Pro
465 470 475 480
Asp Thr Asp Lys Ser Tyr Phe Cys Met Ser Leu Ile Ser Phe Leu Gly
485 490 495
Gly Thr Val Ile Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln
500 505 510
Pro Leu Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro
515 520 525
Cys Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn
530 535 540
Pro Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys
545 550 555 560
Pro Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys
565 570 575
Tyr Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn Pro
580 585 590
Phe Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr
595 600 605
Asn Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln
610 615 620
Asp Ser Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys
625 630 635 640
Val Pro Gly Thr Val Val Arg Thr Leu
645
<210>5
<211>1104
<212>DNA
<213>hpv
<400>5
atggaagcca tcgcgaagag gctcgacgcc tgccaggacc agctgctcga gctgtacgag 60
gagaacagca ttgacatcca taagcacatc atgcactgga agtgcattcg cctggagagc 120
gtgctgttgc acaaggccaa gcagatgggc ctgtcccaca taggccttca ggtggtcccc 180
cctctgaccg tgtcagagac aaagggccat aacgcaatcg agatgcagat gcacctcgag 240
tcgctggcga aaacacagta cggcgtggag ccatggaccc tgcaggacac ctcgtacgaa 300
atgtggctga ccccacctaa gcgatgcttc gccaaacagg gcaacacagt ggaggtgaag 360
ttcgacggct gtgaggataa cgttatggag tatgtcgtgt ggacgcacat ctatctgcag 420
gacaacgaca gttgggtgaa ggtgaccagc tccgtggacg cgaagggcat ctactatacc 480
tgtgggcagt ttaaaaccta ctatgtgaac ttcaacaaag aggcccaaaa gtatggctcc 540
accaaccact gggaggtctg ctatgggagc acggtgattt gctctcccgc cagcgtgtct 600
agcactgtgc gcgaggtgag cattgccgag ccgaccacgt acacccctgc ccagacgacc 660
gctccgaccg tgtctgcttg tactaccgag gacggcgtga gcgctccacc caggaagcgt 720
gcgaggggcc caagcaccaa caacaccctc tgtgtggcga acattcgcag cgtcgacagt 780
accatcaata acatcgtgac ggataactat aacaagcacc agaggcgtaa caactgtcac 840
tctgccgcaa cccccatcgt gcagctccag ggagacagca attgccttaa gtgcttccgc 900
tatcgcctca acgacaagta caagcacctc tttgagctcg cctcgtcgac gtggcactgg 960
gcctcacccg aggcacctca caagaacgcc atcgtcactc tcacttactc cagtgaggag 1020
cagagacagc agtttctgaa cagcgtgaag atcccaccga cgatccgtca taaggtcggc 1080
ttcatgtcac tgcatctcct gtga 1104
<210>6
<211>367
<212>PRT
<213>HPV
<400>6
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Asp Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Ile Asp Ile His Lys His Ile Met His
20 25 30
Trp Lys Cys Ile Arg Leu Glu Ser Va1 Leu Leu His Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Leu Gln Val Val Pro Pro Leu Thr Val
50 55 60
Ser Glu Thr Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Ala Lys Thr Gln Tyr Gly Val Glu Pro Trp Thr Teu Gln Asp
85 90 95
Thr Ser Tyr Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Gln Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val
115 120 125
Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp Ser
130 135 140
Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Asn Lys Glu Ala Gln
165 170 175
Lys Tyr Gly Ser Thr Asn His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Val Arg Glu Val Ser Ile
195 200 205
Ala Glu Pro Thr Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val
210 215 220
Ser Ala Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg
225 230 235 240
Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile Arg
245 250 255
Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn Tyr Asn Lys
260 265 270
His Gln Arg Arg Asn Asn Cys His Ser Ala Ala Thr Pro Ile Val Gln
275 280 285
Leu Gln Gly Asp Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
290 295 300
Asp Lys Tyr Lys His Leu Phe Glu Leu Ala Ser Ser Thr Trp His Trp
305 310 315 320
Ala Ser Pro Glu Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr
325 330 335
Ser Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn Ser Val Lys Ile Pro
340 345 350
Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu
355 360 365
<210>7
<211>1104
<212>DNA
<213>HPV
<400>7
atggaagcca tcgcgaagag gctcgacgcc tgccaggacc agctgctcga gctgtacgag 60
gagaacagca ttgacatcca taagcacatc atgcactgga agtgcattcg cctggagagc 120
gtgctgttgc acaaggccaa gcagatgggc ctgtcccaca taggccttca ggtggtcccc 180
cctctgaccg tgtcagagac aaagggccat aacgcaatcg agatgcagat gcacctcgag 240
tcgctggcga aaacacagta cggcgtggag ccatggaccc tgcaggacac ctcgtacgaa 300
atgtggctga ccccacctaa gcgatgcttc gccaaacagg gcaacacagt ggaggtgaag 360
ttcgacggct gtgaggataa cgttatggag tatgtcgtgt ggacgcacat ctatctgcag 420
gacaacgaca gttgggtgaa ggtgaccagc tccgtggacg cgaagggcat ctactatacc 480
tgtgggcagt ttaaaaccta ctatgtgaac ttcaacaaag aggcccaaaa gtatggctcc 540
accaaccact gggaggtctg ctatgggagc acggtgattt gctctcccgc cagcgtgtct 600
agcactgtgc gcgaggtgag cattgccgag ccgaccacgt acacccctgc ccagacgacc 660
gctccgaccg tgtctgottg tactaccgag gacggcgtga gcgctccacc caggaagcgt 720
gcgaggggcc caagcaccaa caacaccctc tgtgtggcga acattcgcag cgtcgacagt 780
accatcaata acatcgtgac ggataactat aacaagcacc agaggcgtaa caactgtcac 840
tctgccgcaa cccccatcgt gcagctccag ggagacagca attgccttaa gtgcttccgc 900
tatcgcctca acgacaagta caagcacctc tttgagctcg cctcgtcgac gtggcactgg 960
gcctcacccg aggcacctca caagaacgcc atcgtcactc tcacttactc cagtgaggag 1020
cagagacagc agtttctgaa cagcgtgaag atcccaccga cgatccgtca taaggtcggc 1080
ttcatgtcac tgcatctcct gtga 1104
<210>8
<211>367
<212>PRT
<213>HPV
<400>8
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Asp Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Ile Asp Ile His Lys His Ile Met His
20 25 30
Trp Lys Cys Ile Arg Leu Glu Ser Val Leu Leu His Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Leu Gln Val Val Pro Pro Leu Thr Val
50 55 60
Ser Glu Thr Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Ala Lys Thr Gln Tyr Gly Val Glu Pro Trp Thr Leu Gln Asp
85 90 95
Thr Ser Tyr Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Gln Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val
115 120 125
Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp Ser
130 135 140
Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Asn Lys Glu Ala Gln
165 170 175
Lys Tyr Gly Ser Thr Asn His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Val Arg Glu Val Ser Ile
195 200 205
Ala Glu Pro Thr Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val
210 215 220
Ser Ala Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg
225 230 235 240
Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile Arg
245 250 255
Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn Tyr Asn Lys
260 265 270
His Gln Arg Arg Asn Asn Cys His Ser Ala Ala Thr Pro Ile Val Gln
275 280 285
Leu Gln Gly Asp Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
290 295 300
Asp Lys Tyr Lys His Leu Phe Glu Leu Ala Ser Ser Thr Trp His Trp
305 310 315 320
Ala Ser Pro Glu Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr
325 330 335
Ser Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn Ser Val Lys Ile Pro
340 345 350
Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu
355 360 365
<210>9
<211>2206
<212>DNA
<213>HPV
<400>9
atggaagcta ttgccaagcg actggacgcc tgccaggagc agctgctgga gctgtacgag 60
gaaaacagca cagacctcca caagcacgtg ctgcactgga agtgcatgcg ccacgagtca 120
gtgctcctgt acaaggccaa gcagatgggg ctgtcccaca tcgggatgca ggtcgtgccc 180
ccgctgaagg tgagcgaagc caagggccac aacgctatcg agatgcagat gcacctggag 240
agcctgctgc ggaccgaata cagcatggag ccctggactc tccaggagac gtcctacgaa 300
atgtggcaga ctcctccgaa gcgctgtttc gcaaagcgcg gcaagacagt tgaggtgaaa 360
ttcgatgggt gcgcaaac cacgatggac tacgtggtgt ggaccgatgt ct tgtgcag 420
gacaatgaca cctgggtgaa ggtacatagt atggtggatg ccaagggcat ctattacacc 480
tgcgggcagt tcaagacgta ctacgtcaac ttcgtcaagg aagccgaaaa gtatggttcc 540
accaagcact gggaggtgtg ttacgggagt actgtgatct gcagccccgc ctccgtgtcg 600
tccaccaccc aggaagtgag cattccggag agcaccacat acaccccggc ccaaacgagc 660
acgctcgtca gcagcagcac caaggaggac gccgtccaga cgcccccccg gaagagggcc 720
cggggggtcc agcagtctcc ctgcaatgcc ctgtgcgttg ctcacatcgg ccctgtcgat 780
tctgggaacc acaatctcat cacgaacaac cacgaccagc accaaaggcg caacaactct 840
aacagctccg caactccaat agtgcagttc cagggggagt ccaactgcct caagtgtttc 900
cgctaccgcc tcaacgaccg ccaccgccac ctgttcgact tgatcagttc cacgtggcac 960
tgggccagca gcaaggcgcc ccacaaacac gctatcgtga cggtgaccta cgactccgag 1020
gagcagaggc agcagttcct ggacgtcgtg aagattcctc cgacaatcag ccacaagctt 1080
ggcttcatgt ccctgcacct gctgatggaa gccatcgcga agaggctcga cgcctgccag 1140
gaccagctgc tcgagctgta cgaggagaac agcattgaca tccataagca catcatgcac 1200
tggaagtgca ttcgcctgga gagcgtgctg ttgcacaagg ccaagcagat gggcctgtcc 1260
cacataggcc ttcaggtggt cccccctctg accgtgtcag agacaaaggg ccataacgca 1320
atcgagatgc agatgcacct cgagtcgctg gcgaaaacac agtacggcgt ggagccatgg 1380
accctgcagg acacctcgta cgaaatgtgg ctgaccccac ctaagcgatg cttcgccaaa 1440
cagggcaaca cagtggaggt gaagttcgac ggctgtgagg ataacgttat ggagtatgtc 1500
gtgtggacgc acatctatct gcaggacaac gacagttggg tgaaggtgac cagctccgtg 1560
gacgcgaagg gcatctacta tacctgtggg cagtttaaaa cctactatgt gaacttcaac 1620
aaagaggccc aaaagtatgg ctccaccaac cactgggagg tctgctatgg gagcacggtg 1680
atttgctctc ccgccagcgt gtctagcact gtgcgcgagg tgagcattgc cgagccgacc 1740
acgtacaccc ctgcccagac gaccgctccg accgtgtctg cttgtactac cgaggacggc 1800
gtgagcgctc cacccaggaa gcgtgcgagg ggcccaagca ccaacaacac cctctgtgtg 1860
gcgaacattc gcagcgtcga cagtaccatc aataacatcg tgacggataa ctataacaag 1920
caccagaggc gtaacaactg tcactctgcc gcaaccccca tcgtgcagct ccagggagac 1980
agcaattgcc ttaagtgctt ccgctatcgc ctcaacgaca agtacaagca cctctttgag 2040
ctcgcctcgt cgacgtggca ctgggcctca cccgaggcac ctcacaagaa cgccatcgtc 2100
actctcactt actccagtga ggagcagaga cagcagtttc tgaacagcgt gaagatccca 2160
ccgacgatcc gtcataaggt cggcttcatg tcactgcatc tcctga 2206
<210>10
<211>735
<212>PRT
<213>HPV
<400>10
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His
20 25 30
Trp Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val
50 55 60
Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu
85 90 95
Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Arg Gly Lys Thr Val Glu Val Lys Phe Aap Gly Cys Ala Asn Asn Thr
115 120 125
Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr
130 135 140
Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu
165 170 175
Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile
195 200 205
Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser
210 215 220
Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala
225 230 235 240
Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile
245 250 255
Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp
260 265 270
Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val
275 280 285
Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu
290 295 300
Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His
305 310 315 320
Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr
325 330 335
Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile
340 345 350
Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
355 360 365
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Asp Gln Leu Leu
370 375 380
Glu Leu Tyr Glu Glu Asn Ser Ile Asp Ile His Lys His Ile Met His
385 390 395 400
Trp Lys Cys Ile Arg Leu Glu Ser Val Leu Leu His Lys Ala Lys Gln
405 410 415
Met Gly Leu Ser His Ile Gly Leu Gln Val Val Pro Pro Leu Thr Val
420 425 430
Ser Glu Thr Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
435 440 445
Ser Leu Ala Lys Thr Gln Tyr Gly Val Glu Pro Trp Thr Leu Gln Asp
450 455 460
Thr Ser Tyr Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Ala Lys
465 470 475 480
Gln Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val
485 490 495
Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp Ser
500 505 510
Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile Tyr Tyr Thr
515 520 525
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Asn Lys Glu Ala Gln
530 535 540
Lys Tyr Gly Ser Thr Asn His Trp Glu Val Cys Tyr Gly Ser Thr Val
545 550 555 560
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Val Arg Glu Val Ser Ile
565 570 575
Ala Glu Pro Thr Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val
580 585 590
Ser Ala Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg
595 600 605
Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile Arg
610 615 620
Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn Tyr Asn Lys
625 630 635 640
His Gln Arg Arg Asn Asn Cys His Ser Ala Ala Thr Pro Ile Val Gln
645 650 655
Leu Gln Gly Asp Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
660 665 670
Asp Lys Tyr Lys His Leu Phe Glu Leu Ala Ser Ser Thr Trp His Trp
675 680 685
Ala Ser Pro Glu Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr
690 695 700
Ser Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn Ser Val Lys Ile Pro
705 710 715 720
Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu
725 730 735
<210>11
<211>2206
<212>DNA
<213>HPV
<400>11
atggaagcca tcgcgaagag gctcgacgcc tgccaggacc agctgctcga gctgtacgag 60
gagaacagca ttgacatcca taagcacatc atgcactgga agtgcattcg cctggagagc 120
gtgctgttgc acaaggccaa gcagatgggc ctgtcccaca taggccttca ggtggtcccc 180
cctctgaccg tgtcagagac aaagggccat aacgcaatcg agatgcagat gcacctcgag 240
tcgctggcga aaacacagta cggcgtggag ccatggaccc tgcaggacac ctcgtacgaa 300
atgtggctga ccccacctaa gcgatgcttc gccaaacagg gcaacacagt ggaggtgaag 360
ttcgacggct gtgaggataa cgttatggag tatgtcgtgt ggacgcacat ctatctgcag 420
gacaacgaca gttgggtgaa ggtgaccagc tccgtggacg cgaagggcat ctactatacc 480
tgtgggcagt ttaaaaccta ctatgtgaac ttcaacaaag aggcccaaaa gtatggctcc 540
accaaccact gggaggtctg ctatgggagc acggtgattt gctctcccgc cagcgtgtct 600
agcactgtgc gcgaggtgag cattgccgag ccgaccacgt acacccctgc ccagacgacc 660
gctccgaccg tgtctgcttg tactaccgag gacggcgtga gcgctccacc caggaagcgt 720
gcgaggggcc caagcaccaa caacaccctc tgtgtggcga acattcgcag cgtcgacagt 780
accatcaata acatcgtgac ggataactat aacaagcacc agaggcgtaa caactgtcac 840
tctgccgcaa cccccatcgt gcagctccag ggagacagca attgccttaa gtgcttccgc 900
tatcgcctca acgacaagta caagcacctc tttgagctcg cctcgtcgac gtggcactgg 960
gcctcacccg aggcacctca caagaacgcc atcgtcactc tcacttactc cagtgaggag 1020
cagagacagc agtttctgaa cagcgtgaag atcccaccga cgatccgtca taaggtcggc 1080
ttcatgtcac tgcatctcct gatggaagct attgccaagc gactggacgc ctgccaggag 1140
cagctgctgg agctgtacga ggaaaacagc acagacctcc acaagcacgt gctgcactgg 1200
aagtgcatgc gccacgagtc agtgctcctg tacaaggcca agcagatggg gctgtcccac 1260
atcgggatgc aggtcgtgcc cccgctgaag gtgagcgaag ccaagggcca caacgctatc 1320
gagatgcaga tgcacctgga gagcctgctg cggaccgaat acagcatgga gccctggact 1380
ctccaggaga cgtcctacga aatgtggcag actcctccga agcgctgttt cgcaaagcgc 1440
ggcaagacag ttgaggtgaa attcgatggg tgcgcaaaca acacgatgga ctacgtggtg 1500
tggaccgatg tctacgtgca ggacaatgac acctgggtga aggtacatag tatggtggat 1560
gccaagggca tctattacac ctgcgggcag ttcaagacgt actacgtcaa cttcgtcaag 1620
gaagccgaaa agtatggttc caccaagcac tgggaggtgt gttacgggag tactgtgatc 1680
tgcagccccg cctccgtgtc gtccaccacc caggaagtga gcattccgga gagcaccaca 1740
tacaccccgg cccaaacgag cacgctcgtc agcagcagca ccaaggagga cgccgtccag 1800
acgccccccc ggaagagggc ccggggggtc cagcagtctc cctgcaatgc cctgtgcgtt 1860
gctcacatcg gccctgtcga ttctgggaac cacaatctca tcacgaacaa ccacgaccag 1920
caccaaaggc gcaacaactc taacagctcc gcaactccaa tagtgcagtt ccagggggag 1980
tccaactgcc tcaagtgttt ccgctaccgc ctcaacgacc gccaccgcca cctgttcgac 2040
ttgatcagtt ccacgtggca ctgggccagc agcaaggcgc cccacaaaca cgctatcgtg 2100
acggtgacct acgactccga ggagcagagg cagcagttcc tggacgtcgt gaagattcct 2160
ccgacaatca gccacaagct tggcttcatg tccctgcacc tgctga 2206
<210>12
<211>735
<212>PRT
<213>HPV
<400>12
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Asp Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Ile Asp Ile His Lys His Ile Met His
20 25 30
Trp Lys Cys Ile Arg Leu Glu Ser Val Leu Leu His Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Leu Gln Val Val Pro Pro Leu Thr Val
50 55 60
Ser Glu Thr Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Ala Lys Thr Gln Tyr Gly Val Glu Pro Trp Thr Leu Gln Asp
85 90 95
Thr Ser Tyr Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Gln Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val
115 120 125
Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp Ser
130 135 140
Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Asn Lys Glu Ala Gln
165 170 175
Lys Tyr Gly Ser Thr Asn His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Val Arg Glu Val Ser Ile
195 200 205
Ala Glu Pro Thr Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val
210 215 220
Ser Ala Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg
225 230 235 240
Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile Arg
245 250 255
Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn Tyr Asn Lys
260 265 270
His Gln Arg Arg Asn Asn Cys His Ser Ala Ala Thr Pro Ile Val Gln
275 280 285
Leu Gln Gly Asp Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
290 295 300
Asp Lys Tyr Lys His Leu Phe Glu Leu Ala Ser Ser Thr Trp His Trp
305 310 315 320
Ala Ser Pro Glu Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr
325 330 335
Ser Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn ger Val Lys Ile Pro
340 345 350
Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu Met
355 360 365
Glu Ala Ils Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu Glu
370 375 380
Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His Trp
385 390 395 400
Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln Met
405 410 415
Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val Ser
420 425 430
Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu Ser
435 440 445
Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu Thr
450 455 460
Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys Arg
465 470 475 480
Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr Met
485 490 495
Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr Trp
500 505 510
Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr Cys
515 520 525
Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu Lys
530 535 540
Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val Ile
545 550 555 560
Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile Pro
565 570 575
Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser Ser
580 585 590
Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala Arg
595 600 605
Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile Gly
610 615 620
Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp Gln
625 630 635 640
His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val Gln
645 650 655
Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
660 665 670
Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His Trp
675 680 685
Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr Tyr
690 695 700
Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile Pro
705 710 715 720
Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
725 730 735
<210>13
<211>1950
<212>DNA
<213>HPV
<400>13
atggcagacg attccggtac tgagaacgaa ggttctggtt gtaccggttg gttcatggtt 60
gaagcaatcg ttcagcatcc gactggtacc cagatctccg atgacgaaga cgaagaagtt 120
gaagattctg gttacgacat ggttgacttc atcgatgact ccaacatcac tcataactct 180
ctggaagcac aggctctgtt taaccgccag gaagctgata cccattacgc tactgttcag 240
gacctgggag gcaaatatct gggctctccg tacgtttccc cgatcaacac tatcgcagaa 300
gcagttgagt ctgaaatctc cccgcgcctg gacgctatca aactgactcg tcagccgaag 360
aaggttaaac gtcgtctgtt ccagactcgt gaactgaccg actccggtta cggttatagc 420
gaagttgagg ctggcaccgg cacccaggtt gaaaaacacg gtgtaccgga aaacggcggc 480
gacggtcagg aaaaggacac cggccgcgac atcgagggtg aggaacacac cgaagctgaa 540
gctccgacta actctgttcg tgaacacgca ggtactgcgg gtatcctgga actgctgaaa 600
tgcaaagacc tgcgcgcggc tctgctgggc aaattcaaag aatgcttcgg cctgtctttc 660
attgacctga tccgtccgtt taagtctgac aaaactacct gtctggactg ggttgtagca 720
ggcttcggca tccaccactc tatctctgaa gcattccaga aactgatcga gccgctgtct 780
ctgtacgcgc acatccagtg gctgactaac gcttggggta tggttctgct ggtactgctg 840
cgctttaaag taaacaaatc tcgttccact gttgctcgta ctctggctac cctgctgaac 900
atcccggaga accagatgct gatcgaaccg ccgaaaatcc agtctggtgt agctgcactg 960
tactggtttc gtactggcat ctctaacgct agcactgtta tcggtgaagc accggaatgg 1020
atcactcgtc agaccgttat cgaacacggt ctggcagatt ctcagttcaa actgactgaa 1080
atggttcagt gggcatacga caacgacatc tgcgaggaat ctgaaattgc gttcgaatac 1140
gctcagcgtg gcgacttcga ctccaacgct cgtgctttcc tgaacagcaa catgcaggct 1200
aaatacgtaa aagactgcgc taccatgtgc cgtcactaca aacacgcgga aatgcgtaaa 1260
atgtctatca aacagtggat caagcaccgc ggttctaaaa tcgaaggtac cggtaactgg 1320
aaaccgatcg ttcagttcct gcgccatcag aacatcgaat tcatcccgtt cctgaccaaa 1380
ttcaagctgt ggctgcacgg taccccgaaa aaaaactgca tcgctatcgt aggtccaccg 1440
gacactgaca agtcttactt ctgtatgtcc ctgatctctt tcctgggcgg cactgtaatc 1500
tctcacgtta actcttcctc ccatttctgg ctgcagccac tggtagacgc gaaagtagct 1560
ctgctggacg acgcgaccca gccgtgctgg atctacatgg atacttacat gcgcaacctg 1620
ctggacggta acccgatgtc tatcgaccgt aaacacaaag cgctgactct gatcaagtgc 1680
ccgccgctgc tggtaacttc taacatcgac atcaccaagg aagataaata caagtacctg 1740
catacccgtg ttactacctt tactttcccg aacccgttcc cgtttgatcg taacggtaac 1800
gctgtttacg aactgtccaa cactaactgg aaatgcttct tcgagcgtct gtcttcctcc 1860
ctggacatcc aggactctga agatgaagaa gatggttcta actctcaggc tttccgttgt 1920
gttccgggta ctgttgttcg tactctgtga 1950
<210>14
<211>649
<212>PRT
<213>HPV
<400>14
Met Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly
1 5 10 15
Trp Phe Met Val Glu Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile
20 25 30
Ser Asp Asp Glu Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val
35 40 45
Asp Phe Ile Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln
50 55 60
Ala Leu Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln
65 70 75 80
Asp Leu Gly Gly Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn
85 90 95
Thr Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala
100 105 110
Ile Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln
115 120 125
Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala
130 135 140
Gly Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu Asn Gly Gly
145 150 155 160
Asp Gly Gln Glu Lys Asp Thr Gly Arg Asp Ile Glu Gly Glu Glu His
165 170 175
Thr Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr
180 185 190
Ala Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu
195 200 205
Leu Gly Lys Phe Lys Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile
210 215 220
Arg Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu Asp Trp Val Val Ala
225 230 235 240
Gly Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile
245 250 255
Glu Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr Asn Ala Trp
260 265 270
Gly Met Val Leu Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg
275 280 285
Ser Thr Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn
290 295 300
Gln Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala Leu
305 310 315 320
Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr Val Ile Gly Glu
325 330 335
Ala Pro Glu Trp Ile Thr Arg Gln Thr Val Ile Glu His Gly Leu Ala
340 345 350
Asp Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp Asn
355 360 365
Asp Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly
370 375 380
Asp Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala
385 390 395 400
Lys Tyr Val Lys Asp Cys Ala Thr Met Cys Arg His Tyr Lys His Ala
405 410 415
Glu Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly Ser
420 425 430
Lys Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg
435 440 445
His Gln Asn Ile Glu Phe Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp
450 455 460
Leu His Gly Thr Pro Lys Lys Asn Cys Ile Ala Ile Val Gly Pro Pro
465 470 475 480
Asp Thr Asp Lys Ser Tyr Phe Cys Met Ser Leu Ile Ser Phe Leu Gly
485 490 495
Gly Thr Val Ile Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln
500 505 510
Pro Leu Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro
515 520 525
Cys Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn
530 535 540
Pro Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys
545 550 555 560
Pro Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys
565 570 575
Tyr Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn Pro
580 585 590
Phe Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu Ser Ala Thr
595 600 605
Asn Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln
610 615 620
Asp Ser Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys
625 630 635 640
Val Pro Gly Thr Val Val Arg Thr Leu
645
<210>15
<211>1107
<212>DNA
<213>HPV
<400>15
atggaagcta ttgccaagcg actggacgcc tgccaggagc agctgctgga gctgtacgag 60
gaaaacagca cagacctcca caagcacgtg ctgcactgga agtgcatgcg ccacgagtca 120
gtgctcctgt acaaggccaa gcagatgggg ctgtcccaca tcgggatgca ggtcgtgccc 180
ccgctgaagg tgagcgaagc caagggccac aacgctatcg agatgcagat gcacctggag 240
agcctgctgc ggaccgaata cagcatggag ccctggactc tccaggagac gtcctacgaa 300
atgtggcaga ctcctccgaa gcgctgtttc gcaaagcgcg gcaagacagt tgaggtgaaa 360
ttcgatgggt gcgcaaacaa cacgatggac tacgtggtgt ggaccgatgt ctacgtgcag 420
gacaatgaca cctgggtgaa ggtacatagt atggtggatg ccaagggcat ctattacacc 480
tgcgggcagt tcaagacgta ctacgtcaac ttcgtcaagg aagccgaaaa gtatggttcc 540
accaagcact gggaggtgtg ttacgggagt actgtgatct gcagccccgc ctccgtgtcg 600
tccaccaccc aggaagtgag cattccggag agcaccacat acaccccggc ccaaacgagc 660
acgctcgtca gcagcagcac caaggaggac gccgtccaga cgcccccccg gaagagggcc 720
cggggggtcc agcagtctcc ctgcaatgcc ctgtgcgttg ctcacatcgg ccctgtcgat 780
tctgggaacc acaatctcat cacgaacaac cacgaccagc accaaaggcg caacaactct 840
aacagctccg caactccaat agtgcagttc cagggggagt ccaactgcct caagtgtttc 900
cgctaccgcc tcaacgaccg ccaccgccac ctgttcgact tgatcagttc cacgtggcac 960
tgggccagca gcaaggcgcc ccacaaacac gctatcgtga cggtgaccta cgactccgag 1020
gagcagaggc agcagttcct ggacgtcgtg aagattcctc cgacaatcag ccacaagctt 1080
ggcttcatgt ccctgcacct gctgtga 1107
<210>16
<211>368
<212>PRT
<213>HPV
<400>16
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His
20 25 30
Trp Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val
50 55 60
Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu
85 90 95
Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr
115 120 125
Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr
130 135 140
Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu
165 170 175
Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ale Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile
195 200 205
Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser
210 215 220
Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala
225 230 235 240
Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile
245 250 255
Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp
260 265 270
Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val
275 280 285
Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu
290 295 300
Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His
305 310 315 320
Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr
325 330 335
Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile
340 345 350
Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
355 360 365
<210>17
<211>4154
<212>DNA
<213>HPV
<400>17
atggcagacg attccggtac tgagaacgaa ggttctggtt gtaccggttg gttcatggtt 60
gaagcaatcg ttcagcatcc gactggtacc cagatctccg atgacgaaga cgaagaagtt 120
gaagattctg gttacgacat ggttgacttc atcgatgact ccaacatcac tcataactct 180
ctggaagcac aggctctgtt taaccgccag gaagctgata cccattacgc tactgttcag 240
gacctgggag gcaaatatct gggctctccg tacgtttccc cgatcaacac tatcgcagaa 300
gcagttgagt ctgaaatctc cccgcgcctg gacgctatca aactgactcg tcagccgaag 360
aaggttaaac gtcgtctgtt ccagactcgt gaactgaccg actccggtta cggttatagc 420
gaagttgagg ctggcaccgg cacccaggtt gaaaaacacg gtgtaccgga aaacggcggc 480
gacggtcagg aaaaggacac cggccgcgac atcgagggtg aggaacacac cgaagctgaa 540
gctccgacta actctgttcg tgaacacgca ggtactgcgg gtatcctgga actgctgaaa 600
tgcaaagacc tgcgcgcggc tctgctgggc aaattcaaag aatgcttcgg cctgtctttc 660
attgacctga tccgtccgtt taagtctgac aaaactacct gtctggactg ggttgtagca 720
ggcttcggca tccaccactc tatctctgaa gcattccaga aactgatcga gccgctgtct 780
ctgtacgcgc acatccagtg gctgactaac gcttggggta tggttctgct ggtactgctg 840
cgctttaaag taaacaaatc tcgttccact gttgctcgta ctctggctac cctgctgaac 900
atcccggaga accagatgct gatcgaaccg ccgaaaatcc agtctggtgt agctgcactg 960
tactggtttc gtactggcat ctctaacgct agcactgtta tcggtgaagc accggaatgg 1020
atcactcgtc agaccgttat cgaacacggt ctggcagatt ctcagttcaa actgactgaa 1080
atggttcagt gggcatacga caacgacatc tgcgaggaat ctgaaattgc gttcgaatac 1140
gctcagcgtg gcgacttcga ctccaacgct cgtgctttcc tgaacagcaa catgcaggct 1200
aaatacgtaa aagactgcgc taccatgtgc cgtcactaca aacacgcgga aatgcgtaaa 1260
atgtctatca aacagtggat caagcaccgc ggttctaaaa tcgaaggtac cggtaactgg 1320
aaaccgatcg ttcagttcct gcgccatcag aacatcgaat tcatcccgtt cctgaccaaa 1380
ttcaagctgt ggctgcacgg taccccgaaa aaaaactgca tcgctatcgt aggtccaccg 1440
gacactgaca agtcttactt ctgtatgtcc ctgatctctt tcctgggcgg cactgtaatc 1500
tctcacgtta actcttcctc ccatttctgg ctgcagccac tggtagacgc gaaagtagct 1560
ctgctggacg acgcgaccca gccgtgctgg atctacatgg atacttacat gcgcaacctg 1620
ctggacggta acccgatgtc tatcgaccgt aaacacaaag cgctgactct gatcaagtgc 1680
ccgccgctgc tggtaacttc taacatcgac atcaccaagg aagataaata caagtacctg 1740
catacccgtg ttactacctt tactttcccg aacccgttcc cgtttgatcg taacggtaac 1800
gctgtttacg aactgtccaa cactaactgg aaatgcttct tcgagcgtct gtcttcctcc 1860
ctggacatcc aggactctga agatgaagaa gatggttcta actctcaggc tttccgttgt 1920
gttccgggta ctgttgttcg tactctgatg gaagctattg ccaagcgact ggacgcctgc 1980
caggagcagc tgctggagct gtacgaggaa aacagcacag acctccacaa gcacgtgctg 2040
cactggaagt gcatgcgcca cgagtcagtg ctcctgtaca aggccaagca gatggggctg 2100
tcccacatcg ggatgcaggt cgtgcccccg ctgaaggtga gcgaagccaa gggccacaac 2160
gctatcgaga tgcagatg cctggagagc ctgctgcgga ccgaatacag ca ggagccc 2220
tggactctcc aggagacgtc ctacgaaatg tggcagactc ctccgaagcg ctgtttcgca 2280
aagcgcggca agacagttga ggtgaaattc gatgggtgcg caaacaacac gatggactac 2340
gtggtgtgga ccgatgtcta cgtgcaggac aatgacacct gggtgaaggt acatagtatg 2400
gtggatgcca agggcatcta ttacacctgc gggcagttca agacgtacta cgtcaacttc 2460
gtcaaggaag ccgaaaagta tggttccacc aagcactggg aggtgtgtta cgggagtact 2520
gtgatctgca gccccgcctc cgtgtcgtcc accacccagg aagtgagcat tccggagaga 2580
ccacatacac cccggcccaa acgagcacgc tcgtcagcag cagcaccaag gaggacgccg 2640
tccagacgcc cccccggaag agggcccggg gggtccagca gtctccctgc aatgccctgt 2700
gcgttgctca catcggccct gtcgattctg ggaaccacaa tctcatcacg aacaaccacg 2760
accagcacca aaggcgcaac aactctaaca gctccgcaac tccaatagtg cagttccagg 2820
gggagtccaa ctgcctcaag tgtttccgct accgcctcaa cgaccgccac cgccacctgt 2880
tcgacttgat cagttccacg tggcactggg ccagcagcaa ggcgccccac aaacacgcta 2940
tcgtgacggt gacctacgac tccgaggagc agaggcagca gttcctggac gtcgtgaaga 3000
ttcctccgac aatcagccac aagcttggct tcatgtccct gcacctgctg atggaagcca 3060
tcgcgaagag gctcgacgcc tgccaggacc agctgctcga gctgtacgag gagaacagca 3120
ttgacatcca taagcacatc atgcactgga agtgcattcg cctggagagc gtgctgttgc 3180
acaaggccaa gcagatgggc ctgtcccaca taggccttca ggtggtcccc cctctgaccg 3240
tgtcagagac aaagggccat aacgcaatcg agatgcagat gcacctcgag tcgctggcga 3300
aaacacagta cggcgtggag ccatggaccc tgcaggacac ctcgtacgaa atgtggctga 3360
ccccacctaa gcgatgcttc gccaaacagg gcaacacagt ggaggtgaag ttcgacggct 3420
gtgaggataa cgttatggag tatgtcgtgt ggacgcacat ctatctgcag gacaacgaca 3480
gttgggtgaa ggtgaccagc tccgtggacg cgaagggcat ctactatacc tgtgggcagt 3540
ttaaaaccta ctatgtgaac ttcaacaaag aggcccaaaa gtatggctcc accaaccact 3600
gggaggtctg ctatgggagc acggtgattt gctctcccgc cagcgtgtct agcactgtgc 3660
gcgaggtgag cattgccgag ccgaccacgt acacccctgc ccagacgacc gctccgaccg 3720
tgtctgcttg tactaccgag gacggcgtga gcgctccacc caggaagcgt gcgaggggcc 3780
caagcaccaa caacaccctc tgtgtggcga acattcgcag cgtcgacagt accatcaata 3840
acatcgtgac ggataactat aacaagcacc agaggcgtaa caactgtcac tctgccgcaa 3900
cccccatcgt gcagctccag ggagacagca attgccttaa gtgcttccgc tatcgcctca 3960
acgacaagta caagcacctc tttgagctcg cctcgtcgac gtggcactgg gcctcacccg 4020
aggcacctca caagaacgcc atcgtcactc tcacttactc cagtgaggag cagagacagc 4080
agtttctgaa cagcgtgaag atcccaccga cgatccgtca taaggtcggc ttcatgtcac 4140
tgcatctcct gtga 4154
<210>18
<211>1384
<212>PRT
<213>HPV
<400>18
Met Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly
1 5 10 15
Trp Phe Met Val Glu Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile
20 25 30
Ser Asp Asp Glu Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val
35 40 45
Asp Phe Ile Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln
50 55 60
Ala Leu Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln
65 70 75 80
Asp Leu Gly Gly Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn
85 90 95
Thr Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala
100 105 110
Ile Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln
115 120 125
Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala
130 135 140
Gly Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu Asn Gly Gly
145 150 155 160
Asp Gly Gln Glu Lys Asp Thr Gly Arg Asp Ile Glu Gly Glu Glu His
165 170 175
Thr Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr
180 185 190
Ala Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu
195 200 205
Leu Gly Lys Phe Lys Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile
210 215 220
Arg Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu Asp Trp Val Val Ala
225 230 235 240
Gly Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile
245 250 255
Glu Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr Asn Ala Trp
260 265 270
Gly Met Val Leu Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg
275 280 285
Ser Thr Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn
290 295 300
Gln Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala Leu
305 310 315 320
Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr Val Ile Gly Glu
325 330 335
Ala Pro Glu Trp Ile Thr Arg Gln Thr Val Ple Glu His Gly Leu Ala
340 345 350
Asp Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp Asn
355 360 365
Asp Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly
370 375 380
Asp Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala
385 390 395 400
Lys Tyr Val Lys Asp Cys Ala Thr Met Cys Arg His Tyr Lys His Ala
405 410 415
Glu Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly Ser
420 425 430
Lys Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg
435 440 445
His Gln Asn Ile Glu Phe Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp
450 455 460
Leu His Gly Thr Pro Lys Lys Asn Cys Ile Ala Ile Val Gly Pro Pro
465 470 475 480
Asp Thr Asp Lys Ser Tyr Phe Cys Met Ser Leu Ile Ser Phe Leu Gly
485 490 495
Gly Thr Val Ile Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln
500 505 510
Pro Leu Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro
515 520 525
Cys Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn
530 535 540
Pro Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys
545 550 555 560
Pro Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys
565 570 575
Tyr Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn Pro
580 585 590
Phe Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr
595 600 605
Asn Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln
610 615 620
Asp Ser Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys
625 630 635 640
Val Pro Gly Thr Val Val Arg Thr Leu Met Glu Ala Ile Ala Lys Arg
645 650 655
Leu Asp Ala Cys Gln Glu Gln Leu Leu Glu Leu Tyr Glu Glu Asn Ser
660 665 670
Thr Asp Leu His Lys His Val Leu His Trp Lys Cys Met Arg His Glu
675 680 685
Ser Val Leu Leu Tyr Lys Ala Lys Gln Met Gly Leu Ser His Ile Gly
690 695 700
Met Gln Val Val Pro Pro Leu Lys Val Ser Glu Ala Lys Gly His Asn
705 710 715 720
Ala Ile Glu Met Gln Met His Leu Glu Ser Leu Leu Arg Thr Glu Tyr
725 730 735
Ser Met Glu Pro Trp Thr Leu Gln Glu Thr Set Tyr Glu Mer Trp Gln
740 745 750
Thr Pro Pro Lys Arg Cys Phe Ala Lys Arg Gly Lys Thr Val Glu Val
755 760 765
Lys Phe Asp Gly Cys Ala Asn Asn Thr Met Asp Tyr Val Val Trp Thr
770 775 780
Asp Val Tyr Val Gln Asp Asn Asp Thr Trp Val Lys Val His Ser Met
785 790 795 800
Val Asp Ala Lys Gly Ile Tyr Tyr Thr Cys Gly Gln Phe Lys Thr Tyr
805 810 815
Tyr Val Asn Phe Val Lys Glu Ala Glu Lys Tyr Gly Ser Thr Lys His
820 825 830
Trp Glu Val Cys Tyr Gly Ser Thr Val Ile Cys Ser Pro Ala Ser Val
835 840 845
Ser Ser Thr Thr Gln Glu Val Ser Ile Pro Glu Ser Thr Thr Tyr Thr
850 855 860
Pro Ala Gln Thr Ser Thr Leu Val Ser Ser Ser Thr Lys Glu Asp Ala
865 870 875 880
Val Gln Thr Pro Pro Arg Lys Arg Ala Arg Gly Val Gln Gln Ser Pro
885 890 895
Cys Asn Ala Leu Cys Val Ala His Ile Gly Pro Val Asp Ser Gly Asn
900 905 910
His Asn Leu Ile Thr Asn Asn His Asp Gln His Gln Arg Arg Asn Asn
915 920 925
Ser Asn Ser Ser Ala Thr Pro Ile Val Gln Phe Gln Gly Glu Ser Asn
930 935 940
Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn Asp Arg His Arg His Leu
945 950 955 960
Phe Asp Leu Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys Ala Pro
965 970 975
His Lys His Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu Gln Arg
980 985 990
Gln Gln Phe Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser His Lys
995 1000 1005
Leu Gly Phe Met Ser Leu His Leu Leu Met Glu Ala Ile Ala Lys Arg
1010 1015 1020
Leu Asp Ala Cys Gln Asp Gln Leu Leu Glu Leu Tyr Glu Glu Asn Ser
1025 1030 1035 1040
Ile Asp Ile His Lys His Ile Met His Trp Lys Cys Ile Arg Leu Glu
1045 1050 1055
Ser Val Leu Leu His Lys Ala Lys Gln Met Gly Leu Ser His Ile Gly
1060 1065 1070
Leu Gln Val Val Pro Pro Leu Thr Val Ser Glu Thr Lys Gly His Asn
1075 1080 1085
Ala Ile Glu Met Gln Met His Leu Glu Ser Leu Ala Lys Thr Gln Tyr
1090 1095 1100
Gly Val Glu Pro Trp Thr Leu Gln Asp Thr Ser Tyr Glu Met Trp Leu
1105 1110 1115 1120
Thr Pro Pro Lys Arg Cys Phe Ala Lys Gln Gly Asn Thr Val Glu Val
1125 1130 1135
Lys Phe Asp Gly Cys Glu Asp Asn Val Met Glu Tyr Val Val Trp Thr
1140 1145 1150
His Ile Tyr Leu Gln Asp Asn Asp Ser Trp Val Lys Val Thr Ser Ser
1155 1160 1165
Val Asp Ala Lys Gly Ile Tyr Tyr Thr Cys Gly Gln Phs Lys Thr Tyr
1170 1175 1180
Tyr Val Asn Phe Asn Lys Glu Ala Gln Lys Tyr Gly Ser Thr Asn His
1185 1190 1195 1200
Trp Glu Val Cys Tyr Gly Ser Thr Val Ile Cys Ser Pro Ala Ser Val
1205 1210 1215
Ser Ser Thr Val Arg Glu Val Ser Ile Ala Glu Pro Thr Thr Tyr Thr
l220 1225 1230
Pro Ala Gln Thr Thr Ala Pro Thr Val Ser Ala Cys Thr Thr Glu Asp
1235 1240 1245
Gly Val Ser Ala Pro Pro Arg Lys Arg Ala Arg Gly Pro Ser Thr Asn
1250 1255 1260
Asn Thr Leu Cys Val Ala Asn Ile Arg Ser Val Asp Ser Thr Ile Asn
1265 1270 1275 1280
Asn Ile Val Thr Asp Asn Tyr Asn Lys His Gln Arg Arg Asn Asn Cys
1285 1290 1295
His Ser Ala Ala Thr Pro Ile Val Gln Leu Gln Gly Asp Ser Asn Cys
1300 1305 1310
Leu Lys Cys Phe Arg Tyr Arg Leu Asn Asp Lys Tyr Lys His Leu Phe
1315 1320 1325
Glu Leu Ala Ser Ser Thr Trp His Trp Ala Ser Pro Glu Ala Pro His
1330 1335 1340
Lys Asn Ala Ile Val Thr Leu Thr Tyr Ser Ser Glu Glu Gln Arg Gln
1345 1350 1355 1360
Gln Phe Leu Asn Ser Val Lys Ile Pro Pro Thr Ile Arg His Lys Val
1365 1370 1375
Gly Phe Met Ser Leu His Leu Leu
1380
<210>19
<211>4155
<212>DNA
<213>HPV
<400>19
atggaagcta ttgccaagcg actggacgcc tgccaggagc agctgctgga gctgtacgag 60
gaaaacagca cagacctcca caagcacgtg ctgcactgga agtgcatgcg ccacgagtca 120
gtgctcctgt acaaggccaa gcagatgggg ctgtcccaca tcgggatgca ggtcgtgccc 180
ccgctgaagg tgagcgaagc caagggccac aacgctatcg agatgcagat gcacctggag 240
agcctgctgc ggaccgaata cagcatggag ccctggactc tccaggagac gtcctacgaa 300
atgtggcaga ctcctccgaa gcgctgtttc gcaaagcgcg gcaagacagt tgaggtgaaa 360
ttcgatgggt gcgcaaacaa cacgatggac tacgtggtgt ggaccgatgt ctacgtgcag 420
gacaatgaca cctgggtgaa ggtacatagt atggtggatg ccaagggcat ctattacacc 480
tgcgggcagt tcaagacgta ctacgtcaac ttcgtcaagg aagccgaaaa gtatggttcc 540
accaagcact gggaggtgtg ttacgggagt actgtgatct gcagccccgcctccgtgtcg 600
tccaccaccc aggaagtgag cattccggag agcaccacat acaccccggc ccaaacgagc 660
acgctcgtca gcagcagcac caaggaggac gccgtccaga cgcccccccg gaagagggcc 720
cggggggtcc agcagtctcc ctgcaatgcc ctgtgcgttg ctcacatcgg ccctgtcgat 780
tctgggaacc acaatctcat cacgaacaac cacgaccagc accaaaggcg caacaactct 840
aacagctccg caactccaat agtgcagttc cagggggagt ccaactgcct caagtgtttc 900
cgctaccgcc tcaacgaccg ccaccgccac ctgttcgact tgatcagttc cacgtggcac 960
tgggccagca gcaaggcgcc ccacaaacac gctatcgtga cggtgaccta cgactccgag 1020
gagcagaggc agcagttcct ggacgtcgtg aagattcctc cgacaatcag ccacaagctt 1080
ggcttcatgt ccctgcacct gctgatggca gacgattccg gtactgagaa cgaaggttct 1140
ggttgtaccg gttggttcat ggttgaagca atcgttcagc atccgactgg tacccagatc 1200
tccgatgacg aagacgaaga agttgaagat tctggttacg acatggttga cttcatcgat 1260
gactccaaca tcactcataa ctctctggaa gcacaggctc tgtttaaccg ccaggaagct 1320
gatacccatt acgctactgt tcaggacctg ggaggcaaat atctgggctc tccgtacgtt 1380
tccccgatca acactatcgc agaagcagtt gagtctgaaa tctccccgcg cctggacgct 1440
atcaaactga ctcgtcagcc gaagaaggtt aaacgtcgtc tgttccagac tcgtgaactg 1500
accgactccg gttacggtta tagcgaagtt gaggctggca ccggcaccca ggttgaaaaa 1560
cacggtgtac cggaaaacgg cggcgacggt caggaaaagg acaccggccg cgacatcgag 1620
ggtgaggaac acaccgaagc tgaagctccg actaactctg ttcgtgaaca cgcaggtact 1680
gcgggtatcc tggaactgct gaaatgcaaa gacctgcgcg cggctctgct gggcaaattc 1740
aaagaatgct tcggcctgtc tttcattgac ctgatccgtc cgtttaagtc tgacaaaact 1800
acctgtctgg actgggttgt agcaggcttc ggcatccacc actctatctc tgaagcattc 1860
cagaaactga tcgagccgct gtctctgtac gcgcacatcc agtggctgac taacgcttgg 1920
ggtatggttc tgctggtact gctgcgcttt aaagtaaaca aatctcgttc cactgttgct 1980
cgtactctgg ctaccctgct gaacatcccg gagaaccaga tgctgatcga accgccgaaa 2040
atccagtctg gtgtagctgc actgtactgg tttcgtactg gcatctctaa cgctagcact 2100
gttatcggtg aagcaccgga atggatcact cgtcagaccg ttatcgaaca cggtctggca 2160
gattctcagt tcaaactgac tgaaatggtt cagtgggcat acgacaacga catctgcgag 2220
gaatctgaaa ttgcgttcga atacgctcag cgtggcgact tcgactccaa cgctcgtgct 2280
ttcctgaaca gcaacatgca ggctaaatac gtaaaagact gcgctaccat gtgccgtcac 2340
tacaaacacg cggaaatgcg taaaatgtct atcaaacagt ggatcaagca ccgcggttct 2400
aaaatcgaag gtaccggtaa ctggaaaccg atcgttcagt tcctgcgcca tcagaacatc 2460
gaattcatcc cgttcctg caaattcaag ctgtggctgc acggtacccc ga aaaaac 2520
tgcatcgcta tcgtaggtcc accggacact gacaagtctt acttctgtat gtccctgatc 2580
tctttcctgg gcggcactgt aatctctcac gttaactctt cctcccattt ctggctgcag 2640
ccactggtag acgcgaaagt agctctgctg gacgacgcga cccagccgtg ctggatctac 2700
atggatactt acatgcgcaa cctgctggac ggtaacccga tgtctatcga ccgtaaacac 2760
aaagcgctga ctctgatcaa gtgcccgccg ctgctggtaa cttctaacat cgacatcacc 2820
aaggaagata aatacaagta cctgcatacc cgtgttacta cctttacttt cccgaacccg 2880
ttcccgtttg atcgtaacgg taacgctgtt tacgaactgt ccaacactaa ctggaaatgc 2940
ttcttcgagc gtctgtcttc ctccctggac atccaggact ctgaagatga agaagatggt 3000
tctaactctc aggctttccg ttgtgttccg ggtactgttg ttcgtactct gatggaagcc 3060
atcgcgaaga ggctcgacgc ctgccaggac cagctgctcg agctgtacga ggagaacagc 3120
attgacatcc ataagcacat catgcactgg aagtgcattc gcctggagag cgtgctgttg 3180
cacaaggcca agcagatggg cctgtcccac ataggccttc aggtggtccc ccctctgacc 3240
gtgtcagaga caaagggcca taacgcaatc gagatgcaga tgcacctcga gtcgctggcg 3300
aaaacacagt acggcgtgga gccatggacc ctgcaggaca cctcgtacga aatgtggctg 3360
accccaccta agcgatgctt cgccaaacag ggcaacacag tggaggtgaa gttcgacggc 3420
tgtgaggata acgttatgga gtatgtcgtg tggacgcaca tctatctgca ggacaacgac 3480
agttgggtga aggtgaccag ctccgtggac gcgaagggca tctactatac ctgtgggcag 3540
tttaaaacct actatgtgaa cttcaacaaa gaggcccaaa agtatggctc caccaaccac 3600
tgggaggtct gctatgggag cacggtgatt tgctctcccg ccagcgtgtc tagcactgtg 3660
cgcgaggtga gcattgccga gccgaccacg tacacccctg cccagacgac cgctccgacc 3720
gtgtctgctt gtactaccga ggacggcgtg agcgctccac ccaggaagcg tgcgaggggc 3780
ccaagcacca acaacaccct ctgtgtggcg aacattcgca gcgtcgacag taccatcaat 3840
aacatcgtga cggataacta taacaagcac cagaggcgta acaactgtca ctctgccgca 3900
acccccatcg tgcagctcca gggagacagc aattgcctta agtgcttccg ctatcgcctc 3960
aacgacaagt acaagcacct ctttgagctc gcctcgtcga cgtggcactg ggcctcaccc 4020
gaggcacctc acaagaacgc catcgtcact ctcacttact ccagtgagga gcagagacag 4080
cagtttctga acagcgtgaa gatcccaccg acgatccgtc ataaggtcgg cttcatgtca 4140
ctgcatctcc tgtga 4155
<210>20
<211>1384
<212>PRT
<213>HPV
<400>20
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His
20 25 30
Trp Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val
50 55 60
Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu
85 90 95
Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr
115 120 125
Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr
130 135 140
Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu
165 170 175
Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile
195 200 205
Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser
210 215 220
Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala
225 230 235 240
Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile
245 250 255
Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp
260 265 270
Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val
275 280 285
Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu
290 295 300
Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His
305 310 315 320
Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr
325 330 335
Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile
340 345 350
Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
355 360 365
Met Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly
370 375 380
Trp Phe Met Val Glu Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile
385 390 395 400
Ser Asp Asp Glu Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val
405 410 415
Asp Phe Ile Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln
420 425 430
Ala Leu Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln
435 440 445
Asp Leu Gly Gly Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn
450 455 460
Thr Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala
465 470 475 480
Ile Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln
485 490 495
Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala
500 505 510
Gly Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu Asn Gly Gly
515 520 525
Asp Gly Gln Glu Lys Asp Thr Gly Arg Asp Ile Glu Gly Glu Glu His
530 535 540
Thr Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr
545 550 555 560
Ala Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu
565 570 575
Leu Gly Lys Phe Lye Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile
580 585 590
Arg Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu Asp Trp Val Val Ala
595 600 605
Gly Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile
610 615 620
Glu Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr Asn Ala Trp
625 630 635 640
Gly Met Val Leu Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg
645 650 655
Ser Thr Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn
660 665 670
Gln Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala Leu
675 680 685
Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr Val Ile Gly Glu
690 695 700
Ala Pro Glu Trp Ile Thr Arg Gln Thr Val Ile Glu His Gly Leu Ala
705 710 715 720
Asp Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp Asn
725 730 735
Asp Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly
740 745 750
Asp Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala
755 760 765
Lys Tyr Val Lys Asp Lys Ala Thr Met Cys Arg His Tyr Lys His Ala
770 775 780
Glu Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly Ser
785 790 795 800
Lys Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg
805 810 815
His Gln Asn Ile Glu Phe Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp
820 825 830
Leu His Gly Thr Pro Lys Lys Asn Cys Ile Ala Ile Val Gly Pro Pro
835 840 845
Asp Thr Asp Lys Ser Tyr Phe Cys Met Ser Leu Ile Ser Phe Leu Gly
850 855 860
Gly Thr Val Ile Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln
865 870 875 880
Pro Leu Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro
885 890 895
Cys Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn
900 905 910
Pro Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys
915 920 925
Pro Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys
930 935 940
Tyr Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn Pro
945 950 955 960
Phe Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr
965 970 975
Asn Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln
980 985 990
Asp Ser Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys
995 1000 1005
Val Pro Gly Thr Val Val Arg Thr Leu Met Glu Ala Ile Ala Lys Arg
1010 1015 1020
Leu Asp Ala Cys Gln Asp Gln Leu Leu Glu Leu Tyr Glu Glu Asn Ser
1025 1030 1035 1040
Ile Asp Ile His Lys His Ile Met His Trp Lys Cys Ile Arg Leu Glu
1045 1050 1055
Ser Val Leu Leu His Lys Ala Lys Gln Met Gly Leu Ser His Ile Gly
1060 1065 1070
Leu Gln Val Va1 Pro Pro Leu Thr Val Ser Glu Thr Lys Gly His Asn
1075 1080 1085
Ala Ile Glu Met Gln Met His Leu Glu Ser Leu Ala Lys Thr Gln Tyr
1090 1095 1100
Gly Val Glu Pro Trp Thr Leu Gln Asp Thr Ser Tyr Glu Met Trp Leu
1105 1110 1115 1120
Thr Pro Pro Lys Arg Cys Phe Ala Lys Gln Gly Asn Thr Val Glu Val
1125 1130 1135
Lys Phe Asp Gly Cys Glu Asp Asn Val Met Glu Tyr Val Val Trp Thr
1140 1145 1150
His Ile Tyr Leu Gln Asp Asn Asp Ser Trp Val Lys Val Thr Ser Ser
1155 1160 1165
Val Asp Ala Lys Gly Ile Tyr Tyr Thr Cys Gly Gln Phe Lys Thr Tyr
1170 1175 1180
Tyr Val Asn Phe Asn Lys Glu Ala Gln Lys Tyr Gly Ser Thr Asn His
1185 1190 1195 1200
Trp Glu Val Cys Tyr Gly Ser Thr Val Ile Cys Ser Pro Ala Ser Val
1205 1210 1215
Ser Ser Thr Val Arg Glu Val Ser Ile Ala Glu Pro Thr Thr Tyr Thr
1220 1225 1230
Pro Ala Gln Thr Thr Ala Pro Thr Val Ser Ala Cys Thr Thr Glu Asp
1235 1240 1245
Gly Val Ser Ala Pro Pro Arg Lys Arg Ala Arg Gly Pro Ser Thr Asn
1250 1255 1260
Asn Thr Leu Cys Val Ala Asn Ile Arg Ser Val Asp Ser Thr Ile Asn
1265 1270 1275 1280
Asn Ile Val Thr Asp Asn Tyr Asn Lys His Gln Arg Arg Asn Asn Cys
1285 1290 1295
His Ser Ala Ala Thr Pro Ile Val Gln Leu Gln Gly Asp Ser Asn Cys
1300 1305 1310
Leu Lys Cys Phe Arg Tyr Arg Leu Asn Asp Lys Tyr Lys His Leu Phe
1315 1320 1325
Glu Leu Ala Ser Ser Thr Trp His Trp Ala Ser Pro Glu Ala Pro His
1330 1335 1340
Lys Asn Ala Ile Val Thr Leu Thr Tyr Ser Ser Glu Glu Gln Arg Gln
1345 1350 1355 1360
Gln Phe Leu Asn Ser Val Lys Ile Pro Pro Thr Ile Arg His Lys Val
1365 1370 1375
Gly Phe Met Ser Leu His Leu Leu
1380
<210>21
<211>4155
<212>DNA
<213>HPV
<400>21
atggaagcta ttgccaagcg actggacgcc tgccaggagc agctgctgga gctgtacgag 60
gaaaacagca cagacctcca caagcacgtg ctgcactgga agtgcatgcg ccacgagtca 120
gtgctcctgt acaaggccaa gcagatgggg ctgtcccaca tcgggatgca ggtcgtgccc 180
ccgctgaagg tgagcgaagc caagggccac aacgctatcg agatgcagat gcacctggag 240
agcctgctgc ggaccgaata cagcatggag ccctggactc tccaggagac gtcctacgaa 300
atgtggcaga ctcctccgaa gcgctgtttc gcaaagcgcg gcaagacagt tgaggtgaaa 360
ttcgatgggt gcgcaaacaa cacgatggac tacgtggtgt ggaccgatgt ctacgtgcag 420
gacaatgaca cctgggtgaa ggtacatagt atggtggatg ccaagggcat ctattacacc 480
tgcgggcagt tcaagacgta ctacgtcaac ttcgtcaagg aagccgaaaa gtatggttcc 540
accaagcact gggaggtgtg ttacgggagt actgtgatct gcagccccgc ctccgtgtcg 600
tccaccaccc aggaagtgag cattccggag agcaccacat acaccccggc ccaaacgagc 660
acgctcgtca gcagcagcac caaggaggac gccgtccaga cgcccccccg gaagagggcc 720
cggggggtcc agcagtctcc ctgcaatgcc ctgtgcgttg ctcacatcgg ccctgtcgat 780
tctgggaacc acaatctcat cacgaacaac cacgaccagc accaaaggcg caacaactct 840
aacagctccg caactccaat agtgcagttc cagggggagt ccaactgcct caagtgtttc 900
cgctaccgcc tcaacgaccg ccaccgccac ctgttcgact tgatcagttc cacgtggcac 960
tgggccagca gcaaggcgcc ccacaaacac gctatcgtga cggtgaccta cgactccgag 1020
gagcagaggc agcagttcct ggacgtcgtg aagattcctc cgacaatcag ccacaagctt 1080
ggcttcatgt ccctgcacct gctgatggaa gccatcgcga agaggctcga cgcctgccag 1140
gaccagctgc tcgagctgta cgaggagaac agcattgaca tccataagca catcatgcac 1200
tggaagtgca ttcgcctgga gagcgtgctg ttgcacaagg ccaagcagat gggcctgtcc 1260
cacataggcc ttcaggtggt cccccctctg accgtgtcag agacaaaggg ccataacgca 1320
atcgagatgc agatgcacct cgagtcgctg gcgaaaacac agtacggcgt ggagccatgg 1380
accctgcagg acacctcgta cgaaatgtgg ctgaccccac ctaagcgatg cttcgccaaa 1440
cagggcaaca cagtggaggt gaagttcgac ggctgtgagg ataacgttat ggagtatgtc 1500
gtgtggacgc acatctatct gcaggacaac gacagttggg tgaaggtgac cagctccgtg 1560
gacgcgaagg gcatctacta tacctgtggg cagtttaaaa cctactatgt gaacttcaac 1620
aaagaggccc aaaagtatgg ctccaccaac cactgggagg tctgctatgg gagcacggtg 1680
atttgctctc ccgccagcgt gtctagcact gtgcgcgagg tgagcattgc cgagccgacc 1740
acgtacaccc ctgcccagac gaccgctccg accgtgtctg cttgtactac cgaggacggc 1800
gtgagcgctc cacccaggaa gcgtgcgagg ggcccaagca ccaacaacac cctctgtgtg 1860
gcgaacattc gcagcgtcga cagtaccatc aataacatcg tgacggataa ctataacaag 1920
caccagaggc gtaacaactg tcactctgcc gcaaccccca tcgtgcagct ccagggagac 1980
agcaattgcc ttaagtgctt ccgctatcgc ctcaacgaca agtacaagca cctctttgag 2040
ctcgcctcgt cgacgtggca ctgggcctca cccgaggcac ctcacaagaa cgccatcgtc 2100
actctcactt actccagtga ggagcagaga cagcagtttc tgaacagcgt gaagatccca 2160
ccgacgatcc gtcataaggt cggcttcatg tcactgcatc tcctgatggc agacgattcc 2220
ggtactgaga acgaaggttc tggttgtacc ggttggttca tggttgaagc aatcgttcag 2280
catccgactg gtacccagat ctccgatgac gaagacgaag aagttgaaga ttctggttac 2340
gacatggttg acttcatcga tgactccaac atcactcata actctctgga agcacaggct 2400
ctgtttaacc gccaggaagc tgatacccat tacgctactg ttcaggacct gggaggcaaa 2460
tatctgggct ctccgtacgt ttccccgatc aacactatcg cagaagcagt tgagtctgaa 2520
atctccccgc gcctggacgc tatcaaactg actcgtcagc cgaagaaggt taaacgtcgt 2580
ctgttccaga ctcgtgaact gaccgactcc ggttacggtt atagcgaagt tgaggctggc 2640
accggcaccc aggttgaaaa acacggtgta ccggaaaacg gcggcgacgg tcaggaaaag 2700
gacaccggcc gcgacatcga gggtgaggaa cacaccgaag ctgaagctcc gactaactct 2760
gttcgtgaac acgcaggta tgcgggtatc ctggaactgc tgaaatgGaa aga tgcgc 2820
gcggctctgc tgggcaaatt caaagaatgc ttcggcctgt ctttcattga cctgatccgt 2880
ccgtttaagt ctgacaaaac tacctgtctg gactgggttg tagcaggctt cggcatccac 2940
cactctatct ctgaagcatt ccagaaactg atcgagccgc tgtctctgta cgcgcacatc 3000
cagtggctga ctaacgcttg gggtatggtt ctgctggtac tgctgcgctt taaagtaaac 3060
aaatctcgtt ccactgttgc tcgtactctg gctaccctgc tgaacatccc ggagaaccag 3120
atgctgatcg aaccgccgaa aatccagtct ggtgtagctg cactgtactg gtttcgtact 3180
ggcatctcta acgctagcac tgttatcggt gaagcaccgg aatggatcac tcgtcagacc 3240
gttatcgaac acggtctggc agattctcag ttcaaactga ctgaaatggt tcagtgggca 3300
tacgacaacg acatctgcga ggaatctgaa attgcgttcg aatacgctca gcgtggcgac 3360
ttcgactcca acgctcgtgc tttcctgaac agcaacatgc aggctaaata cgtaaaagac 3420
tgcgctacca tgtgccgtca ctacaaacac gcggaaatgc gtaaaatgtc tatcaaacag 3480
tggatcaagc accgcggttc taaaatcgaa ggtaccggta actggaaacc gatcgttcag 3540
ttcctgcgcc atcagaacat cgaattcatc ccgttcctga ccaaattcaa gctgtggctg 3600
cacggtaccc cgaaaaaaaa ctgcatcgct atcgtaggtc caccggacac tgacaagtct 3660
tacttctgta tgtccctgat ctctttcctg ggcggcactg taatctctca cgttaactct 3720
tcctcccatt tctggctgca gccactggta gacgcgaaag tagctctgct ggacgacgcg 3780
acccagccgt gctggatcta catggatact tacatgcgca acctgctgga cggtaacccg 3840
atgtctatcg accgtaaaca caaagcgctg actctgatca agtgcccgcc gctgctggta 3900
acttctaaca tcgacatcac caaggaagat aaatacaagt acctgcatac ccgtgttact 3960
acctttactt tcccgaaccc gttcccgttt gatcgtaacg gtaacgctgt ttacgaactg 4020
tccaacacta actggaaatg cttcttcgag cgtctgtctt cctccctgga catccaggac 4080
tctgaagatg aagaagatgg ttctaactct caggctttcc gttgtgttcc gggtactgtt 4140
gttcgtactc tgtga 4155
<210>22
<211>1384
<212>PRT
<213>HPV
<400>22
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu
1 5 10 15
Glu Leu Tyr Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His
20 25 30
Trp Lys Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln
35 40 45
Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val
50 55 60
Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
65 70 75 80
Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu
85 90 95
Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Ala Lys
100 105 110
Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr
115 120 125
Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr
130 135 140
Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr
145 150 155 160
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu
165 170 175
Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val
180 185 190
Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile
195 200 205
Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser
210 215 220
Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala
225 230 235 240
Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile
245 250 255
Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp
260 265 270
Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val
275 280 285
Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu
290 295 300
Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His
305 310 315 320
Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr
325 330 335
Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile
340 345 350
Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu
355 360 365
Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Gln Asp Gln Leu Leu
370 375 380
Glu Leu Tyr Glu Glu Asn Ser Ile Asp Ile His Lys His Ile Met His
385 390 395 400
Trp Lys Cys Ile Arg Leu Glu Ser Val Leu Leu His Lys Als Lys Gln
405 410 415
Met Gly Leu Ser His Ile Gly Leu Gln Val Val Pro Pro Leu Thr Val
420 425 430
Ser Glu Thr Lya Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu
435 440 445
Ser Leu Ala Lys Thr Gln Tyr Gly Val Glu Pro Trp Thr Leu Gln Asp
450 455 460
Thr Ser Tyr Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Ala Lys
465 470 475 480
Gln Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val
485 490 495
Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp Ser
500 505 510
Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile Tyr Tyr Thr
515 520 525
Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Asn Lys Glu Ala Gln
530 535 540
Lys Tyr Gly Ser Thr Asn His Trp Glu Val Cys Tyr Gly Ser Thr Val
545 550 555 560
Ile Cys Ser Pro Ala Sar Val Ser Ser Thr Val Arg Glu Val Ser Ile
565 570 575
Ala Glu Pro Thr Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val
580 585 590
Ser Ala Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg
595 600 605
Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile Arg
610 615 620
Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn Tyr Asn Lys
625 630 635 640
His Gln Arg Arg Asn Asn Cys His Ser Ala Ala Thr Pro Ile Val Gln
645 650 655
Leu Gln Gly Asp Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu Asn
660 665 670
Asp Lys Tyr Lys His Leu Phe Glu Leu Ala Ser Ser Thr Trp His Trp
675 680 685
Ala Ser Pro Glu Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr
690 695 700
Ser Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn Ser Val Lys Ile Pro
705 710 715 720
Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu Met
725 730 735
Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly Trp
740 745 750
Phe Met Val Glu Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile Ser
755 760 765
Asp Asp Glu Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val Asp
770 775 780
Phe Ile Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln Ala
785 790 795 800
Leu Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln Asp
805 810 815
Leu Gly Gly Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn Thr
820 825 830
Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala Ile
835 840 845
Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln Thr
850 855 860
Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu Ala Gly
865 870 875 880
Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu Asn Gly Gly Asp
885 890 895
Gly Gln Glu Lys Asp Thr Gly Arg Asp Ile Glu Gly Glu Glu His Thr
900 905 910
Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr Ala
915 920 925
Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu Leu
930 935 940
Gly Lys Phe Lys Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile Arg
945 950 955 960
Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu Asp Trp Val Val Ala Gly
965 970 975
Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu Ile Glu
980 985 990
Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr Asn Ala Trp Gly
995 1000 1005
Met Val Leu Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg Ser
1010 1015 1020
Thr Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn Gln
1025 1030 1035 1040
Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala Leu Tyr
1045 1050 1055
Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr Val Ile Gly Glu Ala
1060 1065 1070
Pro Glu Trp Ile Thr Arg Gln Thr Val Ile Glu His Gly Leu Ala Asp
1075 1080 1085
Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp Asn Asp
1090 1095 1100
Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly Asp
1105 1110 1115 1120
Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala Lys
1125 1130 1135
Tyr Val Lys Asp Cys Ala Thr Met Cys Arg His Tyr Lys His Ala Glu
1140 1145 1150
Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly Ser Lys
1155 1160 1165
Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg His
1170 1175 1180
Gln Asn Ile Glu Phe Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp Leu
1165 1190 1195 1200
His Gly Thr Pro Lys Lys Asn Cys Ile Ala Ile Val Gly Pro Pro Asp
1205 1210 1215
Thr Asp Lys Ser Tyr Phe Cys Met Ser Leu Ile Ser Phe Leu Gly Gly
1220 1225 1230
Thr Val Ile Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln Pro
1235 1240 1245
Leu Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro Cys
1250 1255 1260
Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn Pro
1265 1270 1275 1280
Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys Pro
1285 1290 1295
Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Asp Lys Tyr
1300 1305 1310
Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn Pro Phe
1315 1320 1325
Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr Asn
1330 1335 1340
Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln Asp
1345 1350 1355 1360
Ser Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys Val
1365 1370 1375
Pro Gly Thr Val Val Arg Thr Leu
1380
<210>23
<211>23
<212>PRT
<213>HPV
<400>23
Cys Ser Ser Ser Leu Asp Ile Gln Asp Ser Glu Asp Glu Glu Asp Gly
1 5 10 15
Ser Asn Ser Gln Ala Phe Arg
20
<210>24
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激性寡核苷酸
<400>24
tccatgacgt tcctgacgtt 20
<210>25
<211>18
<212>DNA
<213>人工序列
<220>
<223>免疫刺激性寡核苷酸
<400>25
tctcccagcg tgcgccat 18
<210>26
<211>30
<212>DNA
<213>人工序列
<220>
<223>免疫刺激性寡核苷酸
<400>26
accgatgacg tcgccggtga cggcaccacg 30
<210>27
<211>24
<212>DNA
<213>人工序列
<220>
<223>免疫刺激性寡核苷酸
<400>27
tcgtcgtttt gtcgttttgt cgtt 24
<210>28
<211>20
<212>DNA
<213>人工序列
<220>
<223>免疫刺激性寡核苷酸
<400>28
tccatgacgt tcctgatgct 20
Claims (26)
1.一种多核苷酸序列，其编码人乳头状瘤病毒(HPV)多肽，所述多肽具有来自两种不同HPV株的至少两种早期抗原或其片段的表位。
2.权利要求1所述的多核苷酸，其中至少一种抗原源自HPV E1或其片段。
3.权利要求2所述的多核苷酸，其中至少一种抗原源自HPV E2或其片段。
4.根据权利要求1至3任一项所述的多核苷酸序列，其为DNA序列。
5.根据权利要求1至4任一项所述的多核苷酸序列，其编码与宫颈癌、良性皮疣或生殖器疣相关的HPV型或亚型的HPV多肽。
6.根据权利要求1至5任一项所述的多核苷酸序列，其编码1-4、6、7、10、11、16、18、26-29、31、33、35、39、49、51、52、56、58、59和68型之一的HPV多肽。
7.根据6所述的多核苷酸序列，其编码与宫颈癌或生殖器疣相关的HPV型或亚型的HPV多肽。
8.根据权利要求4或5所述的多核苷酸序列，其编码6、11、16、18、33或45型之一的HPV多肽。
9.根据权利要求5所述的多核苷酸序列，其编码选自HPV11、6a或6b的HPV型或亚型的HPV多肽。
10.根据任一前述权利要求的多核苷酸序列，其中编码一种突变的HPV多肽具有减少的生物学功能。
11.根据权利要求1至10任一项所述的多核苷酸序列，其编码一种突变的HPV多肽，所述多肽包含一个或多个点突变，通过所述突变使多肽的一种或多种天然生物学功能失活。
12.根据任一前述权利要求的多核苷酸序列，其具有大于0.4但小于1的人基因密码子使用系数。
13.根据权利要求12所述的多核苷酸序列，其具有大于0.5但小于1的人基因密码子使用系数。
14.一种表达载体，其包含根据任一前述权利要求的多核苷酸序列，所述序列与调控序列可操作连接，所述调控序列能使宿主细胞表达所述多核苷酸序列。
15.根据权利要求14所述的表达载体，其是p7313PLc。
16.一种包含根据权利要求1至13任一项所述多核苷酸序列的药物组合物。
17.一种包含根据权利要求14至15任一项所述载体的药物组合物。
18.根据权利要求16或17所述的药物组合物，其包含众多包被有DNA的金颗粒。
19.根据权利要求16、17或18任一项所述的药物组合物，进一步包含佐剂。
20.根据权利要求19所述的药物组合物，其中所述佐剂与多核苷酸编码的HPV多肽一起编码为融合体。
21.根据权利要求1-13任一项所述的多核苷酸在治疗或预防HPV感染中的应用。
22.根据权利要求14-15任一项所述的载体在治疗或预防HPV感染中的应用。
23.根据权利要求18-20任一项所述的组合物在治疗或预防HPV感染中的应用。
24.根据权利要求1-13任一项所述的多核苷酸、根据权利要求14-15任一项所述的载体或根据权利要求16-20任一项所述的药物组合物在治疗或预防皮(皮肤)疣、生殖器疣、未确定意义的非典型鳞状细胞(ASCUS)、子宫颈发育异常、子宫颈上皮内瘤(CIN)或宫颈癌中的应用。
25.一种治疗或与预防HPV感染或与其相关的症状或疾病的方法，包括施用有效剂量的根据权利要求1-13任一项所述的多核苷酸、根据权利要求14-15任一项所述的载体或根据权利要求16-20任一项所述的药物组合物。
26.一种治疗或与预防HPV感染或与其相关的症状或疾病的方法，包括在带有重组病毒载体的基础-加强接种方案中施用根据权利要求16-20任一项所述的药物组合物、或施用包含根据权利要求1-13任一项所述多核苷酸的非病毒的系统。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0222953.2A GB0222953D0 (en) | 2002-10-03 | 2002-10-03 | Novel Compounds |
| GB0222953.2 | 2002-10-03 |
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| Publication Number | Publication Date |
|---|---|
| CN1720261A true CN1720261A (zh) | 2006-01-11 |
| CN100393878C CN100393878C (zh) | 2008-06-11 |
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| CNB2003801049852A Expired - Fee Related CN100393878C (zh) | 2002-10-03 | 2003-10-01 | 疫苗 |
Country Status (21)
| Country | Link |
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| US (2) | US20060165713A1 (zh) |
| EP (1) | EP1546191A2 (zh) |
| JP (1) | JP2006516386A (zh) |
| KR (1) | KR20050050115A (zh) |
| CN (1) | CN100393878C (zh) |
| AR (1) | AR041515A1 (zh) |
| AU (1) | AU2003294672A1 (zh) |
| BR (1) | BR0314986A (zh) |
| CA (1) | CA2500093A1 (zh) |
| CO (1) | CO5580837A2 (zh) |
| GB (1) | GB0222953D0 (zh) |
| IS (1) | IS7775A (zh) |
| MA (1) | MA27474A1 (zh) |
| MX (1) | MXPA05003558A (zh) |
| NO (1) | NO20051561L (zh) |
| NZ (1) | NZ539154A (zh) |
| PL (1) | PL376534A1 (zh) |
| RU (1) | RU2354701C2 (zh) |
| TW (1) | TW200411055A (zh) |
| WO (1) | WO2004031222A2 (zh) |
| ZA (1) | ZA200503201B (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2503693A1 (en) | 2002-10-29 | 2004-05-13 | Coley Pharmaceutical Group, Ltd. | Use of cpg oligonucleotides in the treatment of hepatitis c virus infection |
| RU2482189C2 (ru) | 2007-01-30 | 2013-05-20 | Трансген С.А. | Полипептиды е2 папилломавируса, применяемые для вакцинации |
| GB0710538D0 (en) * | 2007-06-01 | 2007-07-11 | Glaxo Group Ltd | Vaccine |
| JP2013532971A (ja) | 2010-06-25 | 2013-08-22 | バッシボディ アクスイェ セルスカプ | ホモ二量体タンパク質コンストラクト |
| AU2015335652B2 (en) * | 2014-10-24 | 2020-07-02 | Hpvvax, Llc. | Cancer and skin lesion treatment |
| CN108495649A (zh) | 2016-01-08 | 2018-09-04 | 瓦西博迪公司 | 治疗性抗癌新表位疫苗 |
| US20190134190A1 (en) * | 2016-05-04 | 2019-05-09 | Transgene Sa | Combination therapy with cpg tlr9 ligand |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AUPN443995A0 (en) * | 1995-07-27 | 1995-08-17 | Csl Limited | Papillomavirus polyprotein |
| AU7569501A (en) * | 2000-07-21 | 2002-02-05 | Glaxo Group Ltd | Codon-optimized papilloma virus sequences |
| GB0017990D0 (en) * | 2000-07-21 | 2000-09-13 | Glaxo Group Ltd | Papilloma virus sequences |
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2002
- 2002-10-03 GB GBGB0222953.2A patent/GB0222953D0/en not_active Ceased
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2003
- 2003-10-01 AU AU2003294672A patent/AU2003294672A1/en not_active Abandoned
- 2003-10-01 KR KR1020057005806A patent/KR20050050115A/ko active IP Right Grant
- 2003-10-01 BR BR0314986-2A patent/BR0314986A/pt not_active IP Right Cessation
- 2003-10-01 US US10/529,931 patent/US20060165713A1/en not_active Abandoned
- 2003-10-01 JP JP2004540797A patent/JP2006516386A/ja active Pending
- 2003-10-01 NZ NZ539154A patent/NZ539154A/en unknown
- 2003-10-01 WO PCT/EP2003/011158 patent/WO2004031222A2/en active Application Filing
- 2003-10-01 EP EP03785608A patent/EP1546191A2/en not_active Withdrawn
- 2003-10-01 TW TW092127210A patent/TW200411055A/zh unknown
- 2003-10-01 RU RU2005109155/13A patent/RU2354701C2/ru not_active IP Right Cessation
- 2003-10-01 CA CA002500093A patent/CA2500093A1/en not_active Abandoned
- 2003-10-01 MX MXPA05003558A patent/MXPA05003558A/es not_active Application Discontinuation
- 2003-10-01 CN CNB2003801049852A patent/CN100393878C/zh not_active Expired - Fee Related
- 2003-10-01 PL PL376534A patent/PL376534A1/pl not_active Application Discontinuation
- 2003-10-03 AR ARP030103608A patent/AR041515A1/es unknown
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2005
- 2005-03-23 NO NO20051561A patent/NO20051561L/no not_active Application Discontinuation
- 2005-03-29 IS IS7775A patent/IS7775A/is unknown
- 2005-04-15 MA MA28223A patent/MA27474A1/fr unknown
- 2005-04-20 ZA ZA200503201A patent/ZA200503201B/en unknown
- 2005-04-28 CO CO05040562A patent/CO5580837A2/es not_active Application Discontinuation
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2007
- 2007-06-08 US US11/760,127 patent/US20070264283A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| MXPA05003558A (es) | 2005-06-03 |
| RU2005109155A (ru) | 2006-03-10 |
| WO2004031222A2 (en) | 2004-04-15 |
| EP1546191A2 (en) | 2005-06-29 |
| ZA200503201B (en) | 2006-06-28 |
| KR20050050115A (ko) | 2005-05-27 |
| US20070264283A1 (en) | 2007-11-15 |
| CO5580837A2 (es) | 2005-11-30 |
| JP2006516386A (ja) | 2006-07-06 |
| CA2500093A1 (en) | 2004-04-15 |
| US20060165713A1 (en) | 2006-07-27 |
| MA27474A1 (fr) | 2005-08-01 |
| NZ539154A (en) | 2007-05-31 |
| TW200411055A (en) | 2004-07-01 |
| NO20051561D0 (no) | 2005-03-23 |
| PL376534A1 (pl) | 2006-01-09 |
| CN100393878C (zh) | 2008-06-11 |
| AR041515A1 (es) | 2005-05-18 |
| NO20051561L (no) | 2005-06-02 |
| AU2003294672A1 (en) | 2004-04-23 |
| BR0314986A (pt) | 2005-08-09 |
| RU2354701C2 (ru) | 2009-05-10 |
| IS7775A (is) | 2005-03-29 |
| WO2004031222A3 (en) | 2004-08-19 |
| GB0222953D0 (en) | 2002-11-13 |
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