轮状病毒毒株及其作为疫苗的用途

文档序号：80622 发布日期：2021-10-08 浏览：30次 >En<

阅读说明：本技术 轮状病毒毒株及其作为疫苗的用途 (Rotavirus strains and their use as vaccines ) 是由倪世利邢苗苗李慧娟于菲邹水燕曲衍洪孙萌于 2021-07-09 设计创作，主要内容包括：本发明涉及病毒领域,一种A群轮状病毒G9P[8]毒株的分离、培养、鉴定和作为疫苗的用途。具体来说是A群轮状病毒毒种LSR-8毒株,本发明涉及A群轮状病毒毒种LSR-8毒株的全氨基酸序列如SEQ ID No.1至SEQ ID No.11所示,全核酸序列如SEQ ID No.12至SEQ ID No.22所示。本发明还涉及LSR-8毒株可用于生产灭活型人轮状病毒疫苗、人轮状病毒减毒活疫苗和联合疫苗的用途,可用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病,例如轮状病毒性胃肠炎和腹泻等。(The invention relates to the field of virus, in particular to separation, culture and identification of group A rotavirus G9P [8] strain and application as a vaccine. The invention relates to a group A rotavirus LSR-8 strain, which has the full amino acid sequence shown in SEQ ID No.1 to SEQ ID No.11 and the full nucleic acid sequence shown in SEQ ID No.12 to SEQ ID No. 22. The invention also relates to the use of the LSR-8 strain in the production of inactivated human rotavirus vaccine, attenuated live human rotavirus vaccine and combined vaccine, and can be used for preventing, alleviating or treating rotavirus infection and diseases caused by rotavirus infection, such as rotavirus gastroenteritis, diarrhea and the like.)

轮状病毒毒株及其作为疫苗的用途

技术领域

本发明涉及病毒领域，特别涉及轮状病毒毒株及其作为疫苗的用途。

背景技术

A群轮状病毒能引起90％以上的人类轮状病毒肠胃炎，2016年，全球年龄小于5周岁的儿童死于轮状病毒感染的约128500人，全球年龄小于5周岁的儿童感染轮状病毒约为2.6亿人。A群轮状病毒是通过粪-口途径的高度传染性的人畜共患病原体，也可以气溶胶的形式经呼吸道传播。就轮状病毒的感染率和发病率而言，发达国家和发展中国家相比没有明显的差异，物质、生活环境的差异不是影响抑制轮状病毒感染产生重要的因素。轮状病毒感染无特效治疗药物，疫苗是首选预防和控制轮状病毒感染的手段，世界卫生组织WHO积极推动各国政府进行轮状病毒疫苗的研究与开发，建议全球所有国家应该将轮状病毒疫苗纳入国家计划免疫项目中。

轮状病毒属于呼肠孤病毒科(Reoviridae)，成熟病毒颗粒在电镜下呈光滑车轮状的轴辐式形态的二十面体病毒，直径约为65-75nm左右，有3层蛋白衣壳。其基因组由11个双链RNA片段组成，每一个片段为一个基因，编码一种蛋白。11个基因分别编码病毒的6个结构蛋白：VP1-VP4、VP6和VP7及6个非结构蛋白:NSP1-NSP6。根据轮状病毒VP6蛋白的抗原性，将轮状病毒分为A-G7个群。全部轮状病毒均能在其他动物中引起疾病，但仅有A、B和C三个群感染人，A群轮状病毒的感染占所有轮状病毒感染人类例数的90％。VP4和VP7蛋白作为轮状病毒的中和抗原，均可刺激机体产生中和抗体，从而抑制轮状病毒的感染。VP4蛋白可以在肠道内胰蛋白酶的作用下改变为VP5和VP8两种蛋白，VP8蛋白可以与细胞表面的唾液酸受体结合，从而介导轮状病毒的吸附过程，VP5膜融合位点介导轮状病毒的入胞过程，VP4蛋白决定了轮状病毒的毒性和P型血清型。VP7蛋白为糖基化蛋白，具有Ca2⁺依赖性，它构成了病毒最外层的表面，并决定轮状病毒的P型血清型。根据VP4，轮状病毒被分为35种基因型，当前在全球范围内流行的轮状病毒血清型主要是P[4]和P[8]；根据VP7，轮状病毒被分为27种血清型，当前在全球范围内流行的轮状病毒毒株型血清型主要以G1、G2、G3、G4和G9为主。

灭活轮状病毒疫苗是一种安全有效地预防儿童因感染轮状病毒所引起疾病的疫苗。但至今尚未有灭活轮状病毒疫苗产品上市。目前使用的所有轮状病毒减毒活疫苗的保护效率约为75％，疫苗的保护效果尚待改进；其次，轮状病毒减毒活疫苗在制备、使用和免疫后的排毒使病毒在环境中长期存活、存在安全性隐患，包括毒力返祖、肠套叠的风险等；减毒活疫苗制备过程中的存在外源因子污染风险及不能用于免疫缺陷或者免疫抑制治疗人群等缺点，因此研制灭活轮状病毒疫苗具有重要意义及应用前景。

发明内容

有鉴于此，本发明提供了轮状病毒毒株及其作为疫苗的用途。LSR-8毒株可用于生产灭活型人轮状病毒疫苗、人轮状病毒减毒活疫苗、亚单位疫苗和基因工程重组蛋白疫苗、结合疫苗和联合疫苗的用途，可用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病，例如轮状病毒性胃肠炎和腹泻等。

为了实现上述发明目的，本发明提供以下技术方案：

本发明提供了多肽或多肽组合，所述多肽包括如下序列：

(I)、如SEQ ID No.1～11任意所示的氨基酸序列；

或

(II)、如(I)所述氨基酸序列经取代、缺失或添加一个至多个氨基酸获得的氨基酸序列，且与(I)所述氨基酸序列功能相同或相近的氨基酸序列；

或

(III)、与如SEQ ID No.1～11任意所示的氨基酸序列具有至少99.4％同一性的氨基酸序列；

所述多个为5个。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

与如SEQ ID No.4所示的氨基酸序列具有99.4％以上同一性的氨基酸序列；

与如SEQ ID No.6所示的氨基酸序列具有99.4％以上同一性的氨基酸序列；和/或

与如SEQ ID No.8所示的氨基酸序列具有99.7％以上同一性的氨基酸序列；和/或

与如SEQ ID No.9所示的氨基酸序列具有99.7％以上同一性的氨基酸序列；和/或

与如SEQ ID No.11所示的氨基酸序列具有99.5％以上同一性的氨基酸序列。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

包含与SEQ ID No.4所示的氨基酸序列至少99.4％相同的氨基酸序列的多肽；和/或

包含与SEQ ID No.6所示的氨基酸序列至少99.4％相同的氨基酸序列的多肽；和/或

包含与SEQ ID No.11所示的氨基酸序列至少99.5％相同的氨基酸序列的多肽。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

SEQ ID No.4所示的氨基酸序列；和/或

SEQ ID No.6所示的氨基酸序列；和/或

SEQ ID No.11所示的氨基酸序列。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

包含如SEQ ID No2所示的氨基酸序列的多肽；和

包含如SEQ ID No5所示的氨基酸序列的多肽；

还包括如下序列中的一个或多个：

包含与如SEQ ID No.6所示的氨基酸序列至少99.4％同一性的氨基酸序列的多肽；和/或

包含与如SEQ ID No.7所示的氨基酸序列的多肽；和/或

包含如SEQ ID No.8所示的氨基酸序列至少99.7％同一性的氨基酸序列的多肽；和/或

包含与如SEQ ID No.9所示的氨基酸序列至少99.7％同一性的氨基酸序列的多肽；和/或

包含如SEQ ID No.10所示的氨基酸序列的多肽；和/或

包含与如SEQ ID No.11所示的氨基酸序列至少99.5％同一性的氨基酸序列的多肽；和/或

包含如SEQ ID No.1所示的氨基酸序列的多肽；和/或

包含如SEQ ID No.3所示的氨基酸序列的多肽；和/或

包含与如SEQ ID No.4所示的氨基酸序列至少99.4％同一性的氨基酸序列的多肽。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

包含如SEQ ID NO:2所示的氨基酸序列的多肽；

包含如SEQ ID No.5所示的氨基酸序列的多肽；和

包含与如SEQ ID No.6所示的氨基酸序列至少99.4％同一性的氨基酸序列的多肽；

还包括如下序列中的一个或多个：

包含与如SEQ ID No.7所示的氨基酸序列的多肽；和/或

包含SEQ ID No.8所示的氨基酸序列至少99.7％同一性的氨基酸序列的多肽；和/或

包含与SEQ ID No.9所示的氨基酸序列至少99.7％同一性的氨基酸序列的多肽；和/或

包含SEQ ID No.10所示的氨基酸序列的多肽；和/或

包含与SEQ ID No.11所示的氨基酸序列至少99.5％同一性的氨基酸序列的多肽；和/或

包含SEQ ID No.1所示的氨基酸序列的多肽；和/或

包含SEQ ID No.3所示的氨基酸序列的多肽；和/或

包含与SEQ ID No.4所示的氨基酸序列至少99.4％同一性的氨基酸序列的多肽。

在本发明的一些具体实施方案中，所述多肽包括如下序列：

编码如SEQ ID No.4所示氨基酸序列的多肽的核酸分子第75、76位核苷酸为GA，其对应的第22、23位氨基酸为MK；和/或

编码如SEQ ID No.4所示氨基酸序列的多肽的核酸分子第499位核苷酸为G，其对应的第175位氨基酸为A；和/或

编码如SEQ ID No.4所示氨基酸序列的多肽的核酸分子第772个核苷酸为C，其对应的第266位氨基酸为H；和/或

编码如SEQ ID No.4所示氨基酸序列的多肽的核酸分子第1129个核苷酸为C，其对应的第385位氨基酸为H；和/或

编码如SEQ ID No.6所示氨基酸序列的多肽的核酸分子第404个核苷酸为G，其对应的第119位氨基酸为R；和/或

编码如SEQ ID No.6所示氨基酸序列的多肽的核酸分子第656个核苷酸为C，其对应的第203位氨基酸为S。

本发明还提供了编码所述多肽或多肽组合的核酸分子。

在本发明的一些具体实施方案中，所述核酸分子包括如下序列：

(IV)、编码如SEQ ID No.1～11任意所示氨基酸序列的核苷酸序列；和/或

(V)、编码如SEQ ID No.1～11任意所示氨基酸序列的核苷酸序列的反义互补序列；

(VI)、与编码如SEQ ID No.1～11任意所示氨基酸序列的核苷酸序列在高度严谨情况下杂交的核苷酸序列；

(VII)、如(IV)、(V)或(VI)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(IV)所示的核苷酸序列功能相同或相似的核苷酸序列；

(VIII)、与(IV)、(V)或(VI)所示的核苷酸序列至少有99.4％同一性的核苷酸序列。

所述多个为2个、3个、4个或5个。

在本发明的一些具体实施方案中，所述核酸分子包括如SEQ ID No.12～22任意所示的核苷酸序列。

在本发明的一些具体实施方案中，所述核酸分子包括如下序列：

编码如SEQ ID No.1所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.12所示的核苷酸序列；和/或

编码如SEQ ID No.2所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.13所示的核苷酸序列；和/或

编码如SEQ ID No.3所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.14所示的核苷酸序列；和/或

编码如SEQ ID No.4所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.15所示的核苷酸序列；和/或

编码如SEQ ID No.5所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.16所示的核苷酸序列；和/或

编码如SEQ ID No.6所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.17所示的核苷酸序列；和/或

编码如SEQ ID No.7所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.18所示的核苷酸序列；和/或

编码如SEQ ID No.8所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.19所示的核苷酸序列；和/或

编码如SEQ ID No.9所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.20所示的核苷酸序列；和/或

编码如SEQ ID No.10所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.21所示的核苷酸序列；和/或

编码如SEQ ID No.11所示氨基酸序列的核酸分子，所述核酸分子具有如SEQ IDNo.22所示的核苷酸序列。

本发明还提供了一种表达载体，包括所述的核酸分子。

此外，本发明还提供了转化或转染所述的表达载体的宿主细胞。

更重要的是，本发明还提供了轮状病毒，其特征在于，包括所述的多肽或多肽组合或所述的核酸分子。

在本发明的一些具体实施方案中，所述轮状病毒的保藏编号为CGMCC No.20292。

更重要的是，本发明还提供了所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒在制备预防和/治疗疾病的疫苗和/或药物中的应用；

所述疾病包括但不限于轮状病毒诱发的疾病、白喉、破伤风、百日咳、b型流感嗜血杆菌诱发的疾病、乙型肝炎(HBV)诱发的疾病、脊髓灰质炎和/或肺炎球菌诱发的疾病

本发明还提供了药物，包括所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒以及药学上可接受的辅料。

本发明还提供了药物组合物，包括所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒以及治疗其他疾病的药物。

本发明还提供了疫苗，包括所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒以及可接受的佐剂。

本发明还提供了疾病的预防和/或治疗方法，给予所述的药物、所述的药物组合物或所述的疫苗。

本发明还提供了试剂盒，包括能够扩增所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒。

此外，本发明还提供了试剂盒，包括能够特异性结合所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒。

本发明还提供了疾病的诊断方法，以所述的试剂盒检测病毒表达，根据病毒的表达量判断是否患有疾病。

本发明还提供了轮状病毒灭活疫苗的制备方法，其特征在于，包括以下步骤：

步骤1：将所述的多肽或多肽组合、所述的核酸分子或所述的轮状病毒经适应性培养和噬斑纯化，接种于扩大传代培养的Vero细胞中培养；

步骤2：收获病毒培养液，过滤除杂质后浓缩纯化、灭活得到病毒原液；

步骤3：取步骤2制备的所述病毒原液过滤除菌后加入佐剂既得所述轮状病毒灭活疫苗。

在本发明的一些具体实施方案中，步骤1所述培养为35℃～37℃培养72～120小时。

在本发明的一些具体实施方案中，步骤2所述灭活为用甲醛溶液或B-丙内酯或热灭活。

在本发明的一些具体实施方案中，步骤2所述纯化为阴离子交换层析、分子筛凝胶过滤层析。

在本发明的一些具体实施方案中，步骤3所述佐剂为氢氧化铝溶液。

综上，本发明提供了G9P[8]轮状病毒，具体来说是A群轮状病毒LSR-8毒株。在一些实施方案中，公开了毒株的分离和表征，及毒株分别在MA104细胞和Vero细胞上的适应。

在一些实施方案中，公开了药物组合物，所述药物组合物可包括佐剂和/或灭活的G9P[8]轮状病毒。

所述灭活的轮状病毒也可被包括在药物组合物中以诱导针对其他病原体(例如，但不限于，白喉、破伤风、百日咳、b型流感嗜血杆菌、乙型肝炎(HBV)、脊髓灰质炎和/或肺炎球菌疾病)的反应。

所述免疫反应可为保护性免疫反应。所述受试者可为儿童，例如小于5岁或1岁的人类儿童；所述受试者亦可为成年人。所述方法可包括一次初免和至少一次加强免疫。

本发明公开了分离的来自LSR-8轮状病毒的核酸分子和分离的来自LSR-8轮状病毒的多肽序列。这些分子可被包括在药物组合物中，并且可用于诱导免疫反应。

生物保藏说明

生物材料：LSR-8，分类命名：轮状病毒，于2020年8月31日向中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)进行保藏，地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.20292。

附图说明

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。

图1示VP4和VP7序列与其他轮状病毒的关系，其中，图1A为NC1LSR-8VP4，代表LSR-8的VP4核酸序列，图1B为NC1LSR-8VP7，代表LSR-8的VP7核酸序列；

图2示RNA电泳型的图像；

图3示蚀斑纯化图片；

图4示LSR-8轮状病毒在MA104细胞上的增殖曲线；

图5示LSR-8轮状病毒胶体金试剂条检测图片；

图6示LSR-8轮状病毒在MA104细胞上的免疫荧光图片。

具体实施方式

本发明公开了轮状病毒毒株及其作为疫苗的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。

本发明提供的轮状病毒毒株及其作为疫苗的用途中，所用原料及试剂均可由市场购得。

下面结合实施例，进一步阐述本发明：

实施例1LSR-8毒株的分离和表征

获取病毒样本

收集医院儿科腹泻病患幼儿腹泻排泄物标本，重悬于pH 7.2的0.01M PBS溶液中，8000rpm、4℃离心20分钟取上清液，经胶体金试剂盒检测轮状病毒呈阳性，用0.22μm滤器除菌过滤，分装于1.5ml无菌离心管中，-80℃保存备用。

LSR-8毒株病毒形态噬斑纯化

将轮状病毒收获液加入胰酶于37℃恒温水浴中激活1小时后，用维持液10倍倍比稀释为10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8。

MA104细胞接种6孔板，待细胞长成致密单层时，弃掉其中的生长液，用维持液洗细胞面3次。吸净残余的维持液，每孔加入激活的轮状病毒液500μl，37℃吸附60～120分钟后，吸弃病毒液，用维持液洗细胞面1次。每孔加入含有0.3％琼脂的维持液3ml,每个稀释梯度为2个孔，待琼脂凝固后移入培养箱的倒置培养。72小时后，在原有琼脂层上加盖一层含有0.006％中性红和0.3％琼脂的维持液避光放入培养箱中。每天观察细胞病变情况，挑取合适的稀释度的单个蚀斑的毒种进行培养。

LSR-8毒株病毒在MA104细胞上的适应

将轮状病毒收获液加入终浓度20μg/ml的胰酶，于37℃恒温水浴中激活1小时。

取T25瓶中已长成致密单层的MA104细胞，弃掉其中的生长液，用维持液洗细胞面3次。吸净残余的维持液，加入激活的轮状病毒液500μl，37℃吸附60～120分钟后，加入含有终浓度为4μg/ml胰酶的维持液10ml，于37℃、5％CO₂中培养，观察细胞病变效应CPE。

当以上的细胞出现时75％CPE，收获病毒。将收获病毒培养液-20℃和25℃交替冻融2次。8000rpm、4℃离心20分钟，取上清液，同法接种MA104细胞传代或-80℃保存备用。

经胶体金试剂盒检测轮状病毒抗原，蚀斑法检测LSR-8病毒滴度。

LSR-8毒株基因组核酸带型

LSR-8毒株病毒基因组的核酸提取按GeneJET Viral DNA and RNA PurificationKit病毒基因组RNA提取试剂盒操作手册进行。通过非变性聚丙烯酰胺凝胶电泳，90V，6h后，银染显色观察核酸带型。

LSR-8毒株基因组序列分析

根据克隆测序对提取的RNA进行测序。使用RotaC v2.0在线工具进行全基因分型。用Mega 10.1.8软件使用邻接法生成系统发生树。LSR-8VP4基因与2018年日本分离的毒株和2019年武汉分离的毒株VP4基因高度相似(99.7％)。LSR-8VP7基因与在2018年收集的来自日本东京的G9毒株密切相关。

LSR-8可在Vero细胞中生长至10⁶-10⁷Pfu/ml的滴度并表现出强的稳定性。

LSR-8毒株病毒形态

取10μl经蔗糖垫纯化的病毒悬液滴于铜网上，静置5分钟后用滤纸将多余悬液吸干，静置几十秒，使铜网干燥；取2％磷钨酸染液10μl，滴于铜网上，静置5分钟后用滤纸将多余染液吸干，静置几十秒待铜网干燥后，即可在透射电镜下直接观察病毒形态学特征。

实施例2LSR-8毒株的适应性和传代

LSR-8毒株病毒在VERO细胞上的适应

将轮状病毒收获液加入胰酶，于37℃恒温水浴中激活1小时。

取T25瓶中已长成致密单层的VERO细胞，弃掉其中的生长液，用维持液洗细胞面。吸净残余的维持液，加入激活的轮状病毒液500ul，37℃吸附60～120分钟后，加入含有终浓度为4μlg/ml胰酶的维持液10ml，于37℃、5％CO₂中培养，观察细胞病变效应CPE。

当以上的细胞出现时75％CPE，收获病毒。将收获病毒培养液-20℃和25℃交替冻融2次，8000rpm、4℃离心20分钟，取上清液，同法接种VERO细胞传代或-80℃保存备用。

实施例3LSR-8毒株的鉴定方法

LSR-8毒株病毒滴度测定：

将轮状病毒收获液加入胰酶于37℃恒温水浴中激活1小时，用维持液10倍倍比稀释为10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8。

MA104细胞接种6孔板，待细胞长成致密单层时，弃掉其中的生长液，用维持液洗细胞面3次。吸净残余的维持液，加入激活的轮状病毒液500μl，37℃吸附60-120分钟后，吸弃病毒液，加入含有0.3％琼脂的维持液3ml，每个稀释梯度为2个孔，待琼脂凝固后移入培养箱。72小时后，在原有琼脂层上加盖一层含有0.006％中性红和0.3％琼脂的维持液避光放入培养箱中。37℃继续倒置培养4天，每天观察细胞病变情况，计算滴度。LSR-8毒株可在Vero细胞中生长至10^5.5-10⁷Pfu/ml的滴度,并表现出强的稳定性。

中和鉴别实验

MA104细胞以10⁴细胞/孔接种96孔板，待细胞长成致密单层时，弃掉其中的生长液，用维持液洗细胞面。吸净残余的维持液，将分离毒株病毒抗血清按2倍梯度稀释，按照100ul/孔加入96孔板，加入100Pfu/100μl/孔的病毒激活液，混匀后37℃CO₂孵箱中和2小时后，弃抗血清，加入维持液，37℃CO₂孵箱中培养8～10天，观察结果。

实施例4 LSR-8毒株疫苗的产生和纯化

通过使用大规模生产滚瓶来实现轮状病毒的生产。将滚瓶中Vero细胞的汇合单层用感染复数MOI为0.05-0.5的特定轮状病毒毒株感染。反复冻融收获的病毒液经过滤除杂质后浓缩纯化、灭活得到病毒原液，按照一定的抗原含量进行稀释，加入0-1.6mg/ml氢氧化铝佐剂。

实施例5佐剂

氢氧化铝佐剂对提高轮状病毒灭活疫苗免疫原性有明显作用。将抗体阴性的小鼠肌肉注射不同氢氧化铝佐剂含量的疫苗，免疫2次，间隔28天，免疫后0，14，28，56天分别采血，蚀斑减数中和试验检测小鼠血清中和抗体效价。氢氧化铝对疫苗免疫原性影响实验包括两部分：第一部分不同浓度佐剂对同一疫苗浓度抗原的比较，第二部分在同-佐剂浓度(0.8mg/ml)与不同抗原浓度进行比较，结果显示氢氧化铅佐剂对提高轮状病毒灭活疫苗免疫原性有明显作用。

实施例6 LSR-8毒株的免疫效力评价方法

(1)轮状病毒疫苗样品免疫原性分析

免疫前，小鼠采血，分离血清，通过ELSA检测母鼠是否含有轮状病毒特异性抗体，排除阳性小鼠。将病毒收获液经纯化灭活制备成免疫用疫苗样品，以0EU/100μl、40EU/100μl、80EU/100μl、160EU/100μl4个剂量，每个剂量肌内注射10只小鼠，阴性对照10只，注射PBS100ul，28天后进行一针加强免疫。通过测量在给药后不同时间2、4、6、8周获得的血液样品中的抗体，微量中和测定法测量中和抗体效价。

(2)免疫血清轮状病毒中和抗体效价评价实验

1)将MA104细胞接种在96孔板中，于37℃CO₂孵箱中培养至长成致密单层。

2)将激活的轮状病毒株稀释为1000Pfu/100μl，实验动物免疫后的血清以4倍稀释起，按2倍等比梯度稀释。

3)将激活的轮状病毒株100μl与稀释后的血清1:1混合，37℃CO2孵箱中和1小时。

4)将中和液100μl/孔接种至已用维持液清洗过的96孔培养板中，37℃CO2处理1小时。

5)弃掉未吸附的中和液，用MEM维持液清洗细胞一次，加入维持液，培养7天。

6)将羊抗轮状病毒免疫多克隆抗体(Millipore公司)用碳酸盐缓冲液(pH值9.6)按1：1000稀释，每孔100μl在96孔ELISA板4℃包被过夜；

7)去除包被液，用含0.5％Tween-20的PBS溶液(PBST溶液)洗板3次，每次3min；用含有3％牛血清白蛋白的PBST溶液，100μl孔，37℃封闭2小时；

8)去除封闭液，用PBST溶液，洗板3次，将中和96孔细胞板中的液体完全转移到ELISA板中，37℃温育1h；

9)PBST洗板3次后，加入用PBST缓冲液1：2000稀释的HRP标记的羊抗轮状病毒多克隆抗体，100μl/孔，37℃结合1h；

10)PBST洗板3次后，加入TMB显色液，100μl/孔，在室温显色5min，再加入2MH2SO4，100μl/孔，终止显色；

11)用酶标仪在450nm，630nm(效验波长)波长下检测光吸收值。

Cutoff值：Cutoff阈值＝阴性孔均值×2.1，如果当阴性均值≤0.050，按0.050计算Cutoff阈值。当待测样品的检测值高于阈值，认为该待测样品为阳性孔，抗原量(EU/mL)读值为该阳性最高稀释度×10。

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645 650 655

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

660 665 670

Asp Asp Gln Met Tyr Arg Leu Arg Asp Arg Leu Arg Leu Leu Pro Val

675 680 685

Glu Arg Arg Arg Leu Asp Ile Phe Asn Leu Ile Leu Met Asn Met Glu

690 695 700

Gln Ile Glu Arg Ala Ser Asp Lys Ile Ala Gln Gly Val Ile Ile Ala

705 710 715 720

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

725 730 735

Ile Ala Arg Asn Leu Asp Gly Tyr Gln Gln Ile Asn Leu Glu Glu Leu

740 745 750

Met Arg Thr Gly Asp Tyr Gly Gln Ile Thr Asn Met Leu Leu Asn Asn

755 760 765

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

770 775 780

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

785 790 795 800

Val Lys Leu Arg Lys Val Asp Thr Leu Lys Pro Ile Leu Tyr Lys Ile

805 810 815

Asn Ser Asp Ser Asn Asp Phe Tyr Leu Val Ala Asn Tyr Asp Trp Ile

820 825 830

Pro Thr Ser Thr Thr Lys Val Tyr Lys Gln Val Pro Gln Pro Phe Asp

835 840 845

Phe Arg Ala Ser Met His Met Leu Thr Ser Asn Leu Thr Phe Thr Val

850 855 860

Tyr Ser Asp Leu Leu Ser Phe Val Ser Ala Asp Thr Val Glu Pro Ile

865 870 875 880

Asn Ala Ile Ala Phe Asp Asn Met Arg Ile Met Asn Glu Leu

885 890

<210> 3

<211> 835

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 3

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

1 5 10 15

Thr Gln Thr Tyr Leu His Asp Asp Ser Lys Asp Glu Tyr Glu Asn Ala

20 25 30

Phe Leu Ile Ser Asn Leu Thr Thr His Asn Ile Leu Tyr Leu Asn Tyr

35 40 45

Ser Leu Lys Thr Leu Lys Ile Leu Asn Lys Ser Gly Ile Ala Ala Val

50 55 60

Glu Val Gln Ser Pro Asp Glu Leu Phe Ala Leu Ile Arg Cys Asn Phe

65 70 75 80

Thr Tyr Asp Tyr Glu Asn Asn Ile Ile Tyr Leu His Asp Tyr Ser Tyr

85 90 95

Tyr Thr Asn Asn Glu Ile Arg Thr Asp Gln His Trp Ile Thr Lys Thr

100 105 110

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

115 120 125

Tyr Asn Gly Lys Asn Thr Arg Gly His Tyr Asn Phe Ser Phe Leu Cys

130 135 140

Gln Asn Ala Ala Thr Asp Asp Asp Ile Ile Ile Glu Tyr Ile Tyr Ser

145 150 155 160

Asn Glu Leu Asp Phe Gln Asn Phe Leu Leu Arg Lys Ile Lys Glu Arg

165 170 175

Met Thr Thr Ser Leu Pro Ile Ala Arg Leu Ser Asn Arg Val Phe Arg

180 185 190

Asp Lys Leu Phe Pro Ser Ile Val Asn Met Tyr Lys Lys Val Ile Asn

195 200 205

Val Gly Pro Arg Asn Glu Ser Met Phe Thr Phe Leu Asn Phe Pro Thr

210 215 220

Ile Lys Gln Phe Ser Asn Gly Ala Tyr Ile Val Lys His Thr Ile Lys

225 230 235 240

Leu Lys Gln Glu Lys Trp Leu Gly Lys Arg Val Ser Gln Phe Asp Ile

245 250 255

Gly Gln Tyr Lys Asn Met Leu Asn Val Val Thr Thr Ile Tyr Tyr Tyr

260 265 270

Tyr Asn Leu Tyr His Ser Lys Pro Ile Ile Tyr Met Leu Gly Ser Ala

275 280 285

Pro Ser Tyr Trp Ile Tyr Asp Ile Lys Gln Tyr Ser Asp Phe Thr Phe

290 295 300

Glu Thr Trp Asp Pro Leu Asp Thr Pro Tyr Ser Thr Ile His His Lys

305 310 315 320

Glu Leu Phe Phe Asp Lys Asp Val Asn Lys Leu Lys Asp Asn Ser Val

325 330 335

Leu Tyr Ile Asp Ile Arg Thr Asp Arg Gly Asn Met Asp Trp Lys Glu

340 345 350

Trp Arg Lys Val Val Glu Gln Gln Thr Ile Asn Asn Leu Asn Ile Ala

355 360 365

Tyr Lys Tyr Leu Ala Thr Gly Lys Ala Lys Val Cys Cys Val Lys Leu

370 375 380

Thr Ala Met Asp Leu Glu Leu Pro Ile Thr Ala Lys Leu Leu His His

385 390 395 400

Pro Thr Thr Glu Val Arg Ser Glu Phe Tyr Ala Ile Leu Asp Val Trp

405 410 415

Asp Ile Ile Thr Ile Lys Arg Phe Ile Pro Lys Gly Val Phe Tyr Ala

420 425 430

Phe Ile Asn Asn Ile Ile Thr Glu Asn Val Phe Ile Gln Pro Ser Phe

435 440 445

Lys Leu Lys Val Ser Pro Thr Asp Tyr Ile Val Ala Leu Tyr Ala Leu

450 455 460

Ser Asn Asp Phe Asn Ser Arg Gln Asp Val Ile Asn Leu Ile Asn Lys

465 470 475 480

Gln Lys Gln Ser Leu Ile Thr Val Arg Met Asn Asn Thr Phe Lys Asp

485 490 495

Glu Pro Lys Val Asn Phe Lys Asn Ile Tyr Asp Trp Thr Phe Leu Pro

500 505 510

Thr Asp Phe Glu Leu Lys Asp Ser Ile Ile Thr Ser Tyr Asp Gly Cys

515 520 525

Leu Gly Met Phe Gly Leu Ser Ile Ser Leu Ser Ser Lys Pro Thr Gly

530 535 540

Asn Asn His Leu Phe Ile Ile Asn Gly Thr Asp Lys Tyr Asp Lys Leu

545 550 555 560

Asp Gln Tyr Ala Asn His Met Gly Ile Ser Arg Arg Ser His Gln Ile

565 570 575

Arg Phe Ser Glu Ser Ala Thr Ser Tyr Ser Gly Tyr Ile Phe Arg Asp

580 585 590

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

595 600 605

Val Ser Gly His Val Tyr Asn Ala Leu Ile Tyr Tyr Arg Tyr Asn Tyr

610 615 620

Ala Phe Asp Leu Lys Arg Trp Ile Tyr Leu His Ser Ile Gly Lys Ala

625 630 635 640

Ala Val Glu Gly Gly Arg Tyr Tyr Glu His Ala Pro Ile Glu Leu Ile

645 650 655

Tyr Ala Cys Arg Ser Ala Lys Glu Phe Ala Ile Leu Gln Asp Asp Leu

660 665 670

Thr Val Leu Arg Tyr Ala Asn Glu Ile Glu Glu Tyr Ile Asn Lys Val

675 680 685

Tyr Ser Ile Thr Tyr Ala Asp Asp Pro Asn Tyr Phe Ile Gly Ile Lys

690 695 700

Phe Asn Ser Ile Pro Tyr Glu Tyr Asp Val Lys Val Pro His Leu Thr

705 710 715 720

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

725 730 735

Val Leu Lys Lys Met Lys Thr Glu Leu Phe Lys Thr Glu Ile Ser Thr

740 745 750

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

755 760 765

Gly Val Leu Ser Thr Tyr Phe Lys Leu Tyr Asn Met Phe Tyr Arg Asn

770 775 780

His Ile Thr Phe Gly Gln Ser Arg Met Phe Ile Pro His Ile Thr Leu

785 790 795 800

Ser Phe Ser Asn Lys Gln Thr Val Arg Ile Glu Ser Thr Arg Leu Lys

805 810 815

Ile Asn Ser Ile Tyr Leu Arg Lys Ile Lys Gly Glu Thr Val Phe Asp

820 825 830

Met Ser Glu

835

<210> 4

<211> 775

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 4

Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Ser Val

1 5 10 15

Asp Leu Tyr Asp Glu Met Lys Gln Ile Gly Ser Glu Lys Thr Gln Asn

20 25 30

Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Thr Arg Tyr Ala Pro Val

35 40 45

Asn Trp Gly His Gly Glu Ile Asn Asp Ser Thr Thr Val Glu Pro Ile

50 55 60

Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Thr Asp Tyr

65 70 75 80

Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr

85 90 95

Asn Asn Ser Asp Phe Trp Thr Ala Val Ile Ala Val Glu Pro His Val

100 105 110

Asp Pro Val Asp Arg Gln Tyr Asn Val Phe Gly Glu Asn Lys Gln Phe

115 120 125

Asn Val Arg Asn Asp Ser Asp Lys Trp Lys Phe Leu Glu Met Phe Arg

130 135 140

Gly Ser Ser Gln Ser Asp Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp

145 150 155 160

Thr Lys Leu Val Gly Ile Leu Lys Tyr Gly Gly Arg Ile Trp Ala Phe

165 170 175

His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Asn Thr Ala Asn

180 185 190

Leu Asn Gly Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro

195 200 205

Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu Pro

210 215 220

Pro Ile Gln Asn Thr Arg Asn Val Val Pro Leu Ser Leu Ser Ser Arg

225 230 235 240

Ser Ile Gln Tyr Thr Arg Ala Gln Val Asn Glu Asp Ile Thr Ile Ser

245 250 255

Lys Thr Ser Leu Trp Lys Glu Met Gln His Asn Arg Asp Ile Ile Ile

260 265 270

Arg Phe Lys Phe Gly Asn Ser Ile Ile Lys Leu Gly Gly Leu Gly Tyr

275 280 285

Lys Trp Ser Glu Ile Ser Tyr Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr

290 295 300

Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn

305 310 315 320

Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp Phe

325 330 335

Ser Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val

340 345 350

Asp Tyr Trp Asp Asp Ser Lys Ala Phe Arg Asn Met Val Tyr Val Arg

355 360 365

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

370 375 380

His Phe Ser Ile Pro Val Gly Ala Trp Pro Val Met Asn Gly Gly Ala

385 390 395 400

Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp

405 410 415

Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Asp

420 425 430

Glu Pro Ser Phe Ser Ile Leu Arg Thr Arg Thr Val Asn Leu Tyr Gly

435 440 445

Leu Pro Ala Ala Asn Pro Asn Asn Gly Asn Glu Tyr Tyr Glu Ile Ser

450 455 460

Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Thr Asn Asp Asp Tyr Gln

465 470 475 480

Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln

485 490 495

Leu Thr Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala

500 505 510

Met Ser Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser

515 520 525

Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Leu Thr Lys Ser Met Ala

530 535 540

Thr Ser Val Met Lys Lys Phe Arg Lys Ser Lys Leu Ala Thr Ser Val

545 550 555 560

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

565 570 575

Ser Val Ser Val Arg Ser Asn Ile Ser Ala Phe Ser Asn Trp Thr Asn

580 585 590

Val Ser Asn Asp Val Ser Asn Val Thr Asn Ser Val Asn Asp Ile Ser

595 600 605

Thr Gln Thr Ser Thr Ile Ser Lys Asn Leu Arg Leu Arg Glu Met Ile

610 615 620

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

625 630 635 640

Lys Thr Lys Ile Asp Met Ser Thr Gln Ile Gly Lys Asn Thr Leu Pro

645 650 655

Asp Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Lys Arg Ser Tyr

660 665 670

Arg Ile Leu Lys Asp Asp Glu Val Met Glu Ile Asn Thr Glu Gly Lys

675 680 685

Phe Phe Ala Tyr Lys Ile Asp Thr Leu Asn Glu Val Pro Phe Asp Val

690 695 700

Asn Lys Phe Ala Glu Leu Val Thr Asn Ser Pro Val Ile Ser Ala Ile

705 710 715 720

Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr

725 730 735

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

740 745 750

Asn Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu Gln

755 760 765

Leu Ile Leu Gln Cys Lys Leu

770 775

<210> 5

<211> 397

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 5

Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp

1 5 10 15

Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln

20 25 30

Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr

35 40 45

Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly

50 55 60

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

65 70 75 80

Ala Arg Thr Thr Ile Glu Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys

85 90 95

Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Val Ala Pro Gln

100 105 110

Ser Glu Ala Leu Arg Lys Leu Ala Gly Ile Lys Phe Lys Arg Ile Asn

115 120 125

Phe Asn Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg

130 135 140

Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr

145 150 155 160

Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu

165 170 175

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

180 185 190

Phe Asp Tyr Ser Cys Ala Leu Asn Ala Pro Ala Asn Ile Gln Gln Phe

195 200 205

Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr

210 215 220

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

225 230 235 240

Ala Asp Gly Ala Thr Thr Trp Phe Phe Asn Pro Ile Ile Leu Arg Pro

245 250 255

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

260 265 270

Tyr Gln Ala Arg Phe Gly Thr Ile Val Ala Arg Asn Phe Asp Thr Ile

275 280 285

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

290 295 300

Asn Ala Leu Phe Pro Gln Ala Gln Pro Phe Gln His His Ala Thr Val

305 310 315 320

Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala

325 330 335

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

340 345 350

Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr

355 360 365

Glu Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg

370 375 380

Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys

385 390 395

<210> 6

<211> 326

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 6

Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr Phe Leu Ile Ser Ile

1 5 10 15

Val Leu Leu Asn Tyr Ile Leu Lys Ser Leu Thr Ser Ala Met Asp Phe

20 25 30

Ile Ile Tyr Arg Phe Leu Leu Leu Ile Val Ile Leu Ser Pro Phe Val

35 40 45

Lys Thr Gln Asn Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp

50 55 60

Thr Ala Tyr Ala Asn Ser Ser Gln Gln Glu Thr Phe Leu Thr Ser Thr

65 70 75 80

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

85 90 95

Glu Trp Lys Asn Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro

100 105 110

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

115 120 125

Ile Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Val Val Leu Met Lys His

130 135 140

Asp Ser Thr Leu Glu Leu Asp Met Ser Glu Leu Ala Asp Leu Ile Leu

145 150 155 160

Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln

165 170 175

Gln Thr Asp Glu Ser Asn Lys Trp Ile Ser Met Gly Gln Ser Cys Thr

180 185 190

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

195 200 205

Thr Thr Asn Thr Ala Thr Phe Glu Glu Val Ala Thr Ser Glu Lys Leu

210 215 220

Val Ile Thr Asp Val Val Asp Gly Val Asn His Lys Leu Asp Val Thr

225 230 235 240

Thr Asn Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu

245 250 255

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

260 265 270

Ala Asp Pro Thr Thr Thr Pro Gln Thr Glu Arg Met Met Arg Val Asn

275 280 285

Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Val Val Asp Tyr Ile Asn

290 295 300

Gln Ile Val Gln Val Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala

305 310 315 320

Ala Phe Tyr Tyr Arg Val

325

<210> 7

<211> 486

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 7

Met Ala Thr Phe Lys Asp Ala Cys Tyr Tyr Tyr Lys Arg Ile Asn Lys

1 5 10 15

Leu Asn His Ala Val Leu Lys Leu Gly Val Asn Asp Thr Trp Arg Thr

20 25 30

Ser Pro Pro Thr Lys Tyr Lys Gly Trp Cys Leu Asp Cys Cys Gln His

35 40 45

Thr Asp Leu Thr Tyr Cys Arg Gly Cys Thr Met Tyr His Val Cys Gln

50 55 60

Trp Cys Ser Gln Tyr Asp Arg Cys Phe Leu Asp Asn Gln Pro His Leu

65 70 75 80

Leu Arg Met Arg Thr Phe Lys Asn Glu Val Thr Lys Asn Asp Leu Met

85 90 95

Asn Leu Ile Asp Met Tyr Asn Val Leu Phe Pro Ile Asn Gln Arg Ile

100 105 110

Val Asp Lys Phe Ile Asn Ser Thr Arg Gln His Lys Cys Arg Asn Glu

115 120 125

Cys Met Thr Gln Trp Tyr Asn His Leu Leu Met Pro Ile Thr Leu Gln

130 135 140

Ser Leu Ser Ile Glu Leu Asp Gly Asp Val Tyr Tyr Val Phe Gly Tyr

145 150 155 160

Tyr Asp Ser Met Ser Asp Ile Asn Gln Thr Pro Phe Ser Phe Thr Asn

165 170 175

Leu Ile Gly Met Tyr Asp Lys Leu Leu Leu Asp Asn Val Asn Phe Asn

180 185 190

Arg Met Ser Phe Leu Pro Val Ala Leu Gln Gln Glu Tyr Ala Leu Arg

195 200 205

Tyr Phe Ser Lys Ser Arg Phe Ile Ser Glu Lys Arg Lys Cys Ile Ser

210 215 220

Asp Leu His Phe Ser Thr Asn Val Ile Glu Asn Leu His Asn Pro Ser

225 230 235 240

Phe Lys Ile Gln Ile Thr Arg Asn Cys Ser Glu Leu Ser Ser Asp Trp

245 250 255

Asn Gly Ala Cys Lys Leu Val Lys Asp Met Ser Thr Tyr Phe Asn Val

260 265 270

Leu Lys Thr Ser His Ile Glu Phe Tyr Ser Ile Ser Thr Arg Cys Arg

275 280 285

Ile Phe Thr Gln His Lys Leu Lys Ile Ala Ser Lys His Ile Lys Pro

290 295 300

Asn Tyr Val Thr Ser Asn His Lys Thr Ser Ala Thr Glu Val His Asn

305 310 315 320

Cys Lys Trp Cys Ser Ile Asn Asn Gly Tyr Thr Val Trp Asn Asp Phe

325 330 335

Arg Ile Lys Lys Ile Tyr Asp Asn Ile Phe Asn Phe Leu Arg Ala Leu

340 345 350

Val Lys Ser Asn Ser Asn Ile Gly His Cys Ser Ser Gln Glu Lys Ile

355 360 365

Tyr Glu His Ile Lys Gly Val Leu Asp Ile Cys Asp Asp Glu Lys Trp

370 375 380

Lys Met Ala Val Ala Glu Ile Phe Asn Cys Leu Glu Pro Val Glu Leu

385 390 395 400

Asp Thr Val Lys Tyr Val Leu Phe Asn His Glu Val Asn Trp Asp Val

405 410 415

Ile Asn Leu Leu Val Gln Ser Val Gly Lys Val Pro Gln Ile Leu Thr

420 425 430

Leu Asn Asp Ile Val Ile Ile Met Lys Ser Ile Ile Tyr Asp Trp Phe

435 440 445

Asp Ile Arg Tyr Met Arg Asn Thr Pro Met Thr Thr Phe Thr Val Asp

450 455 460

Lys Leu Arg Gln Leu Cys Thr Gly Val Lys Thr Val Asp Tyr Asp Ser

465 470 475 480

Gly Ile Ser Asp Val Glu

485

<210> 8

<211> 317

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 8

Met Ala Glu Leu Ala Cys Phe Cys Tyr Pro His Leu Glu Asn Asp Ser

1 5 10 15

Tyr Lys Phe Ile Pro Phe Asn Asn Leu Ala Ile Lys Cys Met Leu Thr

20 25 30

Ala Lys Ile Asp Lys Lys Asp Gln Asp Lys Phe Tyr Asn Ser Ile Val

35 40 45

Tyr Gly Ile Ala Pro Pro Pro Gln Phe Arg Lys Arg Tyr Asn Thr Ser

50 55 60

Asp Asn Ser Arg Gly Met Asn Tyr Glu Thr Ile Met Phe Asn Lys Val

65 70 75 80

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

85 90 95

Glu Val Ala Asn Val Leu Ser Arg Val Val Ser Val Arg His Leu Glu

100 105 110

Asn Leu Val Leu Arg Lys Glu Asn His Gln Asp Val Leu Phe His Ser

115 120 125

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

130 135 140

Glu Ile Glu Thr Thr Ala Thr Ala Glu Gly Gly Glu Ile Val Phe Gln

145 150 155 160

Asn Ala Ala Phe Thr Met Trp Lys Leu Thr Tyr Leu Asp His Lys Leu

165 170 175

Met Pro Ile Leu Asp Gln Asn Phe Ile Glu Tyr Lys Ile Thr Leu Asn

180 185 190

Glu Asp Lys Pro Ile Ser Asp Val Cys Val Lys Glu Leu Val Ala Glu

195 200 205

Leu Arg Trp Gln Tyr Asn Arg Phe Ala Ile Ile Thr His Gly Lys Gly

210 215 220

His Tyr Arg Val Val Lys Tyr Ser Ser Val Ala Asn His Ala Asp Arg

225 230 235 240

Val Phe Ala Thr Tyr Lys Asn Asn Ala Lys Ser Gly Asn Thr Thr Asp

245 250 255

Phe Asn Leu Leu Asp Gln Arg Ile Ile Trp Gln Asn Trp Tyr Ala Phe

260 265 270

Thr Ser Ser Met Lys Gln Gly Asn Thr Ile Asp Val Cys Lys Lys Leu

275 280 285

Leu Phe Gln Lys Met Lys Gln Glu Lys Asn Pro Phe Lys Gly Leu Ser

290 295 300

Thr Asp Arg Lys Met Asp Glu Val Ser His Val Gly Ile

305 310 315

<210> 9

<211> 310

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Glu Ser Thr Gln Gln Met Val Ser Ser Ile Ile Asn Thr Ser Phe

1 5 10 15

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

20 25 30

Gln Tyr Asp Tyr Asn Glu Val Phe Thr Arg Val Lys Ser Lys Phe Asp

35 40 45

Tyr Val Met Asp Asp Ser Gly Val Lys Asn Asn Leu Leu Gly Lys Ala

50 55 60

Ile Thr Ile Asp Gln Ala Leu Asn Gly Lys Phe Gly Ser Ala Ile Arg

65 70 75 80

Asn Arg Asn Trp Met Thr Asp Ser Lys Thr Val Ala Lys Leu Asp Glu

85 90 95

Asp Val Asn Lys Leu Arg Met Thr Leu Ser Ser Lys Gly Ile Asp Gln

100 105 110

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

115 120 125

Lys Ser Ser Ser Ile Ile Lys Cys Thr Lys Leu Met Lys Asp Lys Ile

130 135 140

Glu Arg Gly Glu Val Glu Val Asn Asp Ser Tyr Ile Asp Glu Lys Met

145 150 155 160

Glu Ile Asp Thr Ile Asp Trp Lys Ser Arg Tyr Asp Gln Leu Glu Lys

165 170 175

Arg Phe Glu Ser Leu Lys Gln Arg Val Thr Glu Lys Tyr Asn Thr Trp

180 185 190

Val Gln Lys Ala Lys Lys Val Asn Glu Asn Met Tyr Ser Leu Gln Asn

195 200 205

Val Ile Ser Gln Gln Gln Asn Gln Ile Ala Asp Leu Gln Gln Tyr Cys

210 215 220

Asn Lys Leu Glu Thr Asp Leu Gln Gly Lys Phe Ser Ser Leu Val Ser

225 230 235 240

Ser Val Glu Trp Tyr Leu Arg Ser Met Glu Leu Pro Asp Asp Val Lys

245 250 255

Asn Asp Ile Glu Gln Gln Leu Asn Ser Ile Asp Leu Ile Asn Pro Ile

260 265 270

Asn Ala Ile Asp Asp Ile Glu Ser Leu Val Arg Asn Leu Val Gln Asp

275 280 285

Tyr Asp Arg Thr Phe Leu Met Leu Lys Gly Leu Leu Lys Gln Cys Asn

290 295 300

Tyr Glu Tyr Val Tyr Glu

305 310

<210> 10

<211> 175

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Glu Lys Leu Thr Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu

1 5 10 15

Met Asn Ser Thr Leu His Thr Ile Leu Glu Asp Pro Gly Met Ala Tyr

20 25 30

Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys

35 40 45

Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser

50 55 60

Tyr Lys Val Val Lys Tyr Cys Ile Val Thr Ile Phe Asn Thr Leu Leu

65 70 75 80

Lys Leu Ala Gly Tyr Lys Glu Gln Ile Thr Thr Lys Asp Glu Ile Glu

85 90 95

Lys Gln Met Asp Arg Val Val Lys Glu Met Arg Arg Gln Leu Glu Met

100 105 110

Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys

115 120 125

Arg Ile Tyr Asp Lys Leu Met Val Gln Ser Val Gly Glu Ile Asp Met

130 135 140

Thr Lys Glu Ile Asn Gln Lys Asn Val Lys Thr Leu Glu Glu Trp Glu

145 150 155 160

Ser Gly Arg Asn Pro Tyr Glu Pro Lys Glu Val Thr Ala Ala Met

165 170 175

<210> 11

<211> 197

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 11

Met Ser Leu Ser Ile Asp Val Thr Ser Leu Pro Ser Ile Ser Ser Ser

1 5 10 15

Ile Phe Lys Asn Glu Ser Ser Ser Thr Thr Ser Thr Leu Ser Gly Lys

20 25 30

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

35 40 45

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

50 55 60

Ser Ala Ser Asn Asp Pro Leu Thr Asn Phe Ser Ile Arg Ser Asn Ala

65 70 75 80

Val Lys Thr Asn Ala Asp Ala Gly Val Ser Met Asp Ser Ser Thr Gln

85 90 95

Ser Arg Pro Ser Ser Asn Val Gly Cys Asp Gln Leu Asp Phe Ser Leu

100 105 110

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

115 120 125

Ser Thr Asp His Lys Lys Glu Lys Ser Lys Lys Asp Lys Ser Arg Lys

130 135 140

His Tyr Pro Arg Ile Glu Ala Asp Ser Asp Ser Glu Asp Tyr Val Leu

145 150 155 160

Asp Asp Ser Asp Ser Asp Asp Gly Lys Cys Lys Asn Cys Lys Tyr Lys

165 170 175

Lys Lys Tyr Phe Ala Leu Arg Met Arg Met Lys Arg Val Ala Met Gln

180 185 190

Leu Ile Glu Asp Leu

195

<210> 12

<211> 3302

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

ggctattaaa gctgtacaat ggggaagtac aatctaatct tgtcagaata tttatcattt 60

gtttacaatt cacaatctgc agttcaaata ccaatttatt attcttccaa tccagaatta 120

gaaaaaagat gtattgaatt tcatgctaaa tgtgttgaca attctaaaaa aggtatatca 180

ttaaaacctg tatttgaaga gtataaagat gtaatagata atgcaacgtt actatctata 240

ttatcatatt cttatgataa atacaacgct gtggaacgga aactagtcaa ttatgctaaa 300

ggtaaacctt tagaggctga tttaacggca aacgagcttg attatgaaaa taacaaaata 360

acttctgaac tgtttcagtc agctgaagaa tataccgatt cattaatgga tcctgctata 420

ctaacttcgt tatcttcaaa tttaaatgca gttatgtttt ggttagaacg acactcaaat 480

gacgttgctg atgcaaataa aatttataag cgtagactag atttatttac catagtggca 540

tctacaataa ataaatacgg agtacccaga cataatgaaa aatacagata tgaatacgaa 600

gtgatgaagg ataaaccgta ttacttagta acttgggcta actcatctat agaaatgctt 660

atgtcagtgt tttcacatga ggattattta atagcaaaag agttaataat cttatcatat 720

tcaaatagat caacgttagc aaaactagtt tcatctccaa tgtcaatatt agttgcatta 780

atagatatca atggtacgtt tattacgaat gaagaattgg aacttgagtt ttcggataaa 840

tatgttaagg caattgtacc tgatcaaatt ttcgacgaat tacaggaaat gattgacaat 900

atgaagaaag ttggtttagt agacatacca agaatgattc aagaatggtt agttgattgt 960

tcattagaga aatttacact gatgtcaaaa atttattctt ggtcatttca tgttggtttt 1020

agaaaacaaa aaatgattga tgcagcatta gaccaattga agacagagta cactgaagat 1080

gtagatggtg agatgtacaa tgagtacaca atgttaatta gagatgaaat agttaaaatg 1140

ttagaagtac cagttaaaca tgacgatcat ctacttcgtg attcagaatt agctggattg 1200

ttatcaatgt catcagcttc aaatggtgaa tcaaggcaac ttaaatttgg tcgtaaaaca 1260

atattttcaa ctaagaaaaa tatgcatgtt atggatgata ttgcacatgg aaagtatact 1320

cctggtgtca ttcctccagt aaacgtagat agaccaattc cactgggtcg tagagatgtt 1380

cctggacgaa gaacaagaat tatattcata ttgccatacg aatactttat tgcgcagcac 1440

gctgtcgtag aaaaaatgtt atcatacgca aagcatacta gagagtatgc ggaattttac 1500

tcacagtcaa atcaattgct atcatacggt gatgttacaa gattcttatc cagtaattct 1560

atggtgttat acacagacgt ttcacaatgg gattcgtcac aacataacac acagccattt 1620

agaaaaggaa taattatggg tttagatatg ttatctaata tgactaatga tccaaaagta 1680

atacaaacgc taaatttata taaacaaaca caagttaatc tcatggattc atatgttcaa 1740

atacctgatg gtaatgtaat caaaaagatt cagtatggtg ctgttgcttc aggagaaaaa 1800

caaactaagg cagccaattc tatagctaat ttggcactca ttaaaacagt attgtcaagg 1860

attgcaaata aatattcttt tataaccaaa ataatcagag ttgatggtga tgataattat 1920

gcagtactac aatttaacac cgatgttact aaacaaatgg tccaagatgt gtcaaacgat 1980

gtgagacata tatattctag aatgaatgct aaagttaaag cattggtatc tacagtcggt 2040

attgaaatag caaaaagata tatagctgga ggaaaaatat tttttagagc tggtataaac 2100

ttattaaata atgagaagcg tggacaaagt acacaatggg atcaagcagc tattttatat 2160

tcgaactaca ttgttaacaa attacgagga tttgagactg atagagaatt tatattaact 2220

aaaattatac aaatgacatc tgtagccatt actggatcac taaggctatt tccgtcagaa 2280

cgagtgttaa ctactaattc tacattcaaa gtatttgact cagaagattt cattatagag 2340

tatggaacaa ctgacgatga agtatatata cagagagcat ttatgtcatt gtctagtcaa 2400

aagtcaggaa tagctgatga aattgcctct tcacaaacat tcaaaaatta tgttaataaa 2460

ttgtctgacc aactactaat atcaaaaaac gtaattgtat ccaaaggtat agctgtaaca 2520

gaaaaagcaa aattgaattc atatgcacca gtttatttag aaaaacgccg tgcacaaata 2580

tcagcgctat taactatgtt acagaagcca gtgtcattta aatcaaataa aattactatt 2640

aatgacattt tgcgtgacat aaaaccattt tttgttactt ctgaagccaa tttgtcaatt 2700

caatacagaa aatttatgcc aacattacct gataacgtcc aatatgttat acaatgtata 2760

ggatcaagga cgtatcagat agaagatagt gggtcgaaat catcgatttc gaagttaata 2820

tcaaaatact cagtttacaa accatcaatt gaggaactat ataaagttat atctttacga 2880

gaacaagaaa tacagttgta tttagtttca ctaggagttc caccagttga tgcaggcaca 2940

tacgtcggat caagaatata ttctcaagac aaatataaaa tattagaatc ttacgtatac 3000

aatttattat caattaatta tggatgttat caattattcg attttaattc tccagattta 3060

gagaaactta tacgaattcc ttttaaaggt aagataccag ccgtaacatt tatattacat 3120

ctttacgcaa aacttgaaat aataaatcat gccattaaga atggagcatg gattagtttg 3180

ttctgtaatt atccaaaatc tgagatgatt aaactatgga agaaaatgtg gaatataaca 3240

gcattacggt ctccctatac tagtgcgaat ttctttcaag attagagtgc ttagatgtga 3300

cc 3302

<210> 13

<211> 2729

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

ggctattaaa ggctcaatgg cgtacaggaa gcgcggagct aaacgtgaag acttaccaca 60

acaaaatgaa cgtctgcaag aaaaagaaat tgaaaataat acagacgtaa ctatggaaaa 120

taaaaataaa aatagaaata ataatagaaa gcagcaatta tctgacaaag tgctatcaca 180

aaaagaagaa ataataactg atgtacaaga tgatattaaa atagctgatg aggttaaaaa 240

atcatcaaag gaggagtcga aacagttact tgaaatatta aaaacgaaag aagatcatca 300

gaaagaagta caatacgaaa ttctacaaaa aacaataccg acttttgaac caaaagaatc 360

aattttgaaa aaattagagg atataaaacc agagcaagct aagaagcaaa tgaaattatt 420

tagaatcttt gaaccaaggc aattaccaat ataccgagca aatggtgaaa aagaattaag 480

gaatagatgg tattggaaat tgaaaaagga tacgctacca gacggagatt atgacgtacg 540

cgagtatttt ttaaatttat atgatcaaat actgatagaa atgccagatt atttattgct 600

aaaagatatg gctgtagaaa ataaaaactc tagggatgct ggtaaagttg tagattctga 660

gactgcaagt atttgtgatg ctatatttca ggatgaggaa acagagggag ttatcagaag 720

atttatcgca gatatgagac aacaagttca ggccgacaga aatattgtca attatccatc 780

aattttacat ccaattgatc atgcgtttaa cgaatatttt ctgaaccatc aattagttga 840

accattaaat aatgacatta tttttaatta tataccagaa agaataagaa atgacgtcaa 900

ctatattttg aatatggata tgaatttgcc atcaacagca agatatatta gaccaaatct 960

attgcaagat agactaaatt tacatgataa ttttgaatca ttatgggata caataactac 1020

gtcaaattac atattagcaa gatcagtcgt gcctgatttg aaggaaaaag aattagtttc 1080

aactgaagct cagatacaga aaatgtctca agatttgcag cttgaagctt taacaataca 1140

atctgaaacg cagtttctgg ccggcataaa ttcacaagca gcaaatgatt gttttaaaac 1200

actgatagca gctatgttaa gccaacgtac aatgtcatta gattttgtaa ctacgaatta 1260

tatgtcactt atatctggca tgtggctatt gactgttata ccaaatgata tgtttcttcg 1320

tgagtcgcta gttgcatgcg aattggctat aataaacact atagtttacc cagcatttgg 1380

aatgcaaaga atgcattata gaaatggcga tcctcagact ccatttcaaa tagcagagca 1440

acaaatacaa aattttcaag tagctaattg gttacatttt attaataata atagatttag 1500

acaagttgtt attgatggag tgttaaatca aacactaaat gacaacatta gaaatggaca 1560

agttattaat caattaatgg aagcattaat gcagttatcc agacaacaat ttccgactat 1620

gccagttgat tataaaagat caatccaaag aggaatattg ttattatcta atagattggg 1680

tcagttagtt gatttaacaa gattattatc gtataattat gaaactctaa tggcttgtat 1740

aactatgaac atgcaacatg ttcaaactct cactaccgaa agattacagt taacttctgt 1800

cacatcctta tgtatgttaa ttggaaatac cacagtaatt ccaagtccac aaacgctatt 1860

tcactattat aacgtaaatg taaattttca ttcaaattat aatgaacgaa ttaacgatgc 1920

tgtggctatt attacggctg ctaatagact aaacttatat cagaaaaaaa tgaaatcaat 1980

agtcgaggac tttttaaaaa gattgcaaat ttttgatgta cctcgagtac cagatgatca 2040

aatgtatagg ttgagagaca gacttagatt attgccagtt gaaagacgaa gacttgatat 2100

atttaactta atattaatga acatggagca gatcgaacga gcttcagata aaattgctca 2160

aggagtaata attgcttaca gagatatgca actagaaaga gatgagatgt atggatttgt 2220

taacatcgct aggaacctcg atggatatca acaaatcaat ttagaagagt taatgagaac 2280

tggagactat ggtcaaatta ctaatatgct attaaacaac cagcctgtag ctttggtagg 2340

agcattgcca tttgtgaccg attcatcagt tatatcactc attgcaaaat tggatgccac 2400

agtttttgct caaatagtta aacttagaaa agtggacact ttaaaaccga tattatataa 2460

aataaattct gattctaatg atttctactt agttgcaaat tatgattgga taccgacttc 2520

aaccacaaaa gtttacaagc aagtaccaca accttttgat ttcagagcgt caatgcacat 2580

gttaacgtct aatttaactt ttaccgttta ttctgattta ctatctttcg tttctgcaga 2640

cacggttgaa cctattaacg caattgcttt tgacaatatg cgcattatga acgaactgta 2700

aacgccaacc ccactgtgga gatatgacc 2729

<210> 14

<211> 2591

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

ggctattaaa gcaatactag tagtgcgttt tacctctgat ggtgtaagca tgaaagtatt 60

agctttaaga cacagtgtgg ctcaggtgta tgcagatacg caaacttact tgcatgacga 120

ttcgaaagat gagtatgaaa atgcatttct catttctaat ctaactacac ataacatatt 180

atatttaaat tatagtctta aaacattaaa aatattaaat aaatcaggca tagcggcagt 240

tgaagtacaa tctccagatg aattatttgc tttaataaga tgtaatttta catatgatta 300

tgagaataac ataatttatt tacatgatta ttcatattat actaacaatg agataagaac 360

agatcaacat tggataacta aaaccaacat aactgaatat ctactacctg gatggaaatt 420

aacatatgta ggatataatg gaaaaaatac acgtggtcac tataatttct catttttatg 480

tcaaaatgca gctacagatg acgatataat aattgaatat atatattcca atgaattaga 540

ttttcaaaat tttctgttaa gaaaaattaa agagagaatg actacatctc ttccaattgc 600

tagattgtca aatcgggtgt ttagagataa attatttcca tctattgtga acatgtataa 660

aaaagtgata aatgttggac caagaaacga atccatgttc acattcttaa atttcccaac 720

tattaagcaa ttttcaaacg gtgcatacat cgtaaaacat actattaaat taaaacaaga 780

gaaatggctg ggtaaaagag tgtcacaatt tgacattgga caatataaaa atatgttaaa 840

tgtagttact accatctatt actactataa tttataccat tcgaaaccaa taatatacat 900

gctcggttca gctccatctt attggattta tgatattaaa cagtattctg attttacatt 960

tgaaacatgg gatccattag acactccata ttctacaata catcataagg aattattttt 1020

tgataaggac gtcaataaac ttaaagataa ttcagttcta tatattgata tacgaactga 1080

taggggaaat atggattgga aagagtggag aaaagtagtg gaacagcaaa ctatcaacaa 1140

tctaaatatt gcatataaat atctagcaac aggaaaagcc aaagtgtgtt gtgttaaatt 1200

aacagctatg gatctagaat tgccaatcac agcaaaatta cttcatcatc caactactga 1260

agttagatca gaattttatg caatcttaga tgtatgggat attattacta ttaaaagatt 1320

tataccaaag ggtgtatttt atgcttttat aaataatata atcacagaga atgtgtttat 1380

acaaccttct tttaaattga aagtatcacc tactgattat atagtggcat tgtatgcttt 1440

atctaacgat tttaattcaa gacaagatgt tattaattta attaataaac aaaaacaatc 1500

gcttattact gttcgtatga ataacacctt taaagacgaa cctaaagtaa attttaaaaa 1560

tatttatgat tggacgtttc tgccaacaga tttcgaactt aaagattcga taattacatc 1620

gtatgatggt tgtttaggca tgtttggatt atcgatatct ctatcttcta aacctactgg 1680

taataatcat ttatttataa taaatggaac tgacaaatat gataaactag atcagtatgc 1740

aaatcatatg ggtatttcta ggagatcaca tcaaattaga ttttcagagt ctgcgacatc 1800

atattcaggg tatattttta gagatctttc aaataataat ttcaacttga taggtactaa 1860

tgtagaaaat tcagtttcag gacatgttta taatgcattg atttactaca ggtataatta 1920

tgcatttgat cttaaaagat ggatttattt acactcaatt ggaaaagccg ctgtggaagg 1980

tggaagatat tatgaacatg caccaataga attgatatat gcatgccggt cagctaaaga 2040

atttgctata ttgcaagatg acttaacggt attgagatac gctaatgaaa ttgaagagta 2100

cataaacaaa gtatatagta taacttatgc tgatgatcct aattacttta taggaattaa 2160

atttaacagt ataccttatg aatatgatgt taaagttcca catttgacgt taggagtact 2220

atttatatct gataatatga tacatgatgt agttacagta ttgaagaaaa tgaagacaga 2280

actatttaag acggaaatta gtactagtta cacttatatg ctatctgata atatatatgt 2340

agcaaatgcc agcggcgtgt tatcaacgta ttttaaatta tataatatgt tttacagaaa 2400

tcacatcacg tttggacaat cgcgaatgtt tattccacat attacactga gttttagtaa 2460

taaacaaaca gttagaatag aaagcacaag actcaagatt aattcaattt acttaagaaa 2520

gattaaaggg gagaccgtat ttgatatgtc tgagtgagct aaaaacttaa cacactagtc 2580

atgatgtgac c 2591

<210> 15

<211> 2359

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

ggctataaaa tggcttcgct catttataga cagcttctca ctaattcata ttcagtagat 60

ttatatgatg aaatgaagca aattggatca gaaaaaactc aaaatgtaac tataaaccca 120

ggtccatttg ctcaaacaag atatgctcca gtcaattggg gtcatggaga aataaatgat 180

tcaactacag tggaaccaat tttagatggt ccttatcaac ctactacatt tacaccacct 240

actgattact ggatacttat taactcaaat acaaatggag tagtatacga gagtacaaat 300

aatagtgact tttggactgc agtcattgct gttgaaccgc acgtcgatcc agtagataga 360

caatataatg tatttggtga aaataaacaa tttaatgtaa ggaatgattc agataaatgg 420

aagtttttag aaatgtttag aggcagtagt caaagtgact tttataatag acgtacacta 480

acttctgata ctaaactcgt aggaatatta aaatatggtg gaagaatatg ggcatttcat 540

ggtgaaacac caagggctac taccgatagc tcaaacactg caaatttgaa cggtatatca 600

attacaattc attcagaatt ttatattatt ccaagatccc aagagtctaa gtgtaatgaa 660

tatattaata acggtctacc accaattcaa aatactagaa atgtagtacc attatcatta 720

tcatctagat ctatacagta tacgagagca caagttaatg aagacattac aatttcaaag 780

acttcattat ggaaagaaat gcaacataac agagatatta taattaggtt taaatttggc 840

aatagtatta ttaaattagg gggattgggt tataaatggt ctgaaatatc atataaagca 900

gcgaactatc aatataatta tttacgtgat ggtgaacaag taactgcaca tactacctgt 960

tcagtaaacg gagtaaataa ttttagctac aacggaggat ctttgcccac tgattttagt 1020

atttcaagat atgaagttat taaagaaaat tcctatgtat acgtagatta ttgggacgat 1080

tcaaaagcat ttcgaaatat ggtgtatgtt agatcattag cagctaattt aaactcagtg 1140

aaatgtacag gtggaagtta ccattttagt atacctgtgg gtgcatggcc agttatgaat 1200

ggaggtgccg tttcgttgca tttcgctggt gttacattat ctacgcaatt cacagatttc 1260

gtatcattaa attcattacg gtttagattt agtttgacag tagatgaacc atctttttca 1320

atattgagaa cacgtacggt aaatttatat ggattaccag ctgctaatcc aaataatgga 1380

aatgaatatt acgaaatatc aggaaggttt tcgcttattt ctctagttcc aaccaacgat 1440

gattatcaaa ctccaattat gaattcagta acagtaagac aagatttaga acgtcaacta 1500

actgatttac gagaggaatt caattcatta tcacaagaaa tagctatgtc acaattaatt 1560

gatttagcgt tattaccttt agacatgttt tctatgtttt cagggattaa aagcacaatt 1620

gatctaacca aatcaatggc gactagcgta atgaaaaaat ttagaaaatc aaagttagct 1680

acatcagttt cagaaatgac taactcattg tcagacgcag catcatcagc atcaagaagc 1740

gtttctgtta gatcgaatat atccgcattt tcaaactgga ctaatgtttc aaatgatgta 1800

tcaaatgtga ctaattcagt aaatgatatt tcaacgcaaa catctacgat cagtaaaaac 1860

cttagattaa gagaaatgat cactcaaact gaaggaatga gttttgatga tatttcagca 1920

gcagtactaa aaacgaaaat agatatgtct actcaaattg ggaaaaatac tttacctgac 1980

atagtcacag aggcatctga gaaatttatt ccaaaacgct cgtatcgaat actaaaagat 2040

gatgaagtaa tggaaattaa tactgaagga aaattctttg catataaaat tgatacactt 2100

aatgaagtgc catttgatgt aaataaattt gctgaacttg taacaaattc cccagttata 2160

tcagcaataa tcgattttaa aacattgaag aatttgaatg ataattatgg aattacccga 2220

actgaagcgt taaatttaat taaatcaaat ccaaatgtat tacgtaattt tattaaccag 2280

aataatccaa ttataaggaa tagaattgaa cagttaattc tacaatgtaa gttgtgagag 2340

cgctattgag gatgtgacc 2359

<210> 16

<211> 1356

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

ggcttttaaa cgaagtcttc gacatggagg ttctgtattc attgtcaaaa actcttaaag 60

atgctaggga taagattgtt gaaggtacat tatattctaa tgttagtgat cttattcagc 120

aatttaatca aatgatagta accatgaacg gaaatgactt tcaaactgga ggaattggca 180

atttacccat tagaaattgg acattcgact ttggtctact aggaactacg cttttaaatc 240

ttgatgctaa ttatgttgag accgccagaa ctacgattga gtattttatt gatttcattg 300

ataatgtatg tatggatgaa atggcaagag agtctcaaag aaatggagta gctccacaat 360

ctgaggcatt gagaaagcta gccggtatta aatttaaaag aataaacttt aataattcat 420

cagaatatat agaaaattgg aatttacaaa atagaagaca acgtactgga tttgtttttc 480

ataaacctaa tatatttcca tactcagcat catttacttt aaatagatct caaccaatgc 540

atgacaattt aatgggaact atgtggctta atgctggatc agaaattcaa gtggctggat 600

ttgactattc gtgtgctcta aacgctccag caaatattca gcaatttgaa catatcgtcc 660

agctaaggcg tgcgctaact acagctacta taactttgtt acctgatgca gaaagattta 720

gttttccaag agttattaat tcagcagatg gcgcaactac atggttcttt aatccaatca 780

tcctaagacc aaacaacgta gaagtagaat ttttactgaa tggacaaatt attaatacat 840

atcaagctag atttggcact attgtcgcaa gaaatttcga tacaattcgt ctatcattcc 900

aattaatgcg tccaccaaac atgacgccag ccgtaaatgc attatttccg caagcacaac 960

cttttcaaca tcatgcaaca gttggactta cgttacgtat tgagtctgca gtttgtgaat 1020

cagtgcttgc ggatgcaaat gaaactttat tggcgaatgt taccgcagta cgtcaagagt 1080

atgctatacc agttggacca gtatttccac caggcatgaa ttggactgag ctgattacta 1140

actattcacc atctagggaa gataatttgc aacgtgtctt tacagtagcc tctatcagaa 1200

gcatgttgat taagtgagga ccagactaac catctggtat ctaatcttag ttagcatgta 1260

gctatatcaa gtcattcaga ctctataagt aaggacatgg ctccatgttc gctacgtaga 1320

gtaactgtat gaatgatata gtgagaggat gtgacc 1356

<210> 17

<211> 1061

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 17

ggctttaaaa gagagaattt ccgtttggct agcggttagc tccttttaat gtatggtatt 60

gaatatacca cagttctaac ctttctgata tcaatagttt tattgaacta catattaaaa 120

tcactaacta gtgcgatgga ctttataatt tatagatttc ttttacttat tgttattttg 180

tcgccatttg tcaaaacaca aaattatggg ataaatttac caattactgg ctccatggac 240

acagcatatg caaattcgtc acagcaagaa acattcttaa cttcaacgct atgtttatat 300

tatcctactg aagcatcaac tcaaattgga gatactgaat ggaagaatac tctatctcaa 360

ttattcttga ctaaggggtg gccaactgga tcagtctatt ttagagaata tacagatatc 420

gcttctttct caattgatcc acaactttat tgtgattata atgttgtgct aatgaagcat 480

gattcaacgt tagagctaga tatgtctgaa ttagctgatt tgattctaaa tgaatggtta 540

tgcaatccaa tggatataac attatattat tatcagcaaa cagatgaatc gaataaatgg 600

atatcgatgg gacaatcttg taccataaaa gtgtgcccat taaatacaca aacttcagga 660

ataggttgta ctactacaaa tacagcgaca tttgaagaag tagctactag tgagaaatta 720

gtgataactg atgttgttga tggcgtgaat cataaacttg atgtaactac aaatacctgt 780

acaattagaa attgtaagaa gttaggaccg agagaaaatg tagcaattat acaagtcggt 840

ggctcagaag tgttagatat tacagcggat ccaactacca caccacaaac tgagcgtatg 900

atgcgagtaa attggaagaa atggtggcaa gttttctata cagtagtaga ttacattaat 960

cagattgtgc aagttatgtc caaaagatca cggtcattaa attcagcagc tttttattat 1020

agagtctgat atatcttagg ttagaattgg tcgatgtgac c 1061

<210> 18

<211> 1061

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 18