CN114621357A

CN114621357A - 一种带状疱疹亚单位疫苗及其制备方法

Info

Publication number: CN114621357A
Application number: CN202210531577.XA
Authority: CN
Inventors: 张建刚; 司伟雪; 李晓勇; 高荣荣; 苗伟; 朱涛; 邵忠琦; 宇学峰; 毛慧华
Original assignee: CanSino Biologics Inc
Current assignee: CanSino Biologics Inc
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-06-14

Abstract

本发明公开了一种带状疱疹亚单位疫苗及其制备方法和应用。其中，方法包括：将IFNα序列连接至gE膜外区段序列的N端，Fc序列连接至gE膜外区段序列的C端，得到融合蛋白IFNα‑gE‑Fc；本发明制备的带状疱疹亚单位疫苗有效解决了gE亚单位疫苗在实际应用中遇到的半衰期短、免疫原性较弱的问题，为开发安全有效的新型带状疱疹提供候选疫苗。本发明的融合蛋白具有较好的免疫原性，可诱导产生高水平的体液免疫和细胞免疫。因而是具有潜在临床应用价值的疫苗。

Description

一种带状疱疹亚单位疫苗及其制备方法

技术领域

本发明属于生物疫苗技术领域，尤其涉及一种带状疱疹亚单位疫苗及其制备方法。

背景技术

带状疱疹的病原体为水痘-带状疱疹病毒（varicella zoster virus，VZV）。迄今只发现一种血清型，在自然界VZV仅感染人类。VZV的初次感染一般发生在幼儿期，以水痘为主要临床表现，该病毒有极高的自然感染率，据报到全世界＞50岁人群中约有99%的血清VZV呈阳性反应。病毒感染人体后潜伏于机体神经节的后神经元内，呈隐匿性感染。当机体免疫力下降时，病毒再次活化并大量复制，引起以红斑、簇集分布的水泡以及神经痛为主要特征的带状疱疹（病毒学报，36卷，第1期）。

VZV属疱疹病毒亚科，呈球形，直径150~200nm。核衣壳呈二十面体立体对称，最外层是包膜，有糖蛋白刺突，与病毒吸附和穿入宿主细胞有关。VZV基因组为124884bp的线性双链DNA分子，由共价键链接的长片段（L）和短片段（S）组成。长片段约105kb，两端有小片段反向重复序列（TRL和IRL），短片段约5kb，两端有大片段反向重复序列（TRS和IRS），其结构模式为TRL-UL-IRL-IRS-US-TRS。

VZV基因组由71个开放读码框（open readingframe，ORF）组成，约编码69种蛋白，其中ORF42和ORF45接合转录、翻译成一种蛋白产物。另外，ORF62、ORF63、ORF64在TRS和TRS区分别重复一次即形成ORF71、ORF70、ORF69，因此VZV基因组实际包含69个独特基因，编码的糖蛋白（glycoprotein）有8个，分别为gB、gC、gE、gH、gI、gK、gL、gM。

其中gE在病毒包膜含量最丰富，是宿主免疫系统识别的最主要糖蛋白。由ORF68基因编码，位于VZV基因组的短片区，含623个氨基酸，其中亲水膜外区544个，跨膜区17个，胞内区62个。已有研究表明以gE为抗原诱导的免疫反应，能保护动物免受病毒攻击。VZV gE单克隆抗体可介导抗体依赖性的细胞毒性，能中和病毒的感染性。目前的挑战是gE仅可以诱导低水平的体液免疫，无法满足疫苗使用的要求。

干扰素（interferon，IFN）是由不同种细胞分泌生成的具有免疫活性的蛋白质，具有重要的病毒防御和免疫调节功能。根据干扰素抗原性的不同，分成三类二型。三类是：白细胞干扰素（IFN-α）、成纤维干扰素（IFN-β）和免疫干扰素（IFN-γ）；IFN分为Ⅰ型和Ⅱ型两个型别，其中Ⅰ型IFN包括IFN-α和IFN-β，Ⅱ型IFN有IFN-γ。目前己知人类IFN-a可分为2个亚型，20多种。I型干扰素因干扰病毒复制而得名，在临床上成功治疗多种病毒引起的疾病，例如，病毒性肝炎（乙肝，丙肝）、单纯疱疹。IFN-α是能刺激机体抵抗病毒作用的重要细胞因子之一，刺激宿主细胞合成抗病毒蛋白，控制病毒的增殖和扩散；亦可通过激活自然杀伤性细胞（natural killer cell，NK cell）和巨噬细胞（Macrophages，mø）的方法，杀伤破坏被病毒定位的靶细胞，从而发挥抗病毒作用。IFN-α家族在先天免疫和适应性免疫的桥梁中发挥重要作用，目前已应用于新型冠状病毒疫苗中，且在临床试验中取得了耀眼的效果。

Fc受体在许多先天性免疫细胞表面都有表达，免疫球蛋白的Fc段与免疫细胞的Fc受体结合可以发挥多种生物学功能，如介导炎症反应、抗体依赖细胞介导的吞噬作用（ADCP）、补体依赖的细胞毒作用（CDC）、抗体依赖细胞介导的细胞毒作用（ADCC）、促进树突状细胞（DC）成熟等。Fc段同抗原融合后，可以增强抗原免疫呈递效果。Fc段的二硫键链接形成稳定的二聚体，不仅可以提高抗原分子的稳定性，而且增大了融合蛋白分子体积，使抗原更容易被免疫系统识别的同时还延长了抗原在体内的半衰期。Fc段可以特异性结合protein A，提高了Fc融合蛋白的纯化效率。截止2014年9月，已有9种人IgG-Fc融合蛋白药物经美国食品及药品监督管理局（FDA）批准临床使用。

发明内容

针对现有带状疱疹疫苗技术的不足，本发明的目的是提供一种带状疱疹亚单位疫苗及其制备方法，该疫苗既能诱导体液免疫保护，同时又能诱导细胞免疫保护。

本发明将人免疫球蛋白的Fc段、VZV病毒gE的膜外区段和人的IFNα通过linker连接，编码上述融合蛋白的基因插入到真核细胞表达载体pCEP4中，转染CHOS细胞，成功的表达出融合蛋白IFNα-gE-Fc。融合蛋白经Protein A亲和色谱、离子交换色谱、分子筛色谱纯化后，得到高纯度的IFNα-gE-Fc融合蛋白，可诱导小鼠产生高水平的体液免疫和细胞免疫。

为了达到上述目的，本发明采用的技术方案：

本发明第一方面，提供一种融合蛋白，其中包括人免疫球蛋白Fc段氨基酸序列、VZV病毒gE膜外区段氨基酸序列和人IFNα氨基酸序列形成的融合蛋白。

优选的，所述融合蛋白组合方式为IFNα-gE-Fc。

优选的，所述IFNα氨基酸序列、gE膜外区段氨基酸序列和Fc氨基酸序列通过linker连接肽连接，更优选的，所述的融合蛋白linker为GGGGSGGGGSGGGGS和/或EAAAKEAAAKEAAAK。

优选的，所述融合蛋白，其氨基酸序列为SEQ ID No:13所示。

本发明第二方面，提供一种重组基因，其编码上述任一的融合蛋白。

优选的，所述重组基因为DNA或者mRNA。

优选的，所述重组基因包括编码人IFNα的基因序列、编码水痘-带状疱疹病毒（VZV）糖蛋白E（gE）膜外区段的基因序列和编码人免疫球蛋白（IgG）Fc区的基因序列。

本发明将经密码子优化编码人IFNα的基因序列，记为IFNα基因序列，如序列SEQID NO:11所示；将经密码子优化编码水痘-带状疱疹病毒（VZV）糖蛋白E（gE）膜外区段的基因序列，记为gE膜外区段基因序列，如序列SEQ ID NO:1所示；将经密码子优化编码人免疫球蛋白（IgG）Fc区的基因序列，记为Fc基因序列，如序列SEQ ID NO:2所示；

进一步优选的，所述重组基因的DNA序列为SEQ ID No:12。

本发明第三方面，提供一种上述融合蛋白的制备方法，所述制备方法包括步骤：

S1.将所述融合蛋白IFNα-gE-Fc的编码基因导入到表达载体，得到重组质粒；

S2.将所述重组质粒转染细胞，获得高表达IFNα-gE-Fc重组蛋白的细胞株；

S3.培养所述的高表达IFNα-gE-Fc重组蛋白的细胞株，收集发酵物上清进行纯化处理，得到的融合蛋白。

优选的，所述的融合蛋白表达载体为pCEP4，将所述融合蛋白基因IFNα-gE-Fc导入pCEP4质粒，得到重组质粒pCEP4-IFNα-gE-Fc。

优选的，所述的融合蛋白表达系统为CHOS，将所述重组质粒pCEP4-IFNα-gE-Fc转染至CHOS细胞，将能够表达IFNα-gE-Fc重组蛋白的CHOS细胞作为初代IFNα-gE-Fc-CHOS细胞。

进一步地将，初代IFNα-gE-Fc-CHOS细胞用Hygromycin B加压筛选，通过cellpool的方式获得高表达IFNα-gE-Fc重组蛋白的CHOS细胞。

优选的，所述的融合蛋白采用ProteinA亲合色谱粗纯，Q Sepharose 4Fast Flow和Sephacryl S300进行精纯。

优选的，所述的融合蛋白采用抗鼠gE抗体和抗鼠Fc抗体鉴定结果，更优选的，采用Western blot进行鉴定。

本发明第四方面，提供一种上述融合蛋白或者上述重组基因在制备带状疱疹亚单位疫苗中的应用。

本发明第五方面，提供一种带状疱疹亚单位疫苗，所述带状疱疹亚单位疫苗包括上述任一的融合蛋白或者上述任一的重组基因。

优选的，所述的带状疱疹疫苗，其中每个剂量含有融合蛋白10~100μg。

优选的，所述带状疱疹亚单位疫苗还含有铝佐剂。

本发明提供的带状疱疹亚单位疫苗，将Oka株VZV糖蛋白gE膜外区段融合了人免疫球蛋白Fc段，将gE膜外区段与免疫球蛋白Fc融合表达形成二聚体结构，提高了抗原的稳定性，活化了树突状细胞（DC）的抗原呈递作用，同时还提高了纯化效率。进一步融合了Ⅰ型干扰素IFNα，诱导了Th1偏向的细胞免疫，改善了传统佐剂的不足。本发明提供的带状疱疹亚单位疫苗有效解决了gE亚单位疫苗在临床应用中遇到的半衰期短、免疫原性较弱的问题，为开发安全有效的带状疱疹疫苗提供候选疫苗。

附图说明

图1为Protein A纯化后目的蛋白SDS-PAGE图（表达载体筛选）。

图2为Protein A纯化后目的蛋白SDS-PAGE图（连接肽筛选）。

图3为Protein A纯化后IFNα-gE-Fc融合蛋白SDS-PAGE图。

图4为Protein A纯化后IFNα-gE-Fc融合蛋白鼠抗gE Western blot图。

图5为Protein A纯化后IFNα-gE-Fc融合蛋白鼠抗Fc Western blot图。

图6为第一次免疫后的第28天各组小鼠血清中IgG滴度图。

图7为第二次免疫后的第14天各组小鼠血清中IgG滴度图。

图8为各组小鼠CD4+T细胞免疫应答测定结果图。

图9为各组小鼠CD8+T细胞免疫应答测定结果图。

具体实施方式

除非另有定义，本发明中所使用的技术术语与本发明涉及技术领域的技术人员通常理解的含义相同。下面将结合本发明实施例，对本发明进一步详细描述。

实施例1.融合蛋白表达载体的筛选

选择NCBI中Oka株VZV糖蛋白gE膜外区段和人免疫球蛋白（IgG）Fc段蛋白基因序列，根据哺乳动物细胞偏爱的密码子进行密码子优化。由连接肽GGGGSGGGGSGGGGS将Fc序列连接至gE膜外区段序列的C端，得到融合蛋白基因gE-Fc。通过分子生物学技术分别构建至pCDNA3.1、pCEP4、pEE12.4-gE-Fc、pCHO1.0表达载体，获得四种重组质粒。所述四种重组质粒分别命名为pCDNA3.1-gE-Fc、pCEP4-gE-Fc、pEE12.4-gE-Fc、pCHO1.0-gE-Fc。

涉及到的序列如下：

编码水痘-带状疱疹病毒（VZV）糖蛋白E（gE）膜外区段的基因序列，记为gE膜外区段序列，如序列表SEQ ID NO:1所示；编码人免疫球蛋白（IgG）Fc区的基因序列，记为Fc序列，如序列表SEQ ID NO:2所示；gE-Fc融合蛋白DNA序列如序列表SEQ ID NO:3所示；gE-Fc融合蛋白氨基酸序列如序列表SEQ ID NO:4所示。

将四种重组质粒pCDNA3.1-gE-Fc、pCEP4-gE-Fc、pEE12.4-gE-Fc、pCHO1.0-gE-Fc用PCR和酶切鉴定后测序。由PCR鉴定结果、酶切鉴定结果和测序结果鉴定无误后，以终浓度1μg/μl储存于TE Buffer缓冲液中备用。分别取30~60μg pCDNA3.1-gE-Fc、pCEP4-gE-Fc、pEE12.4-gE-Fc、pCHO1.0-gE-Fc重组质粒与1ml ExpiCHO™ Expression轻轻充分混匀，记为溶液1（根据上述质粒不同，依次记为1-1，1-2，1-3，1-4）；取80~120μl ExpiFectamineCHO Reagent与880~920μl预冷的ExpiCHO™ Expression轻轻充分混匀，时间不超过5min，记为溶液2，共4份；溶液1（1-1，1-2，1-3，1-4）对应加入溶液2中，静置3~4min，混合物缓慢滴加入已准备好的细胞中（2~6×10^6 cell/ml）；在转染后24h补加150μl ExpiCHO Enhancer和6ml ExpiCHO Feed，第3天、第7天、第10天补加1ml 5% Cell Boost 5，第12~14天将细胞培养液经8000rpm离心5~10min，收集上清，0.45μm滤膜过滤，将滤液流经20mM PB（pH7.0~7.5）预先平衡的Protein A凝胶色谱柱Mabselect Sure，之后用20mM PB平衡3~5个柱体积至A280回到基线水平；将流动相换为50mM乙酸钠-乙酸缓冲液（pH2.0~4.0）洗脱结合物，将收集到的洗脱物立即用1M Tris调节pH至7.0~7.5，经0.22μm滤膜除菌过滤后保存备用。

通过SDS-PAGE电泳测试表达载体pCDNA3.1、pCEP4、pEE12.4、pCHO1.0对目的蛋白表达能力和二聚体形成能力，结果如图1所示。表达载体pCEP4对目的蛋白的表达量高，且形成二聚体的比例高。后续实验选用pCEP4作为表达载体。

实施例2.融合蛋白间连接肽的筛选

选择NCBI中Oka株VZV糖蛋白gE膜外区段和人免疫球蛋白（IgG）Fc段蛋白基因序列，根据哺乳动物细胞偏爱的密码子进行密码子优化。由连接肽GGGGSGGGGSGGGGS[（G₄S）₃]、EAAAKEAAAKEAAAK[（EAAAK）₃]、APAPAPAPAPAPAPAP[(AP)₈]、KESGSVSSEQLAQFRSLD（scFv）将Fc序列连接至gE序列的C端，得到4种融合蛋白基因gE-(G₄S)₃-Fc、gE-(EAAAK)₃-Fc、gE-(AP)₈-Fc、gE-scFv-Fc。所述融合基因分别通过分子生物学技术分别构建至pCEP4载体，获得四种重组质粒。所述四种重组质粒分别命名为pCEP4-gE-(G₄S)₃-Fc、pCEP4-gE-(EAAAK)₃-Fc、pCEP4-gE-(AP)₈-Fc、pCEP4-gE-scFv-Fc。

涉及到的序列如下：

编码水痘-带状疱疹病毒（VZV）糖蛋白E（gE）膜外区段的基因序列，记为gE膜外区段基因序列，如序列SEQ ID NO:1所示；编码人免疫球蛋白（IgG）Fc区的基因序列，记为Fc基因序列，如序列SEQ ID NO:2所示；gE-(G₄S)₃-Fc融合蛋白DNA序列如序列SEQ ID NO:3所示；gE-(G₄S)₃-Fc融合蛋白氨基酸序列如序列SEQ ID NO:4所示；gE-(EAAAK)₃-Fc融合蛋白DNA序列如序列SEQ ID NO:5所示；gE-(EAAAK)₃-Fc融合蛋白氨基酸序列如序列SEQ ID NO:6所示；gE-(AP)₈-Fc融合蛋白DNA序列如序列SEQ ID NO:7所示；gE-(AP)₈-Fc融合蛋白氨基酸序列如序列SEQ ID NO:8所示；gE-scFv-Fc融合蛋白DNA序列如序列SEQ ID NO:9所示；gE-scFv-Fc融合蛋白氨基酸序列如序列SEQ ID NO:10所示。

将四种重组质粒pCEP4-gE-(G₄S)₃-Fc、pCEP4-gE-(EAAAK)₃-Fc、pCEP4-gE-(AP)₈-Fc、pCEP4-gE-scFv-Fc用PCR和酶切鉴定后测序。由PCR鉴定结果、酶切鉴定结果和测序结果鉴定无误后，以终浓度1μg/μl储存于TE Bμffer缓冲液中备用。分别取30~60μg pCEP4-gE-(G₄S)₃-Fc、pCEP4-gE-(EAAAK)₃-Fc、pCEP4-gE-(AP)₈-Fc、pCEP4-gE-scFv-Fc重组质粒与1mlExpiCHO™ Expression轻轻充分混匀，记为溶液3（根据上述质粒不同，依次记为3-1，3-2，3-3，3-4）；取80~120μl ExpiFectamine CHO Reagent与880~920μl预冷的ExpiCHO™Expression轻轻充分混匀，时间不超过5min，记为溶液4，共4份；溶液3（3-1，3-2，3-3，3-4）对应加入溶液4中，静置3~4min，混合物缓慢滴加入已准备好的细胞中（2~6×10^6 cell/ml）；在转染后24h补加150μl ExpiCHO Enhancer和6ml ExpiCHO Feed，第3天、第7天、第10天补加1ml 5% Cell Boost 5，第12~14天将细胞培养液经8000rpm离心5~10min，收集上清，0.45μm滤膜过滤，将滤液流经20mM PB（pH7.0~7.5）预先平衡的Protein A凝胶色谱柱Mabselect Sure，之后用20mM PB平衡3~5个柱体积至A280回到基线水平；将流动相换为50mM乙酸钠-乙酸缓冲液（pH2.0~4.0）洗脱结合物，将收集到的洗脱物立即用1M Tris调节pH至7.0~7.5，经0.22μm滤膜除菌过滤后保存备用。

通过SDS-PAGE电泳测试连接肽GGGGSGGGGSGGGGS、EAAAKEAAAKEAAAK、APAPAPAPAPAPAPAP、KESGSVSSEQLAQFRSLD对目的蛋白表达能力和二聚体形成能力，结果如图2所示。表达连接肽GGGGSGGGGSGGGGS、EAAAKEAAAKEAAAK对目的蛋白的表达量高，且形成二聚体的比例高。后续实验可以选用GGGGSGGGGSGGGGS和/或EAAAKEAAAKEAAAK作为融合蛋白连接肽。

实施例3.目的基因的合成

根据NCBI中Oka株VZV糖蛋白gE膜外区段、人干扰素（IFNα）和人免疫球蛋白（IgG）Fc段蛋白基因序列，根据哺乳动物细胞偏爱的密码子进行密码子优化，由连接肽GGGGSGGGGSGGGGS和/或EAAAKEAAAKEAAAK将IFNα序列连接至gE膜外区段序列的N端，Fc序列连接至gE膜外区段序列的C端，得到融合蛋白基因IFNα-gE-Fc。所述融合蛋白基因IFNα-gE-Fc通过分子生物学技术构建至pCEP4载体，获得重组质粒pCEP4-IFNα-gE-Fc。

涉及到的序列如下：

编码人IFNα的基因序列，记为IFNα基因序列，如序列SEQ ID NO:11所示；

编码水痘-带状疱疹病毒（VZV）糖蛋白E（gE）膜外区段的基因序列，记为gE膜外区段基因序列，如序列SEQ ID NO:2所示；

编码人免疫球蛋白（IgG）Fc区的基因序列，记为Fc基因序列，如序列SEQ ID NO:3所示；

编码IFNα-gE-Fc融合蛋白的DNA序列如序列SEQ ID NO:12所示；

IFNα-gE-Fc融合蛋白氨基酸序列如序列SEQ ID NO:13所示；

将所述的重组质粒pCEP4-IFNα-gE-Fc用PCR和酶切鉴定后测序。由PCR鉴定结果、酶切鉴定结果和测序结果鉴定无误后，以终浓度1μg/μl储存于TE Buffer缓冲液中备用。

实施例4.目的基因转染及表达

取工作库CHOS细胞株1支，迅速于37℃水浴锅融化后，移至生物安全柜中，拧开冻存管盖，用移液管将细胞全部吸入预先加入9ml ExpiCHO™ Expression培养基的离心管中，800rpm离心5min；弃掉上清，沉淀用9ml预热的ExpiCHO™ Expression培养基重悬，全部移入预先加有21ml ExpiCHO™ Expression培养基的125ml三角摇瓶，37℃、5% CO₂、120rpm培养箱培养。

培养3天后，移至生物安全柜中，进行第一次细胞传代。拧开三角摇瓶盖，用移液管将细胞培养液全部吸入50ml离心管中，800rpm离心5min；弃掉上清，沉淀用20ml预热的ExpiCHO™ Expression培养基重悬，全部移入250ml三角摇瓶，补加40ml ExpiCHO™Expression培养基，37℃、5% CO2、120rpm培养箱培养。连续传代3次以上，细胞用于转染。

取30~60μg质粒与1ml ExpiCHO™ Expression轻轻充分混匀，记为溶液5；取80~120μl ExpiFectamine CHO Reagent与880~920μl预冷的ExpiCHO™ Expression轻轻充分混匀，时间不超过5min，记为溶液6；溶液5加入溶液6中，静置3~4min，混合物缓慢滴加入已准备好的细胞中（2~6×10^6 cell/ml）；

在转染后第24h和96h，分别添加补料初始体积5%的Cell Boost 5，将能够表达IFNα-gE-Fc重组蛋白的CHOS细胞作为初代IFNα-gE-Fc-CHOS细胞。将初代IFNα-gE-Fc-CHOS细胞接种至24孔板，使用cell pool筛选培养基进行Hygromycin B加压筛选。cell pool筛选完成后，收集所有cell pool的上清用鼠抗gE进行ELSIA检测表达水平。选择高表达gE的cell pool采用有限稀释法进行单克隆筛选，选高表达IFNα-gE-Fc融合蛋白且产量稳定的一株建立细胞建库。将所述的高表达IFNα-gE-Fc融合蛋白的CHOS细胞培养预定时间后，收集发酵物上清。

上述Hygromycin B加压筛选经多批次筛选，可重复获得高表达IFNα-gE-Fc融合蛋白的CHOS细胞。

实施例5.目的蛋白纯化

将发酵物清经8000rpm离心5~10min，收集上清，0.45μm滤膜过滤，将滤液流经20mMPB（pH7.0~7.5）预先平衡的Protein A凝胶色谱柱Mabselect Sure，之后用20mM PB平衡3~5个柱体积至A280回到基线水平；将流动相换为50mM乙酸钠-乙酸缓冲液（pH2.0~4.0）洗脱结合物，将收集到的洗脱物立即用1M Tris调节pH至7.0~7.5，经0.45μm滤膜过滤进一步除去不溶性颗粒。之后将收获的含目标产物的蛋白质溶液加载到经20mM PB（pH7.0~7.5）缓冲液平衡好的Q Sepharose 4Fast Flow，加载完成后，使用20mM PB缓冲液平衡色谱柱至A280回到基线水平，依次用不同浓度的NaCl（0~0.5M）溶液进行梯度洗脱，收集目标蛋白IFNα-gE-Fc。由于表达后的蛋白是被糖基化的，SDS-PAGE电泳显示分子量在247kDa左右呈弥散性，如图3所示。将收获的IFNα-gE-Fc再经以生理盐水为流动相的分子筛色谱Sephacryl S300 HR纯化，收集目标产物IFNα-gE-Fc，经0.22μm滤膜除菌过滤后保存备用。

实施例6.IFNα-gE-Fc融合蛋白Western blot检测（鼠抗gE）

将Protein A凝胶色谱柱粗纯的IFNα-gE-Fc融合蛋白，经8％SDS-PAGE分离蛋白后，电转至PVDF膜上，用5％脱脂奶粉（100ml TBST中加入5g脱脂奶粉）于37℃封闭2h或者4℃中过夜封闭；加入经5％脱脂奶粉以1：5000~8000稀释后的一抗鼠抗gE，放置室温孵育2h或者4℃中过夜孵育，用TBST洗涤3次，每次5~10min；加入经5％脱脂奶粉以1：5000~8000稀释后的二抗兔抗鼠IgG，放置37℃中孵育1h，用TBST洗涤6次，每次5~10min；按照显色液试剂盒说明书配制显色液，加到PVDF膜上，荧光扫描仪扫描保存。鼠抗gE Western blot图显示分子量在247kDa左右呈弥散性，如图4所示。

实施例7.IFNα-gE-Fc融合蛋白Western blot检测（鼠抗Fc）

将Protein A凝胶色谱柱粗纯的IFNα-gE-Fc融合蛋白，经8％SDS-PAGE分离蛋白后，电转至PVDF膜上，用5％脱脂奶粉（100ml TBST中加入5g脱脂奶粉）于37℃封闭2h或者4℃中过夜封闭；加入经5％脱脂奶粉以1：5000~8000稀释后的一抗鼠抗Fc，放置室温孵育2h或者4℃中过夜孵育，用TBST洗涤3次，每次5~10min；加入经5％脱脂奶粉以1：5000~8000稀释后的二抗兔抗鼠IgG，放置37℃中孵育1h，用TBST洗涤6次，每次5~10min；按照显色液试剂盒说明书配制显色液，加到PVDF膜上，荧光扫描仪扫描保存。鼠抗Fc Western blot图显示分子量在247kDa左右呈弥散性，如图5所示。

实施例8. gE、Flagellin-gE-Fc抗原制备

gE、Flagellin-gE-Fc融合蛋白抗原分别按照实施例3~7进行制备。gE膜外区段蛋白DNA序列如序列表SEQ ID NO:1所示；gE膜外区段蛋白氨基酸序列如序列表SEQ ID NO:14所示；Flagellin-gE-Fc融合蛋白DNA序列如序列表SEQ ID NO:15所示，Flagellin-gE-Fc融合蛋白氨基酸序列如序列表SEQ ID NO:16所示。

实施例9.融合蛋白的免疫效果评价

选取SPF级，18~22g，6~8周左右C57BL/6雌性小鼠32只随机分组。本实验共设4组（表1），4组实验组，1组阴性对照组，每组8只小鼠。每只小鼠独立编号，血清对应给出编号。采用TCA法对融合蛋白进行蛋白定量，每个实验组gE含量为5μg。

表1 实验动物分组

实验样品准备好后，采用腿部肌肉注射法，按照表2的免疫流程对小鼠进行免疫。

表2 免疫流程

按照表2免疫流程，一免4周后进行二免，二免2周后摘取眼球采全血，采血后处死小鼠分离脾脏细胞。将采集好的全血4℃条件下8000rpm离心6min，分离血清，放置于-20℃以下。血清分装保存，避免反复冻融。小鼠血清用于ELISA检测抗原特异性总IgG。脾脏细胞用于ICS（CD4+T、CD8+T）测定评价。

检测结果如图6、图7、图8、图9所示。

图6显示了第一次免疫后的第28天，各组小鼠的血清中IgG的滴度，生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠的血清IgG滴度分别为1.0×10^2、6.6×10^2、1.8×10^4、1.4×10^4。结果显示，IFNα-gE-Fc组小鼠的血清IgG滴度高于生理盐水组和gE组，具有极显著性差异(P＜0.0001)；IFNα-gE-Fc组小鼠的血清IgG滴度和Flagellin-gE-Fc组无显著差异（P=0.5853）。

图7显示了第二次免疫后的第14天，各组小鼠的血清中IgG的滴度生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠的血清IgG滴度分别为1.0×10^2、2.4×10^5、3.4×10^5、3.2×10^5。结果显示，IFNα-gE-Fc组小鼠的血清IgG滴度高于生理盐水组和gE组，具有极显著性差异(P＜0.0001)；IFNα-gE-Fc组小鼠的血清IgG滴度和Flagellin-gE-Fc组无显著差异（P=0.9875）。

实验结果表明，本发明的IFNα-gE-Fc融合蛋白对小鼠进行免疫后，能够增强体液免疫，提高抗体的滴度。本发明的IFNα-gE-Fc融合蛋白的体液免疫效果明显优于游离gE重组蛋白，与Flagellin-gE-Fc融合蛋白无差异。

图8显示了第二次免疫后的第14天，各组小鼠生产TNF-α、IFN-γ、IL2、IL4和IL5的细胞在总CD4+细胞中的占比。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产TNF-α的细胞在总CD4+细胞中的占比分别为0.019%、0.020%、0.048%、0.18%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内TNF-α表达量高于生理盐水组和gE组，具有极显著性差异(P＜0.001)；IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内TNF-α表达量高于Flagellin-gE-Fc组，具有极显著性差异(P＜0.01)（参见图8左上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IFN-γ的细胞在总CD4+细胞中的占比分别为0.018%、0.013%、0.010%、0.29%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内IFN-γ表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.0001)（参见图8中上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL2的细胞在总CD4+细胞中的占比分别为0.010%、0.031%、0.036%、0.34%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内IL2表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.0001)（参见图8右上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL4的细胞在总CD4+细胞中的占比分别为0.015%、0.015%、0.015%、0.10%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内IL4表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.001)（参见图8左下）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL5的细胞在总CD4+细胞中的占比分别为0.014%、0.020%、0.016%、0.30%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内IL5表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.001)（参见图8中下）。

实验结果表明，本发明的IFNα-gE-Fc融合蛋白对小鼠进行免疫后，能够增加CD4+淋巴T细胞内TNF-α、IFN-γ、IL2、IL4和IL5的表达量，从而增强T细胞介导的细胞免疫效果。

图9显示了第二次免疫后的第14天，各组小鼠生产TNF-α、IFN-γ、IL2、IL4和IL5的细胞在总CD8+细胞中的占比。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产TNF-α的细胞在总CD8+细胞中的占比分别为0.010%、0.035%、0.062%、0.23%。结果显示，IFNα-gE-Fc组小鼠的CD8+淋巴T细胞内TNF-α表达量高于生理盐水组和gE组，具有极显著性差异(P＜0.0001)；IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内TNF-α表达量高于Flagellin-gE-Fc组，具有极显著性差异(P＜0.001)（参见图9左上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IFN-γ的细胞在总CD8+细胞中的占比分别为0.015%、0.022%、0.029%、0.085%。结果显示，IFNα-gE-Fc组小鼠的CD8+淋巴T细胞内IFN-γ表达量高于生理盐水组和gE组，具有显著性差异(P＜0.05)；IFNα-gE-Fc组小鼠的CD8+淋巴T细胞内IFN-γ表达量和Flagellin-gE-Fc组无显著差异（P=0.0518）（参见图9中上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL2的细胞在总CD8+细胞中的占比分别为0.010%、0.032%、0.012%、0.32%。结果显示，IFNα-gE-Fc组小鼠的CD8+淋巴T细胞内IL2表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.001)（参见图9右上）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL4的细胞在总CD8+细胞中的占比分别为0.014%、0.012%、0.022%、0.10%。结果显示，IFNα-gE-Fc组小鼠的CD8+淋巴T细胞内IL4表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有显著性差异(P＜0.05)（参见图9左下）。

生理盐水组、gE组、Flagellin-gE-Fc组、IFNα-gE-Fc组小鼠生产IL5的细胞在总CD8+细胞中的占比分别为0.010%、0.011%、0.011%、0.040%。结果显示，IFNα-gE-Fc组小鼠的CD4+淋巴T细胞内IL5表达量高于生理盐水组、gE组和Flagellin-gE-Fc组，具有极显著性差异(P＜0.0001)（参见图9中下）。

实验结果表明，本发明的IFNα-gE-Fc融合蛋白对小鼠进行免疫后，能够增加CD8+淋巴T细胞内TNF-α、IFN-γ、IL2、IL4和IL5的表达量，从而增强T细胞介导的细胞免疫效果。

综上，本发明提供的IFNα-gE-Fc融合蛋白可用于制备预防由于感染水痘-带状疱疹病毒引起的机体反应的疫苗。

以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。

本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。

本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

序列表

<110> 康希诺生物股份公司

<120> 一种带状疱疹亚单位疫苗及其制备方法

<130> 1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1548

<212> DNA

<213> 人工序列(artificial sequence)

<400> 1

agcgtgctga gatacgacga cttccacatc gacgaggaca agctggacac caacagcgtg 60

tacgagccct actaccacag cgaccacgcc gagagcagct gggtgaacag aggcgagagc 120

agcagaaagg cctacgacca caacagcccc tacatctggc ctagaaacga ctacgacggc 180

ttcctggaga acgcccacga gcaccacggc gtgtacaacc aaggcagagg catcgacagc 240

ggcgagagac tgatgcagcc cacacagatg agcgcccaag aggacctggg cgacgacacc 300

ggcatccacg tgatccccac cctgaacggc gacgacagac acaagatcgt gaacgtggat 360

cagagacagt acggcgacgt gttcaagggc gacctgaacc ccaagcccca agggcagaga 420

ctgatcgagg tgagcgtgga ggagaaccac cccttcaccc tgagagcccc cattcagaga 480

atctacggcg tgagatacac cgagacctgg agcttcctgc cctccctgac ctgcaccggc 540

gacgccgccc ccgccattca gcacatctgc ctgaagcaca ccacctgctt ccaagacgtg 600

gtggtcgacg tcgactgcgc cgagaacacc aaggaggatc agctggccga gatcagctac 660

agattccaag gcaagaagga ggccgatcag ccctggatcg tggtgaacac aagcaccctg 720

ttcgacgagc tggagctgga cccccccgag atcgagcccg gcgtgctgaa ggtgctgaga 780

accgagaagc agtacctggg cgtgtacatc tggaacatga gaggcagcga cggcacaagc 840

acctacgcca ccttcctggt gacctggaag ggcgacgaga agacaagaaa ccccaccccc 900

gccgtgaccc ctcagcctag aggcgccgag ttccacatgt ggaactacca cagccacgtg 960

ttcagcgtgg gcgacacctt cagcctggcc atgcacctgc agtacaagat ccacgaggcc 1020

cccttcgacc tgctcctgga gtggctgtac gtgcccatcg accccacctg tcagcccatg 1080

agactgtaca gcacctgcct gtaccacccc aacgcccccc aatgcctgag ccacatgaac 1140

agcggctgca ccttcacaag cccccacctg gctcagagag tggctagcac cgtgtatcag 1200

aactgcgagc acgccgacaa ctacaccgcc tactgcctgg gcatcagcca catggagcct 1260

agcttcggcc tgatcctgca cgacggcggc accaccctga agttcgtgga cacccccgag 1320

agcctgagcg gcctgtacgt gttcgtggtg tacttcaacg gccacgtgga ggccgtggcc 1380

tacaccgtgg tgagcaccgt ggaccacttc gtcaatgcta ttgaggagag aggcttcccc 1440

cccaccgccg ggcagccccc cgccaccaca aagcccaagg agatcacccc cgtgaacccc 1500

ggcacaagcc ccctgatcag atacgccgcc tggaccggcg gcctggcc 1548

<210> 2

<211> 696

<212> DNA

<213> 人工序列(artificial sequence)

<400> 2

gagcccaagt cctgtgacaa gacccacacc tgtcccccct gccctgctcc cgaactgctg 60

ggcggcccta gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 120

acccccggcg tgacctgcgt ggtcgtggac gtgtcccacg aggaccccga ggtgaagttc 180

aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcctag agaggagcag 240

tacaacagca cctacagagt ggtgagcgtg ctgaccgtgc tgcaccaaga ctggctgaac 300

ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgcccccat cgagaagacc 360

atcagcaagg ccaaggggca gcctagagag ccccaagtgt acaccctgcc ccctagcaga 420

gacgagctga ccaagaacca agtgtccctc acctgcctgg tcaagggctt ctaccctagc 480

gacatcgccg tggagtggga gagcaacggg cagcccgaga acaactacaa gaccaccccc 540

cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 600

agatggcagc aaggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacacaga agagcctgag cctgagcccc ggcaag 696

<210> 3

<211> 2382

<212> DNA

<213> 人工序列(artificial sequence)

<400> 3

atgggcaccg tgaacaagcc cgtggtgggc gtgctgatgg gcttcggcat catcaccggc 60

accctgagaa tcaccaaccc cgtgagagct agcgtgctga gatacgacga cttccacatc 120

gacgaggaca agctggacac caacagcgtg tacgagccct actaccacag cgaccacgcc 180

gagagcagct gggtgaacag aggcgagagc agcagaaagg cctacgacca caacagcccc 240

tacatctggc ctagaaacga ctacgacggc ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc aaggcagagg catcgacagc ggcgagagac tgatgcagcc cacacagatg 360

agcgcccaag aggacctggg cgacgacacc ggcatccacg tgatccccac cctgaacggc 420

gacgacagac acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480

gacctgaacc ccaagcccca agggcagaga ctgatcgagg tgagcgtgga ggagaaccac 540

cccttcaccc tgagagcccc cattcagaga atctacggcg tgagatacac cgagacctgg 600

agcttcctgc cctccctgac ctgcaccggc gacgccgccc ccgccattca gcacatctgc 660

ctgaagcaca ccacctgctt ccaagacgtg gtggtcgacg tcgactgcgc cgagaacacc 720

aaggaggatc agctggccga gatcagctac agattccaag gcaagaagga ggccgatcag 780

ccctggatcg tggtgaacac aagcaccctg ttcgacgagc tggagctgga cccccccgag 840

atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtacatc 900

tggaacatga gaggcagcga cggcacaagc acctacgcca ccttcctggt gacctggaag 960

ggcgacgaga agacaagaaa ccccaccccc gccgtgaccc ctcagcctag aggcgccgag 1020

ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggcc 1080

atgcacctgc agtacaagat ccacgaggcc cccttcgacc tgctcctgga gtggctgtac 1140

gtgcccatcg accccacctg tcagcccatg agactgtaca gcacctgcct gtaccacccc 1200

aacgcccccc aatgcctgag ccacatgaac agcggctgca ccttcacaag cccccacctg 1260

gctcagagag tggctagcac cgtgtatcag aactgcgagc acgccgacaa ctacaccgcc 1320

tactgcctgg gcatcagcca catggagcct agcttcggcc tgatcctgca cgacggcggc 1380

accaccctga agttcgtgga cacccccgag agcctgagcg gcctgtacgt gttcgtggtg 1440

tacttcaacg gccacgtgga ggccgtggcc tacaccgtgg tgagcaccgt ggaccacttc 1500

gtcaatgcta ttgaggagag aggcttcccc cccaccgccg ggcagccccc cgccaccaca 1560

aagcccaagg agatcacccc cgtgaacccc ggcacaagcc ccctgatcag atacgccgcc 1620

tggaccggcg gcctggccgg cgggggcggc tccggcgggg gcggcagcgg ggggggcggc 1680

tccgagccca agtcctgtga caagacccac acctgtcccc cctgccctgc tcccgaactg 1740

ctgggcggcc ctagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 1800

agaacccccg gcgtgacctg cgtggtcgtg gacgtgtccc acgaggaccc cgaggtgaag 1860

ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tagagaggag 1920

cagtacaaca gcacctacag agtggtgagc gtgctgaccg tgctgcacca agactggctg 1980

aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcccgcccc catcgagaag 2040

accatcagca aggccaaggg gcagcctaga gagccccaag tgtacaccct gccccctagc 2100

agagacgagc tgaccaagaa ccaagtgtcc ctcacctgcc tggtcaaggg cttctaccct 2160

agcgacatcg ccgtggagtg ggagagcaac gggcagcccg agaacaacta caagaccacc 2220

ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 2280

agcagatggc agcaaggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 2340

cactacacac agaagagcct gagcctgagc cccggcaagt ga 2382

<210> 4

<211> 793

<212> PRT

<213> 人工序列(artificial sequence)

<400> 4

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

545 550 555 560

Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

565 570 575

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

580 585 590

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys Val

595 600 605

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

610 615 620

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

625 630 635 640

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

645 650 655

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

660 665 670

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

675 680 685

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

690 695 700

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

705 710 715 720

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

725 730 735

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

740 745 750

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

755 760 765

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

770 775 780

Lys Ser Leu Ser Leu Ser Pro Gly Lys

785 790

<210> 5

<211> 2382

<212> DNA

<213> 人工序列(artificial sequence)

<400> 5

atgggcaccg tgaacaagcc cgtggtgggc gtgctgatgg gcttcggcat catcaccggc 60

accctgagaa tcaccaaccc cgtgagagct agcgtgctga gatacgacga cttccacatc 120

gacgaggaca agctggacac caacagcgtg tacgagccct actaccacag cgaccacgcc 180

gagagcagct gggtgaacag aggcgagagc agcagaaagg cctacgacca caacagcccc 240

tacatctggc ctagaaacga ctacgacggc ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc aaggcagagg catcgacagc ggcgagagac tgatgcagcc cacacagatg 360

agcgcccaag aggacctggg cgacgacacc ggcatccacg tgatccccac cctgaacggc 420

gacgacagac acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480

gacctgaacc ccaagcccca agggcagaga ctgatcgagg tgagcgtgga ggagaaccac 540

cccttcaccc tgagagcccc cattcagaga atctacggcg tgagatacac cgagacctgg 600

agcttcctgc cctccctgac ctgcaccggc gacgccgccc ccgccattca gcacatctgc 660

ctgaagcaca ccacctgctt ccaagacgtg gtggtcgacg tcgactgcgc cgagaacacc 720

aaggaggatc agctggccga gatcagctac agattccaag gcaagaagga ggccgatcag 780

ccctggatcg tggtgaacac aagcaccctg ttcgacgagc tggagctgga cccccccgag 840

atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtacatc 900

tggaacatga gaggcagcga cggcacaagc acctacgcca ccttcctggt gacctggaag 960

ggcgacgaga agacaagaaa ccccaccccc gccgtgaccc ctcagcctag aggcgccgag 1020

ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggcc 1080

atgcacctgc agtacaagat ccacgaggcc cccttcgacc tgctcctgga gtggctgtac 1140

gtgcccatcg accccacctg tcagcccatg agactgtaca gcacctgcct gtaccacccc 1200

aacgcccccc aatgcctgag ccacatgaac agcggctgca ccttcacaag cccccacctg 1260

gctcagagag tggctagcac cgtgtatcag aactgcgagc acgccgacaa ctacaccgcc 1320

tactgcctgg gcatcagcca catggagcct agcttcggcc tgatcctgca cgacggcggc 1380

accaccctga agttcgtgga cacccccgag agcctgagcg gcctgtacgt gttcgtggtg 1440

tacttcaacg gccacgtgga ggccgtggcc tacaccgtgg tgagcaccgt ggaccacttc 1500

gtcaatgcta ttgaggagag aggcttcccc cccaccgccg ggcagccccc cgccaccaca 1560

aagcccaagg agatcacccc cgtgaacccc ggcacaagcc ccctgatcag atacgccgcc 1620

tggaccggcg gcctggccga ggctgccgcc aaagaagccg ccgctaagga agccgctgcc 1680

aaggagccca agtcctgtga caagacccac acctgtcccc cctgccctgc tcccgaactg 1740

ctgggcggcc ctagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 1800

agaacccccg gcgtgacctg cgtggtcgtg gacgtgtccc acgaggaccc cgaggtgaag 1860

ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tagagaggag 1920

cagtacaaca gcacctacag agtggtgagc gtgctgaccg tgctgcacca agactggctg 1980

aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcccgcccc catcgagaag 2040

accatcagca aggccaaggg gcagcctaga gagccccaag tgtacaccct gccccctagc 2100

agagacgagc tgaccaagaa ccaagtgtcc ctcacctgcc tggtcaaggg cttctaccct 2160

agcgacatcg ccgtggagtg ggagagcaac gggcagcccg agaacaacta caagaccacc 2220

ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 2280

agcagatggc agcaaggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 2340

cactacacac agaagagcct gagcctgagc cccggcaagt ga 2382

<210> 6

<211> 793

<212> PRT

<213> 人工序列(artificial sequence)

<400> 6

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

Leu Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

545 550 555 560

Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

565 570 575

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

580 585 590

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys Val

595 600 605

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

610 615 620

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

625 630 635 640

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

645 650 655

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

660 665 670

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

675 680 685

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

690 695 700

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

705 710 715 720

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

725 730 735

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

740 745 750

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

755 760 765

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

770 775 780

Lys Ser Leu Ser Leu Ser Pro Gly Lys

785 790

<210> 7

<211> 2385

<212> DNA

<213> 人工序列(artificial sequence)

<400> 7

atgggcaccg tgaacaagcc cgtggtgggc gtgctgatgg gcttcggcat catcaccggc 60

accctgagaa tcaccaaccc cgtgagagct agcgtgctga gatacgacga cttccacatc 120

gacgaggaca agctggacac caacagcgtg tacgagccct actaccacag cgaccacgcc 180

gagagcagct gggtgaacag aggcgagagc agcagaaagg cctacgacca caacagcccc 240

tacatctggc ctagaaacga ctacgacggc ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc aaggcagagg catcgacagc ggcgagagac tgatgcagcc cacacagatg 360

agcgcccaag aggacctggg cgacgacacc ggcatccacg tgatccccac cctgaacggc 420

gacgacagac acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480

gacctgaacc ccaagcccca agggcagaga ctgatcgagg tgagcgtgga ggagaaccac 540

cccttcaccc tgagagcccc cattcagaga atctacggcg tgagatacac cgagacctgg 600

agcttcctgc cctccctgac ctgcaccggc gacgccgccc ccgccattca gcacatctgc 660

ctgaagcaca ccacctgctt ccaagacgtg gtggtcgacg tcgactgcgc cgagaacacc 720

aaggaggatc agctggccga gatcagctac agattccaag gcaagaagga ggccgatcag 780

ccctggatcg tggtgaacac aagcaccctg ttcgacgagc tggagctgga cccccccgag 840

atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtacatc 900

tggaacatga gaggcagcga cggcacaagc acctacgcca ccttcctggt gacctggaag 960

ggcgacgaga agacaagaaa ccccaccccc gccgtgaccc ctcagcctag aggcgccgag 1020

ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggcc 1080

atgcacctgc agtacaagat ccacgaggcc cccttcgacc tgctcctgga gtggctgtac 1140

gtgcccatcg accccacctg tcagcccatg agactgtaca gcacctgcct gtaccacccc 1200

aacgcccccc aatgcctgag ccacatgaac agcggctgca ccttcacaag cccccacctg 1260

gctcagagag tggctagcac cgtgtatcag aactgcgagc acgccgacaa ctacaccgcc 1320

tactgcctgg gcatcagcca catggagcct agcttcggcc tgatcctgca cgacggcggc 1380

accaccctga agttcgtgga cacccccgag agcctgagcg gcctgtacgt gttcgtggtg 1440

tacttcaacg gccacgtgga ggccgtggcc tacaccgtgg tgagcaccgt ggaccacttc 1500

gtcaatgcta ttgaggagag aggcttcccc cccaccgccg ggcagccccc cgccaccaca 1560

aagcccaagg agatcacccc cgtgaacccc ggcacaagcc ccctgatcag atacgccgcc 1620

tggaccggcg gcctggccgc tcccgcccct gctcctgccc ccgctcccgc ccccgctcct 1680

gcccccgagc ccaagtcctg tgacaagacc cacacctgtc ccccctgccc tgctcccgaa 1740

ctgctgggcg gccctagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 1800

agcagaaccc ccggcgtgac ctgcgtggtc gtggacgtgt cccacgagga ccccgaggtg 1860

aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcctagagag 1920

gagcagtaca acagcaccta cagagtggtg agcgtgctga ccgtgctgca ccaagactgg 1980

ctgaacggca aggagtacaa gtgcaaggtg agcaacaagg ccctgcccgc ccccatcgag 2040

aagaccatca gcaaggccaa ggggcagcct agagagcccc aagtgtacac cctgccccct 2100

agcagagacg agctgaccaa gaaccaagtg tccctcacct gcctggtcaa gggcttctac 2160

cctagcgaca tcgccgtgga gtgggagagc aacgggcagc ccgagaacaa ctacaagacc 2220

accccccccg tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac 2280

aagagcagat ggcagcaagg caacgtgttc agctgcagcg tgatgcacga ggccctgcac 2340

aaccactaca cacagaagag cctgagcctg agccccggca agtga 2385

<210> 8

<211> 794

<212> PRT

<213> 人工序列(artificial sequence)

<400> 8

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro

545 550 555 560

Ala Pro Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

565 570 575

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

580 585 590

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys

595 600 605

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

610 615 620

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

625 630 635 640

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

645 650 655

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

660 665 670

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

675 680 685

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

690 695 700

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

705 710 715 720

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

725 730 735

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

740 745 750

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

755 760 765

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

770 775 780

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

785 790

<210> 9

<211> 2391

<212> DNA

<213> 人工序列(artificial sequence)

<400> 9

atgggcaccg tgaacaagcc cgtggtgggc gtgctgatgg gcttcggcat catcaccggc 60

accctgagaa tcaccaaccc cgtgagagct agcgtgctga gatacgacga cttccacatc 120

gacgaggaca agctggacac caacagcgtg tacgagccct actaccacag cgaccacgcc 180

gagagcagct gggtgaacag aggcgagagc agcagaaagg cctacgacca caacagcccc 240

tacatctggc ctagaaacga ctacgacggc ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc aaggcagagg catcgacagc ggcgagagac tgatgcagcc cacacagatg 360

agcgcccaag aggacctggg cgacgacacc ggcatccacg tgatccccac cctgaacggc 420

gacgacagac acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480

gacctgaacc ccaagcccca agggcagaga ctgatcgagg tgagcgtgga ggagaaccac 540

cccttcaccc tgagagcccc cattcagaga atctacggcg tgagatacac cgagacctgg 600

agcttcctgc cctccctgac ctgcaccggc gacgccgccc ccgccattca gcacatctgc 660

ctgaagcaca ccacctgctt ccaagacgtg gtggtcgacg tcgactgcgc cgagaacacc 720

aaggaggatc agctggccga gatcagctac agattccaag gcaagaagga ggccgatcag 780

ccctggatcg tggtgaacac aagcaccctg ttcgacgagc tggagctgga cccccccgag 840

atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtacatc 900

tggaacatga gaggcagcga cggcacaagc acctacgcca ccttcctggt gacctggaag 960

ggcgacgaga agacaagaaa ccccaccccc gccgtgaccc ctcagcctag aggcgccgag 1020

ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggcc 1080

atgcacctgc agtacaagat ccacgaggcc cccttcgacc tgctcctgga gtggctgtac 1140

gtgcccatcg accccacctg tcagcccatg agactgtaca gcacctgcct gtaccacccc 1200

aacgcccccc aatgcctgag ccacatgaac agcggctgca ccttcacaag cccccacctg 1260

gctcagagag tggctagcac cgtgtatcag aactgcgagc acgccgacaa ctacaccgcc 1320

tactgcctgg gcatcagcca catggagcct agcttcggcc tgatcctgca cgacggcggc 1380

accaccctga agttcgtgga cacccccgag agcctgagcg gcctgtacgt gttcgtggtg 1440

tacttcaacg gccacgtgga ggccgtggcc tacaccgtgg tgagcaccgt ggaccacttc 1500

gtcaatgcta ttgaggagag aggcttcccc cccaccgccg ggcagccccc cgccaccaca 1560

aagcccaagg agatcacccc cgtgaacccc ggcacaagcc ccctgatcag atacgccgcc 1620

tggaccggcg gcctggccaa ggagagcggc agcgtgagca gcgagcagct ggctcagttc 1680

agaagcctgg acgagcccaa gtcctgtgac aagacccaca cctgtccccc ctgccctgct 1740

cccgaactgc tgggcggccc tagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 1800

atgatcagca gaacccccgg cgtgacctgc gtggtcgtgg acgtgtccca cgaggacccc 1860

gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagcct 1920

agagaggagc agtacaacag cacctacaga gtggtgagcg tgctgaccgt gctgcaccaa 1980

gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcccgccccc 2040

atcgagaaga ccatcagcaa ggccaagggg cagcctagag agccccaagt gtacaccctg 2100

ccccctagca gagacgagct gaccaagaac caagtgtccc tcacctgcct ggtcaagggc 2160

ttctacccta gcgacatcgc cgtggagtgg gagagcaacg ggcagcccga gaacaactac 2220

aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc 2280

gtggacaaga gcagatggca gcaaggcaac gtgttcagct gcagcgtgat gcacgaggcc 2340

ctgcacaacc actacacaca gaagagcctg agcctgagcc ccggcaagtg a 2391

<210> 10

<211> 796

<212> PRT

<213> 人工序列(artificial sequence)

<400> 10

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

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

545 550 555 560

Arg Ser Leu Asp Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

565 570 575

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

580 585 590

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

595 600 605

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

610 615 620

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

625 630 635 640

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

645 650 655

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

660 665 670

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

675 680 685

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

690 695 700

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

705 710 715 720

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

725 730 735

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

740 745 750

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

755 760 765

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

770 775 780

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

785 790 795

<210> 11

<211> 546

<212> DNA

<213> 人工序列(artificial sequence)

<400> 11

atgaagtggg tgaccttcat cagcctgctg ttcagcagcg cctacagctg cgacctgccc 60

gagacccaca gcctggacaa cagaagaacc ctgatgctgc tggctcagat gagcagaatc 120

agccctagca gctgcctgat ggacagacac gacttcggct tcccccaaga ggagttcgac 180

ggcaatcagt ttcagaaggc ccccgccatc agcgtgctgc acgagctgat tcagcagatc 240

ttcaacctgt tcaccaccaa ggacagcagc gccgcctggg acgaggacct gctggacaag 300

ttctgcaccg agctgtatca gcagctgaac gacctggagg cctgcgtgat gcaagaggag 360

agagtgggcg agacccccct gatgaacgcc gacagcatcc tggccgtgaa gaagtacttc 420

agaagaatca ccctgtacct gaccgagaag aagtacagcc cctgcgcctg ggaggtggtg 480

agagccgaga tcatgagaag cctgagcctc agcaccaacc tgcaagagcg gctcagacgg 540

aaggaa 546

<210> 12

<211> 2883

<212> DNA

<213> 人工序列(artificial sequence)

<400> 12

atgaagtggg tgaccttcat cagcctgctg ttcagcagcg cctacagctg cgacctgccc 60

gagacccaca gcctggacaa cagaagaacc ctgatgctgc tggctcagat gagcagaatc 120

agccctagca gctgcctgat ggacagacac gacttcggct tcccccaaga ggagttcgac 180

ggcaatcagt ttcagaaggc ccccgccatc agcgtgctgc acgagctgat tcagcagatc 240

ttcaacctgt tcaccaccaa ggacagcagc gccgcctggg acgaggacct gctggacaag 300

ttctgcaccg agctgtatca gcagctgaac gacctggagg cctgcgtgat gcaagaggag 360

agagtgggcg agacccccct gatgaacgcc gacagcatcc tggccgtgaa gaagtacttc 420

agaagaatca ccctgtacct gaccgagaag aagtacagcc cctgcgcctg ggaggtggtg 480

agagccgaga tcatgagaag cctgagcctc agcaccaacc tgcaagagcg gctcagacgg 540

aaggaaggcg gcgggggcag cggcgggggc gggagcggcg ggggcggcag cagcgtcctg 600

agatacgacg acttccacat cgacgaggac aagctggaca ccaacagcgt gtacgagccc 660

tactaccaca gcgaccacgc cgagagcagc tgggtgaaca gaggcgagag cagcagaaag 720

gcctacgacc acaacagccc ctacatctgg cctagaaacg actacgacgg cttcctggag 780

aacgcccacg agcaccacgg cgtgtacaac caaggcagag gcatcgacag cggcgagaga 840

ctgatgcagc ccacacagat gagcgcccaa gaggacctgg gcgacgacac cggcatccac 900

gtgatcccca ccctgaacgg cgacgacaga cacaagatcg tgaacgtgga tcagagacag 960

tacggcgacg tgttcaaggg cgacctgaac cccaagcccc aagggcagag actgatcgag 1020

gtgagcgtgg aggagaacca ccccttcacc ctgagagccc ccattcagag aatctacggc 1080

gtgagataca ccgagacctg gagcttcctg ccctccctca cctgtaccgg ggacgccgcc 1140

cctgccattc agcacatctg cctgaagcac accacctgct tccaagacgt ggtcgtggat 1200

gtggactgcg ccgagaacac caaggaggat cagctggccg agatcagcta cagattccaa 1260

ggcaagaagg aggccgatca gccctggatc gtggtgaaca caagcaccct gttcgacgag 1320

ctggagctgg acccccccga gatcgagccc ggcgtgctga aggtgctgag aaccgagaag 1380

cagtacctgg gcgtgtacat ctggaacatg agaggcagcg acggcacaag cacctacgcc 1440

accttcctgg tgacctggaa gggcgacgag aagacaagaa accccacccc cgccgtgacc 1500

cctcagccta gaggcgccga gttccacatg tggaactacc acagccacgt gttcagcgtg 1560

ggcgacacct tcagcctggc catgcacctg cagtacaaga tccacgaggc ccccttcgac 1620

ctgctcctgg agtggctgta cgtgcccatc gaccccacct gtcagcccat gagactgtac 1680

agcacctgcc tgtaccaccc caacgcccct cagtgcctga gccacatgaa cagcggctgc 1740

accttcacaa gcccccacct ggctcagaga gtggctagca ccgtgtatca gaactgcgag 1800

cacgccgaca actacaccgc ctactgcctg ggcatcagcc acatggagcc tagcttcggc 1860

ctgatcctgc acgacggcgg caccaccctg aagttcgtgg acacccccga gagcctgagc 1920

ggcctgtacg tgttcgtggt gtacttcaac ggccacgtgg aggccgtggc ctacaccgtg 1980

gtgagcaccg tggaccactt cgtgaatgcc atcgaggaga gaggcttccc ccccaccgct 2040

gggcagcccc ccgccaccac caagcctaag gagatcaccc ccgtgaaccc cggcacctcc 2100

cctctgattc ggtacgctgc ctggaccggc ggcctcgccg gcgggggcgg gagcggcggg 2160

gggggctccg gcggcggggg cagcgagcct aagagctgcg ataaaacaca cacctgtccc 2220

ccttgccccg cccccgagct gctgggcggg cctagcgtgt tcctgttccc ccccaagccc 2280

aaggacaccc tgatgatcag cagaaccccc ggcgtgacct gcgtggtcgt cgacgtcagc 2340

catgaggacc ccgaggtgaa gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 2400

aagaccaagc ctagagagga gcagtacaac agcacctaca gagtggtgag cgtgctgacc 2460

gtgctgcacc aagactggct gaacggcaag gagtacaagt gcaaggtgag caacaaggcc 2520

ctgcccgccc ccatcgagaa gaccatcagc aaggccaagg ggcagcctag agagccccaa 2580

gtgtacaccc tgccccctag cagagacgag ctgaccaaga accaagtgag cctgacctgc 2640

ctcgtgaagg gcttctaccc tagcgacatc gccgtggagt gggagagcaa cgggcagccc 2700

gagaacaact acaagaccac cccccccgtg ctggacagcg acggcagctt cttcctgtac 2760

agcaagctga ccgtggacaa gagcagatgg cagcaaggca acgtgttcag ctgcagcgtg 2820

atgcacgagg ccctgcacaa ccactacaca cagaagagcc tgagcctgag ccccggcaag 2880

tga 2883

<210> 13

<211> 960

<212> PRT

<213> 人工序列(artificial sequence)

<400> 13

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Ser Ser Ala Tyr Ser

1 5 10 15

Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met

20 25 30

Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp

35 40 45

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

50 55 60

Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile

65 70 75 80

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp

85 90 95

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu

100 105 110

Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

115 120 125

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

130 135 140

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

145 150 155 160

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

165 170 175

Arg Leu Arg Arg Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

180 185 190

Gly Gly Gly Gly Ser Ser Val Leu Arg Tyr Asp Asp Phe His Ile Asp

195 200 205

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

210 215 220

Asp His Ala Glu Ser Ser Trp Val Asn Arg Gly Glu Ser Ser Arg Lys

225 230 235 240

Ala Tyr Asp His Asn Ser Pro Tyr Ile Trp Pro Arg Asn Asp Tyr Asp

245 250 255

Gly Phe Leu Glu Asn Ala His Glu His His Gly Val Tyr Asn Gln Gly

260 265 270

Arg Gly Ile Asp Ser Gly Glu Arg Leu Met Gln Pro Thr Gln Met Ser

275 280 285

Ala Gln Glu Asp Leu Gly Asp Asp Thr Gly Ile His Val Ile Pro Thr

290 295 300

Leu Asn Gly Asp Asp Arg His Lys Ile Val Asn Val Asp Gln Arg Gln

305 310 315 320

Tyr Gly Asp Val Phe Lys Gly Asp Leu Asn Pro Lys Pro Gln Gly Gln

325 330 335

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

340 345 350

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

355 360 365

Phe Leu Pro Ser Leu Thr Cys Thr Gly Asp Ala Ala Pro Ala Ile Gln

370 375 380

His Ile Cys Leu Lys His Thr Thr Cys Phe Gln Asp Val Val Val Asp

385 390 395 400

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

405 410 415

Tyr Arg Phe Gln Gly Lys Lys Glu Ala Asp Gln Pro Trp Ile Val Val

420 425 430

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

435 440 445

Glu Pro Gly Val Leu Lys Val Leu Arg Thr Glu Lys Gln Tyr Leu Gly

450 455 460

Val Tyr Ile Trp Asn Met Arg Gly Ser Asp Gly Thr Ser Thr Tyr Ala

465 470 475 480

Thr Phe Leu Val Thr Trp Lys Gly Asp Glu Lys Thr Arg Asn Pro Thr

485 490 495

Pro Ala Val Thr Pro Gln Pro Arg Gly Ala Glu Phe His Met Trp Asn

500 505 510

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

515 520 525

His Leu Gln Tyr Lys Ile His Glu Ala Pro Phe Asp Leu Leu Leu Glu

530 535 540

Trp Leu Tyr Val Pro Ile Asp Pro Thr Cys Gln Pro Met Arg Leu Tyr

545 550 555 560

Ser Thr Cys Leu Tyr His Pro Asn Ala Pro Gln Cys Leu Ser His Met

565 570 575

Asn Ser Gly Cys Thr Phe Thr Ser Pro His Leu Ala Gln Arg Val Ala

580 585 590

Ser Thr Val Tyr Gln Asn Cys Glu His Ala Asp Asn Tyr Thr Ala Tyr

595 600 605

Cys Leu Gly Ile Ser His Met Glu Pro Ser Phe Gly Leu Ile Leu His

610 615 620

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

625 630 635 640

Gly Leu Tyr Val Phe Val Val Tyr Phe Asn Gly His Val Glu Ala Val

645 650 655

Ala Tyr Thr Val Val Ser Thr Val Asp His Phe Val Asn Ala Ile Glu

660 665 670

Glu Arg Gly Phe Pro Pro Thr Ala Gly Gln Pro Pro Ala Thr Thr Lys

675 680 685

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

690 695 700

Tyr Ala Ala Trp Thr Gly Gly Leu Ala Gly Gly Gly Gly Ser Gly Gly

705 710 715 720

Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr

725 730 735

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

740 745 750

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

755 760 765

Thr Pro Gly Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

770 775 780

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

785 790 795 800

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

805 810 815

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

820 825 830

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

835 840 845

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

850 855 860

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

865 870 875 880

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

885 890 895

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

900 905 910

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

915 920 925

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

930 935 940

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

945 950 955 960

<210> 14

<211> 546

<212> PRT

<213> 人工序列(artificial sequence)

<400> 14

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

Leu Ala

545

<210> 15

<211> 3909

<212> DNA

<213> 人工序列(artificial sequence)

<400> 15

atgggcaccg tgaataagcc cgtggtgggc gtgctgatgg gcttcggcat catcaccggc 60

accctgagaa tcaccaaccc cgtgagagct agcgtgctga gatacgacga cttccacatc 120

gacgaggaca agctggacac caacagcgtg tacgagccct actaccacag cgaccacgcc 180

gagagcagct gggtgaacag aggcgagagc agcagaaagg cctacgacca caacagcccc 240

tacatctggc ctagaaacga ttacgacggc ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc aaggcagagg catcgacagc ggcgagagac tgatgcagcc cacacagatg 360

agcgcccaag aggacctggg cgacgacacc ggcatccacg tgatccccac cctgaacggc 420

gacgacagac acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480

gacctgaacc ccaagcccca agggcagaga ctgatcgagg tgagcgtgga ggaaaaccac 540

cccttcaccc tgagagcccc cattcagaga atctacggcg tgagatacac cgagacctgg 600

agcttcctgc ctagcctcac ctgcaccggc gacgccgccc ccgccattca gcacatctgc 660

ctgaagcaca ccacctgctt ccaagacgtg gtggtcgacg tggactgcgc cgagaacacc 720

aaggaggatc agctggccga gatcagctac agattccaag gcaagaagga ggccgatcag 780

ccctggatcg tggtgaacac aagcaccctg ttcgacgagc tggagctgga cccccccgag 840

atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtacatc 900

tggaacatga gaggcagcga tgggacaagc acctacgcca ccttcctggt gacctggaag 960

ggcgacgaga agacaagaaa ccccaccccc gctgtgacac ctcaacctag aggcgccgag 1020

ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggcc 1080

atgcacctgc agtacaagat ccacgaggcc cccttcgacc tgctcctgga gtggctgtac 1140

gtgcccatcg accccacctg tcagcccatg agactgtaca gcacctgcct gtaccacccc 1200

aacgcccctc agtgtctgtc ccacatgaac agcggctgca ccttcacaag cccccacctg 1260

gctcagagag tggctagcac cgtgtatcag aactgcgagc acgccgacaa ctacaccgcc 1320

tactgcctgg gcatcagcca catggagcct agcttcggcc tgatcctgca cgacggcggc 1380

accaccctga agttcgtgga cacccccgag agcctgagcg gcctgtacgt gttcgtggtg 1440

tacttcaacg gccacgtgga ggccgtggcc tacaccgtgg tgagcaccgt ggaccacttc 1500

gtgaacgcca tcgaggagag aggctttcct cccaccgccg gccaaccccc cgccacaacc 1560

aagcccaaag agatcacccc cgtgaatccc gggacctccc ccctcatcag atatgccgcc 1620

tggaccggcg ggctggccga ggctgccgct aaggaagctg ctgccaagga ggccgctgcc 1680

aaggagccta agtcctgcga caagacccat acctgccctc cctgccccgc ccctgagctg 1740

ctgggcggcc ctagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 1800

agaacccccg gcgtgacctg cgtcgtggtg gacgtgagcc acgaggaccc cgaggtgaag 1860

ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tagagaggag 1920

cagtacaaca gcacctacag agtggtgagc gtgctgaccg tgctgcacca agactggctg 1980

aacggcaagg agtacaagtg caaggtgtcc aacaaggccc tgcccgcccc catcgagaag 2040

accatcagca aggccaaggg gcagcctaga gagccccaag tgtacaccct gccccctagc 2100

agagacgagc tgaccaagaa ccaagtgtcc ctgacctgcc tggtgaaggg cttctaccct 2160

agcgacatcg ccgtggagtg ggagagcaac gggcagcccg agaacaacta caagaccacc 2220

ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 2280

agcagatggc agcaaggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 2340

cactacacac agaaaagcct gtccctgagc cccggcaagg aggctgctgc caaggaagcc 2400

gctgccaaag aagccgccgc taaggcccaa gtgatcaaca ccaacagcct cagcctgctg 2460

acacagaaca acctgaacaa gagccaaagc gccctgggca ccgccatcga gagactgagc 2520

agcggcctga gaatcaacag cgctaaggac gacgccgccg gccaagccat cgccaacaga 2580

ttcaccgcca acatcaaggg cctgacccaa gctagcagaa acgccaatga cgggattagc 2640

atcgctcaga ccaccgaggg cgccctgaac gagatcaaca ataacctgca gagagtgaga 2700

gagctggccg tgcagagcgc caacagcacc aacagccaaa gcgacctgga cagcatccaa 2760

gccgagatca cacagagact gaatgaaatt gacagagtga gcgggcagac acagttcaac 2820

ggcgtgaagg tgctggccca agacaacacc ctgaccatcc aagtgggcgc caacgacggc 2880

gagaccatcg acatcgacct gaagcagatc aacagccaaa ccctgggcct ggacaccctc 2940

aacgtgcagc agaagtacaa ggtgagcgac accgccgcca ccgtgaccgg gtacgctgac 3000

accaccatcg ccctggacaa cagcacattc aaggctagcg ccaccggcct gggcggcacc 3060

gatcagaaga tcgacggcga cctgaagttc gacgacacca ccggcaagta ctacgccaag 3120

gtgaccgtga ccggcggcac cggcaaggac ggctactacg aggtcagcgt cgataaaaca 3180

aatggcgagg tgacactcgc cgggggggcc acaagccccc tgaccggcgg gctgcccgcc 3240

accgccaccg aggacgtgaa gaacgtgcaa gtggccaatg ccgacctcac cgaagccaag 3300

gccgccctga cagccgccgg ggtgaccggc accgctagcg tggtgaagat gagctacacc 3360

gacaacaacg gcaagaccat cgacgggggc ctggccgtga aggtgggcga cgactactac 3420

agcgccacac agaacaagga cggcagcatt tccatcaaca ccaccaagta caccgccgac 3480

gacggcacaa gcaagaccgc cctgaacaag ctgggcggcg ccgacggcaa gaccgaggtg 3540

gtgagcatcg gcggcaagac ctacgccgct agcaaggccg agggccacaa cttcaaagct 3600

cagcccgatc tggctgaagc tgccgccacc acaaccgaga accccctgca gaaaatcgat 3660

gccgccctgg cccaagtgga caccctgaga agcgacctgg gcgccgtgca gaacagattc 3720

aacagcgcca tcaccaacct gggcaacaca gtgaacaatc tgacaagcgc tagaagcaga 3780

atcgaggaca gcgactacgc caccgaagtg agcaatatga gcagagctca gatcctgcag 3840

caagccggca caagcgtgct cgcccaagcc aaccaagtgc ctcagaacgt gctcagcctc 3900

ctgagatga 3909

<210> 16

<211> 1302

<212> PRT

<213> 人工序列(artificial sequence)

<400> 16

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

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

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu

100 105 110

Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp

115 120 125

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

130 135 140

Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr

180 185 190

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

195 200 205

Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

225 230 235 240

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

245 250 255

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

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val

275 280 285

Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro

325 330 335

Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser

340 345 350

Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp

370 375 380

Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

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

420 425 430

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

435 440 445

Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val

515 520 525

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

530 535 540

Leu Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

545 550 555 560

Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

565 570 575

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

580 585 590

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys Val

595 600 605

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

610 615 620

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

625 630 635 640

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

645 650 655

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

660 665 670

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

675 680 685

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

690 695 700

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

705 710 715 720

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

725 730 735

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

740 745 750

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

755 760 765

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

770 775 780

Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Ala Ala Ala Lys Glu Ala

785 790 795 800

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

805 810 815

Leu Ser Leu Leu Thr Gln Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu

820 825 830

Gly Thr Ala Ile Glu Arg Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala

835 840 845

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

850 855 860

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

865 870 875 880

Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu

885 890 895

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

900 905 910

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

915 920 925

Glu Ile Asp Arg Val Ser Gly Gln Thr Gln Phe Asn Gly Val Lys Val

930 935 940

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

945 950 955 960

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

965 970 975

Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr Lys Val Ser Asp Thr Ala

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Val Thr Val Thr Gly Gly Thr Gly Lys Asp Gly Tyr Tyr Glu Val Ser

1045 1050 1055

Val Asp Lys Thr Asn Gly Glu Val Thr Leu Ala Gly Gly Ala Thr Ser

1060 1065 1070

Pro Leu Thr Gly Gly Leu Pro Ala Thr Ala Thr Glu Asp Val Lys Asn

1075 1080 1085

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

1090 1095 1100

Ala Ala Gly Val Thr Gly Thr Ala Ser Val Val Lys Met Ser Tyr Thr

1105 1110 1115 1120

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

1125 1130 1135

Asp Asp Tyr Tyr Ser Ala Thr Gln Asn Lys Asp Gly Ser Ile Ser Ile

1140 1145 1150

Asn Thr Thr Lys Tyr Thr Ala Asp Asp Gly Thr Ser Lys Thr Ala Leu

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

Gln Pro Asp Leu Ala Glu Ala Ala Ala Thr Thr Thr Glu Asn Pro Leu

1205 1210 1215

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

1220 1225 1230

Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile Thr Asn Leu Gly

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

Val Leu Ser Leu Leu Arg

1300

Claims

1.一种融合蛋白，其特征在于，所述融合蛋白包括， IFNα、水痘-带状疱疹病毒gE膜外区段和IgGFc区段。

2.根据权利要求1所述融合蛋白，其特征在于，将所述IFNα氨基酸序列和Fc氨基酸序列连接至所述gE膜外区段氨基酸序列的两端，得到融合蛋白IFNα-gE-Fc。

3.根据权利要求1所述融合蛋白，其特征在于，所述IFNα氨基酸序列、gE膜外区段氨基酸序列和Fc氨基酸序列通过linker连接肽连接。

4.根据权利要求3所述融合蛋白，其特征在于，所述连接肽包括但不限于：GGGGSGGGGSGGGGS连接肽和/或EAAAKEAAAKEAAAK连接肽。

5.根据权利要求4所述融合蛋白，其特征在于，所述融合蛋白的结构如SEQ ID NO:13所示。

6.一种重组基因，其特征在于，所述重组基因编码权利要求1-5任一所述的融合蛋白。

7.根据权利要求6所述重组基因，其特征在于，所述重组基因为DNA或者mRNA。

8.根据权利要求7所述重组基因，其特征在于，所述重组基因的结构如SEQ ID NO:12所示。

9.权利要求1-5任一所述融合蛋白或者权利要求6-8任一所述重组基因在制备带状疱疹亚单位疫苗中的应用。

10.一种带状疱疹亚单位疫苗，其特征在于，所述带状疱疹亚单位疫苗包括权利要求1-5任一所述融合蛋白或者权利要求6-8任一所述重组基因。

11.根据权利要求10所述带状疱疹亚单位疫苗，其特征在于，所述带状疱疹亚单位疫苗的每个剂量单位中含有IFNα-gE-Fc融合蛋白10~100 μg。

12.根据权利要求10所述带状疱疹亚单位疫苗，其特征在于，所述带状疱疹亚单位疫苗还含有铝佐剂。

13.权利要求1-5任一项所述融合蛋白的制备方法，其特征在于，包括步骤：

14.根据权利要求13所述融合蛋白的制备方法，其特征在于，所述细胞为CHOS细胞。

15.根据权利要求13所述融合蛋白的制备方法，其特征在于，所述表达载体为pCEP4。

16.根据权利要求14所述融合蛋白的制备方法，其特征在于，获得高表达IFNα-gE-Fc重组蛋白CHOS细胞株的步骤包括：

将重组质粒pCEP4-IFNα-gE-Fc转染至CHOS细胞，将能够表达IFNα-gE-Fc重组蛋白的CHOS细胞作为初代IFNα-gE-Fc-CHOS细胞，初代IFNα-gE-Fc-CHOS细胞通过细胞池（cellpool）加压筛选的方式获得高表达IFNα-gE-Fc重组蛋白的CHOS细胞。

17.根据权利要求13所述融合蛋白的制备方法，其特征在于，对所述发酵物上清液进行纯化处理的步骤包括：

采用Protein A亲合层析柱对所述发酵物上清液进行粗纯，Q Sepharose 4Fast Flow和Sephacryl S300进行精纯。

18.根据权利要求13所述融合蛋白的制备方法，其特征在于，对所述的融合蛋白采用鼠抗gE抗体和鼠抗Fc抗体进行鉴定。