CN113336838B - 新型冠状病毒肺炎重组痘苗病毒载体疫苗 - Google Patents

新型冠状病毒肺炎重组痘苗病毒载体疫苗 Download PDF

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CN113336838B
CN113336838B CN202110510607.4A CN202110510607A CN113336838B CN 113336838 B CN113336838 B CN 113336838B CN 202110510607 A CN202110510607 A CN 202110510607A CN 113336838 B CN113336838 B CN 113336838B
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步志高
王喜军
温志远
钟功勋
帅磊
王翀
葛金英
刘任强
王金良
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Harbin Veterinary Research Institute of CAAS
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Abstract

本发明提供一种包含改造的SARS‑CoV‑2的刺突蛋白的疫苗组合物，该疫苗组合物将痘苗病毒(Vaccinia virus)Western Reserve(WR)株与新型改造的SARS‑CoV‑2的刺突蛋白(SA/SB)或(S6PA/S6PB)或其免疫原性衍生物结合，构建表达改造的SARS‑CoV‑2的刺突蛋白基因的重组痘苗病毒，用于预防新型冠状病毒的感染，尤其是针对哺乳动物的新型冠状病毒的感染。

Description

新型冠状病毒肺炎重组痘苗病毒载体疫苗
技术领域
本发明涉及针对哺乳动物新型冠状病毒肺炎的预防性疫苗的组合物和方法， 进一步涉及针对哺乳动物新型冠状病毒肺炎的重组痘苗病毒的预防性疫苗组合 物和方法。
背景技术
新型冠状病毒性疾病(COVID-19)是由新型冠状病毒(Severe Acute RespiratorySyndrome Coronavirus 2,SARS-CoV-2)引起的传播能力极强的人兽共患病。而且新型 冠状病毒(SARS-CoV-2)的动物感染谱也较为广泛，蝙蝠、水貂、雪貂、猫科 动物等高度易感。目前，欧洲和北美大量养殖场水貂发生新冠病毒感染，已出 现水貂将新冠病毒传播给人类的个案，近2千万只水貂被扑杀，给欧美水貂产 业带来毁灭性打击，对我国的水貂产业也构成严重威胁。因此迫切需要研制安 全、有效的动物用新冠肺炎疫苗，阻断疫情在易感动物中传播，避免易感动物 成为中间宿主或储存宿主威胁人类的卫生健康和生命安全。到目前为止仍缺少 有效预防COVID-19的方法，其中间宿主和传播途径也有待进一步证实，从而加大了防控COVID-19的难度。疫苗是阻断、消除人间COVID-19最有效最有潜力 的途径。寻找抗原性更优的SARS-CoV-2病毒的抗原依然是目前研究的热点。 进一步制备用于动物的COVID-19的疫苗是阻断、消除COVID-19在动物之间以 及人畜之间传播的最有效的手段。
痘苗病毒(Vaccinia Virus)是痘病毒科(Poxviridae)正痘病毒属(Orthopoxvirus)成员，曾被用作疫苗预防天花。痘苗病毒宿主范围广泛；基因组 大，可插入外源蛋白基因片段的容量可达25kb；病毒在宿主细胞质中复制，不 会与宿主细胞基因整合，无致癌性。因此，痘苗病毒是真核生物基因或外源病毒 基因表达的理想载体和疫苗载体。
从上述内容可以看出，寻找抗原性更优的SARS-CoV-2蛋白提供针对 COVID-19的疫苗，尤其是针对哺乳动物的COVID-19的疫苗是迫切需要的，因 此本发明人在多年工作的积累下开发了这样的新的疫苗来预防新型冠状病毒的 感染，尤其是针对哺乳动物的新的疫苗来预防新型冠状病毒的感染。本发明提 供了这种预防性疫苗。
发明内容
SARS-CoV-2的刺突蛋白(Spike protein,S)是病毒受体结合蛋白和主要毒 力因子之一，决定着病毒的感染谱和致病力，同时也是一个有效的免疫原，是抗 SARS-CoV-2感染过程中诱导宿主免疫反应的主要免疫原。本发明人通过专注研 究冠状病毒S蛋白部分位点氨基酸的改变对S蛋白三聚体的空间构象的影响， 以及Furin裂解位点的突变缺失对S蛋白空间构象的稳定性影响的研究完成了本 发明。
首先，本发明提供了包含改造的SARS-CoV-2的刺突蛋白(SA)的疫苗组合 物，该改造的SARS-CoV-2的刺突蛋白(SA)包含组织型纤溶酶原激活因子信号 肽(tPA)。
在一个实施方案中，本发明提供了包含改造的SARS-CoV-2的刺突蛋白(SA) 或其免疫原性衍生物的疫苗组合物，其中改造的SARS-CoV-2的刺突蛋白(SA) 包括S蛋白基因信号肽替换为组织型纤溶酶原激活因子信号肽(tPA)。
在一个具体实施方案中，该疫苗组合物包含改造的SARS-CoV-2的刺突蛋白 (SA)，该改造的SARS-CoV-2的刺突蛋白(SA)进一步将如下6个位点氨基酸残 基均突变为脯氨酸：F817P、A892P、A899P、A942P、K986P、V987P，获得改 造的SARS-CoV-2的刺突蛋白(S6PA)。
在一个具体实施方案中，该疫苗组合物包含改造的SARS-CoV-2的刺突蛋白 (SA)，该改造的SARS-CoV-2的刺突蛋白(SA)选自命名为SA-tPA-1 (SEQ ID NO：1)的刺突蛋白,进一步SA蛋白Furin裂解位点突变缺失，获得 改造的SARS-CoV-2的刺突蛋白(SB)选自命名为SB-tPA-1(SEQ ID NO：2)的 刺突蛋白。
在一个具体实施方案中，该疫苗组合物包含改造的SARS-CoV-2的刺突蛋白(S6PA)，该改造的SARS-CoV-2的刺突蛋白(S6PA)选自命名为S6PA-tPA-1 (SEQ ID NO：3)的刺突蛋白,进一步S6PA蛋白Furin裂解位点突变缺失，获 得改造的SARS-CoV-2的刺突蛋白(S6PB)选自命名为S6PB-tPA-1(SEQ ID NO： 4)的刺突蛋白。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(SA)或其免疫原性 衍生物包含来自SEQ ID NO：1的氨基酸序列。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(SB)或其免疫原性 衍生物包含来自SEQ ID NO：2的氨基酸序列。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(S6PA)或其免疫原 性衍生物包含来自SEQ ID NO：3的氨基酸序列。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(S6PB)或其免疫原 性衍生物包含来自SEQ ID NO：4的氨基酸序列。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(SA/SB)或(S6PA/S6PB) 或其免疫原性衍生物由病毒载体编码。
在一个实施方案中，该改造的SARS-CoV-2的刺突蛋白(SA/SB)或(S6PA/S6PB) 或其免疫原性衍生物由病毒载体编码，所述病毒载体选自腺病毒载体、腺相关病 毒载体、逆转录病毒载体、痘苗病毒载体、疱疹病毒载体、新城疫病毒载体、流 感病毒载体、狂犬病病毒载体。
在一个具体实施方案中，该疫苗组合物优选包含将痘苗病毒(Vaccinia virus)Western Reserve(WR)与新型改造的SARS-CoV-2的刺突蛋白(SA/SB)或 (S6PA/S6PB)或其免疫原性衍生物结合的重组痘苗病毒。
在一个实施方案中，重组痘苗病毒被命名为重组rWR-SA或rWR-SB或 rWR-S6PA或rWR-S6PB。
在另一个实施方案中，疫苗组合物包含含有改造的SARS-CoV-2的刺突蛋白 (SA/SB)或(S6PA/S6PB)的重组痘苗病毒载体rWR-SA/rWR-SB或 rWR-S6PA/rWR-S6PB。
在一个具体实施方案中，疫苗是口服疫苗，在另一个具体的实施方案中，疫 苗是亚单位疫苗.
在一个方面，本发明的疫苗组合物还包含佐剂。在一个实施方案中，佐剂 选自：水包油佐剂、聚合物和水佐剂、油包水佐剂、氢氧化铝佐剂、维生素E 佐剂及其组合。在一个具体实施方案中，佐剂包含油乳剂，该油乳剂包含聚氧乙 烯-聚氧丙烯嵌段共聚物、角鲨烷、聚氧乙烯山梨醇酐单油酸酯和缓冲盐溶液(SP- 油)。在一个实施方案中，该组合物还包含药学上可接受的载体。
在另一个实施方案中，该疫苗组合物还可以包含至少一种额外的抗原。在某 些实施方案中，至少一种额外的抗原保护性地抵御可以引起哺乳动物疾病的微生 物。
在另一个实施方案中，提供了嵌合核酸分子，其包含编码非致病性痘苗病毒 载体WR株的核酸分子和编码改造的SARS-CoV-2的刺突蛋白(SA/SB)或 (S6PA/S6PB)的核酸分子(SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO： 7、SEQ ID NO：8)。
本发明还提供了保护哺乳动物免受新型冠状病毒SARS-CoV-2感染的方法。该 方法包括向哺乳动物施用免疫有效量的本发明公开的疫苗组合物、嵌合核酸分子、 或病毒重组载体。在多种实施方案中，该方法包括通过选自经口服、胃肠道外、 经鼻、皮内和经皮的一种或多于一种途径向哺乳动物施用疫苗组合物、嵌合核酸 分子或病毒重组载体。在另一个实施方案中，疫苗组合物、嵌合核酸分子或病毒 重组载体以单剂量施用。在另一个实施方案中，组合物与至少一种额外的抗原联 合施用，该额外的抗原保护性地抵御可以引起哺乳动物疾病的微生物。
附图说明
图1：SARS-CoV-2S蛋白变体tPASopti6PB基因的设计
图2：间接免疫荧光检测S6PB蛋白基因在重组病毒rWR-S6PB感染Hela细胞中的表达 (分别用重组病毒rWR-S6PB和野生型WR株病毒感染Hela细胞，36h后以抗SARS-CoV-2 S蛋白鼠血清为一抗，绿色荧光素(FITC)标记的山羊抗鼠IgG为二抗，进行荧光染色。)
图3：Western blot分析S6PB蛋白在重组痘苗病毒感染的细胞中的表达(1：蛋白质Maker； 2：野生型WR株病毒感染的Hela细胞；3：重组病毒rWR-S6PB感染的Hela细胞)。
图4：重组病毒rWR-S6PB在CV-1细胞上的生长动力学曲线(分别用重组病毒rWR-S6PB 和WR株病毒，按MOI为0.01感染生长过夜、密度约为90％单层CV-1细胞，于感染后12h、24h、36h、48h和60h分别收获病毒液，在CV-1细胞上滴定上述不同时间段收获的病毒液， 计算其病毒滴度)。
图5：重组病毒rMVA-tPASopti6PB对小鼠的免疫效力(分别用重组病毒rWR-S6PB和野生 型痘苗病毒WR株病毒，经口服或/和肌肉注射途径，间隔3周两次免疫BALB/c小鼠。在免疫前和免疫后的不同时间点采集、分离血清用于检测SARS-CoV-2中和抗体滴度)。
图6：重组病毒rWR-S6PB在小鼠上的攻毒保护效力评价(首次免疫后6周，按103.6PFU/50 μL/只的剂量，用SARS-CoV-2小鼠适应株HRB26M病毒，经滴鼻途径攻击重组病毒 rWR-S6PB免疫小鼠和野生型病毒WR株免疫小鼠。攻毒后第3天和第5天，每组各剖杀3 只小鼠，用于检测小鼠鼻甲和肺脏中的SARS-CoV-2病毒RNA和感染性病毒)。
具体实施方式
虽然下面详细讨论了本发明各种实施方案的进行和使用，但应该理解，本发 明提供了许多可以在各种特定情况下使用的可适用的发明概念。这里讨论的具体 实施方案仅说明进行和使用本发明的具体方式，并不限制本发明的范围。
S蛋白单体情况下，RBD区存在关闭沉降和上升开放两种状态。RBD的关闭 沉降状态覆盖了S2亚基部分氨基酸，削弱S蛋白的免疫原性；RBD上升开放状 态则是解离S2亚基，可充分发挥其免疫原性。相较野生型S蛋白，K986P、V987P 这两个联合突变可使具有高免疫原性和可移动特性的RBD处于稳定的上升开放 状态，提高表达S蛋白的免疫原性；F817P、A892P、A899P、A942P突变则可使S 蛋白的表达量增高约10倍，并具有耐热、室温贮藏和3次冻融循环的能力； SARS-CoV-2S蛋白基因的Furin裂解位点突变缺失可保留了SB蛋白的预融合构 像，不裂解成S1和S2亚基，提高了SB和S6PB蛋白的抗原性。
本发明通过例举性的实施在新型冠状病毒S蛋白基因密码子优化的基础上， 用组织型纤溶酶原激活因子信号肽(Tissue plasminogen activator signal sequence, tPA)基因序列替换S蛋白信号肽基因序列，同时将6个位点氨基酸残基均突变 为脯氨酸(F817P、A892P、A899P、A942P、K986P、V987P)，再将S蛋白Furin 裂解位点突变缺失，人工合成新型冠状病毒S蛋白变体S6PB基因。以痘苗病毒 Western Reserve(WR)株为疫苗载体，构建了表达新型冠状病毒S蛋白变体S6PB 基因的重组痘苗病毒rWR-S6PB。间接免疫荧光和Westernblot试验结果证实：S 蛋白变体S6PB基因在重组痘苗病毒rWR-S6PB感染的细胞中获得正确表达，表 达的S6PB蛋白具有良好的反应原性。
为了便于理解本发明，下面定义了许多术语。本发明定义的术语具有本发明 相关领域中普通技术人员通常理解的含义。
如本发明所用术语“包含”、“包括”、“含有”旨在表示组合物和方法包括 所列举的要素，但不排除其他要素。
术语“抗原”是指可以在动物中刺激抗体或T细胞应答或两者的产生的化合 物、组合物或免疫原性物质，其包括口服、注射或吸收到动物体内的组合物。可 以对整个分子或分子的一部分(例如表位或半抗原)产生免疫应答。
如本发明所定义的，“免疫原性组合物或免疫组合物”是指包含至少一种抗 原的物质的组合物，该抗原在宿主中引发对感兴趣的组合物或疫苗的细胞的免疫 应答和/或抗体介导的免疫应答。
本发明所用的“佐剂”是指由一种或多于一种增强对抗原的免疫应答的物质 组成的组合物。佐剂如何起作用的机制尚不完全清楚。一些佐剂被认为通过缓慢 释放抗原来增强免疫应答，而其他佐剂本身具有强免疫原性并被认为起到协同作 用。
本发明所用的术语“哺乳动物”是指包括人、及对新型冠状病毒易感的哺 乳动物，例如人、蝙蝠、狮子、老虎、恒河猴、食蟹猴、水貂、雪貂、猫、狗等。
如本文所用术语“药学上可接受的载体”是指用于包含可以口服或注射到 宿主中且没有副作用的疫苗抗原的流体载体。本领域已知的合适的药学上可接受 的载体包括但不限于无菌水、盐水、葡萄糖、右旋糖或缓冲溶液等。载体可包括 助剂包括但不限于稀释剂、稳定剂(糖和氨基酸)、防腐剂、润湿剂、乳化剂、pH 缓冲剂、黏度增强添加剂、着色剂等。
如本文所用术语“疫苗组合物”包括在药学上可接受的载体中的至少一种抗 原或免疫原，其可用于在宿主中诱导免疫应答。疫苗组合物可以按剂量施用并通 过医学或兽医领域技术人员熟知的技术施用，同时考虑例如接受体动物的年龄、 性别、体重、物种和状况、以及给药途径等因素。给药途径可以是经皮(通过皮 内、经皮、皮下、肌内途径而穿过皮肤或通过口腔、鼻腔、肛门、阴道而穿过 黏膜)或通过肠胃外途径(静脉内或腹膜内)。疫苗组合物可以单独施用，或者可 以与其他治疗或疗法共同施用或按顺序施用。给药形式可包括混悬剂、糖浆剂或 酏剂，以及用于肠胃外、皮下、皮内、肌内或静脉内给药(例如注射给药)的制 剂，例如无菌混悬剂或乳剂。疫苗组合物可以以喷雾形式给药或混合于食物和/或水中或与合适的载体、稀释剂、或赋形剂如无菌水、生理盐水、葡萄糖等混合 递送。该组合物可含有辅助物质，如润湿剂或乳化剂、pH缓冲剂、佐剂、凝胶 化或黏度增强添加剂、防腐剂、调味剂、着色剂等，这取决于给药途径和所需 制剂。
为了公开的完整性，包括了以下实施例以说明制备本发明组合物和复合物的 方法以及呈现组合物的某些特征。这些实施例决不旨在限制本公开的范围或教导。
实施例1：材料与方法
1.1病毒株
痘苗病毒(Vaccinia virus)Western Reserve(WR)株由中国农业科学院哈尔 滨兽医研究所重要人兽共患病与烈性外来病创新团队保存。表达绿色荧光蛋白基 因的重组痘苗病毒WR株rWR-EGFP由中国农业科学院哈尔滨兽医研究所重要 人兽共患病与烈性外来病创新团队构建、保存。SARS-CoV-2小鼠适应株 HRB26M是由中国农业科学院国家动物疫病防控高级别生物安全实验室保存。
1.2细胞
BHK细胞(ATCC No.CCL-10)、Hela细胞(ATCC No.CCL-2)和CV-1细 胞(ATCCNo.CCL-70)由中国农业科学院哈尔滨兽医研究所重要人兽共患病与 烈性外来病创新团队保存、培养。BHK细胞培养液为含5％胎牛血清的DMEM； Hela细胞和CV-1细胞培养液均为含10％胎牛血清的DMEM。
1.3质粒与试剂
含有痘苗病毒MVA TK基因同源臂的转移载体pCI-MVATK和含有EGFP基 因、痘苗病毒启动子H6基因的重组转移载体pCI-MVATK-EGFP由中国农业科 学院哈尔滨兽医研究所重要人兽共患病与烈性外来病创新团队构建、保存。高保 真DNA聚合酶(Phanta Max Super-Fidelity DNA Polymerase)、快速克隆试剂盒 (ClonExpress Ultra One Step CloningKit)购自南京诺维赞生物科技股份有限公 司。X-tremeGENETM 9DNA转染试剂购自Merck公司。抗SARS-CoV-2S蛋白 多克隆抗体、抗SARS-CoV-2S蛋白单克隆抗体由中国农业科学院哈尔滨兽医研 究所重要人兽共患病与烈性外来病创新团队制备。绿色荧光素(FITC)标记的山羊抗鼠IgG购自北京中杉金桥生物技术有限公司。红外荧光标记的驴抗鼠IgG 购自Lifetechnologies公司。
按人工合成的方法合成SARS-CoV-2S蛋白变体tPASopti6P(S6PB)基因，并 将其克隆至pBluescriptⅡ(+/-)(Clontech)的EcoRⅤ位点，命名为 pBlue-tPASopti6PB。S6PB基因是在SARS-CoV-2S蛋白基因按哺乳动物密码子 偏嗜性进行优化的基础上，将该蛋白基因2449-2451位、2674-2676位、2695-2697 位、2824-2826位、2956-2958位和2959-2961位碱基均突变为“CCC”，由此将上 述碱基编码的第817位苯丙氨酸(Phenylalanine,Phe,F)、第892位丙氨酸(Alanine, Ala,A)、第899位丙氨酸(Alanine,Ala,A)、第942位丙氨酸(Alanine,Ala,A)、 第986位赖氨酸(Lysine,Lys,K)和第987位缬氨酸(Valine,Val,V)均突变为 脯氨酸(Proline,Pro,P)；将SARS-CoV-2S蛋白裂解位点基因2044-2055位碱基 突变为“GGCTCCGCCTCC”，由此裂解位点氨基酸由“RRAR”突变为“GSAS”[精 氨酸(Arginine,Arg,R)，甘氨酸(Glycine,Gly,G)丝氨酸(Serine,Ser,S)]； 同时用组织型纤溶酶原激活因子(Tissue plasminogen activator,tPA)的信号肽基 因替代SARS-CoV-2S蛋白信号肽基因构成S6PB基因(图1)；在S6PB基因的 起始密码子ATG前插入Kozak序列“GCCGCCACC”。
1.4引物设计与合成
根据S6P蛋白基因序列、痘苗病毒H6启动子基因序列和重组转移载体 pCI-MVATK-EGFP中MVA TK基因同源臂基因序列，设计了构建表达S6PB蛋 白基因重组痘苗病毒的重组转移载体所用引物(表1)，所有引物由吉林省库美 生物科技有限公司合成。
表1表达SARS-CoV-2S蛋白基因重组痘苗病毒构建所用引物
Figure BDA0003060153450000081
注：黑体字部分为转移载体pCI-MVATK中EcoRⅠ位点两侧的MVA TK基因同源臂的同源序列，加方框部分为Kozak序列，斜体字部分为与痘苗病毒启动子H6基因的同源序 列。
1.5表达S6PB蛋白基因重组痘苗病毒的构建
用限制性内切酶EcoRⅠ酶切、线性化转移载体pCI-MVATK，胶回收纯化线 性化转移载体。采用PCR方法，以pCI-MVATK-EGFP为模板，用引物F2-F和 F2-R扩增痘苗病毒启动子H6基因，胶回收纯化启动子H6基因。采用PCR方 法，以pBlue-tPASopti6P为模板，用引物F3-F和F3-R扩增S6PB蛋白基因，胶 回收纯化S6PB蛋白基因。利用快速克隆试剂盒，将启动子H6基因和S6PB蛋 白基因顺序克隆至转移载体pCI-MVATK的EcoRⅠ位点，构成含有S6PB蛋白基因的重组转移载体pCI-MVATK-S6PB。
BHK细胞接种于35mm六孔板中，待过夜生长至80％～90％单层时，用重 组痘苗病毒rWR-EGFP，按M.O.I约0.01的剂量，感染BHK细胞，5％CO2、37℃ 环境感作1小时后，更换新鲜的完全培养基。然后，采用脂质体转染的方法，用 2μg的重组转移载体pCI-MVATK-S6PB转染上述感染过重组痘苗病毒的BHK细 胞。转染后48小时，收获细胞和上清，反复冻融3次后，10倍系列稀释病毒液， 重组痘苗病毒WR株接种Hela细胞，5％CO2、37℃环境感作1小时后，弃去病 毒液，用PBS清洗2次细胞，补加含0.8％低熔点琼脂糖和2％胎牛血清的DMEM， 室温放置至培养液凝固后，5％CO2、37℃温箱培养。2-3天后，荧光显微镜下挑 取不表达绿色荧光蛋白的重组病毒感染形成的噬斑，放置于500μl的无血清 DMEM中，反复冻融3次后，10倍系列稀释病毒液，继续在Hela细胞上纯化重 组病毒。连续纯化3-5代次后，经PCR鉴定正确后，表达S6PB蛋白基因重组痘 苗病毒命名为rWR-S6PB。
1.6间接免疫荧光检测重组真核表达质粒和救获的重组病毒
Hela细胞接种于24孔板中，待生长至80％～90％单层时，分别用重组病毒 rWR-S6PB和野生型WR株病毒按MOI为0.01感染Hela细胞，36h后弃去培养 上清，PBS洗涤细胞2次，冷3％多聚甲醛室温固定30min。分别以1:100倍稀 释抗SARS-CoV-2S蛋白小鼠血清为一抗，作用30min。PBST洗涤后加入1:200 倍稀释绿色荧光素(FITC)标记的山羊抗鼠IgG为二抗，作用30min，PBST洗 涤后荧光显微镜观察结果。
1.7 Western bloting
Hela细胞接种于6孔板中，待生长至80％～90％单层时，分别用重组病毒rWR-S6PB和野生型WR株病毒按MOI为0.01感染Hela细胞，于5％CO2、 37℃培养36-48h后，弃去培养上清，PBS洗涤细胞2次，按每孔80μL体积 加入1×SDS细胞裂解液收集细胞，煮沸30min，10,000×g离心10min，取上清进 行SDS-PAGE电泳，后将蛋白转印到尼龙膜上(Ameresco)，5％脱脂乳封闭过 夜。PBST(0.05％Tween20)洗涤后，以抗SARS-CoV-2S蛋白小鼠血清为一抗，红外荧光标记的驴抗鼠IgG为二抗，通过Odyssey Infrared成像系统成像，并分 析各重组痘苗病毒的蛋白表达情况。
1.8中毒的制备与滴定
重组病毒rWR-S6PB经PCR、IFA和Western blot鉴定无误后，按MOI为 0.01接种过夜生长至90％单层CV-1细胞，5％CO2、37℃培养，48h后收获病 毒液，反复冻融3次后分装，存于-70℃。
将冻存于-70℃冰箱的种毒液取出，待溶化后，将病毒液作10倍连续稀释， 取各稀释度的病毒液稀释100μL接种于96孔板中过夜生长至90％的CV-1细胞， 每个稀释度平行接种8个细胞孔，37℃感作1h，弃去上清，加入100μL 2％的 DMEM。5％CO2、37℃培养，感染后第3天显微镜下观察细胞病变，计算病 毒滴度。病毒滴度用50％组织细胞感染量/mL(TCID50/mL)表示，利用 Reed-Muench法计算。
1.9病毒生长动力学曲线的测定
用重组病毒rWR-S6PB和野生型WR株病毒分别按MOI为0.01接种铺于 12孔板、生长过夜、密度约为90％单层CV-1细胞，37℃感作1h后，PBS洗 2次，加入含2％胎牛血清的DMEM培养液，5％CO2、37℃培养，于感染后 12h、24h、36h、48h和60h分别收获病毒液；每个时间点，每种病毒收获3个 平行孔的培养上清和细胞；将上述不同时间段收获的病毒液冻融3次后，做10 倍连续稀释，分别取100μL各稀释度病毒液接种铺于96孔板、过夜生长至密度 约为90％单层CV-1细胞，37℃感作1h后，PBS洗2次，加入含2％FBS的 DMEM培养液，5％CO2、37℃培养，每个稀释度做8个重复；感染后第3 天显微镜下观察细胞病变，计算病毒滴度，并绘制病毒生长动力学曲线。
1.10重组痘苗病毒对BALB/c小鼠的免疫试验
为了评估表达SARS-CoV-2S蛋白基因重组痘苗病毒rWR-S6PB对小鼠的免 疫原性，随机将6周龄雌性BALB/c小鼠分为6组，每组6只。用重组病毒 rWR-S6PB，按5×107TCID50/100μL/只(高剂量)和1×107TCID50/100μL/只(低 剂量)的剂量，各口服免疫1组小鼠。用重组病毒rWR-S6PB，按1×107 TCID50/100μL/只(高剂量)和1×106TCID50/100μL/只(低剂量)的剂量，经肌 肉注射途径各免疫1组小鼠。同时，用野生型痘苗病毒WR株，按5×107 TCID50/100μL/只(口服)和1×107TCID50/100μL/只(肌肉注射)的剂量，口服+ 肌肉注射联合免疫1组小鼠，作为免疫对照。间隔3周，经相同免疫途径、剂量 进行加强免疫。各组小鼠于免疫前、初次免疫后第3周、第5周和第6周，经眶 下静脉丛采血，分离血清(将每组免疫前、初次免疫后第3周、第5周的血清混 合)，56℃水浴灭活30min，用于SARS-CoV-2中和抗体检测。
1.11重组痘苗病毒rWR-S6PB对BALB/c小鼠的攻毒保护效力试验
初次免疫后第6周，按103.6PFU/50μL/只的剂量，用SARS-CoV-2小鼠适应 株HRB26M病毒，经滴鼻途径，攻击rWR-S6PB免疫小鼠和野生型WR株病毒 免疫小鼠。攻毒后第3天和第5天，每组各剖解3只小鼠，采集鼻甲和肺脏，通 过实时荧光定量PCR和蚀斑计数试验检测样品中的病毒RNA和感染性病毒载量， 从而评价重组痘苗病毒rWR-S6PB对BALB/c小鼠的攻毒保护效力。
1.12实时荧光定量PCR
采用实时荧光定量PCR方法测定组织样品中的病毒载量。针对SARS-CoV-2 N基因的实时荧光定量PCR特异性引物和荧光探针均参照中国疾病预防控制中 心病毒病预防控制所发布的信息(http://nmdc.cn/nCoV)，具体如下：上游引物： 5’-GGG GAA CTT CTC CTGCTA GAA T-3’；下游引物,5’-CAG ACA TTT TGC TCT CAA GCT G-3’，荧光探针为：5’-FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3’。利用病毒RNA提取试剂盒QIAamp vRNA Minikit(Qiagen)提取 病毒RNA后，应用HiScript II Q RT SuperMix for qPCR(Vazyme)试剂盒进行反转 录，再应用DNA聚合酶Premix Ex Taq for probe qPCR(TaRaKa,China)，在实时 荧光定量PCR仪(Applied Biosystems QuantStudio 5Real-Time PCR System, ThermoScientific)上进行qPCR。SARS-CoV-2RNA的拷贝数是通过利用质粒 pBluescript II SK-N(将全长的SARS-CoV-2N基因克隆到pBluescript II SK上构 成)绘制的标准曲线进行计算、校正。该qPCR方法的检测下限为1000拷贝/mL。
1.13中和试验
检测血清中SARS-CoV-2中和抗体的中和试验在96孔板上进行，步骤如下： 首先将血清样品置于56℃水浴30min进行灭活，再将血清样品分别以不完全 DMEM连续倍比稀释，每稀释度体积为50μL，与50μL约含100PFU的 SARS-CoV-2HRB25株病毒液混合，37℃感作1h后，每孔加入约105Vero E6 细胞，5％CO2、37℃培养48h后，置于显微镜下观察SARS-CoV-2HRB25株病 毒感染形成的蚀斑情况。每个血清稀释度做4个重复。血清中SARS-CoV-2中和抗体滴度被定义为能抑制90％蚀斑产生的最高血清稀释倍数。
实施例2：实验结果
2.1表达S6PB蛋白基因重组痘苗病毒的构建
采用PCR方法，以pCI-MVATK-EGFP为模板，用引物F2-F和F2-R扩增痘 苗病毒启动子H6基因；以pBlue-tPASopti6PB为模板，用引物F3-F和F3-R扩 增S6PB蛋白基因。将启动子H6基因和S6PB蛋白基因顺序克隆至转移载体 pCI-MVATK的EcoRⅠ位点，构成含有S6PB蛋白基因重组转移载体 pCI-MVATK-S6PB。酶切和序列测定结果表明，S6PB蛋白基因成功克隆至pCI-MVATK的EcoRⅠ位点。
采用脂质体转染的方法，用重组转移载体pCI-MVATK-S6PB转染预先感染 过重组痘苗病毒rWR-EGFP的BHK细胞，拯救表达S6PB蛋白基因的重组痘苗 病毒。然后，通过蚀斑纯化的方法，在Hela细胞上连续纯化重组病毒。PCR鉴 定结果表明，利用重组转移载体pCI-MVATK-S6PB转染预先感染过重组痘苗病 毒rWR-EGFP的BHK细胞，成功拯救获得表达S6PB蛋白基因重组痘苗病毒 rWR-S6PB。
2.2间接免疫荧光检测救获的重组病毒
分别重组病毒rWR-S6PB和野生型WR株病毒感染Hela细胞，36h后以抗 SARS-CoV-2S蛋白小鼠血清为一抗，以绿色荧光素(FITC)标记的山羊抗鼠IgG 为二抗，进行荧光染色。结果如图2显示：抗SARS-CoV-2S蛋白小鼠血清检测 重组病毒rWR-S6PB感染Hela细胞呈现阳性荧光信号；而抗SARS-CoV-2S蛋 白小鼠血清检测野生型WR株病毒感染Hela细胞呈现的荧光信号为阴性。结果 表明，通过DNA病毒同源重组的方法，成功拯救获得表达S6P蛋白基因的重组 痘苗病毒rWR-S6PB，且S6PB蛋白基因能够在重组痘苗病毒rWR-S6PB感染细 胞中正确表达，且具有良好的免疫原性。
2.3 Western bloting
为了进一步鉴定重组痘苗病毒，分别用重组病毒rWR-S6PB和野生型WR 株病毒感染的Hela细胞制备裂解液，进行SDS-PAGE和Western blot分析。结 果如图3显示：以抗SARS-CoV-2S蛋白小鼠血清检测重组病毒rWR-S6PB在 Hela细胞中表达的蛋白时，显示出特异的蛋白条带，与SARS-CoV-2S蛋白的预 期值相符；而以抗SARS-CoV-2S蛋白小鼠血清分别检测野生型WR株病毒在 Hela细胞中表达的蛋白时，未显示出特异蛋白条带。结果表明，通过DNA病毒 同源重组的方法，成功拯救获得表达S6PB蛋白基因的重组痘苗病毒rWR-S6PB，且S6PB蛋白基因能够在重组痘苗病毒rWR-S6PB感染细胞中正确表达，且具有 良好的反应原性。
2.4重组病毒的体外生长特性
为了比较救获重组病毒rWR-S6PB和野生型WR株病毒在CV-1细胞上的生 长动力学特点，将rWR-S6PB和WR株病毒分别按MOI为0.01接种于生长过 夜、密度约为90％单层CV-1细胞，于感染后12h、24h、36h、48h和60h分别 收获病毒液；在CV-1细胞上滴定上述不同时间段收获的病毒液，计算其病毒滴 度。结果显示，救获重组病毒rWR-S6P在CV-1细胞上的生长动力学曲线与野生 型WR株相似，在感染后36h达到峰值107.87TCID50/mL，略低于亲本株rCDV在 Vero细胞上的最高生长滴度108.09TCID50/mL(图4)。
2.5重组病毒对小鼠的免疫效力
为了评估表达SARS-CoV-2S蛋白的重组痘苗病毒rWR-S6PB对小鼠的免疫 原性，用重组病毒rWR-S6PB高剂量(5×107TCID50/100μL/只)和低剂量(1×107 TCID50/100μL/只)各口服免疫1组小鼠。用重组病毒rWR-S6P高剂量(1×107 TCID50/100μL/只)和低剂量(1×106TCID50/100μL/只)经肌肉注射途径各免疫1 组小鼠。同时，同时，用野生型痘苗病毒WR株，按5×107TCID50/100μL/只(口 服)和1×107TCID50/100μL/只(肌肉注射)的剂量，口服+肌肉注射联合免疫1 组小鼠，作为免疫对照。间隔3周用相同病毒和相同剂量，经相同途径进行加强 免疫。
在整个免疫试验期间，各组免疫小鼠未见明显的疾病相关临床症状，暗示重 组病毒rWR-S6PB具有良好的安全性。中和试验结果显示：初次免疫后3周，在 重组病毒rWR-S6PB口服免疫和肌肉注射免疫的小鼠血清中能检测到 SARS-CoV-2中和抗体，而在野生型WR株病毒免疫小鼠血清中则检测不到 SARS-CoV-2中和抗体(图5)。初次免疫后3周，rWR-S6PB高剂量、低剂量口 服免疫小鼠和高剂量、低剂量肌肉注射免疫小鼠血清中SARS-CoV-2中和抗体滴 度分别为256、256、128和128。第二次免疫后2周，rWR-S6PB高剂量、低剂 量口服免疫小鼠和高剂量、低剂量肌肉注射免疫小鼠血清中SARS-CoV-2中和抗 体滴度分别上升至2048、2048、1024和256。攻毒前，rWR-S6PB免疫的各组 小鼠血清中SARS-CoV-2中和抗体滴度与第二次免疫后2周时相似。结果表明， 重组病毒rWR-S6PB具有良好免疫原性，口服和肌肉注射免疫小鼠均能诱导高水 平的SARS-CoV-2中和抗体反应。
2.6重组病毒对小鼠的攻毒保护效力
为了评估表达SARS-CoV-2S蛋白基因的重组痘苗病毒rWR-S6PB对小鼠的 攻毒保护效力，初次免疫后6周，各选取6只重组病毒rWR-S6PB高剂量或低剂 量口服免疫小鼠和高剂量或低剂量肌肉注射免疫小鼠以及6只野生型WR株口 服免疫小鼠，用于SARS-CoV-2攻击试验。
用SARS-CoV-2HRB26M株病毒，经滴鼻途径攻击rWR-S6PB免疫小鼠和 野生型WR株口服免疫小鼠。口服免疫小鼠攻毒结果如图6所示：攻毒后第3 天，野生型WR株免疫小鼠的鼻甲和肺脏均能检测到高水平的病毒RNA和高滴 度的感染性病毒；而rWR-S6PB高剂量和低剂量口服免疫小鼠的鼻甲和肺脏均检 测不到病毒RNA和感染性病毒。攻毒后第5天，野生型WR株免疫小鼠的鼻甲 和肺脏仍然能检测到高水平的病毒RNA和高滴度的感染性病毒；而rWR-S6PB 高剂量和低剂量口服免疫小鼠的鼻甲和肺脏均检测不到病毒RNA和感染性病毒。结果表明，重组病毒rWR-S6PB口服免疫小鼠诱导的免疫反应能快速、有效地清 除小鼠体内的SARS-CoV-2，能为免疫小鼠提供完全攻毒保护。
肌肉注射免疫小鼠攻毒结果如图6所示：攻毒后第3天，野生型WR株免 疫小鼠的鼻甲和肺脏均能检测到高水平的病毒RNA和高滴度的感染性病毒； rWR-S6PB高剂量免疫组中，2/3只小鼠鼻甲中检测不到病毒RNA，1/3只小鼠 鼻甲能检测到病毒RNA，但病毒RNA载量显著低于野生型WR株免疫小鼠，而 全部3/3小鼠的鼻甲中未检测到感染性病毒，同时3/3小鼠的肺脏中均检测不到 病毒RNA和感染性病毒；rWR-S6PB低剂量免疫组中，全部3/3只小鼠鼻甲中 能检测到病毒RNA和感染性病毒，但载量显著低于野生型WR株免疫小鼠，全 部3/3只小鼠的肺脏中均检测不到病毒RNA和感染性病毒。攻毒后第5天，野 生型WR株免疫小鼠的鼻甲和肺脏均能检测到高水平的病毒RNA和高滴度的感 染性病毒；rWR-S6PB高剂量免疫组中，全部3/3只小鼠鼻甲和肺脏中均未检测 到病毒RNA和感染性病毒；rWR-S6PB低剂量免疫组中，2/3只小鼠的鼻甲中能 检测到病毒RNA，但载量显著低于野生型WR株免疫小鼠，1/3只小鼠的鼻甲中 未检测到病毒RNA，全部3/3只小鼠鼻甲中均未检测到感染性病毒，全部3/3只 小鼠的肺脏中均检测不到病毒RNA和感染性病毒。结果表明，重组痘苗病毒 rWR-S6PB高剂量肌肉注射免疫小鼠诱导的免疫反应能有效清除小鼠体内的 SARS-CoV-2。
以上结果表明，重组痘苗病毒rWR-S6PB具有良好的免疫原性和攻毒保护效 力，能够成为动物用新型冠状病毒肺炎疫苗而且重组痘苗病毒rWR-S6PB具有良 好的安全性、口服免疫原性和攻毒保护效力。
序列表
<110> 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心)
<120> 新冠肺炎重组痘苗病毒载体疫苗
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Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg
210 215 220
Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro
225 230 235 240
Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg
245 250 255
Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala
260 265 270
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys
275 280 285
Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp
290 295 300
Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys
305 310 315 320
Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile
325 330 335
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
340 345 350
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
355 360 365
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
370 375 380
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
385 390 395 400
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
405 410 415
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
420 425 430
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
435 440 445
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
450 455 460
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
465 470 475 480
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
485 490 495
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
500 505 510
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
515 520 525
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
530 535 540
Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly
545 550 555 560
Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly
565 570 575
Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu
580 585 590
Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile
595 600 605
Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp
610 615 620
Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr
625 630 635 640
Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg
645 650 655
Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys
660 665 670
Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr
675 680 685
Asn Ser Pro Gly Ser Ala Ser Ser Val Ala Ser Gln Ser Ile Ile Ala
690 695 700
Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn
705 710 715 720
Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile
725 730 735
Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile
740 745 750
Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser
755 760 765
Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln
770 775 780
Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys
785 790 795 800
Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu
805 810 815
Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu
820 825 830
Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly
835 840 845
Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys
850 855 860
Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile
865 870 875 880
Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp
885 890 895
Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met
900 905 910
Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu
915 920 925
Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile
930 935 940
Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp
945 950 955 960
Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu
965 970 975
Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser
980 985 990
Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr
995 1000 1005
Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg
1010 1015 1020
Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser
1025 1030 1035 1040
Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly
1045 1050 1055
Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1060 1065 1070
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala
1075 1080 1085
Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val
1090 1095 1100
Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1105 1110 1115 1120
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys
1125 1130 1135
Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln
1140 1145 1150
Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1155 1160 1165
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1170 1175 1180
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1185 1190 1195 1200
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1205 1210 1215
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
1220 1225 1230
Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr
1235 1240 1245
Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys
1250 1255 1260
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu
1265 1270 1275 1280
His Tyr Thr
<210> 3
<211> 1283
<212> PRT
<213> 重组蛋白()
<400> 3
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Ala Arg Gln Cys Val Asn Leu Thr Thr Arg Thr
20 25 30
Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr
35 40 45
Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu
50 55 60
Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val
65 70 75 80
Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe
85 90 95
Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg
100 105 110
Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu
115 120 125
Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln
130 135 140
Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys
145 150 155 160
Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys
165 170 175
Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys
180 185 190
Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp
195 200 205
Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg
210 215 220
Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro
225 230 235 240
Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg
245 250 255
Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala
260 265 270
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys
275 280 285
Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp
290 295 300
Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys
305 310 315 320
Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile
325 330 335
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
340 345 350
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
355 360 365
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
370 375 380
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
385 390 395 400
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
405 410 415
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
420 425 430
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
435 440 445
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
450 455 460
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
465 470 475 480
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
485 490 495
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
500 505 510
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
515 520 525
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
530 535 540
Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly
545 550 555 560
Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly
565 570 575
Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu
580 585 590
Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile
595 600 605
Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp
610 615 620
Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr
625 630 635 640
Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg
645 650 655
Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys
660 665 670
Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr
675 680 685
Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala
690 695 700
Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn
705 710 715 720
Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile
725 730 735
Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile
740 745 750
Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser
755 760 765
Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln
770 775 780
Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys
785 790 795 800
Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu
805 810 815
Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Pro Ile Glu Asp Leu Leu
820 825 830
Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly
835 840 845
Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys
850 855 860
Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile
865 870 875 880
Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp
885 890 895
Thr Phe Gly Ala Gly Pro Ala Leu Gln Ile Pro Phe Pro Met Gln Met
900 905 910
Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu
915 920 925
Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile
930 935 940
Gln Asp Ser Leu Ser Ser Thr Pro Ser Ala Leu Gly Lys Leu Gln Asp
945 950 955 960
Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu
965 970 975
Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser
980 985 990
Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr
995 1000 1005
Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg
1010 1015 1020
Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser
1025 1030 1035 1040
Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly
1045 1050 1055
Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1060 1065 1070
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala
1075 1080 1085
Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val
1090 1095 1100
Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1105 1110 1115 1120
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys
1125 1130 1135
Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln
1140 1145 1150
Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1155 1160 1165
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1170 1175 1180
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1185 1190 1195 1200
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1205 1210 1215
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
1220 1225 1230
Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr
1235 1240 1245
Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys
1250 1255 1260
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu
1265 1270 1275 1280
His Tyr Thr
<210> 4
<211> 1283
<212> PRT
<213> 重组蛋白()
<400> 4
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Ala Arg Gln Cys Val Asn Leu Thr Thr Arg Thr
20 25 30
Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr
35 40 45
Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu
50 55 60
Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val
65 70 75 80
Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe
85 90 95
Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg
100 105 110
Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu
115 120 125
Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln
130 135 140
Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys
145 150 155 160
Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys
165 170 175
Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys
180 185 190
Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp
195 200 205
Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg
210 215 220
Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro
225 230 235 240
Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg
245 250 255
Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala
260 265 270
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys
275 280 285
Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp
290 295 300
Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys
305 310 315 320
Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile
325 330 335
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
340 345 350
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
355 360 365
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
370 375 380
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
385 390 395 400
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
405 410 415
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
420 425 430
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
435 440 445
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
450 455 460
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
465 470 475 480
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
485 490 495
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
500 505 510
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
515 520 525
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
530 535 540
Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly
545 550 555 560
Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly
565 570 575
Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu
580 585 590
Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile
595 600 605
Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp
610 615 620
Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr
625 630 635 640
Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg
645 650 655
Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys
660 665 670
Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr
675 680 685
Asn Ser Pro Gly Ser Ala Ser Ser Val Ala Ser Gln Ser Ile Ile Ala
690 695 700
Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn
705 710 715 720
Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile
725 730 735
Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile
740 745 750
Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser
755 760 765
Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln
770 775 780
Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys
785 790 795 800
Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu
805 810 815
Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Pro Ile Glu Asp Leu Leu
820 825 830
Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly
835 840 845
Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys
850 855 860
Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile
865 870 875 880
Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp
885 890 895
Thr Phe Gly Ala Gly Pro Ala Leu Gln Ile Pro Phe Pro Met Gln Met
900 905 910
Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu
915 920 925
Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile
930 935 940
Gln Asp Ser Leu Ser Ser Thr Pro Ser Ala Leu Gly Lys Leu Gln Asp
945 950 955 960
Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu
965 970 975
Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser
980 985 990
Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr
995 1000 1005
Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg
1010 1015 1020
Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser
1025 1030 1035 1040
Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly
1045 1050 1055
Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1060 1065 1070
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala
1075 1080 1085
Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val
1090 1095 1100
Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1105 1110 1115 1120
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys
1125 1130 1135
Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln
1140 1145 1150
Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1155 1160 1165
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1170 1175 1180
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1185 1190 1195 1200
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1205 1210 1215
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
1220 1225 1230
Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr
1235 1240 1245
Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys
1250 1255 1260
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu
1265 1270 1275 1280
His Tyr Thr
<210> 5
<211> 3852
<212> PRT
<213> 重组核苷酸()
<400> 5
Ala Thr Gly Gly Ala Cys Gly Cys Cys Ala Thr Gly Ala Ala Gly Cys
1 5 10 15
Gly Cys Gly Gly Cys Cys Thr Gly Thr Gly Cys Thr Gly Cys Gly Thr
20 25 30
Gly Cys Thr Gly Cys Thr Gly Cys Thr Gly Thr Gly Cys Gly Gly Cys
35 40 45
Gly Cys Cys Gly Thr Gly Thr Thr Cys Gly Thr Gly Thr Cys Cys Gly
50 55 60
Cys Cys Cys Gly Cys Cys Ala Gly Thr Gly Cys Gly Thr Gly Ala Ala
65 70 75 80
Cys Cys Thr Gly Ala Cys Cys Ala Cys Cys Cys Gly Cys Ala Cys Cys
85 90 95
Cys Ala Gly Cys Thr Gly Cys Cys Cys Cys Cys Cys Gly Cys Cys Thr
100 105 110
Ala Cys Ala Cys Cys Ala Ala Cys Thr Cys Cys Thr Thr Cys Ala Cys
115 120 125
Cys Cys Gly Cys Gly Gly Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys
130 135 140
Cys Cys Cys Gly Ala Cys Ala Ala Gly Gly Thr Gly Thr Thr Cys Cys
145 150 155 160
Gly Cys Thr Cys Cys Thr Cys Cys Gly Thr Gly Cys Thr Gly Cys Ala
165 170 175
Cys Thr Cys Cys Ala Cys Cys Cys Ala Gly Gly Ala Cys Cys Thr Gly
180 185 190
Thr Thr Cys Cys Thr Gly Cys Cys Cys Thr Thr Cys Thr Thr Cys Thr
195 200 205
Cys Cys Ala Ala Cys Gly Thr Gly Ala Cys Cys Thr Gly Gly Thr Thr
210 215 220
Cys Cys Ala Cys Gly Cys Cys Ala Thr Cys Cys Ala Cys Gly Thr Gly
225 230 235 240
Thr Cys Cys Gly Gly Cys Ala Cys Cys Ala Ala Cys Gly Gly Cys Ala
245 250 255
Cys Cys Ala Ala Gly Cys Gly Cys Thr Thr Cys Gly Ala Cys Ala Ala
260 265 270
Cys Cys Cys Cys Gly Thr Gly Cys Thr Gly Cys Cys Cys Thr Thr Cys
275 280 285
Ala Ala Cys Gly Ala Cys Gly Gly Cys Gly Thr Gly Thr Ala Cys Thr
290 295 300
Thr Cys Gly Cys Cys Thr Cys Cys Ala Cys Cys Gly Ala Gly Ala Ala
305 310 315 320
Gly Thr Cys Cys Ala Ala Cys Ala Thr Cys Ala Thr Cys Cys Gly Cys
325 330 335
Gly Gly Cys Thr Gly Gly Ala Thr Cys Thr Thr Cys Gly Gly Cys Ala
340 345 350
Cys Cys Ala Cys Cys Cys Thr Gly Gly Ala Cys Thr Cys Cys Ala Ala
355 360 365
Gly Ala Cys Cys Cys Ala Gly Thr Cys Cys Cys Thr Gly Cys Thr Gly
370 375 380
Ala Thr Cys Gly Thr Gly Ala Ala Cys Ala Ala Cys Gly Cys Cys Ala
385 390 395 400
Cys Cys Ala Ala Cys Gly Thr Gly Gly Thr Gly Ala Thr Cys Ala Ala
405 410 415
Gly Gly Thr Gly Thr Gly Cys Gly Ala Gly Thr Thr Cys Cys Ala Gly
420 425 430
Thr Thr Cys Thr Gly Cys Ala Ala Cys Gly Ala Cys Cys Cys Cys Thr
435 440 445
Thr Cys Cys Thr Gly Gly Gly Cys Gly Thr Gly Thr Ala Cys Thr Ala
450 455 460
Cys Cys Ala Cys Ala Ala Gly Ala Ala Cys Ala Ala Cys Ala Ala Gly
465 470 475 480
Thr Cys Cys Thr Gly Gly Ala Thr Gly Gly Ala Gly Thr Cys Cys Gly
485 490 495
Ala Gly Thr Thr Cys Cys Gly Cys Gly Thr Gly Thr Ala Cys Thr Cys
500 505 510
Cys Thr Cys Cys Gly Cys Cys Ala Ala Cys Ala Ala Cys Thr Gly Cys
515 520 525
Ala Cys Cys Thr Thr Cys Gly Ala Gly Thr Ala Cys Gly Thr Gly Thr
530 535 540
Cys Cys Cys Ala Gly Cys Cys Cys Thr Thr Cys Cys Thr Gly Ala Thr
545 550 555 560
Gly Gly Ala Cys Cys Thr Gly Gly Ala Gly Gly Gly Cys Ala Ala Gly
565 570 575
Cys Ala Gly Gly Gly Cys Ala Ala Cys Thr Thr Cys Ala Ala Gly Ala
580 585 590
Ala Cys Cys Thr Gly Cys Gly Cys Gly Ala Gly Thr Thr Cys Gly Thr
595 600 605
Gly Thr Thr Cys Ala Ala Gly Ala Ala Cys Ala Thr Cys Gly Ala Cys
610 615 620
Gly Gly Cys Thr Ala Cys Thr Thr Cys Ala Ala Gly Ala Thr Cys Thr
625 630 635 640
Ala Cys Thr Cys Cys Ala Ala Gly Cys Ala Cys Ala Cys Cys Cys Cys
645 650 655
Cys Ala Thr Cys Ala Ala Cys Cys Thr Gly Gly Thr Gly Cys Gly Cys
660 665 670
Gly Ala Cys Cys Thr Gly Cys Cys Cys Cys Ala Gly Gly Gly Cys Thr
675 680 685
Thr Cys Thr Cys Cys Gly Cys Cys Cys Thr Gly Gly Ala Gly Cys Cys
690 695 700
Cys Cys Thr Gly Gly Thr Gly Gly Ala Cys Cys Thr Gly Cys Cys Cys
705 710 715 720
Ala Thr Cys Gly Gly Cys Ala Thr Cys Ala Ala Cys Ala Thr Cys Ala
725 730 735
Cys Cys Cys Gly Cys Thr Thr Cys Cys Ala Gly Ala Cys Cys Cys Thr
740 745 750
Gly Cys Thr Gly Gly Cys Cys Cys Thr Gly Cys Ala Cys Cys Gly Cys
755 760 765
Thr Cys Cys Thr Ala Cys Cys Thr Gly Ala Cys Cys Cys Cys Cys Gly
770 775 780
Gly Cys Gly Ala Cys Thr Cys Cys Thr Cys Cys Thr Cys Cys Gly Gly
785 790 795 800
Cys Thr Gly Gly Ala Cys Cys Gly Cys Cys Gly Gly Cys Gly Cys Cys
805 810 815
Gly Cys Cys Gly Cys Cys Thr Ala Cys Thr Ala Cys Gly Thr Gly Gly
820 825 830
Gly Cys Thr Ala Cys Cys Thr Gly Cys Ala Gly Cys Cys Cys Cys Gly
835 840 845
Cys Ala Cys Cys Thr Thr Cys Cys Thr Gly Cys Thr Gly Ala Ala Gly
850 855 860
Thr Ala Cys Ala Ala Cys Gly Ala Gly Ala Ala Cys Gly Gly Cys Ala
865 870 875 880
Cys Cys Ala Thr Cys Ala Cys Cys Gly Ala Cys Gly Cys Cys Gly Thr
885 890 895
Gly Gly Ala Cys Thr Gly Cys Gly Cys Cys Cys Thr Gly Gly Ala Cys
900 905 910
Cys Cys Cys Cys Thr Gly Thr Cys Cys Gly Ala Gly Ala Cys Cys Ala
915 920 925
Ala Gly Thr Gly Cys Ala Cys Cys Cys Thr Gly Ala Ala Gly Thr Cys
930 935 940
Cys Thr Thr Cys Ala Cys Cys Gly Thr Gly Gly Ala Gly Ala Ala Gly
945 950 955 960
Gly Gly Cys Ala Thr Cys Thr Ala Cys Cys Ala Gly Ala Cys Cys Thr
965 970 975
Cys Cys Ala Ala Cys Thr Thr Cys Cys Gly Cys Gly Thr Gly Cys Ala
980 985 990
Gly Cys Cys Cys Ala Cys Cys Gly Ala Gly Thr Cys Cys Ala Thr Cys
995 1000 1005
Gly Thr Gly Cys Gly Cys Thr Thr Cys Cys Cys Cys Ala Ala Cys Ala
1010 1015 1020
Thr Cys Ala Cys Cys Ala Ala Cys Cys Thr Gly Thr Gly Cys Cys Cys
1025 1030 1035 1040
Cys Thr Thr Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Thr Thr Cys
1045 1050 1055
Ala Ala Cys Gly Cys Cys Ala Cys Cys Cys Gly Cys Thr Thr Cys Gly
1060 1065 1070
Cys Cys Thr Cys Cys Gly Thr Gly Thr Ala Cys Gly Cys Cys Thr Gly
1075 1080 1085
Gly Ala Ala Cys Cys Gly Cys Ala Ala Gly Cys Gly Cys Ala Thr Cys
1090 1095 1100
Thr Cys Cys Ala Ala Cys Thr Gly Cys Gly Thr Gly Gly Cys Cys Gly
1105 1110 1115 1120
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1125 1130 1135
Cys Ala Ala Cys Thr Cys Cys Gly Cys Cys Thr Cys Cys Thr Thr Cys
1140 1145 1150
Thr Cys Cys Ala Cys Cys Thr Thr Cys Ala Ala Gly Thr Gly Cys Thr
1155 1160 1165
Ala Cys Gly Gly Cys Gly Thr Gly Thr Cys Cys Cys Cys Cys Ala Cys
1170 1175 1180
Cys Ala Ala Gly Cys Thr Gly Ala Ala Cys Gly Ala Cys Cys Thr Gly
1185 1190 1195 1200
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1205 1210 1215
Ala Cys Gly Cys Cys Gly Ala Cys Thr Cys Cys Thr Thr Cys Gly Thr
1220 1225 1230
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1235 1240 1245
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1250 1255 1260
Cys Cys Gly Gly Cys Cys Ala Gly Ala Cys Cys Gly Gly Cys Ala Ala
1265 1270 1275 1280
Gly Ala Thr Cys Gly Cys Cys Gly Ala Cys Thr Ala Cys Ala Ala Cys
1285 1290 1295
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1300 1305 1310
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1315 1320 1325
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1330 1335 1340
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1345 1350 1355 1360
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1365 1370 1375
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1380 1385 1390
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1395 1400 1405
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1410 1415 1420
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1425 1430 1435 1440
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1445 1450 1455
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1460 1465 1470
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1475 1480 1485
Ala Ala Cys Thr Gly Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys Cys
1490 1495 1500
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1505 1510 1515 1520
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1525 1530 1535
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1540 1545 1550
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1555 1560 1565
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1570 1575 1580
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1585 1590 1595 1600
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1605 1610 1615
Gly Thr Cys Cys Ala Cys Cys Ala Ala Cys Cys Thr Gly Gly Thr Gly
1620 1625 1630
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1635 1640 1645
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1650 1655 1660
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1665 1670 1675 1680
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1685 1690 1695
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1700 1705 1710
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1715 1720 1725
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1730 1735 1740
Cys Cys Ala Cys Cys Gly Ala Cys Gly Cys Cys Gly Thr Gly Cys Gly
1745 1750 1755 1760
Cys Gly Ala Cys Cys Cys Cys Cys Ala Gly Ala Cys Cys Cys Thr Gly
1765 1770 1775
Gly Ala Gly Ala Thr Cys Cys Thr Gly Gly Ala Cys Ala Thr Cys Ala
1780 1785 1790
Cys Cys Cys Cys Cys Thr Gly Cys Thr Cys Cys Thr Thr Cys Gly Gly
1795 1800 1805
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1810 1815 1820
Ala Cys Cys Cys Cys Cys Gly Gly Cys Ala Cys Cys Ala Ala Cys Ala
1825 1830 1835 1840
Cys Cys Thr Cys Cys Ala Ala Cys Cys Ala Gly Gly Thr Gly Gly Cys
1845 1850 1855
Cys Gly Thr Gly Cys Thr Gly Thr Ala Cys Cys Ala Gly Gly Ala Cys
1860 1865 1870
Gly Thr Gly Ala Ala Cys Thr Gly Cys Ala Cys Cys Gly Ala Gly Gly
1875 1880 1885
Thr Gly Cys Cys Cys Gly Thr Gly Gly Cys Cys Ala Thr Cys Cys Ala
1890 1895 1900
Cys Gly Cys Cys Gly Ala Cys Cys Ala Gly Cys Thr Gly Ala Cys Cys
1905 1910 1915 1920
Cys Cys Cys Ala Cys Cys Thr Gly Gly Cys Gly Cys Gly Thr Gly Thr
1925 1930 1935
Ala Cys Thr Cys Cys Ala Cys Cys Gly Gly Cys Thr Cys Cys Ala Ala
1940 1945 1950
Cys Gly Thr Gly Thr Thr Cys Cys Ala Gly Ala Cys Cys Cys Gly Cys
1955 1960 1965
Gly Cys Cys Gly Gly Cys Thr Gly Cys Cys Thr Gly Ala Thr Cys Gly
1970 1975 1980
Gly Cys Gly Cys Cys Gly Ala Gly Cys Ala Cys Gly Thr Gly Ala Ala
1985 1990 1995 2000
Cys Ala Ala Cys Thr Cys Cys Thr Ala Cys Gly Ala Gly Thr Gly Cys
2005 2010 2015
Gly Ala Cys Ala Thr Cys Cys Cys Cys Ala Thr Cys Gly Gly Cys Gly
2020 2025 2030
Cys Cys Gly Gly Cys Ala Thr Cys Thr Gly Cys Gly Cys Cys Thr Cys
2035 2040 2045
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2050 2055 2060
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2065 2070 2075 2080
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2085 2090 2095
Cys Cys Ala Gly Thr Cys Cys Ala Thr Cys Ala Thr Cys Gly Cys Cys
2100 2105 2110
Thr Ala Cys Ala Cys Cys Ala Thr Gly Thr Cys Cys Cys Thr Gly Gly
2115 2120 2125
Gly Cys Gly Cys Cys Gly Ala Gly Ala Ala Cys Thr Cys Cys Gly Thr
2130 2135 2140
Gly Gly Cys Cys Thr Ala Cys Thr Cys Cys Ala Ala Cys Ala Ala Cys
2145 2150 2155 2160
Thr Cys Cys Ala Thr Cys Gly Cys Cys Ala Thr Cys Cys Cys Cys Ala
2165 2170 2175
Cys Cys Ala Ala Cys Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr Cys
2180 2185 2190
Cys Gly Thr Gly Ala Cys Cys Ala Cys Cys Gly Ala Gly Ala Thr Cys
2195 2200 2205
Cys Thr Gly Cys Cys Cys Gly Thr Gly Thr Cys Cys Ala Thr Gly Ala
2210 2215 2220
Cys Cys Ala Ala Gly Ala Cys Cys Thr Cys Cys Gly Thr Gly Gly Ala
2225 2230 2235 2240
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2245 2250 2255
Thr Gly Cys Gly Gly Cys Gly Ala Cys Thr Cys Cys Ala Cys Cys Gly
2260 2265 2270
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2275 2280 2285
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2290 2295 2300
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2305 2310 2315 2320
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2325 2330 2335
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2340 2345 2350
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2355 2360 2365
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2370 2375 2380
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2385 2390 2395 2400
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2405 2410 2415
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2420 2425 2430
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2435 2440 2445
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2450 2455 2460
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2465 2470 2475 2480
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2485 2490 2495
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2500 2505 2510
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2515 2520 2525
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2530 2535 2540
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2545 2550 2555 2560
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2565 2570 2575
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2580 2585 2590
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2595 2600 2605
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2610 2615 2620
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2625 2630 2635 2640
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2645 2650 2655
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2660 2665 2670
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2675 2680 2685
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2690 2695 2700
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2705 2710 2715 2720
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2725 2730 2735
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2740 2745 2750
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2755 2760 2765
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2770 2775 2780
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2785 2790 2795 2800
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2805 2810 2815
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2820 2825 2830
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2835 2840 2845
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2850 2855 2860
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2865 2870 2875 2880
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2885 2890 2895
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2900 2905 2910
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2915 2920 2925
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2930 2935 2940
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2945 2950 2955 2960
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2965 2970 2975
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2980 2985 2990
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2995 3000 3005
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3010 3015 3020
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3025 3030 3035 3040
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3045 3050 3055
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3060 3065 3070
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3075 3080 3085
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3090 3095 3100
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3105 3110 3115 3120
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3125 3130 3135
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3140 3145 3150
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3155 3160 3165
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3170 3175 3180
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3185 3190 3195 3200
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3205 3210 3215
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3220 3225 3230
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3235 3240 3245
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3250 3255 3260
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3265 3270 3275 3280
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3285 3290 3295
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3300 3305 3310
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3315 3320 3325
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3330 3335 3340
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3345 3350 3355 3360
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3365 3370 3375
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3380 3385 3390
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3395 3400 3405
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3410 3415 3420
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3425 3430 3435 3440
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3445 3450 3455
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3460 3465 3470
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3475 3480 3485
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3490 3495 3500
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3505 3510 3515 3520
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3525 3530 3535
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3540 3545 3550
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3555 3560 3565
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3570 3575 3580
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3585 3590 3595 3600
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3605 3610 3615
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3620 3625 3630
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3635 3640 3645
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3650 3655 3660
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3665 3670 3675 3680
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3685 3690 3695
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3700 3705 3710
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3715 3720 3725
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3730 3735 3740
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3745 3750 3755 3760
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3765 3770 3775
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3780 3785 3790
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3795 3800 3805
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3810 3815 3820
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3825 3830 3835 3840
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3845 3850
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35 40 45
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50 55 60
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65 70 75 80
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85 90 95
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100 105 110
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115 120 125
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130 135 140
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145 150 155 160
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165 170 175
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180 185 190
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195 200 205
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210 215 220
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225 230 235 240
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245 250 255
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260 265 270
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275 280 285
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290 295 300
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305 310 315 320
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325 330 335
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340 345 350
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355 360 365
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370 375 380
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385 390 395 400
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405 410 415
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420 425 430
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435 440 445
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450 455 460
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465 470 475 480
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485 490 495
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500 505 510
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515 520 525
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530 535 540
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545 550 555 560
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565 570 575
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580 585 590
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595 600 605
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610 615 620
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625 630 635 640
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645 650 655
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660 665 670
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675 680 685
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690 695 700
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705 710 715 720
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725 730 735
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740 745 750
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755 760 765
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770 775 780
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785 790 795 800
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805 810 815
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820 825 830
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835 840 845
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850 855 860
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865 870 875 880
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885 890 895
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945 950 955 960
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980 985 990
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995 1000 1005
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1025 1030 1035 1040
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1075 1080 1085
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1185 1190 1195 1200
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1490 1495 1500
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1925 1930 1935
Ala Cys Thr Cys Cys Ala Cys Cys Gly Gly Cys Thr Cys Cys Ala Ala
1940 1945 1950
Cys Gly Thr Gly Thr Thr Cys Cys Ala Gly Ala Cys Cys Cys Gly Cys
1955 1960 1965
Gly Cys Cys Gly Gly Cys Thr Gly Cys Cys Thr Gly Ala Thr Cys Gly
1970 1975 1980
Gly Cys Gly Cys Cys Gly Ala Gly Cys Ala Cys Gly Thr Gly Ala Ala
1985 1990 1995 2000
Cys Ala Ala Cys Thr Cys Cys Thr Ala Cys Gly Ala Gly Thr Gly Cys
2005 2010 2015
Gly Ala Cys Ala Thr Cys Cys Cys Cys Ala Thr Cys Gly Gly Cys Gly
2020 2025 2030
Cys Cys Gly Gly Cys Ala Thr Cys Thr Gly Cys Gly Cys Cys Thr Cys
2035 2040 2045
Cys Thr Ala Cys Cys Ala Gly Ala Cys Cys Cys Ala Gly Ala Cys Cys
2050 2055 2060
Ala Ala Cys Thr Cys Cys Cys Cys Cys Cys Gly Cys Cys Gly Cys Gly
2065 2070 2075 2080
Cys Cys Cys Gly Cys Thr Cys Cys Gly Thr Gly Gly Cys Cys Thr Cys
2085 2090 2095
Cys Cys Ala Gly Thr Cys Cys Ala Thr Cys Ala Thr Cys Gly Cys Cys
2100 2105 2110
Thr Ala Cys Ala Cys Cys Ala Thr Gly Thr Cys Cys Cys Thr Gly Gly
2115 2120 2125
Gly Cys Gly Cys Cys Gly Ala Gly Ala Ala Cys Thr Cys Cys Gly Thr
2130 2135 2140
Gly Gly Cys Cys Thr Ala Cys Thr Cys Cys Ala Ala Cys Ala Ala Cys
2145 2150 2155 2160
Thr Cys Cys Ala Thr Cys Gly Cys Cys Ala Thr Cys Cys Cys Cys Ala
2165 2170 2175
Cys Cys Ala Ala Cys Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr Cys
2180 2185 2190
Cys Gly Thr Gly Ala Cys Cys Ala Cys Cys Gly Ala Gly Ala Thr Cys
2195 2200 2205
Cys Thr Gly Cys Cys Cys Gly Thr Gly Thr Cys Cys Ala Thr Gly Ala
2210 2215 2220
Cys Cys Ala Ala Gly Ala Cys Cys Thr Cys Cys Gly Thr Gly Gly Ala
2225 2230 2235 2240
Cys Thr Gly Cys Ala Cys Cys Ala Thr Gly Thr Ala Cys Ala Thr Cys
2245 2250 2255
Thr Gly Cys Gly Gly Cys Gly Ala Cys Thr Cys Cys Ala Cys Cys Gly
2260 2265 2270
Ala Gly Thr Gly Cys Thr Cys Cys Ala Ala Cys Cys Thr Gly Cys Thr
2275 2280 2285
Gly Cys Thr Gly Cys Ala Gly Thr Ala Cys Gly Gly Cys Thr Cys Cys
2290 2295 2300
Thr Thr Cys Thr Gly Cys Ala Cys Cys Cys Ala Gly Cys Thr Gly Ala
2305 2310 2315 2320
Ala Cys Cys Gly Cys Gly Cys Cys Cys Thr Gly Ala Cys Cys Gly Gly
2325 2330 2335
Cys Ala Thr Cys Gly Cys Cys Gly Thr Gly Gly Ala Gly Cys Ala Gly
2340 2345 2350
Gly Ala Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Ala Gly Gly
2355 2360 2365
Ala Gly Gly Thr Gly Thr Thr Cys Gly Cys Cys Cys Ala Gly Gly Thr
2370 2375 2380
Gly Ala Ala Gly Cys Ala Gly Ala Thr Cys Thr Ala Cys Ala Ala Gly
2385 2390 2395 2400
Ala Cys Cys Cys Cys Cys Cys Cys Cys Ala Thr Cys Ala Ala Gly Gly
2405 2410 2415
Ala Cys Thr Thr Cys Gly Gly Cys Gly Gly Cys Thr Thr Cys Ala Ala
2420 2425 2430
Cys Thr Thr Cys Thr Cys Cys Cys Ala Gly Ala Thr Cys Cys Thr Gly
2435 2440 2445
Cys Cys Cys Gly Ala Cys Cys Cys Cys Thr Cys Cys Ala Ala Gly Cys
2450 2455 2460
Cys Cys Thr Cys Cys Ala Ala Gly Cys Gly Cys Thr Cys Cys Thr Thr
2465 2470 2475 2480
Cys Ala Thr Cys Gly Ala Gly Gly Ala Cys Cys Thr Gly Cys Thr Gly
2485 2490 2495
Thr Thr Cys Ala Ala Cys Ala Ala Gly Gly Thr Gly Ala Cys Cys Cys
2500 2505 2510
Thr Gly Gly Cys Cys Gly Ala Cys Gly Cys Cys Gly Gly Cys Thr Thr
2515 2520 2525
Cys Ala Thr Cys Ala Ala Gly Cys Ala Gly Thr Ala Cys Gly Gly Cys
2530 2535 2540
Gly Ala Cys Thr Gly Cys Cys Thr Gly Gly Gly Cys Gly Ala Cys Ala
2545 2550 2555 2560
Thr Cys Gly Cys Cys Gly Cys Cys Cys Gly Cys Gly Ala Cys Cys Thr
2565 2570 2575
Gly Ala Thr Cys Thr Gly Cys Gly Cys Cys Cys Ala Gly Ala Ala Gly
2580 2585 2590
Thr Thr Cys Ala Ala Cys Gly Gly Cys Cys Thr Gly Ala Cys Cys Gly
2595 2600 2605
Thr Gly Cys Thr Gly Cys Cys Cys Cys Cys Cys Cys Thr Gly Cys Thr
2610 2615 2620
Gly Ala Cys Cys Gly Ala Cys Gly Ala Gly Ala Thr Gly Ala Thr Cys
2625 2630 2635 2640
Gly Cys Cys Cys Ala Gly Thr Ala Cys Ala Cys Cys Thr Cys Cys Gly
2645 2650 2655
Cys Cys Cys Thr Gly Cys Thr Gly Gly Cys Cys Gly Gly Cys Ala Cys
2660 2665 2670
Cys Ala Thr Cys Ala Cys Cys Thr Cys Cys Gly Gly Cys Thr Gly Gly
2675 2680 2685
Ala Cys Cys Thr Thr Cys Gly Gly Cys Gly Cys Cys Gly Gly Cys Gly
2690 2695 2700
Cys Cys Gly Cys Cys Cys Thr Gly Cys Ala Gly Ala Thr Cys Cys Cys
2705 2710 2715 2720
Cys Thr Thr Cys Gly Cys Cys Ala Thr Gly Cys Ala Gly Ala Thr Gly
2725 2730 2735
Gly Cys Cys Thr Ala Cys Cys Gly Cys Thr Thr Cys Ala Ala Cys Gly
2740 2745 2750
Gly Cys Ala Thr Cys Gly Gly Cys Gly Thr Gly Ala Cys Cys Cys Ala
2755 2760 2765
Gly Ala Ala Cys Gly Thr Gly Cys Thr Gly Thr Ala Cys Gly Ala Gly
2770 2775 2780
Ala Ala Cys Cys Ala Gly Ala Ala Gly Cys Thr Gly Ala Thr Cys Gly
2785 2790 2795 2800
Cys Cys Ala Ala Cys Cys Ala Gly Thr Thr Cys Ala Ala Cys Thr Cys
2805 2810 2815
Cys Gly Cys Cys Ala Thr Cys Gly Gly Cys Ala Ala Gly Ala Thr Cys
2820 2825 2830
Cys Ala Gly Gly Ala Cys Thr Cys Cys Cys Thr Gly Thr Cys Cys Thr
2835 2840 2845
Cys Cys Ala Cys Cys Gly Cys Cys Thr Cys Cys Gly Cys Cys Cys Thr
2850 2855 2860
Gly Gly Gly Cys Ala Ala Gly Cys Thr Gly Cys Ala Gly Gly Ala Cys
2865 2870 2875 2880
Gly Thr Gly Gly Thr Gly Ala Ala Cys Cys Ala Gly Ala Ala Cys Gly
2885 2890 2895
Cys Cys Cys Ala Gly Gly Cys Cys Cys Thr Gly Ala Ala Cys Ala Cys
2900 2905 2910
Cys Cys Thr Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Cys Thr Gly
2915 2920 2925
Thr Cys Cys Thr Cys Cys Ala Ala Cys Thr Thr Cys Gly Gly Cys Gly
2930 2935 2940
Cys Cys Ala Thr Cys Thr Cys Cys Thr Cys Cys Gly Thr Gly Cys Thr
2945 2950 2955 2960
Gly Ala Ala Cys Gly Ala Cys Ala Thr Cys Cys Thr Gly Thr Cys Cys
2965 2970 2975
Cys Gly Cys Cys Thr Gly Gly Ala Cys Ala Ala Gly Gly Thr Gly Gly
2980 2985 2990
Ala Gly Gly Cys Cys Gly Ala Gly Gly Thr Gly Cys Ala Gly Ala Thr
2995 3000 3005
Cys Gly Ala Cys Cys Gly Cys Cys Thr Gly Ala Thr Cys Ala Cys Cys
3010 3015 3020
Gly Gly Cys Cys Gly Cys Cys Thr Gly Cys Ala Gly Thr Cys Cys Cys
3025 3030 3035 3040
Thr Gly Cys Ala Gly Ala Cys Cys Thr Ala Cys Gly Thr Gly Ala Cys
3045 3050 3055
Cys Cys Ala Gly Cys Ala Gly Cys Thr Gly Ala Thr Cys Cys Gly Cys
3060 3065 3070
Gly Cys Cys Gly Cys Cys Gly Ala Gly Ala Thr Cys Cys Gly Cys Gly
3075 3080 3085
Cys Cys Thr Cys Cys Gly Cys Cys Ala Ala Cys Cys Thr Gly Gly Cys
3090 3095 3100
Cys Gly Cys Cys Ala Cys Cys Ala Ala Gly Ala Thr Gly Thr Cys Cys
3105 3110 3115 3120
Gly Ala Gly Thr Gly Cys Gly Thr Gly Cys Thr Gly Gly Gly Cys Cys
3125 3130 3135
Ala Gly Thr Cys Cys Ala Ala Gly Cys Gly Cys Gly Thr Gly Gly Ala
3140 3145 3150
Cys Thr Thr Cys Thr Gly Cys Gly Gly Cys Ala Ala Gly Gly Gly Cys
3155 3160 3165
Thr Ala Cys Cys Ala Cys Cys Thr Gly Ala Thr Gly Thr Cys Cys Thr
3170 3175 3180
Thr Cys Cys Cys Cys Cys Ala Gly Thr Cys Cys Gly Cys Cys Cys Cys
3185 3190 3195 3200
Cys Cys Ala Cys Gly Gly Cys Gly Thr Gly Gly Thr Gly Thr Thr Cys
3205 3210 3215
Cys Thr Gly Cys Ala Cys Gly Thr Gly Ala Cys Cys Thr Ala Cys Gly
3220 3225 3230
Thr Gly Cys Cys Cys Gly Cys Cys Cys Ala Gly Gly Ala Gly Ala Ala
3235 3240 3245
Gly Ala Ala Cys Thr Thr Cys Ala Cys Cys Ala Cys Cys Gly Cys Cys
3250 3255 3260
Cys Cys Cys Gly Cys Cys Ala Thr Cys Thr Gly Cys Cys Ala Cys Gly
3265 3270 3275 3280
Ala Cys Gly Gly Cys Ala Ala Gly Gly Cys Cys Cys Ala Cys Thr Thr
3285 3290 3295
Cys Cys Cys Cys Cys Gly Cys Gly Ala Gly Gly Gly Cys Gly Thr Gly
3300 3305 3310
Thr Thr Cys Gly Thr Gly Thr Cys Cys Ala Ala Cys Gly Gly Cys Ala
3315 3320 3325
Cys Cys Cys Ala Cys Thr Gly Gly Thr Thr Cys Gly Thr Gly Ala Cys
3330 3335 3340
Cys Cys Ala Gly Cys Gly Cys Ala Ala Cys Thr Thr Cys Thr Ala Cys
3345 3350 3355 3360
Gly Ala Gly Cys Cys Cys Cys Ala Gly Ala Thr Cys Ala Thr Cys Ala
3365 3370 3375
Cys Cys Ala Cys Cys Gly Ala Cys Ala Ala Cys Ala Cys Cys Thr Thr
3380 3385 3390
Cys Gly Thr Gly Thr Cys Cys Gly Gly Cys Ala Ala Cys Thr Gly Cys
3395 3400 3405
Gly Ala Cys Gly Thr Gly Gly Thr Gly Ala Thr Cys Gly Gly Cys Ala
3410 3415 3420
Thr Cys Gly Thr Gly Ala Ala Cys Ala Ala Cys Ala Cys Cys Gly Thr
3425 3430 3435 3440
Gly Thr Ala Cys Gly Ala Cys Cys Cys Cys Cys Thr Gly Cys Ala Gly
3445 3450 3455
Cys Cys Cys Gly Ala Gly Cys Thr Gly Gly Ala Cys Thr Cys Cys Thr
3460 3465 3470
Thr Cys Ala Ala Gly Gly Ala Gly Gly Ala Gly Cys Thr Gly Gly Ala
3475 3480 3485
Cys Ala Ala Gly Thr Ala Cys Thr Thr Cys Ala Ala Gly Ala Ala Cys
3490 3495 3500
Cys Ala Cys Ala Cys Cys Thr Cys Cys Cys Cys Cys Gly Ala Cys Gly
3505 3510 3515 3520
Thr Gly Gly Ala Cys Cys Thr Gly Gly Gly Cys Gly Ala Cys Ala Thr
3525 3530 3535
Cys Thr Cys Cys Gly Gly Cys Ala Thr Cys Ala Ala Cys Gly Cys Cys
3540 3545 3550
Thr Cys Cys Gly Thr Gly Gly Thr Gly Ala Ala Cys Ala Thr Cys Cys
3555 3560 3565
Ala Gly Ala Ala Gly Gly Ala Gly Ala Thr Cys Gly Ala Cys Cys Gly
3570 3575 3580
Cys Cys Thr Gly Ala Ala Cys Gly Ala Gly Gly Thr Gly Gly Cys Cys
3585 3590 3595 3600
Ala Ala Gly Ala Ala Cys Cys Thr Gly Ala Ala Cys Gly Ala Gly Thr
3605 3610 3615
Cys Cys Cys Thr Gly Ala Thr Cys Gly Ala Cys Cys Thr Gly Cys Ala
3620 3625 3630
Gly Gly Ala Gly Cys Thr Gly Gly Gly Cys Ala Ala Gly Thr Ala Cys
3635 3640 3645
Gly Ala Gly Cys Ala Gly Thr Ala Cys Ala Thr Cys Ala Ala Gly Thr
3650 3655 3660
Gly Gly Cys Cys Cys Thr Gly Gly Thr Ala Cys Ala Thr Cys Thr Gly
3665 3670 3675 3680
Gly Cys Thr Gly Gly Gly Cys Thr Thr Cys Ala Thr Cys Gly Cys Cys
3685 3690 3695
Gly Gly Cys Cys Thr Gly Ala Thr Cys Gly Cys Cys Ala Thr Cys Gly
3700 3705 3710
Thr Gly Ala Thr Gly Gly Thr Gly Ala Cys Cys Ala Thr Cys Ala Thr
3715 3720 3725
Gly Cys Thr Gly Thr Gly Cys Thr Gly Cys Ala Thr Gly Ala Cys Cys
3730 3735 3740
Thr Cys Cys Thr Gly Cys Thr Gly Cys Thr Cys Cys Thr Gly Cys Cys
3745 3750 3755 3760
Thr Gly Ala Ala Gly Gly Gly Cys Thr Gly Cys Thr Gly Cys Thr Cys
3765 3770 3775
Cys Thr Gly Cys Gly Gly Cys Thr Cys Cys Thr Gly Cys Thr Gly Cys
3780 3785 3790
Ala Ala Gly Thr Thr Cys Gly Ala Cys Gly Ala Gly Gly Ala Cys Gly
3795 3800 3805
Ala Cys Thr Cys Cys Gly Ala Gly Cys Cys Cys Gly Thr Gly Cys Thr
3810 3815 3820
Gly Ala Ala Gly Gly Gly Cys Gly Thr Gly Ala Ala Gly Cys Thr Gly
3825 3830 3835 3840
Cys Ala Cys Thr Ala Cys Ala Cys Cys Thr Ala Ala
3845 3850
<210> 7
<211> 3852
<212> PRT
<213> 重组核苷酸()
<400> 7

Claims (3)

1.一种重组痘苗病毒rWR-S6PB，其是在新型冠状病毒S蛋白基因密码子优化的基础上，用组织型纤溶酶原激活因子信号肽(Tissue plasminogen activator signal sequence,tPA)基因序列替换S蛋白信号肽基因序列，S6PB基因是在SARS-CoV-2S蛋白基因按哺乳动物密码子偏嗜性进行优化的基础上，将该蛋白基因2449-2451位、2674-2676位、2695-2697位、2824-2826位、2956-2958位和2959-2961位碱基均突变为“CCC”，上述碱基编码的第817位苯丙氨酸(Phenylalanine,Phe,F)、第892位丙氨酸(Alanine,Ala,A)、第899位丙氨酸(Alanine,Ala,A)、第942位丙氨酸(Alanine,Ala,A)、第986位赖氨酸(Lysine,Lys,K)和第987位缬氨酸(Valine,Val,V)均突变为脯氨酸(Proline,Pro,P)；将SARS-CoV-2S蛋白裂解位点基因2044-2055位碱基突变为“GGCTCCGCCTCC”，裂解位点氨基酸由“RRAR”突变为“GSAS”[精氨酸(Arginine,Arg,R)，甘氨酸(Glycine,Gly,G)，丝氨酸(Serine,Ser,S)]，并以痘苗病毒Western Reserve(WR)株为疫苗载体，构建了表达新型冠状病毒S蛋白变体S6PB基因的重组痘苗病毒rWR-S6PB。
2.一种疫苗组合物，包含权利要求1所述的重组痘苗病毒rWR-S6PB。
3.如权利要求2所述的疫苗组合物，疫苗是口服疫苗和/或亚单位疫苗。
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