CN113185613B - 新型冠状病毒s蛋白及其亚单位疫苗 - Google Patents
新型冠状病毒s蛋白及其亚单位疫苗 Download PDFInfo
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Abstract
本发明提供了一种新型冠状病毒S蛋白及其亚单位疫苗,所述新型冠状病毒S蛋白的S1/S2两个亚基之间的furin裂解位点682‑RRAR‑685替换为柔性的蛋白linker,所述linker为以甘氨酸G和丝氨酸S构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,n为≥1的整数。所述新型冠状病毒亚单位疫苗包含所述的重组S蛋白以及药学上接受的佐剂。本发明应用获取的具有生物活性的三聚体构象的S蛋白,制备成三聚体亚单位疫苗,免疫小鼠后能够诱导小鼠产生针对SARS‑CoV‑2的具有免疫保护作用的中和抗体,对免疫小鼠进行新型冠状病毒的致死性攻毒感染后,可以提供100%的保护效率。
Description
技术领域
本发明属于生物技术领域,涉及一种新型冠状病毒S蛋白及其亚单位疫苗。
背景技术
2020年1月30日,世卫组织宣布新型冠状病毒(SARS-CoV-2)引发的全球疫情为国际关注的突发公共卫生事件。由于病毒极高的传播潜力,截至2021年3月20日,全球已累计报告超过1.23亿例SARS-CoV-2感染病例,导致2718896例患者死亡。为了应对全球大流行的新型冠状病毒肺炎疫情,各国政府、企业和学术界正在制定各种治疗和预防对策,重中之重是疫苗和抗病毒药物的研发与应用。疫苗是抵抗所有病毒性疾病感染最有效的手段,也是保护未感染人群最有效的措施。
目前在研或已获批上市的SARS-CoV-2疫苗类型有:灭活疫苗、腺病毒载体疫苗、核酸疫苗(包括mRNA疫苗和DNA疫苗)以及亚单位疫苗。虽然已有部分针对SARS-CoV-2的疫苗紧急上市,用于应急使用,但目前的疫苗品种仍有很多问题:1、我国主要使用的灭活疫苗相比于核酸疫苗和亚单位疫苗的免疫原性不够强,灭活试剂会对抗原天然表位产生破环,而病毒颗粒中的其他但蛋白也会干扰S蛋白的免疫原性。2、核酸类疫苗产品是在新冠病毒中首次应用,以前还没有成功上市的核酸类疫苗,其长期的安全性,整合风险都未知。3、腺病毒疫苗的腺病毒载体会受到预存免疫的干扰。4、现有各种疫苗搜身针对早期流行毒株设计的,无法有效应对已经产生的突变株,不具有保守的保护效果。由于亚单位疫苗是采用SARS-CoV-2S蛋白全长或部分氨基酸序列作为抗原,具有强免疫原性,可以诱导高滴度的中和抗体产生,基因重组技术也便于对抗原进行突变株更新和广谱性设计,因此亚单位疫苗在应对SARS-CoV-2突变株具有更大的优势。
SARS-CoV-2是一种基因组约30kb,是有包膜的单股正链RNA病毒,属于冠状病毒家族β属。病毒基因组编码多种蛋白,包括刺突蛋白(S),膜糖蛋白(M)、核衣壳蛋白(N),膜蛋白(E)以及多种非结构蛋白。其中S蛋白是I型病毒融合蛋白,介导病毒附着在细胞表面受体血管紧张素转化酶2(ACE2)上,然后释放基因组进入细胞,因此S蛋白是中和性抗体的靶点,也是疫苗制备的主要有效成分。新型冠状病毒S蛋白由1273个氨基酸组成,包含21-35个N-糖基化位点。S蛋白以三聚体的形式在病毒表面形成特殊的花冠结构,冠状病毒因此而得名。S蛋白在宿主细胞蛋白酶的作用下,通过蛋白中部的RRAR剪切序列被裂解为S1和S2两个亚基,S1主要功能是与宿主细胞表面受体结合,S2亚基介导病毒-细胞以及细胞-细胞膜融合。S蛋白完整的三聚体结构是病毒被宿主细胞核中和性抗体识别的首要结构。
因此,亟需开发一种新的有效的针对新型冠状病毒的疫苗。
发明内容
为了解决所述技术问题,本发明提供了一种新型冠状病毒S蛋白及其亚单位疫苗,S1/S2切割位点RRAR经突变以失去被弗林样蛋白酶切割的能力,以保留完整的S蛋白抗原性。
在本发明的第一方面,提供了一种新型冠状病毒S蛋白,所述新型冠状病毒S蛋白的S1/S2两个亚基之间的Furin裂解位点682-RRAR-685替换为柔性的蛋白linker,所述linker为以甘氨酸G和丝氨酸S构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,其中,n为≥1的整数。
进一步地,所述新型冠状病毒S蛋白具有如下修饰1-修饰4中的至少一种:
修饰1、所述新型冠状病毒S蛋白的原始信号肽替换为tPA信号肽、CD5信号肽和IgG信号肽中的一种;
修饰2、所述新型冠状病毒S蛋白的SEQ ID NO:24所示跨膜区替换为T4噬菌体fibritin三聚体基序或SEQ ID NO:25GCN4多聚体形成基序;
修饰3、所述新型冠状病毒S蛋白的C端结构域删除SEQ ID NO:7所示跨膜结构域;
修饰4、所述新型冠状病毒S蛋白在氨基酸位置817-987处具有一个或多个氨基酸残基突变为脯氨酸的突变,所述突变包括K986P和/或V987P取代。
进一步地,所述新型冠状病毒S蛋白的核苷酸序列如SEQ ID NO:8所示或者SEQ IDNO:10所示或者SEQ ID NO:12所示。
进一步地,所述新型冠状病毒S蛋白的氨基酸序列如SEQ ID NO:9所示或者SEQ IDNO:11所示或者SEQ ID NO:13所示。
在本发明的第二方面,提供了一种核酸分子,所述核酸分子的核苷酸序列如SEQID NO:8所示或者SEQ ID NO:10所示或者SEQ ID NO:12所示。
在本发明的第三方面,提供了一种重组表达载体,所述重组表达载体能够表达所述的新型冠状病毒S蛋白。
进一步地,所述重组表达载体的表达区的核苷酸序列如SEQ ID NO:8所示或者SEQID NO:10所示或者SEQ ID NO:12所示。
在本发明的第四方面,提供了一种工程化细胞,所述工程化细胞包含所述的重组表达载体。
在本发明的第五方面,提供了一种新型冠状病毒S蛋白的制备方法,所述方法包括:
获得所述的重组表达载体;
将所述重组表达载体转染至细胞中,并通过细胞群的谷氨酰胺抗性筛选以及单克隆筛选,获得稳定表达重组S蛋白的细胞株;
将所述细胞株进行分泌表达和纯化,获得纯化的重组新型冠状病毒S蛋白。
在本发明的第六方面,提供了一种新型冠状病毒亚单位疫苗,所述新型冠状病毒亚单位疫苗包含所述的重组S蛋白以及药学上接受的佐剂。
进一步,所述佐剂包括氢氧化铝、卵磷脂、弗氏佐剂、MPL TM、IL-12、氢氧化铝联合CpG ODN复合佐剂、ISA51VG、ISA720VG、MF59、QS21、AS03佐剂中的至少一种。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明提供的新型冠状病毒S蛋白及其亚单位疫苗,本发明的免疫原S蛋白多肽具有稳定的融合前构象,将S1/S2两个亚基之间的Furin裂解位点682-RRAR-685替换为柔性的蛋白linker,如以G(Gly)甘氨酸和S(Ser)丝氨酸构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,通过n来调整linker的长度和效果。在本发明中,我们比较了多种柔性linker,图2B的结果表明,与已经报道的682-QQAQ-685突变相比,GS柔性linker,包括682-GSAS-685,682-GG-685都具有更好的保护剪切的效果,GS突变中S2的剪切更少,能更好地保持不被剪切的S蛋白形式。本发明应用获取的具有生物活性的三聚体构象的S蛋白,制备成三聚体亚单位疫苗,免疫小鼠后能够诱导小鼠产生针对SARS-CoV-2原始株以及目前流行的突变株都具有免疫保护作用的中和抗体,对免疫小鼠进行新型冠状病毒的致死性攻毒感染后,可以提供100%的保护效率。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为哺乳动物细胞分泌型S蛋白序列设计图;
图2为S蛋白在CHO-K1细胞中的优化表达效果;图2A:对比S蛋白原始序列与优化密码子序列蛋白表达水平;图2B:对比S蛋白Furin剪切位点原始序列以及两种不同突变策略的剪切情况;图2C:对比具有原始信号肽与替换为tPA或者IgG信号肽的S蛋白在细胞培养上清中的表达情况;
图3为稳定表达S蛋白的CHO-K1细胞生长曲线;
图4为分泌表达重组S蛋白的表达量分析;
图5为分泌表达重组S蛋白的三聚体分析;
图6为S蛋白三聚体抗原激发特异性抗体水平检测;
图7为S蛋白三聚体抗原免疫小鼠后产生的抗体中和活性检测;图7A和7B分别为采集二次接种疫苗或者PBS的小鼠血清以及收集SARS-CoV-2康复患者血清,稀释不同的梯度,分别与新冠假病毒孵育1h,感染BHK-21ACE2细胞,24h后收样检测萤火虫荧光素酶活性;
图8为重组S蛋白三聚体亚单位疫苗的保护性研究;图8A和图8B分别为二次接种疫苗或者PBS的小鼠通过滴鼻的方式感染SARS-CoV-2,分别记录疫苗组和对照组小鼠的体重变化以及生存的情况;
图9为本发明疫苗对新冠不同突变株假病毒具有极高中和活性的结果;图9A为疫苗二次免疫组小鼠血清针对原始株以及部分目前流行的突变株假病毒具有较高的中和活性结果;图9B为灭活疫苗志愿者血清针对原始株假病毒仅有较低的中和活性,并且对于南非株假病毒完全没有中和活性的结果;
图10为重组S蛋白三聚体亚单位疫苗的攻毒验证;图10A为疫苗组小鼠在感染SARS-CoV-2后体重变化不大的结果;图10B为重组S蛋白三聚体亚单位疫苗可完全抵抗致死剂量的SARS-CoV-2感染结果。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买得到或者可通过现有方法制备得到。
本发明实施例提供的技术方案为解决上述技术问题,总体思路如下:
本申请人经过分析和实验验证发现:覆盖S1和S2的全部序列是最佳的疫苗抗原选择,比单独的S1(或RBD区域)或者单独的S2能诱导更多的抗体种类,更具有广谱性。同时,S蛋白完整的三聚体结构是病毒被宿主细胞核中和性抗体识别的首要结构,因此,如能在体外还原S蛋白三聚体结构,通过体外重组表达核纯化获得三聚体S蛋白,则能够模拟病毒天然构象,激活机体产生最接近天然病毒的识别抗体,也是亚单位重组疫苗的最佳选择。
因此,本发明的目的在于提供一种同时包括S1和S2两个亚基,又能在哺乳动物细胞上清中分泌表达的新型冠状病毒三聚体S蛋白及其基因序列,以及以CHO细胞上清表达的此蛋白做成新型冠状病毒亚单位疫苗所提供的高效保护作用。
根据本发明实施例一种典型的实施方式,提供一种新型冠状病毒S蛋白,所述新型冠状病毒S蛋白的S1/S2两个亚基之间的Furin裂解位点682-RRAR-685替换为柔性的蛋白linker,所述linker为以甘氨酸G和丝氨酸S构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,其中,n为≥1的整数。
n为≥1的整数均在本发明的保护范围之内;作为优选的方案,本申请实施例中n优选取1-3;所述linker为(G)n,时,n优选值可长些,此时n优选取1-10;
本申请的核心在于将S1/S2切割位点RRAR经突变以失去被弗林样蛋白酶切割的能力,以保留完整的S蛋白抗原性,本发明的免疫原S蛋白多肽具有稳定的融合前构象,将S1/S2两个亚基之间的Furin裂解位点682-RRAR-685替换为柔性的蛋白linker,如以G(Gly)甘氨酸和S(Ser)丝氨酸构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,通过n来调整linker的长度和效果。在本发明实施例中,我们比较了多种柔性linker,图2B的结果表明,与已经报道的682-QQAQ-685突变相比,GS柔性linker,包括682-GSAS-685,682-GG-685都具有更好的保护剪切的效果,GS突变中S2的剪切更少,能更好地保持不被剪切的S蛋白形式。
野生的新型冠状病毒S蛋白的核苷酸序列如SEQ ID NO:1所示,野生的新型冠状病毒S蛋白的氨基酸序列如SEQ ID NO:2所示;其中野生的新型冠状病毒S蛋白的原始信号肽的氨基酸序列为MFVFLVLLPLVSS,如SEQ ID NO:15所示;原始信号肽的核苷酸序列如SEQ IDNO:14所示;
为了在真核细胞中高效表达,我们使用了JAVA Codon Adapation软件对S表达基因的密码子进行了哺乳动物偏好的密码子优化,在一些实施方案中,本申请选用了JAVACodon Adapation软件对S表达基因的密码子进行了优化,获得了在哺乳动物细胞中表达效率比天然S基因更高的表达效率。原始信号肽的核苷酸序列优化为如SEQ ID NO:16所示;原始信号肽的氨基酸序列如SEQ ID NO:17所示(氨基酸序列同SEQ ID NO:15);也可采用其他的密码子优化方式,具体可采用如下一种密码子优化方案:
密码子优化方案1:核苷酸序列如SEQ ID NO:3所示;
密码子优化方案2:核苷酸序列如SEQ ID NO:4所示;
密码子优化方案3:核苷酸序列如SEQ ID NO:5所示;
密码子优化方案4:核苷酸序列如SEQ ID NO:6所示;
密码子优化方案5:核苷酸序列如SEQ ID NO:7所示;
并将所述密码子优化方案1-5中S1/S2两个亚基之间的furin裂解位点682-RRAR-685替换为本申请的柔性的蛋白linker的密码子序列即可,所述linker为以甘氨酸G和丝氨酸S构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,其中,n为≥1的整数。
作为一种可选的实施方式,所述新型冠状病毒S蛋白具有如下修饰1-修饰4中的至少一种:
修饰1、所述新型冠状病毒S蛋白的原始信号肽可替换为SEQ ID NO:18所示以及SEQ ID NO:19所示tPA信号肽、SEQ ID NO:20所示以及SEQ ID NO:21所示IgG信号肽、SEQID NO:22所示以及SEQ ID NO:23所示CD5信号肽中的一种;该序列采用分泌性信号肽以保证S蛋白可以在哺乳动物细胞的培养上清中分泌表达,选用tPA信号肽以及S蛋白天然信号肽以及IgG信号肽以及CD5信号肽,最优的选择tPA信号肽。
修饰2、所述新型冠状病毒S蛋白的SEQ ID NO:24所示跨膜区替换为T4噬菌体Fibritin三聚体基序或SEQ ID NO:25GCN4多聚体形成基序。为更优的形成三聚体结构,将S蛋白与T4噬菌体的次要纤维蛋白(Fibritin)的三聚体折叠结构域在C末端进行融合。
修饰3、所述新型冠状病毒S蛋白的C端结构域删除SEQ ID NO:26所示跨膜结构域。目的在于促进所述重组S蛋白的分泌表达。
修饰4、所述新型冠状病毒S蛋白在氨基酸位置817-987处具有一个或多个氨基酸残基突变为脯氨酸的突变,所述突变可包括K986P和/或V987P取代。两个脯氨酸残基的取代提高了预融合构象的稳定性。
以上修饰1-修饰4中的一种或多种,其中任何一种排列组合的方案,均在本发明的保护范围之内。
作为优选地,新型冠状病毒S蛋白的核苷酸序列可采用SEQ ID NO:8、SEQ ID NO:10和SEQ ID NO:12任一所示,其中:
新型冠状病毒S蛋白的核苷酸序列SEQ ID NO:8所示方案中,采用所述密码子优化方案3骨架+原始信号肽+682GSAS685+T4噬菌体Fibritin三聚体基序;氨基酸序列SEQ ID NO:9所示;
新型冠状病毒S蛋白的核苷酸序列SEQ ID NO:10所示方案中,采用所述密码子优化方案3骨架+tPA信号肽+691GSAS694+T4噬菌体Fibritin三聚体基序;氨基酸序列SEQ IDNO:11所示;
新型冠状病毒S蛋白的核苷酸序列SEQ ID NO:12所示方案中,采用所述密码子优化方案3骨架+tPA信号肽+691GG692+T4噬菌体Fibritin三聚体基序;氨基酸序列SEQ ID NO:13所示;
本发明实施例通过多种表达元件的测试和比较,获得了一种可以在哺乳动物细胞中高效分泌表达的S蛋白重组基因序列和蛋白序列。作为一种最优的技术方案,该序列是一种C段截断形式的S蛋白,保留S1和S2两个亚基,除跨膜区外的所有功能区都保留,以最大限度保留S蛋白上的抗体表位。所述最优的技术方案中,所述新型冠状病毒S蛋白的核苷酸序列如SEQ ID NO:8所示或者SEQ ID NO:10所示或者SEQ ID NO:12所示。所述新型冠状病毒S蛋白的氨基酸序列如SEQ ID NO:9所示或者SEQ ID NO:11所示或者SEQ ID NO:13所示。
根据本发明实施例另一种典型的实施方式,提供一种核酸分子,所述核酸分子的核苷酸序列如SEQ ID NO:8所示或者SEQ ID NO:10所示或者SEQ ID NO:12所示。含有所述核酸分子的生物材料也在本发明的保护范围之内,所述生物材料包括重组DNA、质粒载体、噬菌体载体、病毒载体和工程菌中的一种。
根据本发明实施例另一种典型的实施方式,提供一种重组表达载体,所述重组表达载体能够表达所述的新型冠状病毒S蛋白。
根据本发明实施例另一种典型的实施方式,提供一种工程化细胞,所述工程化细胞包含所述的重组表达载体。所述工程化细胞可选用悬浮细胞,包括CHO系列和293、293FT等人用疫苗哺乳动物细胞株都在本发明的保护范围之内,具体地,本发明实施例使用CHO-K1细胞,通过将上述S基因转染该细胞并获得稳定表达细胞株,高效表达重组的S蛋白。
根据本发明实施例另一种典型的实施方式,提供一种新型冠状病毒S蛋白的制备方法,所述方法包括:
获得所述的重组表达载体;
将所述重组表达载体转染至细胞中,并通过细胞群的谷氨酰胺抗性筛选以及单克隆筛选,获得稳定表达重组S蛋白的细胞株;
将所述细胞株进行分泌表达和纯化,获得纯化的重组新型冠状病毒S蛋白。
根据本发明实施例另一种典型的实施方式,提供一种新型冠状病毒亚单位疫苗,所述新型冠状病毒亚单位疫苗包含所述的重组S蛋白以及药学上接受的佐剂。
所述佐剂包括氢氧化铝、卵磷脂、弗氏佐剂、MPL TM、IL-12、氢氧化铝联合CpG ODN复合佐剂、ISA51VG、ISA720VG、MF59、QS21、AS03佐剂中的至少一种。在其他实施方式中,所述佐剂也可选用其他形式的佐剂。
所述新型冠状病毒亚单位疫苗可以制备成滴鼻剂、喷雾剂和肌肉注射剂。
本发明应用获取的具有生物活性的三聚体构象的S蛋白,制备成三聚体亚单位疫苗,免疫小鼠后能够诱导小鼠产生针对SARS-CoV-2的具有免疫保护作用的中和抗体,对免疫小鼠进行新型冠状病毒的致死性攻毒感染后,可以提供100%的保护效率。
下面将结合实施例及实验数据对本申请的效果进行详细说明。
实施例一重组S蛋白载体构建与表达优化
1、哺乳动物细胞上清表达的S蛋白基因的构建
本发明的S蛋白表达基因的构建示意图见图1。图1中哺乳动物细胞分泌型S蛋白序列设计图:序列保留原始信号肽,或者突变为tPA信号肽/CD5信号肽/IgG信号肽;Furin剪切位点由RRAR突变为GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n;C端跨膜区以及胞内区替换为T4噬菌体次要纤维蛋白序列。
首先,为了在真核细胞中高效表达,我们使用了JAVA Codon Adapation软件对S表达基因的密码子进行了哺乳动物偏好的密码子优化,原始信号肽的核苷酸序列优化为如SEQ ID NO:16所示;图2A的结果表明,未经优化的S天然基因在CHO细胞中表达量极低,几乎检测不到;而经过密码子优化的S基因可以在CHO细胞中高效的表达S蛋白。
其次,本发明的免疫原S蛋白多肽具有稳定的融合前构象,将S1/S2两个亚基之间的furin裂解位点682-RRAR-685替换为柔性的蛋白linker,如以G(Gly)甘氨酸和S(Ser)丝氨酸构成的GSAS、GS组合、(GGGS)n或者(GGGGS)n或者(G)n,通过n来调整linker的长度和效果。在本发明中,我们比较了多种柔性linker,图2B的结果表明,与已经报道的682-QQAQ-685突变相比,GS柔性linker,包括682-GSAS-685,682-GG-685都具有更好的保护剪切的效果,GS突变中S2的剪切更少,能更好地保持不被剪切的S蛋白形式。图7的结果表明,完整的S蛋白可以诱导产生高滴度的特异性中和抗体,而仅靶向S蛋白S2亚基的抗体不具有中和病毒的活性,即无法保护机体抵抗SARS-CoV-2的感染。
再次,为了无需裂解细胞而在哺乳动物上清中表达S蛋白,我们去掉了天然S蛋白的跨膜区(TM区),将去掉TM区的S蛋白与T4噬菌体的次要纤维蛋白(Fibritin)的三聚体折叠结构域在C末端进行融合,增强三聚体的形成。在使用分泌信号肽时,我们比较了tPA信号肽,S蛋白自身信号肽以及人IgG信号肽。图2C的结果表明,人组织纤溶酶原激活剂(tPA)信号肽和S蛋白自身信号肽都能在转染的CHO上清中检测到S蛋白,因此优选这两种信号肽进行上清表达。
所述S基因的核苷酸序列如SEQ ID NO:8所示或者SEQ ID NO:10所示或者SEQ IDNO:12所示。
2、分泌表达重组S蛋白的CHO-K1细胞株构建
将上述(1)中构建的S基因(所述S基因的核苷酸序列如SEQ ID NO:8所示)克隆至表达载体载体(由promega公司的pC-neo载体改造,加入GS表达标签)中,得到带有谷氨酰胺合成酶(GS)筛选标签的表达载体。具体步骤为:pC-GS载体利用限制性内切酶NheI以及SmaI(Thermo Fisher)酶切,S基因则用NheI以及EcorV(Thermo Fisher)酶切,然后通过琼脂糖凝胶电泳分别回收已经酶切好的载体以及S基因,再利用T4连接酶(New England Biolabs)链接,随后转化进感受态DH5,挑单克隆并鉴定正确的pC-GS-S克隆,即得到S基因表达质粒。
将上述S基因表达质粒通过电转仪(Biorad公司)电穿孔方式转染至CHO-K1细胞(武汉大学中国典型培养物保藏中心)中。电穿孔转染操作方案:取1×107个细胞到800LCHO-CD1无血清培养基(上海培源生物科技股份有限公司)中重悬,加入40μg质粒混匀,转移上述培养液至电转杯(Biorad公司)中,设置电转程序为电压300V,电容960μF,电阻∞,指数脉冲。电击完毕,取出培养液至CHO-CD1培养基,调整细胞浓度为5×105个/mL。电转后每天检测细胞密度以及存活率,当细胞浓度开始稳定增长至1×106个/mL时,证明细胞株完成建系。通过有限稀释法进行筛选,挑选克隆株分别扩大培养,进一步筛选优势细胞株。优势株扩大培养后,接种细胞进行细胞生长周期检测。
3、CHO-K1-S细胞生长检测
上述优势株细胞转接到20mL CHO-CD1培养基中,细胞终浓度为1×106个/mL,放置到37℃,5%CO2,转速为120rpm的摇床培养箱(Thermo fisher)中。接种当天记第0天,然后每24小时利用血球计数板(Biorad)计数,统计细胞生长情况,计数到细胞数目不在增长或下降为止。实验结果表明,在1×106个/mL接种浓度下,优势株细胞在接种第2-3天进入对数生长期,第6-7天到达平台期,第8天开始进入衰亡期(图3)。图3为稳定表达S蛋白的CHO-K1-S细胞生长曲线:接种2×107个CHO细胞到20mL无血清培养基中,连续培养8天,统计生长情况。
4、分泌表达重组S蛋白的检测
优势株细胞转接到2个含有20mL CHO-CD1培养基的细胞培养瓶中,细胞终浓度分别为1×106个/mL记为A瓶;2×106个/mL记为B瓶。放置到37℃5%CO2,转速为120rpm的摇床培养箱中。连续培养4天后,然后取A瓶以及B瓶的培养液100μL,800rpm离心5分钟,取出上清,加入对应体积的6×SDS loading buffer;离心后的细胞加入40μL 1×SDS loadingbuffer,然后放置100℃金属浴(DLAB)10分钟,SDS-PAGE检测上清中蛋白表达。实验结果表明,优势株细胞上清中可以检测出高表达的S蛋白,并且在接种初始浓度高的样品中,S蛋白表达量更高(图4)。图4为分泌表达重组S蛋白的表达量分析:接种CHO细胞到无血清培养基,根据接种细胞数分别记为A瓶和B瓶,培养4天后,检测细胞和培养基上清S蛋白表达。
上述B瓶细胞培养液离心后取得的上清培养基取出10μL、20μL、40μL、60μL到EP管中,第二份对应地加入2μL、4μL、8μL、12μL 6×Native loading buffer,然后通过Native-PAGE检测上清表达S蛋白的三聚体形式。如图5所示,实验结果表明,优势株细胞上清中可以检测出少量S蛋白单体存在,大部分S蛋白在培养基上清中以二聚体或三聚体形式存在(图5),说明CHO-K1-S细胞株可以大量表达天然多聚体构象的S蛋白,并且上述细胞株分泌表达重组S蛋白能够达到3g/L,可以满足亚单位疫苗生产要求。
实施例二、重组S蛋白三聚体疫苗免疫与效果鉴定
1、小鼠的免疫流程
本实验所用小鼠为K18-hACE雄鼠,6-8周,19-28g,从江苏集萃药康生物科技有限公司购入,所有动物实验操作在SPF实验室进行。实验组接种疫苗+佐剂,共4只小鼠;对照组接种PBS,共4只小鼠。接种第一针疫苗后14天接种第二针,接种第一针后35天(即接种第二针后21天),对所有小鼠进行眼眶取血。取小鼠血清用于检验。
2、ELISA检测流程
(1)用0.1M碳酸盐缓冲液(pH=9.6)稀释RBD(义翘神州科技有限公司),使终浓度为1ng/μL,再向96孔酶标板的各孔加入100μL,使每孔最终含有100ng RBD。37℃孵育3小时。
(2)弃包被液,每孔加250μL的0.05%的PBST(莫纳生物科技有限公司)进行洗涤。洗涤3次,每次5分钟。
(3)再向每孔加入200μL的5%脱脂牛奶(用0.05%PBST配制),进行封闭,37℃孵育3小时。
(4)用5%脱脂牛奶对疫苗组和对照组小鼠的血清样品进行1:500稀释。弃封闭液,每孔加入100μL已经稀释的小鼠血清,每份小鼠血清做三次重复,4℃过夜。
(5)弃血清,每孔加200μL的0.05%的PBST进行洗涤。洗涤3次,每次5分钟。
(6)用5%脱脂牛奶以1:5000比例稀释羊抗鼠IgG二抗(博尔西科技有限公司)。向每孔加入100μL稀释后的二抗。37℃孵育1h。
(7)弃二抗,每孔加200μL的0.05%的PBST进行洗涤。洗涤3次,每次5分钟。
(8)再向每孔加入100μL的酶作用底物(Hcm TMB One),室温避光显色30分钟。每孔加入50μL 1M HCl终止反应。放入酶标仪中检测OD450的值。
3、VSV骨架新冠假病毒包装流程
新冠病毒Spike蛋白表达质粒Sdel-18(尾端删除18个氨基酸,质粒来源夏宁邵教授实验室)转染Vero E6细胞(15μg/10cm dish),转染后48h,用种子病毒VSV-DG-Luc(质粒来源夏宁邵教授实验室)感染细胞(300μL/10cm dish)。感染1h后,吸弃病毒液,换为含有VSV-G抗体(1:1000)的新鲜完全培养基。37℃培养24h,收取细胞上清,分装假病毒,冻存于-80℃备用。
4、新冠假病毒中和实验流程
(1)接种BHK-21ACE2细胞(来自武汉大学中国典型培养物保藏中心)于96孔板中,待细胞聚合度达到90%,进行实验。
(2)小鼠血清56℃,30min去除补体。
(3)用感染培养基(DMEM+2%FBS+1%PS)梯度稀释小鼠血清。
(4)用感染培养基(DMEM+2%FBS+1%PS)稀释假病毒,假病毒(v):总体积(v)=1:10。
(5)血清稀释液与假病毒稀释液等比混合(50L+50L每孔),37℃孵育1h。
(6)用PBS清洗BHK-21ACE2细胞两次。
(7)血清与假病毒混合液加入细胞,37℃培养24h,裂解细胞,加入萤火虫荧光素酶底物(Promega),用Varioskan LUX多功能微孔板读数仪(Thermo fisher)测定萤火虫荧光素酶活性。
5、本发明疫苗免疫的小鼠血清中含有极高的anti-RBD IgG
为了验证设计的疫苗能诱导小鼠产生特异性抗体,将制备的重组S蛋白疫苗免疫小鼠,对照组小鼠接种等体积1×PBS。接种第一针后35天(即接种第二针后21天),对所有小鼠进行眼眶取血。随后将制备的4份疫苗组小鼠血清和4份对照组小鼠血清按1:500稀释,用于酶联免疫反应(ELISA)检测血清中特异性抗体的含量。
实验结果如图6所示。可以看出,在1:500稀释倍数下,疫苗组小鼠血清与新冠病毒RBD蛋白反应的OD450值显著高于对照组小鼠血清与新冠病毒RBD蛋白反应。这说明本发明制备的疫苗能够诱导小鼠产生极高的针对S蛋白RBD区域的特异性抗体。同时,灭活疫苗志愿者血清以1:2000释倍数下,可诱导与本疫苗相似水平的RBD区域抗体反应。
6、本发明疫苗保持S蛋白抗原完整性以及对新冠假病毒具有极高中和活性
为了验证本疫苗产生的抗体对新冠病毒的具有中和能力,将本疫苗二次免疫的小鼠血清和商业化靶向S蛋白的S2亚基抗体(义翘神州科技有限公司)分别进行假病毒中和实验,对比上述两种抗体的中和活性。
取疫苗二次免疫组小鼠(S-55)血清以及对照组(PBS-53)小鼠血清,进行1:100、1:1000、1:10000、1:20000、1:40000和1:80000倍稀释;商业化靶向S蛋白的S2亚基抗体进行1:100、1:500、1:2500、1:5000和1:10000倍稀释。将稀释血清与VSV骨架新冠假病毒进行中和实验,测定小鼠血清和商业化靶向S蛋白的S2亚基特异性抗体的中和活性。
图7A结果显示,疫苗二次免疫小鼠血清在1:100–1:10000稀释度下对新冠假病毒具有中和活性,在稀释度约1:8000时达到50%中和效率。而对照组小鼠血清在所有稀释度下均没有对假病毒中和活性;图7B结果显示,商业化靶向S蛋白的S2亚基特异性抗体仅在低稀释浓度表现出很弱的假病毒中和活性。这表明,本疫苗的重组S蛋白可以保留S蛋白抗原的完整性,并可以诱导产生对新冠病毒中和效果好、高水平的特异性抗体。
7、本发明疫苗对新冠原始株假病毒具有极高中和活性
为了对比本疫苗产生的抗体以及现有新冠灭活疫苗对新冠假病毒的中和活性,将本疫苗二次免疫的小鼠血清以及灭活疫苗志愿者血清进行假病毒中和实验。
取疫苗二次免疫组小鼠血清,进行1:100、1:1000、1:10000、1:20000、1:40000、1:80000、1:160000和1:320000倍稀释;新冠病毒灭活疫苗志愿者血清进行1:102、1:103、1:104、1:105、1:106和1:107倍稀释。对照组小鼠血清作为对照。将稀释血清与VSV骨架新冠假病毒进行中和实验,测定小鼠血清和灭活疫苗志愿者血清中S蛋白特异性抗体的中和活性。
图8A结果显示,疫苗二次免疫小鼠血清在1:100–1:32000稀释度下均对新冠假病毒具有中和活性,在稀释度约为1:6300时达到50%中和效率。而对照组血清在所有稀释度下均没有对假病毒中和活性。图8B结果显示,灭活疫苗志愿者血清1:103–1:105稀释度对新冠假病毒具有低中和活性,且无法达到50%中和效率。这表明,对比现有灭活疫苗,本疫苗可以诱导产生对新冠病毒中和效果更好、高水平的特异性抗体。
8、本发明疫苗对新冠不同突变株假病毒具有极高中和活性
为了对比本疫苗以及现有新冠灭活疫苗产生的抗体对新冠不同突变株假病毒的中和活性,将本疫苗二次免疫的小鼠血清以及灭活疫苗志愿者血清进行突变株假病毒中和实验。
取疫苗二次免疫组小鼠血清以及新冠病毒灭活疫苗志愿者血清,进行1:102、1:103、1:104、1:105、1:106倍稀释。将稀释血清与VSV骨架新冠不同突变株假病毒进行中和实验,测定并统计小鼠血清和灭活疫苗志愿者血清中特异性抗体的50%中和活性或者20%中和活性的稀释度。
图9A结果显示,疫苗二次免疫组小鼠血清针对原始株以及部分目前流行的突变株假病毒具有较高的中和活性,尽管对于南非株假病毒的中和活性有所下降。图9B结果显示,灭活疫苗志愿者血清针对原始株假病毒仅有较低的中和活性,并且对于南非株假病毒完全没有中和活性。这表明本疫苗可以诱导产生对新冠原始株以及目前流行的新冠突变株中和效果更好,且滴度更高的特异性抗体。
实施例三、重组S蛋白三聚体亚单位疫苗的攻毒验证
将上述小鼠适应ABSL-3环境2-3天后进行实验,小鼠对应分为以下2组:对照组:PBS组;实验组:疫苗组;每组4只。
SARS-CoV-2攻毒上述两组小鼠,剂量为2.5×102PFU/只(中国科学院武汉病毒研究所分离的临床毒株,武汉大学ABSL-3实验室扩增)。具体操作流程为:SARS-CoV-2原液初始滴度为6×106PFU/mL,共200μL。准备1.5mL螺帽管,加入714μL 1x PBS以及6μLSARS-CoV-2原液混匀,此时病毒稀释液体积为720μL,滴度为5×104PFU/mL。然后准备2mL螺帽管,加入1800μL 1×PBS以及200μL上述SARS-CoV-2稀释液混匀,此时病毒稀释液体积为2000μL,滴度为5×103PFU/mL,然后将稀释好的SARS-CoV-2放置于冰上待用。用镊子夹出小鼠,使用异氟烷进行吸入麻醉,观察小鼠,待小鼠出现站立不稳昏倒等现象时,用移液枪吸取50μL稀释到滴度为5×103PFU/mL病毒液,向小鼠鼻孔缓慢滴入,使病毒液随小鼠的呼吸自然吸入。数秒后将小鼠放回至饲养笼。攻毒后,每天固定时间点称取小鼠体重,一共记录11天。最后使用Graphpad prism软件绘制每组小鼠体重与生存曲线。
实验结果表明,通过本发明所应用的实验方案获取到的具有天然三聚体结构的重组S蛋白,制备成的疫苗具有优良的免疫原性,疫苗组小鼠在感染SARS-CoV-2后体重变化不大(图10A),并可完全抵抗致死剂量的SARS-CoV-2感染(图10B)。本发明可以作为潜在的SARS-CoV-2重组S蛋白亚单位疫苗。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
序列表
<110> 武汉大学
<120> 新型冠状病毒S蛋白及其亚单位疫苗
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3819
<212> DNA
<213> 新型冠状病毒(SARS-CoV-2 )
<400> 1
atgtttgtgt tcctggtgct gctgccactg gtgtccagcc agtgtgtgaa cctgaccacc 60
aggacccaac ttcctcctgc ctacaccaac tccttcacca ggggagtcta ctaccctgac 120
aaggtgttca ggtcctctgt gctgcacagc acccaggacc tgttcctgcc attcttcagc 180
aatgtgacct ggttccatgc catccatgtg tctggcacca atggcaccaa gaggtttgac 240
aaccctgtgc tgccattcaa tgatggagtc tactttgcca gcacagagaa gagcaacatc 300
atcaggggct ggatttttgg caccaccctg gacagcaaga cccagtccct gctgattgtg 360
aacaatgcca ccaatgtggt gattaaggtg tgtgagttcc agttctgtaa tgacccattc 420
ctgggagtct actaccacaa gaacaacaag tcctggatgg agtctgagtt cagggtctac 480
tcctctgcca acaactgtac ctttgaatat gtgagccaac cattcctgat ggacttggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagtttgtgt tcaagaacat tgatggctac 600
ttcaagattt acagcaaaca cacaccaatc aacctggtga gggacctgcc acagggcttc 660
tctgccttgg aaccactggt ggacctgcca attggcatca acatcaccag gttccagacc 720
ctgctggctc tgcacaggtc ctacctgaca cctggagact cctcctctgg ctggacagca 780
ggagcagcag cctactatgt gggctacctc caaccaagga ccttcctgct gaaatacaat 840
gagaatggca ccatcacaga tgctgtggac tgtgccctgg acccactgtc tgagaccaag 900
tgtaccctga aatccttcac agtggagaag ggcatctacc agaccagcaa cttcagggtc 960
caaccaacag agagcattgt gaggtttcca aacatcacca acctgtgtcc atttggagag 1020
gtgttcaatg ccaccaggtt tgcctctgtc tatgcctgga acaggaagag gattagcaac 1080
tgtgtggctg actactctgt gctctacaac tctgcctcct tcagcacctt caagtgttat 1140
ggagtgagcc caaccaaact gaatgacctg tgtttcacca atgtctatgc tgactccttt 1200
gtgattaggg gagatgaggt gagacagatt gcccctggac aaacaggcaa gattgctgac 1260
tacaactaca aactgcctga tgacttcaca ggctgtgtga ttgcctggaa cagcaacaac 1320
ctggacagca aggtgggagg caactacaac tacctctaca gactgttcag gaagagcaac 1380
ctgaaaccat ttgagaggga catcagcaca gagatttacc aggctggcag cacaccatgt 1440
aatggagtgg agggcttcaa ctgttacttt ccactccaat cctatggctt ccaaccaacc 1500
aatggagtgg gctaccaacc atacagggtg gtggtgctgt cctttgaact gctccatgcc 1560
cctgccacag tgtgtggacc aaagaagagc accaacctgg tgaagaacaa gtgtgtgaac 1620
ttcaacttca atggactgac aggcacagga gtgctgacag agagcaacaa gaagttcctg 1680
ccattccaac agtttggcag ggacattgct gacaccacag atgctgtgag ggacccacag 1740
accttggaga ttctggacat cacaccatgt tcctttggag gagtgtctgt gattacacct 1800
ggcaccaaca ccagcaacca ggtggctgtg ctctaccagg atgtgaactg tactgaggtg 1860
cctgtggcta tccatgctga ccaacttaca ccaacctgga gggtctacag cacaggcagc 1920
aatgtgttcc agaccagggc tggctgtctg attggagcag agcatgtgaa caactcctat 1980
gagtgtgaca tcccaattgg agcaggcatc tgtgcctcct accagaccca gaccaacagc 2040
ccaaggaggg caaggtctgt ggcaagccag agcatcattg cctacacaat gagtctggga 2100
gcagagaact ctgtggctta cagcaacaac agcattgcca tcccaaccaa cttcaccatc 2160
tctgtgacca cagagattct gcctgtgagt atgaccaaga cctctgtgga ctgtacaatg 2220
tatatctgtg gagacagcac agagtgtagc aacctgctgc tccaatatgg ctccttctgt 2280
acccaactta acagggctct gacaggcatt gctgtggaac aggacaagaa cacccaggag 2340
gtgtttgccc aggtgaagca gatttacaag acacctccaa tcaaggactt tggaggcttc 2400
aacttcagcc agattctgcc tgacccaagc aagccaagca agaggtcctt cattgaggac 2460
ctgctgttca acaaggtgac cctggctgat gctggcttca tcaagcaata tggagactgt 2520
ctgggagaca ttgctgccag ggacctgatt tgtgcccaga agttcaatgg actgacagtg 2580
ctgcctccac tgctgacaga tgagatgatt gcccaataca cctctgccct gctggctggc 2640
accatcacct ctggctggac ctttggagca ggagcagccc tccaaatccc atttgctatg 2700
cagatggctt acaggttcaa tggcattgga gtgacccaga atgtgctcta tgagaaccag 2760
aaactgattg ccaaccagtt caactctgcc attggcaaga ttcaggactc cctgtccagc 2820
acagcctctg ccctgggcaa actccaagat gtggtgaacc agaatgccca ggctctgaac 2880
accctggtga agcaactttc cagcaacttt ggagccatct cctctgtgct gaatgacatc 2940
ctgagcagac tggacaaggt ggaggctgag gtccagattg acagactgat tacaggcaga 3000
ctccaatccc tccaaaccta tgtgacccaa caacttatca gggctgctga gattagggca 3060
tctgccaacc tggctgccac caagatgagt gagtgtgtgc tgggacaaag caagagggtg 3120
gacttctgtg gcaagggcta ccacctgatg agttttccac agtctgcccc tcatggagtg 3180
gtgttcctgc atgtgaccta tgtgcctgcc caggagaaga acttcaccac agcccctgcc 3240
atctgccatg atggcaaggc tcactttcca agggagggag tgtttgtgag caatggcacc 3300
cactggtttg tgacccagag gaacttctat gaaccacaga ttatcaccac agacaacacc 3360
tttgtgtctg gcaactgtga tgtggtgatt ggcattgtga acaacacagt ctatgaccca 3420
ctccaacctg aactggactc cttcaaggag gaactggaca aatacttcaa gaaccacacc 3480
agccctgatg tggacctggg agacatctct ggcatcaatg cctctgtggt gaacatccag 3540
aaggagattg acagactgaa tgaggtggct aagaacctga atgagtccct gattgacctc 3600
caagaactgg gcaaatatga acaatacatc aagtggccat ggtacatctg gctgggcttc 3660
attgctggac tgattgccat tgtgatggtg accataatgc tgtgttgtat gacctcctgt 3720
tgttcctgtc tgaaaggctg ttgttcctgt ggctcctgtt gtaagtttga tgaggatgac 3780
tctgaacctg tgctgaaagg agtgaaactg cactacacc 3819
<210> 2
<211> 1273
<212> PRT
<213> 新型冠状病毒(SARS-CoV-2 )
<400> 2
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
1010 1015 1020
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035 1040
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1045 1050 1055
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1060 1065 1070
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His
1075 1080 1085
Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val
1090 1095 1100
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1105 1110 1115 1120
Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1125 1130 1135
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1140 1145 1150
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp
1155 1160 1165
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1170 1175 1180
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1185 1190 1195 1200
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile
1205 1210 1215
Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile
1220 1225 1230
Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val
1250 1255 1260
Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 3
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtttgtgt tcctggtcct gctgcctctt gtgagttcac aatgtgttaa tctgacaacg 60
aggactcagc tcccccccgc ctatacaaat agttttaccc gcggcgtgta ttatccggat 120
aaagtcttca ggtcttctgt gctccacagc acccaggacc tgttcctgcc ttttttttcc 180
aatgtgacct ggttccacgc catccacgtg tctggaacaa acggtaccaa aagattcgat 240
aaccctgtgc tgccctttaa cgatggagtc tactttgcta gcaccgagaa aagcaacatt 300
attagggggt ggatttttgg cactaccctc gacagcaaaa cccagtcatt gcttatcgtc 360
aacaacgcta ccaacgtcgt gattaaggtt tgcgaatttc agttttgcaa tgatcctttc 420
ctcggcgtgt attatcataa gaacaataaa tcttggatgg aatccgagtt ccgagtatat 480
tcaagcgcca acaactgtac ttttgaatat gtgtcccagc cattcctcat ggatctggaa 540
ggcaagcagg ggaactttaa aaatctcaga gagttcgtat tcaagaacat tgacgggtac 600
tttaagatct atagtaagca tacccccatc aaccttgtaa gagacctgcc acaggggttt 660
agtgccctgg agccactcgt ggatctgcca atcggaatca acatcacacg ctttcagact 720
ttgcttgcgc tgcacagaag ctatctgacc ccgggtgata gctcatctgg atggacagcg 780
ggggccgccg cgtactacgt cgggtacctt cagcccagga cgttcctgct gaaatacaac 840
gaaaacggca ccattaccga cgcagtagac tgcgcactcg accccctgag tgaaacaaag 900
tgtacgttga aaagttttac cgtagagaaa ggcatatatc agactagcaa ttttagggtt 960
cagcccacag agtctattgt gcgctttcct aatatcacca atttgtgccc ttttggagaa 1020
gtgtttaatg ccacccgatt tgcgtctgtg tatgcttgga atcgcaaaag gatctcaaac 1080
tgcgtcgccg actattccgt gctgtacaac tctgcttcat ttagcacatt caagtgttat 1140
ggggtgagtc caaccaaatt gaacgacctc tgctttacaa acgtgtacgc tgactcattt 1200
gtcattagag gcgacgaagt gaggcagatt gcccccgggc agacaggaaa aattgcggac 1260
tacaactaca agctccctga tgacttcacg ggctgtgtca tcgcatggaa cagtaacaat 1320
cttgatagca aggtgggcgg caattacaat tacctgtaca gactgtttag aaaatctaat 1380
ctcaaaccct ttgaaaggga catttccact gaaatctatc aggccgggag cactccgtgt 1440
aacggcgtag aggggtttaa ctgctatttc ccactgcagt cctatggatt ccagccaaca 1500
aacggggtgg gctaccaacc ctaccgggta gtggtgctga gctttgaact tctgcatgct 1560
ccggctaccg tctgtggccc aaagaagagc acaaacctcg taaagaacaa gtgtgttaac 1620
ttcaatttta atggcctcac cggaactggc gtcctcactg agtccaataa gaagtttctg 1680
ccgtttcaac agttcggccg ggacatagct gacacgactg acgccgtgag agaccctcaa 1740
accctcgaaa tactggacat cactccttgc tcattcggcg gcgtttctgt gataacacca 1800
ggcacgaaca cttctaatca ggtggctgtg ctttatcagg acgtgaactg cacagaagtg 1860
cctgtcgcca ttcatgccga tcagctcacc cctacttgga gagtttatag caccggctca 1920
aacgtgttcc aaacgagagc aggctgcctt atcggggcag agcacgtgaa caatagctat 1980
gagtgtgata tcccaattgg ggctggcata tgcgctagct accagaccca gacaaactca 2040
cccaggcggg cccggtcagt ggctagccag tctattatcg cctacaccat gtccctgggc 2100
gccgagaaca gtgtcgcgta cagcaataac tccatcgcta tccctaccaa cttcacgatc 2160
tcagtgacga ctgagatatt gccggtttct atgactaaga ccagtgtgga ttgtacaatg 2220
tacatctgtg gtgatagcac agagtgctct aatctcctgc tccaatatgg gagcttttgt 2280
acccagctga acagagcatt gaccgggatt gccgtcgagc aggataagaa cacacaagaa 2340
gtatttgccc aggtgaaaca gatctacaag actcccccta ttaaagactt cggcggcttt 2400
aacttttctc agatactccc cgaccctagc aagcctagca aacggagctt cattgaagat 2460
cttttgttta ataaggtcac attggcggat gccggcttta tcaagcagta cggggattgt 2520
ttgggtgata ttgcggctag ggatctgatt tgtgcccaga agttcaatgg cctgacagtg 2580
ctgccccccc tgcttacaga cgagatgatt gcgcagtaca ccagcgctct gctggcggga 2640
accatcacct ccggctggac ctttggggcc ggagccgcac tccagatccc ttttgccatg 2700
cagatggcct atagattcaa tggaatcggc gtgacacaga acgtcctgta tgagaaccag 2760
aaactcatcg ctaatcagtt taacagcgcc attggcaaaa ttcaggattc tctgagttca 2820
accgcatcag ctttgggtaa actgcaggat gtcgtaaatc agaatgctca ggccctgaat 2880
actcttgtta agcagctctc ctctaacttc ggcgccatca gttctgtgct gaacgacatt 2940
ctgtctagac tggacaaggt ggaggcagag gtacaaatcg accgcctgat caccggacgg 3000
ctgcagtcac tccaaacata cgtgacccaa cagctcatcc gggcagccga aattagagcc 3060
tctgcaaatc tggccgccac aaagatgagt gagtgcgttc tgggtcagtc caaacgagtg 3120
gacttctgcg gcaaaggtta ccacctgatg agtttccccc agtctgcccc gcatggcgtg 3180
gtattcctgc acgtgactta tgtcccagcc caggaaaaga acttcaccac cgccccagca 3240
atttgtcacg atggtaaggc ccacttcccc cgggaaggcg tttttgtgtc caatggcact 3300
cattggttcg tgacacagag aaacttttac gaaccccaaa tcattaccac cgacaacact 3360
ttcgtcagcg ggaattgtga cgtagtaatc gggattgtga acaacaccgt ctatgacccc 3420
ctgcagcccg agcttgactc ctttaaagag gaactggata agtatttcaa gaatcacaca 3480
agccctgatg ttgatctggg cgacatctct ggcattaacg cttcagtggt caacatacaa 3540
aaagagatcg atcgcctcaa tgaagtcgcc aagaatctca atgagtcact catcgatttg 3600
caggaactgg ggaagtacga gcagtatatc aagtggccct ggtacatctg gctgggattt 3660
attgctgggc tcatcgctat cgtaatggtc accattatgt tgtgctgcat gacctcctgt 3720
tgttcctgtc tgaaaggttg ttgtagttgc ggcagttgtt gtaagttcga tgaagatgac 3780
tctgagcctg tgctcaaggg cgtcaagctc cactacaca 3819
<210> 4
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380
ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggcag agacatcgcc gacaccaccg acgccgtgag agacccccag 1740
accctggaga tcctggacat caccccctgc agcttcggcg gcgtgagcgt gatcaccccc 1800
ggcaccaaca ccagcaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
cccgtggcca tccacgccga ccagctgacc cccacctgga gagtgtacag caccggcagc 1920
aacgtgttcc agaccagagc cggctgcctg atcggcgccg agcacgtgaa caacagctac 1980
gagtgcgaca tccccatcgg cgccggcatc tgcgccagct accagaccca gaccaacagc 2040
cccagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280
acccagctga acagagccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400
aacttcagcc agatcctgcc cgaccccagc aagcccagca agagaagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520
ctgggcgaca tcgccgccag agacctgatc tgcgcccaga agttcaacgg cctgaccgtg 2580
ctgccccccc tgctgaccga cgagatgatc gcccagtaca ccagcgccct gctggccggc 2640
accatcacca gcggctggac cttcggcgcc ggcgccgccc tgcagatccc cttcgccatg 2700
cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
accgccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940
ctgagcagac tggacaaggt ggaggccgag gtgcagatcg acagactgat caccggcaga 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagcc 3060
agcgccaacc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttccccc agagcgcccc ccacggcgtg 3180
gtgttcctgc acgtgaccta cgtgcccgcc caggagaaga acttcaccac cgcccccgcc 3240
atctgccacg acggcaaggc ccacttcccc agagagggcg tgttcgtgag caacggcacc 3300
cactggttcg tgacccagag aaacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420
ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccccgacg tggacctggg cgacatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg acagactgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720
tgcagctgcc tgaagggctg ctgcagctgc ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcccg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 5
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
cgcacccagc tgccccccgc ctacaccaac agcttcaccc gcggcgtgta ctaccccgac 120
aaggtgttcc gcagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gcgcttcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atccgcggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt ccgcgtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtgc gcgacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcacccg cttccagacc 720
ctgctggccc tgcaccgcag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagccccgca ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttccgcgtg 960
cagcccaccg agagcatcgt gcgcttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccacccgctt cgccagcgtg tacgcctgga accgcaagcg catcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatccgcg gcgacgaggt gcgccagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtacc gcctgttccg caagagcaac 1380
ctgaagccct tcgagcgcga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctaccgcgtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggccg cgacatcgcc gacaccaccg acgccgtgcg cgacccccag 1740
accctggaga tcctggacat caccccctgc agcttcggcg gcgtgagcgt gatcaccccc 1800
ggcaccaaca ccagcaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
cccgtggcca tccacgccga ccagctgacc cccacctggc gcgtgtacag caccggcagc 1920
aacgtgttcc agacccgcgc cggctgcctg atcggcgccg agcacgtgaa caacagctac 1980
gagtgcgaca tccccatcgg cgccggcatc tgcgccagct accagaccca gaccaacagc 2040
ccccgccgcg cccgcagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280
acccagctga accgcgccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400
aacttcagcc agatcctgcc cgaccccagc aagcccagca agcgcagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520
ctgggcgaca tcgccgcccg cgacctgatc tgcgcccaga agttcaacgg cctgaccgtg 2580
ctgccccccc tgctgaccga cgagatgatc gcccagtaca ccagcgccct gctggccggc 2640
accatcacca gcggctggac cttcggcgcc ggcgccgccc tgcagatccc cttcgccatg 2700
cagatggcct accgcttcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
accgccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940
ctgagccgcc tggacaaggt ggaggccgag gtgcagatcg accgcctgat caccggccgc 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatcc gcgccgccga gatccgcgcc 3060
agcgccaacc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagcgcgtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttccccc agagcgcccc ccacggcgtg 3180
gtgttcctgc acgtgaccta cgtgcccgcc caggagaaga acttcaccac cgcccccgcc 3240
atctgccacg acggcaaggc ccacttcccc cgcgagggcg tgttcgtgag caacggcacc 3300
cactggttcg tgacccagcg caacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420
ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccccgacg tggacctggg cgacatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720
tgcagctgcc tgaagggctg ctgcagctgc ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcccg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 6
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgttcgtgt tcctggtgct cctgcccctg gtgagctctc agtgcgtgaa cctgacaacc 60
cggacacagc tgcctcctgc ctacaccaac tctttcacaa gaggcgtcta ctatcctgat 120
aaggtgttca gaagctctgt gctgcattct acccaagatc tgttcctgcc tttcttcagc 180
aatgtgacat ggttccacgc catccacgtc tctgggacta acggtacaaa gagattcgac 240
aaccccgtac tgcctttcaa cgacggcgtt tacttcgcca gcaccgaaaa atctaacatc 300
atcaggggat ggatctttgg cacaaccctg gacagcaaga cccaatctct gctgatcgtg 360
aacaacgcca ccaacgtggt gataaaggtt tgtgaattcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccataa gaacaacaag agctggatgg aaagcgagtt cagagtgtac 480
agctccgcca acaactgcac attcgagtac gtgtcccagc cttttctgat ggacctggaa 540
ggcaaacaag gcaacttcaa gaacctgaga gagttcgtgt ttaagaacat cgacggctac 600
ttcaagatct actccaagca cacccctatc aacctggttc gggatctgcc tcagggcttt 660
tctgctctgg aacctctggt ggacctgcca atcggcatca acatcacacg cttccagacc 720
ttgctcgccc tgcacagatc ctacctgacc cctggcgact cctctagcgg atggaccgcc 780
ggcgcggccg catactacgt gggatatctg cagcctagaa ccttcctgct gaaatacaac 840
gagaatggca ccatcacaga cgccgtcgat tgcgccctgg accctctgag cgagacaaaa 900
tgtaccctga aaagttttac cgtggaaaag ggcatctacc agaccagcaa ttttagagtg 960
cagcccaccg aaagcatcgt gcggttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtcttcaacg ccaccagatt cgcctctgtc tacgcctgga acagaaagag aatcagcaat 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tctctacgtt caagtgctac 1140
ggcgtaagcc ctaccaagct gaacgacctg tgcttcacca acgtgtacgc cgactccttt 1200
gtgatccggg gagacgaggt gcggcagatt gcccctggcc agaccggcaa gatcgctgac 1260
tacaactaca agctgcccga tgatttcacc ggctgcgtga tcgcttggaa cagcaacaac 1320
cttgactcaa aggtaggagg caattacaac tacctgtaca gactgtttcg gaagagcaac 1380
ctgaagcctt tcgagagaga tatctcgaca gagatctatc aggccggatc tacgccctgt 1440
aatggcgttg aaggctttaa ctgctacttt cccctgcagt cttacggctt tcagcctacc 1500
aatggagttg gttaccagcc ataccgggtg gtggtgctca gcttcgagct gctccacgcc 1560
ccagctaccg tgtgcggccc taagaagtct accaacctcg ttaagaacaa gtgcgtgaac 1620
ttcaatttca acggcctgac cggaaccggc gtgctgaccg agagcaacaa aaagttcctg 1680
ccgttccaac agtttggcag agacatcgcc gataccacag atgccgttag agatcctcag 1740
acactggaaa tcctggatat cacaccttgc agcttcggcg gagtgagcgt gatcaccccc 1800
ggcaccaaca cctctaacca ggtggctgtg ctgtaccagg acgtgaactg caccgaggtc 1860
cccgtcgcca tccacgccga ccaactgacc cccacctggc gggtgtacag caccggcagc 1920
aacgtgttcc agaccagagc cggctgtctg atcggcgccg agcacgtgaa caatagttat 1980
gaatgtgaca tccccatcgg agctggcatt tgcgcttctt accagactca gaccaattct 2040
ccacgcagag ctcggagcgt ggccagccag tccatcatcg cctatactat gagcctgggc 2100
gctgagaaca gcgtggcata cagcaacaac agcatcgcaa tccccaccaa ttttacaatc 2160
agtgtgacca ccgaaatcct gcctgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac agagtgcagc aacctgctgc tgcagtacgg ctccttttgc 2280
acccagctga atagagctct gacaggcatc gctgttgaac aggataagaa cacccaagag 2340
gtgttcgccc aggtaaagca gatctacaag acccctccta tcaaggactt cggcggcttt 2400
aacttcagcc agatcctgcc tgacccaagc aaaccctcca aacggagctt tattgaggat 2460
ctgctgttca acaaggtgac cctggccgac gccggattca tcaagcagta cggcgactgc 2520
ctgggcgaca tcgccgccag agatctgatc tgcgcccaga aattcaacgg gctgacagtg 2580
ctgcctccac tgctgaccga tgagatgatc gcccagtata caagcgccct gctcgctggc 2640
acgatcacca gcggatggac attcggagcc ggcgccgctc tgcaaatccc tttcgccatg 2700
cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ctaaccagtt caatagcgcc atcgggaaga tccaggacag cctgtcatcc 2820
acagccagcg ccctgggcaa gctgcaggac gtggtgaatc aaaacgctca ggcgctgaac 2880
acactggtga agcaactgag cagcaacttc ggcgccatca gctcagtgct gaacgatatt 2940
ctgtctagac tggacaaagt ggaggccgag gtgcagatag atagactgat caccggcaga 3000
ctgcagagcc tgcaaaccta cgtgacccag cagctgatcc gggccgccga aatccgggcc 3060
agcgccaatc tggcagccac taagatgtct gagtgcgtgc tgggccagag caagcgggtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttcccac aatctgcccc tcacggcgtg 3180
gtgttcctac acgtgacata cgtgcctgct caggagaaga atttcacgac cgcccctgct 3240
atctgtcacg acggaaaggc ccacttccct agagaaggcg tctttgtgag caacggaaca 3300
cactggttcg tgacacagag aaacttctac gagcctcaga tcatcacaac tgataacaca 3360
ttcgtgagcg ggaactgcga cgtcgtgatc ggcatcgtga acaataccgt ttacgaccct 3420
ctgcagcctg agctggactc cttcaaagag gaactggata agtacttcaa gaaccacacc 3480
agcccagacg tcgacctggg cgacattagc ggcatcaacg ccagcgtggt caacatccag 3540
aaggaaatcg atagactgaa cgaggtcgcc aagaacctga atgaaagttt gatcgacctg 3600
caggaactgg gcaagtacga gcagtacatc aagtggcctt ggtacatttg gctgggattc 3660
atcgccggcc tgatcgccat cgtgatggtc accatcatgc tgtgttgcat gacaagctgc 3720
tgctcctgcc tgaagggctg ttgttcttgt ggaagctgct gtaaattcga cgaggacgat 3780
tccgagcccg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 7
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgttcgtgt tcctggtgct gctgcccctg gtgtcctctc agtgtgtgaa cctgaccacc 60
agaacacagc tgcctccagc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttcc ggtcctccgt gctgcattct acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccatgtg tctggcacca acggcaccaa gagattcgac 240
aaccccgtgc tgcctttcaa cgacggggtg tactttgcct ccaccgagaa gtccaacatc 300
atcagaggct ggatcttcgg caccacactg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaagtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtct actaccacaa gaacaacaag tcctggatgg aatccgagtt ccgggtgtac 480
tcctccgcca acaactgcac cttcgagtac gtgtcccagc ctttcctgat ggacctggaa 540
ggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt ttaagaacat cgacggctac 600
ttcaagatct actccaagca cacccctatc aacctcgtgc gggatctgcc tcagggcttc 660
tctgctctgg aacccctggt ggatctgccc atcggcatca acatcacccg gtttcagacc 720
ctgctggccc tgcaccggtc ttatttgacc cctggcgact cctcttctgg ctggactgct 780
ggtgccgctg cttactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaatggca ccatcaccga cgccgtggac tgtgctctgg atcctctgtc cgagacaaag 900
tgcaccctga agtccttcac cgtggaaaag ggcatctacc agacctccaa cttccgggtg 960
cagcccaccg agtctatcgt gcggttccct aacatcacca acctgtgtcc tttcggcgag 1020
gtgttcaatg ccaccagatt cgcctctgtg tacgcctgga accggaagcg gatctctaac 1080
tgcgtggccg actacagcgt gctgtacaac tccgcctcct tcagcacctt caagtgctac 1140
ggcgtgtccc ctaccaagct gaacgacctg tgcttcacaa acgtgtacgc cgactccttc 1200
gtgatccggg gagatgaagt gcggcagatc gctcctggac agaccggcaa gatcgccgat 1260
tacaactaca agctgcccga cgacttcacc ggctgtgtga tcgcttggaa ctccaacaac 1320
ctggactcca aagtcggcgg caactacaac tacctgtacc ggctgttccg gaagtctaac 1380
ctgaagcctt tcgagcggga catcagcacc gagatctacc aggctggcag caccccttgt 1440
aacggcgtgg aaggcttcaa ctgctacttc ccactgcagt cctacggctt tcagcctacc 1500
aatggcgtgg gctatcagcc ctacagagtg gtggtgctgt ccttcgagct gctgcatgct 1560
cctgctaccg tgtgcggccc taagaaatct accaacctgg tcaagaacaa atgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgacag agtccaacaa gaagttcctg 1680
ccattccagc agttcggccg ggatatcgcc gataccacag atgccgtcag ggaccctcag 1740
acactggaaa tcctggacat caccccttgc agcttcggcg gagtgtctgt gatcacccca 1800
ggcaccaaca cctctaacca ggtggccgtg ctgtatcagg acgtgaactg taccgaggtg 1860
cccgtggcta tccatgccga tcagctgacc cctacatggc gcgtgtactc caccggctcc 1920
aacgtgttcc agacaagagc tggctgtctg atcggcgctg agcacgtgaa caattcctac 1980
gagtgcgaca tccccatcgg agccggaatc tgcgcctctt atcagaccca gaccaactct 2040
cccagacggg ccagatctgt ggccagccag tctatcattg cttacaccat gagcctgggc 2100
gccgagaact ctgtggccta cagcaacaac tctatcgcta tccccaccaa cttcaccatc 2160
tccgtgacca cagagatcct gcctgtgtcc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgactctac cgagtgctcc aacctgctgc tgcagtacgg ctccttctgc 2280
acccagctga atagagccct gaccggaatc gccgtggaac aggacaagaa cacccaagag 2340
gtgttcgccc aagtgaagca gatctacaag acccctccta tcaaggactt cggcggcttc 2400
aatttctccc agattctgcc cgatcctagc aagccctcca agcggtcttt catcgaggac 2460
ctgctgttca acaaagtgac actggccgac gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca ttgccgccag ggatctgatc tgtgcccaga agtttaacgg actgacagtg 2580
ctgcctcctc tgctgaccga tgagatgatc gcccagtaca cctccgcact gctggctggc 2640
acaatcacct ctggatggac atttggcgct ggcgccgctc tgcagatccc tttcgctatg 2700
cagatggcct accggttcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggaaaga tccaggacag cctgtccagc 2820
accgcttctg ccctgggaaa gctgcaggat gtggtcaacc agaacgctca ggccctgaac 2880
accctcgtga agcagctgtc ctctaacttc ggcgccatct cctctgtgct gaacgatatc 2940
ctgagccggc tggacaaggt ggaagccgag gtgcagatcg acagactgat caccggacgg 3000
ctgcagtccc tgcagaccta tgttacccag cagctgatca gagccgccga gattagagcc 3060
tctgccaatc tggccgccac caagatgtct gagtgtgtgc tgggccagtc caagagagtg 3120
gacttttgcg gcaagggcta ccacctgatg agcttccctc agtctgctcc tcacggcgtg 3180
gtgtttctgc acgtgaccta cgtgcccgct caagagaaga actttaccac cgctcctgcc 3240
atctgccacg acggcaaggc tcactttcct cgagaaggcg tgttcgtgtc taacggcacc 3300
cattggttcg tgacacagcg gaacttctac gagccccaga tcatcaccac cgacaacacc 3360
tttgtgtccg gcaactgcga cgtcgtgatc ggaattgtga acaataccgt gtacgaccct 3420
ctgcagcccg agctggactc cttcaaagag gaactggaca agtactttaa gaaccacaca 3480
agccccgacg tggacctggg agacatctct ggcatcaacg cctccgtggt caacatccag 3540
aaagagatcg accggctgaa cgaggtggcc aagaatctga acgagtccct gatcgacctg 3600
caagaactgg ggaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttt 3660
atcgctggcc tgatcgctat cgtgatggtc acaatcatgc tgtgctgtat gacctcctgc 3720
tgctcttgcc tgaagggctg ctgttcttgc ggctcttgct gcaagttcga cgaggacgac 3780
tctgagcccg tgctgaaagg cgtgaagctg cactacacc 3819
<210> 8
<211> 3708
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
cgcacccagc tgccccccgc ctacaccaac agcttcaccc gcggcgtgta ctaccccgac 120
aaggtgttcc gcagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gcgcttcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atccgcggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt ccgcgtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtgc gcgacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcacccg cttccagacc 720
ctgctggccc tgcaccgcag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagccccgca ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttccgcgtg 960
cagcccaccg agagcatcgt gcgcttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccacccgctt cgccagcgtg tacgcctgga accgcaagcg catcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatccgcg gcgacgaggt gcgccagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtacc gcctgttccg caagagcaac 1380
ctgaagccct tcgagcgcga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctaccgcgtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggccg cgacatcgcc gacaccaccg acgccgtgcg cgacccccag 1740
accctggaga tcctggacat caccccctgc agcttcggcg gcgtgagcgt gatcaccccc 1800
ggcaccaaca ccagcaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
cccgtggcca tccacgccga ccagctgacc cccacctggc gcgtgtacag caccggcagc 1920
aacgtgttcc agacccgcgc cggctgcctg atcggcgccg agcacgtgaa caacagctac 1980
gagtgcgaca tccccatcgg cgccggcatc tgcgccagct accagaccca gaccaacagc 2040
cccggcagcg ccagcagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280
acccagctga accgcgccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400
aacttcagcc agatcctgcc cgaccccagc aagcccagca agcgcagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520
ctgggcgaca tcgccgcccg cgacctgatc tgcgcccaga agttcaacgg cctgaccgtg 2580
ctgccccccc tgctgaccga cgagatgatc gcccagtaca ccagcgccct gctggccggc 2640
accatcacca gcggctggac cttcggcgcc ggcgccgccc tgcagatccc cttcgccatg 2700
cagatggcct accgcttcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
accgccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940
ctgagccgcc tggacaaggt ggaggccgag gtgcagatcg accgcctgat caccggccgc 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatcc gcgccgccga gatccgcgcc 3060
agcgccaacc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagcgcgtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttccccc agagcgcccc ccacggcgtg 3180
gtgttcctgc acgtgaccta cgtgcccgcc caggagaaga acttcaccac cgcccccgcc 3240
atctgccacg acggcaaggc ccacttcccc cgcgagggcg tgttcgtgag caacggcacc 3300
cactggttcg tgacccagcg caacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420
ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccccgacg tggacctggg cgacatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagggctac atccccgagg ccccccgcga cggccaggcc 3660
tacgtgcgca aggacggcga gtgggtgctg ctgagcacct tcctgtga 3708
<210> 9
<211> 1235
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
1010 1015 1020
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035 1040
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1045 1050 1055
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1060 1065 1070
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His
1075 1080 1085
Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val
1090 1095 1100
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1105 1110 1115 1120
Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1125 1130 1135
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1140 1145 1150
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp
1155 1160 1165
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1170 1175 1180
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1185 1190 1195 1200
Gln Glu Leu Gly Lys Tyr Glu Gln Gly Tyr Ile Pro Glu Ala Pro Arg
1205 1210 1215
Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser
1220 1225 1230
Thr Phe Leu
1235
<210> 10
<211> 3735
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agcgcccagt gcgtgaacct gaccacccgc acccagctgc cccccgccta caccaacagc 120
ttcacccgcg gcgtgtacta ccccgacaag gtgttccgca gcagcgtgct gcacagcacc 180
caggacctgt tcctgccctt cttcagcaac gtgacctggt tccacgccat ccacgtgagc 240
ggcaccaacg gcaccaagcg cttcgacaac cccgtgctgc ccttcaacga cggcgtgtac 300
ttcgccagca ccgagaagag caacatcatc cgcggctgga tcttcggcac caccctggac 360
agcaagaccc agagcctgct gatcgtgaac aacgccacca acgtggtgat caaggtgtgc 420
gagttccagt tctgcaacga ccccttcctg ggcgtgtact accacaagaa caacaagagc 480
tggatggaga gcgagttccg cgtgtacagc agcgccaaca actgcacctt cgagtacgtg 540
agccagccct tcctgatgga cctggagggc aagcagggca acttcaagaa cctgcgcgag 600
ttcgtgttca agaacatcga cggctacttc aagatctaca gcaagcacac ccccatcaac 660
ctggtgcgcg acctgcccca gggcttcagc gccctggagc ccctggtgga cctgcccatc 720
ggcatcaaca tcacccgctt ccagaccctg ctggccctgc accgcagcta cctgaccccc 780
ggcgacagca gcagcggctg gaccgccggc gccgccgcct actacgtggg ctacctgcag 840
ccccgcacct tcctgctgaa gtacaacgag aacggcacca tcaccgacgc cgtggactgc 900
gccctggacc ccctgagcga gaccaagtgc accctgaaga gcttcaccgt ggagaagggc 960
atctaccaga ccagcaactt ccgcgtgcag cccaccgaga gcatcgtgcg cttccccaac 1020
atcaccaacc tgtgcccctt cggcgaggtg ttcaacgcca cccgcttcgc cagcgtgtac 1080
gcctggaacc gcaagcgcat cagcaactgc gtggccgact acagcgtgct gtacaacagc 1140
gccagcttca gcaccttcaa gtgctacggc gtgagcccca ccaagctgaa cgacctgtgc 1200
ttcaccaacg tgtacgccga cagcttcgtg atccgcggcg acgaggtgcg ccagatcgcc 1260
cccggccaga ccggcaagat cgccgactac aactacaagc tgcccgacga cttcaccggc 1320
tgcgtgatcg cctggaacag caacaacctg gacagcaagg tgggcggcaa ctacaactac 1380
ctgtaccgcc tgttccgcaa gagcaacctg aagcccttcg agcgcgacat cagcaccgag 1440
atctaccagg ccggcagcac cccctgcaac ggcgtggagg gcttcaactg ctacttcccc 1500
ctgcagagct acggcttcca gcccaccaac ggcgtgggct accagcccta ccgcgtggtg 1560
gtgctgagct tcgagctgct gcacgccccc gccaccgtgt gcggccccaa gaagagcacc 1620
aacctggtga agaacaagtg cgtgaacttc aacttcaacg gcctgaccgg caccggcgtg 1680
ctgaccgaga gcaacaagaa gttcctgccc ttccagcagt tcggccgcga catcgccgac 1740
accaccgacg ccgtgcgcga cccccagacc ctggagatcc tggacatcac cccctgcagc 1800
ttcggcggcg tgagcgtgat cacccccggc accaacacca gcaaccaggt ggccgtgctg 1860
taccaggacg tgaactgcac cgaggtgccc gtggccatcc acgccgacca gctgaccccc 1920
acctggcgcg tgtacagcac cggcagcaac gtgttccaga cccgcgccgg ctgcctgatc 1980
ggcgccgagc acgtgaacaa cagctacgag tgcgacatcc ccatcggcgc cggcatctgc 2040
gccagctacc agacccagac caacagcccc ggcagcgcca gcagcgtggc cagccagagc 2100
atcatcgcct acaccatgag cctgggcgcc gagaacagcg tggcctacag caacaacagc 2160
atcgccatcc ccaccaactt caccatcagc gtgaccaccg agatcctgcc cgtgagcatg 2220
accaagacca gcgtggactg caccatgtac atctgcggcg acagcaccga gtgcagcaac 2280
ctgctgctgc agtacggcag cttctgcacc cagctgaacc gcgccctgac cggcatcgcc 2340
gtggagcagg acaagaacac ccaggaggtg ttcgcccagg tgaagcagat ctacaagacc 2400
ccccccatca aggacttcgg cggcttcaac ttcagccaga tcctgcccga ccccagcaag 2460
cccagcaagc gcagcttcat cgaggacctg ctgttcaaca aggtgaccct ggccgacgcc 2520
ggcttcatca agcagtacgg cgactgcctg ggcgacatcg ccgcccgcga cctgatctgc 2580
gcccagaagt tcaacggcct gaccgtgctg ccccccctgc tgaccgacga gatgatcgcc 2640
cagtacacca gcgccctgct ggccggcacc atcaccagcg gctggacctt cggcgccggc 2700
gccgccctgc agatcccctt cgccatgcag atggcctacc gcttcaacgg catcggcgtg 2760
acccagaacg tgctgtacga gaaccagaag ctgatcgcca accagttcaa cagcgccatc 2820
ggcaagatcc aggacagcct gagcagcacc gccagcgccc tgggcaagct gcaggacgtg 2880
gtgaaccaga acgcccaggc cctgaacacc ctggtgaagc agctgagcag caacttcggc 2940
gccatcagca gcgtgctgaa cgacatcctg agccgcctgg acaaggtgga ggccgaggtg 3000
cagatcgacc gcctgatcac cggccgcctg cagagcctgc agacctacgt gacccagcag 3060
ctgatccgcg ccgccgagat ccgcgccagc gccaacctgg ccgccaccaa gatgagcgag 3120
tgcgtgctgg gccagagcaa gcgcgtggac ttctgcggca agggctacca cctgatgagc 3180
ttcccccaga gcgcccccca cggcgtggtg ttcctgcacg tgacctacgt gcccgcccag 3240
gagaagaact tcaccaccgc ccccgccatc tgccacgacg gcaaggccca cttcccccgc 3300
gagggcgtgt tcgtgagcaa cggcacccac tggttcgtga cccagcgcaa cttctacgag 3360
ccccagatca tcaccaccga caacaccttc gtgagcggca actgcgacgt ggtgatcggc 3420
atcgtgaaca acaccgtgta cgaccccctg cagcccgagc tggacagctt caaggaggag 3480
ctggacaagt acttcaagaa ccacaccagc cccgacgtgg acctgggcga catcagcggc 3540
atcaacgcca gcgtggtgaa catccagaag gagatcgacc gcctgaacga ggtggccaag 3600
aacctgaacg agagcctgat cgacctgcag gagctgggca agtacgagca gggctacatc 3660
cccgaggccc cccgcgacgg ccaggcctac gtgcgcaagg acggcgagtg ggtgctgctg 3720
agcaccttcc tgtga 3735
<210> 11
<211> 1244
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
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 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln
20 25 30
Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro
35 40 45
Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe
50 55 60
Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser
65 70 75 80
Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn
85 90 95
Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly
100 105 110
Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile
115 120 125
Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe
130 135 140
Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser
145 150 155 160
Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr
165 170 175
Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln
180 185 190
Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly
195 200 205
Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp
210 215 220
Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile
225 230 235 240
Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser
245 250 255
Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala
260 265 270
Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr
275 280 285
Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro
290 295 300
Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly
305 310 315 320
Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val
325 330 335
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
340 345 350
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
355 360 365
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
370 375 380
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
385 390 395 400
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
405 410 415
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
420 425 430
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
435 440 445
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
450 455 460
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
465 470 475 480
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
485 490 495
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
500 505 510
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
515 520 525
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
530 535 540
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
545 550 555 560
Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg
565 570 575
Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu
580 585 590
Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr
595 600 605
Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val
610 615 620
Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro
625 630 635 640
Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala
645 650 655
Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp
660 665 670
Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn
675 680 685
Ser Pro Gly Ser Ala Ser Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr
690 695 700
Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser
705 710 715 720
Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu
725 730 735
Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys
740 745 750
Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe
755 760 765
Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp
770 775 780
Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr
785 790 795 800
Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro
805 810 815
Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe
820 825 830
Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp
835 840 845
Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe
850 855 860
Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala
865 870 875 880
Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr
885 890 895
Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala
900 905 910
Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn
915 920 925
Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln
930 935 940
Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val
945 950 955 960
Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser
965 970 975
Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg
980 985 990
Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly
995 1000 1005
Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala
1010 1015 1020
Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu
1025 1030 1035 1040
Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr
1045 1050 1055
His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu
1060 1065 1070
His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro
1075 1080 1085
Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1090 1095 1100
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu
1105 1110 1115 1120
Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp
1125 1130 1135
Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1140 1145 1150
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1155 1160 1165
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser
1170 1175 1180
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1185 1190 1195 1200
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu
1205 1210 1215
Gln Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg
1220 1225 1230
Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
1235 1240
<210> 12
<211> 3729
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agcgcccagt gcgtgaacct gaccacccgc acccagctgc cccccgccta caccaacagc 120
ttcacccgcg gcgtgtacta ccccgacaag gtgttccgca gcagcgtgct gcacagcacc 180
caggacctgt tcctgccctt cttcagcaac gtgacctggt tccacgccat ccacgtgagc 240
ggcaccaacg gcaccaagcg cttcgacaac cccgtgctgc ccttcaacga cggcgtgtac 300
ttcgccagca ccgagaagag caacatcatc cgcggctgga tcttcggcac caccctggac 360
agcaagaccc agagcctgct gatcgtgaac aacgccacca acgtggtgat caaggtgtgc 420
gagttccagt tctgcaacga ccccttcctg ggcgtgtact accacaagaa caacaagagc 480
tggatggaga gcgagttccg cgtgtacagc agcgccaaca actgcacctt cgagtacgtg 540
agccagccct tcctgatgga cctggagggc aagcagggca acttcaagaa cctgcgcgag 600
ttcgtgttca agaacatcga cggctacttc aagatctaca gcaagcacac ccccatcaac 660
ctggtgcgcg acctgcccca gggcttcagc gccctggagc ccctggtgga cctgcccatc 720
ggcatcaaca tcacccgctt ccagaccctg ctggccctgc accgcagcta cctgaccccc 780
ggcgacagca gcagcggctg gaccgccggc gccgccgcct actacgtggg ctacctgcag 840
ccccgcacct tcctgctgaa gtacaacgag aacggcacca tcaccgacgc cgtggactgc 900
gccctggacc ccctgagcga gaccaagtgc accctgaaga gcttcaccgt ggagaagggc 960
atctaccaga ccagcaactt ccgcgtgcag cccaccgaga gcatcgtgcg cttccccaac 1020
atcaccaacc tgtgcccctt cggcgaggtg ttcaacgcca cccgcttcgc cagcgtgtac 1080
gcctggaacc gcaagcgcat cagcaactgc gtggccgact acagcgtgct gtacaacagc 1140
gccagcttca gcaccttcaa gtgctacggc gtgagcccca ccaagctgaa cgacctgtgc 1200
ttcaccaacg tgtacgccga cagcttcgtg atccgcggcg acgaggtgcg ccagatcgcc 1260
cccggccaga ccggcaagat cgccgactac aactacaagc tgcccgacga cttcaccggc 1320
tgcgtgatcg cctggaacag caacaacctg gacagcaagg tgggcggcaa ctacaactac 1380
ctgtaccgcc tgttccgcaa gagcaacctg aagcccttcg agcgcgacat cagcaccgag 1440
atctaccagg ccggcagcac cccctgcaac ggcgtggagg gcttcaactg ctacttcccc 1500
ctgcagagct acggcttcca gcccaccaac ggcgtgggct accagcccta ccgcgtggtg 1560
gtgctgagct tcgagctgct gcacgccccc gccaccgtgt gcggccccaa gaagagcacc 1620
aacctggtga agaacaagtg cgtgaacttc aacttcaacg gcctgaccgg caccggcgtg 1680
ctgaccgaga gcaacaagaa gttcctgccc ttccagcagt tcggccgcga catcgccgac 1740
accaccgacg ccgtgcgcga cccccagacc ctggagatcc tggacatcac cccctgcagc 1800
ttcggcggcg tgagcgtgat cacccccggc accaacacca gcaaccaggt ggccgtgctg 1860
taccaggacg tgaactgcac cgaggtgccc gtggccatcc acgccgacca gctgaccccc 1920
acctggcgcg tgtacagcac cggcagcaac gtgttccaga cccgcgccgg ctgcctgatc 1980
ggcgccgagc acgtgaacaa cagctacgag tgcgacatcc ccatcggcgc cggcatctgc 2040
gccagctacc agacccagac caacagcccc ggcggcagcg tggccagcca gagcatcatc 2100
gcctacacca tgagcctggg cgccgagaac agcgtggcct acagcaacaa cagcatcgcc 2160
atccccacca acttcaccat cagcgtgacc accgagatcc tgcccgtgag catgaccaag 2220
accagcgtgg actgcaccat gtacatctgc ggcgacagca ccgagtgcag caacctgctg 2280
ctgcagtacg gcagcttctg cacccagctg aaccgcgccc tgaccggcat cgccgtggag 2340
caggacaaga acacccagga ggtgttcgcc caggtgaagc agatctacaa gacccccccc 2400
atcaaggact tcggcggctt caacttcagc cagatcctgc ccgaccccag caagcccagc 2460
aagcgcagct tcatcgagga cctgctgttc aacaaggtga ccctggccga cgccggcttc 2520
atcaagcagt acggcgactg cctgggcgac atcgccgccc gcgacctgat ctgcgcccag 2580
aagttcaacg gcctgaccgt gctgcccccc ctgctgaccg acgagatgat cgcccagtac 2640
accagcgccc tgctggccgg caccatcacc agcggctgga ccttcggcgc cggcgccgcc 2700
ctgcagatcc ccttcgccat gcagatggcc taccgcttca acggcatcgg cgtgacccag 2760
aacgtgctgt acgagaacca gaagctgatc gccaaccagt tcaacagcgc catcggcaag 2820
atccaggaca gcctgagcag caccgccagc gccctgggca agctgcagga cgtggtgaac 2880
cagaacgccc aggccctgaa caccctggtg aagcagctga gcagcaactt cggcgccatc 2940
agcagcgtgc tgaacgacat cctgagccgc ctggacaagg tggaggccga ggtgcagatc 3000
gaccgcctga tcaccggccg cctgcagagc ctgcagacct acgtgaccca gcagctgatc 3060
cgcgccgccg agatccgcgc cagcgccaac ctggccgcca ccaagatgag cgagtgcgtg 3120
ctgggccaga gcaagcgcgt ggacttctgc ggcaagggct accacctgat gagcttcccc 3180
cagagcgccc cccacggcgt ggtgttcctg cacgtgacct acgtgcccgc ccaggagaag 3240
aacttcacca ccgcccccgc catctgccac gacggcaagg cccacttccc ccgcgagggc 3300
gtgttcgtga gcaacggcac ccactggttc gtgacccagc gcaacttcta cgagccccag 3360
atcatcacca ccgacaacac cttcgtgagc ggcaactgcg acgtggtgat cggcatcgtg 3420
aacaacaccg tgtacgaccc cctgcagccc gagctggaca gcttcaagga ggagctggac 3480
aagtacttca agaaccacac cagccccgac gtggacctgg gcgacatcag cggcatcaac 3540
gccagcgtgg tgaacatcca gaaggagatc gaccgcctga acgaggtggc caagaacctg 3600
aacgagagcc tgatcgacct gcaggagctg ggcaagtacg agcagggcta catccccgag 3660
gccccccgcg acggccaggc ctacgtgcgc aaggacggcg agtgggtgct gctgagcacc 3720
ttcctgtga 3729
<210> 13
<211> 1242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
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 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln
20 25 30
Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro
35 40 45
Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe
50 55 60
Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser
65 70 75 80
Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn
85 90 95
Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly
100 105 110
Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile
115 120 125
Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe
130 135 140
Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser
145 150 155 160
Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr
165 170 175
Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln
180 185 190
Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly
195 200 205
Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp
210 215 220
Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile
225 230 235 240
Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser
245 250 255
Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala
260 265 270
Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr
275 280 285
Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro
290 295 300
Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly
305 310 315 320
Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val
325 330 335
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
340 345 350
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
355 360 365
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
370 375 380
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
385 390 395 400
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
405 410 415
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
420 425 430
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
435 440 445
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
450 455 460
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
465 470 475 480
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
485 490 495
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
500 505 510
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
515 520 525
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
530 535 540
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
545 550 555 560
Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg
565 570 575
Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu
580 585 590
Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr
595 600 605
Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val
610 615 620
Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro
625 630 635 640
Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala
645 650 655
Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp
660 665 670
Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn
675 680 685
Ser Pro Gly Gly Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met
690 695 700
Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala
705 710 715 720
Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val
725 730 735
Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp
740 745 750
Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr
755 760 765
Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn
770 775 780
Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro
785 790 795 800
Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro
805 810 815
Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys
820 825 830
Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu
835 840 845
Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly
850 855 860
Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr
865 870 875 880
Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly
885 890 895
Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg
900 905 910
Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys
915 920 925
Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser
930 935 940
Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn
945 950 955 960
Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn
965 970 975
Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp
980 985 990
Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu
995 1000 1005
Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu
1010 1015 1020
Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val
1025 1030 1035 1040
Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu
1045 1050 1055
Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1060 1065 1070
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile
1075 1080 1085
Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser
1090 1095 1100
Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1105 1110 1115 1120
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1125 1130 1135
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu
1140 1145 1150
Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser
1155 1160 1165
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val
1170 1175 1180
Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu
1185 1190 1195 1200
Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Gly
1205 1210 1215
Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp
1220 1225 1230
Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
1235 1240
<210> 14
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgtttgttt ttcttgtttt attgccacta gtctctagt 39
<210> 15
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser
1 5 10
<210> 16
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgttcgtgt tcctggtgct gctgcccctg gtgagcagc 39
<210> 17
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser
1 5 10
<210> 18
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agcgcc 66
<210> 19
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
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
20
<210> 20
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgggctggt cctgcatcat cctgttcctg gtcgccaccg ctaccggcgt gcatagc 57
<210> 21
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 22
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg ga 72
<210> 23
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly
20
<210> 24
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys
1 5 10 15
Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
20 25
<210> 25
<211> 31
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr
1 5 10 15
His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu
20 25 30
<210> 26
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val
1 5 10 15
Met Val Thr Ile Met Leu Cys Cys Met
20 25
Claims (5)
1.一种核酸分子,其特征在于,所述核酸分子的核苷酸序列如SEQ ID NO:8所示,所述核酸分子高表达SARS-CoV-2的S蛋白三聚体。
2.一种重组表达载体,其特征在于,所述重组表达载体的表达区的核苷酸序列如SEQID NO:8所示。
3.一种工程化细胞,其特征在于,所述工程化细胞包含权利要求2所述的重组表达载体。
4.一种新型冠状病毒S蛋白的制备方法,其特征在于,所述方法包括:获得权利要求2所述的重组表达载体;
将所述重组表达载体转染至细胞中,并通过细胞群的谷氨酰胺抗性筛选以及单克隆筛选,获得稳定表达重组S蛋白的细胞株;
将所述细胞株进行分泌表达和纯化,获得纯化的重组新型冠状病毒S蛋白。
5.权利要求1所述的核酸分子在制备新型冠状病毒亚单位疫苗中的应用。
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TW202400251A (zh) * | 2022-04-27 | 2024-01-01 | 大陸商瑞可迪(上海)生物醫藥有限公司 | 核酸構建體及其應用 |
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