CN117651557A - 病毒的细胞侵入抑制剂 - Google Patents
病毒的细胞侵入抑制剂 Download PDFInfo
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Abstract
本发明的课题是提供抑制病毒侵入细胞的病毒的细胞侵入抑制剂。本发明包括一种病毒的细胞侵入抑制剂,其是包含选自α‑葡糖基芸香苷、α‑葡糖基橙皮苷以及α‑葡糖基柚皮苷的至少一种成分(A)的病毒的细胞侵入抑制剂,其中,上述病毒具有与血管紧张素转换酶2(ACE2)结合的尖峰蛋白。
Description
技术领域
本发明涉及一种病毒的细胞侵入抑制剂。
背景技术
冠状病毒是从以前开始作为感冒的原因病毒在人类社会蔓延的病毒。但是,近年来,引起SARS(重症急性呼吸综合征)的SARS-CoV、引起COVID-19的SARS-CoV-2等引起全世界范围内疫情的冠状病毒备受瞩目。
根据国际病毒分类委员会(ICTV),冠状病毒被分类为套式病毒目冠状病毒亚目冠状病毒科。冠状病毒科又分为逆转录病毒亚科和原冠状病毒亚科,原冠状病毒亚科包含α冠状病毒、β冠状病毒、γ冠状病毒以及δ冠状病毒四个属。
冠状病毒通常包括尖峰蛋白(也称为S蛋白)、核苷酸蛋白(也称为N蛋白)、膜蛋白(也称为M蛋白)、包膜、包封蛋白(也称为E蛋白)和RNA作为组成部分。
为了使冠状病毒感染宿主细胞,需要上述尖峰蛋白与宿主细胞表面的受体结合吸附,基因组RNA侵入细胞内。此时,冠状病毒的尖峰蛋白结合的宿主细胞的受体根据病毒的种类而不同。例如,被分类为α冠状病毒的感冒病毒之一HCoV-NL63和被分类为β冠状病毒的SARS-CoV和SARS-CoV-2与宿主细胞受体血管紧张素转换酶2(Angiotensin-ConvertingEnzyme 2:ACE2)结合。
另外,在尖峰蛋白与ACE2结合后,病毒要侵入宿主细胞,病毒需要与宿主细胞进行膜融合,此时,被认为重要的是用蛋白分解酶TMPRSS2
(Transmembrane protease serine 2)切断尖峰蛋白。因此,认为通过抑制TMPRSS2的活性,可以抑制膜融合,抑制病毒侵入宿主细胞。作为抑制TMPRSS 2的活性、有效地抑制病毒侵入的药剂,例如可以举出萘莫司他(Nafamostat)、卡莫司他(Camostat)等。
另一方面,作为使用来自天然物的成分的抗病毒剂,专利文献1公开了以啤酒等发泡性酒精饮料的酿造中使用的啤酒花的冷水提取物为有效成分的抗病毒剂。在专利文献1中,该抗病毒剂中含有规定的类黄酮苷,据推测,其会吸附在流感病毒的血红蛋白上,阻碍流感病毒与细胞表面受体的结合,从而阻止流感病毒侵入细胞内。
非专利文献1公开了一种使用计算机的药品靶点评价,通过虚拟筛选,橙皮苷可能与SARS-CoV-2的尖峰蛋白的受体结合位点(Receptor binding domain;RBD)结合。
另外,非专利文献2通过虚拟筛选,将萘莫司他和作为血管紧张素转换酶抑制剂之一的卡托普利(Captopril)用于对照试剂,进行类黄酮苷等天然物来源成分与ACE2的亲和性评价,公开了白藜芦醇、槲皮素、荧光素、柚皮素等与ACE2表现出高亲和力。
但是,在非专利文献1和2中,这些天然物来源成分对SARS-CoV-2的效果,即使在体外也没有得到确认,其效果实际上没有得到验证。
现有技术文献
专利文献
专利文献1:国际公开第2007/099915号
非专利文献
【非专利文献1】Canrong Wu等,Acta Pharmaceutica Sinica B 2020;10(5):766-768
【非特許文献2】Vimal K Maurya等,Virusdisease.2020 Jun;31(2):179-193
发明内容
发明所要解决的技术问题
提供抑制病毒侵入细胞的病毒的侵入细胞抑制剂。
解决技术问题所采用的技术方案
本发明人为了解决上述课题进行了认真研究。结果发现,具有以下构成的病毒的细胞侵入抑制剂能解决上述课题,从而完成了本发明。
即本发明包含以下的[1]~[6]。
[1]一种病毒的细胞侵入抑制剂,其是包含选自α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的至少一种成分(A)的病毒的细胞侵入抑制剂,其中,上述病毒具有与血管紧张素转换酶2(ACE2)结合的尖峰蛋白。
[2]如[1]所述的病毒的细胞侵入抑制剂,其中,上述病毒是选自SARS-CoV、SARS-CoV-2以及HCoV-NL63的任一种。
[3]如[1]或[2]所述的病毒的细胞侵入抑制剂,其中,上述病毒是SARS-CoV-2。
[4]如[1]~[3]中任一项所述的病毒的细胞侵入抑制剂,其中,上述成分(A)包含选自α-单葡糖基芸香苷、α-单葡糖基橙皮苷以及α-单葡糖基柚皮苷的至少一种。
[5]如[1]~[4]中任一项所述的病毒的细胞侵入抑制剂,其中,上述成分(A)的含量为50质量%以上。
[6]如[1]~[5]中任一项所述的病毒的细胞侵入抑制剂,其中,上述成分(A)的含量为65质量%以上。
发明的效果
如果采用本发明,则可提供抑制病毒对细胞的侵入的病毒的细胞侵入抑制剂。
附图说明
图1是显示疑似SARS-CoV-2侵入Caco-2细胞时的基于荧光素酶活性的发光量的图。
图2是显示疑似SARS-CoV-2侵入ACE2表达A549细胞时的基于荧光素酶活性的发光量的图。
具体实施方式
下面,对用于实施本发明的合适的方式进行说明。另外,以下说明的实施方式表示了本发明的代表性实施方式的一例,由此,本发明的范围不会被狭窄地解释。
本发明是一种病毒的细胞侵入抑制剂,其是包含选自α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的至少一种成分(A)的病毒的细胞侵入抑制剂,其中,上述病毒具有与血管紧张素转换酶2(ACE2)结合的尖峰蛋白。
<成分(A)>
本发明的病毒的细胞侵入抑制剂包含选自α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的至少一种成分(A)。成分(A)可以包含α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的任一种或任意选择的2种,也可以包含α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的全部3种。其中从即使是较低浓度也具有较高的病毒的细胞侵入抑制效果出发,作为成分(A),优选包含α-葡糖基芸香苷或α-葡糖基柚皮苷。
成分(A)从抑制病毒的细胞侵入效果的观点出发,优选包含选自α-单葡糖基芸香苷、α-单葡糖基橙皮苷以及α-单葡糖基柚皮苷的至少一种。
病毒的细胞侵入抑制剂所含的成分(A)的含量没有特别限定。例如,作为本发明的病毒的细胞侵入抑制剂所含的成分(A)的含量的下限,例如可例举30质量%、35质量%、40质量%、45质量%、50质量%、55质量%、60质量%、65质量%、70质量%、75质量%、80质量%。此外,作为本发明的病毒的细胞侵入抑制剂所含的成分(A)的含量的上限,例如可例举100质量%、99质量%、98质量%、95质量%、90质量%、85质量%。作为本发明的病毒的细胞侵入抑制剂所含的成分(A)的含量的范围,可以任意设定将上述下限和上限任意组合而成的范围,例如可以设定30~100质量%、60~100质量%、60~90质量%等范围。从抑制病毒的细胞侵入效果的观点出发,本发明的病毒的细胞侵入抑制剂所含的上述成分(A)的含量优选50质量%以上,更优选65质量%以上。
[α-葡糖基芸香苷]
α-葡糖基芸香苷(也称为α葡糖基芸香苷)是在芸香苷具有的芸香糖残基中的葡萄糖残基上通过α1→4键加成1分子以上的葡萄糖的化合物的总称。本发明中的α-葡糖基芸香苷可以是具有这样结构的化合物中的单独一种,也可以是2种以上的混合物。
α-葡糖基芸香苷可用下式(1)表示。式(1)中,n是0或1以上的整数,例如1~19的整数。
[化1]
α-葡糖基芸香苷是在被称为“酶处理芸香苷”(有时也称为“糖转移芸香苷”)的产品中作为主要成分含有的化合物。α-葡糖基芸香苷中,将只结合了一个葡萄糖的称为“α-单葡糖基芸香苷”,将结合了2个以上葡萄糖的称为“α-聚葡糖基芸香苷”。即,式(1)中,α-单葡糖基芸香苷是n为0的化合物,α-聚葡糖基芸香苷通常是n为1~19的化合物。
酶处理芸香苷是通过与芸香苷的糖有关的酶处理而生成的化合物的集合体,通常是与芸香苷结合的葡萄糖个数不同的化合物的混合物,例如,包含由α-单葡糖基芸香苷和α-聚葡糖基芸香苷构成的混合物。此外,酶处理芸香苷通常通过酶处理制造,因此有时还包含未反应的芸香苷或其他衍生物例如异斛皮苷。另外,异斛皮苷(也称为“isoquercitrin”)是在槲皮素骨架的3位的羟基上结合有β-D-葡萄糖的化合物,换而言之,是芸香苷的芸香糖残基中的鼠李糖残基被切断后的化合物。
酶处理芸香苷例如是在作为供糖体的α-葡糖基糖化合物(环糊精、淀粉部分分解物等)的共存下,使糖转移酶(环糊精葡萄糖转移酶(CGTase,EC2.4.1.19)等,具有向芸香苷加成葡萄糖的功能的酶)作用于芸香苷而得的生成物(本说明书中称为“第一酶处理芸香苷”)。
第一酶处理芸香苷是结合的葡萄糖的个数不同的各种α-葡糖基芸香苷,即含有由α-单葡糖基芸香苷以及α-聚葡糖基芸香苷构成的集合体和作为未反应物的芸香苷的组合物。根据需要,例如通过使用多孔性合成吸附材料和适当的溶出液纯化第一酶处理芸香苷,去除供糖体以及其他杂质,进一步减少芸香苷的含量,得到提高了α-葡糖基芸香苷的纯度的第一酶处理芸香苷(α-葡糖基芸香苷纯化物)。
此外,用具有用葡萄糖单元切断α-1,4-葡糖苷键的葡糖淀粉酶活性的酶、例如葡糖淀粉酶(EC3.2.1.3)处理第一酶处理芸香苷,在加成了多个葡萄糖的α-葡糖基芸香苷中,通过仅保留一个直接加成在芸香苷自身的(芸香糖残基中的)葡萄糖残基上的葡萄糖残基并切断除此以外的葡萄糖残基,可以得到含有大量α-单葡糖基芸香苷的酶处理芸香苷(本说明书中称为“第二酶处理芸香苷”。)。通过该酶处理,直接与槲皮素骨架键合的芸香糖残基中的葡萄糖残基不从槲皮素骨架上被切断。
本发明的病毒的细胞侵入抑制剂中,考虑到本发明的效果等,优选使用作为含有α-葡糖基芸香苷的组合物的酶处理芸香苷,可使用第一酶处理芸香苷、以及第二酶处理芸香苷中的任一个含有α-葡糖基芸香苷的组合物。
考虑到本发明的效果等,酶处理芸香苷至少包含α-葡糖基芸香苷,优选进一步包含异斛皮苷的混合物。这样的混合物可通过以下步骤制造:i)制备上述的第一酶处理芸香苷,(ii)用具有葡糖淀粉酶活性的酶处理第一酶处理芸香苷,将α-葡糖基芸香苷几乎全部变换为α-单葡糖基芸香苷,(iii)同时用具有鼠李糖苷酶活性的酶处理,未反应的芸香苷几乎全部变换为异斛皮苷。
作为酶处理芸香苷的市售品,例如可例举东洋精糖株式会社(東洋精糖株式会社)的产品“αGルチンPS”、“αGルチンP”、“αGルチンH”等。“αGルチンPS”是含有65质量%α-单葡糖基芸香苷、15质量%异斛皮苷的组合物。此外,“αGルチンP”是含有60质量%α-葡糖基芸香苷、10质量%芸香苷、1质量%异斛皮苷的组合物。
作为α-葡糖基芸香苷,优选α-单葡糖基芸香苷。由于α-单葡糖基芸香苷的分子量比α-聚葡糖基芸香苷的分子量小,因此α-单葡糖基芸香苷的每单位质量的分子数多,认为在作用效果上是有利的。
酶处理芸香苷中含有的各种α-葡糖基芸香苷、以及其他成分的存在可通过HPLC的色谱图确认,各成分的含量、或所希望的特定的成分的纯度可由色谱图的峰面积算出。
α-葡糖基芸香苷的制造方法没有特别限制,可采用公知的方法。从收率良好、或制造容易的方面出发,如上所述优选通过芸香苷的酶处理制造。芸香苷的获得·制备方法没有特别限定,作为试剂或纯化物可使用通常制造销售的化合物,也可使用从柑橘类(蜜桔、橙子等)的外皮或荞麦(日语:ソバ)的果实等原料提取制备的化合物。
[α-葡糖基橙皮苷]
α-葡糖基橙皮苷(也称为α葡糖基橙皮苷)是橙皮苷的芸香糖单元中的羟基上通过α-1,4键加成了1个分子以上的葡萄糖的化合物的总称。本发明中的α-葡糖基橙皮苷可以是具有这样结构的化合物中的单独一种,也可以是2种以上的混合物。另外,橙皮苷是在橙皮素的7位的羟基上键合了β-芸香糖(6-O-α-L-鼠李糖基-β-D-葡萄糖)的化合物,即橙皮素苷。
α-葡糖基橙皮苷可用下式(2)表示。式(2)中,n是0或1以上的整数,例如1~19的整数。
[化2]
α-葡糖基橙皮苷是在被称为“酶处理橙皮苷”(有时也称为“糖转移橙皮苷”)的物质中作为主要成分含有的化合物。α-葡糖基橙皮苷中,将只结合了一个葡萄糖的称为“α-单葡糖基橙皮苷”,将结合了2个以上葡萄糖的称为“α-聚葡糖基橙皮苷”。即,式(2)中,α-单葡糖基橙皮苷是n为0的化合物,α-聚葡糖基橙皮苷通常是n为1~19的化合物。
酶处理橙皮苷是通过与橙皮苷的糖有关的酶处理而生成的化合物的集合体,通常是与橙皮苷结合的葡萄糖的个数不同的化合物的混合物,例如,包含由α-单葡糖基橙皮苷和α-聚葡糖基橙皮苷构成的混合物。此外,在α-葡糖基橙皮苷之外,也可包含未反应的橙皮苷和7-葡糖基橙皮素等α-葡糖基橙皮苷以外的橙皮苷衍生物(称为其他橙皮苷衍生物)。其中,酶处理橙皮苷中,优选不包含橙皮素。
作为上述橙皮苷的糖相关的酶处理的例,如下所述。
(1)在α-葡糖基糖化合物(例如,环糊精,淀粉部分分解物)等供糖体的共存下在橙皮苷上使糖转移酶起作用,通过在橙皮苷的葡萄糖单元上通过α-1,4键加成葡萄糖,生成α-葡糖基橙皮苷,得到含有未反应的橙皮苷和α-葡糖基橙皮苷的组合物(第一酶处理橙皮苷)。
(2)在由上述(1)生成的α-葡糖基橙皮苷上使葡糖淀粉酶等起作用,通过从与橙皮苷的葡萄糖单元结合的葡萄糖链中只留下1个分子而切断其他葡萄糖,生成α-单葡糖基橙皮苷,得到含有未反应的橙皮苷和α-单葡糖基橙皮苷的组合物(第二酶处理橙皮苷)。
(3)在上述(2)的未反应的橙皮苷上使α-L-鼠李糖苷酶起作用,通过切断橙皮苷的芸香糖单元中含有的鼠李糖而生成7-葡糖基橙皮素,得到含有7-葡糖基橙皮素和α-单葡糖基橙皮苷的组合物(第三酶处理橙皮苷)。
作为第一酶处理的例子,可例举在α-葡糖基糖化合物(例:环糊精,淀粉部分分解物)的共存下使糖转移酶(例:环糊精葡萄糖转移酶(CGTase,EC2.4.1.19)等具有在橙皮苷上加成葡萄糖的功能的酶)作用于橙皮苷。
本发明的病毒的细胞侵入抑制剂中,考虑到本发明的效果等,优选使用作为含有α-葡糖基橙皮苷的组合物的酶处理橙皮苷,可使用第一酶处理橙皮苷、第二酶处理橙皮苷、第三酶处理橙皮苷的任意组合物。
考虑到本发明的效果等,酶处理橙皮苷优选至少包含α-葡糖基橙皮苷,还包含含有橙皮苷以及7-葡糖基橙皮素的任意一方或双方的混合物。
作为酶处理橙皮苷的市售品,例如可例举东洋精糖株式会社的产品“αGヘスペリジンPS-CC”以及“αGヘスペリジンPA-T”。“αGヘスペリジンPS-CC”包含80质量%以上α-单葡糖基橙皮苷并含有7-葡糖基橙皮素。“αGヘスペリジンPA-T”包含75质量%以上α-单葡糖基橙皮苷并含有橙皮苷。
作为α-葡糖基橙皮苷,优选α-单葡糖基橙皮苷。由于α-单葡糖基橙皮苷的分子量比α-聚葡糖基橙皮苷的分子量小,因此α-单葡糖基橙皮苷的每单位质量的分子数多,认为在作用效果上是有利的。
α-单葡糖基橙皮苷可通过使糖水解酶作用于α-聚葡糖基橙皮苷、仅切断1个与橙皮苷键合的葡萄糖来产生(第二酶处理橙皮苷)。作为糖水解酶,可例举具有用葡萄糖单元切断α-1,4-葡糖苷键的葡糖淀粉酶活性的酶,例如葡糖淀粉酶(EC3.2.1.3)。另外,α-葡糖基橙皮苷中的α-单葡糖基橙皮苷的比例能够根据葡糖淀粉酶的酶处理的温度·时间条件等调节,进一步由酶处理橙皮苷的混合物纯化·提取α-单葡糖基橙皮苷的方法也是公知的。
酶处理橙皮苷中含有的各种α-葡糖基橙皮苷、橙皮苷、以及其他成分的存在可通过HPLC的色谱图确认,各成分的含量、或所希望的特定的成分的纯度可由色谱图的峰面积算出。
α-葡糖基橙皮苷的制造方法没有特别限制,可采用公知的方法。从收率良好、或制造容易的方面出发,如上所述优选通过橙皮苷的酶处理制造。橙皮苷的获得·制备方法没有特别限定,作为试剂或纯化物可使用通常制造销售的化合物,也可使用从柑橘类(蜜桔、橙子等)的外皮等原料提取制备的化合物。
[α-葡糖基柚皮苷]
α-葡糖基柚皮苷(也称为α葡糖基柚皮苷)是在柚皮苷所具有的羟基上加成1个分子以上的葡萄糖而成的化合物的总称。柚皮苷是具有柚皮素(5,7,4'-三羟基黄酮)骨架的7位的羟基与新橙皮糖(L-鼠李糖基-(α1→2)-D-葡萄糖)β键合而成的结构的类黄酮的一种。α-葡糖基柚皮苷是其柚皮苷所具有的新橙皮糖残基中的葡萄糖残基的3位(3”位)的羟基以及柚皮素骨架中的苯基的4位(4'位)的羟基的至少一个与1个分子以上的α-葡萄糖键合而成的结构。本发明中的α-葡糖基柚皮苷可以是具有这样结构的化合物中的单独一种,也可以是2种以上的混合物。
α-葡糖基柚皮苷可用下式(3)表示。式(3)中,R1的m以及R2的n分别指与3”位以及4'位键合的α-葡萄糖残基的数量,相互独立地表示0以上、通常为25以下的整数。其中,式(3)表示“α-葡糖基柚皮苷”,满足m+n≥1,即需要柚皮苷与至少1个分子的α-葡萄糖连接(m=n=0的情况下,即R1、R2都为-H的情况下,式(3)表示“柚皮苷”)。
[化3]
α-葡糖基柚皮苷是在被称为“酶处理柚皮苷”(有时也称为“糖转移柚皮苷”)的物质中作为主要成分含有的化合物。α-葡糖基柚皮苷中,将只结合了一个葡萄糖的称为“α-单葡糖基柚皮苷”,将结合了2个以上葡萄糖的称为“α-聚葡糖基柚皮苷”。
酶处理柚皮苷例如是使柚皮苷和供糖体(例如糊精)的混合物与糖转移酶(例如环糊精葡萄糖转移酶)反应而得的生成物(本说明书中称为“第一酶处理柚皮苷”。),是柚皮苷所具有的羟基上加成1个分子以上的葡萄糖而得的各种化合物的集合体。因此,酶处理柚皮苷通常包含与柚皮苷键合的葡萄糖的个数不同的化合物的混合物,例如由α-单葡糖基柚皮苷和α-聚葡糖基柚皮苷构成的混合物。此外,在α-葡糖基柚皮苷之外,也可包含未反应的柚皮苷和7-葡糖基柚皮素等α-葡糖基柚皮苷以外的柚皮苷衍生物(称为其他柚皮苷衍生物)。
第一酶处理柚皮苷的基本的制造方法例如可参照日本专利特开平4-13691号公报。根据需要,例如通过使用多孔性合成吸附材料和适当的溶出液纯化第一酶处理柚皮苷,去除供糖体以及其他杂质,进一步减少柚皮苷的含量,得到提高了α-葡糖基柚皮苷的纯度的第一酶处理柚皮苷(α-葡糖基柚皮苷纯化物)。
作为α-葡糖基柚皮苷,优选在柚皮苷的3”位上连接1个分子α-葡萄糖和/或在4'位上连接1个分子α-葡萄糖的、即选自3”-α-单葡糖基柚皮苷(式(3)中,m=1,n=0)、4'-α-单葡糖基柚皮苷(同样m=0,n=1)、以及3”-4'-α-二葡糖基柚皮苷(同样m=1,n=1)的至少一种α-葡糖基柚皮苷,其中更优选3”-α-单葡糖基柚皮苷。由于α-单葡糖基柚皮苷的分子量比α-聚葡糖基柚皮苷的分子量小,因此每单位质量的α-单葡糖基柚皮苷的分子数多,认为在作用效果上是有利的。
含有大量上述3种α-单/二葡糖基柚皮苷的酶处理柚皮苷(本说明书中称为“第二酶处理柚皮苷”。)例如通过将糖链切断的方法得到,其中该糖链是用具有葡糖淀粉酶活性的酶处理上述的第一酶处理柚皮苷,并由上述糖转移酶转移到柚皮苷的3”位以及/或4'位的2个分子以上的α-葡萄糖通过α-1,4键键合而得的糖链,并以仅留下根部的相当于1个分子的α-葡萄糖残基的方式进行切断。进一步,用具有α-葡糖苷酶活性的酶处理第二酶处理柚皮苷,通过切断与4'位的羟基直接键合的相当于1个分子的α-葡萄糖残基,使3”-α-单葡糖基柚皮苷残存,可得到几乎完全不含有4'-α-单葡糖基柚皮苷以及3”-4'-α-二葡糖基柚皮苷的酶处理柚皮苷(本说明书中称为“第三酶处理柚皮苷”。)。第二以及第三酶处理柚皮苷的基本制造方法例如可参照日本专利特开2002-199896号公报。
进一步,在第三酶处理柚皮苷上使α-L-鼠李糖苷酶起作用,通过切断柚皮苷的芸香糖单元中含有的鼠李糖,生成7-葡糖基柚皮素,可得到含有7-葡糖基柚皮素和α-单葡糖基柚皮苷的酶处理柚皮苷(本说明书中称为“第四酶处理柚皮苷”。)。
此外,如果使转葡糖苷酶作用于第一酶处理柚皮苷,则由于转葡糖苷酶是兼具葡糖淀粉酶活性和α-葡糖苷酶活性的酶,则可不通过如上所述的2阶段的处理,而是通过1阶段的处理得到富含3”-α-单葡糖基柚皮苷的第三酶处理柚皮苷。进一步,根据需要,也可以使具有β-葡糖苷酶活性的酶作用于第三酶处理柚皮苷(也可同时使转葡糖苷酶作用于第一酶处理柚皮苷),进行将少量溶解于水溶液中的未反应的柚皮苷所具有的(没有进行基于糖转移酶的糖链的修饰)新橙皮糖残基从作为其糖苷配基的柚皮素柚皮素切断的处理。通过这样的处理而生成的柚皮素柚皮素比柚皮苷溶解度更低,因此可作为沉淀容易地从水溶液中去除,因此能够从水溶液以更高纯度回收3”-α-单葡糖基柚皮苷。
本发明的病毒的细胞侵入抑制剂中,考虑到本发明的效果等,优选使用含有作为α-葡糖基柚皮苷的组合物的酶处理柚皮苷,可使用第一酶处理柚皮苷、第二酶处理柚皮苷、第三酶处理柚皮苷以及第四酶处理柚皮苷中的任一个含有α-葡糖基柚皮苷的组合物。
考虑到本发明的效果等,酶处理柚皮苷优选至少包含α-葡糖基柚皮苷,还包含含有柚皮苷以及7-葡糖基柚皮素的任意一方或双方的混合物。
酶处理柚皮苷中含有的各种α-葡糖基柚皮苷、柚皮苷、以及其他成分的存在可通过HPLC的色谱图确认,各成分的含量、或所希望的特定的成分的纯度可由色谱图的峰面积算出。
α-葡糖基柚皮苷的制造方法没有特别限制,可采用公知的方法。从收率良好、或制造容易的方面出发,如上所述优选通过柚皮苷的酶处理制造。柚皮苷的获得·制备方法没有特别限定,作为试剂或纯化物可使用通常制造销售的化合物,也可使用从柑橘类(日本夏橙、葡萄柚等)的外皮等原料提取制备的化合物。
<病毒>
本发明中,具有与血管紧张素转换酶2(Angiotensin-Converting Enzyme2:以下称为“ACE2”)结合的尖峰蛋白的病毒没有特别限制,只要是能够藉由病毒的尖峰蛋白和作为宿主细胞的受体的ACE2的结合、病毒的基因组DNA或基因组RNA侵入细胞内的病毒即可,例如可例举SARS-CoV、SARS-CoV-2或HCoV-NL63、BatCoV-WIV1、BatCoV-WIV16、BatCoV-RS4231、BatCoV-RsSHC014等。其中,从本发明的抑制效果的方面出发,优选SARS-CoV,SARS-CoV-2或HCoV-NL63,更优选SARS-CoV-2。
尖峰蛋白(也称为S蛋白、刺突蛋白等)通常是贯穿病毒表面的包膜的糖蛋白。尖峰蛋白只要能够与ACE2结合则没有特别限制,优选具有与ACE2结合的受体结合结构域(RBD)的亚单元或结构域,以及具有用于与宿主细胞膜融合的融合肽的亚单元或结构域。
尖峰蛋白的氨基酸序列及其碱基序列的序列信息例如可从GenBank以及GISAID(Global Initiative on Sharing All Influenza Data)等数据库获得。作为尖峰蛋白的氨基酸序列,例如可例举序列编号1(SARS-CoV)、序列编号2(SARS-CoV-2)或序列编号3(HCoV-NL63)等。
尖峰蛋白只要能够与ACE2结合即可,可以是在其氨基酸序列中缺失、取代或附加了1~3个、优选1~2个、更优选1个氨基酸的突变尖峰蛋白。
尖峰蛋白只要能够与ACE2结合则结构没有特别限制,例如,在受体结合结构域具有下型结构或上型结构的情况下,从与ACE2的亲和性的观点出发,优选受体结合结构域为上型结构。
具有与ACE2结合的尖峰蛋白的病毒也包括上述尖峰蛋白变异的变异株。例如,作为SARS-CoV-2的变异株,可例举N501Y型、E484K型、L452R型、E484Q型、K417N型、H417T型、N439K型、D614G型、A222V型、Y453F型、P681H型、A570D型、T716I型、S982A型、A1708D型、A701V型、D80A型、L18F型、R246I型、D215G型、delH69V70型、delY144型、Q27stop型、以及L242_244L型等变异株。
病毒所侵入的细胞只要在细胞表面具有ACE2则没有特别限制,例如可例举脑、心脏、主动脉、肺、食管、胃、十二指肠、空肠、回肠、盲肠、结肠、直肠、肾脏、骨骼肌、脾脏、胸腺、气管、胎盘、膀胱、子宫、前列腺、精巢、卵巢、胰腺、肾上腺、唾液腺、舌、牙龈、乳腺、骨髓、血管等的细胞。宿主细胞的来源生物没有特别限制,优选来自人、蝙蝠、穿山甲、猪、猫、狗、牛、小鼠、大鼠或鸡,更优选来自人。
<病毒的细胞侵入抑制剂>
病毒的细胞侵入抑制剂只要包含选自α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的至少一种的成分(A)即可,可以仅由上述成分(A)构成,在不妨害上述成分(A)的病毒的细胞侵入抑制效果的情况下,也可以进一步含有赋形剂、稳定剂、润湿剂和乳化剂等公知的任意成分。此外,上述病毒的细胞侵入抑制剂是包含上述成分(A)的组合物,可以是选自酶处理芸香苷、酶处理橙皮苷、酶处理柚皮苷的至少一种,也可以含有选自酶处理芸香苷、酶处理橙皮苷、酶处理柚皮苷的至少一种。
病毒的细胞侵入抑制剂只要具备具有与ACE2结合的尖峰蛋白的病毒与细胞的ACE2结合、吸附,抑制病毒的基因组DNA或基因组RNA对细胞内的侵入的作用即可。病毒的细胞侵入抑制剂的抑制病毒的细胞侵入的作用如实施例所实施的,可在体外的使用疑似病毒的细胞侵入抑制试验中,通过侵入具有ACE2的宿主细胞内的、病毒所具有的荧光素酶活性的发光值的变化进行确认。即,与不使用病毒的细胞侵入抑制剂的组相比,使用病毒的细胞侵入抑制剂的组的荧光素酶活性的发光值小的情况表示病毒向宿主细胞内的侵入被病毒的细胞侵入抑制剂所抑制。
病毒的细胞侵入抑制率没有特别限制,在使用疑似病毒的细胞侵入抑制试验中,在将不添加本发明的病毒的细胞侵入抑制剂时的抑制率作为0%的情况下,添加本发明的病毒的细胞侵入抑制剂时的抑制率优选10%以上,更优选30%以上,进一步优选50%以上。病毒的细胞侵入抑制率可用下式表示。
病毒的细胞侵入抑制率(%)=100×{1-(添加病毒的细胞侵入抑制剂的组的发光值)/(未添加病毒的细胞侵入抑制剂的组的发光值)}
疑似病毒可用公知的方法制作,或者也可购入市售品使用。作为市售品,例如可例举Lenti-X(TM)SARS-CoV-2(宝生物株式会社(タカラバイオ株式会社)制)/SARS-CoV疑似病毒以及HCov-NL63疑似病毒(载体家(Vector Builder)制)等。
[用途]
对病毒的细胞侵入抑制剂的用途没有特别限制,由于病毒的细胞侵入抑制剂具有抑制病毒对细胞内的侵入的作用,因此能够以病毒感染的预防、例如预防SARS-CoV、SARS-CoV-2以及HCoV-NL63的感染为目的使用。
病毒的细胞侵入抑制剂的给药量可根据病毒感染症的种类或给药对象的年龄、性别、人种等适当选择。例如,在口服给药病毒的细胞侵入抑制剂的情况下,作为成分(A)每日优选10mg~1000mg,更优选100mg~300mg。病毒的细胞侵入抑制剂的给药次数可以是单次或多次,每日的给药频率可以例如每日给药1~3次,也可给药2或3次。病毒的细胞侵入抑制剂的给药间隔可根据病毒感染症的种类或给药对象的年龄、性别、人种等适当选择。从病毒的细胞侵入抑制效果的观点出发,优选1天~90天,更优选14天~60天。
[制剂]
病毒的细胞侵入抑制剂可以直接对生物体给药,也可以作为与药学上允许的载体一起掺和而得的制剂给予有效量的病毒的细胞侵入抑制剂。作为制剂,例如可例举饮食品、医药品、准药品。给药方法没有特别限制,可以是口服,或非口服。上述制剂从容易给药的方面出发,优选口服给药。
在口服的情况下,上述制剂可以是饮食品(包括特定保健用食品、营养功能食品、功能性表示食品等保健功能食品、其他所谓的健康食品或补充剂)、医药品、准药品等。
口服用制剂可以制成固体或液体(包括糊状)。对剂型没有限制,具体而言,作为固体制剂,可例举粉末剂、颗粒剂、片剂、胶囊剂、含片等。另外,作为液状制剂,可以例示内用液剂、悬浮剂、乳剂、糖浆剂、饮料剂等,根据目的适当选择这些剂型或其他剂型。
在非口服的情况下,上述制剂可以是注射剂、栓剂等。
上述制剂可根据这些制剂通常使用的方法,通过将病毒的细胞侵入抑制剂添加到制剂中来制造。病毒的细胞侵入抑制剂可以在制剂的制备工序的初期添加,或者在制备工序的中期或末期添加,并且添加方法可以根据制剂形态从混合、混炼、溶解、浸渍、散布、喷雾、涂布等中选择适当的方法。
作为病毒的细胞侵入抑制剂的有效成分的上述成分(A)由于水溶性良好,因此在添加到水或水分多的制剂中时,也可以均匀地溶解或分散。
病毒的细胞侵入抑制剂的对制剂的掺和量可以根据病毒的细胞侵入抑制活性所涉及的疾病的种类和症状的程度适当选择,从给药的容易性和在制剂中的稳定性的观点出发,作为上述成分(A),优选5~98质量%,更优选20~70质量%,进一步优选30~50质量%。
实施例
以下,基于实施例更具体地说明本发明,但本发明并不限定于这些实施例,可以在不变更其主旨的范围内适当变更实施。
[制备例1]
如下制备在包膜表面上具有SARS-CoV-2的尖峰蛋白、侵入宿主细胞内后显示荧光素酶活性的疑似SARS-CoV-2。
(1)fLuc(萤火虫·荧光素酶)表达用慢病毒载体质粒(pCDH-fLuc)的制备
将使用PureLink Expi无内毒素Maxi质粒纯化试剂盒(PureLink ExpiEndotoxin-Free Maxi Plasmid Purification Kit)(赛默飞世尔科技公司(ThermoFisher Scientific)制)纯化的pCDH-EF1-MCS-
(PGK-copGFP-T2A-Puro)质粒(系统生物科学公司(System Biosciences)制)用EcoRI(EcoRI-HF:新英格兰生物实验室(New England Biolabs)制)以及BamHI(BamHI-HF:新英格兰生物实验室制)切断,制备线状质粒载体。
使用In-Fusion(注册商标)HD克隆试剂盒(宝生物株式会社制),根据试剂盒附带的说明书连接上述线状质粒载体柱纯化后的产物和fLuc表达用的合成基因(序列编号4),使用得到的慢病毒制备用载体质粒转化大肠杆菌。
对耐药性的大肠杆菌进行使用各种引物(CD813_F1(序列编号5)、CD813_R1(序列编号6))的菌落PCR,确认包含靶基因的约1.8kbp的扩增产物。使用各种引物(CD813_F1、CD813_R1)对扩增产物进行序列分析,确认pCDH-fLuc具有如设计的序列。
将携带该慢病毒制备用载体质粒的大肠杆菌在液体中培养,通过Maxi-prep制备fLuc表达用慢病毒载体质粒(pCDH-fLuc)。
(2)使SAR-CoV-2表达的质粒(pCD5spCoV2)的制备
用NheI(NheI-HF:新英格兰生物实验室制)、AflII(新英格兰生物实验室制)切断pcDNA3.1/Hygro质粒(赛默飞世尔科技公司制),制备线状质粒载体。在进行该线状质粒载体的柱纯化后,通过In-Fusion HD cloning kit(宝生物株式会社制)将纯化后的该线状质粒载体与连接了CD5的信号序列(序列编号7)以及SARS-CoV-2的S基因(序列编号8)后的合成基因(序列编号9)连接,使用其产物转化大肠杆菌。
对耐药性的大肠杆菌进行使用各种质粒特异性引物(pcDNA3_F1(序列编号10),pcDNA3_R2(序列编号11))的菌落PCR,确认包含靶基因的约4kbp的扩增产物。使用CMV_F4(序列编号12)、SARS-CoV-2_Seq1(序列编号13)、SARS-CoV-2_Seq2(序列编号14)、SARS-CoV-2_Seq3(序列编号15)、pcDNA3_R2的各引物对扩增产物进行序列分析,确认pCD5spCoV2具有如设计的序列。
在液体中培养携带该质粒载体的大肠杆菌,并通过Maxi-prep制备质粒(pCD5spCoV2)。
(3)疑似SARS-CoV-2(疑似新型冠状病毒)的制备
将含5%的FBS、1mM Sodium pyrubate的Opti-MEM(赛默飞世尔科技公司制)作为基础培养基,培养Lenti-X 293T细胞(宝生物株式会社制)。之后,将Lenti-X 293T细胞(1.8×107个细胞)悬浮于34mL的上述基础培养基中,播种在15cm的培养皿中。
第二天,使用Lipofectamine 3000转染试剂(赛默飞世尔科技公司制),进行如下转化(以下混合物的组成表示一个15cm培养皿的组成,并在三个15cm培养皿中进行转化)。
混合3.9mL的Opti-MEM(赛默飞世尔科技公司制)、14.4μg的pPACKH1-GAG(pPACKH1HIV慢载体包装试剂盒(系统生物科学公司制)的组件)、7.2μg的pPACKH1-REV(pPACKH1 HIV慢载体包装试剂盒(系统生物科学公司制)的组件)、11.2μg的上述pCDH-fLuc、12.2μg的上述pCD5spCoV2,以及91μL的P3000试剂(Lipofectamine 3000转染试剂(赛默飞世尔科技公司制)的组件),得到混合物。在得到的混合物中添加3.9mL的Opti-MEM和107μL的Lipofectamine 3000试剂的混合物,在室温下保温10分钟,形成DNA/Lipofectamine 3000复合物。
之后,将包含复合物的溶液(7.8mL)添加到Lenti-X 293T细胞中。在添加复合物的6小时后,更换含有21mL的5%FBS、1mM Sodium pyrubate的Opti-MEM,培养24小时后,回收培养上清(1),在4℃下保存。
将回收培养上清(1)后的培养基更换为含有21mL的5%FBS、1mM Sodium pyrubate的Opti-MEM,培养24小时,回收培养上清(2)。
将培养上清(1)和培养上清(2)的混合物作为疑似SARS-CoV-2溶液得到,用于以下的实验。在-80℃下保存疑似SARS-CoV-2溶液直至使用。
[实施例1]
<使用Caco-2细胞的疑似SARS-CoV-2的细胞侵入抑制试验>
验证在包含αG橙皮苷PA-T、αG芸香苷PS、或αG柚皮苷PS(第四酶处理柚皮苷)的培养基中培养Caco-2细胞(来源于人结肠癌的细胞)和上述疑似SARS-CoV-2时的疑似SARS-CoV-2的细胞侵入抑制效果。
[方法]
样品中,作为α-葡糖基芸香苷(αG芸香苷)使用αG芸香苷PS(东洋精糖株式会社制),作为α-葡糖基橙皮苷(αG橙皮苷)使用αG橙皮苷PA-T(东洋精糖株式会社制),以及作为α-葡糖基柚皮苷(αG柚皮苷)使用αG柚皮苷PS(第四酶处理柚皮苷;α-单葡糖基柚皮苷75质量%,7-葡糖基柚皮素10质量%)。
在Eagle's基本必需培养基(成分:MEM非必需氨基酸溶液)中制备Caco-2细胞(ATCC制)以达到6×103细胞/孔,接种于96孔板,并在37℃、5%CO2环境中培养。播种24小时后,在各孔中添加图1所示的各浓度(培养基中的最终浓度)的样品10μL。
样品添加2小时后,将疑似SARS-CoV-2溶液140μL添加到各孔中,并在不添加疑似SARS-CoV-2溶液的孔中添加上述基础培养基140μL,培养Caco-2细胞46小时。培养后,更换培养基,去除疑似SARS-CoV-2溶液和样品,再培养24小时。
培养后,测定细胞内侵入的疑似SARS-CoV-2的荧光素酶活性的发光值。
[结果]
发光值的结果示于图1。由发光值的值算出的细胞侵入抑制率示于表1。细胞侵入抑制率如下算出。细胞侵入抑制率(%)=100×{1-(有样品的发光值)/(无样品的发光值)}。每个实验条件的N数为3,并使用添加病毒和不添加样品(0ppm)作为对照,使用t检验计算p值,并且p<0.05被认为是显著差异。上述结果表明,各样品在任意浓度下均能有效抑制pseudo-SARS-CoV-2的细胞侵入。
[比较例1]
除了使用萘莫司他(东京化成工业株式会社(東京化成工業株式会社)制)代替上述样品以外,以与实施例1相同的方式测定荧光素酶活性的发光值,算出细胞侵入抑制率(%)。结果示于图1及表1。
[表1]
[实施例2]
<使用ACE2表达A549细胞的疑似SARS-CoV-2的细胞侵入抑制试验>
除了使用通过下述方法修饰为强制表达ACE2的ACE2表达A549细胞来代替Caco-2细胞以外,以与实施例1相同的方式测定荧光素酶活性的发光值,算出细胞侵入抑制率(%)。结果示于图2及表2。上述结果表明,各样品在任意浓度下均能有效抑制疑似SARS-CoV-2的细胞侵入。
[表2]
[制备例2]
如下制备ACE2表达A549细胞(A549-ACE2)。
(1)ACE2质粒载体的制备
用NheI(NheI-HF:新英格兰生物实验室制)、AflII(新英格兰生物实验室制)切断pcDNA3.1/Hygro质粒(赛默飞世尔科技公司制)并进行柱纯化后、将各自的纯化物与ACE2表达用的合成基因(序列编号16)使用In-Fusion HD克隆试剂盒(宝生物株式会社制)根据试剂盒附带的说明书进行连接,使用得到的产物转化大肠杆菌。
对耐药性的大肠杆菌进行菌落PCR,确认包含靶基因的约2.8kbp的扩增产物。使用CMV_F4(序列编号12)、Hs_ACE2_Seq1(序列编号17)、pcDNA3_R2(序列编号11)的各引物对扩增产物进行序列分析,确认如设计的序列(pcDNA3.1-Hyg-ACE2)。在液体中培养携带该质粒载体的大肠杆菌,并通过Maxi-prep制备pcDNA3.1-Hyg-ACE2质粒,制成转染用的DNA。
(2)ACE2表达A549细胞的制备
将pcDNA3.1-Hyg-ACE2质粒转染至A549细胞(JCRB细胞银行(JCRB細胞バンク)制)后,在包含50mg/mL潮霉素B(富士胶片和光纯药株式会社(富士フィルム和光純薬株式会社)制)的增殖培养基中进行选择,得到稳定导入了该pcDNA3.1-Hyg-ACE2质粒的细胞(A549-ACE2)。之后,通过极限稀释法,得到32个克隆后,从克隆细胞和阴性对照(未导入pcDNA3.1-Hyg-ACE2质粒的A549)中提取RNA并纯化,进行cDNA合成。对于A549-ACE2克隆,使用Hs_ACE2_RTF1(序列编号18)、Hs_ACE2_RTR1(序列编号19)的各引物,通过实时PCR对ACE2的mRNA的表达量进行定量分析,确认ACE2的表达。
SEQUENCE LISTING
<110> 东洋精糖株式会社
<120> 病毒的细胞侵入抑制剂
<130> P22001WO-01
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 1255
<212> PRT
<213> SARS 冠状病毒 Tor2
<220>
<223> SARS-CoV的S蛋白
<220>
<221> MISC_FEATURE
<222> (1)..(1255)
<400> 1
Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu
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Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln
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His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg
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Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser
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Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val
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Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn
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Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys
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Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met
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Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr
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Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser
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Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly
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Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp
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Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu
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Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro
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Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr
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Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile
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Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys
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Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn
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Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr
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Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser
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Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr
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Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly
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Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala
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Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly
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Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
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Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser
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Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu
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Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly
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Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp
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Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val
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Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly
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Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn
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Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg
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Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp
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Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys
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Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser
580 585 590
Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr
595 600 605
Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr
610 615 620
Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu
625 630 635 640
His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile
645 650 655
Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys
660 665 670
Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala
675 680 685
Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile
690 695 700
Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys
705 710 715 720
Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu
725 730 735
Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile
740 745 750
Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys
755 760 765
Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe
770 775 780
Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile
785 790 795 800
Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met
805 810 815
Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile
820 825 830
Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr
835 840 845
Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala
850 855 860
Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe
865 870 875 880
Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn
885 890 895
Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala
900 905 910
Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly
915 920 925
Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu
930 935 940
Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
945 950 955 960
Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp
965 970 975
Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln
980 985 990
Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala
995 1000 1005
Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp
1010 1015 1020
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala
1025 1030 1035
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln
1040 1045 1050
Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys
1055 1060 1065
Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser
1070 1075 1080
Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr
1085 1090 1095
Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly
1100 1105 1110
Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp
1115 1120 1125
Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser
1130 1135 1140
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
1145 1150 1155
Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1160 1165 1170
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1175 1180 1185
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile
1190 1195 1200
Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys
1205 1210 1215
Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly
1220 1225 1230
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys
1235 1240 1245
Gly Val Lys Leu His Tyr Thr
1250 1255
<210> 2
<211> 1273
<212> PRT
<213> 重症急性呼吸综合征冠状病毒2
<220>
<223> SARS-CoV的S蛋白-2
<220>
<221> MISC_FEATURE
<222> (1)..(1273)
<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
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
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
1250 1255 1260
Val Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 3
<211> 1356
<212> PRT
<213> 人类冠状病毒NL63
<220>
<223> HCoV-NL63的S蛋白
<220>
<221> MISC_FEATURE
<222> (1)..(1356)
<400> 3
Met Lys Leu Phe Leu Ile Leu Leu Val Leu Pro Leu Ala Ser Cys Phe
1 5 10 15
Phe Thr Cys Asn Ser Asn Ala Asn Leu Ser Met Leu Gln Leu Gly Val
20 25 30
Pro Asp Asn Ser Ser Thr Ile Val Thr Gly Leu Leu Pro Thr His Trp
35 40 45
Phe Cys Ala Asn Gln Ser Thr Ser Val Tyr Ser Ala Asn Gly Phe Phe
50 55 60
Tyr Ile Asp Val Gly Asn His Arg Ser Ala Phe Ala Leu His Thr Gly
65 70 75 80
Tyr Tyr Asp Ala Asn Gln Tyr Tyr Ile Tyr Val Thr Asn Glu Ile Gly
85 90 95
Leu Asn Ala Ser Val Thr Leu Lys Ile Cys Lys Phe Ser Arg Asn Thr
100 105 110
Thr Phe Asp Phe Leu Ser Asn Ala Ser Ser Ser Phe Asp Cys Ile Val
115 120 125
Asn Leu Leu Phe Thr Glu Gln Leu Gly Ala Pro Leu Gly Ile Thr Ile
130 135 140
Ser Gly Glu Thr Val Arg Leu His Leu Tyr Asn Val Thr Arg Thr Phe
145 150 155 160
Tyr Val Pro Ala Ala Tyr Lys Leu Thr Lys Leu Ser Val Lys Cys Tyr
165 170 175
Phe Asn Tyr Ser Cys Val Phe Ser Val Val Asn Ala Thr Val Thr Val
180 185 190
Asn Val Thr Thr His Asn Gly Arg Val Val Asn Tyr Thr Val Cys Asp
195 200 205
Asp Cys Asn Gly Tyr Thr Asp Asn Ile Phe Ser Val Gln Gln Asp Gly
210 215 220
Arg Ile Pro Asn Gly Phe Pro Phe Asn Asn Trp Phe Leu Leu Thr Asn
225 230 235 240
Gly Ser Thr Leu Val Asp Gly Val Ser Arg Leu Tyr Gln Pro Leu Arg
245 250 255
Leu Thr Cys Leu Trp Pro Val Pro Gly Leu Lys Ser Ser Thr Gly Phe
260 265 270
Val Tyr Phe Asn Ala Thr Gly Ser Asp Val Asn Cys Asn Gly Tyr Gln
275 280 285
His Asn Ser Val Val Asp Val Met Arg Tyr Asn Leu Asn Phe Ser Ala
290 295 300
Asn Ser Leu Asp Asn Leu Lys Ser Gly Val Ile Val Phe Lys Thr Leu
305 310 315 320
Gln Tyr Asp Val Leu Phe Tyr Cys Ser Asn Ser Ser Ser Gly Val Leu
325 330 335
Asp Thr Thr Ile Pro Phe Gly Pro Ser Ser Gln Pro Tyr Tyr Cys Phe
340 345 350
Ile Asn Ser Thr Ile Asn Thr Thr His Val Ser Thr Phe Val Gly Ile
355 360 365
Leu Pro Pro Thr Val Arg Glu Ile Val Val Ala Arg Thr Gly Gln Phe
370 375 380
Tyr Ile Asn Gly Phe Lys Tyr Phe Asp Leu Gly Phe Ile Glu Ala Val
385 390 395 400
Asn Phe Asn Val Thr Thr Ala Ser Ala Thr Asp Phe Trp Thr Val Ala
405 410 415
Phe Ala Thr Phe Val Asp Val Leu Val Asn Val Ser Ala Thr Asn Ile
420 425 430
Gln Asn Leu Leu Tyr Cys Asp Ser Pro Phe Glu Lys Leu Gln Cys Glu
435 440 445
His Leu Gln Phe Gly Leu Gln Asp Gly Phe Tyr Ser Ala Asn Phe Leu
450 455 460
Asp Asp Asn Val Leu Pro Glu Thr Tyr Val Ala Leu Pro Ile Tyr Tyr
465 470 475 480
Gln His Thr Asp Ile Asn Phe Thr Ala Thr Ala Ser Phe Gly Gly Ser
485 490 495
Cys Tyr Val Cys Lys Pro His Gln Val Asn Ile Ser Leu Asn Gly Asn
500 505 510
Thr Ser Val Cys Val Arg Thr Ser His Phe Ser Ile Arg Tyr Ile Tyr
515 520 525
Asn Arg Val Lys Ser Gly Ser Pro Gly Asp Ser Ser Trp His Ile Tyr
530 535 540
Leu Lys Ser Gly Thr Cys Pro Phe Ser Phe Ser Lys Leu Asn Asn Phe
545 550 555 560
Gln Lys Phe Lys Thr Ile Cys Phe Ser Thr Val Glu Val Pro Gly Ser
565 570 575
Cys Asn Phe Pro Leu Glu Ala Thr Trp His Tyr Thr Ser Tyr Thr Ile
580 585 590
Val Gly Ala Leu Tyr Val Thr Trp Ser Glu Gly Asn Ser Ile Thr Gly
595 600 605
Val Pro Tyr Pro Val Ser Gly Ile Arg Glu Phe Ser Asn Leu Val Leu
610 615 620
Asn Asn Cys Thr Lys Tyr Asn Ile Tyr Asp Tyr Val Gly Thr Gly Ile
625 630 635 640
Ile Arg Ser Ser Asn Gln Ser Leu Ala Gly Gly Ile Thr Tyr Val Ser
645 650 655
Asn Ser Gly Asn Leu Leu Gly Phe Lys Asn Val Ser Thr Gly Asn Ile
660 665 670
Phe Ile Val Thr Pro Cys Asn Gln Pro Asp Gln Val Ala Val Tyr Gln
675 680 685
Gln Ser Ile Ile Gly Ala Met Thr Ala Val Asn Glu Ser Arg Tyr Gly
690 695 700
Leu Gln Asn Leu Leu Gln Leu Pro Asn Phe Tyr Tyr Val Ser Asn Gly
705 710 715 720
Gly Asn Asn Cys Thr Thr Ala Val Met Thr Tyr Ser Asn Phe Gly Ile
725 730 735
Cys Ala Asp Gly Ser Leu Ile Pro Val Arg Pro Arg Asn Ser Ser Asp
740 745 750
Asn Gly Ile Ser Ala Ile Ile Thr Ala Asn Leu Ser Ile Pro Ser Asn
755 760 765
Trp Thr Thr Ser Val Gln Val Glu Tyr Leu Gln Ile Thr Ser Thr Pro
770 775 780
Ile Val Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Pro Arg Cys
785 790 795 800
Lys Asn Leu Leu Lys Gln Tyr Thr Ser Ala Cys Lys Thr Ile Glu Asp
805 810 815
Ala Leu Arg Leu Ser Ala His Leu Glu Thr Asn Asp Val Ser Ser Met
820 825 830
Leu Thr Phe Asp Ser Asn Ala Phe Ser Leu Ala Asn Val Thr Ser Phe
835 840 845
Gly Asp Tyr Asn Leu Ser Ser Val Leu Pro Gln Arg Asn Ile Arg Ser
850 855 860
Ser Arg Ile Ala Gly Arg Ser Ala Leu Glu Asp Leu Leu Phe Ser Lys
865 870 875 880
Val Val Thr Ser Gly Leu Gly Thr Val Asp Val Asp Tyr Lys Ser Cys
885 890 895
Thr Lys Gly Leu Ser Ile Ala Asp Leu Ala Cys Ala Gln Tyr Tyr Asn
900 905 910
Gly Ile Met Val Leu Pro Gly Val Ala Asp Ala Glu Arg Met Ala Met
915 920 925
Tyr Thr Gly Ser Leu Ile Gly Gly Met Val Leu Gly Gly Leu Thr Ser
930 935 940
Ala Ala Ala Ile Pro Phe Ser Leu Ala Leu Gln Ala Arg Leu Asn Tyr
945 950 955 960
Val Ala Leu Gln Thr Asp Val Leu Gln Glu Asn Gln Lys Ile Leu Ala
965 970 975
Ala Ser Phe Asn Lys Ala Ile Asn Asn Ile Val Ala Ser Phe Ser Ser
980 985 990
Val Asn Asp Ala Ile Thr Gln Thr Ala Glu Ala Ile His Thr Val Thr
995 1000 1005
Ile Ala Leu Asn Lys Ile Gln Asp Val Val Asn Gln Gln Gly Ser
1010 1015 1020
Ala Leu Asn His Leu Thr Ser Gln Leu Arg His Asn Phe Gln Ala
1025 1030 1035
Ile Ser Asn Ser Ile Gln Ala Ile Tyr Asp Arg Leu Asp Ser Ile
1040 1045 1050
Gln Ala Asp Gln Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ala
1055 1060 1065
Ala Leu Asn Ala Phe Val Ser Gln Val Leu Asn Lys Tyr Thr Glu
1070 1075 1080
Val Arg Gly Ser Arg Arg Leu Ala Gln Gln Lys Ile Asn Glu Cys
1085 1090 1095
Val Lys Ser Gln Ser Asn Arg Tyr Gly Phe Cys Gly Asn Gly Thr
1100 1105 1110
His Ile Phe Ser Ile Val Asn Ser Ala Pro Asp Gly Leu Leu Phe
1115 1120 1125
Leu His Thr Val Leu Leu Pro Thr Asp Tyr Lys Asn Val Lys Ala
1130 1135 1140
Trp Ser Gly Ile Cys Val Asp Gly Ile Tyr Gly Tyr Val Leu Arg
1145 1150 1155
Gln Pro Asn Leu Val Leu Tyr Ser Asp Asn Gly Val Phe Arg Val
1160 1165 1170
Thr Ser Arg Val Met Phe Gln Pro Arg Leu Pro Val Leu Ser Asp
1175 1180 1185
Phe Val Gln Ile Tyr Asn Cys Asn Val Thr Phe Val Asn Ile Ser
1190 1195 1200
Arg Val Glu Leu His Thr Val Ile Pro Asp Tyr Val Asp Val Asn
1205 1210 1215
Lys Thr Leu Gln Glu Phe Ala Gln Asn Leu Pro Lys Tyr Val Lys
1220 1225 1230
Pro Asn Phe Asp Leu Thr Pro Phe Asn Leu Thr Tyr Leu Asn Leu
1235 1240 1245
Ser Ser Glu Leu Lys Gln Leu Glu Ala Lys Thr Ala Ser Leu Phe
1250 1255 1260
Gln Thr Thr Val Glu Leu Gln Gly Leu Ile Asp Gln Ile Asn Ser
1265 1270 1275
Thr Tyr Val Asp Leu Lys Leu Leu Asn Arg Phe Glu Asn Tyr Ile
1280 1285 1290
Lys Trp Pro Trp Trp Val Trp Leu Ile Ile Ser Val Val Phe Val
1295 1300 1305
Val Leu Leu Ser Leu Leu Val Phe Cys Cys Leu Ser Thr Gly Cys
1310 1315 1320
Cys Gly Cys Cys Asn Cys Leu Thr Ser Ser Met Arg Gly Cys Cys
1325 1330 1335
Asp Cys Gly Ser Thr Lys Leu Pro Tyr Tyr Glu Phe Glu Lys Val
1340 1345 1350
His Val Gln
1355
<210> 4
<211> 1653
<212> DNA
<213> 人工序列
<220>
<223> fLuc表达的合成基因
<220>
<221> misc_feature
<222> (1)..(1653)
<400> 4
atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctct ggaggatggc 60
accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcc tggcaccatt 120
gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtactt cgagatgtct 180
gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccg catcgtggtg 240
tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgtt catcggagtg 300
gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacag catgggcatt 360
tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcct gaacgtgcag 420
aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccga ctaccagggc 480
ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaa cgagtacgac 540
ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaa cagctctggg 600
tctaccggcc tgcctaaggg cgtggccctg cctcatcgca ccgcctgtgt gcgcttctct 660
cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctat tctgagcgtg 720
gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgat ttgcggcttt 780
cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcct gcaagactac 840
aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaa gagcaccctg 900
atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgc cccactgtct 960
aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccg ccagggctac 1020
ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacga caagcctggc 1080
gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctgga caccggcaag 1140
accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgat tatgtccggc 1200
tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctg gctgcactct 1260
ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccg cctgaagtct 1320
ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctat cctgctgcag 1380
caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgc cggcgagctg 1440
cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaagga gatcgtggac 1500
tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggt gttcgtggac 1560
gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcga gatcctgatc 1620
aaggctaaga aaggcggcaa gatcgccgtg taa 1653
<210> 5
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> CD813_F1
<220>
<221> misc_feature
<222> (1)..(23)
<400> 5
tggatcttgg ttcattctca agc 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> CD813_R1
<220>
<221> misc_feature
<222> (1)..(23)
<400> 6
tagcgtaaaa ggagcaacat agt 23
<210> 7
<211> 72
<212> DNA
<213> 人工序列
<220>
<223> CD5信号序列
<220>
<221> misc_feature
<222> (1)..(72)
<400> 7
atgcctatgg gctctctgca gcctctggcc acactgtatc tgctgggaat gctggtggcc 60
agctgtctgg ga 72
<210> 8
<211> 3777
<212> DNA
<213> 人工序列
<220>
<223> SARS-CoV-2的S基因
<220>
<221> misc_feature
<222> (1)..(3777)
<400> 8
gtgaacctga ccaccagaac acagctgcct ccagcctaca ccaacagctt caccagaggc 60
gtgtactacc ccgacaaggt gttcagatcc agcgtgctgc actctaccca ggacctgttc 120
ctgcctttct tcagcaacgt gacctggttc cacgccatcc acgtgtccgg caccaatggc 180
accaagagat tcgacaaccc cgtgctgccc ttcaacgacg gggtgtactt tgccagcacc 240
gagaagtcca acatcatcag aggctggatc ttcggcacca cactggacag caagacccag 300
agcctgctga tcgtgaacaa cgccaccaac gtggtcatca aagtgtgcga gttccagttc 360
tgcaacgacc ccttcctggg cgtctactac cacaagaaca acaagagctg gatggaaagc 420
gagttccggg tgtacagcag cgccaacaac tgcaccttcg agtacgtgtc ccagcctttc 480
ctgatggacc tggaaggcaa gcagggcaac ttcaagaacc tgcgcgagtt cgtgttcaag 540
aacatcgacg gctacttcaa gatctacagc aagcacaccc ctatcaacct cgtgcgggat 600
ctgcctcagg gcttctctgc tctggaaccc ctggtggatc tgcccatcgg catcaacatc 660
acccggtttc agacactgct ggccctgcac agaagctacc tgacacctgg cgatagcagc 720
tctggatgga cagcaggcgc cgctgcctac tatgtgggat acctgcagcc tagaaccttc 780
ctgctgaagt acaacgagaa cggcaccatc accgacgccg tggattgtgc tctggatcct 840
ctgagcgaga caaagtgcac cctgaagtcc ttcaccgtgg aaaagggcat ctaccagacc 900
agcaacttcc gggtgcagcc caccgaatcc atcgtgcggt tccccaatat caccaatctg 960
tgccccttcg gcgaggtgtt caatgccacc agattcgcct ctgtgtacgc ctggaaccgg 1020
aagcggatca gcaattgcgt ggccgactac tccgtgctgt acaactccgc cagcttcagc 1080
accttcaagt gctacggcgt gtcccctacc aagctgaacg acctgtgctt cacaaacgtg 1140
tacgccgaca gcttcgtgat ccggggagat gaagtgcggc agattgcccc tggacagaca 1200
ggcaagatcg ccgactacaa ctacaagctg cccgacgact tcaccggctg tgtgattgcc 1260
tggaacagca acaacctgga ctccaaagtc ggcggcaact acaattacct gtaccggctg 1320
ttccggaagt ccaatctgaa gcccttcgag cgggacatct ccaccgagat ctatcaggcc 1380
ggcagcaccc cttgtaacgg cgtggaaggc ttcaactgct acttcccact gcagtcctac 1440
ggctttcagc ccacaaatgg cgtgggctac cagccttaca gagtggtggt gctgagcttc 1500
gagctgctgc atgctcctgc cacagtgtgc ggccctaaga aaagcaccaa tctcgtgaag 1560
aacaaatgcg tgaacttcaa cttcaacggc ctgaccggca ccggcgtgct gacagagagc 1620
aacaagaagt tcctgccatt ccagcagttc ggccgggata tcgccgatac cacagatgcc 1680
gtcagagatc cccagacact ggaaatcctg gacatcaccc cttgcagctt cggcggagtg 1740
tctgtgatca cccctggcac caacaccagc aatcaggtgg cagtgctgta ccaggacgtg 1800
aactgtaccg aagtgcccgt ggccattcac gccgatcagc tgacacctac atggcgggtg 1860
tactccaccg gcagcaatgt gtttcagacc agagccggct gtctgatcgg agccgagcac 1920
gtgaacaata gctacgagtg cgacatcccc atcggcgctg gcatctgcgc ctcttaccag 1980
acacagacaa acagccccag acgggccaga tctgtggcca gccagagcat cattgcctac 2040
acaatgtctc tgggcgccga gaacagcgtg gcctactcca acaactctat cgctatcccc 2100
accaacttca ccatcagcgt gaccacagag atcctgcctg tgtccatgac caagaccagc 2160
gtggactgca ccatgtacat ctgcggcgat tccaccgagt gctccaacct gctgctgcag 2220
tacggcagct tctgcaccca gctgaataga gccctgacag ggatcgccgt ggaacaggac 2280
aagaacaccc aagaggtgtt cgcccaagtg aagcagatct acaagacccc tcctatcaag 2340
gacttcggcg gcttcaattt cagccagatt ctgcccgatc ctagcaagcc cagcaagcgg 2400
agcttcatcg aggacctgct gttcaacaaa gtgacactgg ccgacgccgg cttcatcaag 2460
cagtatggcg attgtctggg cgacattgcc gccagggatc tgatttgcgc ccagaagttt 2520
aacggactga cagtgctgcc tcctctgctg accgatgaga tgatcgccca gtacacatct 2580
gccctgctgg ccggcacaat cacaagcggc tggacatttg gagctggcgc cgctctgcag 2640
atcccctttg ctatgcagat ggcctaccgg ttcaacggca tcggagtgac ccagaatgtg 2700
ctgtacgaga accagaagct gatcgccaac cagttcaaca gcgccatcgg caagatccag 2760
gacagcctga gcagcacagc aagcgccctg ggaaagctgc aggacgtggt caaccagaat 2820
gcccaggcac tgaacaccct ggtcaagcag ctgtccagca atttcggcgc catcagctct 2880
gtgctgaacg atatcctgag cagactggac aaggtggaag ccgaggtgca gatcgacaga 2940
ctgatcaccg gaaggctgca gtccctgcag acctacgtta cccagcagct gatcagagcc 3000
gccgagatta gagcctctgc caatctggcc gccaccaaga tgtctgagtg tgtgctgggc 3060
cagagcaaga gagtggactt ttgcggcaag ggctaccacc tgatgagctt ccctcagtct 3120
gcccctcacg gcgtggtgtt tctgcacgtg acatacgtgc ccgctcaaga gaagaatttc 3180
accaccgctc cagccatctg ccacgacggc aaagcccact ttcctagaga aggcgtgttc 3240
gtgtccaacg gcacccattg gttcgtgacc cagcggaact tctacgagcc ccagatcatc 3300
accaccgaca acaccttcgt gtctggcaac tgcgacgtcg tgatcggcat tgtgaacaat 3360
accgtgtacg accctctgca gcccgagctg gactccttca aagaggaact ggataagtac 3420
tttaagaacc acacaagccc cgacgtggac ctgggcgata tcagcggaat caatgccagc 3480
gtcgtgaaca tccagaaaga gatcgaccgg ctgaacgagg tggccaagaa tctgaacgag 3540
agcctgatcg acctgcaaga actggggaag tacgagcagt acatcaagtg gccctggtac 3600
atctggctgg gctttatcgc cggactgatt gccatcgtga tggtcacaat catgctgtgt 3660
tgcatgacca gctgctgtag ctgcctgaag ggctgttgta gctgtggctc ctgctgcaag 3720
ttcgacgagg acgattctga gcccgtgctg aagggcgtga aactgcacta cacctga 3777
<210> 9
<211> 3849
<212> DNA
<213> 人工序列
<220>
<223> 序列7和序列8的连接基因
<220>
<221> misc_feature
<222> (1)..(3849)
<400> 9
atgcctatgg gctctctgca gcctctggcc acactgtatc tgctgggaat gctggtggcc 60
agctgtctgg gagtgaacct gaccaccaga acacagctgc ctccagccta caccaacagc 120
ttcaccagag gcgtgtacta ccccgacaag gtgttcagat ccagcgtgct gcactctacc 180
caggacctgt tcctgccttt cttcagcaac gtgacctggt tccacgccat ccacgtgtcc 240
ggcaccaatg gcaccaagag attcgacaac cccgtgctgc ccttcaacga cggggtgtac 300
tttgccagca ccgagaagtc caacatcatc agaggctgga tcttcggcac cacactggac 360
agcaagaccc agagcctgct gatcgtgaac aacgccacca acgtggtcat caaagtgtgc 420
gagttccagt tctgcaacga ccccttcctg ggcgtctact accacaagaa caacaagagc 480
tggatggaaa gcgagttccg ggtgtacagc agcgccaaca actgcacctt cgagtacgtg 540
tcccagcctt tcctgatgga cctggaaggc aagcagggca acttcaagaa cctgcgcgag 600
ttcgtgttca agaacatcga cggctacttc aagatctaca gcaagcacac ccctatcaac 660
ctcgtgcggg atctgcctca gggcttctct gctctggaac ccctggtgga tctgcccatc 720
ggcatcaaca tcacccggtt tcagacactg ctggccctgc acagaagcta cctgacacct 780
ggcgatagca gctctggatg gacagcaggc gccgctgcct actatgtggg atacctgcag 840
cctagaacct tcctgctgaa gtacaacgag aacggcacca tcaccgacgc cgtggattgt 900
gctctggatc ctctgagcga gacaaagtgc accctgaagt ccttcaccgt ggaaaagggc 960
atctaccaga ccagcaactt ccgggtgcag cccaccgaat ccatcgtgcg gttccccaat 1020
atcaccaatc tgtgcccctt cggcgaggtg ttcaatgcca ccagattcgc ctctgtgtac 1080
gcctggaacc ggaagcggat cagcaattgc gtggccgact actccgtgct gtacaactcc 1140
gccagcttca gcaccttcaa gtgctacggc gtgtccccta ccaagctgaa cgacctgtgc 1200
ttcacaaacg tgtacgccga cagcttcgtg atccggggag atgaagtgcg gcagattgcc 1260
cctggacaga caggcaagat cgccgactac aactacaagc tgcccgacga cttcaccggc 1320
tgtgtgattg cctggaacag caacaacctg gactccaaag tcggcggcaa ctacaattac 1380
ctgtaccggc tgttccggaa gtccaatctg aagcccttcg agcgggacat ctccaccgag 1440
atctatcagg ccggcagcac cccttgtaac ggcgtggaag gcttcaactg ctacttccca 1500
ctgcagtcct acggctttca gcccacaaat ggcgtgggct accagcctta cagagtggtg 1560
gtgctgagct tcgagctgct gcatgctcct gccacagtgt gcggccctaa gaaaagcacc 1620
aatctcgtga agaacaaatg cgtgaacttc aacttcaacg gcctgaccgg caccggcgtg 1680
ctgacagaga gcaacaagaa gttcctgcca ttccagcagt tcggccggga tatcgccgat 1740
accacagatg ccgtcagaga tccccagaca ctggaaatcc tggacatcac cccttgcagc 1800
ttcggcggag tgtctgtgat cacccctggc accaacacca gcaatcaggt ggcagtgctg 1860
taccaggacg tgaactgtac cgaagtgccc gtggccattc acgccgatca gctgacacct 1920
acatggcggg tgtactccac cggcagcaat gtgtttcaga ccagagccgg ctgtctgatc 1980
ggagccgagc acgtgaacaa tagctacgag tgcgacatcc ccatcggcgc tggcatctgc 2040
gcctcttacc agacacagac aaacagcccc agacgggcca gatctgtggc cagccagagc 2100
atcattgcct acacaatgtc tctgggcgcc gagaacagcg tggcctactc caacaactct 2160
atcgctatcc ccaccaactt caccatcagc gtgaccacag agatcctgcc tgtgtccatg 2220
accaagacca gcgtggactg caccatgtac atctgcggcg attccaccga gtgctccaac 2280
ctgctgctgc agtacggcag cttctgcacc cagctgaata gagccctgac agggatcgcc 2340
gtggaacagg acaagaacac ccaagaggtg ttcgcccaag tgaagcagat ctacaagacc 2400
cctcctatca aggacttcgg cggcttcaat ttcagccaga ttctgcccga tcctagcaag 2460
cccagcaagc ggagcttcat cgaggacctg ctgttcaaca aagtgacact ggccgacgcc 2520
ggcttcatca agcagtatgg cgattgtctg ggcgacattg ccgccaggga tctgatttgc 2580
gcccagaagt ttaacggact gacagtgctg cctcctctgc tgaccgatga gatgatcgcc 2640
cagtacacat ctgccctgct ggccggcaca atcacaagcg gctggacatt tggagctggc 2700
gccgctctgc agatcccctt tgctatgcag atggcctacc ggttcaacgg catcggagtg 2760
acccagaatg tgctgtacga gaaccagaag ctgatcgcca accagttcaa cagcgccatc 2820
ggcaagatcc aggacagcct gagcagcaca gcaagcgccc tgggaaagct gcaggacgtg 2880
gtcaaccaga atgcccaggc actgaacacc ctggtcaagc agctgtccag caatttcggc 2940
gccatcagct ctgtgctgaa cgatatcctg agcagactgg acaaggtgga agccgaggtg 3000
cagatcgaca gactgatcac cggaaggctg cagtccctgc agacctacgt tacccagcag 3060
ctgatcagag ccgccgagat tagagcctct gccaatctgg ccgccaccaa gatgtctgag 3120
tgtgtgctgg gccagagcaa gagagtggac ttttgcggca agggctacca cctgatgagc 3180
ttccctcagt ctgcccctca cggcgtggtg tttctgcacg tgacatacgt gcccgctcaa 3240
gagaagaatt tcaccaccgc tccagccatc tgccacgacg gcaaagccca ctttcctaga 3300
gaaggcgtgt tcgtgtccaa cggcacccat tggttcgtga cccagcggaa cttctacgag 3360
ccccagatca tcaccaccga caacaccttc gtgtctggca actgcgacgt cgtgatcggc 3420
attgtgaaca ataccgtgta cgaccctctg cagcccgagc tggactcctt caaagaggaa 3480
ctggataagt actttaagaa ccacacaagc cccgacgtgg acctgggcga tatcagcgga 3540
atcaatgcca gcgtcgtgaa catccagaaa gagatcgacc ggctgaacga ggtggccaag 3600
aatctgaacg agagcctgat cgacctgcaa gaactgggga agtacgagca gtacatcaag 3660
tggccctggt acatctggct gggctttatc gccggactga ttgccatcgt gatggtcaca 3720
atcatgctgt gttgcatgac cagctgctgt agctgcctga agggctgttg tagctgtggc 3780
tcctgctgca agttcgacga ggacgattct gagcccgtgc tgaagggcgt gaaactgcac 3840
tacacctga 3849
<210> 10
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pcDNA3_F1
<220>
<221> misc_feature
<222> (1)..(20)
<400> 10
gatagcggtt tgactcacgg 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pcDNA3_R2
<220>
<221> misc_feature
<222> (1)..(20)
<400> 11
agaaggcaca gtcgaggctg 20
<210> 12
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> CMV_F4
<220>
<221> misc_feature
<222> (1)..(19)
<400> 12
caaatgggcg gtaggcgtg 19
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> SARS-CoV-2_Seq1
<220>
<221> misc_feature
<222> (1)..(20)
<400> 13
cagggcttct ctgctctgga 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> SARS-CoV-2_Seq2
<220>
<221> misc_feature
<222> (1)..(20)
<400> 14
ccactgcagt cctacggctt 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> SARS-CoV-2_Seq3
<220>
<221> misc_feature
<222> (1)..(20)
<400> 15
aatagagccc tgacagggat 20
<210> 16
<211> 2418
<212> DNA
<213> 人工序列
<220>
<223> ACE2表达的合成基因
<220>
<221> misc_feature
<222> (1)..(2418)
<400> 16
atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280
atcttcactg ggatcagaga tcggaagaag aaaaataaag caagaagtgg agaaaatcct 2340
tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400
gttcagacct ccttttag 2418
<210> 17
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Hs_ACE2_Seq1
<220>
<221> misc_feature
<222> (1)..(20)
<400> 17
gcaaagttga tgaatgccta 20
<210> 18
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> Hs_ACE2_RTF1
<220>
<221> misc_feature
<222> (1)..(23)
<400> 18
gtgcgagtgg ctaatttgaa acc 23
<210> 19
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> Hs_ACE2_RTR1
<220>
<221> misc_feature
<222> (1)..(23)
<400> 19
gaaagcatca ttgatacggc tcc 23
Claims (6)
1.一种病毒的细胞侵入抑制剂,其是包含选自α-葡糖基芸香苷、α-葡糖基橙皮苷以及α-葡糖基柚皮苷的至少一种成分(A)的病毒的细胞侵入抑制剂,其中,所述病毒具有与血管紧张素转换酶2(ACE2)结合的尖峰蛋白。
2.如权利要求1所述的病毒的细胞侵入抑制剂,其中,所述病毒是选自SARS-CoV、SARS-CoV-2以及HCoV-NL63的任一种。
3.如权利要求1所述的病毒的细胞侵入抑制剂,其中,所述病毒是SARS-CoV-2。
4.如权利要求1所述的病毒的细胞侵入抑制剂,其中,所述成分(A)包含选自α-单葡糖基芸香苷、α-单葡糖基橙皮苷以及α-单葡糖基柚皮苷的至少一种。
5.如权利要求1所述的病毒的细胞侵入抑制剂,其中,所述成分(A)的含量为50质量%以上。
6.如权利要求1所述的病毒的细胞侵入抑制剂,其中,所述成分(A)的含量为65质量%以上。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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JP2021-110770 | 2021-07-02 | ||
JP2021110770A JP2023007737A (ja) | 2021-07-02 | 2021-07-02 | ウイルスの細胞侵入阻害剤 |
PCT/JP2022/024859 WO2023276811A1 (ja) | 2021-07-02 | 2022-06-22 | ウイルスの細胞侵入阻害剤 |
Publications (1)
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CN117651557A true CN117651557A (zh) | 2024-03-05 |
Family
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CN202280045975.9A Pending CN117651557A (zh) | 2021-07-02 | 2022-06-22 | 病毒的细胞侵入抑制剂 |
Country Status (6)
Country | Link |
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US (1) | US20240285661A1 (zh) |
EP (1) | EP4364744A1 (zh) |
JP (1) | JP2023007737A (zh) |
KR (1) | KR20240031963A (zh) |
CN (1) | CN117651557A (zh) |
WO (1) | WO2023276811A1 (zh) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2926411B2 (ja) * | 1989-03-08 | 1999-07-28 | 株式会社林原生物化学研究所 | α―グリコシル ルチンの製造方法とその用途 |
JP3060232B2 (ja) | 1990-04-29 | 2000-07-10 | 株式会社林原生物化学研究所 | α―グリコシル ナリンジンとその製造方法並びに用途 |
JP3967563B2 (ja) | 2000-05-29 | 2007-08-29 | 株式会社林原生物化学研究所 | 3”−α−モノグルコシルナリンジン含有組成物、その製造方法およびその利用方法 |
JP5171614B2 (ja) | 2006-03-01 | 2013-03-27 | サッポロビール株式会社 | 抗ウイルス剤 |
-
2021
- 2021-07-02 JP JP2021110770A patent/JP2023007737A/ja active Pending
-
2022
- 2022-06-22 EP EP22832959.5A patent/EP4364744A1/en active Pending
- 2022-06-22 WO PCT/JP2022/024859 patent/WO2023276811A1/ja active Application Filing
- 2022-06-22 KR KR1020237044244A patent/KR20240031963A/ko unknown
- 2022-06-22 CN CN202280045975.9A patent/CN117651557A/zh active Pending
- 2022-06-22 US US18/573,549 patent/US20240285661A1/en active Pending
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US20240285661A1 (en) | 2024-08-29 |
KR20240031963A (ko) | 2024-03-08 |
WO2023276811A1 (ja) | 2023-01-05 |
JP2023007737A (ja) | 2023-01-19 |
EP4364744A1 (en) | 2024-05-08 |
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