CN114404584B - 一种新型冠状病毒mRNA疫苗及其制备方法和用途 - Google Patents

一种新型冠状病毒mRNA疫苗及其制备方法和用途 Download PDF

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CN114404584B
CN114404584B CN202210336875.3A CN202210336875A CN114404584B CN 114404584 B CN114404584 B CN 114404584B CN 202210336875 A CN202210336875 A CN 202210336875A CN 114404584 B CN114404584 B CN 114404584B
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王浩猛
李荩
严志红
原晋波
史建明
邓捷
隋秀文
刘健
邱东旭
朱涛
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Abstract

本发明提供了新型冠状病毒mRNA疫苗及其制备方法和用途。本发明的新型冠状病毒mRNA疫苗可以诱导强烈的新型冠状病毒特异性的体液和/或T细胞免疫应答，并可以诱导产生可广泛中和多种变异毒株的中和抗体。本发明提供的新型冠状病毒mRNA疫苗递送载体具有良好的稳定性、递送效率高、安全有效质量可控。

Description

一种新型冠状病毒mRNA疫苗及其制备方法和用途
技术领域
本发明涉及疫苗研制技术领域，具体涉及一种编码新型冠状病毒S蛋白的mRNA在预防新型冠状病毒感染方面的应用。
背景技术
新型冠状病毒肺炎（COVID-19）是新型冠状病毒（SARS-CoV-2）感染导致的肺炎。2020年2月11日世界卫生组织宣布，将新型冠状病毒感染的肺炎正式命名为“COVID-19”(Coronavirus Disease)；同日，国际病毒分类委员会将引起该疾病的病毒正式命名为“SARS-CoV-2”（Severe Acute Respiratory Syndrome Coronavirus 2）。2020年3月11日世界卫生组织宣布，根据评估COVID-19可被称为全球大流行病（pandemic），成为第一个由冠状病毒引起的全球大流行病。鉴于此，接种有效的预防性疫苗仍是防控新型冠状病毒感染的根本措施之一。
已商品化的新型冠状病毒mRNA疫苗的mRNA编码序列都是基于原型株的全长S蛋白，位置986和987处由脯氨酸替代。但是此设计的mRNA疫苗诱导产生的抗体对于一些流行变异株的中和活性有较大程度降低，如对Beta变异株的中和抗体滴度降低10倍以上，对Omicron变异株的中和抗体滴度降低20-50倍。
发明内容
本发明的mRNA疫苗的mRNA编码序列保留了位置986和987处的脯氨酸替代，增加了K417N、E484K或E484A、N501Y突变，这3个突变是Beta变异株在受体结合区（RBD）上的突变，并在其他的重要变异株中反复出现；并含有D614G突变，该突变在目前所有重要变异株中存在；此外还含有弗林酶切位点删除/突变，可以消除S蛋白引起细胞融合的不利，并增强S蛋白的免疫原性。本发明的mRNA疫苗可以诱导产生针对多种流行变异株的高水平的中和抗体。
本发明术语“中性磷脂”术语是指不带电荷的、非磷酸甘油酯的磷脂分子。
本发明术语“聚乙二醇(PEG)-脂质”是指包含脂质部分和聚乙二醇部分的分子。
本发明术语“脂质纳米颗粒”（Lipid nanoparticles，LNP）是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物，在施用后诱导免疫应答，其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。
本发明术语，“N/P”为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
本发明的第一方面，提供了一种mRNA，所述的mRNA编码SARS-CoV-2病毒的刺突（S）蛋白或其衍生蛋白，所述的刺突（S）蛋白的氨基酸序列与SEQ ID NO:1具有至少75%的同源性（例如，与SEQ ID NO:1具有至少80%、85%、90%、95%、99%的同源性），所述刺突（S）蛋白具有相对于SEQ ID NO:1的修饰，所述的修饰位点包括：第417位由天冬酰胺替代；第484位由赖氨酸或丙氨酸替代；第501位由酪氨酸替代；第614位由甘氨酸替代；第682-685位发生氨基酸部分或全部替换和/或缺失；和/或，第986和987位由脯氨酸替代中的一个、多个或全部。
优选的，所述刺突（S）蛋白的修饰还包括将SEQ ID NO:1的第1-13位替换为其他强信号肽，或在N端增加其他强信号肽；所述其他强信号肽包括组织型纤溶酶原激活剂(tPA)的信号肽和血清免疫球蛋白E(lgE)的信号肽。
优选的，所述刺突（S）蛋白的修饰还包括在N端或C端增加用于形成多聚体的片段，所述片段包括Fc片段或噬菌体T4纤维蛋白的三聚化基序，优选的，所述多聚体包括二聚体，或三聚体或更多聚体。
具体的，所述刺突（S）蛋白具有相对于SEQ ID NO:1如下的修饰：
（1）K986P、V987P；或者，
（2）K417N、E484K、N501Y、K986P、V987P；或者，
（3）K417N、E484K、N501Y、R682G、R683S、R685G、K986P、V987P；或者，
（4）K417N、E484K、N501Y、D614G、R682G、R683S、R685G、K986P、V987P；或者，
（5）K417N、E484K、N501Y、D614G、∆R682(删除第682位)、∆R683(删除第683位)、∆R685(删除第685位)、K986P、V987P；或者，
（6）A67V、∆H69(删除第69位)、∆V70(删除第70位)、T95I、G142D、∆V143(删除第143位)、∆Y144(删除第144位)、∆Y145(删除第145位)、N211I、∆L212(删除第212位)、+214EPE（第214位增加EPE三个氨基酸）、G339D、S371L、S373P、S375F、K417N、N440K、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、N954K、N969K、L981F、R682G、R683S、R685G、K986P、V987P。
优选的，所述mRNA序列是天然或修饰的RNA，所述修饰的RNA包括通过用修饰的尿苷部分或全部取代天然尿苷对RNA进行修饰。
更优选的，所述mRNA序列是修饰的RNA，所述修饰的RNA为通过用1-甲基-假尿苷对天然尿苷进行全部替换。
在本发明的具体实施方式中，所述的mRNA的序列选自SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13，或者，所述的mRNA的序列为与上述任意序列至少75%的同源性的序列（例如，与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的同源性），并且所述的mRNA能够编码SARS-CoV-2病毒的刺突（S）蛋白或其衍生蛋白。
本发明的第二方面，提供一种mRNA疫苗，所述mRNA疫苗包含：
1）至少一个信使核糖核酸mRNA，所述mRNA编码至少一种抗原肽或结构蛋白，所述抗原肽或结构蛋白包括SARS-CoV-2冠状病毒的刺突（S）蛋白或衍生蛋白，其中刺突（S）蛋白的序列与SEQ ID NO:1具有至少75%的同源性（例如，与SEQ ID NO:1具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的同源性）；
所述刺突（S）蛋白具有相对于SEQ ID NO:1的修饰，所述的修饰包括：第417位由天冬酰胺替代；第484位由赖氨酸或丙氨酸替代；第501位由酪氨酸替代；第614位由甘氨酸替代；第682-685位发生氨基酸部分或全部替换和/或缺失；和/或，第986和987位由脯氨酸替代中的一个、多个或全部。
优选的，所述mRNA疫苗包括2）脂质纳米颗粒（LNP）；所述mRNA包封于所述脂质纳米颗粒（LNP）中。
优选的，所述抗原肽或结构蛋白还包括包膜蛋白（E），膜蛋白（M）或核衣壳蛋白（N），或其免疫原性片段或免疫原性变体。
优选的，所述mRNA编码的刺突（S）蛋白的修饰包括信号肽变化，例如，可将SEQ IDNO:1的第1-13位替换为其他强信号肽，或在N端增加其他强信号肽，所述其他强信号肽包括组织型纤溶酶原激活剂(tPA)的信号肽和血清免疫球蛋白E(lgE)的信号肽。
优选的，所述mRNA编码的抗原肽或结构蛋白可以是S蛋白全长，或者S蛋白胞外区。
优选的，所述mRNA编码的抗原肽或结构蛋白可以在N端或C端增加用于形成多聚体（例如二聚体，或三聚体，或更多聚体）的片段，所述片段包括Fc片段和噬菌体T4纤维蛋白的三聚化基序。
优选的，所述mRNA可以是天然或修饰的RNA；优选的，可以通过用修饰的尿苷部分或全部取代天然尿苷对RNA进行修饰；更优选的，可以通过用1-甲基-假尿苷对天然尿苷进行全部替换。
优选的，所述的mRNA由若干个结构元件组成，即所述的mRNA除包含上述编码区外还包括5’帽子结构，5’非编码区，3’非编码区和/或多聚腺苷酸尾的mRNA序列。
优选的，所述的mRNA序列的长度为200-10000个核苷酸。进一步优选的，所述的mRNA序列的长度为500-8000个核苷酸。
在本发明的具体实施方式中，所述的mRNA的序列选自SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13，或者，所述的mRNA的序列为与上述任意序列至少75%的同源性的序列（例如，与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的同源性），并且所述的mRNA能够编码SARS-CoV-2病毒的刺突（S）蛋白或其衍生蛋白。
优选的，所述脂质纳米颗粒包含阳离子脂质、中性磷脂、甾族脂质和聚乙二醇(PEG)-脂质。
优选的，所述的阳离子脂质具有以下结构：
Figure 115327DEST_PATH_IMAGE001
或其药物可接受的盐或立体异构体，其中：
G1和G2各自独立地为未取代的C6-C10亚烷基；
G3为未取代的C1-C12亚烷基；
R1和R2各自独立地为C6-C24烷基或C6-C24烯基；
R3为OR5、CN、-C(＝O)OR4、-OC(＝O)R4或–NR5C(＝O)R4；
R4为C1-C12烃基；并且
R5为H或C1-C6烃基。
优选的，阳离子脂质化合物结构：
DLin-MC3-DMA：
Figure 169870DEST_PATH_IMAGE002
ALC-0315：
Figure 130873DEST_PATH_IMAGE003
SM-102：
Figure 903657DEST_PATH_IMAGE004
优选的，所述的聚乙二醇(PEG)-脂质选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA) 或DMG-PEG2000中的一种或多种组合，优选的为DMG-PEG2000。
优选的，所述的中性磷脂选自：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺（DOPE）、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺 (DPPE)、 1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油) (DOPG)、油酰磷脂酰胆碱（POPC）、1-棕榈酰基-2-油酰基磷脂酰乙醇胺（POPE）中的一种或多种组合，优选的为DSPC。
优选的，所述的甾族脂质选自燕麦甾醇、 β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。
优选的，所述的脂质纳米颗粒，其特征在于，所述的阳离子脂质在脂质组分中的摩尔百分含量为20~60%、中性磷脂在脂质组分中的摩尔百分含量为5%~25%、甾族脂质在脂质组分中的摩尔百分含量为25%~55%；聚乙二醇(PEG)-脂质在脂质组分中的摩尔百分含量为0.1%~15%。
优选的，所述的脂质纳米颗粒，其特征在于，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质摩尔比为30-60:5-20:20-50:0.1-10，优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为 40-60:10-20:30-50:1-5，更优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质摩尔比为45:10:43:2或40:10:48:2。
优选的，所述脂质纳米颗粒（LNP）中总脂质（即阳离子脂质、中性磷脂、甾族脂质和聚乙二醇(PEG)-脂质）与mRNA的质量比（w/w）在10-30：1之间。
优选的，所述的mRNA疫苗，所述疫苗中还包含其他辅料，所述其他辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。
优选的，所述的mRNA疫苗，其特征在于，所述脂质纳米颗粒的平均粒径为50~200nm或所述脂质纳米颗粒在中性pH下具有净中性电荷或所述脂质纳米颗粒具有小于0.4的多分散性。
优选的，所述mRNA疫苗为液体制剂或冻干粉剂。更优选的，所述mRNA疫苗为口服制剂、肌肉注射制剂、静脉注射制剂或吸入制剂。进一步优选的，所述mRNA疫苗为雾化吸入剂或干粉吸入剂。
本发明所述的mRNA疫苗还可以包含药学上可接受的赋形剂。所述药学上可接受的赋形剂可以是载体、稀释剂、佐剂或编码佐剂核苷酸序列、增溶剂、粘合剂、润滑剂、助悬剂、转染促进剂等。所述转染促进剂包括但不限于表面活性剂如免疫刺激复合物、费氏(Freunds)不完全佐剂、LPS类似物(例如单磷酰酯A)、胞壁肽、苯醌类似物、角鲨烯、透明质酸、脂质、脂质、钙离子、病毒蛋白质、阳离子、聚阳离子(例如聚-L-谷氨酸(LGS))或纳米粒子或其他已知的转染促进剂。所述的编码佐剂的核苷酸序列为编码如下至少一种佐剂的核苷酸序列：GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF- γ、Lymphotoxin-α、hGH、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、 E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、 ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、、CD40、CD40L、血管生长因子、成纤维细胞生长因子、神经生长因子、血管内皮生长因子、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、 DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、半胱天冬酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、 p38、p65Rel、MyD88、IRAK、TRAF6、IkB、无活性的NIK、SAP K、SAP-1、JNK、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、 NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2以及其功能性片段。
本发明的第三方面，提供一种上述mRNA疫苗的制备方法，所述制备方法包括将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质溶解至溶剂后与核酸混合后制得。
优选的，所述mRNA疫苗的制备方法，其特征在于，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得；优选的，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得；优选的，所述的超滤方式为切向流过滤；更优选的，所述的混合方式可为湍流混合、层流混合或微流体混合。
更优选的，所述稀释液可为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。进一步优选的，所述缓冲液pH为3~6，浓度为6.25~200mM。
优选的，所述的mRNA疫苗的制备方法中，脂质纳米颗粒包封mRNA时的N/P为2-10，优选的N/P为3-9。
优选的，mRNA疫苗的剂型为口服制剂、肌肉注射制剂、静脉注射制剂、吸入制剂、液体制剂、冻干粉剂、雾化吸入剂或干粉吸入剂。
本发明所述的mRNA疫苗还可以包含药学上可接受的赋形剂。所述药学上可接受的赋形剂可以是载体、稀释剂、佐剂或编码佐剂核苷酸序列、增溶剂、粘合剂、润滑剂、助悬剂、转染促进剂等。 所述转染促进剂包括但不限于表面活性剂如免疫刺激复合物、费氏(Freunds)不完全佐剂、LPS类似物(例 如单磷酰酯A)、胞壁肽、苯醌类似物、角鲨烯、透明质酸、脂质、脂质、钙离子、病毒蛋白质、阳离子、聚阳离子(例如聚-L-谷氨酸(LGS))或纳米粒子或其他已知的转染促进剂。所述的编码佐剂的核苷酸 序列为编码如下至少一种佐剂的核苷酸序列：GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁 蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF- γ、Lymphotoxin-α、hGH、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、 E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、 ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、、CD40、CD40L、血管生长因子、成纤维细胞生长因子、 神经生长因子、血管内皮生长因子、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、 DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、半胱天冬酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、 p38、p65Rel、MyD88、IRAK、TRAF6、IkB、无活性的NIK、SAP K、SAP-1、JNK、NFkB、Bax、TRAIL、 TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、 NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2以及其功能性片段。
本发明的第四方面，提供一种上述mRNA或者mRNA疫苗在制备用于预防或治疗病毒感染的疫苗中的应用。
具体地，其中的病毒感染为新型冠状病毒。
本发明相比现有技术的有益效果为：
采用本发明所述的mRNA疫苗制剂稳定性良好。免疫可以诱导产生针对多种流行变异株的高水平的中和抗体，尤其是对现有mRNA疫苗已知有较强逃逸效果的变异株有较好中和效果。
mRNA 新冠疫苗既可诱导人体产生体液免疫，又可引发人体产生细胞免疫，它具有双重免疫作用。
在体内表达mRNA可避免来自病毒和蛋白质等外源因子的污染。且通过修饰mRNA序列和递送系统，可以有效调控mRNA的表达性和体内半衰期，达到高效免疫原性的同时具有长效持续性。
附图说明
图1：不同mRNA序列及不同递送阳离子脂质单免抗体滴度。
图2： HEK293细胞Western-Blot法检测mRNA的体外表达情况，其中标记“1”代表的细胞裂解物样品，标记“阴”代表阴性对照。
图3 ：BALB/c小鼠模型上ELISA法检测S蛋白特异性IgG滴度。
图4 ：BALB/c小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD4+T细胞频数；
图5 ：BALB/c小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD8+T细胞频数。
图6 ：恒河猴模型上ELISA法检测S蛋白特异性IgG滴度。
图7 ：hACE2转基因小鼠模型上qPCR法检测原型株和Beta变异株真病毒攻毒后鼻甲的病毒载量。
图8 ：hACE2转基因小鼠模型上qPCR法检测原型株和Beta变异株真病毒攻毒后肺部的病毒载量。
图9 ：BALB/c小鼠模型上FRNT法检测原型株、Beta变异株、Delta变异株和Omicron变异株真病毒中和抗体滴度。
具体实施方式
下面将结合本发明的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：mRNA序列及递送阳离子脂质匹配筛选
根据下表所示的蛋白序列设计质粒DNA序列。
表1 ：蛋白序列信息汇总
Figure 709939DEST_PATH_IMAGE005
注：∆代表删除突变
人工合成质粒DNA序列，DNA序列含RNA转录相关的元件。质粒转化如大肠杆菌中进行扩增。发酵纯化后的质粒用限制性核酸内切酶BspQ1线性化。用T7体外转录试剂盒进行转录，获得的未加帽的mRNA。分别用DNaseI消化转录模板，并用沉淀法纯化mRNA。用Cap1加帽试剂盒给mRNA加帽，并分别用mRNA纯化试剂盒对加帽后的mRNA进行纯化。将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中，待用。每条mRNA序列分别用含阳离子脂质ALC-0315、SM-102或DLin-MC3-DMA的配方包封；将阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质按摩尔比为45:10:43:2在乙醇中溶解、混合。设定纳米药物制造设备总流速12ml/min。将表1中的mRNA溶液与脂质混合溶液分别按流速比3：1包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶液，得到mRNA疫苗（mRNA-LNP）。BALB/c雌性小鼠按5只组进行随机分组，按5µg/只单免mRNA-LNP，在第14天采血检测S蛋白特异性抗体滴度，检测结果如表2、图1所示，不同mRNA和LNP的组合均可产生较高的抗体滴度，其中RBD/614/GSAG/2P和ALC-0315的组合、RBD/614/A/2P和ALC-0315的组合，以及Omicron/GSAG/2P和ALC-0315的组合的平均抗体滴度明显高于其他种类脂质组合。
表2 ：不同mRNA序列及不同递送阳离子脂质单免抗体平均滴度
Figure 884568DEST_PATH_IMAGE006
实施例2：不同总脂质与mRNA的质量比单免抗体滴度实验
将ALC-0315：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质按摩尔比为45:10:43:2在乙醇中溶解、混合。设定纳米药物制造设备总流速12ml/min。总脂质与编码RBD/614/GSAG/2P，RBD/614/A/2P，Omicron/GSAG/2P的mRNA溶液分别按照总脂质/mRNA(w/w)7:1、10:1、20:1、30:1包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶液，得到mRNA疫苗（mRNA-LNP）。BALB/c雌性小鼠按5只每组进行随机分组，按5ug/只单免mRNA-LNP，在第14天采血检测S蛋白特异性抗体滴度，检测结果如下表所示，当总脂质与mRNA的质量比在10-30：1之间时，单免抗体滴度大于10的5.5次方。
表3：总脂质与mRNA的质量比单免抗体滴度汇总
Figure 750893DEST_PATH_IMAGE007
实施例3：mRNA的制备与检测
包含SEQ ID NO:11的质粒用限制性核酸内切酶BspQ1线性化。用T7体外转录试剂盒进行转录，获得的未加帽的mRNA。分别用DNaseI消化转录模板，并用沉淀法纯化mRNA。用Cap1加帽试剂盒给mRNA加帽，并分别用mRNA纯化试剂盒对加帽后的mRNA进行纯化。将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中，待用。将mRNA用转染试剂转染进入HEK293细胞，24h后收集细胞，用细胞裂解物做Western-Blot检测，可用S蛋白特异性抗体检测到S蛋白在细胞中的表达，如图2所示。
其他序列SEQ ID NO:8、9、10、12、13的mRNA参照上述步骤进行制备，并可检测到S蛋白在细胞中的表达。
实施例4：mRNA疫苗的制备与检测
将ALC-0315：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质按摩尔比为45:10:43:2在乙醇中溶解、混合。设定纳米药物制造设备总流速12ml/min。将实施例1中的RBD/614/A/2P-mRNA溶液与脂质混合溶液按流速比3：1包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶液，得到mRNA疫苗（mRNA-LNP）。取样测得包封率为91.49%，平均粒径为81.99nm，PDI为0.07,Zeta电位为-8.20mV。
实施例5：mRNA疫苗的小鼠免疫原性检测
BALB/c雌性小鼠按10只每组进行随机分组，在第0天和28天按不同免疫剂量（高剂量组10µg/只、低剂量组5µg/只）免疫实施例1 RBD/614/A/2P-mRNA-LNP。在第28天（二免前）和第42天采血；第42天，处死小鼠、收获脾细胞并使用S蛋白的重叠肽库进行刺激检测细胞免疫应答。
一免和二免的抗体滴度如图3所示，一免即可以引起较高的抗体应答，但二免可以使抗体滴度增加约一个数量级；5µg即可在小鼠模型上引起足够高的抗体滴度，增加剂量到10µg并不能提高抗体应答水平。
通过细胞内细胞因子染色流式细胞术（ICS）方法检测受到肽库刺激后分泌IL-2、IFNγ、TNFα、IL-4和IL-5的CD4+T细胞和CD8+T细胞的频数。特异性分泌上述细胞因子的CD4+T细胞频数如图4所示，分泌IL-2、IFNγ和TNFα的CD4+T细胞频数相比阴性组有明显升高。特异性分泌上述细胞因子的CD8+T细胞频数如图5所示，分泌IL-2、IFNγ和TNFα的CD8+T细胞频数相比阴性组有明显升高，分泌IFNγ和TNFα的CD8+T细胞占比达到了5%-10%，展现出很高的细胞应答水平。
实施例6：mRNA疫苗的恒河猴免疫原性检测
恒河猴按6只每组、雌雄各半进行随机分组，在第0天和21天按不同免疫剂量（高剂量组150ug/只、低剂量组50ug/只）免疫实施例1 RBD/614/A/2P-mRNA-LNP。在第21天（二免前）和第28天采血。
一免和二免的抗体滴度如图6所示，一免即可以引起较高的抗体应答，但二免可以使抗体滴度增加约一个数量级；50ug即可在恒河猴模型上引起足够高的抗体滴度，增加剂量到150ug并不能提高抗体应答水平。
实施例7：mRNA疫苗的小鼠保护性研究
hACE2转基因雌性C57BL/6小鼠按16只每组进行随机分组，在第0天和21天按不同免疫剂量（高剂量组10ug/只、低剂量组5ug/只）免疫实施例1 RBD/614/A/2P-mRNA-LNP。二免后第21天，每组小鼠再按8只每组随机分为2组：一组滴鼻感染5×105PFU的原型株的新型冠状病毒（2019-nCoV-WIV04）；一组滴鼻感染5×105PFU的Beta变异株的新型冠状病毒（NPRC2.062100001）。攻毒后第5天，解剖小鼠，分别取小鼠鼻甲和肺，通过RT-qPCR测定病毒载量。小鼠鼻甲的病毒载量如图7所示，mRNA疫苗可以完全或部分阻止病毒在鼻甲的复制。小鼠肺部的病毒载量如图8所示，mRNA疫苗可以完全或部分阻止病毒在肺部的复制。可见本发明的mRNA疫苗对原型株和Beta变异株都有很好的保护效果。
实施例8：mRNA疫苗的交叉保护性研究
BALB/c雌性小鼠按8只每组进行随机分组，在第0天和14天按5ug/只的免疫剂量免疫实施例1 RBD/614/A/2P-mRNA-LNP。在第28天采血，采用斑点减少中和试验（FRNT）评价血清中的中和抗体水平。检测用真病毒包括新型冠状病毒原型株、Beta变异株、Delta变异株及Omicron变异株。中和抗体检测结果表明（图9），二免后14天的小鼠血清具有较好的交叉中和效果，对原型株、Beta变异株、Delta变异株和Omicron变异株的几何平均中和抗体滴度均高于103。本发明的mRNA疫苗两针免疫后血清对Omicron变异株的几何平均中和抗体滴度相比于原型株降低了3.3倍，相对于商品化疫苗对Omicron变异株的交叉中和活性大幅增强。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
序列表
<110> 康希诺生物股份公司 康希诺（上海）生物科技有限公司
<120> 一种新型冠状病毒mRNA疫苗及其制备方法和用途
<130> P0102022030287Y
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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> 2
<211> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Pro Pro 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> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
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
Asn 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 Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr 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 Pro Pro 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> 4
<211> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
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
Asn 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 Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr 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 Gly 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 Pro Pro 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> 5
<211> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
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
Asn 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 Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr 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 Gly 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 Gly 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 Pro Pro 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> 6
<211> 1270
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
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
Asn 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 Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr 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 Gly 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 Ala Ser Val Ala Ser Gln Ser
675 680 685
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr
690 695 700
Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr
705 710 715 720
Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr
725 730 735
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln
740 745 750
Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala
755 760 765
Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln
770 775 780
Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser
785 790 795 800
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu
805 810 815
Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
820 825 830
Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys
835 840 845
Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
850 855 860
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr
865 870 875 880
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
885 890 895
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val
900 905 910
Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile
915 920 925
Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys
930 935 940
Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val
945 950 955 960
Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp
965 970 975
Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg
980 985 990
Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln
995 1000 1005
Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr
1010 1015 1020
Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys
1025 1030 1035 1040
Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly
1045 1050 1055
Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe
1060 1065 1070
Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg
1075 1080 1085
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1090 1095 1100
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser
1105 1110 1115 1120
Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp
1125 1130 1135
Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1140 1145 1150
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1155 1160 1165
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1170 1175 1180
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1185 1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1205 1210 1215
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
1220 1225 1230
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
1235 1240 1245
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
1250 1255 1260
Val Lys Leu His Tyr Thr
1265 1270
<210> 7
<211> 1270
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
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 Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn
130 135 140
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
145 150 155 160
Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly
165 170 175
Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
180 185 190
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Ile Val Arg
195 200 205
Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
355 360 365
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
465 470 475 480
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
485 490 495
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Lys
530 535 540
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln
545 550 555 560
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro
565 570 575
Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val
580 585 590
Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu
595 600 605
Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp
610 615 620
Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe
625 630 635 640
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu Tyr Val Asn Asn Ser
645 650 655
Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln
660 665 670
Thr Gln Thr Lys Ser His Gly Ser Ala Gly Ser Val Ala Ser Gln Ser
675 680 685
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr
690 695 700
Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr
705 710 715 720
Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr
725 730 735
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln
740 745 750
Tyr Gly Ser Phe Cys Thr Gln Leu Lys Arg Ala Leu Thr Gly Ile Ala
755 760 765
Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln
770 775 780
Ile Tyr Lys Thr Pro Pro Ile Lys Tyr Phe Gly Gly Phe Asn Phe Ser
785 790 795 800
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu
805 810 815
Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
820 825 830
Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys
835 840 845
Ala Gln Lys Phe Lys Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
850 855 860
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr
865 870 875 880
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
885 890 895
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val
900 905 910
Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile
915 920 925
Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys
930 935 940
Leu Gln Asp Val Val Asn His Asn Ala Gln Ala Leu Asn Thr Leu Val
945 950 955 960
Lys Gln Leu Ser Ser Lys Phe Gly Ala Ile Ser Ser Val Leu Asn Asp
965 970 975
Ile Phe Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg
980 985 990
Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln
995 1000 1005
Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr
1010 1015 1020
Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys
1025 1030 1035 1040
Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly
1045 1050 1055
Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe
1060 1065 1070
Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg
1075 1080 1085
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1090 1095 1100
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser
1105 1110 1115 1120
Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp
1125 1130 1135
Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1140 1145 1150
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1155 1160 1165
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1170 1175 1180
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1185 1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1205 1210 1215
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
1220 1225 1230
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
1235 1240 1245
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
1250 1255 1260
Val Lys Leu His Tyr Thr
1265 1270
<210> 8
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc agtgcgtgaa cctgaccaca 60
aggacccagc tgccacctgc ctataccaac agcttcacaa gaggcgtgta ctatccggac 120
aaggtgtttc gctccagcgt gctgcactct acacaggacc tgtttctgcc attcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcacaaa gagattcgac 240
aatccagtgc tgcccttcaa cgatggcgtg tacttcgcga gcaccgagaa gtccaacatc 300
atccgcggct ggatctttgg caccacactg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaacaacaag tcctggatgg agagcgagtt tcgcgtgtat 480
tcctctgcca acaattgcac attcgagtac gtgtcccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaatat cgatggctac 600
ttcaagatct actccaagca cacccctatc aacctggtga gggacctgcc acagggcttc 660
tctgccctgg agcctctggt ggatctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggctc tgcaccgcag ctacctgaca cccggcgaca gctcctctgg atggaccgct 780
ggagctgctg cttactatgt gggctatctg cagcctcgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgctgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttccgggtg 960
cagccaaccg agtctatcgt gaggtttcca aatatcacaa acctgtgccc ctttggcgag 1020
gtgttcaacg ctaccaggtt cgccagcgtg tacgcttgga acagaaagcg catctccaac 1080
tgcgtggcgg actacagcgt gctgtacaac agcgcctcct tctctacctt caagtgctat 1140
ggcgtgagcc ctacaaagct gaacgacctg tgctttacca acgtgtacgc cgattccttc 1200
gtgatcagag gcgacgaggt gcgccagatc gctccaggac agacaggcaa gatcgccgac 1260
tacaattaca agctgcccga cgatttcacc ggctgcgtga tcgcctggaa ctccaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgttcag gaagtccaat 1380
ctgaagccct tcgagcggga catctctaca gagatctacc aggccggcag caccccttgc 1440
aatggcgtgg agggcttcaa ctgttatttc cccctgcagt cttacggctt ccagcctaca 1500
aacggcgtgg gctatcagcc atacagggtg gtggtgctgt ctttcgagct gctgcacgct 1560
ccagctacag tgtgcggacc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggactgac cggaacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggccg ggacatcgcc gataccacag acgctgtgag ggacccccag 1740
accctggaga tcctggatat cacaccttgc agcttcggcg gcgtgtccgt gatcacacca 1800
ggaaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860
ccagtggcta tccacgccga tcagctgacc cccacatgga gagtgtactc taccggcagc 1920
aacgtgtttc agacaagggc tggatgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcttcct accagaccca gacaaactct 2040
ccacggcggg cccgcagcgt ggccagccag tccatcatcg cttataccat gtccctgggc 2100
gccgagaatt ctgtggctta ctccaacaat tctatcgcca tccccaccaa cttcacaatc 2160
agcgtgacca cagagatcct gcccgtgagc atgaccaaga caagcgtgga ctgcacaatg 2220
tatatctgtg gcgacagcac cgagtgctcc aacctgctgc tgcagtacgg ctccttttgt 2280
acccagctga atcgcgccct gacaggcatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggcttc 2400
aatttttccc agatcctgcc cgatcccagc aagccttcca agagatcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca tcgctgccag ggacctgatc tgtgctcaga agttcaatgg cctgaccgtg 2580
ctgcctccac tgctgacaga cgagatgatc gctcagtaca caagcgccct gctggctgga 2640
accatcacat ccggctggac cttcggcgct ggagctgccc tgcagatccc atttgccatg 2700
cagatggctt acagattcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt caactccgct atcggcaaga tccaggacag cctgagcagc 2820
acagcttccg ccctgggcaa gctgcaggat gtggtgaatc agaacgctca ggccctgaat 2880
accctggtga agcagctgtc gagcaacttc ggcgctatct cctctgtgct gaacgatatc 2940
ctgagccggc tggaccctcc agaggcggag gtgcagatcg accggctgat cacaggcagg 3000
ctgcagtccc tgcagaccta tgtgacacag cagctgatca gagctgccga gatccgcgct 3060
tctgccaatc tggctgccac caagatgtcc gagtgcgtgc tgggccagtc gaagcgcgtg 3120
gacttttgtg gcaagggcta tcacctgatg tctttcccac agagcgcccc acacggagtg 3180
gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac agctccagcc 3240
atctgccacg atggcaaggc ccactttcca cgggagggcg tgttcgtgag caacggcacc 3300
cactggtttg tgacacagag gaatttctac gagccccaga tcatcaccac agacaatacc 3360
ttcgtgagcg gcaactgcga cgtggtcatc ggcatcgtga acaataccgt gtacgatcct 3420
ctgcagccag agctggactc tttcaaggag gagctggaca agtacttcaa gaatcacacc 3480
agccccgacg tggatctggg cgacatctcc ggcatcaatg cttctgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaatctga acgagagcct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822
<210> 9
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc agtgcgtgaa cctgaccaca 60
aggacccagc tgccacctgc ctataccaac agcttcacaa gaggcgtgta ctatccggac 120
aaggtgtttc gctccagcgt gctgcactct acacaggacc tgtttctgcc attcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcacaaa gagattcgac 240
aatccagtgc tgcccttcaa cgatggcgtg tacttcgcga gcaccgagaa gtccaacatc 300
atccgcggct ggatctttgg caccacactg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaacaacaag tcctggatgg agagcgagtt tcgcgtgtat 480
tcctctgcca acaattgcac attcgagtac gtgtcccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaatat cgatggctac 600
ttcaagatct actccaagca cacccctatc aacctggtga gggacctgcc acagggcttc 660
tctgccctgg agcctctggt ggatctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggctc tgcaccgcag ctacctgaca cccggcgaca gctcctctgg atggaccgct 780
ggagctgctg cttactatgt gggctatctg cagcctcgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgctgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttccgggtg 960
cagccaaccg agtctatcgt gaggtttcca aatatcacaa acctgtgccc ctttggcgag 1020
gtgttcaacg ctaccaggtt cgccagcgtg tacgcttgga acagaaagcg catctccaac 1080
tgcgtggcgg actacagcgt gctgtacaac agcgcctcct tctctacctt caagtgctat 1140
ggcgtgagcc ctacaaagct gaacgacctg tgctttacca acgtgtacgc cgattccttc 1200
gtgatcagag gcgacgaggt gcgccagatc gctccaggac agacaggcaa catcgccgac 1260
tacaattaca agctgcccga cgatttcacc ggctgcgtga tcgcctggaa ctccaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgttcag gaagtccaat 1380
ctgaagccct tcgagcggga catctctaca gagatctacc aggccggcag caccccttgc 1440
aatggcgtga agggcttcaa ctgttatttc cccctgcagt cttacggctt ccagcctaca 1500
tatggcgtgg gctatcagcc atacagggtg gtggtgctgt ctttcgagct gctgcacgct 1560
ccagctacag tgtgcggacc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggactgac cggaacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggccg ggacatcgcc gataccacag acgctgtgag ggacccccag 1740
accctggaga tcctggatat cacaccttgc agcttcggcg gcgtgtccgt gatcacacca 1800
ggaaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860
ccagtggcta tccacgccga tcagctgacc cccacatgga gagtgtactc taccggcagc 1920
aacgtgtttc agacaagggc tggatgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcttcct accagaccca gacaaactct 2040
ccacggcggg cccgcagcgt ggccagccag tccatcatcg cttataccat gtccctgggc 2100
gccgagaatt ctgtggctta ctccaacaat tctatcgcca tccccaccaa cttcacaatc 2160
agcgtgacca cagagatcct gcccgtgagc atgaccaaga caagcgtgga ctgcacaatg 2220
tatatctgtg gcgacagcac cgagtgctcc aacctgctgc tgcagtacgg ctccttttgt 2280
acccagctga atcgcgccct gacaggcatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggcttc 2400
aatttttccc agatcctgcc cgatcccagc aagccttcca agagatcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca tcgctgccag ggacctgatc tgtgctcaga agttcaatgg cctgaccgtg 2580
ctgcctccac tgctgacaga cgagatgatc gctcagtaca caagcgccct gctggctgga 2640
accatcacat ccggctggac cttcggcgct ggagctgccc tgcagatccc atttgccatg 2700
cagatggctt acagattcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt caactccgct atcggcaaga tccaggacag cctgagcagc 2820
acagcttccg ccctgggcaa gctgcaggat gtggtgaatc agaacgctca ggccctgaat 2880
accctggtga agcagctgtc gagcaacttc ggcgctatct cctctgtgct gaacgatatc 2940
ctgagccggc tggaccctcc agaggcggag gtgcagatcg accggctgat cacaggcagg 3000
ctgcagtccc tgcagaccta tgtgacacag cagctgatca gagctgccga gatccgcgct 3060
tctgccaatc tggctgccac caagatgtcc gagtgcgtgc tgggccagtc gaagcgcgtg 3120
gacttttgtg gcaagggcta tcacctgatg tctttcccac agagcgcccc acacggagtg 3180
gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac agctccagcc 3240
atctgccacg atggcaaggc ccactttcca cgggagggcg tgttcgtgag caacggcacc 3300
cactggtttg tgacacagag gaatttctac gagccccaga tcatcaccac agacaatacc 3360
ttcgtgagcg gcaactgcga cgtggtcatc ggcatcgtga acaataccgt gtacgatcct 3420
ctgcagccag agctggactc tttcaaggag gagctggaca agtacttcaa gaatcacacc 3480
agccccgacg tggatctggg cgacatctcc ggcatcaatg cttctgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaatctga acgagagcct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822
<210> 10
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc agtgcgtgaa cctgaccaca 60
aggacccagc tgccacctgc ctataccaac agcttcacaa gaggcgtgta ctatccggac 120
aaggtgtttc gctccagcgt gctgcactct acacaggacc tgtttctgcc attcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcacaaa gagattcgac 240
aatccagtgc tgcccttcaa cgatggcgtg tacttcgcga gcaccgagaa gtccaacatc 300
atccgcggct ggatctttgg caccacactg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaacaacaag tcctggatgg agagcgagtt tcgcgtgtat 480
tcctctgcca acaattgcac attcgagtac gtgtcccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaatat cgatggctac 600
ttcaagatct actccaagca cacccctatc aacctggtga gggacctgcc acagggcttc 660
tctgccctgg agcctctggt ggatctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggctc tgcaccgcag ctacctgaca cccggcgaca gctcctctgg atggaccgct 780
ggagctgctg cttactatgt gggctatctg cagcctcgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgctgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttccgggtg 960
cagccaaccg agtctatcgt gaggtttcca aatatcacaa acctgtgccc ctttggcgag 1020
gtgttcaacg ctaccaggtt cgccagcgtg tacgcttgga acagaaagcg catctccaac 1080
tgcgtggcgg actacagcgt gctgtacaac agcgcctcct tctctacctt caagtgctat 1140
ggcgtgagcc ctacaaagct gaacgacctg tgctttacca acgtgtacgc cgattccttc 1200
gtgatcagag gcgacgaggt gcgccagatc gctccaggac agacaggcaa catcgccgac 1260
tacaattaca agctgcccga cgatttcacc ggctgcgtga tcgcctggaa ctccaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgttcag gaagtccaat 1380
ctgaagccct tcgagcggga catctctaca gagatctacc aggccggcag caccccttgc 1440
aatggcgtga agggcttcaa ctgttatttc cccctgcagt cttacggctt ccagcctaca 1500
tatggcgtgg gctatcagcc atacagggtg gtggtgctgt ctttcgagct gctgcacgct 1560
ccagctacag tgtgcggacc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggactgac cggaacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggccg ggacatcgcc gataccacag acgctgtgag ggacccccag 1740
accctggaga tcctggatat cacaccttgc agcttcggcg gcgtgtccgt gatcacacca 1800
ggaaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860
ccagtggcta tccacgccga tcagctgacc cccacatgga gagtgtactc taccggcagc 1920
aacgtgtttc agacaagggc tggatgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcttcct accagaccca gacaaactct 2040
ccaggctccg ccggcagcgt ggccagccag tccatcatcg cttataccat gtccctgggc 2100
gccgagaatt ctgtggctta ctccaacaat tctatcgcca tccccaccaa cttcacaatc 2160
agcgtgacca cagagatcct gcccgtgagc atgaccaaga caagcgtgga ctgcacaatg 2220
tatatctgtg gcgacagcac cgagtgctcc aacctgctgc tgcagtacgg ctccttttgt 2280
acccagctga atcgcgccct gacaggcatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggcttc 2400
aatttttccc agatcctgcc cgatcccagc aagccttcca agagatcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca tcgctgccag ggacctgatc tgtgctcaga agttcaatgg cctgaccgtg 2580
ctgcctccac tgctgacaga cgagatgatc gctcagtaca caagcgccct gctggctgga 2640
accatcacat ccggctggac cttcggcgct ggagctgccc tgcagatccc atttgccatg 2700
cagatggctt acagattcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt caactccgct atcggcaaga tccaggacag cctgagcagc 2820
acagcttccg ccctgggcaa gctgcaggat gtggtgaatc agaacgctca ggccctgaat 2880
accctggtga agcagctgtc gagcaacttc ggcgctatct cctctgtgct gaacgatatc 2940
ctgagccggc tggaccctcc agaggcggag gtgcagatcg accggctgat cacaggcagg 3000
ctgcagtccc tgcagaccta tgtgacacag cagctgatca gagctgccga gatccgcgct 3060
tctgccaatc tggctgccac caagatgtcc gagtgcgtgc tgggccagtc gaagcgcgtg 3120
gacttttgtg gcaagggcta tcacctgatg tctttcccac agagcgcccc acacggagtg 3180
gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac agctccagcc 3240
atctgccacg atggcaaggc ccactttcca cgggagggcg tgttcgtgag caacggcacc 3300
cactggtttg tgacacagag gaatttctac gagccccaga tcatcaccac agacaatacc 3360
ttcgtgagcg gcaactgcga cgtggtcatc ggcatcgtga acaataccgt gtacgatcct 3420
ctgcagccag agctggactc tttcaaggag gagctggaca agtacttcaa gaatcacacc 3480
agccccgacg tggatctggg cgacatctcc ggcatcaatg cttctgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaatctga acgagagcct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822
<210> 11
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc agtgcgtgaa cctgaccaca 60
aggacccagc tgccacctgc ctataccaac agcttcacaa gaggcgtgta ctatccggac 120
aaggtgtttc gctccagcgt gctgcactct acacaggacc tgtttctgcc attcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcacaaa gagattcgac 240
aatccagtgc tgcccttcaa cgatggcgtg tacttcgcga gcaccgagaa gtccaacatc 300
atccgcggct ggatctttgg caccacactg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaacaacaag tcctggatgg agagcgagtt tcgcgtgtat 480
tcctctgcca acaattgcac attcgagtac gtgtcccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaatat cgatggctac 600
ttcaagatct actccaagca cacccctatc aacctggtga gggacctgcc acagggcttc 660
tctgccctgg agcctctggt ggatctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggctc tgcaccgcag ctacctgaca cccggcgaca gctcctctgg atggaccgct 780
ggagctgctg cttactatgt gggctatctg cagcctcgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgctgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttccgggtg 960
cagccaaccg agtctatcgt gaggtttcca aatatcacaa acctgtgccc ctttggcgag 1020
gtgttcaacg ctaccaggtt cgccagcgtg tacgcttgga acagaaagcg catctccaac 1080
tgcgtggcgg actacagcgt gctgtacaac agcgcctcct tctctacctt caagtgctat 1140
ggcgtgagcc ctacaaagct gaacgacctg tgctttacca acgtgtacgc cgattccttc 1200
gtgatcagag gcgacgaggt gcgccagatc gctccaggac agacaggcaa catcgccgac 1260
tacaattaca agctgcccga cgatttcacc ggctgcgtga tcgcctggaa ctccaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgttcag gaagtccaat 1380
ctgaagccct tcgagcggga catctctaca gagatctacc aggccggcag caccccttgc 1440
aatggcgtga agggcttcaa ctgttatttc cccctgcagt cttacggctt ccagcctaca 1500
tatggcgtgg gctatcagcc atacagggtg gtggtgctgt ctttcgagct gctgcacgct 1560
ccagctacag tgtgcggacc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggactgac cggaacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggccg ggacatcgcc gataccacag acgctgtgag ggacccccag 1740
accctggaga tcctggatat cacaccttgc agcttcggcg gcgtgtccgt gatcacacca 1800
ggaaccaata caagcaacca ggtggccgtg ctgtatcagg gcgtgaattg taccgaggtg 1860
ccagtggcta tccacgccga tcagctgacc cccacatgga gagtgtactc taccggcagc 1920
aacgtgtttc agacaagggc tggatgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcttcct accagaccca gacaaactct 2040
ccaggctccg ccggcagcgt ggccagccag tccatcatcg cttataccat gtccctgggc 2100
gccgagaatt ctgtggctta ctccaacaat tctatcgcca tccccaccaa cttcacaatc 2160
agcgtgacca cagagatcct gcccgtgagc atgaccaaga caagcgtgga ctgcacaatg 2220
tatatctgtg gcgacagcac cgagtgctcc aacctgctgc tgcagtacgg ctccttttgt 2280
acccagctga atcgcgccct gacaggcatc gctgtggagc aggacaagaa cacacaggag 2340
gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggcttc 2400
aatttttccc agatcctgcc cgatcccagc aagccttcca agagatcttt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca tcgctgccag ggacctgatc tgtgctcaga agttcaatgg cctgaccgtg 2580
ctgcctccac tgctgacaga cgagatgatc gctcagtaca caagcgccct gctggctgga 2640
accatcacat ccggctggac cttcggcgct ggagctgccc tgcagatccc atttgccatg 2700
cagatggctt acagattcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt caactccgct atcggcaaga tccaggacag cctgagcagc 2820
acagcttccg ccctgggcaa gctgcaggat gtggtgaatc agaacgctca ggccctgaat 2880
accctggtga agcagctgtc gagcaacttc ggcgctatct cctctgtgct gaacgatatc 2940
ctgagccggc tggaccctcc agaggcggag gtgcagatcg accggctgat cacaggcagg 3000
ctgcagtccc tgcagaccta tgtgacacag cagctgatca gagctgccga gatccgcgct 3060
tctgccaatc tggctgccac caagatgtcc gagtgcgtgc tgggccagtc gaagcgcgtg 3120
gacttttgtg gcaagggcta tcacctgatg tctttcccac agagcgcccc acacggagtg 3180
gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac agctccagcc 3240
atctgccacg atggcaaggc ccactttcca cgggagggcg tgttcgtgag caacggcacc 3300
cactggtttg tgacacagag gaatttctac gagccccaga tcatcaccac agacaatacc 3360
ttcgtgagcg gcaactgcga cgtggtcatc ggcatcgtga acaataccgt gtacgatcct 3420
ctgcagccag agctggactc tttcaaggag gagctggaca agtacttcaa gaatcacacc 3480
agccccgacg tggatctggg cgacatctcc ggcatcaatg cttctgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggcc aagaatctga acgagagcct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720
tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gcaagttcga cgaggacgac 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822
<210> 12
<211> 3813
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc agtgcgtgaa cctgaccaca 60
aggacccagc tgccacctgc ctataccaac agcttcacaa gaggcgtgta ctatccggac 120
aaggtgtttc gctccagcgt gctgcactct acacaggacc tgtttctgcc attcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcacaaa gagattcgac 240
aatccagtgc tgcccttcaa cgatggcgtg tacttcgcga gcaccgagaa gtccaacatc 300
atccgcggct ggatctttgg caccacactg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaacaacaag tcctggatgg agagcgagtt tcgcgtgtat 480
tcctctgcca acaattgcac attcgagtac gtgtcccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaatat cgatggctac 600
ttcaagatct actccaagca cacccctatc aacctggtga gggacctgcc acagggcttc 660
tctgccctgg agcctctggt ggatctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggctc tgcaccgcag ctacctgaca cccggcgaca gctcctctgg atggaccgct 780
ggagctgctg cttactatgt gggctatctg cagcctcgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgctgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttccgggtg 960
cagccaaccg agtctatcgt gaggtttcca aatatcacaa acctgtgccc ctttggcgag 1020
gtgttcaacg ctaccaggtt cgccagcgtg tacgcttgga acagaaagcg catctccaac 1080
tgcgtggcgg actacagcgt gctgtacaac agcgcctcct tctctacctt caagtgctat 1140
ggcgtgagcc ctacaaagct gaacgacctg tgctttacca acgtgtacgc cgattccttc 1200
gtgatcagag gcgacgaggt gcgccagatc gctccaggac agacaggcaa catcgccgac 1260
tacaattaca agctgcccga cgatttcacc ggctgcgtga tcgcctggaa ctccaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgttcag gaagtccaat 1380
ctgaagccct tcgagcggga catctctaca gagatctacc aggccggcag caccccttgc 1440
aatggcgtga agggcttcaa ctgttatttc cccctgcagt cttacggctt ccagcctaca 1500
tatggcgtgg gctatcagcc atacagggtg gtggtgctgt ctttcgagct gctgcacgct 1560
ccagctacag tgtgcggacc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggactgac cggaacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggccg ggacatcgcc gataccacag acgctgtgag ggacccccag 1740
accctggaga tcctggatat cacaccttgc agcttcggcg gcgtgtccgt gatcacacca 1800
ggaaccaata caagcaacca ggtggccgtg ctgtatcagg gcgtgaattg taccgaggtg 1860
ccagtggcta tccacgccga tcagctgacc cccacatgga gagtgtactc taccggcagc 1920
aacgtgtttc agacaagggc tggatgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcttcct accagaccca gacaaactct 2040
ccagccagcg tggccagcca gtccatcatc gcttatacca tgtccctggg cgccgagaat 2100
tctgtggctt actccaacaa ttctatcgcc atccccacca acttcacaat cagcgtgacc 2160
acagagatcc tgcccgtgag catgaccaag acaagcgtgg actgcacaat gtatatctgt 2220
ggcgacagca ccgagtgctc caacctgctg ctgcagtacg gctccttttg tacccagctg 2280
aatcgcgccc tgacaggcat cgctgtggag caggacaaga acacacagga ggtgttcgcc 2340
caggtgaagc agatctacaa gaccccaccc atcaaggact ttggcggctt caatttttcc 2400
cagatcctgc ccgatcccag caagccttcc aagagatctt ttatcgagga cctgctgttc 2460
aacaaggtga ccctggccga tgccggcttc atcaagcagt atggcgattg cctgggcgac 2520
atcgctgcca gggacctgat ctgtgctcag aagttcaatg gcctgaccgt gctgcctcca 2580
ctgctgacag acgagatgat cgctcagtac acaagcgccc tgctggctgg aaccatcaca 2640
tccggctgga ccttcggcgc tggagctgcc ctgcagatcc catttgccat gcagatggct 2700
tacagattca acggcatcgg cgtgacccag aatgtgctgt acgagaacca gaagctgatc 2760
gccaatcagt tcaactccgc tatcggcaag atccaggaca gcctgagcag cacagcttcc 2820
gccctgggca agctgcagga tgtggtgaat cagaacgctc aggccctgaa taccctggtg 2880
aagcagctgt cgagcaactt cggcgctatc tcctctgtgc tgaacgatat cctgagccgg 2940
ctggaccctc cagaggcgga ggtgcagatc gaccggctga tcacaggcag gctgcagtcc 3000
ctgcagacct atgtgacaca gcagctgatc agagctgccg agatccgcgc ttctgccaat 3060
ctggctgcca ccaagatgtc cgagtgcgtg ctgggccagt cgaagcgcgt ggacttttgt 3120
ggcaagggct atcacctgat gtctttccca cagagcgccc cacacggagt ggtgtttctg 3180
cacgtgacct acgtgcctgc ccaggagaag aacttcacca cagctccagc catctgccac 3240
gatggcaagg cccactttcc acgggagggc gtgttcgtga gcaacggcac ccactggttt 3300
gtgacacaga ggaatttcta cgagccccag atcatcacca cagacaatac cttcgtgagc 3360
ggcaactgcg acgtggtcat cggcatcgtg aacaataccg tgtacgatcc tctgcagcca 3420
gagctggact ctttcaagga ggagctggac aagtacttca agaatcacac cagccccgac 3480
gtggatctgg gcgacatctc cggcatcaat gcttctgtgg tgaacatcca gaaggagatc 3540
gaccgcctga acgaggtggc caagaatctg aacgagagcc tgatcgatct gcaggagctg 3600
ggcaagtacg agcagtacat caagtggccc tggtatatct ggctgggctt catcgccggc 3660
ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgta tgacaagctg ctgttcctgc 3720
ctgaagggct gctgttcttg tggcagctgc tgcaagttcg acgaggacga cagcgagcct 3780
gtgctgaagg gcgtgaagct gcactacacc taa 3813
<210> 13
<211> 3813
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgttcgtgt tcctggtcct gctgccactg gtcagcagcc agtgcgtcaa cctgacaacc 60
cgcacccagc tgccaccagc ctacaccaac agcttcaccc ggggcgtgta ctacccagac 120
aaggtgttca gaagcagcgt gctgcacagc acacaggacc tgttcctgcc attcttcagc 180
aacgtgacct ggttccacgt gatcagcggc accaacggca caaagaggtt cgacaaccca 240
gtgctgccat tcaacgacgg cgtgtacttc gccagcatcg agaaaagcaa catcatccgc 300
ggctggatct tcggcaccac actggacagc aagacacaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc attcctggac 420
cacaagaaca acaagagctg gatggagagc gagttcaggg tgtacagcag cgccaacaac 480
tgcaccttcg agtacgtgag ccagccattc ctgatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgcgggagtt cgtgttcaag aacatcgacg gctacttcaa gatctacagc 600
aagcacaccc caatcatcgt gagagagcca gaggacctgc cacagggatt cagcgccctg 660
gagccactgg tggacctgcc aatcggcatc aacatcaccc ggttccagac actgctggcc 720
ctgcacagaa gctacctgac accaggcgac agcagcagcg gatggaccgc aggagcagca 780
gcctactacg tgggctacct gcagccaagg accttcctgc tgaagtacaa cgagaacggc 840
accatcacag acgcagtgga ctgcgcactg gacccactga gcgagacaaa gtgcacactg 900
aaaagcttca ccgtggagaa gggcatctac cagacaagca acttcagggt gcagccaacc 960
gagagcatcg tgcgcttccc aaacatcaca aacctgtgcc cattcgacga ggtgttcaac 1020
gcaaccaggt tcgccagcgt gtacgcatgg aaccggaaga gaatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cctggcccca ttcttcacct tcaaatgcta cggcgtgagc 1140
ccaacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcagg 1200
ggagacgaag tgagacagat cgcaccaggc cagacaggca acatcgccga ctacaactac 1260
aagctgccag acgacttcac cggctgcgtg atcgcctgga acagcaacaa gctggacagc 1320
aaggtgagcg gcaactacaa ctacctgtac aggctgttcc gcaagagcaa cctgaagcca 1380
ttcgagaggg acatcagcac agagatctac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgagg agctacagct tcaggccaac ctacggagtg 1500
ggacaccagc catacagggt ggtggtgctg agcttcgagc tgctgcacgc accagcaaca 1560
gtgtgcggac caaagaaaag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctga agggcaccgg cgtgctgaca gagagcaaca agaagttcct gccattccag 1680
cagttcggca gggacatcgc agacaccaca gacgccgtgc gcgacccaca gaccctggag 1740
atcctggaca tcacaccatg cagcttcggc ggcgtgagcg tgatcacacc aggcaccaac 1800
acaagcaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggca 1860
atccacgcag accagctgac cccaacatgg cgggtgtaca gcaccggcag caacgtgttc 1920
cagacaagag caggatgcct gatcggagca gagtacgtga acaacagcta cgagtgcgac 1980
atcccaatcg gagcaggaat ctgcgcaagc taccagaccc agacaaagag ccacggaagc 2040
gccggcagcg tggcaagcca gagcatcatc gcctacacca tgagcctggg cgccgagaac 2100
agcgtggcct acagcaacaa cagcatcgcc atcccaacca acttcacaat cagcgtgacc 2160
acagagatcc tgccagtgag catgaccaag acaagcgtgg actgcacaat gtacatctgc 2220
ggcgacagca ccgagtgcag caacctgctg ctgcagtacg gcagcttctg cacccagctg 2280
aagcgggccc tgacaggaat cgcagtggag caggacaaga acacacagga ggtgttcgcc 2340
caggtgaagc agatctacaa gaccccacca atcaagtact tcggcggctt caacttcagc 2400
cagatcctgc cagacccaag caagccaagc aagcggagct tcatcgagga cctgctgttc 2460
aacaaggtga ccctggccga cgccggcttc atcaagcagt acggagactg cctgggagac 2520
atcgcagcac gggacctgat ctgcgcccag aagttcaagg gcctgaccgt gctgccacca 2580
ctgctgacag acgagatgat cgcacagtac acaagcgccc tgctggcagg aaccatcaca 2640
agcggatgga ccttcggcgc aggagccgcc ctgcagatcc cattcgccat gcagatggcc 2700
tacagattca acggcatcgg cgtgacccag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc catcggcaag atccaggaca gcctgagcag cacagcaagc 2820
gccctgggaa agctgcagga cgtggtgaac cacaacgccc aggccctgaa caccctggtg 2880
aagcagctga gcagcaagtt cggcgccatc agcagcgtgc tgaacgacat cttcagcagg 2940
ctggacccac cagaggcaga ggtgcagatc gaccggctga tcacaggcag actgcagagc 3000
ctgcagacct acgtgacaca gcagctgatc agggcagcag agatcagggc aagcgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctgggccaga gcaagagagt ggacttctgc 3120
ggcaagggct accacctgat gagcttccca cagagcgccc cacacggagt ggtgttcctg 3180
cacgtgacct acgtgccagc ccaggagaag aacttcacca cagcaccagc aatctgccac 3240
gacggaaagg cacacttccc aagggagggc gtgttcgtga gcaacggcac ccactggttc 3300
gtgacacagc gcaacttcta cgagccacag atcatcacca cagacaacac attcgtgagc 3360
ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc actgcagcca 3420
gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac cagcccagac 3480
gtggacctgg gcgacatcag cggcatcaac gccagcgtgg tgaacatcca gaaggagatc 3540
gaccggctga acgaggtggc caagaacctg aacgagagcc tgatcgacct gcaggagctg 3600
ggcaagtacg agcagtacat caagtggcca tggtacatct ggctgggctt catcgccggc 3660
ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgcagctgc 3720
ctgaagggct gctgcagctg cggcagctgc tgcaagttcg acgaggacga cagcgagcca 3780
gtcctgaaag gggtcaagct gcactacacc taa 3813

Claims (26)

1.一种mRNA疫苗，其特征在于，所述mRNA疫苗包含：
至少一个信使核糖核酸mRNA，所述mRNA编码至少一种抗原肽或结构蛋白，所述抗原肽或结构蛋白包括SARS-CoV-2冠状病毒的刺突蛋白的衍生蛋白，所述刺突蛋白的衍生蛋白的氨基酸序列如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6或者SEQ ID NO:7所示，所述mRNA疫苗还包含脂质纳米颗粒，所述mRNA包封于所述脂质纳米颗粒中，所述脂质纳米颗粒包含阳离子脂质、中性磷脂、甾族脂质和聚乙二醇-脂质，所述阳离子脂质的结构为：ALC-0315：
Figure DEST_PATH_IMAGE001
。
2.根据权利要求1所述的mRNA疫苗，其特征在于，所述刺突蛋白的衍生蛋白还包括将SEQ ID NO:4-7的第1-13位替换为其他强信号肽，或在N端增加其他强信号肽；
所述其他强信号肽包括组织型纤溶酶原激活剂的信号肽和血清免疫球蛋白E的信号肽。
3.根据权利要求1所述的mRNA疫苗，其特征在于，所述刺突蛋白的衍生蛋白还包括在N端或C端增加用于形成多聚体的片段，所述片段包括Fc片段或噬菌体T4纤维蛋白的三聚化基序。
4.根据权利要求1所述的mRNA疫苗，其特征在于，所述mRNA序列是天然或修饰的RNA，所述修饰的RNA包括通过用修饰的尿苷部分或全部取代天然尿苷对RNA进行修饰。
5.根据权利要求4所述的mRNA疫苗，其特征在于，所述mRNA序列是修饰的RNA，所述修饰的RNA为通过用1-甲基-假尿苷对天然尿苷进行全部替换。
6.根据权利要求1所述的mRNA疫苗，其特征在于，所述聚乙二醇-脂质选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)]、PEG-二甾醇基甘油、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺、PEG-二棕榈酰基磷脂酰乙醇胺、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺或DMG-PEG2000中的一种或多种组合。
7.根据权利要求1所述的mRNA疫苗，其特征在于，所述的中性磷脂选自：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱、1,2-二油酰-sn-甘油-3-磷酸乙醇胺、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)、油酰磷脂酰胆碱、1-棕榈酰基-2-油酰基磷脂酰乙醇胺中的一种或多种组合。
8.根据权利要求1所述的mRNA疫苗，其特征在于，所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合。
9.根据权利要求1所述的mRNA疫苗，其特征在于，所述聚乙二醇-脂质为DMG-PEG2000；所述中性磷脂为DSPC；所述的甾族脂质为胆固醇。
10.根据权利要求1所述的mRNA疫苗，其特征在于，所述的阳离子脂质在脂质组分中的摩尔百分含量为20~60%、中性磷脂在脂质组分中的摩尔百分含量为5%~25%、甾族脂质在脂质组分中的摩尔百分含量为25%~55%；聚乙二醇-脂质在脂质组分中的摩尔百分含量为0.1%~15%。
11.根据权利要求1所述的mRNA疫苗，其特征在于，脂质纳米颗粒中总脂质与mRNA的质量比在10-30：1之间。
12.根据权利要求10所述的mRNA疫苗，其特征在于，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇-脂质摩尔比为30-60:5-20:20-50:0.1-10。
13.根据权利要求1所述的mRNA疫苗，其特征在于，所述mRNA疫苗中还包含其他辅料，所述其他辅料包括醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。
14.根据权利要求1所述的mRNA疫苗，其特征在于，所述脂质纳米颗粒的平均粒径为50~200nm；或所述脂质纳米颗粒在中性pH下具有净中性电荷；或所述脂质纳米颗粒具有小于0.4的多分散性。
15.根据权利要求1所述的mRNA疫苗，其特征在于，所述抗原肽或结构蛋白还包括包膜蛋白，膜蛋白或核衣壳蛋白，或其免疫原性片段或免疫原性变体。
16.根据权利要求1所述的mRNA疫苗，其特征在于，所述mRNA疫苗为液体制剂或冻干粉剂。
17.根据权利要求16所述的mRNA疫苗，其特征在于，所述mRNA疫苗为口服制剂、肌肉注射制剂、静脉注射制剂或吸入制剂。
18.根据权利要求17所述的mRNA疫苗，其特征在于，所述mRNA疫苗为雾化吸入剂或干粉吸入剂。
19.根据权利要求1-18任一所述的mRNA疫苗，其特征在于，所述mRNA的序列如SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12或者SEQ ID NO:13所示。
20.一种权利要求1所述的mRNA疫苗的制备方法，其特征在于，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇-脂质溶解至溶剂后与mRNA混合后制得。
21.根据权利要求20所述的mRNA疫苗的制备方法，其特征在于，脂质纳米颗粒包封mRNA时的N/P为2-10。
22.根据权利要求21所述的mRNA疫苗的制备方法，其特征在于，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇-脂质溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得。
23.根据权利要求22所述的mRNA疫苗的制备方法，其特征在于，稀释液可为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。
24.根据权利要求23所述的mRNA疫苗的制备方法，其特征在于，所述缓冲液pH为3~6，浓度为6.25~200mM。
25.一种权利要求1-19任一项所述的mRNA疫苗在制备用于预防新型冠状病毒感染的药物中的应用。
26.根据权利要求25所述的应用，其特征在于，所述的新型冠状病毒为原型株、Beta变异株、Delta变异株或Omicron变异株。
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