CN117625650A - 一种正痘病毒属mRNA疫苗及其制备方法和用途 - Google Patents
一种正痘病毒属mRNA疫苗及其制备方法和用途 Download PDFInfo
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Abstract
本发明提供了正痘病毒属mRNA疫苗及其制备方法和用途。本发明的正痘病毒属mRNA疫苗,可以诱导强烈的猴痘病毒特异性的体液免疫应答,诱导强烈的正痘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。本发明提供的正痘病毒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
技术领域
本发明涉及疫苗技术领域,具体涉及编码正痘病毒属病毒蛋白或其同源蛋白的mRNA的组合物疫苗及其制备方法和在预防痘病毒感染方面的应用。
背景技术
猴痘是一种由猴痘病毒感染引起的罕见疾病。猴痘病毒与能够引起天花的天花病毒属于同一病毒家族。猴痘症状与天花症状相似,但较轻,而且猴痘很少致命(西非突变株的死亡率为1%,而中非突变株的死亡率为10%左右)。猴痘发现于1958年,当时在用于研究的猴子群中爆发了两次类似痘的疾病。尽管被命名为“猴痘”,但这种疾病的来源仍然未知。1970年,刚果(金)发现首例人感染猴痘病例。之后,猴痘病毒主要在西非和中非地区流行,包括刚果(金)、刚果(布)、中非共和国、尼日利亚、喀麦隆等非洲国家。非洲大陆之外的首次猴痘疫情于2003年出现在美国,传染源可追溯至从加纳运到美国的冈比亚鼠和睡鼠。而自2018年以来在以色列、英国、新加坡等国发现的猴痘病毒感染者多为来自尼日利亚的旅客。随着猴痘疫情在全世界多个国家的激增,世卫组织也于2022年5月23日宣布猴痘疫情已构成国际关注的突发公共卫生事件。截至2023年8月9日,全球共有猴痘病例89,308起,而中国猴痘病例达到890起。其中,2023年7月~8月,国内激增524例猴痘病例,呈明显上升趋势。此前有两种天花疫苗被考虑用于应对猴痘的全球疫情,分别是ACAM2000和Jynneos其中,ACAM2000是一种竞争复制型的活病毒疫苗,不适用于免疫力低下的人群使用,也容易造成无意接种或自动接种;接种后会出现皮疹和结痂,而且约0.57%的接种者会出现心肌心包炎。而另一款疫苗Jynneos是一种非复制型的活病毒疫苗,相对比较安全,不会引起严重不良反应,也不会出现自动接种的情况[1]。
猴痘病毒存在两种形式:成熟病毒粒子(mature virions,MV)和细胞外病毒粒子(extracellular virions,EV)。MV是在宿主细胞内形成,并通过细胞裂解释放到胞外;也有一部分胞内MV被转移至高尔基体中包裹上一层额外的包膜,形成EV之后,最终通过胞吐释放到胞外。EV一般游离在胞外基质中,也有小部分附着在细胞表面。MV被认为对病毒在环境中宿主之间的长期稳定性和传播十分重要;而EV被认为对病毒在生物体内细胞间的传播发挥作用。由于EV比MV多了一层外包膜,两者进入细胞的过程也有一定的差异。当MV入侵宿主细胞时,首先通过其膜表面的锚定蛋白与细胞结合,然后由进入融合复合体(entry fusioncomplex,EFC)介导或胞饮作用进入到细胞内。然而EV在进入细胞之前,其外层的包膜首先发生配体诱导的非融合反应而被破裂[3],然后内膜与细胞膜发生融合,过程与MV类似。
猴痘病毒的基因组大小约为197kb,编码190多种蛋白质。猴痘病毒蛋白与牛痘病毒蛋白同一性很高,功能也很保守。比如,牛痘病毒蛋白A27L、A33R、L1R、B5R分别与猴痘病毒蛋白A29L、A35R、M1R、B6R同源。病毒表面的蛋白在其入侵宿主细胞的过程中发挥重要的作用。其中,H3、A27、D8参与MV与细胞的结合[5];蛋白A16、A21、A28、G3、G9、H2、J5、L5、F9、L1、O3、I2共同组成融合复合体EFC,参与EV和MV与细胞的融合[2,3,6];而L1、H3同时也参与MV在细胞内的成熟过程[7]。而B5和A33是EV外包膜所特有的蛋白,其中,,B5参与了EV的形成和外包膜的破裂[3,8,9],而A33与病毒在细胞间的传播有关[10]。另外,A27也参与了胞内MV的转移[11]。疫苗是遏制传染病流行的流行的关键手段之一。Jynneos被认为是现阶段最好的天花或猴痘疫苗,也已经在美国、加拿大、欧盟获批用于预防猴痘病毒。虽然作为非复制型的减毒活疫苗副作用相对较小,但具有接种传染的风险,并且未采用猴痘特异有效抗原。因此,仍需通过其他技术开发更为有效和安全的疫苗。随着mRNA疫苗在新冠病毒预防中成功应用,突显mRNA疫苗的有效性和安全性。另外,mRNA疫苗具有研发周期短、产能增速快等技术优势,同样适合猴痘病毒的预防性疫苗的应急研制与开发。
发明内容
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。
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”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组合物,所述组合物包括编码至少一种抗原肽或结构蛋白,所述抗原肽或结构蛋白选自猴痘病毒H3L蛋白、B6R蛋白、M1R蛋白、A35R蛋白、A29L蛋白、E8L蛋白、A30L蛋白、A5L蛋白、C19L蛋白或其同源蛋白中的一种或者多种,所述同源蛋白与上述蛋白具有至少75%同一性且与其具有相同或基本相同的免疫原性的氨基酸序列的蛋白或其抗原性片段。
优选地,所述抗原肽或结构蛋白包括H3L蛋白或H3L同源蛋白。
优选地,所述同源蛋白与上述蛋白具有至少75%同一性更优选地,具有至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的同一性。例如,
优选地,所述H3L蛋白的序列为SEQ ID NO:4,H3L同源蛋白序列与SEQ ID NO:4具有至少75%的同一性,更优选地,具有至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的同一性。
优选地,所述组合物还包括:编码猴痘蛋白M1R或M1R同源蛋白的mRNA、编码猴痘蛋白B6R或B6R同源蛋白的mRNA、编码A35R或A35R同源蛋白的mRNA中的一种或多种。
优选地,所述M1R蛋白的序列为SEQ ID NO:3,M1R同源蛋白序列与SEQ ID NO:3具有至少75%的同一性,更优选地,具有至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的同一性。
优选地,所述B6R蛋白的序列为SEQ ID NO:1,B6R同源蛋白序列与SEQ ID NO:1具有至少75%的同一性;更优选地,具有至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的同一性。
优选地,所述A35R蛋白的序列为SEQ ID NO:2,A35R同源蛋白序列与SEQ ID NO:2具有至少75%的同一性,更优选地,具有至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的同一性。
优选地,所述抗原肽或结构蛋白包括两种或者两种以上的组合,所述两种或者两种以上的抗原肽或结构蛋白分别表达或者融合表达,
更优选地,所述抗原肽或结构蛋白分别表达,所述组合物包括多个mRNA。
更优选地,所述抗原肽或结构蛋白融合表达,所述多个抗原肽或结构蛋白直接或者通过连接肽间接连接。
优选地,本发明选择了H3L、B6R、M1R 3种抗原以及H3L、B6R、M1R、A35R四种抗原制备mRNA疫苗。
优选地,所述抗原肽或结构蛋白包括如下表1所示的组合:
表1抗原肽或结构蛋白单体或者组合示意
其中,编号10-20,25-34中的抗原肽或结构蛋白分别表达,编号21-24中的抗原肽或结构蛋白融合表达。
下述描述参照上述原则,即“+”表示分别表达,后期混合,“-”表示融合表达。
优选地,或所述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。
优选地,所述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。
优选地,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。
优选地,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。
优选地,所述mRNA编码蛋白的信号肽可替换或串联强信号肽,优选地,为tpA信号肽和/或IgE信号肽。
本发明的第二方面,提供了一种正痘病毒属mRNA疫苗,包括:由递送载体包裹的所述编码正痘病毒属病毒蛋白或同源蛋白的信使核糖核酸(mRNA)。
优选地,所述mRNA如第一方面所限定的mRNA组合物。
优选地,所述组合物包括多个mRNA,所述递送载体分别包裹单个mRNA。
优选地,所述递送载体为脂质纳米颗粒(LNP),所述mRNA包封于所述脂质纳米颗粒(LNP)中。
优选地,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。
优选地,所述的mRNA序列的长度为200-10000个核苷酸。进一步优选地,所述的mRNA序列的长度为500-8000个核苷酸。
本发明的mRNA疫苗,mRNA包封于脂质纳米颗粒中,免疫后可诱导强烈的猴痘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病毒蛋白的中和抗体。
优选地,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。
优选地,所述的阳离子脂质具有以下结构:
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。
优选地,阳离子脂质化合物下结构如下:
优选地,所述的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,更优选地为DMG-PEG2000。
优选地,所述的中性脂质选自: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,更优选地为DSPC。
优选地,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,更优选地为胆固醇。
优选地,所述聚乙二醇(PEG)-脂质为DMG-PEG2000;所述中性磷脂为DSPC;所述的甾族脂质为胆固醇。
优选地,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%;PEG-脂质在脂质组分中的摩尔百分含量为0.1%~15%。
优选地,脂质纳米颗粒(LNP)中总脂质与mRNA的质量比(w/w)在10-30:1之间。
优选地,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:PEG-脂质摩尔比为30-60:1-20:20-50:0.1-10。
优选地,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。
更优选地,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。
优选地,所述的正痘病毒属mRNA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。
优选地,所述的正痘病毒属mRNA疫苗,所述纳米颗粒的平均粒径为50~200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。
优选地,所述正痘病毒属病毒为猴痘病毒。
本发明第三方面,提供一种上述正痘病毒属mRNA疫苗的制备方法,所述制备方法包括由递送载体包裹上述mRNA组合物。
优选地,所述递送载体为脂质纳米颗粒(LNP),所述mRNA包封于所述脂质纳米颗粒(LNP)中,更优选地,所述组合物包括多个mRNA,所述递送载体分别包裹单个mRNA。
优选地,所述脂质纳米颗粒(LNP)包含阳离子脂质、中性磷脂、甾族脂质和聚乙二醇(PEG)-脂质。
更优选地,所述制备方法包括将阳离子脂质、中性磷脂、甾族脂质、PEG-脂质溶解至溶剂后与核酸混合后制得。
优选地,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)-脂质溶解至溶剂后对上述任一所述mRNA组合物进行包封,更优选地,所述原肽或结构蛋白包括两种或者两种以上的组合,所述两种或者两种以上的抗原肽或结构蛋白分别表达或者融合表达,制得多价疫苗。
优选地,所述的正痘病毒属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、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;
优选地,将阳离子脂质、中性磷脂、甾族脂质、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;优选地,所述的超滤方式为切向流过滤;更优选地,所述的混合方式可为湍流混合、层流混合或微流体混合。
所述脂质纳米颗粒(LNP)如第二方面所限定。
优选地,所述的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。
优选地,所述缓冲液pH为3~6,浓度为6.25~200mM。
优选地,脂质包封mRNA时的N/P为2-10,更优选地N/P为3-9。
优选地,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。
本发明一个方面涉及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)-脂质溶解至溶剂后与mRNA混合后制得。
优选地,所述的猴痘病毒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、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;优选地,将阳离子脂质、中性磷脂、甾族脂质、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;优选地,所述的超滤方式为切向流过滤;更优选地,所述的混合方式可为湍流混合、层流混合或微流体混合。
本发明一个方面涉及猴痘病毒mRNA疫苗在制备用于预防病毒感染的疫苗中的应用。
具体地,其中的病毒感染为猴痘病毒。
本发明所述的猴痘病毒脂质纳米颗粒mRNA疫苗可以通过口服、吸入或注射的方式递送所述生物活性物质。
本发明的第四方面,提供了所述的mRNA组合物或mRNA疫苗在制备用于预防或者治疗正痘病毒感染的药物中的应用。
优选地,正痘病毒为猴痘病毒。
本发明所述的猴痘病毒脂质纳米颗粒mRNA疫苗可以通过口服、吸入或注射的方式递送所述生物活性物质。
本发明提供一种疫苗试剂盒,包括使用两剂疫苗,接种间隔14天。
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,效果更强。免疫可以诱导产生针对猴痘病毒的高水平的中和抗体,尤其是比目前获批的针对天花/猴痘的活病毒疫苗Jynneos有更好的抗病毒效果以及更小的副作用。
在非常低的剂量和给药方案下,非常有效地诱导针对编码的蛋白的抗原特异性免疫应答;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;RNA可以大规模生产,并能够生产基于RNA的猴痘疫苗。
附图说明:
图1所示为合成的编码猴痘表面蛋白的mRNA分子的毛细管电泳分析图,其中A、B、C、D、E、F、G、H、I、J、K、L、M分别代表mRNA-B6R、mRNA-A35R、mRNA-M1R、mRNA-H3L、mRNA-A29L、mRNA-E8L、mRNA-A30L、mRNA-A5L、mRNA-C19L、mRNA-H3L-B6R-A35R、mRNA-H3L-B6R-M1R、mRNA-H3L-M1R-B6R-A35R、mRNA-H3L-A35R-M1R;
图2所示为猴痘疫苗的小鼠免疫程序;
图3所示为单价猴痘疫苗免疫28天的小鼠血清中的IgG检测结果;
图4所示为多价猴痘疫苗免疫28天的小鼠血清中的IgG检测结果;
图5所示为只包含MV包膜蛋白(H3L、M1R、E8L、A5L、A30L、A29L)的二价猴痘疫苗免疫14天和28天的小鼠血清中的中和抗体检测;
图6所示为由MV膜蛋白(H3L、M1R)和EV膜蛋白(B6R、A35R)组成三价或四价猴痘疫苗免疫14天和28天的小鼠血清中的中和抗体检测;
图7所示为由MV膜蛋白(H3L、M1R、A29L、A5L、A30L、E8L)和EV膜蛋白(B6R、A35R)组成的四价猴痘疫苗免疫14天和28天的小鼠血清中的中和抗体检测;
图8所示为接种包含MV和EV包膜蛋白的三价或四价猴痘疫苗的小鼠攻毒后的生存曲线
图9所示为攻毒小鼠的体重变化率。
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样品制备
表2蛋白或融合蛋白序列信息汇总表
序列简称 | 蛋白序列编号 |
B6R | SEQ ID NO:1 |
A35R | SEQ ID NO:2 |
M1R | SEQ ID NO:3 |
H3L | SEQ ID NO:4 |
A29L | SEQ ID NO:5 |
E8L | SEQ ID NO:6 |
A30L | SEQ ID NO:7 |
A5L | SEQ ID NO:8 |
C19L | SEQ ID NO:9 |
H3L-B6R-A35R | SEQ ID NO:10 |
H3L-B6R-M1R | SEQ ID NO:11 |
H3L-M1R-B6R-A35R | SEQ ID NO:12 |
H3L-A35R-M1R | SEQ ID NO:13 |
将编码B6R(SEQ ID NO:1)、A35R(SEQ ID NO:2)、M1R(SEQ ID NO:3)、H3L(SEQ IDNO:4)、A29L(SEQ ID NO:5)、E8L(SEQ ID NO:6)、A30L(SEQ ID NO:7)、A5L(SEQ ID NO:8)、A19L(SEQ ID NO:9)、H3L-B6R-A35R(SEQ ID NO:10)、H3L-B6R-M1R(SEQ ID NO:11)、H3L-M1R-B6R-A35R(SEQ ID NO:12)、H3L-A35R-M1R(SEQ ID NO:13)蛋白或融合蛋白的基因序列进行密码子优化,优化策略主要基于人源密码子使用频率、GC含量、重复序列的长度、限制性酶切位点的去除等因素,优化后的M1R、C19L、E8L、H3L、A29L、A30L、A5L、B6R、A35R目的核甘酸序列的密码子适应指数分别达到0.93、0.95、0.94、0.96、0.97、0.96、0.96、0.94、0.95。其中,融合蛋白H3L-B6R-A35R(SEQ ID NO:10)、H3L-B6R-M1R(SEQ ID NO:11)、H3L-M1R-B6R-A35R(SEQ ID NO:12)、H3L-A35R-M1R(SEQ ID NO:13)的每个抗原之间通过3个重复的GGGGS(SEQ ID NO:14)氨基酸序列连接。将优化后的基因序列通过无缝克隆的方法插入到含有T7启动子、UTR、PolyA的载体中,通过sanger测序确定构建质粒的正确性。
表3编码蛋白或融合蛋白的mRNA序列信息汇总表
序列简称 | mRNA序列编号 |
B6R | SEQ ID NO:15 |
A35R | SEQ ID NO:16 |
M1R | SEQ ID NO:17 |
H3L | SEQ ID NO:18 |
A29L | SEQ ID NO:19 |
E8L | SEQ ID NO:20 |
A30L | SEQ ID NO:21 |
A5L | SEQ ID NO:22 |
C19L | SEQ ID NO:23 |
H3L-B6R-A35R | SEQ ID NO:24 |
H3L-B6R-M1R | SEQ ID NO:25 |
H3L-M1R-B6R-A35R | SEQ ID NO:26 |
H3L-A35R-M1R | SEQ ID NO:27 |
其中连接肽(GGGGS)3对应的DNA序列如
GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC(SEQ ID NO:28)所示。
将质粒转化至大肠杆菌中进行扩增并提取质粒;将获得的质粒用限制性核酸内切酶BspQ I酶切线性化并纯化得到线性化质粒;以线性化的质粒为模板,通过T7体外转录试剂盒进行体外转录;然后DNase I消化转录模板,通过LiCl沉淀法纯化转录产物并得到目的mRNA;将得到的mRNA沉淀溶于RNA储存液中,备用。本实验通过共转录的方式进行加帽和加尾,因此mRNA转录产物中带有添加5’帽子和3’尾巴。通过毛细血管电泳检测mRNA原液的完整性,结果见表4和图1。
表4毛细血管电泳检测mRNA原液完整性
SEQ ID NO:15 | 95.7% |
SEQ ID NO:16 | 96.6% |
SEQ ID NO:17 | 96.4% |
SEQ ID NO:18 | 97.1% |
SEQ ID NO:19 | 97.8% |
SEQ ID NO:20 | 96.6% |
SEQ ID NO:21 | 95.8% |
SEQ ID NO:22 | 91.6% |
SEQ ID NO:23 | 90.8% |
SEQ ID NO:24 | 94.3% |
SEQ ID NO:25 | 96.2% |
SEQ ID NO:26 | 85.8% |
SEQ ID NO:27 | 95.2% |
实施例2:mRNA-LNP的制备与检测
mRNA-LNP的制备过程如下:首先将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质按45:10:43:2的摩尔比溶解于乙醇,配制得到脂质混合溶液,同时使用50mM pH=4醋酸钠缓冲溶液将mRNA原液稀释至135ng/ul。使用上述脂质将稀释后的mRNA溶液进行包封,设定纳米药物制造设备总流速12ml/min,mRNA溶液和脂质混合溶液的流速比为3:1。包封完成后,使用20mM Tris,10.7mM醋酸钠,pH7.5缓冲溶液,使得溶液的总体积将包装样品稀释10倍,经超滤、换液、浓缩得到mRNA-LNP中间品。随后使用含20mM Tris,10.7mM醋酸钠,87mg/ml蔗糖,pH7.5的缓冲溶液将mRNA-LNP中间品稀释成浓度为30ng/ul的成品。取样检测成品的包封率、浓度、粒径和电位,如表5所示。
混合型多价猴痘疫苗的制备过程是先将mRNA分别进行包封,然后将不同的mRNA-LNP mRNA疫苗按等质量比进行混合。
Control组为0.85%NaCl溶液。
表5mRNA-LNP成品检测
实施例3:猴痘mRNA疫苗的特异性IgG抗体反应
通过后腿肌肉注射途径免疫6-8周龄的BALB/c雌性小鼠,在第0和14天各免疫一次(免疫剂量如表6所示),检测首次免疫后28天的小鼠血清中的抗原特异性IgG,免疫程序如图2所示。将M1R、B6R、A35R、H3L、A29L、E8L、A30L、A5L、C19L重组蛋白包被于96孔板上,封闭后通过酶联免疫吸附法检测IgG抗体效价。
表6免疫实验组别及剂量
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图3和图4显示了单价和多价猴痘mRNA疫苗免疫第28天(加强免疫后),小鼠血清中的IgG抗体水平。其中,A29L、B6R、A35R、M1R、H3L、E8L、A30L、A5L、C19L的单价mRNA疫苗均能够诱导产生高水平的特异性IgG抗体(图3)。同样的,多价猴痘病毒mRNA疫苗同样能够产生针对各组分抗原的特异性IgG(图4),且各组分之间的相容性良好。
实施例4:猴痘mRNA疫苗诱导的中和抗体水平检测
通过蚀斑减少中和试验(PRNT)检测多价猴痘疫苗免疫后小鼠血清对于痘病毒的中和效价。具体地,使用活病毒与稀释后血清混合,37℃孵育1h;将血清-病毒混合液加入到Vero细胞中,37℃培养48h,然后使用ELISpot计数设备对显示的斑点计数,根据不同血清稀释梯度下的斑点相比阳性对照减少的比例绘制抑制率曲线,以该曲线的IC50(半抑制浓度)值作为该血清样品的PRNT50值。需要指出的是,该方法检测是IMV病毒颗粒的中和活性。
首先对由猴痘病毒MV表面抗原组成的二价疫苗诱导的中和抗体水平进行了评估,涉及的抗原包括H3L、M1R、E8L、A5L、A30L、A29L。从图5可以看出首次免疫14天,各组二价疫苗诱导微弱的中和抗体滴度,各疫苗组之间无显著性差异,但高于对照组。而首次免疫28天(加强免疫)后,二价混合疫苗均可诱导较高的中和抗体滴度,相对于对照组均有显著性差异。尤其以H3L和M1R混合的二价苗诱导中和抗体水平达到956,显著高于其他二价疫苗E8L+A5L(396)、A30L+E8L(438)、A29L+A5L(588)、A30L+A29L(516)诱导的中和抗体滴度。
后续使用同样的方法评价了MV包膜抗原(如H3L、E8L、A29L、M1R、A5L、A30L)和EV包膜抗原(B6R、A35R)组成的三价或四价疫苗的免疫原性,结果如图6和7所示。图6显示的是由MV膜蛋白(H3L、M1R)和EV膜蛋白(B6R、A35R)组成三价或四价猴痘疫苗免疫第14天和第28天的小鼠血清中的中和抗体水平。从图6可以看出免疫第14天,H3L+B6R+A35R、H3L+M1R+B6R、H3L+M1R+B6R+A35R免疫小鼠血清中检测到微弱中和抗体滴度,但高于阴性对照组(基线);而免疫第28天(加强免疫后),各组疫苗免疫小鼠血清中的中和抗体水平得到进一步提升。无论是分别表达方式还是融合表达方式,均可产生高滴度的中和抗体,相对于对照组均有显著性的差异。Two way anova统计分析显示H3L+M1R+B6R三价疫苗或H3L+M1R+B6R+A35R四价疫苗诱导的中和抗体水平显著高于H3L+B6R+A35R。这一结果表明在H3L+B6R+A35R三价猴痘疫苗的基础上额外添加M1R组分后,可以进一步提高中和抗体水平。
图7显示了H3L+M1R+B6R+A35R四价疫苗与其他四价疫苗的效果比较,结果同样表明H3L+M1R+B6R+A35R四价疫苗诱导的中和抗体不劣于其他抗原组成的四价疫苗,包括A5L+M1R+B6R+A35R、H3L+A29L+B6R+A35R、H3L+A5L+B6R+A35R、A30L+M1R+B6R+A35R、E8L+A30L+B6R+A35R、A29L+E8L+B6R+A35R、E8L+A5L+B6R+A35R、A29L+M1R+B6R+A35R、E8L+M1R+B6R+A35R,本发明中的多价疫苗具有良好的保护效果。
实施例5:致死攻毒保护性
首次免疫28天(加强免疫两周)后,对接种和未接种疫苗的小鼠进行攻毒挑战,并记录小鼠的存活状况及体重变化(图8和图9)。表7为小鼠攻毒后的平均体重变化率。
表7免疫小鼠攻毒后的体重变化率
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由实验结果可知,阴性对照组小鼠在攻毒9天内全部死亡;接种H3L+M1R二价疫苗的小鼠存活率为83%;H3L+B6R+A35R、H3L+M1R+B6R、H3L+M1R+B6R+A35R、A29L+M1R+B6R+A35R多价猴痘疫苗免疫的小鼠存活率达到100%(图8)。这一结果表明相对于对照组,本发明的疫苗可以提供抗病毒保护,尤其是H3L与B6R、A35R、M1R抗原组成的三价或四价疫苗可以提供100%的抗病毒保护。
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。
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。
参考文献
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14.参考专利:US7393533B1、US8535687B2、US6620412B2。
序列表:
SEQ ID NO:1猴痘病毒蛋白B6R氨基酸序列为:
MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLP
SEQ ID NO:2猴痘病毒蛋白A35R氨基酸序列为:
MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT
SEQ ID NO:3猴痘病毒蛋白M1R氨基酸序列为:
MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN
SEQ ID NO:4猴痘病毒蛋白H3L氨基酸序列为:
MAAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFI
SEQ ID NO:5猴痘病毒蛋白A29L氨基酸序列为:
MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKF EQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE
SEQ ID NO:6猴痘病毒蛋白E8L氨基酸序列为:
MPQQLSPINIETKKAISDARLKTLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNEYVLSTIHIYW
GKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGIIIIAIFLQVSDHKNVYFQKIVNQ
LDSIRSANMSAPFDSVFYLDNLLPSTLDYFTYLGTTINHSADAAWIIFPTPINIHSDQLSKFRTLLSSSN
HEGKPHYITENYRNPYKLNDDTQVYYSGEIIRAATTSPVRENYFMKWLSDLREACFSYYQKYIEGNK
TFAIIAIVFVFILTAILFLMSQRYSREKQN
SEQ ID NO:7猴痘病毒蛋白A30L氨基酸序列为:
MNSLSIFFIVVATAAVCLLFIQSYSIYENYGNIKEFNATHAAFEYSKSIGGTPALDRRVQDVNDTISDVKQKWRCVVYPGNGFVSASIFGFQAEVGPNNTRSIRKFNTMRQCIDFTFSDVINIDIYNPCIAPNINNTECQFLKSVL
SEQ ID NO:8猴痘病毒蛋白A5L氨基酸序列为:
MDFFNKFSQGLAESSTPKSSIYYSEEKDPDTKKDEAIEIGLKSQESYYQRQLREQLARDNMMTASRQPTQPLQPTIHITPQPVPTPTPAPILLPSSTAPVLKPRQQTNTSSDMSNLFDWLSTDTDAPASTLLPALTPSNTVQDIISKFNKDQKMTTPPSTQPSQTLPTTTCTQQSDGSISCTTPTVTPLQPPIVATVCTPTPTGGTVCTTAQQNPNPGAASQQNLDDMTLKDLMSSVEKDMRQLQAETNDLVTNVYDAREYTRRAIDQILQLVKGFERFQK
SEQ ID NO:9猴痘病毒蛋白C19L氨基酸序列为:
MWPFVSVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKL
KEVSEKGIKIIVLLDERGKRNLGELQSHSPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFT
GGSIHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFT
DSPEHLLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGSSYYWPDIYNSIIEAAINRGVKIRLL
VGNWDKNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGF
VSFNSIDKQLVSEAKKIFERDWVSSHSKSLKI
SEQ ID NO:10猴痘病毒蛋白H3L-B6R-A35R氨基酸序列为:
MAAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFIGGGGSGGGGSGGGGSKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLPGGGGSGGGGSGGGGSMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT
SEQ ID NO:11猴痘病毒蛋白H3L-B6R-M1R氨基酸序列为:
MAAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFIGGGGSGGGGSGGGGSKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLPGGGGSGGGGSGGGGSGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN
SEQ ID NO:12猴痘病毒蛋白H3L-M1R-B6R-A35R氨基酸序列为:
MAAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQ
WTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDI
TFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIK
SGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGV
MYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFIGGGGSGGGGSGGGGSGA
AASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSA
ATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYG
APGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKR
MLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQNGGGGSGGGGSGGGGSKTISVVTLL
CVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKM
CTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQ
PVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSC
KSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHI
IIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLPGGGGSGGGGSGGGGSMTPENDEEQTSVFSAT
VYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHR
KVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDT
WGSDGNPITKTTSDYQDSDVSQEVRKYFCT
SEQ ID NO:13猴痘病毒蛋白H3L-A35R-M1R氨基酸序列为:
MAAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFIGGGGSGGGGSGGGGSMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTGGGGSGGGGSGGGGSGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN
SEQ ID NO:14(GGGGS)3连接肽的氨基酸序列为:
GGGGSGGGGSGGGGS
SEQ ID NO:15编码猴痘病毒B6R蛋白的多核苷酸分子为:(mRNA为将T替换为U,以下序列相同原则)
ATGAAAACCATCTCCGTGGTGACCCTCCTGTGCGTGCTGCCCGCGGTGGTGTACAGCACGTGCACGGTGCCCACCATGAACAACGCCAAGCTCACCAGCACCGAGACCAGCTTCAACGACAAGCAGAAGGTCACCTTCACGTGCGACAGCGGCTACCACTCCCTGGACCCCAACGCCGTCTGCGAGACCGACAAGTGGAAGTACGAGAACCCCTGCAAGAAGATGTGCACCGTGTCCGACTACGTGTCCGAGCTGTACGACAAGCCCCTCTACGAGGTGAACAGCACCATGACCCTCTCCTGCAACGGGGAGACCAAGTACTTCCGCTGCGAGGAGAAGAACGGGAACACCAGCTGGAACGACACCGTCACGTGCCCCAACGCCGAGTGCCAGCCGCTGCAGCTGGAGCACGGCTCCTGCCAGCCGGTGAAGGAGAAGTACAGCTTCGGGGAGTACATGACCATCAACTGCGACGTGGGCTACGAGGTCATCGGGGTCTCCTACATCTCCTGCACGGCCAACAGCTGGAACGTCATCCCCAGCTGCCAGCAGAAGTGCGACATCCCCAGCCTGTCCAACGGCCTCATCAGCGGCAGCACCTTCTCCATCGGGGGCGTGATCCACCTGTCCTGCAAGAGCGGCTTCACCCTCACCGGCAGCCCCAGCAGCACGTGCATCGACGGGAAGTGGAACCCCATCCTGCCCACGTGCGTGCGGTCCAACGAGGAGTTCGACCCGGTGGACGACGGCCCGGACGACGAGACCGACCTGTCCAAGCTGTCCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGTCCCTGGAGGCCACCTACCACATCATCATCATGGCCCTCACCATCATGGGGGTCATCTTCCTCATCTCCATCATCGTGCTGGTCTGCAGCTGCGACAAGAACAACGACCAGTACAAGTTCCACAAGCTGCTGCCCTAA
SEQ ID NO:16编码猴痘病毒A35R蛋白的多核苷酸分子为:
ATGATGACGCCGGAGAACGACGAGGAGCAGACCAGCGTGTTCAGCGCCACCGTGTACGGGGACAAGATCCAGGGCAAGAACAAGCGGAAGCGGGTCATCGGCCTCTGCATCCGCATCTCCATGGTCATCTCCCTGCTGAGCATGATCACCATGAGCGCCTTCCTGATCGTCCGCCTGAACCAGTGCATGAGCGCCAACAAGGCCGCCATCACGGACAGCGCGGTGGCCGTGGCCGCCGCCAGCAGCACCCACCGGAAGGTGGTGTCCAGCACCACCCAGTACGACCACAAGGAGTCCTGCAACGGGCTCTACTACCAGGGCTCCTGCTACATCCTGCACAGCGACTACAAGTCCTTCGAGGACGCCAAGGCCAACTGCGCGGCGGAGAGCTCCACGCTGCCCAACAAGTCCGACGTCCTCACCACCTGGCTGATCGACTACGTGGAGGACACCTGGGGCTCCGACGGGAACCCCATCACCAAGACCACCAGCGACTACCAGGACTCCGACGTGTCCCAGGAGGTGCGCAAGTACTTCTGCACGTAA
SEQ ID NO:17编码猴痘病毒M1R蛋白的多核苷酸分子为:
ATGGGGGCGGCCGCCAGCATCCAGACCACCGTGAACACGCTCTCCGAGCGCATCTCCTCCAAGCTGGAGCAGGAGGCCAACGCCAGCGCCCAGACCAAGTGCGACATCGAGATCGGGAACTTCTACATCCGCCAGAACCACGGCTGCAACATCACGGTCAAGAACATGTGCAGCGCGGACGCGGACGCCCAGCTGGACGCGGTGCTGTCCGCCGCCACCGAGACCTACTCCGGCCTCACCCCCGAGCAGAAGGCCTACGTGCCGGCCATGTTCACGGCCGCCCTGAACATCCAGACCAGCGTCAACACGGTGGTGCGGGACTTCGAGAACTACGTGAAGCAGACGTGCAACTCCAGCGCGGTGGTGGACAACAAGCTGAAGATCCAGAACGTCATCATCGACGAGTGCTACGGGGCCCCCGGCAGCCCCACCAACCTGGAGTTCATCAACACGGGCTCCTCCAAGGGCAACTGCGCCATCAAGGCCCTCATGCAGCTCACCACCAAGGCCACCACCCAGATCGCCCCGCGCCAGGTGGCCGGCACGGGGGTGCAGTTCTACATGATCGTCATCGGGGTCATCATCCTGGCGGCCCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTCGCCAACAAGGAGAACGTGCACTGGACCACCTACATGGACACCTTCTTCCGGACCAGCCCCATGATCATCGCCACCACCGACATCCAGAACTAA
SEQ ID NO:18编码猴痘病毒H3L蛋白的多核苷酸分子为:
ATGGCGGCGGTCAAGACCCCGGTCATCGTGGTGCCCGTGATCGACCGGCCGCCCAGCGAGACCTTCCCCAACGTGCACGAGCACATCAACGACCAGAAGTTCGACGACGTGAAGGACAACGAGGTGATGCAGGAGAAGCGGGACGTGGTGATCGTCAACGACGACCCCGACCACTACAAGGACTACGTGTTCATCCAGTGGACGGGGGGCAACATCCGGGACGACGACAAGTACACCCACTTCTTCAGCGGCTTCTGCAACACCATGTGCACGGAGGAGACCAAGCGCAACATCGCCCGGCACCTGGCGCTCTGGGACTCCAAGTTCTTCACCGAGCTGGAGAACAAGAACGTGGAGTACGTGGTGATCATCGAGAACGACAACGTCATCGAGGACATCACCTTCCTGCGCCCGGTGCTGAAGGCCATCCACGACAAGAAGATCGACATCCTGCAGATGCGGGAGATCATCACCGGGAACAAGGTCAAGACGGAGCTGGTGATCGACAAGGACCACGCCATCTTCACCTACACGGGGGGCTACGACGTGTCCCTCAGCGCCTACATCATCCGCGTCACCACCGCCCTCAACATCGTGGACGAGATCATCAAGTCCGGGGGCCTCAGCAGCGGCTTCTACTTCGAGATCGCCCGGATCGAGAACGAGATGAAGATCAACCGCCAGATCATGGACAACAGCGCCAAGTACGTGGAGCACGACCCCCGGCTGGTGGCCGAGCACCGCTTCGAGACCATGAAGCCCAACTTCTGGTCCCGCATCGGGACGGTGGCCGCCAAGCGCTACCCCGGGGTCATGTACACCTTCACCACCCCCCTCATCTCCTTCTTCGGCCTGTTCGACATCAACGTCATCGGCCTGATCGTCATCCTGTTCATCATGTTCATGCTGATCTTCAACGTGAAGTCCAAGCTGCTGTGGTTCCTCACCGGCACCTTCGTCACGGCCTTCATCTAA
SEQ ID NO:19编码猴痘病毒A29L蛋白的多核苷酸分子为:
ATGGACGGGACCCTGTTCCCCGGGGACGACGACCTCGCCATCCCGGCGACCGAGTTCTTCAGCACGAAGGCCGCCAAGAACCCCGAGACCAAGCGGGAGGCCATCGTCAAGGCCTACGGCGACGACAACGAGGAGACCCTGAAGCAGCGCCTCACCAACCTGGAGAAGAAGATCACGAACATCACCACCAAGTTCGAGCAGATCGAGAAGTGCTGCAAGCGGAACGACGAGGTGCTGTTCCGGCTGGAGAACCACGCGGAGACCCTGCGCGCCGCCATGATCTCCCTCGCCAAGAAGATCGACGTGCAGACGGGCCGCCACCCCTACGAGTAA
SEQ ID NO:20编码猴痘病毒E8L蛋白的多核苷酸分子为:
ATGCCGCAGCAGCTCAGCCCCATCAACATCGAGACCAAGAAGGCCATCTCCGACGCGCGGCTCAAGACGCTGGACATCCACTACAACGAGTCCAAGCCCACCACCATCCAGAACACCGGGAAGCTGGTCCGGATCAACTTCAAGGGGGGCTACATCTCCGGGGGCTTCCTGCCCAACGAGTACGTCCTGAGCACCATCCACATCTACTGGGGGAAGGAGGACGACTACGGCTCCAACCACCTGATCGACGTGTACAAGTACAGCGGCGAGATCAACCTGGTGCACTGGAACAAGAAGAAGTACTCCAGCTACGAGGAGGCCAAGAAGCACGACGACGGCATCATCATCATCGCCATCTTCCTCCAGGTGTCCGACCACAAGAACGTGTACTTCCAGAAGATCGTCAACCAGCTGGACTCCATCCGGAGCGCCAACATGAGCGCGCCCTTCGACTCCGTGTTCTACCTGGACAACCTCCTGCCCAGCACGCTGGACTACTTCACCTACCTGGGCACCACCATCAACCACAGCGCGGACGCCGCCTGGATCATCTTCCCCACGCCCATCAACATCCACAGCGACCAGCTGTCCAAGTTCCGCACCCTGCTGAGCAGCAGCAACCACGAGGGGAAGCCGCACTACATCACCGAGAACTACCGCAACCCCTACAAGCTCAACGACGACACCCAGGTCTACTACAGCGGCGAGATCATCCGGGCCGCCACCACCAGCCCGGTGCGCGAGAACTACTTCATGAAGTGGCTGTCCGACCTCCGCGAGGCCTGCTTCTCCTACTACCAGAAGTACATCGAGGGGAACAAGACCTTCGCCATCATCGCCATCGTGTTCGTGTTCATCCTGACGGCCATCCTCTTCCTCATGTCCCAGCGCTACTCCCGGGAGAAGCAGAACTAA
SEQ ID NO:21编码猴痘病毒A30L蛋白的多核苷酸分子为:
ATGAACAGCCTGAGCATTTTCTTCATCGTGGTCGCCACCGCCGCTGTGTGCCTGCTGTTCATCCAGAGCTACTCTATCTATGAGAACTACGGCAACATCAAGGAATTCAACGCCACACACGCCGCCTTCGAGTACAGCAAGTCTATCGGCGGCACCCCTGCTCTGGACAGAAGAGTGCAGGACGTGAACGACACAATTAGCGATGTTAAGCAGAAATGGCGGTGCGTGGTGTACCCTGGAAATGGCTTCGTGTCCGCCAGCATCTTCGGCTTTCAGGCCGAAGTGGGACCTAACAACACAAGAAGCATCAGAAAGTTCAATACCATGCGGCAGTGCATCGACTTCACCTTCAGCGACGTGATCAACATCGATATCTACAACCCCTGTATCGCCCCAAATATCAACAACACCGAGTGCCAATTTCTGAAGTCCGTGCTGTAA
SEQ ID NO:22编码猴痘病毒A5L蛋白的多核苷酸分子为:
ATGGACTTCTTTAACAAGTTCAGCCAAGGCCTCGCCGAGAGCAGCACCCCCAAGAGCAGCATCTACTACAGCGAGGAAAAGGACCCTGATACCAAGAAGGACGAGGCCATCGAGATCGGCCTGAAGAGTCAGGAGTCCTACTATCAGAGGCAGCTGAGAGAACAGCTGGCTAGAGATAACATGATGACCGCCTCTAGACAGCCTACACAGCCTCTGCAGCCTACCATCCACATCACCCCCCAGCCCGTGCCTACCCCTACCCCCGCTCCTATCCTGCTGCCTTCTAGCACCGCCCCTGTGCTGAAGCCCAGACAGCAGACAAACACCAGCTCCGACATGAGCAATCTGTTCGACTGGCTGAGCACAGACACCGATGCCCCTGCCTCTACACTGCTGCCTGCTCTGACCCCTTCCAACACCGTGCAGGACATCATCAGCAAGTTTAACAAGGATCAGAAGATGACCACACCACCTAGCACCCAGCCTAGCCAGACCCTGCCAACTACCACCTGTACACAGCAGTCTGATGGCAGCATCAGCTGCACCACCCCTACAGTGACCCCACTGCAACCCCCAATTGTGGCCACCGTCTGCACACCCACCCCTACAGGCGGAACAGTGTGCACCACAGCCCAGCAGAACCCTAACCCCGGAGCCGCTAGCCAGCAAAACCTGGACGACATGACACTGAAAGATCTGATGAGCAGCGTGGAAAAAGACATGCGGCAACTGCAGGCCGAAACCAACGACCTGGTGACAAATGTGTACGACGCCAGAGAGTACACCAGACGGGCCATCGACCAGATCCTGCAGCTGGTTAAGGGCTTCGAGCGGTTCCAGAAATAA
SEQ ID NO:23编码猴痘病毒C19L蛋白的多核苷酸分子:
ATGTGGCCCTTCGTGAGCGTGCCCGCCGGAGCCAAATGCAGACTGGTCGAGACACTGCCC
GAAAACATGGACTTCCGGTCTGACCACCTGACCACATTCGAGTGCTTTAACGAGATCATT
ACATTGGCTAAAAAGTACATCTACATCGCCTCTTTTTGTTGTAATCCTCTGTCTACCACCC
GGGGCGCCCTTATTTTCGACAAACTGAAGGAAGTGTCTGAGAAGGGCATCAAGATCATC
GTGCTGCTGGATGAAAGAGGCAAGAGAAACCTGGGCGAGCTGCAAAGCCACAGCCCCGA
TATCAATTTCATCACTGTGAACATCGACAAGAAGAACAACGTCGGCCTGCTCCTGGGCTG
CTTCTGGGTGTCCGACGACGAACGGTGCTACGTGGGCAACGCCAGCTTTACCGGCGGAA
GCATCCACACAATCAAGACCCTGGGCGTTTATTCTGATTACCCTCCTCTGGCAACCGACC
TGAGGCGGCGGTTCGATACCTTCAAGGCCTTCAATTCTGCTAAAAATAGCTGGCTGAACC
TGTGTAGCGCTGCCTGCTGCCTGCCTGTGAGCACCGCCTACCACATCAAGAACCCAATCG
GCGGCGTGTTCTTCACCGATAGCCCTGAGCATCTGCTGGGATATTCCAGAGACCTGGACA
CCGACGTGGTGATCGATAAGCTGAAAAGCGCCAAGACAAGCATCGACATCGAACACCTG
GCCATCGTGCCTACAACCAGAGTGGACGGCAGCAGCTACTACTGGCCTGACATCTACAAC
AGCATCATCGAGGCCGCTATCAACAGAGGCGTGAAGATTAGACTGCTGGTGGGAAACTG
GGACAAGAACGATGTGTACAGCATGGCCACAGCCAGAAGCCTGGACGCCCTGTGCGTGC
AGAATGATCTGTCCGTTAAGGTGTTCACCATCCAGAACAACACCAAGCTGCTGATCGTGG
ATGACGAGTACGTGCATATCACCAGCGCCAACTTCGACGGCACCCACTACCAGAACCAC
GGCTTCGTGTCCTTTAACAGCATCGACAAGCAGCTGGTGTCTGAAGCCAAGAAGATCTTC
GAGAGAGACTGGGTCTCCAGCCACAGCAAGAGCCTGAAGATCTAA
SEQ ID NO:24编码猴痘病毒H3L-B6R-A35R融合蛋白的多核苷酸分子:ATGGCGGCGGTCAAGACCCCGGTCATCGTGGTGCCCGTGATCGACCGGCCGCCCAGCGAGACCTTCCCCAACGTGCACGAGCACATCAACGACCAGAAGTTCGACGACGTGAAGGACAACGAGGTGATGCAGGAGAAGCGGGACGTGGTGATCGTCAACGACGACCCCGACCACTACAAGGACTACGTGTTCATCCAGTGGACGGGGGGCAACATCCGGGACGACGACAAGTACACCCACTTCTTCAGCGGCTTCTGCAACACCATGTGCACGGAGGAGACCAAGCGCAACATCGCCCGGCACCTGGCGCTCTGGGACTCCAAGTTCTTCACCGAGCTGGAGAACAAGAACGTGGAGTACGTGGTGATCATCGAGAACGACAACGTCATCGAGGACATCACCTTCCTGCGCCCGGTGCTGAAGGCCATCCACGACAAGAAGATCGACATCCTGCAGATGCGGGAGATCATCACCGGGAACAAGGTCAAGACGGAGCTGGTGATCGACAAGGACCACGCCATCTTCACCTACACGGGGGGCTACGACGTGTCCCTCAGCGCCTACATCATCCGCGTCACCACCGCCCTCAACATCGTGGACGAGATCATCAAGTCCGGGGGCCTCAGCAGCGGCTTCTACTTCGAGATCGCCCGGATCGAGAACGAGATGAAGATCAACCGCCAGATCATGGACAACAGCGCCAAGTACGTGGAGCACGACCCCCGGCTGGTGGCCGAGCACCGCTTCGAGACCATGAAGCCCAACTTCTGGTCCCGCATCGGGACGGTGGCCGCCAAGCGCTACCCCGGGGTCATGTACACCTTCACCACCCCCCTCATCTCCTTCTTCGGCCTGTTCGACATCAACGTCATCGGCCTGATCGTCATCCTGTTCATCATGTTCATGCTGATCTTCAACGTGAAGTCCAAGCTGCTGTGGTTCCTCACCGGCACCTTCGTCACGGCCTTCATCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGAAAACCATCTCCGTGGTGACCCTCCTGTGCGTGCTGCCCGCGGTGGTGTACAGCACGTGCACGGTGCCCACCATGAACAACGCCAAGCTCACCAGCACCGAGACCAGCTTCAACGACAAGCAGAAGGTCACCTTCACGTGCGACAGCGGCTACCACTCCCTGGACCCCAACGCCGTCTGCGAGACCGACAAGTGGAAGTACGAGAACCCCTGCAAGAAGATGTGCACCGTGTCCGACTACGTGTCCGAGCTGTACGACAAGCCCCTCTACGAGGTGAACAGCACCATGACCCTCTCCTGCAACGGGGAGACCAAGTACTTCCGCTGCGAGGAGAAGAACGGGAACACCAGCTGGAACGACACCGTCACGTGCCCCAACGCCGAGTGCCAGCCGCTGCAGCTGGAGCACGGCTCCTGCCAGCCGGTGAAGGAGAAGTACAGCTTCGGGGAGTACATGACCATCAACTGCGACGTGGGCTACGAGGTCATCGGGGTCTCCTACATCTCCTGCACGGCCAACAGCTGGAACGTCATCCCCAGCTGCCAGCAGAAGTGCGACATCCCCAGCCTGTCCAACGGCCTCATCAGCGGCAGCACCTTCTCCATCGGGGGCGTGATCCACCTGTCCTGCAAGAGCGGCTTCACCCTCACCGGCAGCCCCAGCAGCACGTGCATCGACGGGAAGTGGAACCCCATCCTGCCCACGTGCGTGCGGTCCAACGAGGAGTTCGACCCGGTGGACGACGGCCCGGACGACGAGACCGACCTGTCCAAGCTGTCCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGTCCCTGGAGGCCACCTACCACATCATCATCATGGCCCTCACCATCATGGGGGTCATCTTCCTCATCTCCATCATCGTGCTGGTCTGCAGCTGCGACAAGAACAACGACCAGTACAAGTTCCACAAGCTGCTGCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGATGACGCCGGAGAACGACGAGGAGCAGACCAGCGTGTTCAGCGCCACCGTGTACGGGGACAAGATCCAGGGCAAGAACAAGCGGAAGCGGGTCATCGGCCTCTGCATCCGCATCTCCATGGTCATCTCCCTGCTGAGCATGATCACCATGAGCGCCTTCCTGATCGTCCGCCTGAACCAGTGCATGAGCGCCAACAAGGCCGCCATCACGGACAGCGCGGTGGCCGTGGCCGCCGCCAGCAGCACCCACCGGAAGGTGGTGTCCAGCACCACCCAGTACGACCACAAGGAGTCCTGCAACGGGCTCTACTACCAGGGCTCCTGCTACATCCTGCACAGCGACTACAAGTCCTTCGAGGACGCCAAGGCCAACTGCGCGGCGGAGAGCTCCACGCTGCCCAACAAGTCCGACGTCCTCACCACCTGGCTGATCGACTACGTGGAGGACACCTGGGGCTCCGACGGGAACCCCATCACCAAGACCACCAGCGACTACCAGGACTCCGACGTGTCCCAGGAGGTGCGCAAGTACTTCTGCACGTAA
SEQ ID NO:25编码猴痘病毒H3L-B6R-M1R融合蛋白的多核苷酸分子:
ATGGCGGCGGTCAAGACCCCGGTCATCGTGGTGCCCGTGATCGACCGGCCGCCCAGCGAGACCT
TCCCCAACGTGCACGAGCACATCAACGACCAGAAGTTCGACGACGTGAAGGACAACGAGGTGA
TGCAGGAGAAGCGGGACGTGGTGATCGTCAACGACGACCCCGACCACTACAAGGACTACGTGTT
CATCCAGTGGACGGGGGGCAACATCCGGGACGACGACAAGTACACCCACTTCTTCAGCGGCTTC
TGCAACACCATGTGCACGGAGGAGACCAAGCGCAACATCGCCCGGCACCTGGCGCTCTGGGACT
CCAAGTTCTTCACCGAGCTGGAGAACAAGAACGTGGAGTACGTGGTGATCATCGAGAACGACAA
CGTCATCGAGGACATCACCTTCCTGCGCCCGGTGCTGAAGGCCATCCACGACAAGAAGATCGAC
ATCCTGCAGATGCGGGAGATCATCACCGGGAACAAGGTCAAGACGGAGCTGGTGATCGACAAGG
ACCACGCCATCTTCACCTACACGGGGGGCTACGACGTGTCCCTCAGCGCCTACATCATCCGCGTC
ACCACCGCCCTCAACATCGTGGACGAGATCATCAAGTCCGGGGGCCTCAGCAGCGGCTTCTACTT
CGAGATCGCCCGGATCGAGAACGAGATGAAGATCAACCGCCAGATCATGGACAACAGCGCCAAG
TACGTGGAGCACGACCCCCGGCTGGTGGCCGAGCACCGCTTCGAGACCATGAAGCCCAACTTCT
GGTCCCGCATCGGGACGGTGGCCGCCAAGCGCTACCCCGGGGTCATGTACACCTTCACCACCCC
CCTCATCTCCTTCTTCGGCCTGTTCGACATCAACGTCATCGGCCTGATCGTCATCCTGTTCATCATG
TTCATGCTGATCTTCAACGTGAAGTCCAAGCTGCTGTGGTTCCTCACCGGCACCTTCGTCACGGC
CTTCATCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGAAAACCATC
TCCGTGGTGACCCTCCTGTGCGTGCTGCCCGCGGTGGTGTACAGCACGTGCACGGTGCCCACCAT
GAACAACGCCAAGCTCACCAGCACCGAGACCAGCTTCAACGACAAGCAGAAGGTCACCTTCAC
GTGCGACAGCGGCTACCACTCCCTGGACCCCAACGCCGTCTGCGAGACCGACAAGTGGAAGTAC
GAGAACCCCTGCAAGAAGATGTGCACCGTGTCCGACTACGTGTCCGAGCTGTACGACAAGCCCC
TCTACGAGGTGAACAGCACCATGACCCTCTCCTGCAACGGGGAGACCAAGTACTTCCGCTGCGA
GGAGAAGAACGGGAACACCAGCTGGAACGACACCGTCACGTGCCCCAACGCCGAGTGCCAGCC
GCTGCAGCTGGAGCACGGCTCCTGCCAGCCGGTGAAGGAGAAGTACAGCTTCGGGGAGTACATG
ACCATCAACTGCGACGTGGGCTACGAGGTCATCGGGGTCTCCTACATCTCCTGCACGGCCAACAG
CTGGAACGTCATCCCCAGCTGCCAGCAGAAGTGCGACATCCCCAGCCTGTCCAACGGCCTCATC
AGCGGCAGCACCTTCTCCATCGGGGGCGTGATCCACCTGTCCTGCAAGAGCGGCTTCACCCTCAC
CGGCAGCCCCAGCAGCACGTGCATCGACGGGAAGTGGAACCCCATCCTGCCCACGTGCGTGCGG
TCCAACGAGGAGTTCGACCCGGTGGACGACGGCCCGGACGACGAGACCGACCTGTCCAAGCTG
TCCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGTCCCTGGAGGCCACCTACCACATCATCA
TCATGGCCCTCACCATCATGGGGGTCATCTTCCTCATCTCCATCATCGTGCTGGTCTGCAGCTGCG
ACAAGAACAACGACCAGTACAAGTTCCACAAGCTGCTGCCCGGCGGCGGCGGCAGCGGCGGCG
GCGGCAGCGGCGGCGGCGGCAGCATGGGGGCGGCCGCCAGCATCCAGACCACCGTGAACACGC
TCTCCGAGCGCATCTCCTCCAAGCTGGAGCAGGAGGCCAACGCCAGCGCCCAGACCAAGTGCGA
CATCGAGATCGGGAACTTCTACATCCGCCAGAACCACGGCTGCAACATCACGGTCAAGAACATGT
GCAGCGCGGACGCGGACGCCCAGCTGGACGCGGTGCTGTCCGCCGCCACCGAGACCTACTCCG
GCCTCACCCCCGAGCAGAAGGCCTACGTGCCGGCCATGTTCACGGCCGCCCTGAACATCCAGAC
CAGCGTCAACACGGTGGTGCGGGACTTCGAGAACTACGTGAAGCAGACGTGCAACTCCAGCGC
GGTGGTGGACAACAAGCTGAAGATCCAGAACGTCATCATCGACGAGTGCTACGGGGCCCCCGGC
AGCCCCACCAACCTGGAGTTCATCAACACGGGCTCCTCCAAGGGCAACTGCGCCATCAAGGCCC
TCATGCAGCTCACCACCAAGGCCACCACCCAGATCGCCCCGCGCCAGGTGGCCGGCACGGGGGT
GCAGTTCTACATGATCGTCATCGGGGTCATCATCCTGGCGGCCCTGTTCATGTACTACGCCAAGCG
GATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTCGCCAACAAGGAGAACGTGCAC
TGGACCACCTACATGGACACCTTCTTCCGGACCAGCCCCATGATCATCGCCACCACCGACATCCA
GAACTAA
SEQ ID NO:26编码猴痘病毒H3L-M1R-B6R-A35R融合蛋白的多核苷酸分子:ATGGCGGCGGTCAAGACCCCGGTCATCGTGGTGCCCGTGATCGACCGGCCGCCCAGCGAGACCTTCCCCAACGTGCACGAGCACATCAACGACCAGAAGTTCGACGACGTGAAGGACAACGAGGTGATGCAGGAGAAGCGGGACGTGGTGATCGTCAACGACGACCCCGACCACTACAAGGACTACGTGTTCATCCAGTGGACGGGGGGCAACATCCGGGACGACGACAAGTACACCCACTTCTTCAGCGGCTTCTGCAACACCATGTGCACGGAGGAGACCAAGCGCAACATCGCCCGGCACCTGGCGCTCTGGGACTCCAAGTTCTTCACCGAGCTGGAGAACAAGAACGTGGAGTACGTGGTGATCATCGAGAACGACAACGTCATCGAGGACATCACCTTCCTGCGCCCGGTGCTGAAGGCCATCCACGACAAGAAGATCGACATCCTGCAGATGCGGGAGATCATCACCGGGAACAAGGTCAAGACGGAGCTGGTGATCGACAAGGACCACGCCATCTTCACCTACACGGGGGGCTACGACGTGTCCCTCAGCGCCTACATCATCCGCGTCACCACCGCCCTCAACATCGTGGACGAGATCATCAAGTCCGGGGGCCTCAGCAGCGGCTTCTACTTCGAGATCGCCCGGATCGAGAACGAGATGAAGATCAACCGCCAGATCATGGACAACAGCGCCAAGTACGTGGAGCACGACCCCCGGCTGGTGGCCGAGCACCGCTTCGAGACCATGAAGCCCAACTTCTGGTCCCGCATCGGGACGGTGGCCGCCAAGCGCTACCCCGGGGTCATGTACACCTTCACCACCCCCCTCATCTCCTTCTTCGGCCTGTTCGACATCAACGTCATCGGCCTGATCGTCATCCTGTTCATCATGTTCATGCTGATCTTCAACGTGAAGTCCAAGCTGCTGTGGTTCCTCACCGGCACCTTCGTCACGGCCTTCATCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGGGGGCGGCCGCCAGCATCCAGACCACCGTGAACACGCTCTCCGAGCGCATCTCCTCCAAGCTGGAGCAGGAGGCCAACGCCAGCGCCCAGACCAAGTGCGACATCGAGATCGGGAACTTCTACATCCGCCAGAACCACGGCTGCAACATCACGGTCAAGAACATGTGCAGCGCGGACGCGGACGCCCAGCTGGACGCGGTGCTGTCCGCCGCCACCGAGACCTACTCCGGCCTCACCCCCGAGCAGAAGGCCTACGTGCCGGCCATGTTCACGGCCGCCCTGAACATCCAGACCAGCGTCAACACGGTGGTGCGGGACTTCGAGAACTACGTGAAGCAGACGTGCAACTCCAGCGCGGTGGTGGACAACAAGCTGAAGATCCAGAACGTCATCATCGACGAGTGCTACGGGGCCCCCGGCAGCCCCACCAACCTGGAGTTCATCAACACGGGCTCCTCCAAGGGCAACTGCGCCATCAAGGCCCTCATGCAGCTCACCACCAAGGCCACCACCCAGATCGCCCCGCGCCAGGTGGCCGGCACGGGGGTGCAGTTCTACATGATCGTCATCGGGGTCATCATCCTGGCGGCCCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTCGCCAACAAGGAGAACGTGCACTGGACCACCTACATGGACACCTTCTTCCGGACCAGCCCCATGATCATCGCCACCACCGACATCCAGAACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGAAAACCATCTCCGTGGTGACCCTCCTGTGCGTGCTGCCCGCGGTGGTGTACAGCACGTGCACGGTGCCCACCATGAACAACGCCAAGCTCACCAGCACCGAGACCAGCTTCAACGACAAGCAGAAGGTCACCTTCACGTGCGACAGCGGCTACCACTCCCTGGACCCCAACGCCGTCTGCGAGACCGACAAGTGGAAGTACGAGAACCCCTGCAAGAAGATGTGCACCGTGTCCGACTACGTGTCCGAGCTGTACGACAAGCCCCTCTACGAGGTGAACAGCACCATGACCCTCTCCTGCAACGGGGAGACCAAGTACTTCCGCTGCGAGGAGAAGAACGGGAACACCAGCTGGAACGACACCGTCACGTGCCCCAACGCCGAGTGCCAGCCGCTGCAGCTGGAGCACGGCTCCTGCCAGCCGGTGAAGGAGAAGTACAGCTTCGGGGAGTACATGACCATCAACTGCGACGTGGGCTACGAGGTCATCGGGGTCTCCTACATCTCCTGCACGGCCAACAGCTGGAACGTCATCCCCAGCTGCCAGCAGAAGTGCGACATCCCCAGCCTGTCCAACGGCCTCATCAGCGGCAGCACCTTCTCCATCGGGGGCGTGATCCACCTGTCCTGCAAGAGCGGCTTCACCCTCACCGGCAGCCCCAGCAGCACGTGCATCGACGGGAAGTGGAACCCCATCCTGCCCACGTGCGTGCGGTCCAACGAGGAGTTCGACCCGGTGGACGACGGCCCGGACGACGAGACCGACCTGTCCAAGCTGTCCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGTCCCTGGAGGCCACCTACCACATCATCATCATGGCCCTCACCATCATGGGGGTCATCTTCCTCATCTCCATCATCGTGCTGGTCTGCAGCTGCGACAAGAACAACGACCAGTACAAGTTCCACAAGCTGCTGCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGATGACGCCGGAGAACGACGAGGAGCAGACCAGCGTGTTCAGCGCCACCGTGTACGGGGACAAGATCCAGGGCAAGAACAAGCGGAAGCGGGTCATCGGCCTCTGCATCCGCATCTCCATGGTCATCTCCCTGCTGAGCATGATCACCATGAGCGCCTTCCTGATCGTCCGCCTGAACCAGTGCATGAGCGCCAACAAGGCCGCCATCACGGACAGCGCGGTGGCCGTGGCCGCCGCCAGCAGCACCCACCGGAAGGTGGTGTCCAGCACCACCCAGTACGACCACAAGGAGTCCTGCAACGGGCTCTACTACCAGGGCTCCTGCTACATCCTGCACAGCGACTACAAGTCCTTCGAGGACGCCAAGGCCAACTGCGCGGCGGAGAGCTCCACGCTGCCCAACAAGTCCGACGTCCTCACCACCTGGCTGATCGACTACGTGGAGGACACCTGGGGCTCCGACGGGAACCCCATCACCAAGACCACCAGCGACTACCAGGACTCCGACGTGTCCCAGGAGGTGCGCAAGTACTTCTGCACGTAA
SEQ ID NO:27编码猴痘病毒H3L-A35R-M1R融合蛋白的多核苷酸分子:ATGGCGGCGGTCAAGACCCCGGTCATCGTGGTGCCCGTGATCGACCGGCCGCCCAGCGAGACCTTCCCCAACGTGCACGAGCACATCAACGACCAGAAGTTCGACGACGTGAAGGACAACGAGGTGATGCAGGAGAAGCGGGACGTGGTGATCGTCAACGACGACCCCGACCACTACAAGGACTACGTGTTCATCCAGTGGACGGGGGGCAACATCCGGGACGACGACAAGTACACCCACTTCTTCAGCGGCTTCTGCAACACCATGTGCACGGAGGAGACCAAGCGCAACATCGCCCGGCACCTGGCGCTCTGGGACTCCAAGTTCTTCACCGAGCTGGAGAACAAGAACGTGGAGTACGTGGTGATCATCGAGAACGACAACGTCATCGAGGACATCACCTTCCTGCGCCCGGTGCTGAAGGCCATCCACGACAAGAAGATCGACATCCTGCAGATGCGGGAGATCATCACCGGGAACAAGGTCAAGACGGAGCTGGTGATCGACAAGGACCACGCCATCTTCACCTACACGGGGGGCTACGACGTGTCCCTCAGCGCCTACATCATCCGCGTCACCACCGCCCTCAACATCGTGGACGAGATCATCAAGTCCGGGGGCCTCAGCAGCGGCTTCTACTTCGAGATCGCCCGGATCGAGAACGAGATGAAGATCAACCGCCAGATCATGGACAACAGCGCCAAGTACGTGGAGCACGACCCCCGGCTGGTGGCCGAGCACCGCTTCGAGACCATGAAGCCCAACTTCTGGTCCCGCATCGGGACGGTGGCCGCCAAGCGCTACCCCGGGGTCATGTACACCTTCACCACCCCCCTCATCTCCTTCTTCGGCCTGTTCGACATCAACGTCATCGGCCTGATCGTCATCCTGTTCATCATGTTCATGCTGATCTTCAACGTGAAGTCCAAGCTGCTGTGGTTCCTCACCGGCACCTTCGTCACGGCCTTCATCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGATGACGCCGGAGAACGACGAGGAGCAGACCAGCGTGTTCAGCGCCACCGTGTACGGGGACAAGATCCAGGGCAAGAACAAGCGGAAGCGGGTCATCGGCCTCTGCATCCGCATCTCCATGGTCATCTCCCTGCTGAGCATGATCACCATGAGCGCCTTCCTGATCGTCCGCCTGAACCAGTGCATGAGCGCCAACAAGGCCGCCATCACGGACAGCGCGGTGGCCGTGGCCGCCGCCAGCAGCACCCACCGGAAGGTGGTGTCCAGCACCACCCAGTACGACCACAAGGAGTCCTGCAACGGGCTCTACTACCAGGGCTCCTGCTACATCCTGCACAGCGACTACAAGTCCTTCGAGGACGCCAAGGCCAACTGCGCGGCGGAGAGCTCCACGCTGCCCAACAAGTCCGACGTCCTCACCACCTGGCTGATCGACTACGTGGAGGACACCTGGGGCTCCGACGGGAACCCCATCACCAAGACCACCAGCGACTACCAGGACTCCGACGTGTCCCAGGAGGTGCGCAAGTACTTCTGCACGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGGGGGCGGCCGCCAGCATCCAGACCACCGTGAACACGCTCTCCGAGCGCATCTCCTCCAAGCTGGAGCAGGAGGCCAACGCCAGCGCCCAGACCAAGTGCGACATCGAGATCGGGAACTTCTACATCCGCCAGAACCACGGCTGCAACATCACGGTCAAGAACATGTGCAGCGCGGACGCGGACGCCCAGCTGGACGCGGTGCTGTCCGCCGCCACCGAGACCTACTCCGGCCTCACCCCCGAGCAGAAGGCCTACGTGCCGGCCATGTTCACGGCCGCCCTGAACATCCAGACCAGCGTCAACACGGTGGTGCGGGACTTCGAGAACTACGTGAAGCAGACGTGCAACTCCAGCGCGGTGGTGGACAACAAGCTGAAGATCCAGAACGTCATCATCGACGAGTGCTACGGGGCCCCCGGCAGCCCCACCAACCTGGAGTTCATCAACACGGGCTCCTCCAAGGGCAACTGCGCCATCAAGGCCCTCATGCAGCTCACCACCAAGGCCACCACCCAGATCGCCCCGCGCCAGGTGGCCGGCACGGGGGTGCAGTTCTACATGATCGTCATCGGGGTCATCATCCTGGCGGCCCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTCGCCAACAAGGAGAACGTGCACTGGACCACCTACATGGACACCTTCTTCCGGACCAGCCCCATGATCATCGCCACCACCGACATCCAGAACTAA
SEQ ID NO:28编码(GGGGS)3连接肽的多核苷酸分子:
GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC
Claims (12)
1.一种mRNA组合物,其特征在于,所述组合物包括编码至少一种抗原肽或结构蛋白,所述抗原肽或结构蛋白选自猴痘病毒H3L蛋白、B6R蛋白、M1R蛋白、A35R蛋白、A29L蛋白、E8L蛋白、A30L蛋白、A5L蛋白、C19L蛋白或其同源蛋白中的一种或者多种,所述同源蛋白与上述蛋白具有至少75%同一性且与其具有相同或基本相同的免疫原性的氨基酸序列的蛋白或其抗原性片段,优选地,所述抗原肽或结构蛋白包括H3L蛋白或其同源蛋白。
2.根据权利要求1所述的mRNA组合物,其特征在于,所述抗原肽或结构蛋白包括两种或者两种以上的组合,所述两种或者两种以上的抗原肽或结构蛋白分别表达或者融合表达,优选地,所述抗原肽或结构蛋白分别表达,所述组合物包括多个mRNA。
3.根据权利要求1-2任一所述的mRNA组合物,其特征在于,所述抗原肽或结构蛋白包括如下表所示的组合:
其中,编号10-20,25-34中的抗原肽或结构蛋白分别表达,编号21-24中的抗原肽或结构蛋白融合表达。
4.根据权利要求1-3任一项所述的mRNA组合物,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;优选地,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(N1-Methyl-Pseudo-UTP)对天然尿苷进行全部替换。
5.根据权利要求1-4任一项所述的mRNA组合物,其特征在于,所述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。
6.根据权利要求1-5任一项所述的mRNA组合物,其特征在于,所述抗原肽或结构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。
7.一种正痘病毒属mRNA疫苗,其特征在于,由递送载体包裹的权利要求1-6任一项所述的组合物;优选地,所述递送载体为脂质纳米颗粒(LNP),所述mRNA包封于所述脂质纳米颗粒(LNP)中,优选地,所述组合物包括多个mRNA,所述递送载体分别包裹单个mRNA。
8.根据权利要求7所述的正痘病毒属mRNA疫苗,其特征在于,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。
9.根据权利要求7-8任一项所述的正痘病毒属mRNA疫苗,其特征在于,所述阳离子脂质:中性磷脂:甾族脂质:PEG-脂质摩尔比为30-60:1-20:20-50:0.1-10。
10.一种正痘病毒属mRNA疫苗的制备方法,其特征在于,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)-脂质溶解至溶剂后对权利要求1-6任一项所述mRNA组合物进行包封,优选地,所述原肽或结构蛋白包括两种或者两种以上的组合,所述两种或者两种以上的抗原肽或结构蛋白分别表达或者融合表达,制得多价疫苗。
11.根据权利要求10所述的制备方法,其特征在于,包封完成后,使用Tris缓冲液、乙酸盐缓冲液、柠檬酸盐缓冲液或磷酸盐缓冲液对包装样品进行稀释。
12.一种权利要求1-6任一项所述的mRNA组合物或权利要求7-9任一项所述的mRNA疫苗在制备用于预防或者治疗正痘病毒感染的药物中的应用;优选地,所述正痘病毒为猴痘病毒。
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