CN117815377A - mRNA的用途 - Google Patents
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Abstract
本发明属于生物领域,公开了一种核苷酸序列如SEQ ID NO.1所示mRNA作为制备预防和治疗禽流感感染的哺乳动物用疫苗的用途。本发明的H5亚型禽流感mRNA疫苗对禽流感毒株具有良好的保护作用。小鼠动物试验表明,以0.5μg剂量的mRNA疫苗免疫两次,即可诱导对异源禽流感毒株感染有100%的保护,显著降低攻毒后小鼠肺脏的载毒量。证明了本发明H5亚型的禽流感mRNA‑LNPs疫苗具有有效性和一定的广谱性,可以为我国禽流感的防控提供重要的技术储备。
Description
技术领域
本发明涉及生物领域,具体为一种mRNA的用途。
背景技术
禽流感病毒自发现以来,就对家禽及野禽的生产和健康产生巨大的影响。H5亚型禽流感病毒,尤其是高致病性H5亚型禽流感病毒,对家禽生产的影响是所有禽流感病毒亚型中最为严重的,其不仅导致家禽严重发病,而且部分病毒还可以感染人并引发严重临床症状和死亡,使得全球对H5亚型禽流感病毒保持高度戒备。我国现有的禽流感防控策略是采取动物疫病防控的三大措施:疫苗免疫、疫情监测和染疫动物捕杀。
目前我国用于防控禽流感的疫苗主要是全病毒灭活疫苗。全病毒灭活疫苗能够诱导机体产生有效的免疫应答反应,在以往控制疫情蔓延和扩散过程中发挥了重要作用。但是接种灭活疫苗的机体产生针对病毒内部结构蛋白(如NP)的特异性抗体,而这类抗体是进行疾病流行病学调查时的主要判定标准,所以灭活疫苗的应用将导致无法区分自然感染和疫苗接种,从而干扰禽流感疫情的检测和流行病学调查。
mRNA疫苗是一种将RNA在体外进行相关的修饰后传递至机体细胞内表达并产生蛋白抗原,从而导致机体产生针对该抗原的免疫应答。mRNA疫苗在体内产生中和抗体主要通过进入三类细胞:1)体细胞(包括肌肉细胞和皮下细胞等)、2)注射部位的组织驻留免疫细胞、3)二级淋巴组织的免疫细胞(包括淋巴结和脾脏),进而发挥体液和细胞的双重免疫作用。
与传统灭活疫苗相比,mRNA疫苗由于其特殊的抗原表达和展示机制,能有效激活细胞和体液免疫反应,免疫效果优良;在新冠疫情期间,两款SARS-Cov-19mRNA疫苗(BNT162b2和mRNA-1273)被快速开发并广泛使用,显示出了出色的免疫保护作用。与灭活疫苗、亚单位和DNA疫苗等其他疫苗相比,mRNA疫苗生产过程不涉及活病毒增殖,不需要佐剂,不含病毒成分,无感染风险,不会整合进宿主基因组,因此具有优良的安全性。此外,mRNA疫苗还具有研发快速、易于规模化生产等优点。因此,mRNA疫苗在应对传染性疾病,尤其是新发、突发传染性疾病方面具有独特优势。
一般来说,禽用mRNA疫苗和哺乳动物用mRNA疫苗是不同的,本项目的初衷是用来研制禽用mRNA疫苗,在研究过程中,我们发现了一些新的现象,基于此提出本案。
本案解决的技术问题是:如何开发出一种适用于哺乳动物的H5亚型的mRNA疫苗。
发明内容
本发明的第一目的在于提供一种mRNA的用途,本发明的H5亚型禽流感mRNA疫苗对禽流感毒株具有良好的保护作用。小鼠动物试验表明,以0.5μg剂量的mRNA疫苗免疫两次,即可诱导对异源禽流感毒株感染有100%的保护,显著降低攻毒后小鼠肺脏的载毒量。证明了本发明H5亚型的禽流感mRNA-LNPs疫苗具有有效性和一定的广谱性,可以为我国禽流感的防控提供重要的技术储备。
作为本领域技术人员公知的,当一款疫苗在小鼠身上表现出较好的免疫原性时,其在人、猪、马、狗、猫、水貂等身上至少针对同种病毒抗原具有同等的免疫原性。
本发明提供如下技术方案:
核苷酸序列如SEQ ID NO.1所示mRNA作为制备预防和治疗禽流感感染的哺乳动物用疫苗的用途。
更为具体来说,所述哺乳动物用疫苗包括脂质体混合物和包裹在脂质体内的mRNA;所述疫苗中的脂质体的平均粒径为50-150nm。
优选地,所述哺乳动物用疫苗中的脂质体的平均粒径为70-100nm。
更为优选地,所述脂质体混合物包括阳离子脂、DSPC、胆固醇、PEG200-DMG,阳离子脂、DSPC、胆固醇、PEG200-DMG的摩尔比为50:10:38.5:1.5。
在上述的用途中,所述哺乳动物用疫苗用于免疫鼠、人、猪、马、狗、猫或水貂。
与现有技术相比,本发明的突出特点在于:
本发明选取禽流感病毒的HA抗原编码序列并进行优化设计,构建了禽流感mRNA疫苗。将H5亚型禽流感的HA蛋白编码序列进行密码子优化后,序列前端添加T7启动子、5’端非翻译区(5’UTR)和Kozak序列,后端添加3’端非翻译区(3’UTR)和多聚腺苷酸尾(Poly A),构成mRNA疫苗模板DNA序列。将该序列分别连接在质粒载体上构成重组质粒pUC57-H5-HA。通过将重组质粒进行线性化、转录、加帽,获得mRNA,并用LNP技术包装,制成H5亚型禽流感mRNA疫苗。
本发明的H5亚型禽流感mRNA疫苗对禽流感毒株具有良好的保护作用。小鼠动物试验表明,以0.5μg剂量的mRNA疫苗免疫两次,即可诱导对异源禽流感毒株感染有100%的保护,显著降低攻毒后小鼠肺脏的载毒量。证明了本发明H5亚型的禽流感mRNA-LNPs疫苗具有有效性和一定的广谱性,可以为我国禽流感的防控提供重要的技术储备。
通过和本申请人的在先申请CN202310509346.3,主题为一种禽流感病毒mRNA疫苗及其制备方法和应用中所涉及的同类mRNA疫苗对比可以发现,本发明的mRNA疫苗作用于哺乳动物后,能够在更低剂量的时候表现出更高的抗体水平。
附图说明
图1为实施例1中重组质粒pUC57-H5-HA的质粒图谱;
图2为实施例2中重组质粒线性化酶切产物的电泳鉴定图;
图3为实施例2中转录加帽后mRNA的电泳鉴定图;
图4为实施例3中HA蛋白的WB检测结果;
图5为实施例3中HA蛋白的三聚体WB鉴定结果;
图6为实施例4中mRNA-LNPs粒径分析结果;
图7为实施例5的小鼠免疫后血清HI效价结果;
图8为实施例5的小鼠攻毒后平均体重变化结果;
图9为实施例5的小鼠肺脏排毒量检测结果;
图10为实施例5的小鼠肺脏细胞因子的检测结果;
图11为实施例1的HA基因序列进行密码子优化前后对比图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。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。
本发明实施例中涉及的H5N1亚型禽流感病毒为A/Chicken/Shandong/WFZC/2017(H5N1)(H5N1-SD57),由华南农业大学兽医学院国家禽流感专业实验室(广州)提供,已在中国专利申请(申请号为“202310079761.X”、申请名称为“一种抗H5N1亚型禽流感的病毒样颗粒疫苗及其制备方法和应用”)中公开(文中命名为:H5N1-SD57)。
本发明实施例中涉及的H5N1-D889毒株为A/Chicken/Guangdong/D889/2015(H5N1),由华南农业大学兽医学院国家禽流感专业实验室(广州)提供,已在中国专利申请(申请号为“201910117092.4”、申请名称为“基于MultiBac杆状病毒表达系统的禽流感疫苗及制备与应用”)中公开(文中命名为:H5N1-D889)。
实施例1禽流感病毒mRNA疫苗的抗原表达载体的构建
禽流感病毒mRNA疫苗序列包含以下元件:T7启动子、5’端非翻译区(5’UTR)、HA抗原编码基因、3’端非翻译区(3’UTR)和多聚腺苷酸(Poly A),并且在Poly A尾结构下游连接有质粒线性化的酶切位点(Eco31I)。
参考禽流感病毒A/Chicken/Shandong/WFZC/2017(H5N1)(H5N1-SD57)的HA基因编码序列,设计本发明中mRNA疫苗的HA抗原编码序列。在HA蛋白的C端添加His标签。将源于A/Chicken/Shandong/WFZC/2017(H5N1)(H5N1-SD57)的HA基因序列进行密码子优化。优化前后的对比图可见图11;
mRNA疫苗的抗原表达序列(SEQ ID NO.2)如下:
TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAGGCCGCCACCATGGAAAAAATTGTTCTGCTGTTTGCTACCATTAGCTTAGTGAAATCAGATCATATTTGCATCGGCTACCACGCCAATAATTCCACCGAGCAAGTGGACACTATCATGGAGAAGAATGTGACTGTCACTCATGCAAAGGATATTTTGGAGAAGACACACAACGGAAAGCTGTGTGACCTCAATGGGGTCAAACCCCTAATTCTCAAAGATTGCAGTGTAGCTGGTTGGCTGCTGGGAAACCCTCTCTGTGATGAGTTCACCAATGTACCAGAATGGTCTTATATAGTCGAGAAAGCTAATCCTGCAAACGATCTCTGCTACCCTGGGAAGTTTAATGACTATGAAGAACTCAAGCACCTCTTATCTAGAATAAATCACTTCGAGAAGATCCAGATTATACCTAAGGACAGCTGGAGTGACCATGAAGCCTCTCTGGGAGTTAGCGCAGCATGCTCATATCAAGGATCCAGTTCCTTTTTCAGGAATGTTGTTTGGCTAATCAAAAAAGACAACGCCTATCCCACGATCAAGAAGTCATACAATAACACGAATCGAGAGGATTTACTGATCCTCTGGGGAATTCATCACCCGAATGATGAAGCAGAGCAGACAAAGCTTTACCAAAACCCCACCACATACATCAGCATTGGTACTTCAACACTGAACCAGAGGCTGGTGCCAAAGATTGCCACCCGCAGCAAGATCAACGGCCAGAGTGGGCGCATCGACTTCTTCTGGACTATACTTAAACCAAACGACGCCATTCATTTTGAGTCTAATGGGAACTTCATCGCACCAGAATATGCCTACAAAATTGTAAAAAAGGGAGATAGTACCATCATGAGGAGTGAGGTGGAGTATGGCAACTGTAACACGAGATGCCAGACGCCCGTGGGTGCGATTAATTCAAGCATGCCTTTTCACAATATCCATCCGCTTACCATAGGAGAATGTCCCAAATACGTGAAAAGCAACAAACTGGTGCTGGCGACTGGCCTGCGGAACTCTCCACAAAGAGAGAGCAGAGGGCTTTTTGGTGCAATCGCTGGCTTCATTGAGGGAGGTTGGCAGGGCATGGTGGATGGTTGGTACGGCTATCACCATTCTAATGAACAAGGGAGCGGCTACGCAGCCGATAAGGAATCGACACAGAAAGCTATAGATGGTGTGACAAACAAGGTAAACAGCATCATCGACAAGATGAATACTCAGTTCGAGGCAGTCGGCAGGGAATTCAACAATCTGGAGCGTCGGATTGAAAACTTAAATAAAAAAATGGAAGATGGATTTCTCGACGTTTGGACATACAATGCGGAACTGTTGGTCCTGATGGAAAATGAGAGGACTTTGGACTTTCATGATAGTAACGTGAAAAACCTATATGACAAAGTTCGATTACAGCTGAAGGACAATGCTAAAGAGCTTGGAAATGGATGTTTCGAATTTTATCACAAGTGCAACAACGAGTGCATGGAGTCTGTCCGGAACGGAACATATGATTACCCACAGTACAGCGAGGAAGCTCGTCTGAAGCGCGAGGAAATATCAGGGGTGAAGCTTGAATCCATCGGTATCTACCAGATCCTGAGCATATACAGTACAGTGGCCTCATCCTTGGTCTTGGCCATTATGATGGCTGGGCTTTCTCTGTGGATGTGTTCCAACGGCTCGCTACAGTGCAGAATATGTATTCACCACCATCATCACCACTGAGGTACCGATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGATCTAGAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGACC
疫苗转录后对应的mRNA序列(SEQ ID NO.1)如下:
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCGCCACCAUGGAAAAAAUUGUUCUGCUGUUUGCUACCAUUAGCUUAGUGAAAUCAGAUCAUAUUUGCAUCGGCUACCACGCCAAUAAUUCCACCGAGCAAGUGGACACUAUCAUGGAGAAGAAUGUGACUGUCACUCAUGCAAAGGAUAUUUUGGAGAAGACACACAACGGAAAGCUGUGUGACCUCAAUGGGGUCAAACCCCUAAUUCUCAAAGAUUGCAGUGUAGCUGGUUGGCUGCUGGGAAACCCUCUCUGUGAUGAGU
UCACCAAUGUACCAGAAUGGUCUUAUAUAGUCGAGAAAGCUAAUCCUGCAAACG
AUCUCUGCUACCCUGGGAAGUUUAAUGACUAUGAAGAACUCAAGCACCUCUUAUC
UAGAAUAAAUCACUUCGAGAAGAUCCAGAUUAUACCUAAGGACAGCUGGAGUGA
CCAUGAAGCCUCUCUGGGAGUUAGCGCAGCAUGCUCAUAUCAAGGAUCCAGUUCC
UUUUUCAGGAAUGUUGUUUGGCUAAUCAAAAAAGACAACGCCUAUCCCACGAUC
AAGAAGUCAUACAAUAACACGAAUCGAGAGGAUUUACUGAUCCUCUGGGGAAUU
CAUCACCCGAAUGAUGAAGCAGAGCAGACAAAGCUUUACCAAAACCCCACCACAU
ACAUCAGCAUUGGUACUUCAACACUGAACCAGAGGCUGGUGCCAAAGAUUGCCAC
CCGCAGCAAGAUCAACGGCCAGAGUGGGCGCAUCGACUUCUUCUGGACUAUACUU
AAACCAAACGACGCCAUUCAUUUUGAGUCUAAUGGGAACUUCAUCGCACCAGAAU
AUGCCUACAAAAUUGUAAAAAAGGGAGAUAGUACCAUCAUGAGGAGUGAGGUGG
AGUAUGGCAACUGUAACACGAGAUGCCAGACGCCCGUGGGUGCGAUUAAUUCAA
GCAUGCCUUUUCACAAUAUCCAUCCGCUUACCAUAGGAGAAUGUCCCAAAUACGU
GAAAAGCAACAAACUGGUGCUGGCGACUGGCCUGCGGAACUCUCCACAAAGAGAG
AGCAGAGGGCUUUUUGGUGCAAUCGCUGGCUUCAUUGAGGGAGGUUGGCAGGGC
AUGGUGGAUGGUUGGUACGGCUAUCACCAUUCUAAUGAACAAGGGAGCGGCUAC
GCAGCCGAUAAGGAAUCGACACAGAAAGCUAUAGAUGGUGUGACAAACAAGGUA
AACAGCAUCAUCGACAAGAUGAAUACUCAGUUCGAGGCAGUCGGCAGGGAAUUC
AACAAUCUGGAGCGUCGGAUUGAAAACUUAAAUAAAAAAAUGGAAGAUGGAUUU
CUCGACGUUUGGACAUACAAUGCGGAACUGUUGGUCCUGAUGGAAAAUGAGAGG
ACUUUGGACUUUCAUGAUAGUAACGUGAAAAACCUAUAUGACAAAGUUCGAUUA
CAGCUGAAGGACAAUGCUAAAGAGCUUGGAAAUGGAUGUUUCGAAUUUUAUCAC
AAGUGCAACAACGAGUGCAUGGAGUCUGUCCGGAACGGAACAUAUGAUUACCCA
CAGUACAGCGAGGAAGCUCGUCUGAAGCGCGAGGAAAUAUCAGGGGUGAAGCUU
GAAUCCAUCGGUAUCUACCAGAUCCUGAGCAUAUACAGUACAGUGGCCUCAUCCU
UGGUCUUGGCCAUUAUGAUGGCUGGGCUUUCUCUGUGGAUGUGUUCCAACGGCU
CGCUACAGUGCAGAAUAUGUAUUCACCACCAUCAUCACCACUGAGGUACCGAUAU
CUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCC
CAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG
AUCUAGAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AA
其中包含T7启动子序列为:
TAATACGACTCACTATAGG。
5’UTR序列为:
GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCC TCGAG。
3’UTR序列为:
GGTACCGATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCT TGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAA TAAAGTCTGA。
poly A序列为50-150A,优选为101A,序列为:
AAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
HA基因核苷酸序列(SEQ ID NO.3)如下:
ATGGAAAAAATTGTTCTGCTGTTTGCTACCATTAGCTTAGTGAAATCAGATCAT
ATTTGCATCGGCTACCACGCCAATAATTCCACCGAGCAAGTGGACACTATCATGGAG
AAGAATGTGACTGTCACTCATGCAAAGGATATTTTGGAGAAGACACACAACGGAAA
GCTGTGTGACCTCAATGGGGTCAAACCCCTAATTCTCAAAGATTGCAGTGTAGCTGG
TTGGCTGCTGGGAAACCCTCTCTGTGATGAGTTCACCAATGTACCAGAATGGTCTTA
TATAGTCGAGAAAGCTAATCCTGCAAACGATCTCTGCTACCCTGGGAAGTTTAATGA
CTATGAAGAACTCAAGCACCTCTTATCTAGAATAAATCACTTCGAGAAGATCCAGAT
TATACCTAAGGACAGCTGGAGTGACCATGAAGCCTCTCTGGGAGTTAGCGCAGCAT
GCTCATATCAAGGATCCAGTTCCTTTTTCAGGAATGTTGTTTGGCTAATCAAAAAAG
ACAACGCCTATCCCACGATCAAGAAGTCATACAATAACACGAATCGAGAGGATTTA
CTGATCCTCTGGGGAATTCATCACCCGAATGATGAAGCAGAGCAGACAAAGCTTTA
CCAAAACCCCACCACATACATCAGCATTGGTACTTCAACACTGAACCAGAGGCTGG
TGCCAAAGATTGCCACCCGCAGCAAGATCAACGGCCAGAGTGGGCGCATCGACTTC
TTCTGGACTATACTTAAACCAAACGACGCCATTCATTTTGAGTCTAATGGGAACTTC
ATCGCACCAGAATATGCCTACAAAATTGTAAAAAAGGGAGATAGTACCATCATGAG
GAGTGAGGTGGAGTATGGCAACTGTAACACGAGATGCCAGACGCCCGTGGGTGCGA
TTAATTCAAGCATGCCTTTTCACAATATCCATCCGCTTACCATAGGAGAATGTCCCA
AATACGTGAAAAGCAACAAACTGGTGCTGGCGACTGGCCTGCGGAACTCTCCACAA
AGAGAGAGCAGAGGGCTTTTTGGTGCAATCGCTGGCTTCATTGAGGGAGGTTGGCA
GGGCATGGTGGATGGTTGGTACGGCTATCACCATTCTAATGAACAAGGGAGCGGCT
ACGCAGCCGATAAGGAATCGACACAGAAAGCTATAGATGGTGTGACAAACAAGGT
AAACAGCATCATCGACAAGATGAATACTCAGTTCGAGGCAGTCGGCAGGGAATTCA
ACAATCTGGAGCGTCGGATTGAAAACTTAAATAAAAAAATGGAAGATGGATTTCTC
GACGTTTGGACATACAATGCGGAACTGTTGGTCCTGATGGAAAATGAGAGGACTTT
GGACTTTCATGATAGTAACGTGAAAAACCTATATGACAAAGTTCGATTACAGCTGA
AGGACAATGCTAAAGAGCTTGGAAATGGATGTTTCGAATTTTATCACAAGTGCAAC
AACGAGTGCATGGAGTCTGTCCGGAACGGAACATATGATTACCCACAGTACAGCGA
GGAAGCTCGTCTGAAGCGCGAGGAAATATCAGGGGTGAAGCTTGAATCCATCGGTA
TCTACCAGATCCTGAGCATATACAGTACAGTGGCCTCATCCTTGGTCTTGGCCATTA
TGATGGCTGGGCTTTCTCTGTGGATGTGTTCCAACGGCTCGCTACAGTGCAGAATAT
GTATTCACCACCATCATCACCACTGA
HA基因mRNA序列如下:
AUGGAAAAAAUUGUUCUGCUGUUUGCUACCAUUAGCUUAGUGAAAUCAGAU
CAUAUUUGCAUCGGCUACCACGCCAAUAAUUCCACCGAGCAAGUGGACACUAUCA
UGGAGAAGAAUGUGACUGUCACUCAUGCAAAGGAUAUUUUGGAGAAGACACACA
ACGGAAAGCUGUGUGACCUCAAUGGGGUCAAACCCCUAAUUCUCAAAGAUUGCA
GUGUAGCUGGUUGGCUGCUGGGAAACCCUCUCUGUGAUGAGUUCACCAAUGUAC
CAGAAUGGUCUUAUAUAGUCGAGAAAGCUAAUCCUGCAAACGAUCUCUGCUACCC
UGGGAAGUUUAAUGACUAUGAAGAACUCAAGCACCUCUUAUCUAGAAUAAAUCA
CUUCGAGAAGAUCCAGAUUAUACCUAAGGACAGCUGGAGUGACCAUGAAGCCUC
UCUGGGAGUUAGCGCAGCAUGCUCAUAUCAAGGAUCCAGUUCCUUUUUCAGGAA
UGUUGUUUGGCUAAUCAAAAAAGACAACGCCUAUCCCACGAUCAAGAAGUCAUA
CAAUAACACGAAUCGAGAGGAUUUACUGAUCCUCUGGGGAAUUCAUCACCCGAA
UGAUGAAGCAGAGCAGACAAAGCUUUACCAAAACCCCACCACAUACAUCAGCAUU
GGUACUUCAACACUGAACCAGAGGCUGGUGCCAAAGAUUGCCACCCGCAGCAAGA
UCAACGGCCAGAGUGGGCGCAUCGACUUCUUCUGGACUAUACUUAAACCAAACGA
CGCCAUUCAUUUUGAGUCUAAUGGGAACUUCAUCGCACCAGAAUAUGCCUACAAA
AUUGUAAAAAAGGGAGAUAGUACCAUCAUGAGGAGUGAGGUGGAGUAUGGCAAC
UGUAACACGAGAUGCCAGACGCCCGUGGGUGCGAUUAAUUCAAGCAUGCCUUUUC
ACAAUAUCCAUCCGCUUACCAUAGGAGAAUGUCCCAAAUACGUGAAAAGCAACAA
ACUGGUGCUGGCGACUGGCCUGCGGAACUCUCCACAAAGAGAGAGCAGAGGGCUU
UUUGGUGCAAUCGCUGGCUUCAUUGAGGGAGGUUGGCAGGGCAUGGUGGAUGGU
UGGUACGGCUAUCACCAUUCUAAUGAACAAGGGAGCGGCUACGCAGCCGAUAAG
GAAUCGACACAGAAAGCUAUAGAUGGUGUGACAAACAAGGUAAACAGCAUCAUC
GACAAGAUGAAUACUCAGUUCGAGGCAGUCGGCAGGGAAUUCAACAAUCUGGAG
CGUCGGAUUGAAAACUUAAAUAAAAAAAUGGAAGAUGGAUUUCUCGACGUUUGG
ACAUACAAUGCGGAACUGUUGGUCCUGAUGGAAAAUGAGAGGACUUUGGACUUU
CAUGAUAGUAACGUGAAAAACCUAUAUGACAAAGUUCGAUUACAGCUGAAGGAC
AAUGCUAAAGAGCUUGGAAAUGGAUGUUUCGAAUUUUAUCACAAGUGCAACAAC
GAGUGCAUGGAGUCUGUCCGGAACGGAACAUAUGAUUACCCACAGUACAGCGAG
GAAGCUCGUCUGAAGCGCGAGGAAAUAUCAGGGGUGAAGCUUGAAUCCAUCGGU
AUCUACCAGAUCCUGAGCAUAUACAGUACAGUGGCCUCAUCCUUGGUCUUGGCCA
UUAUGAUGGCUGGGCUUUCUCUGUGGAUGUGUUCCAACGGCUCGCUACAGUGCA
GAAUAUGUAUUCACCACCAUCAUCACCACUGA
HA基因对应的氨基酸序列:
MEKIVLLFATISLVKSDHICIGYHANNSTEQVDTIMEKNVTVTHAKDILEKTHNGKL
CDLNGVKPLILKDCSVAGWLLGNPLCDEFTNVPEWSYIVEKANPANDLCYPGKFNDYE
ELKHLLSRINHFEKIQIIPKDSWSDHEASLGVSAACSYQGSSSFFRNVVWLIKKDNAYPTI
KKSYNNTNREDLLILWGIHHPNDEAEQTKLYQNPTTYISIGTSTLNQRLVPKIATRSKING
QSGRIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTIMRSEVEYGNCNTRCQTP
VGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRESRGLFGAIAGFIEGGWQ
GMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNN
LERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLKDN
AKELGNGCFEFYHKCNNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLVLAIMMAGLSLWMCSNGSLQCRICIHHHHHH.
mRNA疫苗序列委托南京金斯瑞生物科技公司合成,并克隆至pUC57-Kan载体。疫苗序列包含T7启动子、5’端非翻译区(5’UTR)、经密码子优化的HA基因、3’端非翻译区(3’UTR)和多聚腺苷酸(Poly A)。
所合成的DNA序列经测序比对验证后,使用质粒提取试剂盒提取质粒(购自天根生化科技有限公司,Cat.#DP103-03),并将质粒命名为pUC57-H5-HA(质粒图谱如图1所示),使用N50超微量紫外可见光分光光度计测量质粒浓度,并保存在-20℃备用。
实施例2mRNA转录验证试验
1.质粒pUC57-H5-HA的线性化
将实施例1中所获得的质粒用FastDigest Eco31I(IIs型)限制性核酸内切酶进行酶切(购自赛默飞世尔科技,货号FD0294),反应体系如下表1所示:
表1限制性内切酶FastDigest Eco31I酶切体系反应
轻柔混匀后,瞬离。37℃反应30min,65℃反应5min,灭活内切酶。随后用1%核酸凝胶电泳鉴定质粒,150V 20min。结果如图2所示,目的条带约5000bp,与预期大小一致。最后使用胶回收试剂盒回收线性化质粒(Gel Extraction Kit,购自OMEGA BIO-TEK),使用/>N50超微量紫外可见光分光光度计测量质粒浓度,并保存在-20℃备用。
2.线性化质粒的转录
使用mRNA转录试剂盒T7 High Yield RNA Transcription Kit(N1-Me-PseudoUTP),购自南京诺唯赞生物科技公司。按照产品说明书转录mRNA,将除T7 RNA PolymeraseMix外的组分振荡混匀,短暂离心收集于管底,冰上储存备用。依次加入以下各组分:
线性化质粒体外转录体系
用移液器轻轻混匀各组分,并短暂离心收集,37℃孵育4h。在反应体系中加入2μLDNase I,37℃孵育30min,消化转录的DNA模板。
3.mRNA的纯化
用氯化锂法沉淀纯化mRNA(8M LiCl(DNase/RNase free),购自上海碧云天生物技术有限公司),具体纯化步骤如下:取30μL DEPC水加入上述转录体系中,然后加入22.7μL8MLiCl(DNase/RNase free)以调节LiCl至工作浓度2.5M,置于-20℃孵育30min。将产物转移至1.5mL EP管,15,000g离心10min。弃去上清,加入1mL 70%乙醇溶液,上下颠倒数次以洗涤RNA团块,15,000g离心10min。重复上述洗涤步骤。根据RNA团块大小,加入适量体积DEPC水,室温孵育5min以充分溶解RNA,然后用移液器吹打数次,最后分装1-2μL,使用N50超微量紫外可见光分光光度计测量质粒浓度,并保存在-20℃备用。
4.mRNA的加帽反应
将纯化后RNA使用加帽试剂盒进行加帽(Vaccinia Capping System和mRNACap2'-O-Methyltransferase),加帽试剂盒购自南京诺唯赞生物科技公司,按照产品说明书转录mRNA,具体步骤如下:加帽反应效率受RNA 5’端结构影响,因此通过热变性(65℃加热5min,冰上放置5min)打开RNA 5’端的高级结构。依次加入以下各组分:
加帽反应体系
反应条件:37℃反应1h。
5.mRNA的纯化与大小鉴定
用氯化锂法沉淀纯化mRNA(8M LiCl(DNase/RNase free),购自上海碧云天生物技术有限公司),具体纯化步骤与本实施例步骤3相同。使用N50超微量紫外可见光分光光度计测量质粒浓度,并保存在-20℃备用。
将纯化后的mRNA与2×RNA上样缓冲液1:1混匀(2×RNA上样缓冲液购自NEB),将混合液先用70℃金属浴热变性10min,再冰浴5min,使用变性RNA核酸凝胶电泳(180V,10min)验证产物,结果如图3所示,目的mRNA约为2000nt,条带大小与预期相符。
实施例3HA蛋白表达验证试验
1.mRNA的细胞转染与制样
使用RNA转染试剂盒(-mRNA Transfection Kit,MIR2225购自Mirus-bio),将所得的mRNA转染至12孔板中的HEK-293T细胞中,每孔转染1μg mRNA。按照转染试剂说明书具体步骤如下:前一天调节细胞浓度为2×105/mL,接种HEK-293T细胞于12孔板中。第二天待HEK-293T细胞生长至80-90%,进行转染。取100uL MEM,加入1μgmRNA,轻轻吹打混匀。加入2μL mRNA Boost试剂,轻轻吹打混匀。加入2μLTransIT-mRNA转染试剂,轻轻吹打混匀,室温孵育5min。将生长至80-90%的HEK-293T细胞,更换新鲜的完全培养基,并将配置好的转染试剂和mRNA混合物逐滴加入。转染24h后,弃去细胞上清液,PBS洗涤一次,使用裂解液裂解在4℃裂解2-4h(RIPA裂解液+1%蛋白酶抑制剂,现用现配,购自北京鼎国昌盛生物技术有限公司)。加入蛋白Loading Buffer,沸水浴10min。
2.HA蛋白表达的WB鉴定
首先配制10%的蛋白胶进行SDS-PAGE电泳,条件为恒压80V 20min,随后是恒压120V 70min。转膜,条件为恒流200mA 80min。转膜后用5%脱脂奶粉室温封闭2h,PBS洗涤3次,每次5min。用1:10,000的比例稀释H5亚型流感单克隆抗体,室温孵育1h。PBST洗涤3次,每次10min。最后用PBS洗涤1次,每次10min。用以1:10,000的比例稀释辣根酶标记羊抗鼠IgG,室温孵育1h。PBST洗涤3次,每次10min。最后用PBS洗涤1次,每次10min(辣根酶标记羊抗鼠IgG购自北京鼎国昌盛生物技术有限公司)。最后使用SuperKineTM增强型ECL发光液(购自亚科因(abbkine)生物技术有限公司),按照说明书进行操作ECL发光。
结果如图4所示,与对照组相比,转染了H5亚型mRNA的试验组的细胞裂解物泳道中出现了约75kDa的明显的条带。结果表明,体外转录的mRNA可以在HEK-293T中表达。
3.HA蛋白的三聚体结构的WB鉴定
取部分细胞上清和裂解后的细胞产物,加入非还原性蛋白5×Loading Buffer(购自上海碧云天生物技术有限公司)。配制10%的蛋白胶进行蛋白凝胶电泳,条件为恒压80V20min,随后是恒压120V 70min。转膜,条件为恒流200mA 80min。转膜后用5%脱脂奶粉室温封闭2h,PBS洗涤3次,每次5min。用1:10,000的比例稀释H5亚型流感单克隆抗体,室温孵育1h。PBST洗涤3次,每次10min。最后用PBS洗涤1次,每次10min。用以1:10,000的比例稀释辣根酶标记羊抗鼠IgG,室温孵育1h。PBST洗涤3次,每次10min。最后用PBS洗涤1次,每次10min(辣根酶标记羊抗鼠IgG购自北京鼎国昌盛生物技术有限公司)。最后使用SuperKineTM增强型ECL发光液(购自亚科因(abbkine)生物技术有限公司),按照说明书进行操作ECL发光。
结果如图5所示,与对照组相比,转染了H5亚型mRNA的试验组的细胞裂解物泳道中出现了大于180kDa明显的条带。结果表明,mRNA在HEK-293T中表达的HA蛋白以三聚体形式存在。
实施例4mRNA-LNPs疫苗的制备
对于实施例2中所获得mRNA,分别使用脂质体包装技术进行包装,制备mRNA脂质体纳米颗粒疫苗。将脂质按阳离子脂:DSPC:胆固醇:DMG-2000=50:10:38.5:1.5的比例溶于无水乙醇,mRNA溶于50mM的柠檬酸缓冲液(pH4.0),随后醇相和水相按1:3的比例用微流控系统进行混合包裹,获得制剂后用PBS稀释,并用中空纤维换液进行换液,将所得制剂加入蔗糖保护液后,用0.22μm滤膜,并于4℃储存备用。并鉴定疫苗的LNP粒径、Zeta电位和包封率。采用粒度分析仪检测,结果如图6所示最终制剂的粒径为84.88nm,分散系数(PDI)为0.070,包封率约为96%。
实施例5mRNA-LNPs疫苗小鼠效力评估
1.小鼠免疫与攻毒
为验证HA mRNA-LNPs疫苗免疫效果,实施小鼠动物试验。将6周龄SPF BALB/c小鼠随机分为5组,每组12只小鼠。第1组免疫10μg mRNA-LNPs疫苗,第2组免疫5μgmRNA-LNPs疫苗,第3组免疫2μg mRNA-LNPs疫苗,第4组免疫0.5μg mRNA-LNPs疫苗,第5组免疫PBS作为空白对照。所有分组首次免疫3周后实施相同剂量的加强免疫。
在首次免疫后第3、5周,采血进行HI试验分析特异性抗体水平,并在第5周使用禽流感病毒A/Chicken/Guangdong/D889/2015(H5N1)毒株以106EID50剂量对各试验小鼠进行滴鼻攻毒。攻毒之后,每日记录小鼠体重变化,连续记录14天。并观察小鼠死亡情况,最后分析疫苗的保护率。攻毒后第3天,第3组和第5组各随机取3只小鼠,通过qPCR检测小鼠肺脏细胞因子水平。攻毒后第5天每组处死三只小鼠,检测肺脏病毒载量。
2.免疫后抗体水平检测
免疫后第3,5周采血并分离血清。通过眼眶静脉丛采集血液,采集后在37℃孵育2-3h,3,000g离心10min,收集并小量分装血清,避免反复冻融,正常血清为透亮淡黄色。然后将15μL血清加入至45μL RDE溶液中(日本生研受体破坏酶RDE,购自北京兰易科技有限公司),并在37℃孵育18-20h,然后在56℃孵育30-60min。首先用H5N1-SD57毒株制备4单位抗原(4HAU),将25μL预处理血清的两倍系列稀释液与等体积的4HAU,在室温下孵育1h。然后,向每个孔中加入25μL 1%鸡红细胞(RBC)悬浮液,并在室温下孵育30min。HI滴度表示为完全抑制病毒血凝的最高血清稀释度的倒数。
小鼠HI效价结果如图6所示,不同分组小鼠HI效价与免疫剂量呈正相关,并且加强免疫后小鼠血清HI效价明显升高。10μg组、5μg组、2μg组和0.5μg组1免后3周平均HI效价为4log2、3.17log2、0.67log2和0.58log2。10μg组、5μg组、2μg组和0.5μg组1免后5周平均HI效价为9.5log2、8.5log2、7.92log2和6.4log2。
3.攻毒保护率
疫苗攻毒保护率结果如图7所示,攻毒后,PBS组小鼠体重持续降低,并在攻毒后第5天全部死亡。mRNA疫苗免疫组小鼠存活率为100%,并且在观察期内,小鼠平均体重恢复至攻毒前的水平。结果表明以0.5μg免疫两次即可100%保护致死剂量的禽流感病毒的攻毒。
4.小鼠肺脏排毒检测
攻毒后第5天每组处死三只小鼠,检测肺脏病毒载量。摘眼球处死小鼠,将小鼠浸泡在75%酒精中10min,打开小鼠胸腔,取出肺脏,称重后按照每0.1g加入100μL双抗PBS。研磨后,取100μL肺脏混悬液用PBS溶液依次作10倍稀释(10-1 -10-11),进行鸡胚尿囊腔接种,每个稀释度接种3枚,0.1mL/枚,同时设置空白对照,置37℃培养箱孵化48h,弃去24h内死亡胚,收获24h后死亡胚及48h活胚的尿囊液,测试尿囊液的血凝活性,并计算EID50。结果如图8所示,PBS组均检测到排毒,平均EID50为10-6.33/mL。mRNA疫苗组小鼠均未检测到排毒,表明mRNA疫苗免疫后能够有效的抑制排毒。
5.细胞因子检测
攻毒后第3天检测小鼠肺脏细胞因子,摘眼球处死小鼠,将小鼠浸泡在75%酒精中10min,打开小鼠胸腔,取出肺脏,称重后按照每0.1g加入100μL双抗PBS。研磨后,随后使用RNAfast200总RNA极速抽提试剂盒提取RNA(购自上海飞捷生物技术有限公司)。使用N50超微量紫外可见光分光光度计测量RNA浓度,并保存在-20℃备用。
将提取的总RNA样品使用购自Takara的Premix型反转录试剂PrimeScriptTM RTMaster Mix(Perfect Real Time)(RR036A)转录为cDNA。使用购自南京诺唯赞生物科技股份有限公司的ChamQ Universal SYBR qPCR Master Mix通过qPCR检测淋巴细胞的细胞因子IFN-γ,TNF-α,IL-6,IL-4,和IL-13的表达量。
qPCR反应体系
qPCR的引物序列
qPCR反应程序
*荧光信号采集。
qPCR检测细胞因子结果如图9所示,mRNA疫苗组小鼠肺脏的IFN-γ水平高于PBS组,IFN-γ具有抗病毒的作用,表明mRNA疫苗免疫组能够提高小鼠的抗病毒能力。mRNA疫苗组提高了与炎症相关细胞因子TNF-α的水平,适度的炎症有利于机体对抗病毒的感染。IL-6主要免疫相关功能包括刺激B细胞增殖,分泌抗体,刺激T细胞增殖及CTL活化。mRNA疫苗组小鼠肺脏的IL-6水平高于PBS,表明mRNA疫苗免疫组能够有效的激活了小鼠的免疫反应。而与Th2型免疫相关的细胞因子IL-4和IL-13,mRNA疫苗组与PBS组相比细胞因子IL-4和IL-13的平均水平大致相同,表明mRNA疫苗免疫小鼠主要引起偏向Th1型的免疫反应。
上述所有的动物试验结果表明:本发明提供的H5N1亚型mRNA疫苗可诱导交叉保护抵抗异源H5N1亚型高致病性禽流感病毒的攻击。本发明制备的H5N1亚型mRNA疫苗为防控H5N1亚型禽流感提供新的疫苗选择。
对比例1
参考CN202310509346.3,主题为一种禽流感病毒mRNA疫苗及其制备方法和应用,其是本申请人的在先申请,其同样为mRNA疫苗,该mRNA疫苗的制备策略为:
首先将禽流感2.3.2.1c分支毒株的HA蛋白信号肽编码序列替换为强信号肽编码序列,去掉HA蛋白跨膜区,并添加3×GGGGS的柔性Linker,以实现HA蛋白的分泌表达;然后将序列进行禽源密码子优化后,序列前端添加T7启动子、5’端非翻译区(5’UTR)和Kozak序列,序列后端添加3’端非翻译区(3’UTR)和多聚腺苷酸尾(poly A),构成mRNA疫苗候选序列;再进一步预测各候选序列的二级结构,挑选出2条二级结构中5’UTR处于相对游离状态的mRNA序列,将对应的DNA序列分别连接在质粒载体上构成重组质粒,并将重组质粒进行线性化、转录、加帽,并用LNP技术包装,制成禽流感mRNA疫苗。
该疫苗的免疫方式为:
将15只6周龄SPF BALB/c小鼠随机分成3组,每组5只小鼠。第1-2组小鼠经小鼠左和右后腿内侧肌肉分别注射免疫不同剂量的pUC-H5N1-HA-3mRNA疫苗,免疫剂量分别为10+10μg和2+2μg,第3组小鼠经小鼠左和右后腿肌肉分别注射与mRNA疫苗组等体积的PBS缓冲液作为空白对照。首次免疫2周后进行加强免疫。首次免疫后10天以及加强免疫后10天通过小鼠眼眶静脉丛采血,采集后在4℃孵育过夜,3000×g离心10分钟,收集并小量分装血清,-20℃保存。将采集血清,加入3倍体积的受体破坏酶(RDE)(购自北京兰易科技有限公司)(1:3稀释),37℃孵育18h,56℃灭活30min。
在微量反应板的第1孔至11孔中分别加入25μl PBS,第12孔加入50μl PBS;吸取25μl的血清加入到第一孔,混匀,依次倍比稀释至第10孔,弃25μl混合液;然后在第1至11孔均加入4HAU抗原液25μl,室温放置1h;每孔均加入25μl 1%鸡红细胞悬液或每孔均加入50μl0.5%鸡红细胞悬液,振荡混匀,室温放置1h(40min-1h)观察结果,对照红细胞将呈纽扣状沉于孔底。以完全抑制4个HAU抗原的血清最高稀释倍数为HI滴度;只有阴阳性对照组均成立的情况下,实验结果才有效。
其结果为:实验结果表明,初免10天,所有实验组均未检测到抗体;加强免疫后10天,10μg组均检测到抗体HI效价,HI效价分别为5log2,5log2,4log2,8log2,3log2;2μg组有两只小鼠检测到抗体HI效价,HI效价分别为5log2,4log2。
结果分析:
相比现有技术中的mRNA疫苗,本发明的mRNA疫苗能够在更低剂量的时候表现出更高的效价;通过和对比例1的对比可见,本发明的mRNA疫苗免疫剂量低、免疫0.5μg平均HI效价(6.42log2)就可以超过对比例1中免疫10μg组的平均HI效价(4.5log2)。
Claims (5)
1.核苷酸序列如SEQ ID NO.1所示mRNA作为制备预防和治疗禽流感感染的哺乳动物用疫苗的用途。
2.根据权利要求1所述的用途,其特征在于,所述哺乳动物用疫苗包括脂质体混合物和包裹在脂质体内的mRNA;所述疫苗中的脂质体的平均粒径为50-150nm。
3.根据权利要求2所述的用途,其特征在于,所述哺乳动物用疫苗中的脂质体的平均粒径为70-100nm。
4.根据权利要求2所述的用途,其特征在于,所述脂质体混合物包括阳离子脂、DSPC、胆固醇、PEG200-DMG,阳离子脂、DSPC、胆固醇、PEG200-DMG的摩尔比为50:10:38.5:1.5。
5.根据权利要求1所述的用途,其特征在于,所述哺乳动物用疫苗用于免疫鼠、猪、人、马、狗、猫或水貂。
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