CN110714015A - 一种mRNA狂犬病疫苗 - Google Patents
一种mRNA狂犬病疫苗 Download PDFInfo
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- CN110714015A CN110714015A CN201911042634.2A CN201911042634A CN110714015A CN 110714015 A CN110714015 A CN 110714015A CN 201911042634 A CN201911042634 A CN 201911042634A CN 110714015 A CN110714015 A CN 110714015A
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- rabies virus
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Abstract
本发明涉及核酸疫苗领域，具体而言，提供了一种mRNA狂犬病疫苗。本发明提供一种优化RVG mRNA，转录其的核苷酸序列如SEQ ID NO.1所示。发明人通过对狂犬病毒CTN‑1毒株G蛋白(RVG)编码区进行优化，得到核苷酸序列如SEQ ID NO.1所示的优化RVG mRNA的序列。该核苷酸序列使得转录后的mRNA结构更稳定，在哺乳动物和人体内的目标蛋白翻译效率更高。本发明提供的狂犬病毒核酸疫苗，包括疫苗载体和优化RVG mRNA，实现使用极小剂量就能达到足够的保护效果，在安全性、有效性方面优于现有的狂犬病疫苗技术。
Description
技术领域
本发明涉及核酸疫苗领域，具体而言，涉及一种mRNA狂犬病疫苗。
背景技术
狂犬病是世界上影响人类健康最古老的疾病之一，最早的记载见于4300年前，该病是由狂犬病病毒(Rabies Virus，RV)引起的一种病毒性动物传染病。其病毒的传播途径主要是通过未接种或接种失败的犬、猫等动物，目前，在兽用狂犬疫苗上，主要为Vero细胞灭活疫苗，虽然这些疫苗的出现可以大大降低狂犬病的发生率，但该种疫苗的生产过程仍存在很多的技术难点，例如，细胞大规模悬浮培养技术、病毒的扩大生产等，因此，在世界范围内正在广泛研制新型的狂犬病疫苗。
目前市售的狂犬病疫苗主要为狂犬病毒灭活疫苗，该疫苗生产成本高，最主要的原因是由于生产出来的狂犬病毒滴度低，而每毫升疫苗中所含的有效抗原量较高，故需生产更高滴度的狂犬病毒。
狂犬病毒为单链RNA病毒，具有两种主要抗原：一种是病毒外膜上的糖蛋白抗原，能与乙酰胆碱受体结合使病毒具有神经毒性，并使体内产生中和抗体及血凝抑制抗体，中和抗体具有保护作用；另一种为内层的核蛋白抗原，可使体内产生补体结合抗体和沉淀素，无保护作用。虽然可通过免疫具有功能的糖蛋白抗原，不但可以减少非特异性免疫还能降低生产成本。但目前，在进行体外生产亚单位疫苗的过程中，如何选择合适的表达系统及表达产物的纯化工艺成为整个研发和生产过程的技术难点，因此，目前市场尚未见到有类似疫苗。
目前市场上存在的狂犬病毒疫苗通常在其应用时有许多缺点，特别是：1)制备工艺复杂，需要用细胞培养的方法生产病毒，存在一定的安全性隐患。2)质量控制和工艺放大要求高，控制不好会引发产品质量事故。3)病毒产生的有效抗原量低，为了保证效果，要求提高病毒滴度，从而增加了生产成本。
有鉴于此，特提出本发明。
发明内容
本发明的第一目的在于提供一种优化RVG mRNA，以缓解现有技术中用于核酸疫苗的抗原活性低，翻译效率差，稳定性有待提高的技术问题。
本发明的第二目的在于提供上述优化RVG mRNA的应用。
本发明的第三目的在于提供一种狂犬病毒核酸疫苗，以缓解现有技术中狂犬病疫苗病毒滴度低，有效抗原量少，生产成本高的问题。
本发明的第四目的在于提供上述狂犬病毒核酸疫苗的制备方法，提供一种操作简便，产率高的方法。
为了实现本发明的上述目的，特采用以下技术方案：
一种优化RVG mRNA，转录所述优化RVG mRNA的核苷酸序列如SEQ ID NO.1所示。
进一步地，转录所述优化RVG mRNA的核苷酸序列还包括如下元件：5’-帽结构、5’-UTR、3’-聚腺苷酸序列和3’-UTR中的至少一种；
优选地，所述5’-UTR为10-200个核苷酸，更优选为15-100个核苷酸；
优选地，所述5’-UTR为DNAH2 5’-UTR序列或KOZAK序列；
优选地，所述KOZAK序列如SEQ ID NO.3所示：
5’-GGCTAGCGCCGCCACC-3’(SEQ ID NO.3)；
优选地，所述DNAH2 5’-UTR序列如SEQ ID NO.4所示：
5’-GAGACCCAAGCTGGCTAGCGGGAGAAAGCTTACC-3’(SEQ ID NO.4)；
优选地，所述3’-聚腺苷酸序列优选为60-120个A，更优选为80-110个A，进一步优选为100个A；
优选地，所述3’-UTR包括β－珠蛋白3’-UTR序列或血红蛋白HBA23’-UTR序列；
优选地，所述血红蛋白HBA2 3’-UTR序列如SEQ ID NO.5所示：
5’-GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCC AACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAGTCTGAGTGGGCAGC-3’(SEQ ID NO.5)。
进一步地，所述优化RVG mRNA的序列如SEQ ID NO.2所示。
上述优化RVG mRNA在制备狂犬病毒核酸疫苗中的应用。
一种狂犬病毒核酸疫苗，所述狂犬病毒核酸疫苗包括疫苗载体和本发明提供的优化RVG mRNA。
进一步地，所述疫苗载体包括阳离子脂质体、阳离子蛋白、阳离子聚合物或阳离子脂质纳米颗粒中的一种，优选为阳离子脂质体或阳离子脂质纳米颗粒；
优选地，阳离子脂质纳米颗粒与优化RVG mRNA的质量比为10-30:1，优选为15-25:1，更优选为20:1；
优选地，阳离子脂质体与优化RVG mRNA的质量比为1-8:1，优选为1-4:1，更优选为2:1。
进一步地，所述疫苗载体为阳离子脂质纳米颗粒，按摩尔百分比计，包括20-50％可质子化阳离子脂质、20-50％结构脂质、5-20％辅助脂质和1-5％表面活性剂，其中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％；
优选地，所述可质子化阳离子脂质包括Dlin-MC3-DMA、Dlin-KC2-DMA、DODMA、c12-200或DlinDMA，优选为Dlin-MC3-DMA；
优选地，所述结构脂质包括胆固醇、胆固醇酯、固醇类激素、固醇类维生素或胆汁酸，优选为胆固醇；
优选地，所述辅助脂质包括DSPC，DOPE，DOPC或DOPS，优选为DSPC；
优选地，所述表面活性剂包括PEG-DMG或PEG-DSPE，优选为PEG-DMG；
优选地，所述狂犬病毒核酸疫苗的给药方式包括静脉注射、肌肉注射或皮内注射，优选为肌肉注射。
上述狂犬病毒核酸疫苗的制备方法，将疫苗载体和本发明提供的优化RVG mRNA混合，得到狂犬病毒核酸疫苗。
进一步地，所述疫苗载体为上述狂犬病毒核酸疫苗中的阳离子脂质纳米颗粒，制备方法包括：
(a)按配方比例将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液，得到有机相；
(b)将优化RVG mRNA溶于PBS或柠檬酸盐溶液中，得到水相；
(c)将(a)得到的有机相和(b)得到的水相混匀得到混合液，替换混合液中的溶媒为缓冲液，得到狂犬病毒核酸疫苗；
优选地，有机溶液包括无水乙醇、四氢呋喃、丙酮或DMSO，优选为无水乙醇；
优选地，有机相中可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的总浓度为5-7mg/ml；
优选地，水相中优化RVG mRNA的浓度为0.8-1.2mg/ml；
优选地，有机相和水相的体积比为1：2-4；
优选地，混匀采用微流控设备，流速范围为6-24ml/min，优选为12.0ml/min；
优选地，替换混合液中的溶媒为缓冲液包括：用缓冲液将混合液稀释50-100倍，再浓缩。
进一步地，所述制备方法还包括将液态的狂犬病毒核酸疫苗制备成冻干制剂的过程；
优选地，在狂犬病毒核酸疫苗中加入5-30w/v％药学上可接受的冻干保护剂得到待冻干制剂，待冻干制剂经冻干得到冻干制剂；
优选地，待冻干制剂中还包括0.005-0.05w/v％药学上可接受的表面活性剂；
优选地，冻干过程包括：真空度为20μbar-80μbar的条件下，依次为，-40℃至-60℃保持1-8h，-30℃至-50℃保持10-30h，4℃至20℃保持10-30h。
与现有技术相比，本发明的有益效果为：
本发明提供一种优化RVG mRNA，转录其的核苷酸序列如SEQ ID NO.1所示。发明人对狂犬病毒CTN-1毒株G蛋白(RVG)编码区进行优化，序列优化包括：针对在人体内表达时的密码子偏好性进行调整、常用密码子的使用频率进行调整、提高序列的GC含量等，得到核苷酸序列如SEQ ID NO.1所示的优化RVG mRNA的序列。该核苷酸序列使得转录后的mRNA结构更稳定，在哺乳动物和人体内的目标蛋白翻译效率更高。
本发明提供的狂犬病毒核酸疫苗，包括疫苗载体和优化RVG mRNA。该核酸疫苗将体外转录合成的优化RVG mRNA，递送至体内产生狂犬病毒糖蛋白(即抗原蛋白)，实现使用极小剂量就能达到足够的保护效果，降低生产成本，在有效性方面优于现有的狂犬病疫苗技术。
本发明提供上述狂犬病毒核酸疫苗的制备方法，将疫苗载体和优化RVG mRNA混合，得到狂犬病毒核酸疫苗。该方法简单易操作，降低了疫苗生产的工艺难度，可以大规模进行生产，批间差易于控制，极大降低了生产成本。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例3中不同RVG mRNA转染HEK293细胞的表达量结果；
图2为实施例5中不同给药方式递送mRNA的结果图；
图3为实施例7中比格犬试验狂犬病毒核酸疫苗的中和抗体滴度结果；
图4为实施例8中冻干前后狂犬病毒核酸疫苗产生抗体效果结果图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
一种优化RVG mRNA，转录优化RVG mRNA的核苷酸序列如SEQ ID NO.1所示：
5’-ATGATTCCTCAGGCTCTGCTGTTCGTCCCACTGCTGGTCTTCCCACTGTGCTTCGGCAAGTTCCCCATCTACACTATTCCTGACAAACTGGGCCCTTGGTCCCCAATCGACATCCACCACCTGTCCTGTCCCAACAATCTGGTGGTGGAGGACGAGGGCTGTACCAATCTGAGCGGCTTCTCCTACATGGAGCTGAAGGTGGGCTACATCTCCGCCATCAAGGTGAACGGCTTCACATGTACCGGCGTGGTGACAGAGGCCGAGACATACACCAACTTCGTGGGCTATGTGACCACAACCTTCAAGCGGAAGCACTTCAGACCAACCCCTGATGCCTGTCGGTCCGCCTATAACTGGAAGATGGCCGGCGACCCACGGTATGAGGAGAGCCTGCACAACCCATACCCCGATTACCACTGGCTGAGGACAGTGAAGACAACCAAGGAGTCCGTGGTAATCATCTCTCCAAGCGTGGCCGATCTGGACCCATACGATAAGTCCCTGCACAGCAGAGTGTTCCCCCGCGGCAAGTGTTCTGGCATCACAGTGAGCTCCGCCTATTGCTCTACCAACCACGACTACACCATCTGGATGCCTGAGAATCCAAGACTGGGCACCTCCTGTGACATCTTTACCAACTCTCGGGGCAAGAGAGCCTCCAAGGGCTCCAAGACATGTGGCTTCGTGGACGAGAGAGGCCTGTATAAGTCCCTGAAGGGCGCCTGCAAGCTGAAGCTGTGCGGCGTGCTGGGCCTGAGGCTGATGGACGGCACCTGGGTGGCCATCCAGACATCCAACGAGACCAAGTGGTGCCCTCCTGATCAGCTGGTGAACCTGCACGACTTTCACAGCGACGAGATCGAGCACCTGGTGGTGGAGGAGCTGGTGAAGAAGAGAGAGGAGTGCCTGGATGCCCTGGAGTCCATCATGACCACCAAGAGCGTGTCCTTTAGACGGCTGAGCCACCTGAGAAAGCTGGTGCCCGGCTTTGGCAAGGCCTACACCATCTTCAACAAGACACTGATGGAGGCCGATGCCCACTACAAGTCCGTGAGAACCTGGAACGAGATCATCCCTAGCAAGGGCTGTCTGAGGGTGGGCGGCAGATGTCACCCTCACGTGAATGGCGTGTTCTTTAATGGCATCATCCTGGGCCCAGATGGCCACGTGCTGATCCCAGAGATGCAGTCCTCTCTGCTGCAGCAGCACATGGAGCTGCTGGAGTCTAGCGTGATCCCTCTGATGCACCCACTGGCCGATCCAAGCACAGTGTTCAAGGACGGCGACGAGGTGGAGGACTTTGTGGAGGTGCACCTGCCAGATGTGCACAAGCAGGTGTCCGGCGTGGATCTGGGCCTGCCAAATTGGGGCAAGGACGTGCTGATGGGCGCCGGCGTGCTGACCGCCCTGATGCTGATGATCTTCCTGATGACCTGCTGTAGACGGACAAATAGAGCCGAGAGCATCCAGCACTCCCTGGGCGAGACAGGCCGGAAGGTGTCTGTGACCAGCCAGAGTGGACGAGTGATCTCCTCATGGGAATCCTACAAAAGCGGCGGCGAGACCAAACTGTGA-3’(SEQ IDNO.1)。
发明人对狂犬病毒CTN-1毒株G蛋白(RVG)编码区进行优化，序列优化包括：针对在人体内表达时的密码子偏好性进行调整、常用密码子的使用频率进行调整、提高序列的GC含量等，得到核苷酸序列如SEQ ID NO.1所示的转录优化RVG mRNA的序列。该核苷酸序列使得转录后的mRNA结构更稳定，在哺乳动物和人体内的目标蛋白翻译效率更高。
在优选地实施方式中，转录优化RVG mRNA的核苷酸序列还包括如下元件：5’-帽结构、5’-UTR、3’-聚腺苷酸序列和3’-UTR中的至少一种。5’-帽结构、5’-UTR、3’-聚腺苷酸序列和3’-UTR序列元件的添加均可以进一步提高序列的稳定性，避免降解。
在优选地实施方式中，5’-UTR为10-200个核苷酸，更优选为15-100个核苷酸。
在优选地实施方式中，5’-UTR为DNAH2 5’-UTR序列或KOZAK序列；
在优选地实施方式中，KOZAK序列如SEQ ID NO.3所示：
5’-GGCTAGCGCCGCCACC-3’(SEQ ID NO.3)。
在优选地实施方式中，DNAH2 5’-UTR序列如SEQ ID NO.4所示：
5’-GAGACCCAAGCTGGCTAGCGGGAGAAAGCTTACC-3’(SEQ ID NO.4)。
在优选地实施方式中，3’-聚腺苷酸序列优选为60-120个A，更优选为80-110个A，进一步优选为100个A。
在优选地实施方式中，3’-UTR包括β－珠蛋白3’-UTR序列或血红蛋白HBA2 3’-UTR序列。
在优选地实施方式中，血红蛋白HBA2 3’-UTR序列如SEQ ID NO.5所示：
5’-GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAGTCTGAGTGGGCAGC-3’(SEQ ID NO.5)。
在优选地实施方式中，优化RVG mRNA的序列如SEQ ID NO.2所示：
5’-GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGGUACCGAGCUCGGAUCCACUAGUCCAGUGUGGUGGAAUUCGGGAGAAAGCUUACCAUGAUUCCUCAGGCUCUGCUGUUCGUCCCACUGCUGGUCUUCCCACUGUGCUUCGGCAAGUUCCCCAUCUACACUAUUCCUGACAAACUGGGCCCUUGGUCCCCAAUCGACAUCCACCACCUGUCCUGUCCCAACAAUCUGGUGGUGGAGGACGAGGGCUGUACCAAUCUGAGCGGCUUCUCCUACAUGGAGCUGAAGGUGGGCUACAUCUCCGCCAUCAAGGUGAACGGCUUCACAUGUACCGGCGUGGUGACAGAGGCCGAGACAUACACCAACUUCGUGGGCUAUGUGACCACAACCUUCAAGCGGAAGCACUUCAGACCAACCCCUGAUGCCUGUCGGUCCGCCUAUAACUGGAAGAUGGCCGGCGACCCACGGUAUGAGGAGAGCCUGCACAACCCAUACCCCGAUUACCACUGGCUGAGGACAGUGAAGACAACCAAGGAGUCCGUGGUAAUCAUCUCUCCAAGCGUGGCCGAUCUGGACCCAUACGAUAAGUCCCUGCACAGCAGAGUGUUCCCCCGCGGCAAGUGUUCUGGCAUCACAGUGAGCUCCGCCUAUUGCUCUACCAACCACGACUACACCAUCUGGAUGCCUGAGAAUCCAAGACUGGGCACCUCCUGUGACAUCUUUACCAACUCUCGGGGCAAGAGAGCCUCCAAGGGCUCCAAGACAUGUGGCUUCGUGGACGAGAGAGGCCUGUAUAAGUCCCUGAAGGGCGCCUGCAAGCUGAAGCUGUGCGGCGUGCUGGGCCUGAGGCUGAUGGACGGCACCUGGGUGGCCAUCCAGACAUCCAACGAGACCAAGUGGUGCCCUCCUGAUCAGCUGGUGAACCUGCACGACUUUCACAGCGACGAGAUCGAGCACCUGGUGGUGGAGGAGCUGGUGAAGAAGAGAGAGGAGUGCCUGGAUGCCCUGGAGUCCAUCAUGACCACCAAGAGCGUGUCCUUUAGACGGCUGAGCCACCUGAGAAAGCUGGUGCCCGGCUUUGGCAAGGCCUACACCAUCUUCAACAAGACACUGAUGGAGGCCGAUGCCCACUACAAGUCCGUGAGAACCUGGAACGAGAUCAUCCCUAGCAAGGGCUGUCUGAGGGUGGGCGGCAGAUGUCACCCUCACGUGAAUGGCGUGUUCUUUAAUGGCAUCAUCCUGGGCCCAGAUGGCCACGUGCUGAUCCCAGAGAUGCAGUCCUCUCUGCUGCAGCAGCACAUGGAGCUGCUGGAGUCUAGCGUGAUCCCUCUGAUGCACCCACUGGCCGAUCCAAGCACAGUGUUCAAGGACGGCGACGAGGUGGAGGACUUUGUGGAGGUGCACCUGCCAGAUGUGCACAAGCAGGUGUCCGGCGUGGAUCUGGGCCUGCCAAAUUGGGGCAAGGACGUGCUGAUGGGCGCCGGCGUGCUGACCGCCCUGAUGCUGAUGAUCUUCCUGAUGACCUGCUGUAGACGGACAAAUAGAGCCGAGAGCAUCCAGCACUCCCUGGGCGAGACAGGCCGGAAGGUGUCUGUGACCAGCCAGAGUGGACGAGUGAUCUCCUCAUGGGAAUCCUACAAAAGCGGCGGCGAGACCAAACUGUGAGGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAU-3’(SEQ ID NO.2)。
本发明提供上述优化RVG mRNA在制备狂犬病毒核酸疫苗中的应用。
一种狂犬病毒核酸疫苗，狂犬病毒核酸疫苗包括疫苗载体和本发明提供的优化RVG mRNA。
该狂犬病毒核酸疫苗将体外转录合成的优化RVG mRNA，递送至体内产生狂犬病毒糖蛋白(即抗原蛋白)，实现使用极小剂量就能达到足够的保护效果，在安全性、有效性方面优于现有的兽用狂犬病疫苗技术。可以理解的是，疫苗载体可以为本领域常用的适用于核酸疫苗制备的疫苗载体。
在优选地实施方式中，疫苗载体包括阳离子脂质体、阳离子蛋白、阳离子聚合物或阳离子脂质纳米颗粒中的一种，优选为阳离子脂质体或阳离子脂质纳米颗粒。
在优选地实施方式中，阳离子脂质纳米颗粒与优化RVG mRNA的质量比为10-30:1，优选为15-25:1，更优选为20:1。阳离子脂质纳米颗粒与优化RVG mRNA的质量比典型但非限制性的为10:1、15:1、20:1、25:1或30:1。
在优选地实施方式中，阳离子脂质体与优化RVG mRNA的质量比为1-8:1，优选为1-4:1，更优选为2:1。阳离子脂质体与优化RVG mRNA的质量比典型但非限制性的为1:1、3:1、5:1、7:1或8:1。
在优选地实施方式中，疫苗载体为阳离子脂质纳米颗粒，按摩尔百分比计，包括20-50％可质子化阳离子脂质、20-50％结构脂质、5-20％辅助脂质和1-5％表面活性剂，其中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％。需要说明的是，按摩尔百分比计，可质子化阳离子脂质典型但非限制性的为20％、25％、30％、35％、40％、45％或50％；结构脂质典型但非限制性的为20％、25％、30％、35％、40％、45％或50％；辅助脂质典型但非限制性的为5％、10％、15％或20％；表明活性剂典型但非限制性的为1％、2％、3％、4％或5％。
在优选地实施方式中，可质子化阳离子脂质包括Dlin-MC3-DMA、Dlin-KC2-DMA、DODMA、c12-200或DlinDMA，优选为Dlin-MC3-DMA。
在优选地实施方式中，结构脂质包括胆固醇、胆固醇酯、固醇类激素、固醇类维生素或胆汁酸，优选为胆固醇。
在优选地实施方式中，辅助脂质包括DSPC，DOPE，DOPC或DOPS，优选为DSPC。需要说明的是，DOPE全称为1,2-dioleoyl-sn-glycero-3-phosphoethanolamine中文名为二油酰基磷脂酰乙醇胺；DOPC全称1,2-dioleoyl-sn-glycero-3-phosphocholine中文名为二油酰基卵磷脂，cholesterol为胆固醇；DSPC为二硬脂酰基磷脂酰胆碱；DOPS二油酰基磷脂酰丝氨酸。
在优选地实施方式中，表面活性剂包括PEG-DMG或PEG-DSPE，优选为PEG-DMG；需要说明的是，PEG-DMG是1,2-二肉豆蔻酸甘油酯的聚乙二醇(PEG)衍生物，PEG长度为2000或5000，优选2000；PEG-DSPE是二硬脂酰基磷脂酰乙醇胺的聚乙二醇(PEG)衍生物，PEG长度为2000或5000，优选2000。
在优选地实施方式中，狂犬病毒核酸疫苗的给药方式包括静脉注射、肌肉注射或皮内注射，优选为肌肉注射。
本发明提供上述狂犬病毒核酸疫苗的制备方法，将疫苗载体和本发明提供的优化RVG mRNA混合，得到狂犬病毒核酸疫苗。该方法简单易操作，极大降低了生产成本。
在优选地实施方式中，疫苗载体为本发明提供的狂犬病毒核酸疫苗中的阳离子脂质纳米颗粒，狂犬病毒核酸疫苗的制备方法包括：
(a)按配方比例将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液，得到有机相；
(b)将优化RVG mRNA溶于PBS或柠檬酸盐溶液中，得到水相；
(c)将(a)得到的有机相和(b)得到的水相混匀得到混合液，替换混合液中的溶媒为缓冲液，得到狂犬病毒核酸疫苗。可以理解的是，(c)中的溶媒是指有机相和水相中的溶剂，缓冲液可以为本领域常用的缓冲液，优选为pH值7-8的PBS溶液。
在优选地实施方式中，有机溶液包括无水乙醇、四氢呋喃、丙酮或DMSO，优选为无水乙醇。无水乙醇的效果最好。
在优选地实施方式中，有机相中可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的总浓度为5-7mg/ml。
在优选地实施方式中，水相中优化RVG mRNA的浓度为0.8-1.2mg/ml。
在优选地实施方式中，有机相和水相的体积比为1：2-4。
在优选地实施方式中，混匀采用微流控设备，流速范围为6-24ml/min，优选为12.0ml/min。
在优选地实施方式中，替换混合液中的溶媒为缓冲液包括：用缓冲液将混合液稀释50-100倍，再浓缩。需要说明的是，替换混合液中的溶媒可以采用本领域置换缓冲液的常规方法，例如切向流过滤等。最后将得到的狂犬病毒核酸疫苗的浓度调整为本领域常规浓度。
在优选地实施方式中，制备方法还包括将液态的狂犬病毒核酸疫苗制备成冻干制剂的过程。制成冻干制剂，有利于延长产品的有效期。
在优选地实施方式中，在狂犬病毒核酸疫苗中加入5-30w/v％药学上可接受的冻干保护剂得到待冻干制剂，待冻干制剂经冻干得到冻干制剂。需要说明的是，冻干保护剂可以为本领域常用的冻干保护剂，本申请不做特殊限定，优选为蔗糖或海藻糖。
在优选地实施方式中，待冻干制剂中还包括0.005-0.05w/v％药学上可接受的表面活性剂。需要说明的是，表面活性剂可以为本领域常用的表面活性剂，本申请不做特殊限定，优选为吐温20或吐温80。
在优选地实施方式中，冻干过程包括：真空度为20μbar-80μbar的条件下，依次为，-40℃至-60℃保持1-8h，-30℃至-50℃保持10-30h，4℃至20℃保持10-30h。
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。
实施例1狂犬病毒核酸疫苗的制备方法
将SEQ ID NO.2的mRNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相。
将配方比例Dlin-MC3-DMA、胆固醇、DSPC和PEG-DMG溶于无水乙醇，并将总脂质浓度调整至6mg/ml，得到有机相。
利用微流控设备混合的方式将有机相与水相以1:3的体积比混合。利用微流控设备混合时流速12.0ml/min。
混合液立刻用pH7.4的PBS溶液稀释50-100倍，并使用切向流过滤(TFF)除去溶液中乙醇成分并浓缩至mRNA浓度约100μg/ml，得到包裹mRNA的脂质纳米颗粒，即为狂犬病毒核酸疫苗。
实施例2冻干制剂
将实施例1中的狂犬病毒核酸疫苗制备成冻干制剂，添加冻干保护剂蔗糖和表面活性剂吐温20，工艺包括预冻，一次冻干及二次冻干：预冻温度为-50℃,温度保持5小时。一次冻干温度为-40℃24小时，二次冻干温度为10℃保持17小时，冻干过程真空度为40μbar。
实施例3 RVG mRNA的序列优化
本实施例中的RVG mRNA的序列优化方案如表1所示，具体如下：
RVG-A mRNA序列关键元件是采用DNAH2 5’UTR、CTN-1毒株的编码G蛋白的开放阅读框核苷酸(表1中称为“天然序列”，如SEQ ID NO.6所示)、HBA2 3’UTR和polyA序列以及后续元件(包括64个聚腺苷酸、36个聚胞苷酸和组蛋白茎环结构，参考Curevec狂犬病毒mRNA疫苗专利(CN105517569A))。
RVG-B mRNA和RVG-C mRNA与RVG-A mRNA的非编码区域(UTR)部分一致，只是在ORF部分改用了优化后的序列(分别对应表1中的“优化ORF-1”和“优化ORF-2”)，这3个ORF最终翻译出来的蛋白序列一致，为CTN-1毒株的G蛋白，具体为SEQ NO.7所示。
RVG-D mRNA是在RVG-C mRNA的序列基础上，把polyA以及后续元件部分采用100个聚腺苷酸来代替。
表1
体外转录工艺制备出来的上述RVG-A mRNA、RVG-B mRNA、RVG-C mRNA和RVG-DmRNA分别转染HEK293细胞。首先采用4×105个细胞/ml的密度进行HEK293细胞的铺板，24小时后细胞融合状态大约为80％时进行细胞转染。转染时加入6孔板的1个孔的HEK293细胞中的转染体系中包括2μg mRNA和转染试剂Lipofectamine MessagerMAX(ThermoFisherScientific)，具体转染操作按照转染试剂产品说明书进行。转染24小时后的HEK293细胞用狂犬病毒G蛋白抗体进行免疫标记，然后使用流式细胞仪检测G蛋白表达量。荧光素mRNA(Luc mRNA)转染细胞作为阴性对照。结果如图1所示，从图中结果可以看出，RVG-B mRNA和RVG-C mRNA转染细胞中的G蛋白表达量高于RVG-A转染组，可见优化ORF密码子可以提高蛋白表达量，RVG-D mRNA转染细胞的G蛋白含量远远高于RVG-C转染组，可见在其他元件不变的情况下，100个A比含有64A+36C+histone stem loop更能提高目标蛋白表达量。RVG-DmRNA即为本发明提供的序列为SEQ ID NO.2的mRNA。
需要说明的是，表1中的“天然序列”同GenBank:JN234418.1：
5’-ATGATTCCTCAAGCTCTGTTGTTTGTACCTCTTCTGGTTTTTCCATTGTGTTTCGGGAAATTCCCCATTTACACGATACCAGACAAACTCGGCCCCTGGAGTCCCATCGATATACATCACCTCAGCTGTCCGAACAATCTGGTTGTGGAGGACGAAGGATGTACCAATCTGTCAGGATTCTCATACATGGAGCTTAAAGTAGGATATATTTCGGCCATAAAGGTGAACGGGTTCACTTGTACGGGTGTGGTAACGGAAGCAGAAACCTACACTAACTTTGTCGGTTATGTCACCACCACGTTTAAGAGAAAGCACTTCCGACCAACACCGGATGCATGCAGATCAGCATACAATTGGAAGATGGCAGGTGACCCCAGATATGAAGAGTCTCTGCACAATCCCTATCCTGATTATCATTGGCTCCGGACTGTAAAAACCACCAAAGAGTCTGTTGTTATCATATCTCCAAGTGTGGCAGACTTAGACCCGTACGATAAATCACTTCATTCGAGAGTTTTTCCTAGAGGAAAATGCTCAGGAATAACGGTGTCTTCTGCCTACTGCTCTACCAACCATGATTATACCATCTGGATGCCTGAAAATCCTAGACTGGGGACCTCTTGTGATATTTTCACCAACAGCAGAGGGAAGAGAGCATCCAAAGGGAGCAAGACCTGTGGATTTGTGGATGAGAGAGGCTTGTACAAATCTCTAAAAGGAGCATGCAAACTGAAGCTGTGTGGAGTTCTTGGACTTAGGCTTATGGACGGAACCTGGGTCGCGATTCAGACATCAAACGAGACCAAGTGGTGCCCTCCTGATCAACTAGTGAATCTACATGACTTTCATTCAGATGAGATTGAACATCTTGTTGTGGAGGAGTTGGTTAAGAAGAGGGAGGAGTGTCTAGATGCACTGGAGTCCATCATGACCACCAAGTCCGTGAGTTTCAGACGTCTCAGTCACTTGAGGAAGCTTGTGCCTGGATTTGGAAAAGCATACACCATATTCAACAAGACCTTAATGGAGGCTGATGCTCACTACAAATCGGTCCGAACTTGGAATGAGATCATCCCCTCGAAAGGGTGTTTAAGAGTCGGGGGGAGATGTCATCCTCATGTGAACGGAGTATTTTTCAATGGTATCATCCTAGGCCCTGACGGCCATGTCTTAATCCCGGAAATGCAGTCATCCCTCCTCCAGCAGCATATGGAGTTGTTGGAATCCTCGGTCATCCCCTTAATGCATCCCTTGGCAGATCCATCAACGGTTTTTAAAGATGGTGACGAGGTGGAGGATTTTGTTGAGGTTCACCTTCCAGATGTGCATAAGCAGGTCTCAGGGGTTGATCTCGGTCTCCCAAACTGGGGGAAGGATGTGTTGATGGGCGCAGGCGTTTTGACGGCACTGATGTTGATGATTTTCCTAATGACGTGTTGCCGAAGGACTAATAGAGCAGAATCAATACAACACAGTCTTGGAGAGACAGGGAGGAAAGTGTCGGTGACCTCCCAAAGCGGGAGGGTCATATCTTCATGGGAGTCATATAAAAGCGGAGGTGAGACCAAGCTGTAA-3’(SEQ IDNO.6)。
CTN-1毒株的G蛋白同GenBank:ACR39382.1：
MIPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDKSLHSRVFPRGKCSGITVSSAYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTSQSGRVISSWESYKSGGETKL(SEQ NO.7)。
实施例4疫苗载体的优化
利用荧光素酶作为报告基因，通过活体荧光成像技术研究不同疫苗载体的配方(如下表2所示，“MC3”是指Dlin-MC3-DMA，“+”：给药后通过小动物活体荧光成相系统检测到小鼠体内荧光素酶表达)在小鼠体内递送编码荧光素酶基因的mRNA(实施例3中的RVG-DmRNA)的效率，并检测不同复合制剂(制备方法参见实施例1)的理化指标，结果如表2所示。通过研究发现，提高脂质与mRNA质量比有利于增加mRNA在脂质纳米颗粒中的包封率从而使其具有更高的稳定性，此外适度提高配方中聚乙二醇(PEG)的含量有利于提高mRNA在体内的表达效率。因此综合考虑mRNA包封率以及mRNA体内递送效率等因素，选取了4号配方用于后续mRNA狂犬病疫苗的研究。
表2
实施例5不同给药方式的影响
利用实施例4中4号配方包裹荧光素酶mRNA通过不同给药途径(静脉注射、肌肉注射和皮内注射)对小鼠给药后2小时，利用荧光活体成像技术分别在小鼠不同组织部位检测到荧光素酶基因的表达。结果如图2所示(图中，I.V.：静脉注射，I.M.：肌肉注射，I.D.：皮内注射)，表明本发明提供的核酸疫苗可以通过多种给药途径对小鼠递送mRNA。其中，小鼠腿部肌肉注射递送效果较高，并且考虑到给药难易程度，药物研发难易程度以及患者感受等因素，最终选取了肌肉注射给药作为最优给药方式用于后续狂犬病疫苗的开发。
实施例6中和抗体滴度测定
mRNA选用实施例3中的RVG-D mRNA进行小鼠免疫实验，疫苗载体分别选取：阳离子脂质纳米颗粒应用实施例4中4号配方制备，阳离子脂质体制剂使用相应商品化试剂用作阳性对照所用商品化试剂为lipofectamine2000，制备时参考相关产品说明书，将mRNA利用OptiMEM稀释至0.1mg/ml，再按照lipofectamine2000/mRNA(v/w)＝2:1的比例准备lipofectamine2000试剂并利用OptiMEM将其体积稀释到与mRNA稀释液相同的体积，缓慢将二者混合并于室温静置15min即可用于小鼠给药实验，每只小鼠按照5μg的给药量注射，注射体积为100μl/只。商品化疫苗为灭活全病毒疫苗(瑞比克)作为阳性对照(≥106.3FAID50/ml，给药体积为100μl/只)，鱼精蛋白制剂参考现有技术中鱼精蛋白递送mRNA的方法制备得到(鱼精蛋白/mRNA(w/w)＝2:1，每只小鼠按照5μg的给药量注射，注射体积为100μl/只。)，阴性对照为等体积生理盐水注射液。以上各种疫苗制剂一次给药后14天检测小鼠血清狂犬病毒中和抗体滴度结果如下表3所示：
从表中结果可以看出，单次注射后阳离子脂质体制剂及脂质纳米颗粒制剂均诱导产生远高于市售狂犬病疫苗的中和抗体滴度，同时实验发现鱼精蛋白制剂在该给药剂量下无法诱导产生有效中和抗体，说明本发明技术优于现有技术。
实施例7动物实验
应用实施例4中4号配方制备得到的狂犬病毒核酸疫苗，mRNA选用实施例3中的RVG-D mRNA。将制备好的疫苗制剂测定浓度并最终浓缩用于靶动物：比格犬的免疫实验，给药量为20μg/只，给药体积为500μl/只。商品化疫苗为灭活全病毒疫苗(瑞比克)作为阳性对照(≥106.3FAID50/ml，给药体积为1ml/只)，阴性对照为等体积生理盐水注射液。一次给药后比格犬体内中和抗体滴度变化结果如图3所示，其中，RNA001为本发明提供的核酸疫苗。从图中可以看出，给药后检测6个月，本发明提供的核酸疫苗发现相比商业化全病毒疫苗能够诱导产生更高的中和抗体滴度，且能在6个月时间内一直处于达标线以上。
实施例8冻干工艺
为了核酸疫苗制剂能够实现长期保存，我们将本发明提供的核酸疫苗(实施例4中的4号配方制备成的核酸疫苗)开发成冻干制剂使其能在低温下长期保存。纳米颗粒冻干制剂最大的难点在于冻干过程会对纳米颗粒质量造成影响，所以冻干制剂在复水后其理化性质及药效都会比冻干前样本有一定差异。因此为了降低冻干过程对核酸疫苗理化及药效的影响，我们自主开发了冻干处方及工艺，具体流程参照实施例2，并通过动物实验验证冻干前后核酸疫苗药效的变化，结果如图4所示。从图中可以看出，冻干前后核酸疫苗的药效无明显差异，此外，冻干复水后核酸疫苗的粒径相比冻干前增加约20-60nm，mRNA包封率下降约2％-15％，表明冻干工艺开发成功。
尽管已用具体实施例来说明和描述了本发明，然而应意识到，在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
SEQUENCE LISTING
<110> 珠海丽凡达生物技术有限公司
<120> 一种mRNA狂犬病疫苗
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1575
<212> DNA
<213> 人工序列
<400> 1
atgattcctc aggctctgct gttcgtccca ctgctggtct tcccactgtg cttcggcaag 60
ttccccatct acactattcc tgacaaactg ggcccttggt ccccaatcga catccaccac 120
ctgtcctgtc ccaacaatct ggtggtggag gacgagggct gtaccaatct gagcggcttc 180
tcctacatgg agctgaaggt gggctacatc tccgccatca aggtgaacgg cttcacatgt 240
accggcgtgg tgacagaggc cgagacatac accaacttcg tgggctatgt gaccacaacc 300
ttcaagcgga agcacttcag accaacccct gatgcctgtc ggtccgccta taactggaag 360
atggccggcg acccacggta tgaggagagc ctgcacaacc cataccccga ttaccactgg 420
ctgaggacag tgaagacaac caaggagtcc gtggtaatca tctctccaag cgtggccgat 480
ctggacccat acgataagtc cctgcacagc agagtgttcc cccgcggcaa gtgttctggc 540
atcacagtga gctccgccta ttgctctacc aaccacgact acaccatctg gatgcctgag 600
aatccaagac tgggcacctc ctgtgacatc tttaccaact ctcggggcaa gagagcctcc 660
aagggctcca agacatgtgg cttcgtggac gagagaggcc tgtataagtc cctgaagggc 720
gcctgcaagc tgaagctgtg cggcgtgctg ggcctgaggc tgatggacgg cacctgggtg 780
gccatccaga catccaacga gaccaagtgg tgccctcctg atcagctggt gaacctgcac 840
gactttcaca gcgacgagat cgagcacctg gtggtggagg agctggtgaa gaagagagag 900
gagtgcctgg atgccctgga gtccatcatg accaccaaga gcgtgtcctt tagacggctg 960
agccacctga gaaagctggt gcccggcttt ggcaaggcct acaccatctt caacaagaca 1020
ctgatggagg ccgatgccca ctacaagtcc gtgagaacct ggaacgagat catccctagc 1080
aagggctgtc tgagggtggg cggcagatgt caccctcacg tgaatggcgt gttctttaat 1140
ggcatcatcc tgggcccaga tggccacgtg ctgatcccag agatgcagtc ctctctgctg 1200
cagcagcaca tggagctgct ggagtctagc gtgatccctc tgatgcaccc actggccgat 1260
ccaagcacag tgttcaagga cggcgacgag gtggaggact ttgtggaggt gcacctgcca 1320
gatgtgcaca agcaggtgtc cggcgtggat ctgggcctgc caaattgggg caaggacgtg 1380
ctgatgggcg ccggcgtgct gaccgccctg atgctgatga tcttcctgat gacctgctgt 1440
agacggacaa atagagccga gagcatccag cactccctgg gcgagacagg ccggaaggtg 1500
tctgtgacca gccagagtgg acgagtgatc tcctcatggg aatcctacaa aagcggcggc 1560
gagaccaaac tgtga 1575
<210> 2
<211> 1843
<212> RNA
<213> 人工序列
<400> 2
gggagaccca agcuggcuag cguuuaaacu uaagcuuggu accgagcucg gauccacuag 60
uccagugugg uggaauucgg gagaaagcuu accaugauuc cucaggcucu gcuguucguc 120
ccacugcugg ucuucccacu gugcuucggc aaguucccca ucuacacuau uccugacaaa 180
cugggcccuu gguccccaau cgacauccac caccuguccu gucccaacaa ucugguggug 240
gaggacgagg gcuguaccaa ucugagcggc uucuccuaca uggagcugaa ggugggcuac 300
aucuccgcca ucaaggugaa cggcuucaca uguaccggcg uggugacaga ggccgagaca 360
uacaccaacu ucgugggcua ugugaccaca accuucaagc ggaagcacuu cagaccaacc 420
ccugaugccu gucgguccgc cuauaacugg aagauggccg gcgacccacg guaugaggag 480
agccugcaca acccauaccc cgauuaccac uggcugagga cagugaagac aaccaaggag 540
uccgugguaa ucaucucucc aagcguggcc gaucuggacc cauacgauaa gucccugcac 600
agcagagugu ucccccgcgg caaguguucu ggcaucacag ugagcuccgc cuauugcucu 660
accaaccacg acuacaccau cuggaugccu gagaauccaa gacugggcac cuccugugac 720
aucuuuacca acucucgggg caagagagcc uccaagggcu ccaagacaug uggcuucgug 780
gacgagagag gccuguauaa gucccugaag ggcgccugca agcugaagcu gugcggcgug 840
cugggccuga ggcugaugga cggcaccugg guggccaucc agacauccaa cgagaccaag 900
uggugcccuc cugaucagcu ggugaaccug cacgacuuuc acagcgacga gaucgagcac 960
cugguggugg aggagcuggu gaagaagaga gaggagugcc uggaugcccu ggaguccauc 1020
augaccacca agagcguguc cuuuagacgg cugagccacc ugagaaagcu ggugcccggc 1080
uuuggcaagg ccuacaccau cuucaacaag acacugaugg aggccgaugc ccacuacaag 1140
uccgugagaa ccuggaacga gaucaucccu agcaagggcu gucugagggu gggcggcaga 1200
ugucacccuc acgugaaugg cguguucuuu aauggcauca uccugggccc agauggccac 1260
gugcugaucc cagagaugca guccucucug cugcagcagc acauggagcu gcuggagucu 1320
agcgugaucc cucugaugca cccacuggcc gauccaagca caguguucaa ggacggcgac 1380
gagguggagg acuuugugga ggugcaccug ccagaugugc acaagcaggu guccggcgug 1440
gaucugggcc ugccaaauug gggcaaggac gugcugaugg gcgccggcgu gcugaccgcc 1500
cugaugcuga ugaucuuccu gaugaccugc uguagacgga caaauagagc cgagagcauc 1560
cagcacuccc ugggcgagac aggccggaag gugucuguga ccagccagag uggacgagug 1620
aucuccucau gggaauccua caaaagcggc ggcgagacca aacugugagg acuaguuaua 1680
agacugacua gcccgauggg ccucccaacg ggcccuccuc cccuccuugc accgagauua 1740
auaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aau 1843
<210> 3
<211> 16
<212> DNA
<213> 人工序列
<400> 3
ggctagcgcc gccacc 16
<210> 4
<211> 34
<212> DNA
<213> 人工序列
<400> 4
gagacccaag ctggctagcg ggagaaagct tacc 34
<210> 5
<211> 109
<212> DNA
<213> 人工序列
<400> 5
gctggagcct cggtagccgt tcctcctgcc cgctgggcct cccaacgggc cctcctcccc 60
tccttgcacc ggcccttcct ggtctttgaa taaagtctga gtgggcagc 109
<210> 6
<211> 1575
<212> DNA
<213> CTN-1毒株
<400> 6
atgattcctc aagctctgtt gtttgtacct cttctggttt ttccattgtg tttcgggaaa 60
ttccccattt acacgatacc agacaaactc ggcccctgga gtcccatcga tatacatcac 120
ctcagctgtc cgaacaatct ggttgtggag gacgaaggat gtaccaatct gtcaggattc 180
tcatacatgg agcttaaagt aggatatatt tcggccataa aggtgaacgg gttcacttgt 240
acgggtgtgg taacggaagc agaaacctac actaactttg tcggttatgt caccaccacg 300
tttaagagaa agcacttccg accaacaccg gatgcatgca gatcagcata caattggaag 360
atggcaggtg accccagata tgaagagtct ctgcacaatc cctatcctga ttatcattgg 420
ctccggactg taaaaaccac caaagagtct gttgttatca tatctccaag tgtggcagac 480
ttagacccgt acgataaatc acttcattcg agagtttttc ctagaggaaa atgctcagga 540
ataacggtgt cttctgccta ctgctctacc aaccatgatt ataccatctg gatgcctgaa 600
aatcctagac tggggacctc ttgtgatatt ttcaccaaca gcagagggaa gagagcatcc 660
aaagggagca agacctgtgg atttgtggat gagagaggct tgtacaaatc tctaaaagga 720
gcatgcaaac tgaagctgtg tggagttctt ggacttaggc ttatggacgg aacctgggtc 780
gcgattcaga catcaaacga gaccaagtgg tgccctcctg atcaactagt gaatctacat 840
gactttcatt cagatgagat tgaacatctt gttgtggagg agttggttaa gaagagggag 900
gagtgtctag atgcactgga gtccatcatg accaccaagt ccgtgagttt cagacgtctc 960
agtcacttga ggaagcttgt gcctggattt ggaaaagcat acaccatatt caacaagacc 1020
ttaatggagg ctgatgctca ctacaaatcg gtccgaactt ggaatgagat catcccctcg 1080
aaagggtgtt taagagtcgg ggggagatgt catcctcatg tgaacggagt atttttcaat 1140
ggtatcatcc taggccctga cggccatgtc ttaatcccgg aaatgcagtc atccctcctc 1200
cagcagcata tggagttgtt ggaatcctcg gtcatcccct taatgcatcc cttggcagat 1260
ccatcaacgg tttttaaaga tggtgacgag gtggaggatt ttgttgaggt tcaccttcca 1320
gatgtgcata agcaggtctc aggggttgat ctcggtctcc caaactgggg gaaggatgtg 1380
ttgatgggcg caggcgtttt gacggcactg atgttgatga ttttcctaat gacgtgttgc 1440
cgaaggacta atagagcaga atcaatacaa cacagtcttg gagagacagg gaggaaagtg 1500
tcggtgacct cccaaagcgg gagggtcata tcttcatggg agtcatataa aagcggaggt 1560
gagaccaagc tgtaa 1575
<210> 7
<211> 524
<212> PRT
<213> CTN-1毒株
<400> 7
Met Ile Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu
1 5 10 15
Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro
20 25 30
Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val
35 40 45
Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu
50 55 60
Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys
65 70 75 80
Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr
85 90 95
Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala
100 105 110
Cys Arg Ser Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu
115 120 125
Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val
130 135 140
Lys Thr Thr Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala Asp
145 150 155 160
Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg Gly
165 170 175
Lys Cys Ser Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn His
180 185 190
Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys
195 200 205
Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Lys
210 215 220
Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly
225 230 235 240
Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp
245 250 255
Gly Thr Trp Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro
260 265 270
Pro Asp Gln Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile Glu
275 280 285
His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp
290 295 300
Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu
305 310 315 320
Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile
325 330 335
Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg
340 345 350
Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly
355 360 365
Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu
370 375 380
Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu
385 390 395 400
Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His
405 410 415
Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu
420 425 430
Asp Phe Val Glu Val His Leu Pro Asp Val His Lys Gln Val Ser Gly
435 440 445
Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Asp Val Leu Met Gly Ala
450 455 460
Gly Val Leu Thr Ala Leu Met Leu Met Ile Phe Leu Met Thr Cys Cys
465 470 475 480
Arg Arg Thr Asn Arg Ala Glu Ser Ile Gln His Ser Leu Gly Glu Thr
485 490 495
Gly Arg Lys Val Ser Val Thr Ser Gln Ser Gly Arg Val Ile Ser Ser
500 505 510
Trp Glu Ser Tyr Lys Ser Gly Gly Glu Thr Lys Leu
515 520
Claims (10)
1.一种优化RVG mRNA，其特征在于，转录所述优化RVG mRNA的核苷酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的优化RVG mRNA，其特征在于，转录所述优化RVG mRNA的核苷酸序列还包括如下元件：5’-帽结构、5’-UTR、3’-聚腺苷酸序列和3’-UTR中的至少一种；
优选地，所述5’-UTR为10-200个核苷酸，更优选为15-100个核苷酸；
优选地，所述5’-UTR为DNAH2 5’-UTR序列或KOZAK序列；
优选地，所述KOZAK序列如SEQ ID NO.3所示；
优选地，所述DNAH2 5’-UTR序列如SEQ ID NO.4所示；
优选地，所述3’-聚腺苷酸序列优选为60-120个A，更优选为80-110个A，进一步优选为100个A；
优选地，所述3’-UTR包括β－珠蛋白3’-UTR序列或血红蛋白HBA23’-UTR序列；
优选地，所述血红蛋白HBA2 3’-UTR序列如SEQ ID NO.5所示。
3.根据权利要求2所述的优化RVG mRNA，其特征在于，所述优化RVG mRNA的序列如SEQID NO.2所示。
4.权利要求1-3任一项所述的优化RVG mRNA在制备狂犬病毒核酸疫苗中的应用。
5.一种狂犬病毒核酸疫苗，其特征在于，所述狂犬病毒核酸疫苗包括疫苗载体和权利要求1-3任一项所述的优化RVG mRNA。
6.根据权利要求5所述的狂犬病毒核酸疫苗，其特征在于，所述疫苗载体包括阳离子脂质体、阳离子蛋白、阳离子聚合物或阳离子脂质纳米颗粒中的一种，优选为阳离子脂质体或阳离子脂质纳米颗粒；
优选地，阳离子脂质纳米颗粒与优化RVG mRNA的质量比为10-30:1，优选为15-25:1，更优选为20:1；
优选地，阳离子脂质体与优化RVG mRNA的质量比为1-8:1，优选为1-4:1，更优选为2:1。
7.根据权利要求5或6所述的狂犬病毒核酸疫苗，其特征在于，所述疫苗载体为阳离子脂质纳米颗粒，按摩尔百分比计，包括20-50％可质子化阳离子脂质、20-50％结构脂质、5-20％辅助脂质和1-5％表面活性剂，其中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％；
优选地，所述可质子化阳离子脂质包括Dlin-MC3-DMA、Dlin-KC2-DMA、DODMA、c12-200或DlinDMA，优选为Dlin-MC3-DMA；
优选地，所述结构脂质包括胆固醇、胆固醇酯、固醇类激素、固醇类维生素或胆汁酸，优选为胆固醇；
优选地，所述辅助脂质包括DSPC，DOPE，DOPC或DOPS，优选为DSPC；
优选地，所述表面活性剂包括PEG-DMG或PEG-DSPE，优选为PEG-DMG；
优选地，所述狂犬病毒核酸疫苗的给药方式包括静脉注射、肌肉注射或皮内注射，优选为肌肉注射。
8.权利要求5所述的狂犬病毒核酸疫苗的制备方法，其特征在于，将疫苗载体和权利要求1-3任一项所述的优化RVG mRNA混合，得到狂犬病毒核酸疫苗。
9.根据权利要求8所述的制备方法，其特征在于，所述疫苗载体为权利要求7所述狂犬病毒核酸疫苗中的阳离子脂质纳米颗粒，制备方法包括：
(a)按配方比例将可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂溶于有机溶液，得到有机相；
(b)将优化RVG mRNA溶于PBS或柠檬酸盐溶液中，得到水相；
(c)将(a)得到的有机相和(b)得到的水相混匀得到混合液，替换混合液中的溶媒为缓冲液，得到狂犬病毒核酸疫苗；
优选地，有机溶液包括无水乙醇、四氢呋喃、丙酮或DMSO，优选为无水乙醇；
优选地，有机相中可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的总浓度为5-7mg/ml；
优选地，水相中优化RVG mRNA的浓度为0.8-1.2mg/ml；
优选地，有机相和水相的体积比为1：2-4；
优选地，混匀采用微流控设备，流速范围为6-24ml/min，优选为12.0ml/min；
优选地，替换混合液中的溶媒为缓冲液包括：用缓冲液将混合液稀释50-100倍，再浓缩。
10.根据权利要求8或9所述的制备方法，其特征在于，所述制备方法还包括将液态的狂犬病毒核酸疫苗制备成冻干制剂的过程；
优选地，在狂犬病毒核酸疫苗中加入5-30w/v％药学上可接受的冻干保护剂得到待冻干制剂，待冻干制剂经冻干得到冻干制剂；
优选地，待冻干制剂中还包括0.005-0.05w/v％药学上可接受的表面活性剂；
优选地，冻干过程包括：真空度为20μbar-80μbar的条件下，依次为，-40℃至-60℃保持1-8h，-30℃至-50℃保持10-30h，4℃至20℃保持10-30h。
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