CN116655749A - 一种EBV的Rta截短mRNA相关疫苗及其制备方法与应用 - Google Patents
一种EBV的Rta截短mRNA相关疫苗及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了一种EBV的Rta截短mRNA相关疫苗及其制备方法与应用,具体公开了一种蛋白质。本发明提供一种EBV的Rta截短的蛋白质,以及基于编码截短蛋白质的mRNA制备的相关mRNA疫苗。首先申请人分析Rta蛋白(BRLF1编码)结构以及MHC(主要组织相容性复合体)结合序列;筛选出免疫原性较强的序列:截短体1和截断体2。并基于截短体1和截断体2制备mRNA疫苗,发现截短序列和全长序列表现出了相似的肿瘤抑制作用和免疫原性,且截短体激发了更强的细胞免疫,综合考虑生产成本及治疗效果后,截短体序列可以用于EBV相关肿瘤的治疗疫苗的开发。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种EBV的Rta截短mRNA相关疫苗及其制备方法与应用。
背景技术
EBV属人类γ-疱疹病毒亚科淋巴潜隐病毒属,是第一种被鉴定的人类致癌病毒,与鼻咽癌、淋巴瘤、部分胃癌等肿瘤发生密切相关。EBV是双链DNA囊膜病毒,基因组大小约为172kb,可以编码85个基因和多种miRNA。这些病毒的糖蛋白和miRNA在病毒入侵机体,潜伏期凋亡逃避,增殖期的免疫逃避过程中发挥了至关重要的作用。据报道,至少90%以上的鼻咽癌在流行病学上证实与EBV相关,因此以EBV为靶点开发药物和疫苗具有开阔的前景。虽然EBV从发现至今已经过去了40年,但是截止目前为止仍然没有EBV疫苗上市,表明疫苗开发难度大开发周期长的特点。目前每年约有20万新发鼻咽癌病例,开发行之有效的预防和治疗疫苗已刻不容缓。
mRNA疫苗是是继灭活疫苗、减毒活疫苗、亚单位疫苗和病毒载体疫苗后的第三代疫苗,它通过将含有编码抗原蛋白的mRNA导入人体,直接进行翻译生成相应的抗原蛋白,从而诱导机体产生特异性免疫应答,达到预防免疫的作用。因此mRNA疫苗具有不带有病毒成分,没有感染风险,疫苗整体的安全性要远高于蛋白疫苗。同时由于疫苗的研发周期短,生产成本低,可以针对病毒新兴的变异株在短时间内设计和生产出特异性疫苗。本发明旨在设计和开发针对EBV感染关键性蛋白的mRNA治疗性疫苗。
发明内容
本发明第一方面的目的,在于提供一种蛋白质。
本发明第二方面的目的,在于提供编码本发明第一方面的蛋白质的核酸分子。
本发明第三方面的目的,在于提供与本发明第二方面的核酸分子相关的生物材料。
本发明第四方面的目的,在于提供一种mRNA疫苗组合物。
本发明第五方面的目的,在于提供本发明第一方面的蛋白质、第二方面的核酸分子、第三方面的生物材料和/或第四方面的mRNA疫苗组合物在制备产品中的应用。
本发明第六方面的目的,在于提供一种产品。
为了实现上述目的,本发明所采取的技术方案是:
本发明第一个方面,在于提供一种蛋白质,所述蛋白质的氨基酸序列为a)或b):
a)如SEQ ID NO:1或SEQ ID NO:4所示;
b)a)所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
优选地,所述蛋白质的氨基酸序列还可为与a)或b)所限定的氨基酸序列具有99%以上、95%以上或者90%以上同源性且具有相同功能的氨基酸序列。
本发明第二个方面,在于提供编码本发明第一方面的蛋白质的核酸分子。
优选地,所述核酸分子的核苷酸序列如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:6所示。
本发明第三个方面,在于提供与本发明第二方面的核酸分子相关的生物材料,所述生物材料包括(1)~(3)中的至少一种:
(1)表达盒,含有本发明第二方面的核酸分子;
(2)重组载体,含有本发明第二方面的核酸分子或(1)中表达盒;
(3)重组细胞,含有本发明第二方面的核酸分子、(1)中表达盒或(2)中重组载体。
优选地,所述重组载体为质粒载体、病毒载体或细胞载体。
优选地,所述质粒载体可以是任选的质粒,所述病毒载体可以任选的病毒,所述细胞载体不包括繁殖材料。
优选地,所述细胞为昆虫细胞和/或哺乳动物细胞。
本发明第四个方面,在于提供一种mRNA疫苗组合物,所述mRNA疫苗组合物包括本发明第一方面的蛋白质、本发明第二方面的核酸分子或本发明第三方面的生物材料。
优选地,所述疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
优选地,所述载体为脂质体。
优选地,所述载体为阳离子脂质体,包括阳离子脂质和辅助脂质。
优选地,所述阳离子脂质包括DOTAP、DOTMA、DOEPC、DC-Chol、DDAB、DODMA和DLinDMA中的至少一种。
优选地,所述辅助脂质为中性辅助脂质。
优选地,所述中性辅助脂质包括DSPC、DOPE、DOPC、DOPG、DOPS和胆固醇中的至少一种。
优选地,所述脂质体为DOTMA和DOPA,两者的摩尔比为1~10:1;进一步为2:1。
优选地,所述疫苗的制备方法为:将mRNA与脂质体混合,其中mRNA与脂质体的质量比为1~5:1,进一步为2~3:1。
优选地,所述疫苗以生理可给药的形式提供,并且适合于口服、肌内、静脉内、皮下或皮肤注射应用。
本发明第五个方面,在于提供本发明第一方面的蛋白质、第二方面的核酸分子、第三方面的生物材料和/或第四方面的mRNA疫苗组合物在制备产品中的应用。
优选地,所述产品的功能为(b1)~(b5)中的任一种:
(b1)预防或治疗EB病毒感染相关疾病;
(b2)预防或治疗肿瘤;
(b3)抑制肿瘤生长;
(b4)引起免疫反应;
(b5)制备EB病毒抗体。
优选地,所述产品包括试剂和/或试剂盒。
优选地,所述产品为药物,进一步为疫苗。
本发明第六个方面,在于提供一种产品,包含本发明第一方面的蛋白质、第二方面的核酸分子、第三方面的生物材料和/或第四方面的mRNA疫苗组合物。
优选地,所述产品的功能为(b1)~(b5)中的任一种:
(b1)预防或治疗EB病毒感染相关疾病;
(b2)预防或治疗肿瘤;
(b3)抑制肿瘤生长;
(b4)引起免疫反应;
(b5)制备EB病毒抗体。
优选地,所述产品包括试剂和/或试剂盒。
优选地,所述产品为药物,进一步为疫苗。
本发明的有益效果是:
本发明提供一种EBV的Rta截短的蛋白质,以及基于编码截短蛋白质的mRNA制备的相关mRNA疫苗。首先申请人分析Rta蛋白(BRLF1编码)结构以及MHC(主要组织相容性复合体)结合序列;筛选出免疫原性较强的序列:截短体1和截断体2。并基于截短体1和截断体2制备mRNA疫苗,发现截短序列和全长序列表现出了相似的肿瘤抑制作用和免疫原性,且截短体激发了更强的细胞免疫,综合考虑生产成本及治疗效果后,截短体序列可以用于EBV相关肿瘤的治疗疫苗的开发。
附图说明
图1为不同抗原截短体在293T细胞表达情况,图中,A为BRLF1全长序列(1~605aa),B为截断体1(1~440aa),C为截断体2(16~440aa),内参蛋白为Actin。
图2为mRNA疫苗和脂质体的Zeta电位检测结果图。
图3为mRNA疫苗和脂质体的粒径检测结果图。
图4为不同组小鼠的成瘤率统计结果图。
图5为不同组小鼠的肿瘤大小统计结果图。
图6为不同组小鼠的外周血淋巴细胞流式分析结果图。
图7为不同组小鼠的外周血总IgG浓度检测结果图。
具体实施方式
现结合具体实施例对本发明进行详细说明,但不限制本发明的范围。
本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的材料和试剂。
实施例1抗原体截短体的筛选
发明人基于Rta蛋白质结构预测筛选出结构较稳定的部分,然后通过MHC(主要组织相容性复合体)分子结合预测以及免疫原性预测,筛选出最佳的序列进行构建和表达。共筛选出了2个免疫原性较强的序列(截断体1和截断体2),其具体氨基酸序列和核苷酸序列如下:
截断体1的氨基酸序列(位于Rta蛋白全长序列(NCBI Reference Sequence:YP_401674.1)第1~440aa)为:
MRPKKDGLEDFLRLTPEIKKQLGSLVSDYCNVLNKEFTAGSVEITLRSYKICKAFINEAKAHGREWGGLMATLNICNFWAILRNNRVRRRAENAGNDACSIACPIVMRYVLDHLIVVTDRFFIQAPSNRVMIPATIGTAMYKLLKHSRVRAYTYSKVLGVDRAAIMASGKQVVEHLNRMEKEGLLSSKFKAFCKWVFTYPVLEEMFQTMVSSKTGHLTDDVKDVRALIKTLPRASYSSHAGQRSYVSGVLPACLLSTKSKAVETPILVSGADRMDEELMGNDGGASHTEARYSESGQFHAFTDELESLPSPTMPLKPGAQSADCGDSSSSSSDSGNSDTEQSEREEARAEAPRLRAPKSRRTSRPNRGQTPCPSNAAEPEQPWIAAVHQESDERPIFPHPSKPTFLPPVKRKKGLRDSREGMFLPKPEAGSAISDVFEGR(SEQ IDNO:1)。
截断体1的DNA序列为:
ATGAGGCCTAAAAAGGATGGCTTGGAAGACTTTCTGAGGCTAACTCCTGAAATCAAAAAGCAGCTGGGCTCTCTGGTCTCTGACTACTGCAACGTCCTCAACAAGGAATTTACAGCCGGGAGTGTGGAGATTACTCTGAGATCCTACAAAATATGCAAGGCATTTATAAATGAGGCCAAGGCCCACGGGCGAGAATGGGGCGGGCTAATGGCCACGCTCAACATCTGCAATTTTTGGGCCATTCTCCGAAACAACAGGGTAAGAAGACGGGCTGAGAATGCCGGCAACGACGCATGTTCCATCGCGTGCCCTATAGTGATGCGCTACGTGTTAGACCACCTGATAGTGGTCACTGACAGATTCTTCATCCAGGCCCCCAGCAACCGGGTGATGATTCCTGCCACCATAGGCACCGCTATGTACAAGCTCCTAAAACACAGTCGGGTGCGGGCCTACACCTACAGCAAGGTGCTGGGCGTGGACCGCGCGGCCATCATGGCCTCCGGCAAGCAGGTAGTGGAACACCTGAACAGGATGGAGAAGGAAGGCCTCCTAAGCTCCAAGTTCAAGGCCTTTTGCAAGTGGGTGTTCACCTATCCCGTCCTCGAGGAGATGTTCCAGACTATGGTCTCGTCCAAGACAGGCCATCTGACGGACGATGTTAAGGATGTCAGGGCTCTGATTAAGACACTGCCCCGGGCCTCCTACTCCAGCCACGCCGGACAGAGGAGCTACGTGAGCGGCGTGCTTCCCGCGTGCCTGCTGTCAACCAAGTCCAAGGCAGTGGAAACTCCTATCCTCGTGTCCGGAGCCGACAGGATGGACGAGGAGCTCATGGGGAATGATGGGGGTGCCTCTCACACCGAGGCCCGCTACTCGGAGTCCGGACAGTTTCATGCTTTTACAGATGAACTCGAAAGTCTCCCGAGCCCGACCATGCCCCTGAAGCCCGGTGCCCAAAGCGCCGACTGCGGTGACAGCAGTTCCAGCAGCAGTGACTCGGGCAACAGTGACACCGAGCAGAGCGAGCGGGAAGAGGCCAGGGCCGAGGCCCCGCGCCTGCGGGCCCCAAAGTCGCGCCGGACATCCAGGCCCAACCGTGGTCAAACTCCATGTCCTTCCAACGCGGAGGAACCTGAACAGCCTTGGATAGCAGCGGTCCACCAAGAGAGCGATGAGAGACCCATATTCCCCCACCCCTCAAAGCCCACCTTTCTTCCTCCCGTTAAAAGGAAGAAGGGCCTCAGGGATAGCCGGGAAGGTATGTTCCTGCCAAAGCCGGAAGCGGGCAGTGCCATATCTGACGTGTTCGAGGGGCG A(SEQ ID NO:2)。
截断体1的mRNA序列为:
AUGAGGCCUAAAAAGGAUGGCUUGGAAGACUUUCUGAGGCUAACUCCUGA
AAUCAAAAAGCAGCUGGGCUCUCUGGUCUCUGACUACUGCAACGUCCUCAACA
AGGAAUUUACAGCCGGGAGUGUGGAGAUUACUCUGAGAUCCUACAAAAUAUGC
AAGGCAUUUAUAAAUGAGGCCAAGGCCCACGGGCGAGAAUGGGGCGGGCUAAU
GGCCACGCUCAACAUCUGCAAUUUUUGGGCCAUUCUCCGAAACAACAGGGUAA
GAAGACGGGCUGAGAAUGCCGGCAACGACGCAUGUUCCAUCGCGUGCCCUAUA
GUGAUGCGCUACGUGUUAGACCACCUGAUAGUGGUCACUGACAGAUUCUUCAU
CCAGGCCCCCAGCAACCGGGUGAUGAUUCCUGCCACCAUAGGCACCGCUAUGU
ACAAGCUCCUAAAACACAGUCGGGUGCGGGCCUACACCUACAGCAAGGUGCUG
GGCGUGGACCGCGCGGCCAUCAUGGCCUCCGGCAAGCAGGUAGUGGAACACCU
GAACAGGAUGGAGAAGGAAGGCCUCCUAAGCUCCAAGUUCAAGGCCUUUUGCA
AGUGGGUGUUCACCUAUCCCGUCCUCGAGGAGAUGUUCCAGACUAUGGUCUCG
UCCAAGACAGGCCAUCUGACGGACGAUGUUAAGGAUGUCAGGGCUCUGAUUAA
GACACUGCCCCGGGCCUCCUACUCCAGCCACGCCGGACAGAGGAGCUACGUGA
GCGGCGUGCUUCCCGCGUGCCUGCUGUCAACCAAGUCCAAGGCAGUGGAAACU
CCUAUCCUCGUGUCCGGAGCCGACAGGAUGGACGAGGAGCUCAUGGGGAAUGA
UGGGGGUGCCUCUCACACCGAGGCCCGCUACUCGGAGUCCGGACAGUUUCAUG
CUUUUACAGAUGAACUCGAAAGUCUCCCGAGCCCGACCAUGCCCCUGAAGCCC
GGUGCCCAAAGCGCCGACUGCGGUGACAGCAGUUCCAGCAGCAGUGACUCGGG
CAACAGUGACACCGAGCAGAGCGAGCGGGAAGAGGCCAGGGCCGAGGCCCCGC
GCCUGCGGGCCCCAAAGUCGCGCCGGACAUCCAGGCCCAACCGUGGUCAAACUC
CAUGUCCUUCCAACGCGGAGGAACCUGAACAGCCUUGGAUAGCAGCGGUCCAC
CAAGAGAGCGAUGAGAGACCCAUAUUCCCCCACCCCUCAAAGCCCACCUUUCU
UCCUCCCGUUAAAAGGAAGAAGGGCCUCAGGGAUAGCCGGGAAGGUAUGUUCC
UGCCAAAGCCGGAAGCGGGCAGUGCCAUAUCUGACGUGUUCGAGGGGCGA(SEQ ID NO:3)。
截断体2的氨基酸序列(位于Rta蛋白全长序列第16~440aa):PEIKKQLGSLVSDYCNVLNKEFTAGSVEITLRSYKICKAFINEAKAHGREWGGLMATLNICNFWAILRNNRVRRRAENAGNDACSIACPIVMRYVLDHLIVVTDRFFIQAPSNRVMIPATIGTAMYKLLKHSRVRAYTYSKVLGVDRAAIMASGKQVVEHLNRMEKEGLLSSKFKAFCKWVFTYPVLEEMFQTMVSSKTGHLTDDVKDVRALIKTLPRASYSSHAGQRSY VSGVLPACLLSTKSKAVETPILVSGADRMDEELMGNDGGASHTEARYSESGQFHAFTDELESLPSPTMPLKPGAQSADCGDSSSSSSDSGNSDTEQSEREEARAEAPRLRAPKSRRTSRPNRGQTPCPSNAAEPEQPWIAAVHQESDERPIFPHPSKPTFLPPVKRKKGLRDSREGMFLPKPEAGSAISDVFEGR(SEQ ID NO:4)。
截断体2的DNA序列为:
ATGCCTGAAATCAAAAAGCAGCTGGGCTCTCTGGTCTCTGACTACTGCAACGTCCTCAACAAGGAATTTACAGCCGGGAGTGTGGAGATTACTCTGAGATCCTACAAAATATGCAAGGCATTTATAAATGAGGCCAAGGCCCACGGGCGAGAATGGGGCGGGCTAATGGCCACGCTCAACATCTGCAATTTTTGGGCCATTCTCCGAAACAACAGGGTAAGAAGACGGGCTGAGAATGCCGGCAACGACGCATGTTCCATCGCGTGCCCTATAGTGATGCGCTACGTGTTAGACCACCTGATAGTGGTCACTGACAGATTCTTCATCCAGGCCCCCAGCAACCGGGTGATGATTCCTGCCACCATAGGCACCGCTATGTACAAGCTCCTAAAACACAGTCGGGTGCGGGCCTACACCTACAGCAAGGTGCTGGGCGTGGACCGCGCGGCCATCATGGCCTCCGGCAAGCAGGTAGTGGAACACCTGAACAGGATGGAGAAGGAAGGCCTCCTAAGCTCCAAGTTCAAGGCCTTTTGCAAGTGGGTGTTCACCTATCCCGTCCTCGAGGAGATGTTCCAGACTATGGTCTCGTCCAAGACAGGCCATCTGACGGACGATGTTAAGGATGTCAGGGCTCTGATTAAGACACTGCCCCGGGCCTCCTACTCCAGCCACGCCGGACAGAGGAGCTACGTGAGCGGCGTGCTTCCCGCGTGCCTGCTGTCAACCAAGTCCAAGGCAGTGGAAACTCCTATCCTCGTGTCCGGAGCCGACAGGATGGACGAGGAGCTCATGGGGAATGATGGGGGTGCCTCTCACACCGAGGCCCGCTACTCGGAGTCCGGACAGTTTCATGCTTTTACAGATGAACTCGAAAGTCTCCCGAGCCCGACCATGCCCCTGAAGCCCGGTGCCCAAAGCGCCGACTGCGGTGACAGCAGTTCCAGCAGCAGTGACTCGGGCAACAGTGACACCGAGCAGAGCGAGCGGGAAGAGGCCAGGGCCGAGGCCCCGCGCCTGCGGGCCCCAAAGTCGCGCCGGACATCCAGGCCCAACCGTGGTCAAACTCCATGTCCTTCCAACGCGGAGGAACCTGAACAGCCTTGGATAGCAGCGGTCCACCAAGAGAGCGATGAGAGACCCATATTCCCCCACCCCTCAAAGCCCACCTTTCTTCCTCCCGTTAAAAGGAAGAAGGGCCTCAGGGATAGCCGGGAAGGTATGTTCCTGCCAAAGCCGGAAGCGGGCAGTGCCATATCTGACGTGTTCGAGGGGCGA(SEQ IDNO:5)。
截断体2的mRNA序列为:
AUGCCUGAAAUCAAAAAGCAGCUGGGCUCUCUGGUCUCUGACUACUGCAACGUCCUCAACAAGGAAUUUACAGCCGGGAGUGUGGAGAUUACUCUGAGAUCCUACAAAAUAUGCAAGGCAUUUAUAAAUGAGGCCAAGGCCCACGGGCGAGAAUGG GGCGGGCUAAUGGCCACGCUCAACAUCUGCAAUUUUUGGGCCAUUCUCCGAAACAACAGGGUAAGAAGACGGGCUGAGAAUGCCGGCAACGACGCAUGUUCCAUCGCGUGCCCUAUAGUGAUGCGCUACGUGUUAGACCACCUGAUAGUGGUCACUGACAGAUUCUUCAUCCAGGCCCCCAGCAACCGGGUGAUGAUUCCUGCCACCAUAGGCACCGCUAUGUACAAGCUCCUAAAACACAGUCGGGUGCGGGCCUACACCUACAGCAAGGUGCUGGGCGUGGACCGCGCGGCCAUCAUGGCCUCCGGCAAGCAGGUAGUGGAACACCUGAACAGGAUGGAGAAGGAAGGCCUCCUAAGCUCCAAGUUCAAGGCCUUUUGCAAGUGGGUGUUCACCUAUCCCGUCCUCGAGGAGAUGUUCCAGACUAUGGUCUCGUCCAAGACAGGCCAUCUGACGGACGAUGUUAAGGAUGUCAGGGCUCUGAUUAAGACACUGCCCCGGGCCUCCUACUCCAGCCACGCCGGACAGAGGAGCUACGUGAGCGGCGUGCUUCCCGCGUGCCUGCUGUCAACCAAGUCCAAGGCAGUGGAAACUCCUAUCCUCGUGUCCGGAGCCGACAGGAUGGACGAGGAGCUCAUGGGGAAUGAUGGGGGUGCCUCUCACACCGAGGCCCGCUACUCGGAGUCCGGACAGUUUCAUGCUUUUACAGAUGAACUCGAAAGUCUCCCGAGCCCGACCAUGCCCCUGAAGCCCGGUGCCCAAAGCGCCGACUGCGGUGACAGCAGUUCCAGCAGCAGUGACUCGGGCAACAGUGACACCGAGCAGAGCGAGCGGGAAGAGGCCAGGGCCGAGGCCCCGCGCCUGCGGGCCCCAAAGUCGCGCCGGACAUCCAGGCCCAACCGUGGUCAAACUCCAUGUCCUUCCAACGCGGAGGAACCUGAACAGCCUUGGAUAGCAGCGGUCCACCAAGAGAGCGAUGAGAGACCCAUAUUCCCCCACCCCUCAAAGCCCACCUUUCUUCCUCCCGUUAAAAGGAAGAAGGGCCUCAGGGAUAGCCGGGAAGGUAUGUUCCUGCCAAAGCCGGAAGCGGGCAGUGCCAUAUCUGACGUGUUCGAGGGGCGA(SEQID NO:6)。
实施例2
1.过表达质粒的构建
根据实施例1筛选得到的靶点蛋白序列(截断体1和截断体2,同时以全长的Rta蛋白作为对照),经过密码子优化后得到对应的DNA序列;分别合成带有特异性DNA序列的质粒,并在各序列的C端增加一个Flag标签(5’-GATTACAAGGATGACGACGATAAG-3’(SEQ ID NO:13)),根据截断序列设计不同的特异性引物(引物序列如表1),以质粒为模板进行PCR扩增,PCR扩增反应体系和反应程序如表2和表3所示。将得到的PCR产物通过琼脂糖凝胶进行分离纯化,切胶并回收特异性DNA片段,具体为:核酸凝胶电泳结束后切下目的片段胶块装入1.5mL无菌离心管中,每1mg凝胶加入1μL Buffer MB混匀后置于55℃至完全溶解,冷却至室温后,转移到离心吸附柱内静置1min;室温下12000rpm离心1min,弃除收集管中的废液,加入600μL Buffer MW于离心吸附柱中;室温下12000rpm离心1min,弃除收集管中的废液,加入600μL Buffer MW于离心吸附柱中;室温下12000rpm离心1min,弃除收集管中的废液,室温下12000rpm离心3min,弃除收集管,将吸附柱转移到新的无菌EP管中;加入适量BufferEB至吸附柱中央室温静置5min,12000rpm室温离心1min,弃去吸附柱;收集获得的DNA片段于-20℃长期保存。按照表4配制反应体系将mRNA疫苗质粒载体(自构,序列已提交至Genebank,BankIt2714872pcDNA3.1(+)-mRNA-vaccine-vector.dna OR147959)的较大的酶切片段(约5800bp)与回收的DNA片段进行重组,混匀各组分后37℃孵育30min完成重组,置于冰上保存。将重组的质粒通过DH5α感受态细胞进行转化,涂板筛选单克隆进行DNA测序,根据测序结果选择序列一致的单克隆进行扩增,获得相应的目的质粒。
表1特异性引物序列
表2PCR体系
表3PCR扩增程序
表4重组反应体系
2.质粒蛋白表达验证
对上述构建的目的质粒进行蛋白表达验证,具体如下:将293T细胞使用添加10%胎牛血清的DMEM培养基复苏,传代至细胞培养六孔板中待密度长至60~70%;将目的质粒和聚乙二醇(PEI)按照1:3的比例在200μL Opti-mem培养基中混合静置;按照每孔加入2μg质粒的比例加入混合液,6~8小时后进行换液;待细胞长满后,吸弃细胞上清,加入1mL/孔PBS溶液清洗后吸弃;加入200μL/孔RIPA细胞裂解液,冰上裂解10min后转移至1.5mLEP管;加入50μL 5X上样缓冲液,充分混匀后100℃变性10min,待样品冷却到室温后上样到浓度为8%聚丙烯酰胺凝胶加样孔内,每孔上样量为40μL,70V恒压电泳30min后110V恒压电泳1h进行转膜;将PVDF膜(0.22μm,5.5*8.5cm)用无水甲醇进行活化1mim,然后浸入转膜液(弗德生物,货号FD1033)中,使用Bio-Rad的标准湿式转膜装置,110V恒压转膜时间为1小时;转膜完毕后,将PVDF膜放入预先加入5%脱脂牛奶TBST溶液的Western洗膜盒中,置于摇床上室温封闭2小时;一抗孵育:按照1:2000加入抗体稀释液(弗德生物,货号FD0040)稀释好的一抗(anti-Flag,sigma或者anti-beta-actin,CST),4℃缓慢摇动孵育过夜;将PVDF膜取出置于另一加入TBST溶液的Western洗膜盒中在摇床上洗涤4次,10min/次;二抗孵育:按照1:4000加入抗体稀释液稀释好的二抗(anti-mouse,CST,或者anti-rabbit,CST),室温缓慢摇动孵育1小时;将PVDF膜取出置于另一加入TBST溶液的Western洗膜盒中在摇床上洗涤4次,10min/次;蛋白检测:将显色液A,B液混合,将上述PVDF膜浸入显色液内,1min后放入化学发光仪(Biorad)内进行成像。
检测结果如图1所示,目的条带小大合适,表明已将目的片段成功表达。
实施例3疫苗的合成和包装
为了保证mRNA在体内的稳定性使其可以实现有效的递送,发明人采用脂质纳米颗粒(Lipid nanoparticles,LNPs)对合成的mRNA进行包装得到Rta-mRNA脂质体颗粒(mRNA疫苗),具体过程如下:
mRNA疫苗合成:使用限制性核酸内切酶对构建并验证后的质粒进行酶切;琼脂糖凝胶电泳后切胶回收特异性DNA片段;使用T7体外转录试剂盒进行体外扩增(体系如表5),混匀各组分,并短暂离心收集,37℃孵育过夜。反应结束后加入DNase I,37℃孵育15min,消化转录中残留的DNA,得到产物溶液。使用氯化锂对RNA产物进行初步纯化:将产物溶液进行混匀分装,加入LiCl沉淀溶液(7.5M)至终浓度为2.5M,混匀后放入-20℃放置30min;12000rpm离心15min,去上清,收集沉淀;RNase Free Water复溶后检测RNA浓度。对纯化后的RNA产物进行RNA加帽反应:根据RNA浓度将其稀释为0.75μg/μL,稀释好的RNA置于65℃加热5min,结束后冰上放置5min;按表6配制mRNA加帽体系,37℃反应30min,即加帽反应完成。再次使用氯化锂对RNA产物进行纯化,得到加帽的mRNA。
表5mRNA体外转录体系
表6mRNA加帽体系
mRNA包装:
脂质体制备(参见如下文献进行:Systemic RNA delivery to dendritic cellsexploits antiviral defence for cancer immunotherapy DOI:10.1038/nature18300):将100mg DOTMA和40.92mg DOPE溶解于680μL无水乙醇中,然后将乙醇溶液缓慢滴加在34mL蒸馏水中,以200rpm的转速不断搅拌1h后通过0.45μm滤膜过滤,得到LNP溶液,4℃存放。
将新鲜配置的PBS溶液以及LNP溶液使用0.22μm滤器过滤除杂质;以100μL体系为例,取无菌EP管加入100μL PBS溶液;按照mRNA(加帽的mRNA):LNP=25:11的质量比例依次加入mRNA和LNP,混匀离心后置于冰上30min完成mRNA包装得到Rta-mRNA脂质体颗粒(mRNA疫苗),即得到截断体1疫苗、截断体2疫苗和全长序列疫苗。
对Rta-mRNA脂质体颗粒的粒径大小及Zeta电位进行检测,结果如图3所示,Rta-mRNA脂质体颗粒(全长序列)的直径大于脂质体,表明成功将脂质体包装在mRNA上。包装后Rta-mRNA脂质体颗粒的Zeta电位数见图2。
实施例4疫苗治疗效果检测
为了评估实施例3所制备得到mRNA疫苗对肿瘤的治疗效果,发明人进一步构建了表达BRLF1全长的B16-luc单克隆细胞稳株,并以C57小鼠(6~8周龄)为实验对象,每只小鼠皮下注射6*104个B16-luc单克隆细胞稳株构建小鼠荷瘤模型。将C57小鼠随机分为4组:截断体1疫苗组(注射实施例3制备得到的截断体1疫苗)、截断体2疫苗组(注射实施例3制备得到的截断体2疫苗)、全长序列疫苗组(注射实施例3制备得到的全长序列疫苗)和对照组(实施例3中制备的脂质体)。在种瘤后的第4、9、14、19、24天,按照分组分别对各组小鼠进行尾静脉注射(40μg/只/次),以评估mRNA疫苗对肿瘤的治疗效果。
其中,表达BRLF1全长的B16-luc单克隆细胞稳株的构建方法如下:
(1)单克隆稳株的质粒构建
本实验使用PLVX质粒进行稳株的构建;使用限制性核酸内切酶EcoRI和XbaI对PLVX质粒37℃酶切过夜,根据基因序列以及酶切位点设计特异性引物(上游引物:5’-CTACCGGACTCAGATCTCGAGAYGCATCATCATCATCATCATAGGCCTAAAAAGG AT-3’(SEQ ID NO:14);下游引物:5’-TACCCGGTAGAATTATCTAGACTAAAATAAGCTGGTGTCAA-3’(SEQ ID NO:15))同时在N端引入Flag序列;PCR反应特异性扩增目的基因片段,反应体系如表2;将PCR产物以及酶切产物通过琼脂糖凝胶进行分离纯化,切胶并回收特异性DNA片段过程同实施例2;按照表7所示配制反应体系,将酶切片段和PCR产物进行同源重组;使用DH5α感受态细胞进行转化实验,使用氨苄青霉素抗性板进行涂板,筛选单克隆菌落,挑选单克隆菌落送测序确定质粒序列,将序列正确的单菌落进行扩增和质粒提取,即得到单克隆稳株的质粒。
表7质粒酶切反应体系
(2)B16-luc单克隆细胞稳株构建
培养293T细胞、B16细胞(DMEM培养基+10%胎牛血清),将293T细胞扩增至直径15cm的细胞培养皿,进行慢病毒包被,慢病毒包被体系如表8(以单个15cm皿为例)所示,体系混合均匀后室温静置20min。将培养皿中置于细胞培养箱中培养6~8小时后换液,换液36h后收集细胞培养基上清使用0.45μm滤膜过滤。过滤后的上清转移至高离管中配平(每管约40mL),100000g 4℃离心2h,弃上清,使用1640培养基进行重悬(40mL离心后用400μL重悬)。B16细胞在六孔细胞培养板培养至密度60~70%,每孔加入浓缩的病毒液400μL,病毒感染48小时后换液。孔板内细胞长满后传代至新的六孔细胞培养板中,按照1:500的比例加入嘌呤霉素(1mg/mL),筛选48~72h至阴性对照组细胞(阴性对照组由于不具有有抗性所以会死亡)全部死亡,将剩余细胞传代至细胞培养瓶中扩大培养。取一部分细胞提取蛋白质进行靶点蛋白表达量检测:将六孔细胞培养板内培养基弃去,加入预冷的PBS溶液(1mL/孔)进行洗涤,吸弃PBS溶液,加入500μL/孔RIPA细胞裂解液,冰上裂解20min后转移至1.5mL EP管中,加入100μL5X SDS loading buffer充分混匀95℃变性10min后得到蛋白样品。将检测无误的细胞稳株按照1个细胞/孔铺96孔细胞培养板,培养14~21天后挑选单克隆细胞株传至细胞培养板中扩大培养,提取不同单克隆细胞株的蛋白质进行检测,选择高表达目的蛋白的细胞株为实验用B16-luc单克隆细胞稳株。
表8慢病毒包被体系
在种瘤后第7、11、17、19、21、24、26、28、30天分别记录各小组小鼠的肿瘤生长情况,并计算小鼠的肿瘤成瘤率,以及利用游标卡尺测量各小鼠的肿瘤体积。结果显示,与脂质体对照组相比,注射mRNA疫苗可有效控制肿瘤的成瘤率,延缓成瘤时间,尤其是截断体2疫苗的效果尤为突出(图4)。与脂质体对照组相比,注射mRNA疫苗可有效控制肿瘤体积的变大,各mRNA疫苗在控制肿瘤体积方面的效果没有显著性差异(图5)。
进一步采用流式染色法对实验结束后(第30天)的各组小鼠的外周血淋巴细胞进行检测,并检测各组小鼠的外周血的总IgG浓度。外周血淋巴细胞检测具体如下:使用抗凝管收集各组小鼠的外周血200~500μL,颠倒混匀后冰上放置,鼠外周血淋巴细胞分离试剂盒(天津灏洋,LTS1092)分离淋巴细胞,使用样本稀释液将血液稀释至4mL;15mL离心管中加入4mL外周血淋巴细胞分离液,将稀释后的血液沿壁缓缓加入离心管中,保持分界不被破坏;500g室温离心30min,小心吸取中间的淋巴细胞分层转至新的15mL离心管中,心管中加入适量无菌PBS溶液补充至10mL,400g室温离心10min;使用无血清1640培养基重悬后加入48孔细胞培养板,入PMA刺激剂(终浓度为100ng/mL),置于细胞培养箱中刺激4~6h;刺激结束每孔加入1mL PBS溶液进行中止,吸取适量细胞悬液转至EP管中,800g 4℃离心5min弃上清;使用1:200稀释后的CD4和CD8流式抗体溶液重悬沉淀,避光置于4℃染色30min。染色结束后加入1mL PBS溶液/管中止染色,800g 4℃离心5min弃上清;使用200μL IC固定液来固定细胞重悬沉淀(100μL/管),避光置于4℃固定30min;每管加入1mL 1×破膜液(Thermo,货号:88-8824-00)进行中止,800g 4℃离心5min弃上清,使用1×破膜液稀释的IFNγ抗体溶液(1:200)重悬沉淀(100μL/管),避光置于4℃固定30min;染色结束后每管加入1mL1X破膜液进行中止,1000g 4℃离心5min弃上清,使用适量PBS溶液重悬后进行流式上机。总IgG浓度检测方法如下:采集的外周血3000g离心10min取上清转移至新的EP管中,使用样本稀释液将血清稀释10000倍用作待测样品,使用小鼠免疫球蛋白G ELISA试剂盒(CSB-E07980m华美生物)进行抗体检测将预先包被抗体的Elisa板置于室温平衡1h;将IgG标准品溶液(试剂盒附带)以及待测样品加入Elisa板中50μL/孔,设置三个重复;将HRP结合的底物溶液加入Elisa板中50μL/孔,空白孔不加,封板膜封板后置于37℃孵育1h;孵育结束后弃去液体,同时加入洗涤液(试剂盒附带)200μL/孔,静置2min弃去板内液体,重复洗板五次;将孔板内水分充分甩干,加入TMB溶液70μL/孔,37℃孵育10min;加入中止液,50μ/孔,于酶标仪中测定450nm处的OD值。
检测结果显示,截短序列和全长序列表现出了相似的肿瘤抑制作用和免疫原性,且截短体激发了更强的细胞免疫(图6和图7)。
上面结合附图对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种蛋白质,所述蛋白质的氨基酸序列为a)或b):
a)如SEQ ID NO:1或SEQ ID NO:4所示;
b)a)所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后且功能相同或相似的氨基酸序列。
2.编码权利要求1所述的蛋白质的核酸分子,所述核酸分子的核苷酸序列优选如SEQID NO:2、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:6所示。
3.与权利要求2所述的核酸分子相关的生物材料,所述生物材料包括(1)~(3)中的至少一种:
(1)表达盒,含有权利要求2所述的核酸分子;
(2)重组载体,含有权利要求2所述的核酸分子或(1)中表达盒;
(3)重组细胞,含有权利要求2所述的核酸分子、(1)中表达盒或(2)中重组载体。
4.一种mRNA疫苗组合物,所述mRNA疫苗组合物包括权利要求1所述的蛋白质、权利要求2所述的核酸分子或权利要求3所述的生物材料。
5.根据权利要求4所述的mRNA疫苗组合物,其特征在于,所述疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
6.根据权利要求5所述的mRNA疫苗组合物,其特征在于,所述载体为脂质体。
7.根据权利要求6所述的mRNA疫苗组合物,其特征在于,所述疫苗以生理可给药的形式提供,并且适合于口服、肌内、静脉内、皮下或皮肤注射应用。
8.权利要求1所述的蛋白质、权利要求2所述的核酸分子、权利要求3所述的生物材料和/或权利要求4~7中任一项所述的mRNA疫苗组合物在制备产品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述产品的功能为(b1)~(b5)中的任一种:
(b1)预防或治疗EB病毒感染相关疾病;
(b2)预防或治疗肿瘤;
(b3)抑制肿瘤生长;
(b4)引起免疫反应;
(b5)制备EB病毒抗体。
10.一种产品,包含权利要求1所述的蛋白质、权利要求2所述的核酸分子、权利要求3所述的生物材料和/或权利要求4~7中任一项所述mRNA疫苗组合物。
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