WO2022037095A1 - 优化的表达新型冠状病毒抗原的核苷酸序列及其应用 - Google Patents
优化的表达新型冠状病毒抗原的核苷酸序列及其应用 Download PDFInfo
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Abstract
提供了一种优化的能表达对新型冠状病毒SARS-CoV-2引起免疫原性的多肽的核酸序列,该多肽为SARS-CoV-2的S蛋白S1亚基受体结合区第331-524位,其氨基酸序列如SEQ ID NO:2所示,编码其的核苷酸序列与SEQ ID NO:1所示的核苷酸序列具有至少80%同源性。优化的核苷酸序列可以利用流感载体有限的外源基因容纳量,表达对SARS-CoV-2引起免疫原性的多肽。
Description
本发明生物技术领域,具体涉及优化的表达新型冠状病毒抗原的核苷酸序列及其应用,特别涉及一种携带新型冠状病毒抗原的流感载体及其应用。
已有研究表明新型冠状病毒SARS-CoV-2的潜伏期可长达24天,传染性强,与引发非典的SARS病毒不同,部分病例潜伏期具有传染性,另有一些病毒携带者没有表现出任何明显症状。这对疫情的防控增加了难度。截止2020年8月19日,全球累计确诊病例数超过2200万,死亡人数接近78万。迄今尚未发现COVID-19的特效药物。为控制疫情,多数国家实行隔离措施,关闭非必要的设施,造成了巨大的经济损失。因此,快速研制出能够提升群体免疫水平并阻断病毒传播的预防疫苗已成为当前最为紧迫的重大需求。
针对新型冠状病毒,目前国内外尚无明确验证的特效抗病毒药物和预防性疫苗。因此做好预防,阻断病毒的传播是控制疫情的关键。而疫苗的应用已在消除多种传染病中发挥了不可替代的作用。已公布的十几个SARS-CoV-2病毒基因组比对的结果显示病毒之间的差异非常小,目前还没发现发生变异。因此,如果SARS-CoV-2疫苗研发成功,必将能在很大程度上抑制新疫情的暴发。
使用SARS-CoV-2的S蛋白作为抗原,有望开发出相应的疫苗。然后发明人之前的研究表明,野生型的SARS-CoV-2 S蛋白序列在人源细胞并不能很好的表达,其表达甚至可能在检测限之下。对其序列进行优化,提高其在人源细胞中的表达量,是获得高效价疫苗的关键所在。
流感病毒为单负链RNA病毒,因此直接将病毒基因组vRNA注入细胞内不能产生有感染性的病毒颗粒。反向遗传学拯救系统方面的进展使得从cDNA直接得到有感染性的病毒粒子成为可能,一方面加快了流感病毒疫苗开发进程,另一方面大大提高了流感病毒生物学性质研究速度,也为将流感病毒改造成为有用的基因载体提供了强有力的技术支持。流感病毒反向遗传学拯救系统的发展为流感病毒载体的开发提供了方便而又有力的技术支撑。纵观流感病毒载体的开发历程,反向遗传学系统是载体能否研发成功的关键技术之一。
在不断探索中,现今最常用的反向遗传学系统使用人PolI作为vRNA启动子而以mTer作为终止子,并在PolI-mTer两端加上反向的CMV-BGH表达盒。该系统在流感病毒生物学 研究以及疫苗及载体开发方面均获得广泛应用。
现有技术:
(1)活病毒载体
第一类活病毒载体利用流感病毒HA或NA基因片段容纳一个或者几个外源抗原表位,作为疫苗载体。第二类活病毒载体在NA片段内部引入一个额外的3’NCR组成双表达框。第三类活病毒载体在NA片段3’NCR与编码区之间放置外源基因,而用2A蛋白自剪切序列连接与NA编码区N端。第四类活病毒载体利用其它病毒的蛋白来行使流感病毒HA蛋白和NA蛋白功能,从而空余NA片段来携带外源基因。
(2)复制缺陷载体
正常细胞内不能复制的流感病毒载体缺失了某重要基因的部分或者全部功能,因此生长复制依赖于能够提供这些功能的特殊细胞系。主要有以下几类:第一类利用缺陷的流感病毒NS片段来携带外源基因。第二类复制缺陷型流感病毒载体缺失了某些重要基因如M2、NA等的功能,需要在特殊细胞系中才能生长、复制。还有不改变流感病毒本身,而改造生产细胞系如MDCK细胞的方式来生产流感病毒载体。
SARS-CoV-2作为一种新型的RNA病毒,其蛋白分为:刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白),和核衣壳蛋白(N蛋白)。S蛋白(刺突蛋白,spike protein)是冠状病毒最重要的表面膜蛋白,含有两个亚基(subunit),S1和S2。S蛋白可识别宿主细胞受体并介导膜融合,对于病毒颗粒进入细胞至关重要,是病毒感染宿主细胞的关键因子。流感病毒HA基因容纳外源基因的能力有限,据文献报道,插入HA的最大外源基因长度约为700bp,过大的外源片段存在丢失的可能。如何利用流感病毒有限的外源基因插入量,表达出具有免疫原活性的多肽,依然是一项具有挑战性的工作。
发明内容
本发明的目的在于克服现有技术的至少一个不足,提供一种优化的表达新型冠状病毒抗原的核苷酸序列及其应用,特别提供一种携带新型冠状病毒抗原的流感载体及其应用。
本发明所采取的技术方案是:
本发明的第一个方面,提供:
一种优化的SARS-CoV-2抗原核苷酸序列,其与SEQ ID NO.:1具有至少80%,优选85%、90%、95%、100%的同源性,且可以表达SEQ ID NO.:2所示的氨基酸序列。
在一些实例中,所述SARS-CoV-2抗原核苷酸序列:
可在人体中诱导免疫应答;或
产生生物报告分子;或
用于检测的分子;或
能调节基因功能;或
成为治疗性分子。
本发明的第二个方面,提供:
一种表达载体,其含有本发明第一个方面所述的SARS-CoV-2抗原核苷酸序列。
在一些实例中,所述的载体为病毒载体。
在一些实例中,所述的病毒载体为流感病毒载体。
在一些实例中,所述的SARS-CoV-2抗原核苷酸序列插入在流感载体的HA基因上。
在一些实例中,所述的SARS-CoV-2抗原核苷酸序列插入在流感病毒载体的HA基因的第360个氨基酸位置。
在一些实例中,所述流感载体包括但不限于PR8(A/Puerto Rico/8/1934(H1N1))、A/California/7/09(H1N1)、A/Brisbane/02/2018(H1N1)、A/Guangdong-Maonan/SWL1536/2019(H1N1)等甲型H1N1流感病毒,优选的,载体系统通用pM系统。
本发明的第三个方面,提供:
一种表达细胞,所述表达细胞可基于本发明第一个方面所述的SARS-CoV-2抗原核苷酸序列表达多肽;或包括本发明第二个方面所述的表达载体。
本发明的第四个方面,提供:
一种组合物,所述组合物包括:本发明第一个方面所述的SARS-CoV-2抗原核苷酸序列;或本发明第二个方面所述的表达载体。
在一些实例中,所述组合物包括药学上可接受的佐剂、载体、稀释剂或赋形剂中的至少一种。
本发明的第五个方面,提供:
含有本发明第一个方面所述的SARS-CoV-2抗原核苷酸序列的组合物在制备COVID-19疫苗中的应用。
在一些实例中,所述组合物包括本发明第二个方面所述的表达载体。
在一些实例中,所述组合物为本发明第四个方面所述的组合物。
本发明的第六个方面,提供:
一种SARS-CoV-2抗原流感载体的构建方法,包括:将本发明第一个方面所述的SARS-CoV-2抗原核苷酸序列插入流感载体中表达。
在一些实例中,所述SARS-CoV-2抗原核苷酸序列插入流感载体HA基因的第360个氨基酸位置。
本发明的有益效果是:
本发明的一些实例,其优化后的核苷酸序列,可以很好地在人源细胞中表达,同时表达产物具有良好的免疫原性。
本发明的一些实例,可以很好地利用流感载体有限的外源基因容纳量,高效表达出优化后的核苷酸序列。
本发明的一些实例,同时保留了SARS-CoV-2的RBD功能和构象,可以带来良好的免疫效果。
图1是本发明一些实例的流感载体构建设计示意图;
图2是pMPR8-RBD-HA(360)构建流程图;
图3是pMPR8-RBD-HA(360)骨架与片段的PCR扩增结果;
图4是pMPR8-RBD-HA(360)骨架与片段同源重组后的菌液PCR扩增结果;
图5是中提质粒pMPR8-RBD-HA(360)的PCR扩增鉴定结果;
图6是反向遗传技术拯救流感病毒示意图;
图7是质粒pMPR8-RBD-HA(360)转染293T细胞后收取细胞蛋白并进行WB检测,HA与RBD融合蛋白的表达验证结果;
图8是本发明对比载体构建方法的流感载体构建设计示意图;
图9是pMPR8-RBD-HA(565)构建流程图;
图10是pMPR8-RBD-HA(565)骨架与片段的PCR扩增结果;
图11是pMPR8-RBD-HA(565)骨架与片段同源重组后的菌液PCR扩增结果;
图12是中提质粒pMPR8-RBD-HA(565)的酶切鉴定结果;
图13是质粒pMPR8-RBD-HA(565)转染293T细胞后收取细胞蛋白并进行WB检测,HA与RBD融合蛋白的表达验证结果。
SARS-CoV-2的蛋白分为:刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白),和核衣壳蛋白(N蛋白)。S蛋白(刺突蛋白,spike protein)是冠状病毒最重要的表面膜蛋白,含有两个亚基(subunit),S1和S2。S蛋白可识别宿主细胞受体并介导膜融合,对于病毒颗粒进入细胞至关重要,是病毒感染宿主细胞的关键因子。其中S1主要包含有受体 结合区(receptor binding domain,RBD)(S1蛋白的319-591氨基酸),负责识别细胞的受体,RBD直接参与了宿主受体的识别,该区域的氨基酸变异会导致病毒的种属嗜性和感染特性的变化。完整的RBD序列全长819bp,无法装载到流感病毒载体中。发明人通过自有技术进一步分析及实验,意外发现选择特定的截短RBD(S1蛋白的331-524氨基酸,582bp,蛋白序列如SEQ ID NO.:2所示)既可以很好地保持RBD的功能,又可以维持其构象。进一步通过自有技术进行密码子优化,得到582bp的碱基片段,即SEQ ID NO.:1所示的核苷酸序列,记为RBD序列。
SEQ ID NO.:1如下:
SEQ ID NO.:2如下:
HA又名血凝素,主要功能是受体结合和膜融合。它是一个典型的Ⅰ型跨膜蛋白,呈三聚体结构突出于病毒粒子表面,HA蛋白三聚体在结构上分为球状的头部和长的纤维状的颈部,其中头部由3个HA1分子构成,茎部则由三个HA2分子构成。HA1和HA2分子之间以一个二硫键相连,其连接处有一个酶切位点,为了保护其功能,我们选取距离HA2 16aa位置,即HA第360个氨基酸位置处插入外源基因(图1)。此时,HA2不只发挥原本的功能,还作为一个基座,达到将RBD带到流感病毒表面的目的。
本发明确立了一种携带新型冠状病毒抗原的流感载体的构建方法,通过研究流感病毒与新冠病毒的特性,选取HA作为承载外源基因的片段,选取RBD作为插入HA的外源基因,制备RBD与HA的融合基因,即能表达RBD-HA的融合蛋白。
下面结合实验,进一步说明本发明的技术方案。
一种携带新型冠状病毒抗原的流感载体的构建方法,包含如下步骤:
S1)对SARS-CoV-2抗原S基因进行设计、优化与合成,得到SARS-CoV-2抗原核苷酸序列,记为RBD序列;
S2)在流感载体PR8的HA基因的第360个氨基酸处对应的核苷酸处插入上述RBD序列;
S3)设计引物,使得扩增RBD的上下游引物与扩增PR8-HA的上下游引物有约15-20bp的同源臂;
S4)以pMPR8-NA-RBD为模板,以RBD(360)-F和RBD(360)-R为引物PCR扩增获得RBD(612bp)基因片段,做胶回收纯化;
S5)以pMPR8-HA为模板,以pM-HA(360)-F和pM-HA(360)-R为引物PCR扩增获得PR8-HA(4806bp)骨架片段,做胶回收纯化;
S6)将RBD基因片段与PR8-HA基因片段用Exnase酶进行同源重组,转化Top10接种Amp抗性LB固体培养基,得到菌落产物pMPR8-RBD-HA(360);
S7)挑取单克隆接种于Amp抗性LB液体培养基,小提质粒;
S8)将质粒送测序,获得阳性克隆;
S9)将阳性克隆接种Amp抗性LB液体培养基,扩大培养,中提质粒;
S10)对质粒进行PCR鉴定及测序鉴定,鉴定正确即为目的质粒pMPR8-RBD-HA(360);
S11)将质粒pMPR8-RBD-HA(360)以2.5ug的量转染293T细胞,48h后收取细胞蛋白并进行WB检测,使用PR8-HA兔多抗作为一抗检测到HA与RBD蛋白的融合表达。
实施例1:构建pMPR8-RBD-HA(360)质粒
pMPR8-RBD-HA(360)构建流程图如图2所示。
以pMPR8-HA为模板,以PM-HA(360)-F和PM-HA(360)-R为引物扩增获得骨架pMPR8-HA片段;以pMPR8-NA-RBD为模板,以RBD(360)-F和RBD(360)-R为引物PCR扩增获得RBD基因片段,将此两片段用Exnase酶同源重组连接,连接产物转化Top10感受态细胞,将转化产物接种于氨苄抗性(Amp+)的LB固体平板培养基上,37℃过夜培养。培养基长出菌落,挑取单个菌落接种于氨苄抗性(Amp+)的LB液体培养基里,以转速220rpm37℃摇晃过夜,获得未提纯菌液产物pMPR8-RBD-HA(360)。
PM-HA(360)-F:gtgggcggaggttccggcatagatggatggtatggttatcatcatcagaatgaac(SEQ ID NO.:3)
PM-HA(360)-R:attaccgctgcctccacccattccagtccatcccccttcaataaaaccggca(SEQ ID NO.:4)
PCR条件:98℃,3min;98℃,10s;60℃,5s;72℃,30s;cycles 30;72℃,5min;12℃保存。
RBD(360)-F:ggtggaggcagcggtaatatcaccaacctgtgcccttttggcgaggtgttca(SEQ ID NO.:5)
RBD(360)-R:gccggaacctccgcccacggttgcaggtgcgtgcagcagctcaaa(SEQ ID NO.:6)
PCR条件:98℃,3min;98℃,10s;60℃,5s;72℃,10s;cycles 30;72℃,5min;12℃保存。
在此载体构建过程中,中间产物pMPR8-RBD-HA(360)PCR扩增骨架与RBD基因片段、骨架与RBD基因片段同源重组后的菌液进行PCR扩增鉴定,鉴定结果分别如图3和图4所示。结果显示载体构建是成功的,与预期的结果相符。
实施例2:质粒pMPR8-RBD-HA(360)的菌液PCR鉴定
以将实施例1中的未提纯菌液产物pMPR8-RBD-HA(360)为模板,以RBD(360)-F、RBD(360)-R为引物,进行pMPR8-RBD-HA(360)的菌液PCR鉴定,鉴定为阳性的菌液进行质粒抽提,得到阳性质粒pMPR8-RBD-HA(360)。
RBD(360)-F:ggtggaggcagcggtaatatcaccaacctgtgcccttttggcgaggtgttca
RBD(360)-R:gccggaacctccgcccacggttgcaggtgcgtgcagcagctcaaa
PCR条件:98℃,3min;98℃,10s;60℃,5s;72℃,10s;cycles 30;72℃,5min;12℃保存。
中提阳性质粒pMPR8-RBD-HA(360)的PCR鉴定结果如图5所示,与预期的结果相符。
实施例3:反向遗传技术拯救流感病毒
利用本实验室的反向遗传学系统(也可以采用其他已知方法),将阳性质粒pMPR8-RBD-HA(360)与流感病毒其他7个片段质粒(pMPR8-PB2、pMPR8-PB1、pMPR8-PA、
pMPR8-NP、pMPR8-NA、pMPR8-M、pMPR8-NS)共转染至293T与MDCK混合生长细胞,60h后收取细胞与上清混合液。将此混合液接种至9日龄SPF鸡胚,置于37℃生化培养箱培养,72h后测定血凝效价(HA)并收取鸡胚尿囊液,即为拯救病毒(图6)。
实施例4:质粒pMPR8-RBD-HA(360)转染293T细胞后的RBD蛋白表达验证
将质粒pMPR8-RBD-HA(360)以2.5ug的量转染至293T细胞,48h后收取细胞蛋白并进行WB检测,使用PR8-HA兔多抗作为一抗检测到HA与RBD蛋白的融合表达。用PBS清洗三遍以完全去除培养基。将细胞置于冰上,用裂解液处理细胞,加入5×Loading Buffer(含有β-巯基乙醇),沸水浴10min,得到线性化蛋白产物。配制蛋白胶(5%浓缩胶+10%分离胶),将制备的样品上样到蛋白胶里,经过跑胶-转膜-封闭-洗膜-孵育一抗-孵育二抗-显 色等过程,得到质粒pMPR8-RBD-HA(360)转染293T细胞后的RBD蛋白表达验证结果。
验证结果如图7所示,结果表明,质粒pMPR8-RBD-HA(360)转染293T细胞后收取的细胞蛋白中,能检测到RBD与HA蛋白的融合表达。
对比载体pMPR8-RBD-HA(565)的构建:
主要操作同上,不同之处在于在HA第565个氨基酸位置处插入外源基因RBD序列,即HA末尾基因以linker连接RBD基因。包含如下步骤:
S1)对SARS-CoV-2抗原S基因进行设计、优化与合成,得到SARS-CoV-2抗原核苷酸序列,记为RBD序列;
S2)在流感载体PR8的HA基因的第565个氨基酸处对应的核苷酸处(即HA末尾基因以linker连接RBD基因)插入上述RBD序列;
S3)设计引物,使得扩增RBD的上下游引物与扩增PR8-HA的上下游引物有约15-20bp的同源臂;
S4)以pMPR8-NA-RBD为模板,以RBD(565)-F和RBD(565)-R为引物PCR扩增获得RBD(612bp)基因片段,做胶回收纯化;
S5)以pMPR8-HA为模板,以pM-HA(565)-F和pM-HA(565)-R为引物PCR扩增获得PR8-HA(4800bp)骨架片段,做胶回收纯化;
S6)将RBD基因片段与PR8-HA基因片段用Exnase酶进行同源重组,转化Top10接种Amp抗性LB固体培养基,得到菌落产物pMPR8-RBD-HA(565);
S7)挑取单克隆接种于Amp抗性LB液体培养基,小提质粒;
S8)将质粒送测序,获得阳性克隆;
S9)将阳性克隆接种Amp抗性LB液体培养基,扩大培养,中提质粒;
S10)对质粒进行PCR鉴定及测序鉴定,鉴定正确即为目的质粒pMPR8-RBD-HA(565)。
具体操作参照实施例1~4进行。
图8是本发明对比载体构建方法的流感载体pMPR8-RBD-HA(565)构建设计示意图;
图9是pMPR8-RBD-HA(565)构建流程图;
图10是pMPR8-RBD-HA(565)骨架与片段的PCR扩增结果;
图11是pMPR8-RBD-HA(565)骨架与片段同源重组后的菌液PCR扩增结果;
图12是中提质粒pMPR8-RBD-HA(565)的酶切鉴定结果;
图13是质粒pMPR8-RBD-HA(565)转染293T细胞后收取细胞蛋白并进行WB检测,HA与RBD融合蛋白的表达验证结果。
将质粒pMPR8-RBD-HA(565)以2.5ug的量转染293T细胞,48h后收取细胞蛋白并进行WB检测,使用RBD兔多抗作为一抗未检测到HA与RBD蛋白的融合表达。
对比载体pMPR8-RBD-HA(17)的构建:
主要操作同上,不同之处在于在HA第17个氨基酸位置处插入外源基因RBD序列,即HA首部基因以linker连接RBD基因。
将质粒pMPR8-RBD-HA以2.5ug的量转染293T细胞,48h后收取细胞蛋白并进行WB检测,使用RBD兔多抗作为一抗未检测到HA与RBD蛋白的融合表达。
通过对比可知,pMPR8-RBD-HA(360)可以很好地表达RBD蛋白。而在HA的首部或末尾插入RBD序列,均不能很好地表达RBD蛋白。
Claims (10)
- 一种优化的SARS-CoV-2抗原核苷酸序列,其与SEQ ID NO.:1具有至少80%,优选85%、90%、95%、100%的同源性,且可以表达SEQ ID NO.:2所示的氨基酸序列。
- 根据权利要求1所述的SARS-CoV-2抗原核苷酸序列,其特征在于,所述核苷酸序列:可在人体中诱导免疫应答;或产生生物报告分子;或用于检测的分子;或能调节基因功能;或成为治疗性分子。
- 一种表达载体,其特征在于,所述的载体含有权利要求1所述的SARS-CoV-2抗原核苷酸序列。
- 根据权利要求3所述的表达载体,其特征在于:所述的载体为病毒载体。
- 根据权利要求4所述的表达载体,其特征在于:所述的病毒载体为流感病毒载体。
- 根据权利要求5所述的表达载体,其特征在于:所述的SARS-CoV-2抗原核苷酸序列插入在流感病毒载体的HA基因的第360个氨基酸位置。
- 一种表达细胞,其特征在于:所述表达细胞可基于权利要求1所述的核酸序列表达多肽;或包括权利要求3~5任一项所述的表达载体。
- 一种组合物,其特征在于:所述组合物包括:权利要求1所述的SARS-CoV-2抗原核苷酸序列;或权利要求3~6任一项所述的表达载体。
- 根据权利要求8所述的组合物,其特征在于:还包括药学上可接受的佐剂、载体、稀释剂或赋形剂中的至少一种。
- 含有权利要求1所述的SARS-CoV-2抗原核苷酸序列的组合物在制备COVID-19疫苗中的应用。
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