CN113461828B - 针对2019-nCoV的重组蛋白疫苗及其制备方法 - Google Patents
针对2019-nCoV的重组蛋白疫苗及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种融合蛋白、编码所述融合蛋白的基因序列、包含所述基因序列的真核细胞表达载体、表达所述融合蛋白的细胞以及以包含所述融合蛋白的重组蛋白疫苗。本发明提供的重组蛋白疫苗既可以预防2003Sars‑CoV引起的疾病,也能够预防2019‑nCoV引起的疾病,能够有效诱导机体产生细胞免疫和体液免疫,抗原在机体内的半衰期长,并且可以快速制备,适用于新型冠状病毒2019‑nCoV的预防。
Description
技术领域
本发明涉及流行病预防及疫苗生产领域,具体涉及一种针对2019-nCoV的重组蛋白疫苗及其制备方法。
背景技术
新型冠状病毒2019-nCoV(WHO命名)是一种以前尚未在人类中发现的新型冠状病毒。国际病毒分类委员会于2020年2月将新型冠状病毒(2019-nCoV)的正式分类名确定为严重急性呼吸综合征冠状病毒2,英文缩写SARS-CoV-2(severe acute respiratorysyndrome coronavirus 2)。WHO于2020年2月将由新型冠状病毒2019-nCoV引发的疾病命名为2019冠状病毒病,英文缩写COVID-19(Corona Virus Disease 2019)。
冠状病毒属于套氏病毒目,冠状病毒科,冠状病毒属,是一类具有囊膜、基因组为线性单股正链RNA的病毒,是自然界中广泛存在的一大类病毒,引起的疾病患者表现为从普通感冒到严重肺部感染等不同的临床症状,例如中东呼吸综合征(MERS)和严重急性呼吸道综合症(SARS)。2019-nCoV与SARS-CoV同属于冠状病毒β家族。现亟需开发能够有效预防引起新型冠状病毒2019-nCoV感染的疫苗。
目前应用于人体的预防病毒感染的疫苗主要包括采用病毒经减毒后制备的减毒活疫苗,如水痘减毒活疫苗、麻疹减毒活疫苗等;以及灭活全病毒颗粒疫苗,如肠道病毒EV71型灭活疫苗、甲型肝炎疫苗等。然而,减毒疫苗要费很长时间使病毒繁殖许多代,然后逐渐减除病毒的大部分活性,难以满足急性传染病的预防需要。灭活疫苗则在免疫病理方面存在许多难以解决的问题,例如有些灭活病毒进入人体会在病毒入侵时加重病情。
重组蛋白疫苗是将病毒的目的抗原基因构建在表达载体上,再转化到细菌、酵母、哺乳动物或昆虫细胞中,在一定的诱导条件下,表达出大量的抗原蛋白,通过纯化后制备的疫苗。重组蛋白疫苗能够诱导机体产生体液免疫和细胞免疫,并且可以快速制备,适用于新型冠状病毒的预防。
发明内容
本发明的第一目的是提供一种融合蛋白,该融合蛋白包含(1)2019-nCoV的S蛋白的受体结合结构域和(2)IgG的Fc片段。
2019-nCoV的蛋白包括RNA聚合酶蛋白、纤突蛋白、包膜蛋白、膜蛋白和核衣壳蛋白。其中,纤突蛋白(Spike蛋白,本发明简称S蛋白)为糖蛋白,是诱导保护性免疫应答的决定因素。本发明提供的融合蛋白包含2019-nCoV的S蛋白的受体结合结构域(receptorbinding domain,RBD)。该结构域与2003 Sars-CoV的ACE2受体结合结构域高度保守,是引起免疫反应的重要抗原决定簇,既可以预防2003 Sars-CoV引起的疾病,也能够预防2019-nCoV引起的疾病,能够有效诱导机体产生细胞免疫和体液免疫,还能够与病毒竞争结合宿主细胞的受体(例如:血管紧张素转换酶2,ACE2),从而起到疾病的治疗作用。
本发明提供的融合蛋白还包含有免疫球蛋白G(本发明简称IgG)的Fc片段,Fc可以辅助诱导机体产生更加强烈的免疫反应,并且大大提高了抗原在机体内的半衰期,减少了免疫程序;同时,Fc使得疫苗的纯化步骤大大缩小,增加了纯化效率。本发明所述Ig的Fc片段优选为人源IgG的Fc片段。
作为本发明的一种优选方案,所述融合蛋白具有SEQ ID NO. 1所示的氨基酸序列,或与其相似度为90%以上的氨基酸序列。
SEQ ID NO. 1所示的氨基酸序列参见本发明的序列表,具体如下所示:
本发明的第二目的是提供编码所述融合蛋白的基因序列。
作为本发明的一种优选方案,所述基因序列是经过密码子优化后的DNA序列。
本发明的第三目的是提供一种包含编码所述融合蛋白的基因序列的真核细胞表达载体。其是将所述基因序列插入到真核细胞表达载体中构建而成。
本发明采用真核表达载体在哺乳动物细胞中进行表达。
所述真核细胞表达载体优选为可以用于人的表达载体。
作为本发明的一种优选方案,所述真核细胞表达载体为pCHO1.0载体。所述pCHO1.0载体的示意图如图1所示。
作为本发明的另一种优选方案,所属的真核细胞载体为pCAGGS载体。所述pCAGGS载体的示意图如图2所示。
本发明的第四目的是提供表达所述融合蛋白的细胞,所述细胞中外源转入了包含所述基因序列的真核细胞表达载体。
作为本发明的一种优选方案,所述细胞为CHO细胞。CHO细胞(Chinese hamsterovary cell)是1957年从中国地鼠卵巢细胞得到。该细胞是上皮样细胞,通常贴壁生长,也可悬浮生长,被广泛地用于表达重组蛋白。
在选用CHO细胞进行抗原表达的情况下,本发明优选将所述目的基因序列经过CHO细胞密码子优化后克隆到所述真核细胞表达载体中,再转入CHO细胞中。
本发明的另一种优选方案,所述的细胞为293F细胞。293F细胞是从HEK293细胞中分离出的一种适应于悬浮生长的细胞,被广泛地用于表达重组蛋白。
在选用293F细胞进行抗原表达的情况下,本发明优选将所述目的基因序列经过293F细胞密码子优化后克隆到所述真核细胞表达载体中,再转入293F细胞中。
本发明的第五目的是提供针对2019-nCoV的重组蛋白疫苗,其包含所述融合蛋白。所述融合蛋白作为疫苗的抗原。
作为本发明的一种优选方案,所述重组蛋白疫苗中抗原有效成分的含量为10~100μg/剂。具体而言,所述重组蛋白疫苗中抗原有效成分的含量可以为12.5μg/剂,25μg/剂,50μg/剂或100μg/剂。在本发明的一些具体实施方式中,所述抗原有效成分的含量是指疫苗产品中疫苗原液的含量。
本发明提供的疫苗中还可以含有免疫佐剂。所述免疫佐剂可以选用无机佐剂,如氢氧化铝佐剂等。所述免疫佐剂还可以选用有机佐剂,如脂多糖、细胞因子等。
本发明提供的疫苗优选为液体疫苗,可以采用多种剂型形式。具体而言,所述重组蛋白疫苗可以是肌肉内液体注射剂、静脉内液体注射剂、鼻腔内液体注射剂、皮内液体注射剂或皮下液体注射剂。在实际应用时,可以根据转染效率、局部免疫监视等临床需要进行调整和选择,如选择单一的剂型进行注射免疫,或者选择多种混合剂型进行注射免疫。
本发明的第六目的是提供所述重组蛋白疫苗的制备方法,包括如下步骤:将包含所述基因序列的真核细胞表达载体转入细胞中表达,取上清液纯化,得疫苗原液。
作为本发明的一种优选方案,所述细胞为CHO细胞。在选用CHO细胞进行抗原表达的情况下,本发明优选将所述目的基因序列经过CHO细胞密码子优化后克隆到所述真核细胞表达载体中,再转入CHO细胞中。
作为本发明的一种优选方案,所述细胞为293F细胞。在选用293F细胞进行抗原表达的情况下,本发明优选将所述目的基因序列经过293F细胞密码子优化后克隆到所述真核细胞表达载体中,再转入293F细胞中。
所述纯化可采用本领域常规的疫苗原液纯化方法。
作为本发明的一种优选方案,所述纯化包括:将所述上清液进行超滤浓缩后,进行分子筛和阴离子交换两步层析,所得层析液经滤膜过滤之后,即为疫苗原液。所述滤膜优选为0.22μm滤膜。
作为本发明的一种优选方案,所述方法还包括:将所述疫苗原液配制成疫苗。所述疫苗原液的用量优选为10~100 μg/剂。具体而言,所述重组蛋白疫苗中疫苗原液的含量可以为12.5μg/剂,25μg/剂,50μg/剂或100μg/剂。
本发明的第七目的是提供所述融合蛋白、所述基因序列、所述真核细胞表达载体、所述细胞、所述重组蛋白疫苗或所述方法制备而成的重组蛋白疫苗在制备用于预防新型冠状病毒2019-nCoV感染引起的疾病的药物中的应用。所述疾病即为世界卫生组织命名的2019冠状病毒病,英文缩写COVID-19。
优选地,所述疾病为新型冠状病毒2019-nCoV感染引起的严重急性呼吸道感染,肠道疾病,心脏衰竭,肾衰竭或严重急性呼吸道综合征。
本发明提供的融合蛋白包含有2019-nCoV Spike蛋白的受体结合结构域,与2003Sars-CoV高度保守,且能够与ACE2结合,是引起免疫反应的重要抗原决定簇。本发明提供的含有该融合蛋白的疫苗既可以预防2003 Sars-CoV引起的疾病,也能够预防2019-nCoV引起的疾病,能够有效诱导机体产生细胞免疫和体液免疫,且可以快速制备,适用于新型冠状病毒2019-nCoV的预防。本发明提供的融合蛋白还包含有IgG的Fc片段,该片段可以辅助诱导机体产生更加强烈的免疫反应,并且大大提高了抗原在机体内的半衰期,减少了免疫程序;同时,Fc片段使得疫苗的纯化步骤大大缩小,增加了纯化效率。
附图说明
图1为pCHO1.0载体的示意图。
图2为pCAGGS载体的示意图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
本实施例提供了2019-nCoV重组蛋白疫苗原液的制备方法,具体为:
(1)将编码SEQ ID NO. 1的基因序列进行CHO细胞密码子优化后,插入到pCHO1.0载体上,同源重组到CHO细胞,进行蛋白的重组表达。
(2)取细胞培养的上清液,进行超滤浓缩后,进行分子筛和阴离子交换两步层析,所得层析液经0.22μm滤膜过滤,得疫苗原液。
实施例2
(1)将编码SEQ ID NO. 1的基因序列进行293细胞密码子优化后,插入到pCAGGS载体上,同源重组到293F细胞,进行蛋白的重组表达。
(2)取细胞培养的上清液,进行超滤浓缩后,进行分子筛和阴离子交换两步层析,所得层析液经0.22μm滤膜过滤,得疫苗原液。
实施例3
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液12.5μg/剂以及氢氧化铝佐剂50μg/剂。将本实施例所得疫苗编号记为20200201。
实施例4
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液25μg/剂以及氢氧化铝佐剂50μg/剂。将本实施例所得疫苗编号记为20200202。
实施例5
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液50μg/剂以及氢氧化铝佐剂50μg/剂。将本实施例所得疫苗编号记为20200203。
实施例6
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液100μg/剂以及氢氧化铝佐剂50μg/剂。将本实施例所得疫苗编号记为20200204。
实施例7
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液12.5μg/剂以及氢氧化铝佐剂200μg/剂。将本实施例所得疫苗编号记为20200205。
实施例8
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液25μg/剂以及氢氧化铝佐剂200μg/剂。将本实施例所得疫苗编号记为20200206。
实施例9
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液50μg/剂以及氢氧化铝佐剂200μg/剂。将本实施例所得疫苗编号记为20200207。
实施例10
本实施例提供了一种2019-nCoV重组蛋白疫苗,该疫苗为液体疫苗,其中含有实施例1提供的疫苗原液100μg/剂以及氢氧化铝佐剂200μg/剂。将本实施例所得疫苗编号记为20200208。
实施例11:疫苗免疫小鼠效果
将实施例3~10提供的8组疫苗分别做1:4、1:16、1:64稀释,接种BALB/c小鼠,每组10只,按照0,7天免疫,每只腹腔注射一个剂量,第1次免疫后4周采血,检测中和抗体。中和抗体效价GMT大于8的视为阳性,视为有保护效果。结果如表1所示。
结果显示,上述不同有效成分和佐剂含量的2019-nCoV疫苗免疫BALB/c小鼠,均能产生足够高的中和抗体效价。佐剂含量高的疫苗免疫原性相对高于含量低的同样有效成份的疫苗。本发明提供疫苗均能诱导BALB/c小鼠产生有保护能力的中和抗体。
实施例12:疫苗免疫豚鼠效果
将实施例2、5、6、9提供的4组疫苗分别接种豚鼠,每组4只,每只肌肉注射1.0ml(2个剂量),按照0,7天免疫,28天采血的免疫程序,检测血清中和抗体效价。结果如表2所示。
结果显示,不同批次2019-nCoV疫苗免疫豚鼠,能够产生不同水平的中和抗体效价。本发明提供的疫苗均能诱导豚鼠产生良好的中和抗体。
实施例13:细胞因子检测
将编号为20200201、20200202、20200203和20200204的疫苗按照0、7天免疫,28天采血的程序分别免疫BALB/c小鼠,采集细胞上清进行细胞免疫检测。采用细胞内细胞因子染色法和流式细胞法检测血清中特异性表达S蛋白细胞因子的CD4+T细胞。
结果显示,20200201、20200202、20200203和20200204均能诱导机体产生细胞免疫。虽然有效成份含量高的疫苗能产生更高水平的细胞免疫,但是在本发明提供的抗原含量范围内的疫苗均能引起良好的细胞免疫。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 北京科兴中维生物技术有限公司
<120> 针对2019-nCoV的重组蛋白疫苗及其制备方法
<130> RYP2010607.7
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 441
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白氨基酸序列
<400> 1
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Arg Val Gln
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Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
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Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
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Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
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Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
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Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
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Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
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Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
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Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
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Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
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Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
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Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
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Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
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Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
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Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly Gly
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Ser Gly Gly Gly Gly Ser Ile Ser Arg Thr Pro Glu Val Thr Cys Val
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Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
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Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
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Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
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Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
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Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
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Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
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Claims (21)
1.一种融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO. 1所示。
2.编码权利要求1所述融合蛋白的基因序列。
3.根据权利要求2所述的基因序列,其特征在于,是经过密码子优化后的DNA序列。
4.包含权利要求2或3所述基因序列的真核细胞表达载体。
5.根据权利要求4所述的真核细胞表达载体,其特征在于,所述真核细胞表达载体为用于人的表达载体。
6.根据权利要求4所述的真核细胞表达载体,其特征在于,所述真核细胞表达载体为pCHO1.0载体或pCAGGS载体。
7.表达权利要求1所述融合蛋白的细胞,所述细胞中外源转入了权利要求4~6任意一项所述真核细胞表达载体。
8.根据权利要求7所述的细胞,其特征在于,所述细胞为CHO细胞或293F细胞。
9.针对2019-nCoV的重组蛋白疫苗,其包含权利要求1所述融合蛋白。
10. 根据权利要求9所述的重组蛋白疫苗,其特征在于,所述重组蛋白疫苗中抗原有效成分的含量为10~100 μg/剂。
11.根据权利要求9所述的重组蛋白疫苗,其特征在于,所述疫苗中还含有免疫佐剂。
12.根据权利要求9所述的重组蛋白疫苗,其特征在于,所述疫苗中还含有氢氧化铝佐剂。
13.根据权利要求9~12任意一项所述的重组蛋白疫苗,其特征在于,所述疫苗为液体疫苗。
14.根据权利要求13所述的重组蛋白疫苗,其特征在于,所述疫苗为肌肉内液体注射剂、静脉内液体注射剂、鼻腔内液体注射剂、皮内液体注射剂或皮下液体注射剂。
15.针对2019-nCoV的重组蛋白疫苗的制备方法,其特征在于,包括如下步骤:将权利要求4~6任意一项所述真核细胞表达载体转入细胞中表达,取上清液纯化,得疫苗原液。
16.根据权利要求15所述的制备方法,其特征在于,所述细胞为CHO细胞或293F细胞。
17.根据权利要求15所述的制备方法,其特征在于,所述纯化包括:将所述上清液进行超滤浓缩后,进行分子筛和阴离子交换两步层析,所得层析液经滤膜过滤。
18.根据权利要求15所述的制备方法,其特征在于,所述方法还包括:将所述疫苗原液配制成疫苗。
19. 根据权利要求15所述的制备方法,其特征在于,所述方法还包括:将所述疫苗原液按照10~100 μg/剂的用量配制成疫苗。
20.权利要求1所述融合蛋白、权利要求2或3所述基因序列、权利要求4~6任意一项所述真核细胞表达载体、权利要求7或8所述细胞、权利要求9~14任意一项所述重组蛋白疫苗或权利要求15~19任意一项所述方法制备而成的重组蛋白疫苗在制备用于预防新型冠状病毒2019-nCoV感染引起的疾病的药物中的应用。
21.根据权利要求20所述的应用,其特征在于,所述疾病为新型冠状病毒2019-nCoV感染引起的严重急性呼吸道感染,肠道疾病,心脏衰竭,肾衰竭或严重急性呼吸道综合征。
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