CN113912708B - 单域重链抗体及其编码基因和制备方法及应用和药物组合物 - Google Patents
单域重链抗体及其编码基因和制备方法及应用和药物组合物 Download PDFInfo
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- CN113912708B CN113912708B CN202111019238.5A CN202111019238A CN113912708B CN 113912708 B CN113912708 B CN 113912708B CN 202111019238 A CN202111019238 A CN 202111019238A CN 113912708 B CN113912708 B CN 113912708B
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Abstract
本发明涉及生物技术领域领域,公开了一种单域重链抗体及其编码基因和制备方法及应用和药物组合物。本发明提供的单域重链抗体能够特异性结合中东呼吸综合征冠状病毒的受体结合区,并且对其具有良好的中和活性,具有用于中东呼吸综合征防治的抗病毒产品中作为活性组分的潜力。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种单域重链抗体及其编码基因和制备方法及应用和药物组合物。
背景技术
冠状病毒最先于1937年从鸡身上分离出来,经研究发现,冠状病毒仅能感染脊椎动物,其中,中东呼吸综合征冠状病毒(Middle East Respiratory SyndromeCoronavirus,MERS-CoV)最早于2012年9月被发现,是目前已知7种能够感染人的冠状病毒之一。MERS-CoV具有致病性强、致死率高的特点,并且具有人传人的传播能力,是继严重急性呼吸综合征冠状病毒(SARS-CoV)之后又一个对人类健康和发展产生严重威胁的冠状病毒。虽然目前我国尚未发生MERS-CoV大规模感染事件,但是其仍存在发生严重疫情,对社会和经济发展造成重大影响的风险。
疫苗是针对病毒感染造成的疫情最有效的防控措施之一,然而,目前暂未报道有针对MERS-CoV的疫苗上市推广。而且,有些研究的结果表明,MERS减活、灭活疫苗等传统疫苗在实验中表现出的免疫保护效果并不理想。因此,不仅MERS疫苗开发工作迫在眉睫,积极拓展新的疫苗开发思路也十分重要。
发明内容
本发明的目的是为了克服现有技术存在的缺乏针对MERS-CoV的疫苗产品,以及MERS疫苗开发困难等问题,提供一种单域重链抗体及其编码基因和制备方法及应用和药物组合物。本发明提供的单域重链抗体能够特异性结合MERS-CoV受体结合区,对MERS-CoV具有良好的中和性,具有制备用于MERS预防和治疗的产品的潜力。
为了实现上述目的,本发明一方面提供一种单域重链抗体,所述抗体包括:
(1)氨基酸序列如SEQ ID NO:1所示的蛋白质;
(2)氨基酸序列如SEQ ID NO:1所示的蛋白质经改造获得的与改造前具有相同活性的衍生蛋白质。
本发明第二方面提供编码前述单域重链抗体的基因。
本发明第三方面提供一种制备如前所述的单域重链抗体的方法,所述方法包括:将含有前述基因的重组载体导入宿主细胞,表达获得所述单域重链抗体。
本发明第四方面提供含有如前所述的基因的重组载体、表达盒和表达载体。
本发明第五方面提供扩增如前所述的基因或其片段的引物。
本发明第六方面提供如前所述的单域重链抗体、基因、重组载体、表达盒和表达载体或引物在制备针对严重急性呼吸综合征冠状病毒的抗病毒产品中的应用。
本发明第七方面提供一种药物组合物,所述药物组合物包括如前所述的单域重链抗体、基因或重组载体、表达盒和表达载体。
本发明提供的单域重链抗体通过分子生物学方法构建获得,也可以通过大肠杆菌等生物工程菌进行表达生产,制备方法简单。经实验验证,其与MERS-CoV的受体结合区(receptor-binding domain,RBD)具有很强的反应性,且对MERS-CoV具有良好的中和活性。这表明该单域重链抗体能够(单独或与其他抗体配合)作为活性组分,发挥对MERS-CoV的防治作用。
附图说明
图1为本发明实施例1中获得的表达载体pCold I-Nano-Anti-MRBD的PCR鉴定结果。
图2为本发明实施例1中获得的表达载体pCold I-Nano-Anti-MRBD的测序结果。
图3为本发明实施例1中诱导表达实验中获得的SDS-PAGE结果。
图4为本发明实施例1中表达形式鉴定实验中获得的SDS-PAGE结果。
图5为本发明实施例1中纯化样品的SDS-PAGE结果。
图6为本发明实施例1中浓缩蛋白样品的SDS-PAGE结果。
图7为本发明实施例2中获得的ELISA检测结果示意图。
图8为本发明实施例3中获得的对MERS-CoV假病毒的中和活性检测结果示意图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
本发明中,“MERS”为“中东呼吸综合征”的简写形式,其二者含义相同,可以互换使用。“MERS-CoV”为“中东呼吸综合征冠状病毒”的简写形式,其二者含义相同,可以互换使用。
单域重链抗体(the variable domain of the heavy-chain of heavy-chainantibody,VHH)是由重链可变区结合抗原的单一结构域构成的小分子抗体,是具有完整的抗原结合活性的单一折叠单元。单域重链抗体的制备方法简单,能够采用大肠杆菌等本领域常用表达载体进行表达生产,而且与具有轻链和重链的完整结构的抗体相比,单域重链抗体的分子(量)小,更易通过血管壁,更利于发挥治疗作用,而且能与分布于病毒表面凹槽的抗原结合,更利于病毒性疾病的防治。
本发明的发明人在研究的过程中巧妙地通过分子生物学方法构建了一种针对MERS-CoV的单域重链抗体(氨基酸序列如SEQ ID NO:1所示),该抗体能够与MERS-CoV的受体结合域特异性结合,并且具有良好的MERS-CoV中和性,从而具有应用于MERS-CoV防治相关产品研发的潜力。
本发明一方面提供一种单域重链抗体,所述抗体包括:
(1)氨基酸序列如SEQ ID NO:1所示的蛋白质;
(2)氨基酸序列如SEQ ID NO:1所示的蛋白质经改造获得的与改造前具有相同活性的衍生蛋白质。
本发明提供的单域重链抗体可以是在具有相同活性的情况下,将上述蛋白质(1)采用任意本领域现有方式进行改造获得的衍生蛋白质,对于其改造方法和改造后的衍生蛋白质的具体序列和其他特性没有特别限定。所述“其他特性”是指改造获得的衍生蛋白质所具有的除与蛋白质(1)具有相同活性之外的任意其他生物学特性。
根据本发明的优选实施方式,其中,所述改造可以包括:
i、使SEQ ID NO:1所示的氨基酸序列中的一个或几个氨基酸残基发生取代、缺失和添加中的至少一种;和/或
ii、在SEQ ID NO:1所示的氨基酸序列的N末端和/或C末端连接便于纯化的标签和/或利于蛋白分泌表达的信号肽序列;和/或
iii、将SEQ ID NO:1所示的氨基酸序列进行人源化改造。
本发明中,改造方式ii中所采用的便于纯化的标签可以为任意本领域现有的能够方便蛋白质纯化的标签。优选地,所述便于纯化的标签选自Poly-Arg、Poly-His、FLAG、Strep-tag II和c-myc中的至少一种。上述标签的具体氨基酸序列没有特别限制,例如可以为如下表1中所示的序列。
表1便于纯化的标签序列
标签 | 残基数/个 | 序列 |
Poly-Arg | 5* | RRRRR(SEQ ID NO:5) |
Poly-His | 6** | HHHHHH(SEQ ID NO:6) |
FLAG | 8 | DYKDDDDK(SEQ ID NO:7) |
Strep-tag II | 8 | WSHPQFEK(SEQ ID NO:8) |
c-myc | 10 | EQKLISEEDL(SEQ ID NO:9) |
*Poly-Arg可以由5-6个精氨酸残基组成,表1中仅列出了通常采用的5个精氨酸残基组成的Poly-Arg标签,但是6个精氨酸残基组成的Poly-Arg标签也可适用于本发明。
**Poly-His可以由2-10个组氨酸残基组成,表1中仅列出了通常采用的6个组氨酸残基组成的Poly-His标签,但是2-10个组氨酸组成的Poly-His标签均可适用于本发明。
本发明中,改造方式ii中所采用的信号肽序列可以为任意本领域现有的能够利于蛋白质分泌表达的信号肽序列。优选地,所述信号肽序列选自人IL-2SP、CD5 SP、人IgG2 HSP、人胰凝乳蛋白酶原SP、人胰蛋白酶原-2SP和人胰岛素SP中的至少一种。
本发明中,改造方式iii可以采用任意本领域现有的对蛋白质(尤其是单域重链抗体)进行人源化改造的方式。优选地,可以通过将SEQ ID NO:1所示的氨基酸序列的编码基因进行人源化改造实现。
本发明提供的单域重链抗体可以通过将氨基酸序列如SEQ ID NO:1所示的蛋白质经过上述改造方式i、ii和iii中的任意一种或几种的组合处理获得,只要改造后的衍生蛋白质具有与氨基酸序列如SEQ ID NO:1所示的蛋白质相同的活性即可。
本发明第二方面提供编码如上所述的单域重链抗体的基因。
任意能够编码如上所述的单域重链抗体的基因均属于本发明的内容。根据本发明的优选实施方式,其中,所述基因包括:
a、编码区核苷酸序列如SEQ ID NO:2所示的DNA分子;和/或
b、编码区核苷酸序列如SEQ ID NO:3所示的DNA分子;和/或
c、与DNA分子a或b具有至少70%同源性,并编码具有相同功能的蛋白质的DNA分子;和/或
d、DNA分子a或b经突变或改造获得的编码具有相同功能的蛋白质的DNA分子。
本发明中,上述DNA分子a可以仅包含SEQ ID NO:2所示的序列,也可以是以SEQ IDNO:2所示的序列为编码区,并额外添加其他组件的DNA分子。所述其他组件可以包括任意本领域用于人工合成基因序列并通过表达载体进行表达时所需的组件,例如启动子、增强子、Kozak序列等。
本发明中,上述DNA分子b可以仅包含SEQ ID NO:3所示的序列,也可以是以SEQ IDNO:3所示的序列为编码区,并额外添加其他组件的DNA分子。所述其他组件可以包括任意本领域用于人工合成基因序列并通过表达载体进行表达时所需的组件,例如启动子、增强子、Kozak序列等。
优选地,上述DNA分子c与DNA分子a或b具有至少75%、至少80%、至少85、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%同源性,并且编码具有相同功能的蛋白质。所述“具有相同功能的蛋白质”的氨基酸序列与DNA分子a或b所编码的蛋白质的氨基酸序列可以相同,也可以不同。
本发明中,上述DNA分子d可以经过任意本领域现有的基因突变或改造方式获得,只要最终获得的DNA分子d所表达的蛋白质与DNA分子a或b所表达的蛋白质的功能相同即可。
根据本发明的一种优选实施方式,其中,所述DNA分子d可以通过将其他DNA分子在严格杂交条件下与DNA分子a或b杂交获得。
优选地,所述其他DNA分子可以为任意不影响DNA分子a或b功能,且杂交获得的DNA分子d的表达产物仅为与DNA分子a或b表达产物具有相同功能的蛋白质的DNA分子。
优选地,所述严格杂交条件可以为在60-70℃(最优选65℃)的温度下,于杂交溶液中进行杂交,然后用洗膜溶液进行洗膜。
更优选地,所述杂交溶液为含有0.1-1重量%十二烷基硫酸钠(SDS)的柠檬酸钠缓冲液(SSC缓冲液),优选所述SCC缓冲液为氯化钠和柠檬酸钠的水溶液,其中氯化钠浓度为0.8-1M,柠檬酸钠浓度为0.08-0.1M。
优选地,所述洗膜采用分步操作的方式进行,且每步采用不同的洗膜溶液。更优选采用如下方式进行分步洗膜:
第一洗膜:采用含有0.01-0.2重量%SDS的SCC缓冲液作为第一洗膜溶液洗膜一次,所述SCC缓冲液中氯化钠浓度为0.2-0.4M,柠檬酸钠的浓度为0.02-0.04M。
第二洗膜:采用含有0.01-0.2重量%SDS的SCC缓冲液作为第二洗膜溶液洗膜一次,所述SCC缓冲液中氯化钠浓度为0.1-0.2M,柠檬酸钠的浓度为0.01-0.02M。
更优选地,第一洗膜溶液和第二洗膜溶液中的SDS含量相同。
更优选地,第一洗膜溶液采用的SCC缓冲液浓度高于第二洗膜溶液,优选第一洗膜溶液采用的SCC缓冲液的浓度是第二洗膜溶液采用的SCC缓冲液浓度的1.5-2.5倍。
本发明第三方面提供一种制备如前所述的单域重链抗体的方法,所述方法包括:将含有如上所述的基因的重组载体导入宿主细胞,表达获得所述单域重链抗体。
任意本领域中能够插入上述基因,并在宿主细胞(即表达载体)中表达所述单域重链抗体的重组载体均可适用于本发明。根据本发明的优选实施方式,其中,所述重组载体选自pCold I载体、pQE30载体、pET32a载体、pcDNA3.1载体和pPICZ载体中的至少一种。优选为pCold I载体。
任意本领域用于表达外源性基因的宿主细胞均可适用于本发明。根据本发明的优选实施方式,其中,所述宿主细胞选自大肠杆菌、293T细胞和酵母菌中的至少一种。
根据本发明的优选实施方式,其中,所述方法还包括对表达获得的单域重链抗体进行纯化的方法。
任意本领域用于纯化宿主细胞表达的外源性蛋白的方法均可适用于本发明。例如,可以利用载体中或所述单域重链抗体中带有的便于纯化的标签(如表1中的标签)进行纯化。
本发明第四方面提供含有如前所述的基因的重组载体、表达盒和表达载体。
本发明中,任意含有如前所述的基因的重组载体或表达盒均属于本发明的保护范围,本领域技术人员可以根据实际情况对其进行具体选择和调整。
本发明中,任意含有如前所述的基因的表达载体均属于本发明的保护范围。优选地,所述表达载体包括(含有如前所述的基因的)转基因细胞系、重组菌和重组酵母中的至少一种。
本发明第五方面提供扩增如前所述的基因或其片段的引物。
本发明中,所述引物用于扩增如前所述的基因(例如前述DNA分子a、DNA分子b、DNA分子c和DNA分子d)的全长序列,或用于扩增如前所述的基因中的任意长度的片段(例如扩增其中的编码区,即核苷酸序列如SEQ ID NO:2或SEQ ID NO:3所示的DNA分子,或扩增其中含有编码区和部分非编码区的片段)。
本发明第六方面提供如前所述的单域重链抗体、基因、重组载体、表达盒和表达载体或引物在制备针对MERS-CoV的抗病毒产品中的应用。所述“针对MERS-CoV的抗病毒产品”指的是用于MERS-CoV防治的相关产品,例如MERS-CoV疫苗、抗病毒药物等。
本发明中,所述在制备针对MERS-CoV的抗病毒产品中的应用可以包括利用如前所述的单域重链抗体、基因、重组载体、表达盒和表达载体或引物进行所述抗病毒产品的研发和/或生产。
本发明第七方面提供一种药物组合物,所述药物组合物包括如前所述的单域重链抗体、基因或重组载体、表达盒和表达载体。
优选地,所述药物组合物还包括药学上可接受的辅料。所述辅料可以为任意本领域现有的用于药物组合物制备的辅料,只要该辅料不影响活性组分(即如前所述的单域重链抗体、基因或重组载体、表达盒和表达载体)的功能和作用即可。
本发明提供的药物组合物中,可以仅以如前所述的单域重链抗体(或其编码基因或重组载体、表达盒和表达载体)作为活性组分,也可以将其与其他抗体(或药物)配合作为活性组分。所述其他抗体(或药物)可以是本领域中任意抗MERS-CoV的抗体(或药物),也可以是本领域中任意能够调节前述单域重链抗体功能的抗体(或药物),还可以是与MERS相关的衍生疾病防治有关的抗体(或药物)。
以下将通过实施例对本发明进行详细描述。应当能够理解的是,以下实施例仅用于示例性地解释和说明本发明的内容,而不用于限制本发明的范围。
以下实施例中采用的基因片段均为委托金斯瑞公司合成。未经特殊说明的情况下,采用的试剂均购自正规化学或生物试剂供应商,纯度为分析纯。
实施例1
本实施例用于说明单域重链抗体Nano-Anti-MRBD的表达和鉴定。
(一)表达载体构建
发明人设计了SEQ ID NO:2所示的核苷酸序列作为单域重链抗体Nano-Anti-MRBD(氨基酸序列如SEQ ID NO:1所示)的编码基因,其中,5’末端第1-6位为BamH I的识别位点,第7-363位(即SEQ ID NO:3所示的核苷酸序列)为Nano-Anti-MRBD编码序列,第364-366位为终止密码子,第367-372位为Xba I的识别位点。
采用BamH I和Xba I(均购自Thermofisher公司,牌号分别为FD0055和FD0685)分别对单域重链抗体Nano-Anti-MRBD的编码基因和pCold I载体(购自TAKARA公司,牌号为D3361,其表达蛋白带有Poly-His标签(该标签的氨基酸序列如SEQ ID NO:6所示)进行双酶切,并将酶切产物连接,获得表达载体pCold I-Nano-Anti-MRBD。
利用表达载体pCold I-Nano-Anti-MRBD转化大肠杆菌BL21(DE3)(购自全式金公司,牌号为C601)感受态细胞,获得重组大肠杆菌pCold I-Nano-Anti-MRBD/BL21(DE3),利用含100μg/mL氨苄青霉素的LB固体培养基进行培养,挑选氨苄青霉素抗性克隆,提取质粒进行PCR鉴定和测序鉴定(委托诺赛公司完成)。PCR鉴定结果如图1所示,1号泳道为Marker,2号泳道为PCR产物条带(长度250-500bp)。测序结果如图2所示,其中用方框圈出了BamH I和Xba I的酶切位点。
理论而言,表达载体pCold I-Nano-Anti-MRBD的序列应为将pCold I载体中BamHI和Xba I的酶切位点间的DNA片段替换为Nano-Anti-MRBD的编码基因(SEQ ID NO:2)的核苷酸序列。经比对,该测序结果与理论序列完全一致,说明表达载体构建成功。
(二)单域重链抗体的表达和纯化
以下实验中采用的LB液体培养基购自全式金公司,牌号为GG101-01,其中含有100μg/mL氨苄青霉素。
实验重组菌:含有正确重组质粒的重组大肠杆菌pCold I-Nano-Anti-MRBD/BL21(DE3)
阴性对照菌:含有pCold I空质粒的重组大肠杆菌pCold I/BL21(DE3)
诱导表达实验
分别取实验重组菌和阴性对照菌接种于5mL LB液体培养基中,37℃震荡培养10h,分别获得实验重组菌种子液和阴性对照菌种子液。
将上述种子液分别以1:100的体积比接种于新鲜的5mL LB液体培养基中,37℃振荡培养3h,至OD600达到0.5±0.1。加入异丙基-β-D-硫代半乳糖苷(IPTG,购自TAKARA公司,牌号为D9030A),加入量使得IPTG的终浓度为0.4mM,15℃诱导培养12h。
分别取1mL诱导培养后的实验重组菌和阴性对照菌菌液作为诱导实验组,同时分别取1mL未经诱导培养的实验重组菌和阴性对照菌菌液作为诱导对照组,12000rpm离心10min收集菌体沉淀,使用生理盐水洗涤后,加入125μL生理盐水和125μL 5×SDS上样缓冲液(购自康为世纪公司,牌号为CW0027S),混匀后100℃煮沸10min,自然冷却至室温后,12000rpm离心1min,取40μL上清液进行SDS-PAGE凝胶电泳检测。检测条件:分离胶(购自金斯瑞公司,牌号为M01210)浓度为12%,恒定电压140V,电泳时间1h,电泳结束后考马斯亮蓝染色观察。
结果如图3所示,图中泳道1为蛋白Marker(购自Thermo公司,牌号为26616),泳道2为诱导实验组实验重组菌表达产物,泳道3为诱导对照组实验重组菌表达产物,泳道4为诱导对照组阴性对照菌表达产物,泳道5位诱导实验组阴性对照菌表达产物。根据图3中的结果可以看出,只有经诱导培养的实验重组菌pCold I-Nano-Anti-MRBD/BL21(DE3)能够表达Nano-Anti-MRBD(箭头所示,大小约为17kDa)。
表达形式鉴定实验
取3mL实验重组菌的诱导表达菌液,12000rpm离心1min,弃上清,用PBS缓冲液洗涤2次,而后用500μL无菌水重悬菌体,置于1.5mL离心管中。冰浴条件下超声破碎重悬菌体,具体超声条件:功率100W,采用超声5s停歇10s的间歇超声模式处理10min。4℃下,12000rpm离心超声后菌体10min,获得超声上清液和粗制包涵体沉淀。分别对全菌样品(即超声破碎后的菌液混合物)、超声上清液以及粗制包涵体沉淀进行SDS-PAGE电泳检测。检测条件:分离胶(购自金斯瑞公司,牌号为M01210)浓度为12%,恒定电压140V,电泳时间1h,电泳结束后考马斯亮蓝染色观察。
结果如图4所示,图中泳道1为蛋白Marker(购自Thermo公司,牌号为26616),泳道2为全菌样品,泳道3为粗制包涵体沉淀,泳道4为超声上清液。通过图4的结果可以看出,Nano-Anti-MRBD在超声上清液和粗制包涵体沉淀中均有存在,属于部分可溶性表达。
单域重链抗体的纯化
按照前述方式进行实验重组菌种子液制备。
将种子液以1:100的体积比接种于新鲜的LB液体培养基(500mL)中,37℃震荡培养3h至OD600达到0.5±0.1。加入异丙基-β-D-硫代半乳糖苷(IPTG,购自TAKARA公司,牌号为D9030A),加入量使得IPTG的终浓度为0.4mM,15℃诱导培养12h。
12000rpm离心10min收集菌体沉淀,使用50mL无菌水重悬菌体沉淀,加入适量蛋白酶抑制剂(50×,蛋白酶抑制剂混合物,购自普利莱,P1265,用量约为1ml),超声破碎重悬菌体,超声条件:300W,采用超声4s停歇6s的间歇超声模式处理60min。4℃下,13000rpm离心15min,将上清液转入新的离心管中再在4℃下13000rpm离心15min,彻底去除沉淀。采用0.22μm滤器(购自PALL公司,牌号为PN4612)过滤离心上清于50mL细离心管中,获得超声上清样品。
利用pCold I载体表达蛋白所带的Poly-His标签,采用Ni亲和层析的方法需超声上清样品进行纯化。所用的Ni柱为HisTrap HP层析柱(购自GE Healthcare公司,牌号为17524701),其为5mL柱体积的预装柱,按照以下方法进行纯化:使用结合缓冲液Buffer A平衡镍柱5个柱体积,平衡流速为5mL/min;将超声上清样品上样,上样流速为1mL/min;用结合缓冲液Buffer A再洗5个柱体积,流速为5mL/min;分别使用5%、10%、20%、100%(对应含25mM、50mM、100mM、500mM咪唑,pH7.5)浓度的洗脱缓冲液Buffer B(50mM HEPES,500mMNaCl,500mM咪唑,pH 7.5)洗脱蛋白,流速为3mL/min,分别对应收集各洗脱峰1管(即为纯化样品),最后用纯水洗5个柱体积,再用20%的乙醇洗5个柱床体积,流速为3mL/min,柱子置于4℃环境中保存。
将收集获得的纯化样品进行SDS电泳鉴定,结果如图5所示。图5中,泳道1为蛋白Marker(购自Thermo公司,牌号为26616),泳道2为全菌样品,泳道3为粗制包涵体沉淀样品,泳道4为超声上清样品,泳道5为穿柱液样品,泳道6-9分别为25mM、50mM、100mM和500mM咪唑洗脱的纯化样品,由图5可看出,500mM咪唑洗脱的纯化样品纯度最高。
使用3kDa超滤管(购自Merck Millipore公司,牌号为UFC900396)对Nano-Anti-MRBD蛋白(即纯化样品)进行浓缩,具体步骤如下:取15mL 500mM咪唑洗脱的纯化样品与8mLPBS缓冲液混合获得稀释液,将稀释液转入3kDa超滤管,在4℃下4000×g离心60min;将被截留的液体(约2.5mL)与12mL PBS缓冲液混合,而后再于4℃下4000×g离心60min,获得的被截留的液体即为浓缩Nano-Anti-MRBD蛋白样品,体积约1.5mL。取少量浓缩Nano-Anti-MRBD蛋白样品进行浓度测定及SDS电泳鉴定,其余分装后于-70℃冻存。
蛋白浓度检测结果显示浓缩Nano-Anti-MRBD蛋白样品的浓度为1.88mg/mL。
SDS电泳鉴定结果如图6所示,其中,泳道1为蛋白Marker(购自Thermo公司,牌号为26616),泳道2为浓缩Nano-Anti-MRBD蛋白样品。
实施例2
本实施例用于说明单域重链抗体Nano-Anti-MRBD与MERS-CoV的结合特性。
采用间接ELISA法检测Nano-Anti-MRBD与MERS-CoV RBD的反应性,具体方法如下:
使用MERS-CoV RBD-Fc蛋白(MERS-CoV S蛋白RBD功能区与人IgG Fc片段的融合蛋白,其氨基酸序列如SEQ ID NO:4所示)包被96孔酶标板,包被浓度为1μg/mL,每孔50μL。用封闭液(含3重量%BSA的PBS溶液)实验组将实施例1获得的浓缩Nano-Anti-MRBD蛋白样品配制成不同浓度的蛋白溶液作为一抗,使用HRP-His鼠单克隆抗体(购自康为世纪公司,牌号为CW0285M)作为二抗。对照组采用SARS-CoV抗体(制备方法参考Generation andCharacterization of a Nanobody Against SARS-CoV;DOI:10.1007/s12250-021-00436-1)作为一抗,HRP-His鼠单克隆抗体作为二抗。
ELISA检测结果如图7所示,图中M34和Negative分别代表实验组和对照组的检测结果。由图中可以看出,单域重链抗体Nano-Anti-MRBD与MERS-CoV RBD-Fc蛋白反应性很好,说明其能够很好地结合,进一步的结果显示其EC50值为0.744ng/mL。
实施例3
本实施例用于说明单域重链抗体Nano-Anti-MRBD对MERS-CoV假病毒的中和活性。
本实施例中采用的Huh-7细胞购自ATCC,采用的MERS-CoV假病毒(含萤火虫荧光素酶报告基因)的制备方法参照:A safe and convenient pseudovirus-based inhibitionassay to detect neutralizing antibodies and screen for viral entry inhibitorsagainst the novel human coronavirus MERS-CoV.Zhao G,Du L,Ma C,et al.VirolJ.2013;10:266.doi:10.1186/1743-422X-10-266。
实验抗体:实施例1获得的浓缩Nano-Anti-MRBD蛋白样品
对照抗体:抗SARS-CoV中和抗体(制备方法参考Generation andCharacterization of a Nanobody Against SARS-CoV;DOI:10.1007/s12250-021-00436-1)
实验组:采用不同浓度的抗体(利用DMEM高糖培养基对实验抗体或对照抗体进行稀释获得)进行处理
对照组I:以等体积DMEM高糖培养基替代抗体进行处理,其他操作与实验组相同,即病毒对照组
对照组II:以等体积DMEM高糖培养基替代抗体和假病毒(溶液),其他操作与实验组相同,即细胞对照组
使用96孔板,分别将50μL/孔不同浓度的实验抗体和对照抗体与500TCID50的MERS-CoV假病毒(加入量为100μL/孔)混合,于37℃孵育1小时。向孵育后的混合液中加入100μL的Huh-7细胞(约4×104个细胞/孔),即培养体系为250μL/孔,37℃培养48小时。
而后吸弃部分培养基(150μL/孔),加入显色底物(购自Perkin Elmer,牌号为6066761),加入量为100μL/孔,反应2分钟,然后将反应混合物(200μL/孔)转移至96孔白板中,测定其相对光单位(Relative Light Unit,RLU)的强度,采用以下公式计算抗体对MERS-CoV假病毒进入细胞的抑制率:
抑制率=(病毒对照孔RLU-样品孔RLU)/(病毒对照孔RLU-细胞对照孔RLU)×100%
式中,样品孔RLU即为实验组RLU。
结果如图8所示,图中M34和Negative对应的曲线分别代表实验抗体和对照抗体的检测结果。从图中可以看出,单域重链抗体Nano-Anti-MRBD对MERS-CoV假病毒具有较好的中和活性,能够有效抑制MERS-CoV假病毒进入Huh-7细胞,进一步的结果显示其IC50为10.7ng/mL。相比之下,对照抗体则基本对MERS-CoV假病毒不产生抑制作用。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
SEQUENCE LISTING
<110> 中国人民解放军军事科学院军事医学研究院
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Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
245 250 255
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
260 265 270
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
275 280 285
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
325 330 335
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
340 345 350
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
355 360 365
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
370 375 380
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
385 390 395 400
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
405 410 415
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
420 425 430
Leu Ser Leu Ser Pro Gly Lys
435
<210> 5
<211> 5
<212> PRT
<213> 人工序列
<400> 5
Arg Arg Arg Arg Arg
1 5
<210> 6
<211> 6
<212> PRT
<213> 人工序列
<400> 6
His His His His His His
1 5
<210> 7
<211> 8
<212> PRT
<213> 人工序列
<400> 7
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 8
<211> 8
<212> PRT
<213> 人工序列
<400> 8
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 9
<211> 10
<212> PRT
<213> 人工序列
<400> 9
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
Claims (11)
1. 一种针对中东呼吸综合征冠状病毒的单域重链抗体,其特征在于,所述抗体为:
(1)氨基酸序列如SEQ ID NO:1所示的蛋白质;或者
(2)氨基酸序列如SEQ ID NO:1所示的蛋白质经改造获得的与改造前具有相同活性的衍生蛋白质,所述改造为:
i、在氨基酸序列如SEQ ID NO:1所示的蛋白质的N末端和/或C末端连接便于纯化的标签和/或利于蛋白分泌表达的信号肽序列;和/或
ii、将氨基酸序列如SEQ ID NO:1所示的蛋白质进行人源化改造。
2. 根据权利要求1所述的单域重链抗体,其中,所述便于纯化的标签选自Poly-Arg、Poly-His、FLAG、Strep-tag II和c-myc中的至少一种;
和/或,所述信号肽序列选自人IL-2 SP、CD5 SP、人IgG2 H SP、人胰凝乳蛋白酶原SP、人胰蛋白酶原-2 SP和人胰岛素SP中的至少一种。
3.编码权利要求1或2所述的单域重链抗体的基因。
4. 根据权利要求3所述的基因,其中,所述基因包括:
a、编码区核苷酸序列如SEQ ID NO:2所示的DNA分子;或
b、编码区核苷酸序列如SEQ ID NO:3所示的DNA分子。
5.一种制备权利要求1或2所述的单域重链抗体的方法,其特征在于,所述方法包括:将含有权利要求3或4所述的基因的重组载体导入宿主细胞,表达获得所述单域重链抗体。
6. 根据权利要求5所述的方法,其中,所述重组载体选自pCold I载体、pQE30载体、pET32a载体、pcDNA3.1载体和pPICZ载体中的至少一种;
和/或,所述宿主细胞选自大肠杆菌、293T细胞和酵母菌中的至少一种。
7.含有权利要求3或4所述的基因的重组载体、表达盒和表达载体。
8.根据权利要求7所述的重组载体、表达盒和表达载体,其中,所述表达载体包括转基因细胞系、重组菌和重组酵母中的至少一种。
9.权利要求1或2所述的单域重链抗体、权利要求3或4所述的基因或权利要求7或8所述的重组载体、表达盒和表达载体在制备针对中东呼吸综合征冠状病毒的抗病毒产品中的应用。
10.一种药物组合物,其特征在于,所述药物组合物包括权利要求1或2所述的单域重链抗体、权利要求3或4所述的基因或权利要求7或8所述的重组载体、表达盒和表达载体。
11.根据权利要求10所述的药物组合物,其中,所述药物组合物还包括药学上可接受的辅料。
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