CN112940111A - 一种基于新型冠状病毒s蛋白的纳米抗体及其应用 - Google Patents
一种基于新型冠状病毒s蛋白的纳米抗体及其应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,尤其涉及一种基于新型冠状病毒S蛋白的纳米抗体及其应用。本发明利用纳米抗体文库,以2019新型冠状病毒的Spike S1+S2ECD为靶点,筛选获得一种针对2019新型冠状病毒的纳米抗体,经ELISA检测,不仅能够特异性识别2019新型冠状病毒的Spike S1+S2ECD靶点,还同时能够识别Spike RBD靶点,且结合信号较强。将相应抗体序列构建至原核表达载体中进行表达纯化,成功表达出目标抗体,纯化后纯度大于90%;且经VHH抗体ELISA检测发现,纯化后的纳米抗体对两种靶点均具有较高的亲和力。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种基于新型冠状病毒S蛋白的纳米抗体及其应用。
背景技术
2019新型冠状病毒(2019-nCoV)是一类呈球形、表面有突起、电镜下观察形似皇冠的病毒,病毒基因为连续线性单链RNA,直径在75-160nm。国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)发表声明,正式将2019新型冠状病毒(2019-nCoV)更名为严重急性呼吸综合征冠状病毒2(severe acute respiratorysyndrome coronavirus 2,SARS-CoV-2)。国务院联防联控机制发布会上表示,新型冠状病毒感染的肺炎统一称谓为新型冠状病毒肺炎,简称新冠肺炎(Novel coronaviruspneumonia,NCP)。世界卫生组织(World Health Organization,WHO)宣布,将新型冠状病毒肺炎命名为“COVID-19”:其中字母CO代表“冠状”(corona),字母VI代表“病毒”(virus),字母D代表“疾病”(disease),数字19代表该疾病发现时间为2019年。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭,甚至死亡。
2019-新型冠状病毒基因组依次编码棘突蛋白(spike protein)、包膜蛋白(Envelope protein)、膜蛋白(Membrane protein)和核衣壳蛋白(Nucleocapsid)。S蛋白(棘突蛋白,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。其中S1主要包含有受体结合区(receptorbinding domain,RBD),负责识别细胞的受体。S2含有膜融合过程所需的基本元件。S1亚基可进一步分成两个相对独立的区域(domain),分别是N-端区域(N-terminal domain,NTD)和C-端区域(C-terminal domain,CTD)。S1包含有受体结合域(receptor binding domain,RBD),大部分的冠状病毒S蛋白的RBD都位于CTD,例如SARS-CoV和MERS-CoV等。只有少部分β冠状病毒的RBD位于NTD(通常NTD结合糖类受体,CTD结合蛋白类受体)。S2亚基通过跨膜区锚定在膜上,它含有膜融合过程所需要的基本元件,包括:一个内在的膜融合肽(fusion peptide,FP),两个7肽重复序列(heptad repeat,HR)、一个跨膜临近区(juxamembrain domain,JMD)和一个跨膜结构域(transmembrane domain,TMD),以及C末端的胞浆区域(cytoplasmic domain,CD)。S蛋白是宿主中和抗体的重要作用位点。S蛋白是疫苗设计的关键靶点:所有冠状病毒都具有保守的功能motif,分别位于S1(RBD序列具有高度保守性)和S2(S2比S1区域氨基酸序列更为保守)结构域,对RBD及S2区的研究,有助于设计病毒疫苗和开发新的抗冠状病毒药物。
纳米抗体又称为单域重链抗体(Nanobody,VHH)是一种特殊的重链抗体的可变区,是一种优秀阻断剂的潜力。纳米抗体首次发现存在于骆驼血液,羊驼、鲨鱼等动物体内也存在。由于纳米抗体与单克隆抗体等抗体的结合方式与结合位点不同,因此在一些特殊的靶点中与抗原的结合要优于单克隆抗体。纳米抗体可以使用原核细胞等表达系统进行表达,可以极大的降低抗体的生产成本。可以对纳米抗体进行基因编辑、对纳米抗体进行修饰,达到更好地效果。纳米抗体不容易引起机体的免疫反应,可以很好的作为抗体应用进行应用。
特异性的纳米抗体是通过筛选纳米抗体的噬菌体库来获取的。纳米抗体的噬菌体文库分为免疫库和非免疫库。免疫库是使用蛋白免疫羊驼、骆驼等动物制备的。非免疫库是根据纳米抗体的恒定区与可变区的结构,保留一定的恒定区对可变区进行随机编辑制备的。当纳米抗体库的库容达到107以上时,就可以获取到针对抗原的特异性纳米抗体。非免疫库的使用节约了时间,避免了免疫动物以及采集动物血液对动物造成的伤害。
目前,新型冠状病毒SARS-CoV-2在全球范围内迅速传播,危及全球人民的生命安全,扰乱了全球经济。因此,针对新型冠状病毒SARS-CoV-2开展抗体的研发工作迫在眉睫。
发明内容
针对现有技术存在的问题,本发明提供了一种基于新型冠状病毒S蛋白的纳米抗体及其应用,目的在于解决现有技术中的一部分问题或至少缓解现有技术中的一部分问题。
本发明是这样实现的,一种基于新型冠状病毒S蛋白的纳米抗体,包括sdAb抗体片段,所述sdAb抗体片段氨基酸序列如SEQ ID NO.6所示。
进一步地,所述sdAb抗体片段核苷酸序列见SEQ ID NO.7所示。
一种表达载体,包括如上述的核苷酸序列。
进一步地,所述表达载体的氨基酸序列见SEQ ID NO.2所示。
进一步地,所述表达载体的核苷酸序列见SEQ ID NO.1所示。
一种宿主表达菌株,包括如上述的表达载体。
进一步地,所述载体为原核表达载体。
本发明还提供了一种如上述的基于新型冠状病毒S蛋白的纳米抗体的制备方法,包括:
将编码所述纳米抗体的核苷酸序列插入pET28a-SUMO表达载体的BamHI和XhoI酶切位点之间,获得表达载体;
将表达载体转化至E.coli BL21(DE3)菌株,挑取单克隆进行扩大培养;
诱导表达后,破胞收集纯化抗体蛋白。
进一步地,所述pET28a-SUMO表达载体SUMO的N端添加了6×His tag。
本发明还提供了如上述的基于新型冠状病毒S蛋白的纳米抗体或表达载体在制备治疗和/或诊断SARS-CoV-2新型冠状病毒感染的试剂中的应用。
综上所述,本发明的优点及积极效果为:
本发明以新冠病毒上的结构Spike S1+S2 ECD蛋白为靶点,筛选纳米抗体预制文库,在噬菌体水平获得纳米抗体。由于其中Spike S1+S2 ECD蛋白包含Spike RBD蛋白。因此,结合于其中一个靶点的纳米抗体也可能结合于另一个靶点Spike RBD。这两种靶点不但大小不一,位置有所区别,而且所使用的表达系统不同,充分的说明了纳米抗体对2019新型冠状病毒的结合力。
本发明筛选出的针对2019新型冠状病毒的纳米抗体,经ELISA检测,不仅能够特异性识别2019新型冠状病毒的SPIKE S1+S2 ECD靶点,还同时能够识别Spike RBD靶点,且结合信号较强。将相应抗体序列构建至原核表达载体中进行表达纯化,成功表达出目标抗体,纯化后纯度大于90%;且经VHH(VHH是纳米抗体的英文简写)抗体ELISA检测发现,纯化后的纳米抗体对两种靶点均具有较高的亲和力。
发明人使用假病毒证明纳米抗体阻断2019新型冠状病毒感染细胞,证明具有结合2019新型冠状病毒的能力,并有阻止2019新型冠状病毒感染细胞的能力;使用金斯瑞试剂盒SARS-CoV-2Surrogate Virus Neutralization Test Kit验证纳米抗体具有结合病毒中和位点的能力。
附图说明
图1是pMECS-纳米抗体phagemid图谱;
图2是SPIKE S1+S2 ECD蛋白SDS-PAGE电泳结果;
图3是Monoclonal phage ELISA检测结果;
图4是pET28a-SUMO-纳米抗体载体图谱;
图5是纳米抗体片段PCR扩增产物电泳结果;
图6是纳米抗体序列;
图7是SUMO-纳米抗体样品的SDS-PAGE分析;
图8是新冠中和抗体ELISA检测试剂盒检测纳米抗体中和活性实验结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明,各实施例及试验例中所用的设备和试剂如无特殊说明,均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
根据本申请包含的信息,对于本领域技术人员来说可以轻而易举地对本发明的精确描述进行各种改变,而不会偏离所附权利要求的精神和范围。应该理解,本发明的范围不局限于所限定的过程、性质或组分,因为这些实施方案以及其他的描述仅仅是为了示意性说明本发明的特定方面。实际上,本领域或相关领域的技术人员明显能够对本发明实施方式作出的各种改变都涵盖在所附权利要求的范围内。
本发明披露了一种基于新型冠状病毒S蛋白的纳米抗体及其应用,具体如下实施例所示。
实施例
1、靶点蛋白的选择
本发明本实施例所用靶点蛋白为购自北京义翘神州科技有限公司的SARS-CoV-2(2019-nCoV)Spike S1+S2 ECD-His Recombinant Protein新冠S蛋白(货号:40589-V08B1,全长胞外段,130kd,昆虫细胞表达,代号:SPIKE S1+S2 ECD)。
2、纳米抗体文库的选择
使用预制文库纳米抗体文库。
1)纳米抗体文库展示系统简介
纳米抗体Library为M13噬菌体展示系统构建的文库,该系统由pMECS噬菌粒载体、E.coli TG1和M13KO7辅助噬菌体组成。pMECS噬菌粒载体的结构如图1所示:Pst I酶切位点之前的序列为pelB分泌信号肽和抗体第一个框架区部分氨基酸的编码序列,pelB信号肽在成功将后续纳米抗体引导至周质腔后,信号肽酶作用于AQPAMA序列末尾,将pelB信号肽切除;Not I酶切位点之后是HA和6×His标签的编码序列,可用于融合蛋白的纯化或检测。紧随其后的序列编码噬菌体pIII衣壳蛋白。6×His标签和gene III序列之间有一个琥珀终止密码子,在琥珀终止密码子抑制型菌株(例如E.coli TG1)中有10%~20%的琥珀终止密码子能够翻译为谷氨酸(Glu,或E),导致纳米抗体与pIII蛋白融合表达。当利用辅助噬菌体M13KO7拯救后,纳米抗体得以展示在噬菌体尾部pIII蛋白的N末端。
3、纳米抗体的筛选与鉴定
1)利用SDS-PAGE分析目标蛋白
取3μg SPIKE S1+S2 ECD,变性后上样至10%SDS-PAGE凝胶,进行电泳分析,结果如图2所示:(SPIKE S1+S2 ECD)有三条带,分子量分别在60kDa、100kDa和150kDa附近;样品未发生降解,其纯度亦符合筛选要求。
2)筛选策略及效价
表1.SPIKE S1+S2 ECD蛋白的纳米抗体的筛选方法
3)S30和SPIKE S1+S2 ECD单克隆噬菌体ELISA结果
从(SPIKE S1+S2 ECD)第三轮筛选后的Output克隆中,随机挑取个单克隆,进行phage rescue后,在包被了靶点(SPIKE S1+S2 ECD)(200ng/孔)的ELISA wells进行monoclonal phage ELISA检测,以No Coating作对照。详细结果见表2,在R3克隆中,有个特异性识别靶点(SPIKE S1+S2 ECD)。
表2 R3克隆的SPIKE S1+S2 ECD单克隆噬菌体ELISA结果
4)阳性克隆的序列分析
将SPIKE S1+S2 ECD筛选得到的特异性识别靶点蛋白克隆进行测序,测序引物为MP57(TTATGCTTCCGGCTCGTATG)。经对测序序列进行分析和排布,得到unique的VHH抗体序列。结果见表3所示。
表3阳性克隆的DNA序列汇总
Items | Clones with unique DNA sequence |
3 | 22 |
5)阳性克隆的可溶性ELISA实验
将特异性识别(SPIKE S1+S2 ECD)的克隆(in E.coli TG1)在30℃进行IPTG诱导表达,离心后收集菌体,进行周质腔抽提。将周质腔抽提样品用0.5×blocker稀释10倍备用。在96孔ELISA板上包被(SPIKE S1+S2 ECD)(200ng/孔),以No Coating作对照,针对上述稀释后的周质腔提取样品进行ELISA检测。Anti-HA mouse McAb作为ELISA检测的二抗,HRP-conjugated Goat Anti-Mouse IgG(H+L)为三抗。结果如表4所示,克隆能够特异性结合靶点蛋白。
表4.纳米抗体在噬菌体中与(SPIKE S1+S2 ECD)蛋白结合的ELISA实验
6)针对SPIKE RBD靶点进行ELISA检测
本发明本实施例中使用购自北京义翘神州科技有限公司的SARS-CoV-2(2019-nCoV)Spike RBD-His Recombinant Protein(货号:40592-V08B,20kd,293细胞表达,代号:Spike RBD)进行ELISA检测。
将上述结合(SPIKE S1+S2 ECD)靶点的克隆的周质腔提取样品,针对(SPIKE RBD)进行检测,检测方法同上述可溶性ELISA。结果如表5所示:识别(SPIKE S1+S2 ECD)的克隆能检测到与(SPIKE RBD)的结合信号。
Table 5.纳米抗体在噬菌体中与(SPIKE RBD)蛋白结合的ELISA实验
分组 | 包被(SPIKE RBD)蛋白 | 空白对照 |
OD450 | 0.7424 | 0.0836 |
4、筛选后的单克隆噬菌体ELISA检测
1)ELISA方法
包被:用1×PBS pH 7.4稀释靶点蛋白至2μg/mL,100μL/well加入ELISA板孔,4℃包被过夜;
封闭:PBST(0.1%Tween)洗涤ELISA板1次,每孔加入300μL 5%BSA作为封闭液,37℃孵育2h;
孵育phage上清:PBST(0.1%Tween)洗涤ELISA板1次,对应的ELISA well中加入100μL phage上清,37℃孵育2h;
孵育检检测抗体:PBST(0.1%Tween)洗涤ELISA板3次,利用2.5%BSA稀释anti-M13-HRP(1:5000),100μL/well加入对应的ELIS板孔,37℃孵育1h;
TMB显色:PBST(0.1%Tween)洗板3次,PBS洗板2次,100μL/well TMB显色,37℃孵育约30min,50μL/well 2M H2SO4终止。
酶标仪读数:利用酶标仪检测OD450nm处的吸光值。
2)检测结果
结果见图3所示,其中A:SPIKE RBD靶点检测,B:SPIKE S1+S2 ECD靶点检测。
5、纳米抗体的获取
1)阳性克隆原核表达载体构建及表达纯化
SUMO标签蛋白是一种小分子泛素样修饰蛋白(Small ubiquitin-likemodifier),分子量大小约为11.2kDa,作为重组蛋白表达的融合标签时,可以提高融合蛋白的表达量,具有抗蛋白酶水解、促进靶蛋白正确折叠,以及提高重组蛋白可溶性等功能。为了获得可溶性表达的纳米抗体抗体,将纳米抗体编码序列插入pET28a-SUMO表达载体的BamHI和XhoI酶切位点之间,使其与SUMO标签融合表达,SUMO的N端添加了6×His tag,可用于融合蛋白的纯化,纳米抗体的C端添加了HA tag可用于检测。pET28a-SUMO-纳米抗体的载体图谱见图4。
将识别(SPIKE S1+S2 ECD)的克隆(pMECS in TG1)接种至2YT-AG培养基,37℃过夜培养,以克隆的菌液作为模板,SUMOVHH-F和SUMOVHH-R为引物。
表PCR体系
PCR反应条件
PCR扩增纳米抗体基因片段,PCR产物的琼脂糖凝胶电泳结果如图5所示:目的片段得到了有效扩增,片段大小约为400bp,与预期一致。将PCR产物分别上样至1.2%琼脂糖凝胶,进行电泳分离,并利用凝胶回收试剂盒对目的片段进行切胶纯化回收。
SUMOVHH-F:cacagagaacagattggtggatccCAGGTGCAGCTGCAGG,见SEQ ID NO.3;
SUMOVHH-R:cagtggtggtggtggtggtgctcgagtcaGGAACCGTAGTCCGGAAC,见SEQ IDNO.4。
将纳米抗体抗体合成到pET28a-SUMO表达质粒中。
SUMO-纳米抗体的表达cassette序列见SEQ ID NO.1所示,氨基酸序列见SEQ IDNO.2所示;同时见图6所示,其中His、SUMO、纳米抗体和HA tag各结构区分别用不用灰度或下划线区别开,并与名称中的灰度或下划线部分对应。
将SPIKE S1+S2 ECD SUMO-纳米抗体表达载体分别转化E.coli BL21(DE3)菌株,涂布Kan抗性平板,过夜培养后,挑取单克隆接种至200mL 2YT-K培养基,培养至对数生长中期,随后加入终浓度为1mM的IPTG溶液,30℃诱导表达过夜。离心收集菌体沉淀,超声波破碎菌体后,利用Ni离子亲和层析柱纯化SUMO-纳米抗体抗体,采用Millipore浓缩管,进行样品浓缩并将缓冲液置换成PBS(pH7.4)。用0.22μm滤膜过滤除菌,加入5%终浓度的无菌甘油,分装后冻存。使用Nanodrop对纯化后的SUMO-纳米抗体样品进行定量分析,结果如表6所示。最后,通过SDS-PAGE电泳对上述样品进行检测,结果如图7所示:SPIKE S1+S2 ECD-纳米抗体纯化样品,仅在28kDa附近有一条目的条带,其纯度大于90%。
表6样品浓度信息
6、VHH抗体ELISA检测
为了验证特异性及亲和力,在96孔ELISA板上分别包被SPIKE S1+S2 ECD和SPIKERBD两种靶点(200ng/孔),以No Coating作对照,针对SUMO-纳米抗体进行浓度梯度ELISA检测,Anti-HA mouse McAb作为ELISA检测的二抗,HRP-conjugated Goat Anti-Mouse IgG(H+L)为三抗。结果如表7所示:SUMO-纳米抗体对两种靶点(SPIKE S1+S2 ECD和SPIKE RBD)均有结合活性,两种SPIKE S1+S2 ECD SUMO-纳米抗体对两种靶点(SPIKE S1+S2 ECD和SPIKERBD)的结合活性相当,而两种SPIKE RBD SUMO-纳米抗体对SPIKE RBD靶点的结合信号强于SPIKE S1+S2 ECD靶点。
表7种SUMO-纳米抗体抗体的ELISA检测结果
7、新冠中和抗体ELISA检测试剂盒检测纳米抗体中和活性实验
具体操作如下:
按1:1体积比预先将阳性对照,阴性对照(本实施例中阳性对照是金斯瑞公司试剂盒L00847 SARS-CoV-2Surrogate Virus Neutralization Test Kit里带的新冠病毒的中和抗体;阴性对照为人的IgG)和样品分别与HRP-RBD溶液的混合。例如:60μL阳性对照加入60μL HRP-RBD溶液,37℃孵育30min。在酶标板中加100μL阳性对照混合液,阴性对照混合液和样品混合液。用盖板膜盖板,然后在37℃下孵育15分钟。取下盖板膜,并用260μL 1×洗液洗板4次。在洗涤后,将板拍在纸巾上除去孔中残留液体。在酶标板每孔中加入100μL TMBSolution,并在20-25℃下避光孵育10-15分钟(从开始加入TMB时开始计时)。移去盖板膜,每孔加入终止液50μL。终止后立即用酶标仪在450nm处测量吸光值。
结果:根据试剂盒的检测结果表明,纳米抗体具有中和活性。数据见图8。
8、SARS-CoV-2假病毒测定纳米抗体中和活性实验
本实施例中假病毒由南方医科大学黎诚耀构建并提供。具体操作如下:
1)样品准备:制备好的纳米抗体测定浓度。
2)第1列(细胞对照CC)加入DMEM完全培养基100μl/孔,第2列(病毒对照VC)列加入DMEM完全培养基50μl/孔,在第三列中加入培养基90μl/孔,其余孔加入培养基50μl/孔。
3)加入待测样品纳米抗体10μl
4)对第三列孔中液体轻柔的反复吹吸6-8次,然后转移50μl液体至对应的孔,之后所有孔都2倍倍比稀释。
5)用DMEM完全培养基将SARS-CoV-2假病毒稀释至1.28×104TCID50/mL,于第3~11列每孔加50μl,即640TCID50/孔。
6)将上述96孔板置于细胞培养箱中(37℃,5%CO2)孵育1小时。
7)待孵育30min后,开始消化ACE2-293T细胞,将细胞浓度稀释至3×105cells/mL。
8)孵育结束后,每孔加入100μl细胞,使每孔细胞为3×104cells。
9)放入37℃,5%CO2细胞培养箱中培养48小时。
10)培养完毕后,吸弃100μl上清,加入100μl Bright-GloTM荧光素酶检测试剂(promega),室温避光反应5min后,反复吹打,转移200μl液体至白板中。
11)使用PerkinElmer EnSight多功能成像酶标仪读取发光值(RLU)。
12)计算中和抑制率:
中和抗体滴度抑制率为50%时对应的抗体浓度。则根据开始测定的纳米抗体浓度及其加入的量,计算出对应的中和抗体滴度。
结果:纳米抗体对假病毒具有中和活性,且随着浓度的增加对假病毒的抑制率逐渐增加。结果见下表。
表8纳米抗体对假病毒的抑制率
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 石河子大学
<120> 一种基于新型冠状病毒S蛋白的纳米抗体及其应用
<150> 202011001280X
<151> 2020-09-22
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Claims (9)
1.一种基于新型冠状病毒S蛋白的纳米抗体,其特征在于:包括sdAb抗体片段,所述sdAb抗体片段氨基酸序列如SEQ ID NO.6所示。
2.根据权利要求1所述的一种基于新型冠状病毒S蛋白的纳米抗体,其特征在于:所述sdAb抗体片段核苷酸序列见SEQ ID NO.7所示。
3.一种表达载体,其特征在于,包括如权利要求2中所述的核苷酸序列。
4.根据权利要求3所述的表达载体,其特征在于,所述表达载体的氨基酸序列见SEQ IDNO.2所示。
5.根据权利要求3所述的表达载体,其特征在于:所述表达载体的核苷酸序列见SEQ IDNO.1所示。
6.一种宿主表达菌株,其特征在于,包括如权利要求3-5任一所述的表达载体。
7.一种如权利要求3-5任一所述的表达载体的制备方法,其特征在于,包括:
将编码所述纳米抗体的核苷酸序列插入pET28a-SUMO表达载体的BamHI和XhoI酶切位点之间,获得表达载体;
将表达载体转化至E.coli BL21(DE3)菌株,挑取单克隆进行扩大培养;
诱导表达后,破胞收集纯化抗体蛋白。
8.根据权利要求7所述的制备方法,其特征在于:所述pET28a-SUMO表达载体SUMO的N端添加了6×His tag。
9.一种如权利要求1或2所述的基于新型冠状病毒S蛋白的纳米抗体或权利要求3-5任一所述的表达载体在制备治疗和/或诊断SARS-CoV-2新型冠状病毒感染的试剂中的应用。
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CN112062840A (zh) | 2020-12-11 |
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