CN112225800A - 纳米抗体及其应用 - Google Patents
纳米抗体及其应用 Download PDFInfo
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- CN112225800A CN112225800A CN202011123691.6A CN202011123691A CN112225800A CN 112225800 A CN112225800 A CN 112225800A CN 202011123691 A CN202011123691 A CN 202011123691A CN 112225800 A CN112225800 A CN 112225800A
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Abstract
本发明涉及生物医药领域，特别涉及针对RSV病毒F蛋白的纳米抗体、其编码序列在诊断和治疗中的应用。本发明提供的RSV纳米抗体具有独特的CDR1、2和3区序列，使所述抗体对RSV抗原具有特异的识别和结合能力。而与其他非特异性蛋白无交叉反应。通过中和活性检测，其显示了良好的中和活性，可单独或与其他药物或抗体等联合应用，用于制备RSV治疗性药物。
Description
技术领域
本发明涉及生物医药领域，特别涉及针对于RSV病毒F蛋白的纳米抗体、其编码序列在诊断和治疗中的应用。
背景技术
呼吸道合胞病毒(respiratory syncytial virus，RSV)是导致婴幼儿、老年人和免疫力低下的个体严重下呼吸道疾病的主要病因。RSV感染呼吸道黏膜上皮细胞，病毒蛋白能在胞内引起复杂的信号传导，导致各种细胞因子表达异常，这可能是导致严重下呼吸道疾病的原因。相关灭活疫苗加重疾病的重要特征是诱导相关细胞因子大量分泌，从而引起不平衡的体液免疫和细胞免疫应答。
RSV属副粘病毒科肺炎病毒属，基因组为单股负链RNA，编码11种蛋白质，其中3个为跨膜包膜糖蛋白F蛋白(fusion protein)、G蛋白(attachmentprotein)和SH蛋白(smallhydrophobic protein)，根据G蛋白抗原性的不同，将RSV分为A、B两个亚型。F蛋白通过介导RSV与宿主细胞膜融合导致RSV核糖核蛋白穿入到细胞浆。感染后，表达在细胞膜上的F蛋白可促进感染细胞与邻近细胞细胞膜融合形成合胞体。
F蛋白在RSV不同亚型间具有高度的抗原同源性，是主要的交叉保护性抗原，同时还是细胞毒性T淋巴细胞(CTL)的主要靶抗原，因此，F蛋白是机体免疫系统重要的靶抗原，可介导产生保护性抗体和细胞免疫反应，且能提供交叉保护作用。F是RSV最主要的表面结构蛋白之一，作为最重要的中和抗原，F蛋白具有大量的中和抗体识别表位。
单克隆抗体在一些应用方面仍存在缺陷：分子质量大(150kDa)限制其组织穿透能力；在体内能够引起免疫反应，进而中和抗体活性；半衰期较长限制其在分子成像中的应用；生产操作复杂、生产成本高。
1993年，科学家在骆驼体内发现天然缺失轻链的重链抗体(Heavy chainantibodies，HcAbs)，其抗原结合域可变区，也被称为VHH或纳米抗体(nanobody，Nb)，为抗体小型化研究提供了新的发展方向。随后，软体鱼类体内也发现了骆驼体内类似的抗原受体(new antigenreceptor，NAR)。与其他小分子抗体相比，纳米抗体具有如分子质量小(12～15kDa)、亲和力高、免疫源性低、可溶性好、稳定性好、表达量高等独特的性质，使其在疾病诊断及治疗方面更具优势。因此，提供针对于RSV病毒F蛋白的纳米抗体、其编码序列在诊断和治疗中的应用具有重要的现实意义。
发明内容
有鉴于此，本发明提供了针对RSV病毒F蛋白的纳米抗体、其编码序列在诊断和治疗中的应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了纳米抗体，所述纳米抗体包括三个互补决定区CDR1、CDR2、CDR3；其中
(I)、所述纳米抗体的互补决定区CDR1具有如SEQ ID No.1所示的氨基酸序列；和/或
所述纳米抗体的互补决定区CDR2具有如SEQ ID No.2所示的氨基酸序列；和/或
所述纳米抗体的互补决定区CDR3具有如SEQ ID No.3所示的氨基酸序列；
或(II)、如(I)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(I)所述氨基酸序列功能相同的氨基酸序列；
或(III)、与(I)或(II)所述氨基酸序列具有80％以上同一性的氨基酸序列。
在本发明的一些具体实施方案中，所述纳米抗体还包括四个恒定区FR1、FR2、FR3、FR4，其中，
(IV)、所述纳米抗体的恒定区FR1具有如SEQ ID No.4所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR2具有如SEQ ID No.5所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR3具有如SEQ ID No.6所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR4具有如SEQ ID No.7所示的氨基酸序列；
或(V)、如(IV)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(IV)所述氨基酸序列功能相同的氨基酸序列；
或(VI)、与(IV)或(V)所述氨基酸序列具有80％以上同一性的氨基酸序列。
在本发明的一些具体实施方案中，
(VII)、所述纳米抗体具有如SEQ ID No.8所示的氨基酸序列；
或
(VIII)、如(VII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(VII)所述氨基酸序列功能相同的氨基酸序列；
或
(IX)、与(VII)或(VIII)所述氨基酸序列具有80％以上同一性的氨基酸序列。
本发明还提供了所述的纳米抗体的二聚体，
(X)、所述纳米抗体的二聚体具有如SEQ ID No.9所示的氨基酸序列；
或
(XI)、如(X)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(X)所述氨基酸序列功能相同的氨基酸序列；
或
(XII)、与(X)或(XI)所述氨基酸序列具有80％以上同一性的氨基酸序列。
本发明还提供了人源化RSV纳米抗体，
(XIII)、所述人源化RSV纳米抗体具有如SEQ ID No.10所示的氨基酸序列；
或
(XIV)、如(XIII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(XIII)所述氨基酸序列功能相同的氨基酸序列；
或
(XV)、与(XIII)或(XIV)所述氨基酸序列具有80％以上同一性的氨基酸序列。
本发明中，所述多个为2个、3个、4个或5个。
基于上述研究，本发明还提供了编码所述的纳米抗体、编码所述的纳米抗体的二聚体或编码所述的人源化RSV纳米抗体的核苷酸。
本发明还提供了一种表达载体，包括所述的核苷酸。
此外，本发明还提供了转化或转染所述的表达载体的宿主细胞。
本发明还提供了结合物，包含经化学标记或生物标记的所述的纳米抗体、所述的纳米抗体的二聚体或所述的人源化RSV纳米抗体。
在本发明中，所述化学标记为同位素、免疫毒素和/或化学药物；所述生物标记为生物素、亲和素或酶标记。所述酶标记优选为辣根过氧化物酶或碱性磷酸酶。所述免疫毒素优选为黄曲霉毒素、白喉毒素、绿脓杆菌外毒素、蓖麻毒蛋白、相思子毒蛋白、槲寄生凝集素、蒴莲根毒素、PAP、造草素、白树毒素或丝瓜毒素。
基于上述研究，本发明还提供了所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体或所述的结合物与固体介质或半固体介质偶联制得的偶联物。
在本发明中，所述固体介质或非固体介质选自胶体金、聚苯乙烯平板或珠粒。
本发明还提供了所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体、所述的结合物和/或所述的偶联物在制备RSV病毒诊断试剂和/或试剂盒中的应用。
本发明还提供了所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体、所述的结合物和/或所述的偶联物在制备预防和/或治疗RSV病毒相关疾病的药物中的应用。
基于上述研究，本发明还提供了RSV病毒诊断试剂，包括所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体、所述的结合物和/或所述的偶联物以及可接受的助剂。
本发明还提供了RSV病毒诊断试剂盒，包括所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体、所述的结合物和/或所述的偶联物以及可接受的助剂和/或载体。
本发明还提供了疾病的诊断方法，以所述的RSV病毒诊断试剂或所述的RSV病毒诊断试剂盒检测RSV表达，根据表达量判断是否患有疾病。
基于上述研究，本发明还提供了预防和/或治疗RSV病毒相关疾病的药物，包括所述的纳米抗体、所述的纳米抗体的二聚体、所述的人源化RSV纳米抗体、所述的结合物和/或所述的偶联物以及可接受的辅料。
此外，本发明还提供了疾病的预防和/或治疗方法，给予本发明所述的药物。
本发明首先将RSV基因工程重组F蛋白免疫骆驼，经过4次免疫之后分离该免疫骆驼外周血淋巴细胞，并构建特异性的RSV基因工程重组F蛋白重链抗体基因文库。将重组F蛋白包被在酶标板上，利用噬菌体展示技术，从免疫后的纳米抗体库中筛选得到了抗重组F蛋白的纳米抗体。构建的纳米抗体原核表达载体，将其导入大肠杆菌，表达后纯化，纯化后的RSV纳米抗体能够与重组F蛋白特异性结合，并能有效中和RSV病毒。
本发明提供的RSV纳米抗体具有独特的CDR1、2和3区序列，使所述抗体对RSV抗原具有特异的识别和结合能力。而与其他非特异性交叉反应性蛋白没有反应。通过中和活性检测，其显示了良好的中和活性，可单独或与其他药物或抗体等联合应用，用于制备RSV治疗性药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示单域抗体PCR产物1.5％凝胶鉴定结果；
图2示载体双酶切1％凝胶鉴定结果；
图3示单菌落进行菌液PCR，测定的阳性率结果；
图4示抗体亲和纯化后电泳图谱；
图5示抗体与F蛋白结合活性检测；
图6示单域抗体中和活性检测
图7示单域抗体人源化和二聚体检测结果；
图8示RSV病毒阴性、阳性荧光鉴定照片；其中，a示RSV病毒阴性(-)照片；b示RSV病毒阳性(+)照片。
具体实施方式
本发明公开了针对RSV病毒F蛋白的纳米抗体、其编码序列在诊断和治疗中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明首先将RSV基因工程重组F蛋白免疫骆驼，经过4次免疫之后分离该免疫骆驼外周血淋巴细胞，并构建特异性的RSV基因工程重组F蛋白重链抗体基因文库。将重组F蛋白包被在酶标板上，利用噬菌体展示技术，从免疫后的纳米抗体库中筛选得到了抗重组F蛋白的纳米抗体。构建的纳米抗体原核表达载体，将其导入大肠杆菌，表达后纯化，纯化后的RSV纳米抗体能够与重组F蛋白特异性结合，并能有效中和RSV病毒。
本发明提供一种可以特异性结合RSV病毒F蛋白的纳米抗体编码序列、其制备方法及应用。
具体而言：
所述纳米抗体可变区具有3个互补决定区CDR1、CDR2、CDR3，其中，
CDR1序列由SEQ ID No.1所述氨基酸序列组成；(不是全人源，是否CDR可以交叉)
CDR2序列由SEQ ID No.2所述氨基酸序列组成；
CDR3序列由SEQ ID No.3所述氨基酸序列组成。
所述纳米抗体还具有恒定区FR1、FR2、FR3、FR4，其中，
FR1序列由SEQ ID No.4所述氨基酸序列组成；
FR2序列由SEQ ID No.5所述氨基酸序列组成；
FR3序列由SEQ ID No.6所述氨基酸序列组成；
FR4序列由SEQ ID No.7所述氨基酸序列组成。
本发明还提供了一种编码上述纳米抗体的氨基酸编码序列，所述编码序列由SEQID No.8。
本发明还提供了一种编码上述纳米抗体二聚体序列，所述编码序列含有SEQ IDNo.9。
本发明还提供了一种编码上述人源化RSV纳米抗体的氨基酸序列，所述编码序列含有SEQ ID No.10。
本发明提供了上述纳米抗体在制备诊断RSV病毒诊断试剂中的应用。
本发明提供了上述纳米抗体在制备RSV治疗性药物中的应用。
Sequence:
FR1:EVQLQASGGGLVQPGGSLRLS CTASG SEQ No.4
FR2:WYRQAPGKQRELVA SEQ No.5
FR3:AD SVKGRFTISR DNAKNTVNLQ MNNLNPEDTG VYYC SEQ No.6
FR4:WGQ GTQVTVGS EQKLISEEDL N SEQ No.7
第七组
>9-BF-16
CDR1:SISQFN GIG SEQ No.1
CDR2:AIT DGGSKKY SEQ No.2
CDR3:SIGDYYD GYYYQSGGY SEQ No.3
SEQ No.8EVQLQASGGGLVQPGGSLRLSCTASG SISQFNGIG WYRQAPGKQRELVAAITDGGSKKY ADSVKGRFTISRDNAKNTVNLQMNNLNPEDTGVYYC SIGDYYDGYYYQSGGYWGQGTQVTVGSEQKLISEEDLN
SEQ No.9EVQLQASGGGLVQPGGSLRLSCTASG SISQFNGIG WYRQAPGKQRELVAAITDGGSKKY ADSVKGRFTISRDNAKNTVNLQMNNLNPEDTGVYYC SIGDYYDGYYYQSGGYWGQGTQVTVGSEQKLISEEDLN AAA EVQLQASGGGLVQPGGSLRLSCTASG SISQFNGIGWYRQAPGKQRELVA AITDGGSKKY ADSVKGRFTISRDNAKNTVNLQMNNLNPEDTGVYYCSIGDYYDGYYYQSGGY WGQGTQVTVGSEQKLISEEDLN
SEQ No.10EVQLQASGGGLVQPGGSLRLSCTASG SISQFNGIG WYRQAPGKQRELVAAITDGGSKKY ADSVKGRFTISRDNAKNTVNLQMNNLNPEDTGVYYC SIGDYYDGYYYQSGGYWGQGTQVTVGSEQKLISEEDLN DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
本发明提供的RSV纳米抗体具有独特的CDR1、2和3区序列，使所述抗体对RSV抗原具有特异的识别和结合能力。而与其他非特异性交叉反应性蛋白没有反应。通过中和活性检测，其显示了良好的中和活性，可单独或与其他药物或抗体等联合应用，用于制备RSV治疗性药物。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in MolecularBiology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
“抗体”是指由能特异结合抗原的一种或多种多肽构成的蛋白质。抗体的一种形式构成了抗体的基本结构单元。这种形式是四聚物，它由两对完全相同的抗体链构成，每一对都有一个轻链和一个重链。在每对抗体链中，轻链和重链的可变区联合在一起共同负责结合抗原，而恒定区则负责抗体的效应器功能。
“可变区”是该链的N端成熟区域。目前已知的抗体类型包括κ和λ轻链，以及α，γ(IgG1，IgG2，IgG3，IgG4)，δ，ε和μ重链或它们的其它类型等价物。全长的免疫球蛋白“轻链”(大约25kDa或大约214个氨基酸)包含一个由NH2-末端上大约110个氨基酸形成的可变区，以及一个COOH-末端上的κ或λ恒定区。全长的免疫球蛋白“重链”(大约50kDa或大约446个氨基酸)，同样包含一个可变区(大约116个氨基酸)，以及重链恒定区之一，例如γ(大约330个氨基酸)。
“抗体”包括任何同型体的抗体或免疫球蛋白，或保持与抗原特异结合的抗体片段，包括但不限于Fab，Fv，scFv和Fd片段、嵌合抗体、人源化抗体、单链抗体以及包含抗体的抗原结合部分和非抗体蛋白质的融合蛋白质。抗体可以被标记和检测，例如，可以通过放射性同位素、能产生可检测物的酶、荧光蛋白质、生物素等等进行标记并被检测。抗体还可以结合于固相载体，包括但不限于聚苯乙烯平板或珠粒等等。
“人源化抗体”是指一种抗体，其包含来源于非人源抗体的CDR区、并且该抗体分子的其他部分来源于一种(或几种)人抗体。而且，为了保留结合亲和力，可以修饰骨架(称为FR)区段的一些残基。
所述“纳米抗体”系指在羊驼外周血液中存在一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却不像人工改造的单链抗体片段(scFv)那样容易相互沾粘，甚至聚集成块。更重要的是单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。VHH晶体为2.5nm，长4nm，分子量只有15KD，因此也被称作纳米抗体(Nanobody，Nb)。VHH可溶性极高，不易聚集，能耐高温、强酸、强碱等致变性条件，适合于原核表达和各种真核表达系统，广泛用于开发治疗性抗体药物、诊断试剂、亲和纯化基质和科学研究等领域。纳米抗体诸多方面均优于传统抗体。基于羊驼重链抗体的VHH单域抗体的特殊结构，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，逐渐成为新一代治疗性生物医药与临床诊断试剂中的新兴力量。相比于常规抗体，纳米抗体的优势有：1.分子量小，可穿透血脑屏障；2.原核或真核系统中高表达；3.特异性强，亲和力高；4.对人的免疫原性弱。
所述药物中含有至少一种功能成分，还包括可药用的载体。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
本文使用的“CDR区”或“CDR”是指免疫球蛋白的重链和轻链的高变区，如Kabat etal.所定义(Kabat et al.，Sequences ofproteins ofimmunological interest，5th Ed.，U.S.Department ofHealth and Human Services，NIH，1991，以及以后版本)。存在三个重链CDR和三个轻链CDR。根据情况，本文所用术语CDR或CDRs是为了指示这些区域之一、或者这些区域的几个或者甚至全部，所述区域包含通过抗体对抗原或其识别表位的亲和力而负责结合的大部分氨基酸残基。
本文使用的“FR区”或“FR”是指骨架区，在免疫球蛋白的H和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V)和恒定区(C)。VH和VL。各有3个区域的氨基酸组成和排列顺序高度变化，称为高变区(HVR)或互补决定区(CDR)，分别为CDRl、CDR2和CDR3。CDR以外区域的氨基酸组成和排列顺序相对不易变化，称为骨架区(FR)。VH和VL，各有113和107个氨基酸残基，组成4个FR(分别为FRl、FR2、FR3和FR4)和3个CDR(CDR1、CDR2、CDR3)。
本发明提供的纳米抗体及其应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1动物免疫
将基因工程重组F蛋白与弗氏佐剂等体积混合并乳化均匀，免疫羊驼，每两周免疫一次，共免疫4次，第一次使用弗氏完全佐剂，其余几次使用不完全佐剂。
实施例2免疫动物血清效价检测
以0.05M碳酸盐包被液稀释基因工程重组F蛋白至2μg/ml，每孔100μl，4℃过夜；次日弃包被液，洗涤3次，每次5min；加入3％BSA封闭液，每孔200μl，37℃放置2h，洗涤后真空干燥备用。每孔加入100μl以样本稀释液稀释的待检血清，37℃孵育1h，洗板5次，每次5min；再加入HRP标记的二抗，37℃45min，洗板5次，每次5min；加入TMB底物每孔100μl，37℃15min。于各反应孔中加入2M硫酸50μl终止反应。结果如表1所示
表1.免疫动物血清效价ELISA检测结果
实施例3RSV纳米抗体文库的构建
取适量淋巴细胞进行RNA提取。以提取的RNA为模板进行反转录。以cDNA为模板，单域特异性引物进行两轮PCR扩增，1.2％琼脂糖凝胶鉴定。第一轮回收600bp左右的抗体基因片段，并将小于400bp单域抗体基因片段进行回收(图1)。用限制性内切酶对片段及载体进行酶切过夜，载体回收4000bp左右片段(图2)。用T4连接酶将回收片段与载体16℃连接过夜。TG1电转化，取电转后总菌液中的30μl稀释1000倍涂布150μl于15cm平板，培养过夜，计数。库容＝单克隆×稀释倍数×总体积÷涂板体积。
单菌落个数为85个，总体积75ml，根据稀释倍数计算的库容量为4.1×107cfu。
将达到库容量的平板上的菌刮下，加入70％甘油，使菌液终浓度在15％的甘油内，－80℃冻存。随机挑选单菌落进行阳性率测定(图3)。随机挑选单菌落进行测序，查看序列重复率。
单域抗体PCR产物1.5％凝胶鉴定结果如(图1)，回收1#约600bp和2#，3#400bp左右条带。
载体双酶切1％凝胶鉴定结果如(图2)，回收4000bp左右条带。
实施例4噬菌体抗体库淘筛
包被呼吸道合胞病毒F蛋白，对噬菌体库进行三轮“吸附-洗脱-扩增”的亲和筛选。由于该抗原包含一段无关区域，故采用无关蛋白先进行噬菌体库吸附后再与F蛋白结合，减少非特异抗体的吸附。
表2.噬菌体抗体库淘筛回收率
第二轮筛选后扩增噬菌体抗体库进行无关蛋白吸附(百克PA90)
表3.噬菌体抗体库进行无关蛋白吸附结果
以呼吸道合胞病毒F蛋白为目的抗原进行亲和筛选，第二轮筛选回收率较第一轮提高了93倍，第三轮较第二轮提高了0.451倍，文库到达富集目的。由于该抗原存在一段无关蛋白区域，故又将第二轮扩增后的噬菌体抗体库先进行无关蛋白吸附后，再与F蛋白结合，洗脱，扩增(进行两轮筛选)。均有不同程度的富集。
实施例5构建原核表达库
使用高保真酶进行PCR反应，目的基因400bp左右，纯化回收。双酶切PCR产物、载体pSJF2，纯化回收。按照连接酶的说明书进行，所加入量分子比vector:PCR＝1:3。TG1电转化，取100ul/板涂布LB/Amp平板，32℃培养过夜。菌落PCR鉴定转化效率。随机挑取96个单克隆在96孔板培养并进行ELISA阳性克隆筛选。阳性克隆培养保存，送样测序。根据测序结果CDR1，CDR2，CDR3的不同分型归类。分型后选择需表达纯化的单克隆。
实施例6RSV纳米抗体的表达与分离纯化
RSV纳米抗体的表达
将纳米抗体的基因序列插入表达载体pET23a，并转入大肠杆菌BL21细胞；用LB培养基。在温度37℃条件下培养。在菌体浓度达到OD6001-1.5时，加入浓度为1M的IPTG进行诱导；继续培养，3-4h后去上行收集菌体。用裂解缓冲液，重悬菌体，超声裂解细胞。收集上清液以及沉淀。用15％的SDS-PAGE电泳检测纳米抗体。
RSV纳米抗体的纯化
采用Ni-NTA亲和层析柱纯化蛋白：
(1)层析柱用上样缓冲液(20mMPBS，50mM咪唑，500mMNaCl)
平衡缓冲液后，加入离心收集的上清液；
(2)先用清洗缓冲液(20mMPBS，100mM咪唑，500mMNaCl)清
洗去除杂蛋白，再用洗脱缓冲液(20mMPBS，500mM咪唑，500mMNaCl)洗脱并收集洗脱液；
(3)纯化后的抗体用15％的SDS-PAGE电泳检测蛋白的纯化情况。如图4所示，抗体分子量在15～20KD之间。
实施例7RSV纳米抗体的结合活性
将RSV基因工程重组F蛋白100倍稀释后包被96孔ELISA板，4℃过夜，100ul/孔；封闭液37℃封闭2h。将纳米抗体按3倍系列稀释，37℃孵育1h，洗涤3遍；用HRP标记的抗His标签抗体作为二抗，37℃孵育1h，洗涤5遍；加入底物显色液100μl/孔；37℃避光显色15min；用2M硫酸终止反应；用450nm波长进行比色检测，分析结果。如图5和表4所示，检测结果的OD值与抗体浓度呈现明显的剂量依赖效应。
表4.抗体与F蛋白结合活性检测
实施例8RSV纳米抗体的中和活性测定
(1)加抗体，取一块96孔板横向使用，对待检抗体进行3倍系列稀释，每孔加入50μl样品稀释液。每孔加入相应样品25μl，排枪由第一行混匀后吸25μl至第二行混匀，依次混匀稀释至最后一行弃去25μl。
(2)稀释RSV病毒A2株(确定病毒2×104PFU/ml)：稀释后向96孔板加入50μl/孔，细胞阴性不加病毒。
(3)将细胞培养板轻拍混匀，放至37℃，5％CO2中孵育2h。
(4)用消化液消化细胞，制备细胞悬液浓度为1-2×105个/ml，每孔分别加入0.1mL细胞悬液，混匀，放入37℃，5％CO2孵箱中孵育培养。
(5)使用倒置显微镜每天观察CPE，以抑制50％细胞病变的血清最高稀释度的倒数为终点效价，培养6d后判定结果。
(6)染色法：细胞观察后也可以进行染色法判断，弃去上清液，加入结晶紫染色固定液100μl/孔，染色24h。
(7)去除染色液，用流水冲洗培养板，放置晾干。
(8)测定中和抗体滴度，中和抗体滴度定义为有50％以上完整HEP2细胞的最后一个抗体稀释度。
中和活性如图6和表5所示，9-BF-16的中和活性为4.2U/mg。
表5.单域抗体中和活性检测
| 抗体类别 | Positive | 9-BF-16 | Negative |
| 中和活性 | 850 | 3.1 | 0.03 |
实施例9制备RSV病毒检测制剂的应用(ELISA)
(1)将RSV基因工程重组F蛋白50-100ug/剂与弗氏佐剂乳化均匀免疫新西兰大白兔，第一次使用弗氏完全佐剂，其余三次使用不完全佐剂；
(2)将获得的F蛋白抗血清300倍稀释后包被96孔ELISA板，4℃过夜，100ul/孔；
(3)封闭液37℃封闭2h。将F蛋白按2倍系列稀释，37℃孵育1h，洗涤3遍；
(4)加入1ug/ml浓度的纳米抗体，37℃孵育1h，洗涤5遍；
(5)加入HRP标记的抗His标签抗体，37℃孵育1h，洗涤5遍；加入底物显色液100μl/孔；
(6)37℃避光显色15min；用2M硫酸终止反应；用450nm波长进行比色检测，分析结果。
如表6所示，检测结果的OD值与F蛋白抗原结果浓度呈现明显的剂量依赖效应，非常适合F蛋白抗原浓度检测。
表6夹心ELISA检测F蛋白结果
| F蛋白稀释倍数 | 4 | 8 | 16 | 32 | 64 | 128 | 256 |
| OD1 | 2.473 | 1.987 | 1.343 | 0.818 | 0.447 | 0.237 | 0.114 |
| OD2 | 1.852 | 1.359 | 0.846 | 0.545 | 0.310 | 0.173 | 0.088 |
实施例10抗体衍生物的应用
单域抗体的分子量较小通过AAA作为Linker将VHH连接形成同源二聚体参照实施例6RSV抗体的表达与分离纯化，制备9-BF-16的单体(Monomer)与二聚体(Dimer)。
人源化的单域抗体采用瞬时转染的方式表达制备
1、9-BF-16质粒制备
1)设计、优化并合成9-BF-16DNA序列；
2)将完整序列亚克隆到pcDNA3.4载体中；
3)转染级质粒制备用于表达Expi293F细胞。
2、细胞培养和瞬时转染：
1)Expi293F细胞在无血清Expi293中生长表达介质(Thermo FisherScientific)。
2)细胞保存在37℃的Erlenmeyer烧瓶中，8％的CO2放在震摇培养箱上。
3)在转染前一天，将细胞以适当的密度接种于培养皿中。
4)在转染当天，DNA和ExpiFectamineTM293试剂以最佳比例混合，然后加入细胞准备转染的烧瓶中。
5)目的抗体重组质粒瞬时共转染悬液中Expi293F细胞培养。
6)第6天收集的细胞培养上清用于纯化。
3、9-BF-16纯化
1)细胞培养液离心后过滤。
2)过滤后的细胞培养上清以适当的流速装载到亲和纯化(MonofinityAResin)柱上。
3)用适当的缓冲液洗涤和洗脱后，将洗脱的部分混合并交换缓冲液最终配方缓冲液。
将所获得的单域抗体二聚体与人源化抗体采用实施例8RSV抗体的中和活性测定，检测结果如图7和表7所示，表明单域抗体通过二聚化或人源化后抗体的中和活性都有显著提升，对RSV病毒具有良好的中和作用。
表7.单域抗体人源化和二聚体检测结果
实施例11抗体及其衍生物动物水平中和活性检测
(1)病毒悬液制备:RSV病毒A2株接种于Hep-2细胞上，按常规培养，在细胞融合病变最明显时，用力振荡细胞瓶，使细胞从瓶壁脱落，反复冻融3次，4℃离心(800×g，10min)，去除细胞散片。收集病毒悬液，进行病毒滴定，用于动物接种。
(2)动物接种过程:选用鼻腔接种法。豚鼠(每组3只)麻醉完全后，鼻腔内缓慢滴入50TCID50病毒与50个活性单位的RSV抗体悬液0.3ml；阴性对照组动物滴入PBS液0.3ml；阳性对照组缓慢滴入50TCID50病毒悬液0.3ml。每天1次，连滴3天。
(3)肺组织标本处理:采用急性失血法处死豚鼠。无菌条件下，取出部分肺组织置于灭菌Hank's液中，用于病毒分离。
(4)肺组织病毒分离:无菌条件下，剪碎新鲜的肺组织标本，研磨后接种到Hep-2细胞瓶中，观察细胞病变，待细胞融合病变时，收取标本，用免疫荧光法鉴定RSV。
免疫荧光法:含有病毒抗原的Hep-2细胞制成抗原片，冷丙酮固定20分钟，取出晾干。每孔加入1滴RSV/FITC抗体试剂，37℃温盒孵育30分钟，晾干后用基质液封片，移至荧光显微镜下观察。检测结果如表8和图8所示，豚鼠体内中和活性检测结果表明，单域抗体及其衍生物在动物水平表现出良好的中和活性。
表8.单域抗体及其衍生物豚鼠体内中和活性检测结果
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
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<120> 纳米抗体及其应用
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Claims (17)
1.纳米抗体，其特征在于，所述纳米抗体包括三个互补决定区CDR1、CDR2、CDR3；其中
(I)、所述纳米抗体的互补决定区CDR1具有如SEQ ID No.1所示的氨基酸序列；和/或
所述纳米抗体的互补决定区CDR2具有如SEQ ID No.2所示的氨基酸序列；和/或
所述纳米抗体的互补决定区CDR3具有如SEQ ID No.3所示的氨基酸序列；
或(II)、如(I)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(I)所述氨基酸序列功能相同的氨基酸序列；
或(III)、与(I)或(II)所述氨基酸序列具有80％以上同一性的氨基酸序列。
2.如权利要求1所述的纳米抗体，其特征在于，所述纳米抗体还包括四个恒定区FR1、FR2、FR3、FR4，其中，
(IV)、所述纳米抗体的恒定区FR1具有如SEQ ID No.4所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR2具有如SEQ ID No.5所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR3具有如SEQ ID No.6所示的氨基酸序列；和/或
所述纳米抗体的恒定区FR4具有如SEQ ID No.7所示的氨基酸序列；
或(V)、如(IV)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(IV)所述氨基酸序列功能相同的氨基酸序列；
或(VI)、与(IV)或(V)所述氨基酸序列具有80％以上同一性的氨基酸序列。
3.如权利要求1所述的纳米抗体，其特征在于，
(VII)、所述纳米抗体具有如SEQ ID No.8所示的氨基酸序列；
或
(VIII)、如(VII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(VII)所述氨基酸序列功能相同的氨基酸序列；
或
(IX)、与(VII)或(VIII)所述氨基酸序列具有80％以上同一性的氨基酸序列。
4.如权利要求1至3任一项所述的纳米抗体的二聚体，其特征在于，
(X)、所述纳米抗体的二聚体具有如SEQ ID No.9所示的氨基酸序列；
或
(XI)、如(X)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(X)所述氨基酸序列功能相同的氨基酸序列；
或
(XII)、与(X)或(XI)所述氨基酸序列具有80％以上同一性的氨基酸序列。
5.人源化RSV纳米抗体，其特征在于，
(XIII)、所述人源化RSV纳米抗体具有如SEQ ID No.10所示的氨基酸序列；
或
(XIV)、如(XIII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(XIII)所述氨基酸序列功能相同的氨基酸序列；
或
(XV)、与(XIII)或(XIV)所述氨基酸序列具有80％以上同一性的氨基酸序列。
6.编码如权利要求1～3任一项所述的纳米抗体、编码如权利要求4所述的纳米抗体的二聚体或编码如权利要求5所述的人源化RSV纳米抗体的核苷酸。
7.一种表达载体，包括如权利要求6所述的核苷酸。
8.转化或转染如权利要求7所述的表达载体的宿主细胞。
9.结合物，其特征在于，包含经化学标记或生物标记的如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体或如权利要求5所述的人源化RSV纳米抗体。
10.如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体或如权利要求5所述的人源化RSV纳米抗体或如权利要求9所述的结合物与固体介质或半固体介质偶联制得的偶联物。
11.如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体、如权利要求5所述的人源化RSV纳米抗体、如权利要求9所述的结合物和/或如权利要求10所述的偶联物在制备RSV病毒诊断试剂和/或试剂盒中的应用。
12.如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体、如权利要求5所述的人源化RSV纳米抗体、如权利要求9所述的结合物和/或如权利要求10所述的偶联物在制备预防和/或治疗RSV病毒相关疾病的药物中的应用。
13.RSV病毒诊断试剂，其特征在于，包括如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体、如权利要求5所述的人源化RSV纳米抗体、如权利要求9所述的结合物和/或如权利要求10所述的偶联物以及可接受的助剂。
14.RSV病毒诊断试剂盒，其特征在于，包括如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体、如权利要求5所述的人源化RSV纳米抗体、如权利要求9所述的结合物和/或如权利要求10所述的偶联物以及可接受的助剂和/或载体。
15.疾病的诊断方法，其特征在于，以如权利要求13所述的RSV病毒诊断试剂或如权利要求14所述的RSV病毒诊断试剂盒检测RSV表达，根据表达量判断是否患有疾病。
16.预防和/或治疗RSV病毒相关疾病的药物，其特征在于，包括如权利要求1～3任一项所述的纳米抗体、如权利要求4所述的纳米抗体的二聚体、如权利要求5所述的人源化RSV纳米抗体、如权利要求9所述的结合物和/或如权利要求10所述的偶联物以及可接受的辅料。
17.疾病的预防和/或治疗方法，其特征在于，给予如权利要求16所述的药物。
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