CN118184773A - 靶向s蛋白的纳米抗体制备及其应用 - Google Patents
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Abstract
本发明涉及一种可识别和中和MERS‑CoV的多肽NbM67，包括3个互补决定区CDR1‑3，序列如SEQ ID NO:1‑3所示。本发明针对MERS‑CoV进行纳米抗体药物开发，通过制备MERS‑CoV S蛋白、免疫双峰骆驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合MERS‑CoV的纳米抗体VHH，鉴定了其CDR序列，并构建了人源化的VHH‑huFc1；同时利用假病毒中和实验评估NbM67在治疗MERS‑CoV感染的疗效，结果显示当抗体浓度在0.0627μg/ml时，抑制率能达到90％。因此，本发明为MERS‑CoV感染的防治提供了潜在的检测剂和治疗药物。
Description
技术领域
本发明涉及生物医药领域。更特别地，涉及一种可靶向结合MERS-CoV的S蛋白的多肽，还涉及所述多肽在制备MERS-CoV感染的治疗药物和诊断剂中的应用。
背景技术
中东呼吸综合征(Middle East Respiratory Syndrome,MERS)首先发现于2012年6月一名死亡的60岁沙特阿拉伯男性患者。该病的潜伏期为2至14天，典型表现为急性呼吸道感染，起病急，高热(39-40℃)，可伴有畏寒、寒战、咳嗽、胸痛、头痛、全身肌肉关节酸痛、乏力、食欲减退等症状。目前尚无可用的疫苗和特异性治疗方法。虽然该病最初发生在中东地区，但随着贸易、旅游、宗教等的活动的开展，逐渐蔓延到欧洲、非洲、亚洲和北美洲等27个国家。因此，开发安全有效的中和抗体在MERS-CoV疫情防控中有重要的公共卫生意义。
MERS-CoV属于β冠状病毒属(β-CoV)的一种有包膜的单股正链RNA病毒，其单链RNA基因组的大小约30kb,共有10个开放阅读框(ORF)，编码16种非结构蛋白(nsp1-16)和四种结构蛋白：刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和核衣壳蛋白(N)。四种结构蛋白组成球形、似王冠样病毒颗粒，其中S和N具有很强的免疫原性。S蛋白为三聚体状态的Ⅰ型跨膜糖蛋白，位于病毒膜表面，介导病毒附着宿主细胞及病毒-细胞膜的融合，是细胞靶向性和发病机制的主要决定因子。S蛋白能够诱导机体产生中和抗体，该抗体在预防MERS-CoV感染中发挥关键作用。因此，以S蛋白免疫动物，并从中筛选有中和活性的抗体将是治疗MERS-CoV感染的有效策略之一。
1993年，一种来源于骆驼科的新型天然抗体被发现。该抗体天然缺失轻链而只由重链组成，其重链包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(Variableheavy chain domain，VHH，即抗原结合位点)，该重链可变区的相对分子质量约为13KDa，仅为常规抗体的1/10，且分子高度和直径均在纳米级别，是目前可获得的最小的功能性抗体片段，因此又被称为纳米单抗(Nanobody，Nb)。因纳米单抗稳定性高(90℃条件下仍不会降解)、亲和力高、与人源抗体同源性超过80％、毒性和免疫原性均较低等特点，最近纳米单抗被广泛用于免疫诊断试剂盒研发、影像学研发以及针对肿瘤、炎症、传染病和神经系统疾病等领域的抗体药物研发。
我们期望通过纳米抗体新技术，筛选获得能够有效预防和治疗MERS-CoV的中和抗体。
发明内容
本发明通过用抗原免疫骆驼，获取骆驼源纳米单抗，用于检测和治疗MERS-CoV感染。基于这些研究，本发明提供了一种可结合MERS-CoV的多肽，包括3个互补决定区CDR1-3，序列如SEQ ID NO:1-3所示。
在一个具体实施方案中，所述多肽为纳米抗体。
在一个具体实施方案中，所述多肽还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3按顺序交错排列。例如，可将FR1-4序列设计为如SEQ ID NO:4-7所示(羊驼源)，但本发明的范围不限于此。抗体的特异性识别和结合能力主要由CDR区序列决定，FR序列影响不大，可根据物种来设计，这是本领域公知的。可设计人源、鼠源或骆驼源的FR区序列来连接上述CDR，从而得到一个可结合MERS-CoV的纳米抗体。
在一个具体实施方案中，所述多肽为骆驼源的VHH或人源化的VHH。
本发明还提供了上述多肽在制备MERS-CoV的检测剂或MERS-CoV的S蛋白的检测剂中的应用。
本发明还提供了上述多肽在制备MERS-CoV治疗药物中的应用。
本发明还提供了编码上述多肽的核酸。
本发明还提供了上述核酸在制备MERS-CoV治疗药物中的应用。
本发明针对MERS-CoV进行纳米抗体药物开发，通过制备MERS-CoV S蛋白、免疫双峰骆驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合MERS-CoV的纳米抗体VHH，鉴定了其CDR序列，并构建了人源化的VHH-huFc1；同时利用假病毒中和实验评估NbM67在治疗MERS-CoV感染的疗效，为MERS-CoV感染的防治提供了潜在的检测剂和治疗药物。
附图说明
图1为MERS-CoV-S蛋白第4次免疫羊驼一周后的抗血清效价检测曲线。
图2为不同稀释度的第4次免疫骆驼一周后的抗血清抑制MERS-CoV假病毒体外感染ghost细胞的曲线，以免疫前的血清为对照。
图3为MERS-VHH噬菌体抗体文库的淘选鉴定，其中，A为噬菌体文库针对MERS-CoV-S蛋白淘选后ELISA检测统计图；B为从第一轮(1st)第二轮(2nd)和第三轮(3rd)淘选后的噬菌体抗体文库各挑选24,24个和40个克隆进行噬菌体ELISA检测统计图。
图4为原核表达的VHH抗体的ELISA检测统计图，每个点代表一个克隆，纵坐标为针对S蛋白的OD450/空白对照的OD450，比值大于5.0定义为阳性。
图5为NbM67抗体的SPR检测统计图。
图6为ELISA检测不同纯化浓度的NbM67抗体与MERS-S蛋白结合的OD450统计图。
图7为NbM67抗体中和MERS-CoV假病毒感染实验曲线图。
具体实施方式
1.羊驼免疫与抗血清的获得
用250μg MERS-S蛋白与250μl弗氏完全佐剂的乳化混合物对羊驼进行初免，在第14天、28天、42天用MERS-S蛋白与250μl弗氏不完全佐剂加强免疫3次，第2次和第3次免疫1周后，采血检测抗血清滴度；第4次免疫1周后，采血200ml用于噬菌体抗体库的构建。
抗血清效价通过ELISA检测，用浓度为0.5μg/ml的MERS-S-his蛋白包被检测板，每孔加入梯度稀释的抗血清或者纯化的抗体100μl(对照为免疫前骆驼血清)，37℃孵育1.5h，洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的Goat anti-Llamma IgG(H+L)二抗，37℃孵育1h，洗涤4-6次后，加100μl TMB底物，37℃孵育10min，50μl 0.2M的H2SO4中止反应，测定OD 450nm。ELISA检测血清效价规定为在OD450是空白对照的2倍以上并且大于0.2的最高稀释倍数。
结果如图1所示，4免的抗血清效价分别为3.28×106。由此可见，该抗原可诱导骆驼产生特异性针对MERS-S蛋白的高滴度抗血清。
为了进一步验证该高滴度的骆驼抗血清是否能有效阻止MERS-CoV病毒感染，进行病毒感染的中和实验。将不同稀释浓度的抗血清和免疫前血清分别与MERS-CoV假病毒共同孵育60min，随后转移到vero细胞，48h后通过Glo Max化学发光酶标仪(Promega)检测病毒载量并计算中和效果。中和实验结果显示，MERS-S蛋白诱导的抗血清抑制90％MERS-CoV感染的ID90为1000倍稀释度以上(图2)。综上所述，MERS-S蛋白诱导了高滴度抗血清，同时该抗血清具有高效抑制MERS-CoV假病毒感染的能力。
2.VHH噬菌体库构建及淘选
收集200ml免疫后骆驼的外周血，利用淋巴细胞分离液(GE Ficoll-Paque Plus)分离获得骆驼的PBMC，根据TRIzol操作手册，提取RNA，并利用oligo(dT)反转为cDNA,通过引物扩增，以及分子克隆等技术，将骆驼的VHH基因克隆至phagemid质粒，转化TG1细菌，得到VHH噬菌体库。
为了进一步鉴定MERS-CoV-VHH噬菌体库是否构建成功，通过PCR扩增免疫MERS-S蛋白骆驼的VHH目的基因，可以看出目的条带为450bp，大小符合预期，说明该MERS-CoV-VHH噬菌体抗体文库里含有VHH基因。挑选25个克隆进行测序，21个克隆有目的片段插入，插入率约为84％。测序结果显示，这21个克隆没有完全一致的重复序列，文库多样性为100％。比对结果显示，差异序列大多在CDR结合区。经检测，该构建了一个CD4-VHH噬菌体抗体文库的库容为1.37×109。
在M13KO7辅助噬菌体的帮助下，用VHH-phagemid转化的细菌，进行噬菌体抗体库的复苏，并用PEG/NaCl进行沉淀。将包被有50μg/ml的MERS-S-His蛋白进行三次富集噬菌体抗体库。将富集的噬菌体，洗脱、转化、涂板、挑取单克隆进行噬菌体与CD4蛋白ELISA的结合鉴定，将结合读值>1.0的克隆进行测序，并克隆至表达载体pcDNA3.1，转染293tt细胞表达生产纳米单抗。
淘选后的文库与MERS-S蛋白进行结合检测。噬菌体ELISA结果显示，没有富集前的CD4-VHH噬菌体文库与CD4蛋白的结合读值为0.78，经过一轮、二轮、三轮富集后的噬菌体文库读值分别为0.97、2.59、3.34(图3A)。为了进一步验证富集后的文库中结合MERS-CoV-VHH蛋白的阳性噬菌体率，从第1、2和3轮富集后的文库里各挑选了24、24和40个克隆进行单个噬菌体ELISA检测。结果显示，第2轮文库里，50％的单个噬菌体克隆为阳性，第3轮文库里75％的噬菌体克隆克隆为阳性，而且Target/Blank比值均＞5(图3B)，通过MERS-S蛋白淘选成功的富集了高结合力的MERS-CoV-VHH噬菌体文库。
3.VHH原核表达文库的构建及VHH表达
对上述二轮和三轮淘选富集后的2nd-MERS-CoV-VHH和3rd-MERS-CoV-VHH噬菌体抗体文库进行PCR扩增；获取并纯化抗体库中所有VHH的基因片段，将VHH的基因片段克隆至原核表达载体，转化SS320菌株，构建VHH的原核表达抗体库；将原核表达抗体库涂布平板，过夜培养，次日随机挑选单克隆菌落182个，使用IPTG诱导表达抗体上清，对抗体上清与S蛋白进行ELISA结合检测。
结果显示，有49个细菌上清与S蛋白结合，同时不与空白对照结合，S蛋白结合的读值/空白对照的读值大于5.0(图4)。其中,筛选出抗体NbM67,CDR1-3的序列如SEQ ID NO:1-3所示,FR1-4的序列如SEQ ID NO:4-7所示。
4.VHH-huFc(NbM67)真核表达
通过分子克隆技术，将NbM67基因融合人的Fc基因并插入在pCDNA3.4真核表达载体，构建形成NbM67-huFc-pCDNA3.4表达质粒。将构建好的NbM67-huFc-pCDNA3.4，转染293tt细胞，表达生产NbM67-huFc(4NB)。收集细胞上清进行ELISA测定。
对抗体NbM67与MERS-S蛋白的亲和力测定试验。应用Fortebio生物分子相互作用平台检测亲和力。将抗体固化至Anti-human IgG Fc Capture Biosensors(AHC)探头上，固化时间400s,再结合抗原CD4-his蛋白，结合时间180s,解离时间180s，观察抗体-抗原的结合解离情况，由仪器拟合曲线，导出数据。亲和力测定结果如表1所示，大部分的抗体的亲和力能达到10-12(picomole级)，结合解离曲线如图5所示。由此可见，我们获得了具有高亲和力抗体。
表1NbM67亲和力数据
| Clone ID | Ka(1/MS) | Kd(1/S) | KD(M) | Response(nm) |
| NbM67 | 6.52E+05 | ＜1E-07 | ＜1E-12 | 0.1551 |
5.抗体梯度稀释ELISA
用0.5μg/ml的MERS-S蛋白包被检测板，每孔100μl，37℃孵育2h，洗涤2-4次，4％牛血清封闭，每孔250μl，37℃孵育1h，洗涤2-4次，每孔加入梯度稀释的纯化抗体100μl，37℃孵育1.5h，洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的anti-human抗体100μl，37℃孵育1h，洗涤4-6次后，加100μl TMB底物，37℃孵育10min，50μl 0.2M的H2SO4中止反应，测定OD450nm。结果如图6所示，当抗体NbM67浓度低至0.00188μg/ml，其与MERS-S蛋白结合的OD450/空白对照的OD450，比值仍大于2。
5.NbM67中和MERS-CoV假病毒
通过将表达萤火虫荧光素酶(pNL43R-E-luciferase)和pcDNA3.1(Invitrogen)表达载体共转染到293T细胞(ATCC)中，生成了MERS-CoV假病毒。48h后收集病毒上清。病毒滴度通过相对光单位的荧光素酶活性测定(Bright-Glo荧光素酶检测载体系统，PromegaBiosciences)。对照单克隆抗体为抗SFTSV抗体SNB02(1mg/ml),对NbM67进行体外中和实验。将抗体梯度稀释为不同的浓度，与MERS-CoV假病毒一起，于5％CO2、37℃下共孵育1个小时，添加1×104个vero细胞，5％CO2、37℃温箱培养48小时后，通过检测荧光素酶活性测定评估单克隆抗体的半最大抑制浓度(IC50)
结果如图7所示，NbM67具有很好的中和活性，当抗体浓度在0.0627μg/ml时，抑制率能达到90％。
由以上实验结果可知，本发明的抗体NbM67及其人源化形式均可对特异性地识别、结合MERS-CoV及其S蛋白，并且可中和MERS-CoV，阻断其感染，从而用来治疗MERS。
以上所述仅为本发明的较佳实施例，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、改进等，均应包含在本发明的保护范围之内。
Claims (8)
1.一种可结合MERS-CoV的多肽，其特征在于，包括3个互补决定区CDR1-3，序列如SEQID NO:1-3所示。
2.根据权利要求1所述的多肽，其特征在于，所述多肽为纳米抗体。
3.根据权利要求3所述的多肽，其特征在于，还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3按顺序交错排列。
4.根据权利要求3所述的多肽，其特征在于，所述多肽为骆驼源的VHH或人源化的VHH。
5.权利要求1-4中任一项所述的多肽在制备MERS-CoV的检测剂或MERS-CoV的S蛋白的检测剂中的应用。
6.权利要求1-4中任一项所述的多肽在制备MERS-CoV感染的治疗药物中的应用。
7.一种核酸，其特征在于，编码权利要求1-4中任一项所述的多肽。
8.权利要求7所述的核酸在制备MERS-CoV感染的治疗药物中的应用。
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