CN114634565B - 一种抗裂谷热病毒的单克隆抗体e44及应用 - Google Patents
一种抗裂谷热病毒的单克隆抗体e44及应用 Download PDFInfo
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Abstract
本发明公开了一种抗裂谷热病毒的单克隆抗体，所述抗体通过流式分选‑单细胞PCR技术筛选获得，具有独特的CDR分区，本发明还公开了所述抗体在制备裂谷热治疗药物中的应用。本发明公开的单克隆抗体具有高效、特异的抗裂谷热病毒活性，还具有高表达、人源化程度高、稳定性好的特点，适合产业化生产。
Description
技术领域
本发明公开了一种抗体，属于微生物学和免疫学领域。
背景技术
裂谷热是由裂谷热病毒引起的一种人兽共患蚊媒传染病，因最早分离自东非大裂谷而得名。主要在非洲区域流行，亚洲地区有输入性病例报告。2016年，我国发现了首例输入性裂谷热病例。裂谷热跨区域传播的潜在风险，也对我国的生物安全提出了挑战。WHO分别于2015年、2016年和2018年发布当前最需研究的病毒清单，裂谷热病毒位列其中。1997年7月，生物武器缔约国政府专家特设小组第7次会议将裂谷热与埃博拉、马尔堡等一起列入攻击人的病毒类战剂。裂谷热临床症状为发热、头痛和肌肉关节痛，重症病例可表现为多脏器受累，病死率高。2020年9月毛里塔尼亚暴发裂谷热疫情，报告确诊病例75例，死亡25例，病死率33.3%。裂谷热的治疗以对症支持治疗为主，目前没有有效的治疗手段。相较于其他类型药物，单克隆抗体药物具有靶标明确，作用特异，起效迅速，副作用较小等优点，因而单抗成为抗病毒领域的研究热点。研制特异性单抗治疗药物对于裂谷热病毒相关的疫情防控、防生、反生物恐怖均具有重要意义。
截止2021年2月，FDA许可的100种抗体新药中有6个抗感染单抗新药，可以说抗感染单抗占有一席之地，其中包括针对炭疽的单抗2个，针对埃博拉的单抗2个。 随着抗体研发技术的不断进步和全球对突发公共卫生事件关注，获批抗感染抗体新药加速增加。新冠大流行更是将抗感染抗体新药研发推向新的高度，特别是变异株的不断出现，吸引越来越多的关注。美国再生元和VIR公司在抗埃博拉和SARS-COV-2病毒抗体研发上均有抗埃博拉单抗获批上市，新冠单抗获EUA许可。我国目前没有抗感染单抗获批上市。
目前，国内外无裂谷热疫苗和中和单抗获批上市。美国陆军传染病研究所研制的减毒疫苗MP-12完成II期临床。裂谷热中和单抗研发处于实验室研究阶段，且主要针对于裂谷热病毒表面糖蛋白（Gn和Gc蛋白）进行研发。其中，Gn蛋白主要介导病毒的识别宿主细胞的受体，Gc蛋白主要介导病毒与宿主细胞的膜融合。2018年英国牛津大学Thomas A.Bowden等用流式分选获得靶向Gn蛋白具有中和活性的兔单克隆抗体，并在小鼠模型上验证了单抗的保护活性。德国科学家2020年报道筛选获得2株靶向Gn蛋白的鼠单抗Gn3和Gn32，如果使用Gn3单抗进行小鼠体内保护实验，保护率58%，使用Gn3和Gn32的“鸡尾酒”疗法获得完全保护，提示联用具有更好的治疗前景。高福、严景华团队在一例RVFV感染的康复病人体内率先分离到高效中和RVFV感染的单克隆抗体。该抗体在小鼠模型上能有效治疗RVFV感染，有望成为治疗其感染的候选药物。
本发明的目的就是提供一种具有高中和活性的单抗，并进一步提供制备裂谷热治疗药物中的应用。
发明内容
基于上述发明目的，本发明首先构建了腺病毒载体的裂谷热候选疫苗，通过免疫恒河猴，流式分选-单细胞PCR技术筛选到了一种抗裂谷热的单克隆抗体，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:1第26-34、52-59、98-108位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:5第26-33、51-53、90-100位氨基酸序列所示。
在一个优选的技术方案中，所述抗体的重链可变区的氨基酸序如SEQ ID NO:1所示，所述轻链可变区的氨基酸序列如SEQ ID NO:5所示。在本发明中，具有所述重链和轻链可变区的一个具体的抗体被命名为“E44”。
在一个更为优选的技术方案中，所述抗体的重链恒定区的氨基酸序如SEQ ID NO:3所示，所述轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9所示，其中，SEQ IDNO:7为κ链恒定区的序列，SEQ ID NO:9为λ链恒定区的序列，所述抗体的重链恒定区和轻链恒定区为人源。
第二，本发明还提供了一种编码上述单克隆抗体重链和轻链的碱基编码序列，所述抗体的重链可变区的碱基编码序列由SEQ ID NO:2所示，所述抗体的轻链可变区的碱基编码序列由SEQ ID NO:6所示。
在一个优选的技术方案中，所述抗体的重链恒定区的碱基编码序列由SEQ ID NO:4所示，所述抗体的轻链恒定区的碱基编码序列由SEQ ID NO:8或者SEQ ID NO:10所示。
第三，本发明还提供了一种表达上述编码单克隆抗体重链和轻链的核苷酸编码序列的功能元件，这种功能元件可以是传统的表达载体。
在一个优选的技术方案中，所述功能元件为线性表达框。
第四，本发明还提供了一种含有上述线性表达框的宿主细胞。
在一个优选的技术方案中，所述细胞为Expi 293F细胞或CHO-S细胞。
最后，本发明还提供了上述单克隆抗体在制备裂谷热治疗药物中的应用。
本发明提供的单克隆抗体显示出对裂谷热病毒感染细胞良好的中和保护效果。本发明的研究结果显示，所述抗体在制备裂谷热治疗药物中具有广泛应用的前景。本发明公开的单克隆抗体还具有以下的技术优势:（1）高结合活性，重组单抗与Gn蛋白KD 2.58nM；（2）高中和活性 在细胞模型上IC50为0.38nM；在致死小鼠模型上，200 ug/只可提供完全保护，20 ug/可提供60%的保护率。（3）稳定性好 因为抗体基因来自恒河猴的同一个细胞，是天然配对的。
附图说明
图1. 免疫后不同时间点恒河猴体内针对Gn和Gc蛋白IgG抗体效价；
图2. Gn和Gc蛋白特异的记忆B细胞的分选策略；
图3. 重链二轮巢式PCR扩增产物核酸电泳检测；
图4. 轻链二轮巢式PCR扩增产物核酸电泳检测；
图5. 重链和轻链可变区扩增产物核酸电泳检测；
图6. 前导序列和恒定区-多聚A尾的扩增；
图7. 重链和轻链线性表达框的核酸电泳检测；
图8. 线性表达框转染表达抗体结合活性检测；
图9. 表达载体pAb-H和pAb-λ双酶切线性化核酸电泳检测；
图10. 表达载体的双酶切鉴定核酸电泳检测；
图11. 亲和层析纯化后的单抗SDS-PAGE检测；
图12. 单抗与Gn蛋白的结合活性检测；
图13. 单抗与Gn蛋白的亲和力检测；
图14. 单抗在细胞模型上的IC50测定；
图15. 单抗对感染RVFV小鼠治疗后生存情况的监测；
图16. 单抗对感染RVFV小鼠治疗后体重变化的监测；
图17. 感染RVFV小鼠在单抗治疗后肝脏中病毒基因拷贝数检测；
图18. 感染RVFV小鼠在单抗治疗后脾脏中病毒基因拷贝数检测；
图19. 单抗对RVFV病毒粒子与Vero细胞结合的阻断作用。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明权利要求所限定的范围构成进一步的限定。
实施例1抗裂谷热单克隆抗体的筛选和制备
1.1表达裂谷热病毒GnGc蛋白的人5型重组腺病毒（HAdV5-GnGcopt）的包装
将重组GnGc蛋白编码基因（GenBank: DQ380208.1）克隆至表达载体pDC316后，与AdMax腺病毒系统的骨架质粒（pBHGlox_E1，Cre）共转染至HEK293细胞中，待细胞病变明显时，收取细胞上清，接种于293F细胞进行扩繁后，测定病毒滴度储存于-80℃备用（构建方法参见CN105483140A）。
1.2恒河猴免疫
首先在免疫前通过静脉采集恒河猴血液2 mL并分离血清做阴性血清对照，同时将1×108 IFUs的HAdV5-GnGcopt以肌肉注射的方式免疫恒河猴。在免疫后第28天以同样方式和同样剂量的HAdV5-GnGcopt再次免疫恒河猴，并在免前采集血液2 mL。在首免后的第56天和182天将0.25 mg Gn蛋白和0.25 mg Gc蛋白的混合物与0.5 mg铝佐剂充分混匀后，以肌肉注射的方式对恒河猴进行加强免疫，并在免前采集血液2mL。
1.3测定血清中Gn和Gc蛋白结合抗体的效价
取纯化的Gn和Gc蛋白（GenBank: DQ380208.1）包被酶联板（2 μg/mL，100 μL/孔）4℃过夜，PBST洗涤3次后，用2% BSA于37℃封闭1h后；洗涤3次。首孔用PBST将待测血清以1:100的比例稀释，然后以1:3的比例进行梯度稀释，然后置于37℃孵育1h；洗涤三次后加入HRP标记的羊抗人二抗，37 ℃孵育1 h；洗涤3次后，加入100 μL TMB单组份显色液，室温显色5 min，再加入50 μL终止液，最后用酶标仪读取OD450nm-OD630nm值。最后，使用GraphPadPrism软件计算针对Gn和Gc蛋白结合抗体的效价。
在对恒河猴完成HAdV5-GnGcopt的首次免疫后的28天内，恒河猴体内针对Gn和Gc蛋白的IgG结合抗体的水平有明显提升。在完成HAdV5-GnGcopt二免后的28天内（首免后第56天），恒河猴体内针对Gn和Gc蛋白的IgG结合抗体的水平有了进一步提升。在首免后第56天时，用Gn和Gc蛋白的混合物对该恒河猴进行加强免疫。两周后，恒河猴体内针对Gn和Gc蛋白IgG结合抗体水平达到顶峰，并在随后四周之内缓慢下降，并保持平稳。在首免后第182天，用Gn和Gc蛋白的混合物对该恒河猴进行再一次加强免疫，在随后四周内，恒河猴体内针对Gn和Gc蛋白IgG结合抗体水平再一次达到顶峰，且比首免后第70天的峰值有了进一步的提高。在整个免疫过程中，恒河猴体内针对Gn蛋白的抗体水平整体都略低于针对Gc蛋白的抗体水平（见图1）。
1.4 恒河猴外周血单核淋巴细胞分离
从恒河猴前肢采集抗凝血10 mL，用10 mL PBS将全血进行等体积稀释。然后在50mL离心管中加入10 mL的分离液，将稀释后的全血缓慢平铺至分离液上方，保证二者液面分界清晰。将离心管缓慢放置于水平离心机中，并将离心机加减速度设置为最低档，然后在室温中800 g离心30 min。离心结束后，血浆位于最上层，分离液位于中间层，红细胞则位于管底。单核细胞层位于血浆层与分离液层之间，将单核细胞小心吸取至新的50 mL离心管中。然后加入10 mL PBS，轻轻混匀清洗单核细胞后，将离心管放置于水平离心机中300 g，离心10 min后弃上清。然后再重复清洗一次。用细胞冻存液（10% DMSO和90% FBS）将离心至管底的单核细胞重悬后，保存于-80 ℃备用。
1.5 Gn和Gc蛋白特异性记忆B细胞的染色
将上述冻存的PBMCs置于37 ℃水浴锅中，迅速解冻复苏。然后在室温中500 g离心5 min。弃上清后，用5 mL FPBS重悬后将细胞转移至流式管中，500 g离心5min，弃上清。重复清洗一次。弃掉上清后，用500 μL FPBS（2% FBS溶于PBS）重悬后，对细胞进行计数。具体染色过程如下：
为了调节分选过程中各个荧光通道的补偿，首先对细胞进行单荧光染色，取5个流式细胞管，每管加入1×105个细胞，然后根据表1中提供的所需染料量，分别将相应体积的染料加入至5个流式细胞管，其中2个流式细胞管分别加入带有Strep标签的Gn和Gc蛋白各2μg，用FPBS将细胞补足至100 μL，并轻轻混匀后，置于4 ℃避光孵育30min。然后每管加入3mL FPBS轻轻重悬清洗细胞后，500 g离心5 min，弃上清，重复清洗2次。用100 μL FPBS重悬细胞后，加入1 μL APC-anti Strep-tag荧光抗体轻轻混匀后，置于4 ℃避光孵育30 min，清洗三次后，用1mL FPBS重悬备用。
表1.PBMCs染色所需染料体积
为了进行单细胞流式分选，首先对细胞进行多荧光染色，取1×105个细胞加入至一个细胞流式管中，然后根据表1中提供的所需染料量，将包括Gn和Gc蛋白在内的5种染料加入至流式细胞管中轻轻混匀后，置于4 ℃避光孵育30 min。然后加入3 mL FPBS轻轻重悬清洗细胞后，500 g离心5 min，弃上清，重复清洗2次。用100 μL FPBS重悬细胞后，加入2 μLAPC-anti Strep-tag荧光抗体轻轻混匀后，置于4 ℃避光孵育30 min，清洗三次后，用1 mLFPBS重悬备用。
从对恒河猴体内Gn和Gc蛋白结合抗体水平的持续监测中，可以看到在四免第四周时，恒河猴体内针对Gn和Gc蛋白的抗体水平达到了最高点。因此，我们选定四免第四周的血液样本取进行单细胞分选。在用流式分选单个记忆B细胞之前，我们首先利用密度梯度离心法将PBMCs从恒河猴外周血中分离出来。然后利用Beckman MoFlo-XDP单细胞流式分选仪对单个Gn和Gc蛋白特异的记忆B细胞进行分选。在分选中，我们首先通过前向角散射光（forward scatter，FSC）和侧向角散射光（side scatter，SSC）将淋巴细胞、单核细胞及细胞碎片分离（图2，R1）。其中，淋巴细胞占总细胞数量为27%。由于T细胞表面表达CD3分子，而B细胞表面不表达CD3分子，在R1的基础上，我们通过CD3分子将B细胞及T细胞区分开。同时由于记忆B细胞可以表达CD19分子，我们通过CD19分子进一步圈定了记忆B细胞（图2，R2），且这部分细胞占总细胞的8.04%。由于记忆B细胞表面还可以表达非特异的表面IgG和特异抗原的表面IgG，因此，我们最终圈定了Gn和Gc蛋白特异的记忆B细胞（图2，R3），且这部分细胞约占分选前总细胞数的0.2%。最终分选获得1253个记忆B细胞。
1.6 流式分选Gn和Gc蛋白特异性记忆B细胞
将细胞分选到96孔PCR板前，在每个孔中加入20 μL去RNA酶蒸馏水和20 UnitsRNA酶抑制剂的混合液，并置于4 ℃备用。
进行单个Gn和Gc蛋白特异性记忆B细胞分选。在上样前，需将所有的细胞样品用300目细胞筛进行过滤以去除细胞聚集形成的团块。然后使用上述制备的5个单荧光染色的细胞进行各个荧光染料之间的补偿调节。完成调节补偿后，开始上样多种染料染色的细胞。首先，通过FSC-H和SSC-H圈选出淋巴细胞，并设定为门1。以门1圈定的细胞为基础，通过Alex Flour700和PerCP圈选出CD19+/CD3- 的细胞，并设定为门2。以门2圈定的细胞为基础，通过APC和PE圈选出Gn+/Gc+/IgG+的细胞，并设定为门3。门3中的细胞群（CD19+/CD3-/IgG+/Gn+/Gc+）为Gn和Gc蛋白特异的记忆B细胞。然后将流式细胞的分选仪的分选模式设置为“单细胞模式”。将门3中的细胞以每孔一个细胞的方式分选至上述96孔PCR板。将分选后的96孔PCR板用封板膜封闭后，立即置于液氮中冷冻，并保存于-80℃备用。
1.7单细胞PCR扩增抗体可变区基因
单细胞反转录PCR：
利用反转录试剂盒SUPERSCRIPT III将上述分选所得单个记忆B细胞进行反转录PCR反应。反应体系如表2：
表2. 单细胞反转录PCR反应体系
反应程序如表3：
表3.单细胞反转录PCR反应程序
单细胞PCR扩增及轻重链配对抗体基因的筛选：
（1）第一轮PCR扩增
将表2中的多条H链引物溶于ddH2O后，然后以相同摩尔量均匀混合成引物Mix。将κ链引物和λ链引物以同样的方式分别混合成引物Mix。然后以反转录PCR产物为模板，以表4中的引物Mix作为第一轮PCR引物，利用TranStart TaqDNA聚合酶对抗体基因进行第一轮PCR扩增。
反应体系如表4：
表4. 第一轮PCR扩增反应体系
反应程序如表5：
表5. 第一轮PCR扩增反应程序
（2）第二轮PCR扩增
首先，将表6中的多条H链引物溶于ddH2O后，然后以相同摩尔量均匀混合成引物Mix。将κ链引物和λ链引物以同样的方式分别混合成引物Mix。然后以第一轮PCR产物为模板，以表6中的引物Mix作为第二轮PCR引物，利用TranStart TaqDNA聚合酶对抗体基因进行第二轮PCR扩增。反应体系及反应程序同第一轮PCR。
表6. 第一轮PCR引物
表7. 第二轮PCR引物
（3）轻、重链配对抗体基因的筛选
将经过两轮PCR扩增的产物用QIAxcel DNA Fast Analysis Kit 进行毛细管核酸电泳。将轻链和重链均为阳性的单细胞克隆记作配对成功的阳性克隆，并将其对应的PCR产物挑选出来存于-20℃。抗体E44重链二轮巢式PCR扩增条带大小与预期结果相符，结果见图3，F5泳道；轻链二轮巢式PCR扩增条带大小也与预期结果相符，结果见图4，E9泳道。
轻重链配对抗体可变区基因的PCR扩增：
首先，将表9中的多条H链可变扩增引物溶于ddH2O后，然后以相同摩尔量均匀混合成引物Mix。将κ链引物和λ链引物以同样的方式分别混合成引物Mix。然后，以上述轻重链配对成功的PCR产物为模板，以表9中的引物Mix为引物，利用TranStart TaqDNA聚合酶对抗体的可变区基因进行PCR扩增。反应体系同巢式PCR。反应程序如表8所示：
表8. 可变区基因的PCR扩增反应程序
表9.可变区扩增引物
为了能从上述分选所得的Gn特异的记忆B细胞中扩增出抗体基因，我们首先通过反转录试剂盒将282个记忆B细胞中的抗体基因反转录成为cDNA。由于单个记忆B细胞中抗体基因的转录本的量较少，因此，我们利用恒河猴抗体基因的特异引物通过两轮的PCR将记忆B细胞中抗体的基因扩增放大，并进行轻重链配对抗体的筛选，共有204株抗体轻重链配对成功，其中轻链为κ型的抗体有122株，轻链为λ型的抗体有82株，总配对成功率约为72.3%。为了将筛选出来的恒河猴源单克隆抗体轻重链的可变区克隆至人源IgG轻重链的恒定区，我们将轻重链配对成功的单细胞巢式第二轮PCR产物挑选出来，并以此为模板，利用特异的抗体引物对这些抗体的可变区基因进行扩增，结果显示抗体重链和轻链可变区大小与预期相符，且扩增效率为100%。E44重链可变区PCR扩增条带大小（约450bp）与预期结果相符，结果见附图5泳道1，轻链可变区PCR扩增条带大小（约450bp）也与预期结果相符结果见附图5泳道10。
1.8单克隆抗体基因线性表达框的构建
利用重叠延伸PCR将抗体的可变区基因克隆至包含启动子及信号肽的前导序列与包含抗体恒定区与终止子的恒定区-多聚A尾片段之间。
前导序列的扩增：
以质粒pCDNA-H为模板，通过上下游引物CMV-UP/3'Leader H扩增抗体重链基因的前导序列；以pCDNA-λ为模板，通过上下游引物CMV-UP/3'Leader L扩增抗体轻链基因的前导序列，引物序列见表10。反应体系与反应程序同可变区扩增PCR。
表10.前导序列扩增引物
恒定区-多聚A尾片段的扩增
以质粒pCDNA-H为模板，通过上下游引物5'CH/TK-POLYA扩增抗体重链基因的恒定区-多聚A尾片段；以pCDNA-κ为模板，通过上下游引物5'Cκ / TK-POLYA扩增抗体κ链基因的恒定区-多聚A尾片段；以pCDNA-λ为模板，通过上下游引物5'Cλ / TK-POLYA扩增抗体λ链基因的恒定区-多聚A尾片段，引物序列见表11。反应体系与反应程序同可变区扩增PCR。
表11.恒定区-多聚A尾片段扩增引物
抗体基因线性表达框的扩增
以上述扩增后的前导序列，抗体轻重链的可变区基因和恒定区-多聚A尾片段为模板，以CMV-UP/TK-POLYA为上下游引物，利用重叠延伸PCR分别扩增出包含抗体H，κ和λ链全长基因的线性表达框。反应程序同可变区扩增PCR。反应体系如表12所示：
表12. 抗体基因线性表达框的扩增反应体系
为了将上述扩增后的抗体轻重链可变区克隆至人源IgG轻重链的恒定区以及后续能够快速筛选Gn和Gc蛋白的结合抗体，我们通过重叠延伸PCR将抗体轻重链的可变区基因克隆至包含CMV启动子及信号肽的前导序列与包含抗体恒定区与终止子的恒定区-多聚A尾片段之间从而生成包含抗体轻重链全长基因的线性表达框。首先，我们成功扩增了抗体H链，κ链和λ链所对应的前导序列及恒定区-多聚A尾片段。其中，重和轻链的前导序列大小均为750 bp（图6泳道1和泳道2），重链恒定区-多聚A尾片段大小为1900 bp（图6泳道3），κ链和λ链恒定区-多聚A尾片段大小均为为1350 bp（图6泳道4和泳道5）。然后，我们通过重叠延伸PCR成功扩增了E44抗体轻重链的线性表达框。其中，重链的线性表达框大小约为3200 bp（图7泳道1），轻链的线性表达框大小约为2500 bp（图7泳道10）。
1.9单克隆抗体基因线性表达框的回收与纯化
为了能够高通量完成上述扩增完成的抗体基因线性表达框PCR产物的纯化，我们利用天根公司的N96 DNA产物纯化试剂盒进行胶回收。具体步骤如下：
1）96孔吸附板的平衡：将96孔吸附板CB2放置于96孔深孔板上，每孔加入500 μL平衡液BL，置于水平离心集中，室温3500 rpm离心4 min，弃掉深孔板中废液，将吸附板重新放置于深孔板。
2）将线性表达框PCR产物与结合液PB按1:3的比例均匀混合，室温静置3 min。
3）将混合液转移至平衡后的吸附板中，室温3500 rpm离心6 min，弃掉深孔板中废液，将吸附板重新放置于深孔板。
4）向离心后的吸附板中加入700 μL的漂洗液PW，室温3500 rpm离心5 min，弃掉深孔板中废液，将吸附板重新放置于深孔板。重复漂洗一次。
5）将漂洗后的吸附板于室温3500 rpm离心10 min，然后将吸附室温静置5 min，将残余的漂洗液去除。
6）将吸附板放置于新的深孔板中，向吸附柱中的孔膜中央滴加80 μL ddH2O，室温静置5 min后，3500 rpm离心15 min，收集纯化后的线性表达框PCR产物，并用紫外分光光度计测定浓度后，存于-20 ℃备用。
1.10 配对抗体基因线性表达框的共转染293T细胞
在转染前24 h，将293T细胞以4×104个/孔的密度铺于96孔细胞板中，置于37 ℃、5% CO2的细胞培养箱中培养过夜。然后，将配对的轻重链线性表达框各以0.1μg/孔的量加入20 μL Opti-MEM培养基中，并轻轻混匀。再将0.4 μL/孔TurboFect转染试剂加入稀释后的线性表达框中，室温孵育18min。然后利用排枪将孵育后的混合液轻轻加入96孔细胞板中，并置于37 ℃、5% CO2的细胞培养箱中培养48 h。
1.11 ELISA快速筛选针对Gn蛋白的结合抗体
取纯化的截短型Gn蛋白包被于ELISA板（2 μg/mL，100 μL/孔）4 ℃过夜，PBST洗涤3次后，用2% BSA于37 ℃封闭1 h后；PBST洗涤3次。每孔加入上述293T细胞表达上清50 μL，用稀释液补齐至100 μL，置于37 ℃孵育1 h后，PBST洗涤三次。每孔加入100 μLHRP标记的羊抗人IgG二抗（1:10000稀释），置于37 ℃孵育1 h后，PBST洗涤三次。每孔加入100 μL TMB单组份显色液，室温显色5 min，再加入50 μL终止液，最后用酶标仪读取OD450nm-OD630nm值。
为了快速筛选针对Gn蛋白的结合抗体，我们将上述204株扩增成功的抗体轻重链线性表达框共转染293T细胞，培养48h后，收取上清。然后通过ELISA检测上清中是否含有针对Gn蛋白的结合抗体筛选到针对Gn蛋白的结合抗体有47株，其中轻链为κ型的抗体有25株，轻链为λ型的抗体有22株。针对Gn蛋白的总结合抗体的阳性率为23.03%。E44结合的OD值为2.3495，结果见图8。
1.12 Gn蛋白结合抗体真核表达质粒的构建
为了提高Gn蛋白结合抗体的表达量，将阳性抗体轻重链的可变区基因通过同源重组的方式分别克隆至抗体重链和轻链真核表达质粒pAb-H和pAb-λ（构建方法见CN114480501A）。
抗体重链和轻链真核表达质粒pAb-H和pAb-λ的双酶切线性化及回收纯化
使用限制性内切酶Hpa I和Srf I对重链真核表达质粒pAb-H进行双酶切，使其线性化；使用限制性内切酶Kpn I和Pml I对轻链真核表达质粒pAb-λ进行双酶切，使其线性化。酶切体系如表13：
表13. 抗体表达质粒酶切反应体系
37 ℃酶切2 h，结果见图9，泳道1和2分别为酶切前和后的pAb-H；泳道3和4分别为酶切前和后的pAb-λ；然后进行胶回收纯化，并置于-20 ℃备用。
连接，转化及阳性克隆筛选：
目的片段与载体的连接是使用NEBuilder组装试剂盒，按说明书进行操作，连接目的片段。反应体系如表14所示。
表14. NEBuilder组装体系
然后轻微混匀，50 ℃连接15 min，随后将体系置于冰上。取2 μL连接产物加入Top10感受态中，冰浴30 min，42 ℃热激90 s，冰浴3 min 后，使用无抗性LB培养基，振荡培养60分钟，8000 rpm离心1分钟，取沉淀重悬液100 μL，涂板，37℃温箱孵育过夜。用无菌的枪头挑取生长状态良好的单克隆菌落到装有600 μL Amp/LB液体培养基的1.5 mL无菌离心管中，置于37 ℃摇床培养12 h。最后挑取4个单克隆菌液进行测序验证。
E44真核表达质粒双酶切鉴定
使用限制性内切酶EcoR I和BamH I对E44轻重链表达质粒进行双酶切鉴定。酶切体系如表15。
表15. E44轻重链表达质粒进行双酶切体系
37 ℃酶切2 h后，进行电泳，鉴定结果见图10，泳道1为E44抗体重链质粒双酶切结果，泳道2为E44抗体轻链质粒双酶切结果。
1.13 Gn蛋白结合抗体真核表达与纯化
将测序正确且配对的轻重链质粒按照说明书共转染至Expi293F细胞中。收取连续培养108 h后的细胞上清，首先以4 ℃，1500 g离心15 min，然后再以3000 g离心15 min，将上清转移至新的离心管中，最后再以12000 rpm高速离心10 min后，将高速离心后的上清转移至新的离心管中，置于4 ℃备用。
为了将Expi293F细胞表达的抗体从细胞培养基中分离出来，我们通过HiTraprProteinA亲和柱对抗体进行了纯化，步骤如下：
1）首先，将HiTrap rProteinA亲和柱以低流速接于蛋白纯化仪AKTA pure中，然后走5个柱体积的纯水将rProteinA亲和柱中20%的乙醇冲洗干净，再走平衡缓冲液（PBS），直至UV走平后将UV设置调零。
2）将上述高速离心后的细胞上清以正常流速上样，并收集柱后，上样完成后，继续用PBS平衡直至UV走平。
3）用pH 2.7的0.1 M甘氨酸洗脱亲和柱中抗体，并收集洗脱峰。并用pH 9.0的Tris-HCl溶液将洗脱液中和至pH 7.0。然后通过SDS-PAGE对纯化后抗体还原与非还原的状态进行分析，E44抗体表达量较高且纯度较好，结果见图11，泳道1为非还原状态下纯化后的E44抗体，泳道2为还原状态下纯化后的E44抗体。
4）将纯化成功的抗体通过50kDa的超滤管进行浓缩换液至PBS中，离心条件为4℃，3000 g离心30 min。最后用BCA试剂盒进行浓度测定，30mL悬浮细胞可获得抗体约为2.5mg。
1.14 ELISA测定纯化后Gn蛋白单克隆抗体的结合活性
取纯化的截短型Gn蛋白包被于ELISA板（2 μg/mL，100 μL/孔）4 ℃过夜，PBST洗涤3次后，用2% BSA于37 ℃封闭1 h后；PBST洗涤3次。首孔加150 μL浓度为10 μg/mL的抗体，然后进行三倍梯度稀释，共稀释12个稀释度，然后置于37 ℃孵育1 h后，PBST洗涤三次。每孔加入100 μL HRP标记的羊抗人IgG二抗（1:10000稀释），置于37 ℃孵育1 h后，PBST洗涤三次。每孔加入100 μL TMB单组份显色液，室温显色5 min，再加入50 μL终止液，最后用酶标仪读取OD450nm-OD630nm值。最后利用Graphpad Prism软件对每个单克隆抗体的结合曲线进行四参数，E44与Gn蛋白结合活性好，EC50为2.659ng/mL，结果见图12。
1.15. 单克隆抗体E44的序列测定结果如下：
所述单克隆抗体的氨基酸序如SEQ ID NO:1所示，其中，。重链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:1第26-34、52-59、98-108位氨基酸序列所示；所述抗体重链可变区的核苷酸编码序列如SEQ ID NO:2所示，重链恒定区的氨基酸序如SEQ IDNO:3所示，重链恒定区的的核苷酸编码序列如SEQ ID NO:4所示。所述重链恒定区为人源。
轻链可变区的氨基酸序列如SEQ ID NO:5所示，其中，轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:5第26-33、51-53、90-100位氨基酸序列所示，所述抗体轻链可变区的核苷酸编码序列如SEQ ID NO:6所示。本发明所制备的抗体的轻链恒定区可选择地为κ链恒定区或λ链恒定区，其中，κ链恒定区的氨基酸序列如SEQ ID NO:7所示，核苷酸编码序列如SEQ ID NO:8所示，λ链恒定区的序列如SEQ ID NO:9所示，核苷酸编码序列如SEQ ID NO:10所示。所述轻链恒定区均为人源。
本领域技术人员能够明确，单克隆抗体的可变区是决定抗原抗体特异性结合和免疫反应的关键部位，不论E44轻链恒定区是κ链或者λ链都可以实现本发明的以下药效学实验。
实施例2.药效学研究
2.1中和抗体亲和力的测定
通过Biacore T200仪器利用Protein A芯片检测E44与Gn蛋白亲和力。首先将抗体稀释至0.5 μg/mL，以10μL/min的流速通过Protein A芯片对抗体进行捕获。然后将Gn蛋白稀释至320nM，160nM，80nM，40nM和20nM，并以30μL/min的流速通过已捕获E44的Protein A芯片并结合120s测定结合速率（Ka），然后解离600s测定解离速率（Kd）。最后通过Ka和Kd的比值来计算待测抗体的亲和力（KD）。最后测得E44与Gn蛋白的亲和力为2..58nM，结果见图13及表16，从图表中可以看出E44与Gn蛋白的结合速度较快且解离速度较慢，因此E44与Gn蛋白的亲和力较好。
表16. E44抗体与Gn蛋白亲和力分析
2.2 中和抗体的中和活性测定
将单克隆抗体E44在96孔细胞板中进行三倍梯度稀释，共稀释10个梯度，每个稀释度做3个重复。然后每孔加入等体积的表达eGFP的RVFV（100 TCID50/well），并置于37 ℃孵育60 min。将Vero E6细胞以2×104/孔的量加入96孔细胞板中，置于37℃，5% CO2细胞培养箱继续培养48 h。将细胞上清弃掉，加入300 μL/well 4%多聚甲醛，室温固定3小时。用PBS清洗三次后，用DAPI对细胞核进行染色，用PBS清洗三次，利用Nexcelom Celigo对RVFV感染的细胞（表达eGFP的细胞）进行计数。单克隆抗体的中和活性通过公式（1-感染细胞数抗体/感染细胞数病毒）×100计算。最后利用软件Graphpad Prism8.0通过四参数计算方法去拟合中和曲线，计算得出E44的IC50为56.37ng/mL（0.38nM），结果见图14。
2.3 致死小鼠模型效力评价
在治疗活性评价实验中，将5-6周龄的IFNR1-/-小鼠随机分为3组，分别为高剂量给药组、低剂量给药组及PBS对照组，每组5只。将RVFV MP-12稀释至2×105TCID50/mL，以腹腔注射的方式对每只小鼠攻毒100μL。攻毒24小时后，以腹腔注射的方式对每只小鼠进行给药。高剂量给药组每只小鼠给药200μg，低剂量给药组每只小鼠给药20μg以及对照组注射等体积PBS。在攻毒后每隔24小时对观察小鼠生存情况及体重变化情况，观察14天。E44高剂量（200μg/只）可以完全保护小鼠且体重变化较小，低剂量（20μg/只）对小鼠的保护率达到了60%，且显著延长了小鼠的生存时间，结果分别见图15和图16。
在观察结束后，将给药组小鼠安乐死后，取出全部实验小鼠的脾脏及肝脏，利用Qaigen RNeasy Mini Kit（货号：74106）试剂提取肝脏及脾脏总RNA，利用绝对荧光定量PCR法测定组织中病毒基因组的拷贝数，引物及探针见表17。相对于PBS对照组，给药组小鼠组织中病毒基因组的拷贝显著降低，肝脏和脾脏中病毒RNA基因组的拷贝数结果分别见图17和图18。
表17. 荧光定量PCR引物及探针序列
2.4抗体中和作用机制检测
将10μg抗体分别与2000TCID50，1000TCID50，500TCID50RVFV-SeGFP等体积混合后，置于37℃孵育1小时。同时，将铺于96孔细胞板的Vero E6细胞置于4℃预冷15分钟后，弃掉上清；将抗体与病毒混合物加入96孔细胞板中，4℃孵育2小时，使病毒充分吸附至细胞表面。弃掉上清，用预冷的PBS清洗细胞3次，洗掉游离的病毒。加入5%FBS DMEM培养基，置于37℃，5%CO2培养箱中培养48小时。弃掉上清，加入5%多聚甲醛室温固定3小时，用PBS清洗3次，用DAPI对细胞核进行染色，用PBS清洗三次，用Nexcelom Celigo对RVFV感染的细胞（表达eGFP的细胞）进行计数。最后，将抗体组以PBS组进行归一化。相对于无关抗体4A8（新冠病毒中和抗体）对照组，E44可以有效阻断病毒吸附至细胞表面，且与阳性对照抗体R15效果相当。结果见图19。图19中，1为抗体E44，2为抗体R15，3为抗体4A8，4为PBS。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
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ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcctccatc tcgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210> 5
<211> 110
<212> PRT
<213> Macaca mulatta
<400> 5
Asn Phe Met Leu Thr Gln Pro Pro Ser Val Ser Ala Asp Gln Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Phe Asn Val Arg Arg Tyr
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Ala Ala Pro Lys Leu Leu
35 40 45
Ile Ser Asp Val Asn Lys Arg Pro Ser Gly Val Ser Asp Arg Phe Ser
50 55 60
Gly Ser Gln Ser Gly Thr Ser Ala Thr Leu Gly Ile Ser Gly Leu Arg
65 70 75 80
Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp Asp Ser Ser Leu
85 90 95
Gly Ala Arg Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu
100 105 110
<210> 6
<211> 330
<212> DNA
<213> Macaca mulatta
<400> 6
aattttatgc tgactcagcc gccctcagtg tctgcggacc aaggacagag ggtcaccatc 60
tcctgctctg gaagcagttt caacgtcagg aggtattatg tgtcctggta ccagcaggtc 120
ccaggagcag cccccaaact cctcatctct gatgttaata agcgaccctc aggggtttct 180
gaccgattct ctggatccca gtctggcacc tcagccaccc tgggcatcag tggactccgg 240
cctgaggatg aggccgacta ttattgctca gtatgggata gcagcctggg tgctcgggtt 300
ttcggcggag ggactcggct gaccgtccta 330
<210> 7
<211> 107
<212> PRT
<213> Homo sapiens
<400> 7
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 8
<211> 321
<212> DNA
<213> Homo sapiens
<400> 8
cggaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaaca ggggagagtg t 321
<210> 9
<211> 106
<212> PRT
<213> Homo sapiens
<400> 9
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 10
<211> 318
<212> DNA
<213> Homo sapiens
<400> 10
ggtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa 60
gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120
gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180
caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag 240
tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300
gcccctacag aatgttca 318
Claims (12)
1.一种抗裂谷热的单克隆抗体，其特征在于，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:1第26-34、52-59、98-108位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列如SEQ ID NO:5第26-33、51-53、90-100位氨基酸序列所示。
2.根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列分别如SEQ ID NO: 5所示。
3.根据权利要求2所述的单克隆抗体，其特征在于，所述单克隆抗体的重链恒定区的氨基酸序列如SEQ ID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9所示。
4.一种编码权利要求1-3任一所述单克隆抗体的重链和轻链的多核苷酸分子，其特征在于，编码所述单克隆抗体的重链可变区的多核苷酸分子的序列由SEQ ID NO:2所示，编码所述单克隆抗体的轻链可变区的多核苷酸分子的序列由SEQ ID NO:6所示。
5.根据权利要求4所述的多核苷酸分子，其特征在于，编码所述单克隆抗体的重链恒定区的多核苷酸分子的序列由SEQ ID NO:4所示，编码所述单克隆抗体的轻链恒定区的多核苷酸分子的序列由SEQ ID NO:8或者SEQ ID NO:10所示。
6.一种表达权利要求5所述编码单克隆抗体的重链和轻链的多核苷酸分子的编码序列的功能元件。
7.根据权利要求6所述的功能元件，其特征在于，所述功能元件为线性表达框。
8.根据权利要求6所述的功能元件，其特征在于，所述功能元件为哺乳动物表达载体。
9.一种含有权利要求7所述功能元件的宿主细胞。
10.根据权利要求9所述的宿主细胞，其特征在于，所述细胞为Expi 293F细胞。
11.根据权利要求9所述的宿主细胞，其特征在于，所述细胞为CHO-S细胞。
12.权利要求1-3任一所述的单克隆抗体在制备裂谷热治疗药物中的应用。
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