CN112094340B - 植物作为宿主在表达新型冠状病毒肺炎中和抗体b38抗体和/或h4抗体中的应用 - Google Patents
植物作为宿主在表达新型冠状病毒肺炎中和抗体b38抗体和/或h4抗体中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,特别涉及植物作为宿主在表达新冠(COVID‑19)B38抗体和/或H4中和抗体中的应用。本发明利用植物例如生菜作为重组蛋白质生产的有效表达平台,利用简单以及有效的农杆菌介导真空渗透方法来表达B38中和抗体以及H4中和抗体。该表达体系确定植物外源蛋白在农杆菌侵染4d后就可以收集。利用SDS‑PAGE法确定重组B38以及H4抗体成功表达。细胞体外实验证明植物生产新冠中和抗体B38以及H4抗体具有阻断新冠病毒COVID‑19S‑蛋白‑RBD与宿主细胞表面ACE2受体结合,从而阻断病毒入侵宿主细胞的生物学活性。
Description
技术领域
本发明涉及生物技术领域,特别涉及植物作为宿主在表达新型冠状病毒肺炎(COVID-19)中和抗体B38抗体和/或H4抗体中的应用。
背景技术
2019年冠状病毒病(COVID-19)疫情给中国和全世界带来严重的危害,在这种肺炎的早期阶段,严重的急性呼吸道感染症状出现了,一些患者迅速发展为急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)、急性呼吸衰竭和其他的严重并发症。有些患者出现严重的肺炎,肺水肿,ARDS或多器官功能衰竭和死亡。
Spike蛋白(含S1和S2两个亚基)是冠状病毒最重要的致病蛋白,其帮助病毒和人体细胞膜上的跨膜受体蛋白质结合,从而帮助自己进入细胞内部。研究表明,新型冠状病毒与SARS病毒的Spike(S)蛋白RBD区域的保守性更高,且通过体外实验证明只要细胞表达ACE2,新型冠状病毒就可以感染细胞;相反,如果细胞上没有ACE2这个蛋白,新型冠状病毒就不会感染。因此,我们有理由相信新型冠状病毒就是通过Spike蛋白RBD区域与ACE2蛋白的结合介导进入细胞内部,ACE2蛋白或将成为研究新型冠状病毒的突破口。
新型冠状病毒(SARS-CoV-2)可能是迄今为止科研人员遇到的最棘手的冠状病毒,它具有与普通流感相等的传染性,但是致死率却远高于普通流感。尽管已经有一些关于新冠肺炎(COVID-19)疫苗的好消息传来,但是距离疫苗投入使用仍有一段较长的时间。在此之前,找到有效疗法是应对COVID-19大流行的关键。
目前,全球多个研究团队正在寻找能够治疗甚至预防COVID-19的方法,其中特异性强的中和抗体被认为是治疗COVID-19的潜在“特效药”。最近的一项研究报告称,两种单克隆抗体B38和H4抗体,可以阻止病毒S蛋白受体结合区域RBD与人体细胞受体ACE2之间的结合。在对每种抗体阻断病毒RBD与ACE2结合的能力进行评估后,研究人员发现仅B38和H4可以阻断二者的结合。于是,研究人员进行了小鼠治疗实验,发现这两种抗体可以有效降低受感染小鼠肺部的病毒滴度,说明B38和H4有望成为预防和治疗新冠病毒的候选抗体。
现阶段有利用动物细胞生产B38以及H4中和抗体。但是动物细胞培养需要价格昂贵的培养液,严格的厂房条件,操作复杂,时间周期至少两周,而且动物细胞生产能力低,造成成本极高。有时候动物细胞所带的病毒可以侵染人类,造成安全性低。
发明内容
有鉴于此,本发明提供了植物作为宿主在表达B38抗体和/或H4抗体中的应用。本发明利用植物尤其是生菜作为重组蛋白生产的高效平台技术,表达了B38和H4抗体。并且在温和的条件下成功分离出有活性的外源蛋白,证明植物尤其是生菜表达平台可以成功用来生产B38和H4抗体蛋白。时间短(4d),纯化简单,生产便捷。消除基因污染,消除感染人体的潜在病虫害等。大大降低生产成本,提高产品安全性。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了植物作为宿主在表达新型冠状病毒肺炎(COVID-19)B38抗体和/或H4抗体中的应用。优选的,所述抗体为中和抗体。
在本发明的一些具体实施方案中,所述植物选自生菜、烟草、白菜、水稻、玉米、大豆或小麦;所述植物的器官选自种子、叶、根茎或整株植物。本发明还提供了一种表达载体,包括以下各项中的任意一项以及载体:
ⅰ.B38的重链序列或轻链序列;
ⅱ.H4的重链序列或轻链序列。
在本发明的一些具体实施方案中,所述B38的重链序列或轻链序列为将B38重链、B38轻链的密码子优化为植物偏好的密码子,获得的优化的B38的重链序列或优化的B38的轻链序列;
所述H4的重链序列或轻链序列为将H4重链、H4轻链的密码子优化为植物偏好的密码子,获得的优化的H4的重链序列或优化的H4的轻链序列。
在本发明的一些具体实施方案中,所述优化的B38的重链序列如SEQ ID No.1所示;所述优化的B38的重链的核苷酸序列如SEQ ID No.2所示;
所述优化的B38的轻链序列如SEQ ID No.3所示;所述优化的B38的轻链的核苷酸序列如SEQ ID No.4所示;
所述优化的H4的重链序列如SEQ ID No.5所示;所述优化的H4的重链的核苷酸序列如SEQ ID No.6所示;
所述优化的H4的轻链序列如SEQ ID No.7所示;所述优化的H4的轻链的核苷酸序列如SEQ ID No.8所示。
B38重链:氨基酸序列如SEQ ID No.1所示;
EVQLVESGGGLVQPGGSLRLSCAASGFIVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRHNSKNTLYLQMNSLRAEDTAVYYCAREAYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
B38重链:核苷酸序列如SEQ ID No.2所示;
B38轻链:氨基酸序列如SEQ ID No.3所示;
B38轻链:核苷酸序列如SEQ ID No.4所示;
H4重链:氨基酸序列如SEQ ID No.5所示;
H4重链:核苷酸序列如SEQ ID No.6所示;
H4轻链:氨基酸序列如SEQ ID No.7所示;
H4轻链:核苷酸序列如SEQ ID No.8所示;
在本发明的一些具体实施方案中,所述载体为双元植物载体。
在本发明的一些具体实施方案中,所述表达载体的构建方法包括如下步骤:
步骤1:分别将B38重链、B38轻链、H4重链、H4轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的B38的重链序列;
ⅱ.优化的B38的轻链序列;
ⅲ.优化的H4的重链序列;
ⅳ.优化的H4的轻链序列;
步骤2:在所述优化的B38的重链序列、优化的B38的轻链序列、优化的H4的重链序列或优化的H4的轻链序列的5’末端以及在3’末端分别加入Xmal限制性酶切位点,克隆到pUC57载体中,分别获得pB38H,pB38L,pH4H或pH4L克隆载体;
步骤3:通过Xmal分别从步骤2所得的克隆载体中获得基因片段,通过同源重组质粒构建法克隆至双元植物载体pCam35S,分别获得表达载体p35S-B38H,p35S-B38L,p35S-H4H或p35S-H4L。
具体的,为了提供外援蛋白在植物中的高效表达,本发明将人B38重链,轻链,H4重链,轻链,蛋白序列反翻译软件将其其密码子优化为植物偏好的密码子,进行基因合成。在优化的B38轻重链序列,以及H4轻重链序列5’末端以及3’末端分别加入Xmal限制性酶切位点。并克隆到克隆载体中(图1A),分别生成pB38H,pB38L,pH4H和pH4L克隆载体。基因片段通过Xma分别从克隆载体中分离(图1B),并由同源重组手段克隆到双元植物载体pCam35S,分别产生植物表达载体p35S-B38H,p35S-B38L,p35S-H4H和p35S-H4L。
本发明还提供了所述的表达载体在表达B38抗体和/或H4抗体中的应用。
此外,本发明还提供了一种植物作为宿主表达B38抗体和/或H4抗体的方法,将本发明提供共的表达载体转化到农杆菌中,通过农杆菌介导真空渗透入植物组织后,提取、分离蛋白质,获得B38抗体和/或H4抗体。
具体的,将四种植物表达载体p35S-B38H,p35S-B38L,p35S-H4H和p35S-H4L分别通过用Multiporator(Eppendorf,Hamburg,Germany)电穿孔转化到根癌土壤杆菌GV3101中。将所得菌株均匀地铺展在含有卡那霉素抗生素(50mg/L)的选择性LB平板上。在黑暗中28℃孵育2d后,挑取单菌落接种到0.5L YEB(酵母提取物肉汤,5g/L蔗糖,5g/L胰蛋白胨,6g/L酵母提取物,0.24g/L MgSO4,pH7.2)并补充抗生素液体培养基(50mg/L卡那霉素)。将接种的培养物在振荡器(220rpm)中以25~28℃孵育72h。通过添加YEB培养基测量OD600值并调节至3.5~4.5。然后收集培养液,离心(4500转速)10min。将农杆菌细胞重悬在渗透培养基(10mM MES,10mM MgSO4)中至O.D.600为0.5。
将制备好的含有p35S-B38H以及p35S-B38L农杆菌等量混匀至O.D.600为0.5;同样将含有p35S-H4H以及p35S-H4L的农杆菌等量混匀至O.D.600为0.5。将培养悬浮液置于2L烧杯,并置于干燥器中。将本实验室保存的生菜倒置(核心向上)并轻轻地旋转于细菌悬浮液中,将干燥器密封。将真空泵(Welch Vacuum,Niles,IL,USA)打开以抽空,并且可见渗透液在叶片组织中。保持压力状态30~60s。快速打开该系统以释放压力,使渗透液渗入组织内的空间。该过程重复2~3次,直到清晰可见渗透液在生菜组织中扩散明显。然后将生菜组织从渗透液中轻轻取出,并用蒸馏水连续冲洗三次,然后转移到塑料膜覆盖的容器中。将处理的样品在黑暗中保持4d。
在本发明的一些具体实施方案中,所述农杆菌介导真空渗透包括如下步骤:
步骤1:抽真空25~45s;
步骤2:保持真空(-95kPa)压力30~60s;
步骤3:释放压力使得渗透液渗入所述植物组织;
重复上述步骤2~3次,避光处理4d。
在本发明的一些具体实施方案中,农杆菌具体为根癌土壤杆菌GV3101。
本发明所述克隆pB38H,pB38L,pH4H和pH4L基因片段(图2A,B),并且构建四种双元植物表达载体p35S-B38H,p35S-B38L,p35S-H4H和p35S-H4L。在完成构建体后,用特异性限制酶消化证实基因片段是完整的。渗透后,绝大多数生菜组织在真空浸润过程中淹没,除了坚固的中肋区域外,其余部分均在真空渗透4天后显示淡黄褐色区域。
提取、分离蛋白质具体为:将经农杆菌真空渗透的生菜样品用搅拌器搅拌,并用体积比为1:1比例的提取缓冲液(100mM KPi,pH7.8;5mM EDTA;10mMβ-巯基乙醇)搅拌机中高速匀浆1~2min。将匀浆物调节至pH8.0,用纱布过滤,过滤物在4℃以10,000g离心15min以除去细胞碎片。收集上清液,与硫酸铵(50%)混合,并在冰上摇动孵育60min。通过离心机(10,000g)在4℃下再次分离15min。将得到的上清液进行第二轮硫酸铵(70%)沉淀,冰上摇动悬浮60min,再次在4℃下以10,000g离心15min。然后,弃去上清液,将处理样品沉淀蛋白质溶于5mL缓冲液(20mM KPi,pH 7.8;2mM EDTA;10mMβ-巯基乙醇)中并在4℃下储存。
SDS-PAGE凝胶电泳具体为:收集从农杆菌真空渗透生菜提取的纯化蛋白质,取样品(5μL)热变性(95℃)加载缓冲液(Biorad,Hercules,CA,USA)在4-12%Bis-TrisPlus SDS-变性凝胶(ThermoFisher Scientific,Waltham,MA,USA)跑电泳。同样,在非变性凝胶电泳中检测抗体的亲和程度。然后用考马斯蓝G250(Biorad)染色后再次对凝胶进行拍照。
植物来源的重组蛋白质的下游加工通常难于并且昂贵,因为纤维素细胞壁难以裂解以及次级植物代谢产物。我们用搅拌机搅拌匀浆,大大节省匀浆成本以及工艺。重组B38抗体以及H4抗体经过变性凝胶SDS-PAGE分离我们在泳道中观察到估计分子量大约分别为23kDa以及48kDa条带(图3A),符合B38以及H4抗体轻重链的蛋白大小。在非变性的凝胶电泳中观察到大约147kDa(图3B)的条带,证明生菜重组轻重链成功的结合为抗体结构,符合B38以及H4的抗体蛋白分子量。基于Bradford测定法和光密度测定对照组测定纯化样品的B38以及H4抗体蛋白含量分别大约为0.59mg/g和0.58mg/g。
最近的一项研究报告称,两种单克隆抗体B38和H4抗体,可以阻止病毒S蛋白受体结合区域RBD与人体细胞受体ACE2之间的结合。本发明利用体外实验对每种抗体阻断病毒RBD与ACE2结合的能力进行评估。将生物素化RBD(0.3μg/mL,Sino Biological Inc.)固定于ELISA板上。标准洗涤后,将0.05μg/mL His标记的hACE2蛋白加入微孔中,然后立即加入稀释的植物源单克隆抗体B38和H4并分别混合。室温孵育2小时后,洗板,加入0.08μg/mL抗His/HRP。室温孵育1h后,以显色剂溶液为底物,用微孔板阅读器测定450nm处的吸光度。与mAbs阴性对照组比较,计算ACE2/RBD结合抑制率。结果表明,B38和H4可以阻断二者的结合。说明B38和H4有望成为预防和治疗新冠病毒的候选抗体。
这些结果表明,通过植物系统瞬间表达的外源B38以及H4抗体具有生物学活性并且可以阻断COVID-19 ACE2/RBD结合。结果表明,植物尤其生菜是一种生产B38以及H4抗体的合适的生物反应器。
本发明利用生菜来瞬时表达B38以及H4抗体,在较短的时间内(4d)可产生高含量的蛋白质。生菜是高等植物,可以进行翻译后修饰过程,即表达的蛋白自动具有活性。而且这种方法最大限度地减少了生物安全问题,因为处理过的生菜组织通常是在完全封闭的设施或容器中开发,不存在生物污染问题。生菜基本不含有植物有毒物质,而且其本身纤维少,利于下游的蛋白纯化。利用生菜系统生产B38以及H4中和抗体,可以大大缩短生产周期和生产成本。
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(A)示克隆载体pB38H,pB38L,pH4H或pH4L示意图;
图1(B)基因片段通过Xma分别从克隆载体中分离;
图2(A)示B38植物双元表达载体p35S-B38H(重链)以及p35S-B38L(轻链)构建流程;利用限制性内切酶Xmal酶切,从图1克隆载体分别切下B38H重链,同源重组连接入pCam35S的Xmal位点,生成植物双元表达载体p35S-B38H;利用限制性内切酶(Xmal)酶切,从图1克隆载体分别切下B38H重链,连接入pCam35S的Xmal位点,生成植物双元表达载体p35S-B38L;
LB andRB:Ti质粒左右边界;35S,具有烟草花叶病毒(TMV)5‘UTR的CaMV 35S启动子;NPT II,用于卡那霉素抗性的编码nptII基因的表达;Nos3’,终止子;
图2(B)示H4植物双元表达载体p35S-H4H(重链)以及p35S-H4L(轻链)构建流程;利用限制性内切酶(XmalI)酶切,从图1克隆载体分别切下H4H以及H4L轻重链片段,连接入pCam35S的Xmal位点,生成植物双元表达载体p35S-H4H以及p35S-H4L;
LB andRB:Ti质粒左右边界;35S,具有烟草花叶病毒(TMV)5‘UTR的CaMV 35S启动子;NPT II,用于卡那霉素抗性的编码nptII基因的表达;Nos3’,终止子;
图3示凝胶电泳结果,其中A示SDS-PAGE凝胶电泳结果;泳道1:B38重组抗体;泳道2:H4重组抗体;B示非变性凝胶电泳结果;泳道3:B38重组抗体;泳道4:H4重组抗体。
具体实施方式
本发明公开了植物作为宿主在表达B38抗体和/或H4抗体中的应用,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。
本发明通过实验发现,植物系统尤其是生菜系统是更加经济、高效的表达平台,是一种快速的瞬时表达重组蛋白质的方法。本发明描述的真空农杆菌渗透方法简单,快速,而且可以提高重组蛋白产量。生菜可以通过承受真空压力而增加蛋白质产量,并允许每片叶子更完整的渗透。由于生菜易于生长并且可商业上大量生产,因此比其他瞬时表达植物,如烟草等更容易获得并且更便宜,并且由于不需要复杂的特殊生产设备,成本可显著降低。综上所述,本发明可以利用生菜系统短时间内大规模生产B38以及H4中和抗体。
本发明提供的植物作为宿主在表达B38抗体和/或H4抗体中的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植物瞬时表达载体的构建
为了提供外援蛋白在植物中的高效表达,将人B38重链,轻链;H4重链,轻链,蛋白序列反翻译软件将其其密码子优化为植物偏好的密码子,进行基因合成。在优化的B38轻重链序列,以及H4轻重链序列5'末端以及3'末端分别加入Xmal位点,基因合成后克隆到pUC57载体中,分别生成pB38H,pB38L,pH4H和pH4L克隆载体(图1A,B)。基因片段通过Xmal分别从克隆载体中分离,利用同源重组法克隆到双元植物载体,pCam35S,分别产生植物表达载体p35S-B38H,p35S-B38L,p35S-H4H和p35S-H4L。将四种植物表达载体分别通过用Multiporator(Eppendorf,Hamburg,Germany)电穿孔转化到根癌土壤杆菌GV3101中。将所得菌株均匀地铺展在含有卡那霉素抗生素(50mg/L)的选择性LB平板上。在黑暗中28℃孵育2d后,挑取单菌落接种到0.5L YEB(酵母提取物肉汤,5g/L蔗糖,5g/L胰蛋白胨,6g/L酵母提取物,0.24g/L MgSO4,pH7.2)并补充抗生素液体培养基(50mg/L卡那霉素)。将接种的培养物在振荡器(220rpm)中以25~28℃孵育72h。通过添加YEB培养基测量OD600值并调节至3.5~4.5。然后收集培养液,离心(4500转速)10min。将农杆菌细胞重悬在渗透培养基(10mMMES,10mM MgSO4)中至O.D.600为0.5。
克隆获得pB38H,pB38L,pH4H和pH4L基因片段(图2A,B),并且构建四种双元植物表达载体p35S-B38H,p35S-B38L,p35S-H4H和p35S-H4L。在完成构建体后,用特异性限制酶消化证实基因片段是完整的。渗透后,绝大多数生菜组织在真空浸润过程中淹没,除了坚固的中肋区域外,其余部分均在真空渗透4天后显示淡黄褐色区域。
实施例2农杆菌介导的真空渗透
将制备好的含有p35S-B38H以及p35S-B38L农杆菌等量混匀至O.D.600为0.5;同样将含有p35S-H4H以及p35S-H4L的农杆菌等量混匀至O.D.600为0.5。将培养悬浮液置于2L烧杯,并置于干燥器中。将本实验室保存的生菜倒置(核心向上)并轻轻地旋转于细菌悬浮液中,将干燥器密封。将真空泵(Welch Vacuum,Niles,IL,USA)打开以抽空,并且可见渗透液在叶片组织中。保持压力状态30-60秒。快速打开该系统以释放压力,使渗透液渗入组织内的空间。该过程重复2至3次,直到清晰可见渗透液在生菜组织中扩散明显。然后将生菜组织从渗透液中轻轻取出,并用蒸馏水连续冲洗三次,然后转移到塑料膜覆盖的容器中。将处理的样品在黑暗中保持4天。
实施例3蛋白质提取和分离
经农杆菌真空渗透的生菜样品用搅拌器搅拌,并用体积比为1:1比例的提取缓冲液(100mM KPi,pH7.8;5mM EDTA;10mMβ-巯基乙醇)搅拌机中高速匀浆1-2分钟。将匀浆物调节至pH 8.0,用纱布过滤,过滤物在4℃以10,000g离心15分钟以除去细胞碎片。收集上清液,与硫酸铵(50%)混合,并在冰上摇动孵育60分钟。通过离心机(10,000g)在4℃下再次分离15分钟。将得到的上清液进行第二轮硫酸铵(70%)沉淀,冰上摇动悬浮60分钟,再次在4℃下以10,000g离心15分钟。然后,弃去上清液,将处理样品沉淀蛋白质溶于5mL缓冲液(20mM KPi,pH 7.8;2mM EDTA;10mMβ-巯基乙醇)中并在4℃下储存。
植物来源的重组蛋白质的下游加工通常难于并且昂贵,因为纤维素细胞壁难以裂解以及次级植物代谢产物。我们用搅拌机搅拌匀浆,大大节省匀浆成本以及工艺。重组B38抗体以及H4抗体经过变性凝胶SDS-PAGE分离我们在泳道中观察到估计分子量大约分别为23kDa以及47kDa条带(图3A),符合B38以及H4抗体轻重链的蛋白大小。在非变性的凝胶电泳中观察到大约145kDa(图3B)的条带,证明生菜重组轻重链成功的结合为抗体结构,符合B38以及H4的抗体蛋白分子量。基于Bradford测定法和光密度测定对照组测定纯化样品的B38以及H4抗体蛋白含量分别为0.59mg/g和0.58mg/g。
实施例4SDS-PAGE凝胶电泳
收集从农杆菌真空渗透生菜提取的纯化蛋白质,取样品(5μL)热变性(95℃)加载缓冲液(Biorad,Hercules,CA,USA)在4~12%Bis-Tris Plus SDS-变性凝胶(ThermoFisher Scientific,Waltham,MA,USA)跑电泳。同样,在非变性凝胶电泳中检测抗体的亲和程度。然后用考马斯蓝G250(Biorad)染色后再次对凝胶进行拍照。
实施例5体外RBD与ACE2结合抑制实验
利用体外实验对每种抗体阻断病毒RBD与ACE2结合的能力进行评估。将生物素化RBD(0.3μg/mL,Sino Biological Inc.)固定于ELISA板上。标准洗涤后,将0.05μg/mL His标记的hACE2蛋白加入微孔中,然后立即加入稀释的植物源单克隆抗体B38和H4并分别混合。室温孵育2小时后,洗板,加入0.08μg/mL抗His/HRP。室温孵育1h后,以显色剂溶液为底物,用微孔板阅读器测定450nm处的吸光度。与mAbs阴性对照组比较,计算ACE2/RBD结合抑制率。结果表明,B38和H4可以阻断二者的结合。说明B38和H4有望成为预防和治疗新冠病毒的候选抗体。这些结果表明,通过生菜系统瞬间表达的外源B38以及H4抗体具有生物学活性并且可以阻断ACE2/RBD结合。生菜系统是一种生产B38以及H4抗体的合适的生物反应器。
实施例6
对照组1:利用动物生产B38
对照组2:利用动物生产H4抗体
实验组A1:本发明提供的植物生产B38抗体;
实验组A2:本发明提供的植物生产H4抗体;
实验组B1:利用烟叶生产B38和H4抗体;
实验组B2:利用烟叶生产H4抗体;
表1 B38以及H4抗体
*示与对照组相比P≤0.05;**示与对照组相比P≤0.01;
#示与实验组A相比P≤0.05;##示与实验组A相比P≤0.01;
由表1可知,与对照组的动物系统相比,本发明提供的生菜瞬时表达B38和H4抗体,极显著(P≤0.01)缩短了生产周期,极显著(P≤0.01)提高了蛋白含量,显著(P≤0.05)提高了蛋白活性,简化了蛋白纯化的难易程度,极显著(P≤0.01)降低了生产成本。
与实验组B的烟叶系统相比,生菜瞬时表达B38和H4抗体,显著(P≤0.05)缩短了生产周期,显著(P≤0.05)提高了蛋白含量,显著(P≤0.05)提高了蛋白活性,简化了蛋白纯化的难易程度,极显著(P≤0.01)降低了生产成本。
实验组B与对照组相比,烟叶瞬时表达B38和H4抗体比动物系统显著(P≤0.05)缩短了生产周期,显著(P≤0.05)提高了蛋白含量,显著(P≤0.05)提高了蛋白活性,简化了蛋白纯化的难易程度,显著(P≤0.05)降低了生产成本。
综合上述试验结果表明,植物系统尤其是生菜系统是更加经济、高效的表达平台。能够快速瞬时表达重组蛋白质,可以在短时间内大规模生产B38以及H4中和抗体。
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
<110> 王跃驹
<120> 植物作为宿主在表达新型冠状病毒肺炎中和抗体B38抗体和/或H4 抗体中的应用
<130> MP2013260
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 445
<212> PRT
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
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Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
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Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
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Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
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Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
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Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
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Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
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Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
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Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
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<210> 2
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gaggttcaat tggtcgaatc cggtggaggt cttgttcaac ctggcggatc cttgcgtctc 60
tcatgcgctg cttctggatt cattgtgtca tcaaattata tgtcttgggt acgtcaggca 120
ccgggaaagg gacttgaatg ggtctctgtc atctactctg gtggttcaac atactatgca 180
gattcagtga agggtcggtt taccataagc agacataatt ctaagaatac tctttattta 240
cagatgaact cattgcgtgc tgaagatact gctgtatact actgtgctcg tgaagcatat 300
ggaatggatg tatggggaca gggtaccact gtgacggtaa gttcagcctc tacaaaaggc 360
ccatctgtct ttcctttggc tcccagtagc aaatctactt ctggtggtac tgccgccctt 420
ggctgtcttg ttaaagatta ttttcctgag cctgtaaccg taagttggaa ttctggtgct 480
ttaacaagcg gcgttcatac gttcccagct gttcttcaaa gttctggttt gtacagcttg 540
tcatccgttg tcactgtgcc ttctagctca cttgggaccc aaacttatat ctgcaatgtg 600
aatcataaac catcaaatac aaaggtggat aagaaggctg aacctaaaag ctgcgataag 660
acgcacacat gcccaccatg tccagcacct gaactcttgg gtggtccgtc agtatttttg 720
ttcccgccaa aaccaaaaga tacccttatg atttcaagaa caccagaggt tacttgtgtg 780
gtcgtggatg tttctcacga agacccagaa gtgaaattta actggtatgt tgatggtgtg 840
gaagttcata atgcaaagac aaagccccga gaagagcagt ataattccac ctatagggtc 900
gtgtctgtat tgactgtgct tcaccaagat tggttgaacg gaaaggaata caagtgcaag 960
gtaagtaata aggcactgcc agcccccatt gaaaaaacga ttagtaaggc taagggtcag 1020
cctagggagc cacaggtata cactttgcca ccttctagag atgaacttac gaagaaccaa 1080
gtttcattaa cctgtttagt caaagggttc tacccaagcg acatcgccgt agaatgggag 1140
tctaatggac aaccggagaa taactacaaa acgacacctc cagttttaga cagcgacggg 1200
agtttctttc tctattccaa gcttaccgta gataagtcta ggtggcaaca ggggaacgtc 1260
ttcagttgta gtgtgatgca cgaagcactg cataaccatt atacacagaa atctttgtca 1320
cttagtcctg gcaag 1335
<210> 3
<211> 215
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 4
<211> 645
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatatagtca tgacacaatc accgtccttt ctttctgcct ccgtaggaga tcgtgtcact 60
attacctgtc gtgcttccca ggggatctct agttacttgg cctggtatca gcagaagcct 120
ggtaaagctc caaaattatt gatttacgcc gctagtacac tccaatcagg tgtgccgtct 180
aggttctccg gtagtggctc aggaaccgaa tttacgctta caatttcctc tctgcaaccc 240
gaagattttg ctacttacta ttgtcaacaa ctgaattcat atcctccata caccttcggt 300
caaggaacta agcttgagat caagcgaaca gttgctgccc ctagtgtctt tatctttcct 360
ccttccgacg aacaacttaa gtctggtaca gcttctgtcg tgtgtctgct gaataatttt 420
tacccacggg aagcaaaggt gcaatggaag gttgataatg cactccaatc cggaaatagc 480
caggaaagcg tgacagaaca agatagtaag gatagcacct attctttatc tagcacactg 540
accttgtcaa aggctgatta tgagaaacat aaagtgtatg cttgtgaagt gacccatcaa 600
ggtttaagta gtccggttac taaatctttt aatcgaggtg agtgt 645
<210> 5
<211> 456
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr
100 105 110
Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala
210 215 220
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
355 360 365
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 6
<211> 1368
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caggtccagc tcgttcaaag cggagctgaa gttaaaaaac ctggagctag cgttaaggtt 60
tcttgtaagg ctagtggata cacattcacc ggttattata tgcactgggt tagacaggct 120
cctggtcagg gtttggagtg gatgggaaga atcaacccaa acagtggagg gaccaattat 180
gctcagaaat ttcaaggccg ggtgactatg actcgggata cgtccatatc aactgcttac 240
atggagctct caagattgag gagtgatgat acagctgtat actattgcgc aagggttcca 300
tattgttcaa gcacgagttg tcatcgagac tggtattttg acctttgggg taggggaacc 360
cttgtcaccg tttcatcagc atccactaag ggaccctctg ttttccctct tgctcctagt 420
agtaagagca ccagtggagg cacagctgct ctgggttgtt tagttaagga ttattttcct 480
gaacctgtaa cagtgtcctg gaattctgga gctttaactt ctggggtaca tactttccca 540
gctgttcttc agagtagcgg cttgtacagt cttagttctg tcgtcactgt tccttcatct 600
agtcttggaa cgcagaccta catttgtaac gtgaatcaca agcccagtaa tacaaaggtg 660
gataagaagg ctgagccaaa gtcatgtgat aaaacccaca cttgtccgcc atgtcctgct 720
cctgaactgt tgggtggccc aagcgttttc ctttttcctc cgaaaccaaa ggacaccttg 780
atgattagta ggactcctga ggtcacttgc gtggttgttg atgtgtctca tgaggacccg 840
gaggtgaagt tcaactggta tgttgacgga gtggaagtgc ataacgctaa gactaagcca 900
cgtgaggagc aatacaactc cacatataga gtggtgtctg ttttgacagt cctgcatcaa 960
gattggctga acggcaagga gtataaatgt aaggtgtcta acaaagcttt acctgcccca 1020
atcgaaaaaa ccattagcaa ggccaaaggc caacctcgtg aaccacaggt ctacactctc 1080
ccgcctagta gagatgagtt gactaaaaat caagtttcct tgacatgctt agtgaaagga 1140
ttctatccta gtgatatcgc tgtggaatgg gaaagcaatg gccagcccga gaataattat 1200
aagacaactc ctcctgtgtt agatagtgat ggttcttttt tcctttattc aaagcttact 1260
gtggataaat ctcgatggca gcaagggaat gttttttcct gctctgttat gcatgaggct 1320
cttcataacc actatacaca aaaatcttta tcattgtccc ctggaaaa 1368
<210> 7
<211> 220
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
85 90 95
Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 8
<211> 660
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatatccaaa tgactcagtc tccactctct cttcctgtaa cacctggaga accagctagt 60
attagttgta gaagttccca gtctttgtta gactcagatg atggaaatac gtatctcgac 120
tggtacctgc agaagccagg tcagtcacca caattgttga tatacacttt gtcatataga 180
gcaagcggcg tacctgatag gtttagcggc agtggttccg gcacagattt taccttaaag 240
atttctcgag ttgaagccga agatgttggc gtttattatt gcatgcagcg tattgagttt 300
cccttgacat tcggcggtgg gaccaaggtt gaaataaagc gaacagtggc agccccatca 360
gttttcatct tcccaccttc agatgaacaa cttaaatctg ggaccgcaag cgtggtctgc 420
cttcttaata atttctatcc tcgggaggct aaggtccagt ggaaagtaga taacgctttg 480
cagagcggaa actctcaaga atcagtcacg gagcaagact caaaggactc aacttatagt 540
cttagttcaa ctcttacact cagtaaggca gattatgaaa agcataaggt ctatgcttgt 600
gaagttacac accaggggtt gagtagtcca gtgacaaaat catttaatag aggagaatgt 660
Claims (1)
1.一种植物作为宿主表达B38抗体和H4 抗体的方法,其特征在于,将表达载体转化到农杆菌中,通过农杆菌介导真空渗透入植物组织后,提取、分离蛋白质,获得B38抗体和H4抗体;
所述植物为生菜;
所述B38的重链序列或轻链序列为将B38重链、B38轻链的密码子优化为植物偏好的密码子,获得的优化的B38的重链序列或优化的B38的轻链序列;
所述H4的重链序列或轻链序列为将H4重链、H4轻链的密码子优化为植物偏好的密码子,获得的优化的H4的重链序列或优化的H4的轻链序列;
所述优化的B38的重链序列如SEQ ID No.1所示;所述优化的B38的重链的核苷酸序列如SEQ ID No.2所示;
所述优化的B38的轻链序列如SEQ ID No.3所示;所述优化的B38的轻链的核苷酸序列如SEQ ID No.4所示;
所述优化的H4的重链序列如SEQ ID No.5所示;所述优化的H4的重链的核苷酸序列如SEQ ID No.6所示;
所述优化的H4的轻链序列如SEQ ID No.7所示;所述优化的H4的轻链的核苷酸序列如SEQ ID No.8所示;
所述表达载体的构建方法包括如下步骤:
步骤1:分别将B38重链、B38轻链、H4重链、H4轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的B38的重链序列;
ⅱ.优化的B38的轻链序列;
ⅲ.优化的H4的重链序列;
ⅳ.优化的H4的轻链序列;
步骤2:在所述优化的B38的重链序列、优化的B38的轻链序列、优化的H4的重链序列或优化的H4的轻链序列的5’末端以及在3’末端分别加入Xmal 限制性酶切位点,克隆到pUC57载体中,分别获得pB38H,pB38L,pH4H或pH4L克隆载体;
步骤3:通过Xmal分别从步骤2所得的克隆载体中获得基因片段,通过同源重组质粒构建法克隆至双元植物载体pCam35S,分别获得表达载体p35S-B38H,p35S-B38L,p35S-H4H或p35S-H4L;
所述农杆菌介导真空渗透包括如下步骤:
步骤1:抽真空25~45s;
步骤2:保持真空-95kPa压力30~60s;
步骤3:释放压力使得渗透液渗入所述植物组织;
重复上述步骤2~3次,避光处理4d。
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