CN107723310B - 植物作为宿主在表达卡那抗体中的应用 - Google Patents
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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卡那抗体的应用。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卡那单克隆抗体(Canakinumab,Ilaris,ACZ885,人抗白介素‑1β单克隆抗体)。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法确定重组卡那抗体成功表达。多行核白细胞抑制实验证明生菜生产的卡那抗体具有抑制白介素‑1β以及抑制中性粒细胞的生物学活性。
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卡那抗体中的应用。
背景技术
癌症是世界各地死亡的主要原因,由于人口的增长和老龄化以及其他因素的普遍存在,如空气,食品等污染,其发生率正在上升。在治疗方法中,手术,化疗和放射性照射依然是现阶段治疗各种肿瘤类型和阶段的主要手段。然而,由于对肿瘤细胞缺乏选择性,化疗方法的成功率有限,且会导致全身毒性和耐药性。放射性疗法也不能全部杀死癌细胞,而且导致病人病体更加虚弱。因为癌细胞具有扩散性,手术可以切除发病部位,但是也不能阻挡癌细胞的传播。目前较先进的疗法是根据肿瘤细胞的分子特征,设计出更好的靶向治疗来阻止其生长和传播。这些疗法中的大多数都是基于容易进入肿瘤细胞的小分子药物或与其表面上的特定靶标结合的单克隆抗体(mAb)。
基于mAb的靶向治疗是针对不同目标的免疫疗法,例如阻断致癌途径,随后对细胞生长和凋亡的影响,阻断新血管形成,调节对肿瘤细胞的免疫应答,破骨细胞功能的调节或细胞毒性药物的递送来杀死肿瘤细胞。自从美国食品和药物管理局(FDA)批准第一种单克隆抗体()以来,其中包括鼠,嵌合和人源化抗体在内的数百种抗体已被开发用于癌症治疗。这些单克隆抗体中的一些已经被FDA批准,并且已经可用于日常实践中的临床应用,作为单一疗法或与标准化疗方案组合,而许多其它单克隆抗体仍在不同的临床试验中进行测试。
卡那抗体(Canakinumab,Ilaris,ACZ885,人抗白介素-1β单克隆抗体)是诺华公司生产的一种完全人源性单克隆抗体。其选择性地阻滞I L-1β与IL-1受体的相互作用,使其活性失效,与其他具有I L-1特性的家族性成员无交叉反应。主要用于治疗因为基因突变而引起的冷吡啉相关周期性综合征(cryopyrin-associated periodic syndromes CAPS),治疗幼年特发性关节炎(JIA),急性痛风性关节炎,也可以显著降低既往心梗合并高C反应蛋白、SCAD患者的心血管事件发生风险。美国在2009年批准卡那单抗用于临床治疗冷吡啉相关周期性综合征(CAPS),包括罕有但有虚弱症状的终身自体炎症疾病。卡那单抗是首个获准治疗2种类型的CAPS的药品:家族冷自主炎症综合征(FamilialColdAuto-InflammatorySyndrome,FCAS)和穆-韦二氏综合征(Muckle-WellsSyndrome,MWS)。是一可快速和选择性阻滞IL-1β的全人单抗。本品给药方案是八周1次,较目前其它上市的治疗药大大地减少了给药次数。90%以上患者未出现注射部位反应。CAPS是由导致白介素1β(IL-1β)过度产生的单一基因突变引起的,造成虚弱乏力、潮红、发热、头痛、关节痛和结膜炎,可出现在初生儿或婴儿,可在患者一生中每日出现,长期可造成严重疾病和可能致命,包括耳聋、骨和关节变形、中枢神经***损伤导致失明和淀粉样变造成肾衰竭。
现阶段主要利用动物细胞生产卡那单克隆抗体。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卡那抗体中的应用。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,表达了卡那抗体。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卡那抗体蛋白。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卡那抗体中的应用。优选的,所述抗体为单克隆抗体。所述植物选自生菜、白菜、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,包括卡那的重链序列或轻链序列以及载体。
在本发明的一些具体实施方案中,所述卡那的重链序列或轻链序列为将卡那重链、卡那轻链的密码子优化为植物偏好的密码子,获得的优化的卡那的重链序列或优化的卡那的轻链序列。
在本发明的一些具体实施方案中,所述优化的卡那的重链序列如SEQ ID No.1所示;所述优化的卡那的重链的核苷酸序列如SEQ ID No.2所示;
所述优化的卡那的轻链序列如SEQ ID No.3所示;所述优化的卡那的轻链的核苷酸序列如SEQ ID No.4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:分别将卡那重链、卡那轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的卡那的重链序列;
ⅱ.优化的卡那的轻链序列;
步骤2:在所述优化的卡那的重链序列的5’末端加入Xbal限制性酶切位点,在3’末端加入Sac I位点;
在所述优化的卡那的轻链序列的5’末端加入Xbal限制性酶切位点,在3’末端加入Sac I位点;
由金斯瑞克隆到pUC57载体中,分别获得pCana-H克隆载体,pCana-L克隆载体;
步骤3:通过Xbal/Sacl分别从步骤2所得的克隆载体中获得基因片段,克隆至双元植物载体pCam35S,分别获得表达载体p35S-Cana-H,p35S-Cana-L。
具体的,为了提供外援蛋白在植物中的高效表达,本发明将人卡那重链,轻链(https://www.drugbank.ca/drugs/DB06168)蛋白序列反翻译软件(https://www.idtdna.com/CodonOpt)将其其密码子优化为植物偏好的密码子,由金斯瑞公司(南京,中国)合成。在优化的卡那重链序列5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入Sacl位点。在卡那轻链序列5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入SacI位点。并由金斯瑞克隆到pUC57载体中(图1),分别获得pUC57-Cana-H,pUC57-Cana-L克隆载体。基因片段通过XbaI/Sacl分别从克隆载体中分离,并克隆到双元植物载体pCam35S,分别产生植物表达载体p35S-Cana-H,p35S-Cana-L。
本发明还提供了所述的表达载体在表达卡那抗体中的应用。
此外,本发明还提供了一种植物作为宿主表达卡那抗体的方法,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,获得卡那抗体。
具体的,将两种植物表达载体p35S-Cana-H,p35S-Cana-L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/LMgSO4,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-Cana-H和p35S-Cana-L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***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pUC57-Cana-H,pUC57-Cana-L基因片段,并且构建两种双元植物表达载体p35S-Cana-H,p35S-Cana-L(图2),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;10m Mβ-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困难,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。重组卡那抗体经过变性凝胶SDS-PAGE分离我们在泳道中观察到估计分子量大约分别为23kDa(轻链)以及50kDa(重链)(图3A),符合卡那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150kDa(图3B)的条带,证明生菜重组轻重链成功的结合为抗体结构,符合卡那抗体蛋白分子量。基于Bradford测定法和光密度测定对照组测定纯化样品的蛋白含量大约为0.86mg/g。
植物来源的卡那抗体生物学活性通过多行核白细胞抑制实验证明。具体实施方案为:在OF-1雌性小鼠背部注射空气形成皮下空气袋,六天后,通过腹膜内(i.p.)注射卡那抗体或对照生理盐水(PBS)。一天以后,将表达人IL-1β的重组NIH3T3细胞注射到空气袋中以诱导炎症反应。24小时后,测定观察气囊灌洗液中的PMN数。结果表明,没有任何处理的PMN细胞数目增多,生长良好。相比之下,使用纯化的重组卡那抗体培养的PMN细胞数目受到明显抑制。这些结果表明,通过生菜***瞬间表达的外源卡那抗体具有生物学活性并且抑制PMN细胞的增长。结果表明,植物尤其生菜是一种生产卡那抗体的合适的生物反应器。
本发明利用植物尤其是生菜来瞬时表达卡那抗体,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***生产卡那单克隆抗体,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。
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示克隆载体pUC57示意图;
图2示卡那植物双元表达载体p35S-Cana-H(重链)以及p35S-Cana-L(轻链)构建流程;利用限制性内切酶(Xbal/SacI)双酶切,从图1克隆载体分别切下卡那H重链,连接入pCam35S的Xbal/SacI位点,生成植物双元表达载体p35S-Cana-H;利用限制性内切酶(Xbal/SacI)双酶切,从图1克隆载体分别切下卡那L轻链,连接入pCam35S的Xbal/SacI位点,生成植物双元表达载体p35S-Cana-L;
LB and RB: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:卡那重组抗体;B示非变性凝胶电泳结果;
图4示多行核白细胞抑制实验证明生菜生产的卡那抗体生物学活性。
具体实施方式
本发明公开了植物作为宿主在表达卡那抗体中的应用,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***尤其是生菜***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***短时间内大规模生产卡那单克隆抗体。
本发明提供的植物作为宿主在表达卡那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,将人卡那重链,轻链,(https://www.drugbank.ca/drugs/DB06168)蛋白序列反翻译软件(https://www.idtdna.com/CodonOpt)将其其密码子优化为植物偏好的密码子,由金斯锐公司(南京,中国)合成。在优化的卡那重链序列5'末端分别加入Xbal限制性酶切位点,在3'末端分别加入SacI位点。在卡那轻链序列5'末端分别加入Xbal限制性酶切位点,在3'末端分别加入Sacl位点。并由金斯瑞公司克隆到pUC57载体中(图1),分别生成pUC57-Cana-H,pUC57-Cana-L克隆载体。基因片段通过Xbal/Sacl分别从克隆载体中分离,并克隆到双元植物载体,pCam35S,分别产生植物表达载体p35S-Cana-H,p35S-Cana-L。将两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(10mM MES,10mMMgSO4)中至O.D.600为0.5。
克隆获得pUC57-Cana-H,pUC57-Cana-L基因片段,并且构建两种双元植物表达载体p35S-Cana-H,p35S-Cana-L(图2)。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-Cana-H以及p35S-Cana-L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***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pH8.0,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(20mMKPi,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非常困难并且昂贵,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。重组卡那抗体经过变性凝胶SDS-PAGE分离我们在泳道中观察到估计分子量大约为23kDa(轻链)以及50kDa(重链)条带(图3A),符合卡那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150kDa(图3B)的条带,证明生菜重组轻重链成功的结合为抗体结构,符合卡那抗体蛋白分子量。基于Bradford测定法和光密度测定对照组测定纯化样品的蛋白含量大约为0.86mg/g。
实施例4 SDS-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-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。
实施例5多行核白细胞抑制实验
植物来源的卡那抗体生物学活性通过多行核白细胞抑制实验证明。具体实施方案为:在OF-1雌性小鼠背部注射空气形成皮下空气袋,六天后,通过腹膜内(i.p.)注射卡那抗体或对照生理盐水(PBS)。一天以后,将表达人IL-1β的重组NIH3T3细胞注射到空气袋中以诱导炎症反应。24小时后,测定观察气囊灌洗液中的PMN数。由图4结果表明,没有任何处理的PMN细胞数目增多,生长良好。相比之下,使用纯化的重组卡那抗体培养的PMN细胞数目受到明显抑制。这些结果表明,通过生菜***瞬间表达的外源卡那抗体具有生物学活性并且抑制PMN细胞的增长。结果表明,植物尤其生菜是一种生产卡那抗体的合适的生物反应器。
实施例6
对照组:利用动物细胞生产卡那抗体;
实验组1:本发明提供的植物生产卡那抗体;
实验组2:利用烟叶生产卡那抗体;
表1卡那抗体
*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2相比P≤0.05;##示与实验组2相比P≤0.01;
由表1可知,实验组1与对照组的动物***相比,本发明提供的生菜瞬时表达卡那抗体,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,极显著(P≤0.01)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1与实验组2的烟叶***相比,生菜瞬时表达卡那抗体,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。
综合上述试验结果表明,植物***尤其是生菜***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卡那单克隆抗体。
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> 深圳惠升生物科技有限公司
<120> 植物作为宿主在表达卡那抗体中的应用
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atgcaagtgc aactcgttga atccggcggt ggcgtagttc agcctggccg aagcctgcgt 60
ttgtcctgtg ccgcttctgg atttacattt agcgtttatg gaatgaattg ggttaggcag 120
gcacctggga aaggacttga gtgggtagct atcatctggt acgatggcga caaccaatac 180
tacgcagatt ctgtgaaggg ccgatttacg atctcccgtg ataactctaa gaacacactg 240
tatctgcaaa tgaacggact tagggctgaa gataccgccg tctactattg cgcaagagat 300
ttaagaactg ggcccttcga ctactggggt caggggactt tagtcacggt ttcttcagcc 360
tcaacaaaag gtccaagcgt tttccctctc gctccatcct caaaatctac gagtggcggg 420
acagctgcac tgggatgctt agttaaggat tatttccctg aaccggttac agtaagttgg 480
aactctgggg ctcttacttc aggcgtacat acgttcccag cagtccttca gagctccggg 540
ttgtacagtc tgtcctcagt tgtgaccgtg ccgtcttcta gccttggtac acaaacgtat 600
atctgcaatg tcaatcacaa accgtccaac actaaggttg acaagcgagt cgagcccaaa 660
tcttgcgata agacccacac atgtcctcca tgccccgctc ccgaactctt agggggcccg 720
tccgtgttcc tgtttccacc gaagcccaag gacacactca tgatttcccg tacgccggaa 780
gttacttgcg tggttgtgga tgtctcacac gaagacccag aggttaaatt taattggtat 840
gtagatgggg tagaggtgca taatgcaaaa acgaaaccga gggaggagca gtataactct 900
acttatagag tagtaagcgt tttgacggtg ttacatcagg actggcttaa tggcaaagag 960
tataagtgta aagtaagtaa caaagcctta cctgccccca tagaaaagac tatatctaaa 1020
gctaaaggtc aacctcgtga acctcaggtc tatacgctgc ctccgtctag ggaagaaatg 1080
actaagaatc aagtttccct gacgtgcctt gttaaaggct tctatccaag cgacatcgct 1140
gtcgagtggg aaagtaatgg gcaacctgag aacaactaca aaacgactcc accggtttta 1200
gactctgacg gctccttctt tttgtacagt aagttaacgg tagacaagtc taggtggcag 1260
cagggaaacg tcttttcatg ctctgtcatg catgaagccc tgcacaatca ttatacacag 1320
aagagtttaa gtctcagtcc gggtaaataa 1350
<210> 2
<211> 448
<212> PRT
<213> Heavy chain of Canakinumab
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 3
<211> 648
<212> DNA
<213> Light chain of Canakinumab
<400> 3
atggagatag tattgacaca atcccctgat ttccaatctg taaccccaaa ggaaaaagtc 60
accatcacat gccgtgcttc ccagagcatt ggttcctcac tccattggta tcaacaaaag 120
cccgaccagt ctcccaagct gctgatcaaa tatgcttctc aaagtttttc aggtgtgcca 180
tccaggttct ctgggtctgg gagtggcacg gatttcactc ttactataaa cagtcttgaa 240
gctgaggatg ctgccgccta ctactgtcac caaagcagca gcctgccttt tacgtttggc 300
cccggaacaa aagtggatat taagagaacc gtcgccgccc catcagtatt tatctttcct 360
cccagcgatg agcagttaaa atctggtacg gcaagcgtgg tctgcctctt aaacaatttt 420
tacccacgtg aggctaaagt tcaatggaaa gtagacaacg ctcttcagtc tggaaatagc 480
caggaaagcg tgactgagca agatagcaag gattccacat acagtctttc atccacactg 540
acattatcaa aggccgacta tgaaaaacat aaagtttatg cttgcgaagt cacacatcag 600
ggtctctcct cacccgtgac caaaagtttc aataggggtg aatgttaa 648
<210> 4
<211> 214
<212> PRT
<213> Light chain of Canakinumab
<400> 4
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
Claims (1)
1.一种植物作为宿主表达卡那抗体的方法,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,获得卡那抗体;
所述植物为生菜;
所述表达载体包括卡那的重链序列或轻链序列以及载体;
所述卡那的重链序列或轻链序列为将卡那重链、卡那轻链的密码子优化为植物偏好的密码子,获得的优化的卡那的重链序列或优化的卡那的轻链序列;
所述优化的卡那的重链序列如SEQ ID No.1所示;所述优化的卡那的重链的核苷酸序列如SEQ ID No.2所示;
所述优化的卡那的轻链序列如SEQ ID No.3所示;所述优化的卡那的轻链的核苷酸序列如SEQ ID No.4所示;
所述载体为双元植物载体;
所述表达载体的构建方法包括如下步骤:
步骤1:分别将卡那重链、卡那轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的卡那的重链序列;
ⅱ.优化的卡那的轻链序列;
步骤2:在所述优化的卡那的重链序列的5’末端加入Xbal限制性酶切位点,在3’末端加入SacI位点;
在所述优化的卡那的轻链序列的5’末端加入Xbal限制性酶切位点,在3’末端加入SacI位点;
克隆到pUC57载体中,分别获得pCana-H克隆载体,pCana-L克隆载体;
步骤3:通过Xbal/Sacl分别从步骤2所得的克隆载体中获得基因片段,克隆至双元植物载体pCam35S,分别获得表达载体p35S-Cana-H,p35S-Cana-L;
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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CN110218257A (zh) * | 2019-06-24 | 2019-09-10 | 王跃驹 | 植物作为宿主在表达Antis15抗体中的应用 |
CN110229847A (zh) * | 2019-06-24 | 2019-09-13 | 王跃驹 | 生菜作为宿主在表达乙肝疫苗中的应用 |
CN110302366A (zh) * | 2019-07-05 | 2019-10-08 | 王跃驹 | 植物源蚓激酶胶囊及其生产方法 |
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