CN110241132B - 植物作为宿主在表达Dulaglutide中的应用 - Google Patents
植物作为宿主在表达Dulaglutide中的应用 Download PDFInfo
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- CN110241132B CN110241132B CN201910549732.9A CN201910549732A CN110241132B CN 110241132 B CN110241132 B CN 110241132B CN 201910549732 A CN201910549732 A CN 201910549732A CN 110241132 B CN110241132 B CN 110241132B
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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Dulaglutide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Dulaglutide。该表达体系确定植物外源蛋白在农杆菌侵染4天后就可以收集。利用Western Blot蛋白杂交法确定Dulaglutide成功表达,用AKTA蛋白纯化系统成功纯化出Dulaglutide。生物活性测试结果表明利用该平台技术生产的Dulaglutide显著降低了狗血液中的血糖浓度。
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Dulaglutide中的应用。
背景技术
糖尿病是以慢性高血糖为特征的常见病、多发病,是由体内胰岛素分泌或作用缺陷,或二者同时存在而引起的糖、脂肪、蛋白质代谢紊乱。临床上主要有胰岛素依赖型(IDDM,I型)和非胰岛素依赖型(NIDDM,II型)两种类型。随着生活水平提高,无论在发达国家还是在发展中国家,糖尿病的发病率都在逐年上升。糖尿病作为一种严重的非传染性慢性疾病现已成为全世界各国密切关注的重大公共卫生问题之一,是全球范围内继心血管和肿瘤疾病之后的第三号杀手。从世界卫生组织公布的数据来看,1995年全球糖尿病患者仅3000万人左右,而到了1997年已增至1.35亿,到2030年将有3亿II型糖尿病患者,患者增幅最快的地区为亚洲和非洲。II型糖尿病患者的传统治疗模式一般是遵循饮食控制、口服抗糖尿病药物和外源性胰岛素的阶梯式治疗。但目前糖尿病治疗领域仍存在着诸多尚待解决的重要问题,而且还存在一些副作用和限制。
胰高血糖素样肽-1(Glucagon-like peptide-1,GLP-1)是由肠道内分泌细胞分泌的肠降血糖素,是胰高血糖素原基因翻译后加工产物,在体内有多种存在形式。它具有以下生理作用:以葡萄糖依赖方式作用于胰岛β细胞,促进胰岛素基因的转录,增加胰岛素的生物合成和分泌;刺激β细胞的增殖和分化,抑制β细胞凋亡,从而增加胰岛β细胞数量,抑制胰高血糖素的分泌,抑制食欲及摄食,延缓胃内容物排空等。这些功能都有利于降低餐后血糖并使血糖维持在恒定水平。
尽管天然GLP-1在治疗糖尿病上有诸多优点,但它的体内半衰期仅为2分钟左右,限制了其在临床上的直接应用。
发明内容
有鉴于此,本发明提供了植物作为宿主在表达Dulaglutide(杜拉鲁肽)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Dulaglutide。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Dulaglutide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Dulaglutide中的应用;所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草;所述植物的器官选自种子、叶、根茎或整株植物。
植物瞬时表达技术是在植物生长到一定阶段,利用多种不同的技术方式将含有目标蛋白的质粒转移到植物细胞中,在植物细胞中建立高效、可控的表达系统,获得该基因短暂的可控表达的技术。与稳定表达相比,瞬时表达所需时间短,不需要将外源基因整合到宿主植物染色体中,仅需几天的时间就可拿到实验结果。与细菌表达系统相比,植物表达系统可以对所表达的蛋白进行正确的折叠、加工、修饰,所生产的蛋白活性高于细菌表达系统;与动物细胞表达系统相比,植物表达系统的成本非常低,仅为其千分之一到千分之二。
在上述研究的基础上,本发明还提供了一种表达载体,包括Dulaglutide的序列以及双元植物载体。
在本发明的一些具体实施方案中,所述Dulaglutide的序列为将Dulaglutide的密码子优化为植物偏好的密码子获得的优化Dulaglutide的序列。
在本发明的一些具体实施方案中,所述优化的Dulaglutide的序列中核苷酸序列如SEQ ID No.1所示;所述优化的Dulaglutide的序列中氨基酸序列如SEQ ID No.2所示;
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:将Dulaglutide的密码子优化为植物偏好的密码子,获得优化的Dulaglutide的序列;
步骤2:在所述优化的Dulaglutide的序列的5’末端加入Xbal限制性酶切位点,在3’末端加入Sac I位点;克隆到pUC57载体中,获得pDula克隆载体;
步骤3:通过Xbal/Sacl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,获得表达载体p35S-Dula。
本发明还提供了所述的表达载体在表达Dulaglutide中的应用。
本发明还提供了一种植物作为宿主表达Dulaglutide的方法,将所述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Dulaglutide。
在本发明的一些具体实施方案中,所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草;所述植物的器官选自种子、叶、根茎或整株植物。
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。
将天然的GLP-1中的某些氨基酸突变后可以在保证其活性的条件下延长它的半衰期,做到每周一次给药就可以保持正常的血糖水平。本发明采用植物系统生产Dulaglutide融合蛋白是一种将突变后的GLP短肽融合HAS以延长其效益期的原创新药。
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Dulaglutide。该表达体系确定植物外源蛋白在农杆菌侵染4天后就可以收集。利用Western Blot蛋白杂交法确定Dulaglutide成功表达,用AKTA蛋白纯化系统成功纯化出Dulaglutide。生物活性测试结果表明利用该平台技术生产的Dulaglutide显著降低了狗血液中的血糖浓度。
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示克隆载体pDula示意图;
图2示Dulaglutide植物双元表达载体p35S-Dula构建流程;利用限制性内切酶(Xbal/SacI)双酶切,从图1克隆载体切下Dulaglutide,连接入pCam35S的Xbal/SacI位点,生成植物双元表达载体p35S-Dula;
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;Nos 3’,终止子;
图3示SDS-PAGE凝胶电泳结果;泳道1:非侵染生菜;泳道2:生菜表达的Dulaglutide;泳道3:Dulaglutide商业化产品。
具体实施方式
本发明公开了植物作为宿主在表达Dulaglutide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。
有鉴于此,本发明提供了植物作为宿主在表达Dulaglutide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Dulaglutide。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Dulaglutide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Dulaglutide中的应用。优选的,所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草;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Dulaglutide的序列以及载体。
在本发明的一些具体实施方案中,所述Dulaglutide的序列为将Dulaglutide的密码子优化为植物偏好的密码子,获得的优化的Dulaglutide序列。
在本发明的一些具体实施方案中,所述优化的Dulaglutide的序列如SEQ ID No.1所示;所述优化的Dulaglutide的序列如SEQ ID No.2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:将Dulaglutide的密码子优化为植物偏好的密码子,获得:优化的Dulaglutide的序列;
步骤2:在所述优化的Dulaglutide的序列的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,获得pDula克隆载体;
步骤3:通过Xbal/Sacl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,获得表达载体p35S-Dula。
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Dulaglutide氨基酸序列利用反翻译软件(https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/)得到核苷酸序列,并将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Dulaglutide序列5’末端加入Xbal限制性酶切位点,在3’末端加入Sacl位点。并由金斯瑞克隆到pUC57载体中,获得pDula克隆载体(图1)。基因片段通过XbaI/Sacl从克隆载体中分离,并克隆到双元植物载体pCam35S,产生植物表达载体p35S-Dula(图2)。
本发明还提供了所述的表达载体在表达Dulaglutide中的应用。
此外,本发明还提供了一种植物作为宿主表达Dulaglutide的方法,将本发明提供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Dulaglutide。
具体的,将植物表达载体p35S-Dula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-Dula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pDula基因片段,并且构建双元植物表达载体p35S-Dula(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Dulaglutide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32.6kDa(图3)的条带,符合Dulaglutide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1.81mg/g。
本发明利用生菜来瞬时表达Dulaglutide,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。生菜不含有植物有毒物质,而且其本身蛋白含量少,利于下游的蛋白纯化。利用生菜系统生产Dulaglutide,可以大大缩短生产周期和生产成本。
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Dulaglutide。
本发明提供的植物作为宿主在表达Dulaglutide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
为了将外源蛋白在植物中的高效表达,将Dulaglutide氨基酸序列利用反翻译软件(https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/)得到核苷酸序列,并将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Dulaglutide序列5'末端加入Xbal限制性酶切位点,在3'末端加入Sacl位点。并由金斯瑞公司克隆到pUC57载体中,获得pDula克隆载体(图1),基因片段通过Xbal/Sacl从克隆载体中分离,并克隆到双元植物载体,pCam35S,产生植物表达载体p35S-Dula(图2)。将植物表达载体通过用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。
实施例2农杆菌介导的真空渗透
将制备好的含有p35S-Dula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;10m Mβ-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。
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-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。重组Dulaglutide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32.6kDa(图3)的条带,符合Dulaglutide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1.81mg/g。
实施例5 Dulaglutide蛋白活性检测
在持续七周的稳定期后,将狗随机分为两个治疗组,每组3只,接收含有降糖蛋白(实施例4制得的Dulaglutide蛋白)及不含降糖蛋白两种实验胶囊中的一种,做第一次重复。再次将狗随机分组,接受另一种不同的实验饮食,做第二次重复。重复I和II至少持续2周,在每次重复结束后检测血糖反应。
在血糖检测开始前,狗禁食24小时。剃去导管插入部位的毛,无菌处理,导管插入右头静脉。大约间隔5分钟采集两基线样品。在采集最后一个基线样品后,立即给狗喂相当其体重1%的饮食并含有1片或3片降糖胶囊,至多允许其吃15分钟。如果在15分钟内狗不吃实验饮食,则当天不检测其血糖反应,次日重新检测。在进食后10、20、30、45、60、120、180和240分钟,采集额外的血液样品。血液样品1300×g离心15分钟,在采集后两小时内将每个时间点1ml血浆之两等分样品冻存。应用己糖激酶方法测定血浆葡萄糖浓度(mg/dl)。
表1狗血液中糖浓度的实验结果
注:*示与无降糖对照组比较具有显著差异(P<0.05);**示与无降糖对照组比较具有极显著差异(P<0.01)。
本发明利用生菜来瞬时表达抗体,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。生菜不含有植物有毒物质,而且其本身蛋白含量少,利于下游的蛋白纯化。利用生菜系统生产,可以大大缩短生产周期和生产成本
实施例6动物毒性试验
将7周大小的实验用小白鼠随机分为三个治疗组,每组10只,分别接收含有降糖蛋白(按照体重喂食500ng/g)(本发明得到的Dulaglutide蛋白),商业用索玛鲁泰Semaglutid(按照体重喂食500ng/g)作为阳性对照,及不含降糖蛋白两种实验胶囊中的一种,接受相同的实验饮食。连续喂食10天,每次喂食后实进行观察,每天需要连续观察6小时以上,并没有看小鼠处于兴奋状态还是抑制状态,没有出现行动迟缓等现象,也没有出现腹泻等情况。证明索玛鲁泰Semaglutide与蚓激酶的融合蛋白胶囊口服安全性高。
实施例7
对照组:利用动物细胞生产Dulaglutide;
实验组1:本发明提供的植物生产Dulaglutide;
实验组2:利用烟叶生产Dulaglutide;
表2 Dulaglutide
*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2可知,实验组1与对照组的动物系统相比,本发明提供的生菜瞬时表达Dulaglutide,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,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的烟叶系统相比,生菜瞬时表达Dulaglutide,显著(P≤0.05)缩短了生产周期,显著(P≤0.05)提高了蛋白含量,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2与对照组相比,烟叶瞬时表达Dulaglutide比动物系统显著(P≤0.05)缩短了生产周期,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Dulaglutide。
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Dulaglutide中的应用
<130> MP1906700
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 900
<212> DNA
<213> Dulaglutide(Dulaglutide)
<400> 1
atgggcatca agatggaaag ccagatccag gtgttcgttt tcgtgttcct ttggctgtct 60
ggtgtggatg gtcatggtga gggaaccttt acctccgacg tgtcatctta ccttgaggaa 120
caggctgcca aagagttcat tgcctggctt gtgaaaggtg gtggtggcgg tggtggatct 180
ggtggcggag gtagcggagg tggtggaagt gctgaatcta agtatggtcc tccttgtccg 240
ccttgtcctg ctcctgaagc tgctggtggt ccttctgttt ttctgttccc acctaagcct 300
aaggacaccc tgatgatttc taggactcct gaggttacct gcgtcgtggt tgatgtgtct 360
caagaggatc cagaggtgca gttcaattgg tacgttgacg gtgttgaggt gcacaacgct 420
aagactaagc ctcgagagga acagttcaac agcacctaca gggttgtgtc tgtgcttact 480
gtgcttcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgag caacaagggc 540
ctgccgtcct ctattgaaaa gaccatctct aaggctaagg gccagcctag agaaccgcaa 600
gtttacactc ttccgccgag ccaagaagag atgaccaaga atcaggtgag ccttacctgc 660
cttgtgaagg gcttttaccc ttccgatatc gctgttgagt gggagtctaa tggtcagccc 720
gagaacaact acaagactac ccctcctgtg ctggattccg atggttcatt cttcctgtac 780
agcaggctga ccgtggataa gtcaagatgg caagagggca acgtgttcag ctgctctgtt 840
atgcatgagg ctctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggttga 900
<210> 2
<211> 299
<212> PRT
<213> Dulaglutide(Dulaglutide)
<400> 2
Met Gly Ile Lys Met Glu Ser Gln Ile Gln Val Phe Val Phe Val Phe
1 5 10 15
Leu Trp Leu Ser Gly Val Asp Gly His Gly Glu Gly Thr Phe Thr Ser
20 25 30
Asp Val Ser Ser Tyr Leu Glu Glu Gln Ala Ala Lys Glu Phe Ile Ala
35 40 45
Trp Leu Val Lys Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly
50 55 60
Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro
65 70 75 80
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
85 90 95
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
100 105 110
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
115 120 125
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
130 135 140
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
145 150 155 160
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
165 170 175
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
180 185 190
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
195 200 205
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
210 215 220
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
225 230 235 240
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
245 250 255
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
260 265 270
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
275 280 285
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
290 295
Claims (1)
1.一种植物作为宿主表达Dulaglutide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Dulaglutide;
所述表达载体包括Dulaglutide的编码序列以及双元植物载体;
所述Dulaglutide的编码序列为将Dulaglutide的密码子优化为植物偏好的密码子获得的优化的核苷酸序列;
所述优化的核苷酸序列如SEQ ID No.1所示;所述优化的核苷酸序列编码的氨基酸序列如SEQ ID No.2所示;
所述表达载体的构建方法包括如下步骤:
步骤1:将Dulaglutide的密码子优化为植物偏好的密码子,获得优化的Dulaglutide的序列;
步骤2:在所述优化的Dulaglutide的序列的5’末端加入Xbal限制性酶切位点,在3’末端加入Sac I位点;克隆到pUC57载体中,获得pDula克隆载体;
步骤3:通过Xbal/ Sacl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,获得表达载体p35S-Dula;
所述植物为生菜;
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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