CN109777824A - 植物作为宿主在表达hiv中和抗体中的应用 - Google Patents
植物作为宿主在表达hiv中和抗体中的应用 Download PDFInfo
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- CN109777824A CN109777824A CN201910129295.5A CN201910129295A CN109777824A CN 109777824 A CN109777824 A CN 109777824A CN 201910129295 A CN201910129295 A CN 201910129295A CN 109777824 A CN109777824 A CN 109777824A
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- neutralizing antibody
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- hiv neutralizing
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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HIV广谱中和抗体的应用。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HIV广谱中和抗体。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WesternBlot蛋白杂交法确定HIV广谱中和抗体成功表达,对HIV毒株SF162的中和活性测试表明利用生菜生产的HIV抗体具有中和HIV的生物活性。
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HIV中和抗体中的应用。
背景技术
艾滋病病毒(Human immunodeficiency virus,HIV)的传播是全球关注的主要公共健康问题之一。目前全球HIV感染者约有3500万人,每年新增感染者约为200万人。尽管高效抗反转录病毒治疗(Highly active antiretroviral therapy,HAART)可以有效抑制HIV复制,近20年成功地用于临床治疗,但是仍无法彻底清除感染者体内的病毒,而且长期服药提高了耐药株产生的概率。为了进一步降低全球新发感染率,清除储藏库病毒,亟待寻找更加有效的HIV防治方法。
HIV-1广谱中和抗体(Broadly neutralizing antibodies)在预防感染和补充现有治疗方案中均显现出重要作用,在体外实验中,HIV-1广谱中和抗体可以阻断病毒在细胞间的传播。2018年3月7日,FDA批准创新药物ibalizumab-uiyk/TMB-355(商品名Trogarzo)上市,作为一种全新的抗逆转录病毒疗法,Trogarzo用于治疗现有多种疗法均无法起效的成人艾滋病病毒(HIV)感染者。使用用方法是,患者每14天静脉内注射一次,也可与其他同类药物联合使用。
Trogarzo(ibalizumab-uiyk)是一种静脉滴注的人源化免疫球蛋白G4单克隆抗体,它与CD4+T细胞受体的第二个胞外区域结合,阻止HIV病毒入侵这些细胞,和PRO140类似都是一种“病毒侵入抑制剂”。Trogarzo是10多年来首款具有全新作用机制的抗逆转录病毒疗法,ibalizumab曾获得美国FDA颁发的快速通道资格(Fast Track)、优先审评资格(Priority Review)、突破性疗法认定(Breakthrough Therapy designations)、以及孤儿药资格(Orphan Drug)。
Trogarzo的安全性与疗效在一项临床试验中已得到了验证。该试验招募了40名感染有多重耐药性HIV的患者,他们均重度经治,有些患者甚至已经接受过10种或更多的抗逆转录病毒疗法。然而,研究人员发现,患者即便接受了大量治疗,他们血液中的病毒水平(HIV-RNA)依旧很高。但在原有疗法中,加入Trogarzo的治疗一周后,大部分患者血液中的HIV-RNA水平就有显著下降。24周后,43%的患者其HIV-RNA水平得到了抑制。
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。
发明内容
有鉴于此,本发明提供了植物作为宿主在表达HIV中和抗体Ibalizumab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HIV中和抗体Ibalizumab。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Ibalizumab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Ibalizumab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、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Ibalizumab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Ibalizumab的重链序列或轻链序列为将Ibalizumab重链、Ibalizumab轻链的密码子优化为植物偏好的密码子,获得的优化的Ibalizumab的重链序列或优化的Ibalizumab的轻链序列。
在本发明的一些具体实施方案中,所述优化的Ibalizumab的重链序列如SEQ IDNo.2所示;所述优化的Ibalizumab的重链的核苷酸序列如SEQ ID No.1所示;
所述优化的Ibalizumab的轻链序列如SEQ ID No.4所示;所述优化的Ibalizumab的轻链的核苷酸序列如SEQ ID No.3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Ibalizumab重链、Ibalizumab轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的Ibalizumab的重链序列;
ⅱ.优化的Ibalizumab的轻链序列;
步骤2:在所述优化的Ibalizumab的重链序列的5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入Sac I位点;
在所述优化的Ibalizumab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pIbali-H,pIbali-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-Ibali-H,p35S-Ibali-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Ibalizumab重链以及轻链氨基酸序列利用反翻译软件得到核苷酸序列,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Ibalizumab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Ibalizumab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,分别获得pIbali-H,pIbali-L克隆载体(图1)。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-Ibali-H,p35S-Ibali-L(图2)。
本发明还提供了所述的表达载体在表达Ibalizumab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Ibalizumab抗体的方法,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Ibalizumab抗体。
具体的,将两种植物表达载体p35S-Ibali-H,p35S-Ibali-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/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。
将制备好的含有p35S-Ibali-H和p35S-Ibali-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pIbali-H,pIbali-L基因片段(图1),并且构建两种双元植物表达载体p35S-Ibali-H,p35S-Ibali-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(20mMKPi,pH 7.8;2mMEDTA;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Ibalizumab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50kDa(图3A,泳道2),与商业Ibalizumab抗体跑带大小一致(图3A,泳道3),符合Ibalizumab抗体轻重链的蛋白大小。而非侵染的生菜提取液当中没有发现条带。在非变性的凝胶电泳中观察到大约150kDa(图3B,泳道2)的条带,证明生菜重组轻重链成功的结合为抗体结构,符合Ibalizumab抗体蛋白分子量(图3B,泳道3,商业Ibalizumab抗体)。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1.78mg/g。
采用HIV假病毒感染系统评价Ibalizumab的抗病毒活性。将表达HIV毒株SF162或者HXB2的ENV蛋白的质粒转染293T细胞,假病毒分泌至上清中。收获大量的假病毒后,测试Ibalizumab对假病毒的中和活性。结果如图4所示,Ibalizumab对SF162的中和活性为2.23ug/ml,对HIV病毒株HXB2的中和活性为5.72ug/ml,该结果说明Ibalizumab具有HIV中和活性。
本发明利用生菜来瞬时表达Ibalizumab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Ibalizumab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。
图1示克隆载体pIbali-H(见图1(A))以及pIbali-L示意图(见图1(B));
图2示Ibalizumab植物双元表达载体p35S-Ibali-H(重链)以及p35S-Ibali-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Ibalizumab重链,连接入pCam35S的Xbal/SacI位点,生成植物双元表达载体p35S-Ibali-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Ibalizumab抗体轻链,重链,连接入pCam35S的Xbal/SacI位点,生成植物双元表达载体p35S-Ibali-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;Nos 3’,终止子;
图3(A)示SDS-PAGE凝胶电泳结果;图3(B)示非变性凝胶电泳结果;泳道1:非侵染生菜;泳道2:生菜表达的Ibalizumab抗体;泳道3:Ibalizumab抗体阳性对照;
图4示通过中和实验证明生菜纯化的Ibalizumab对HIV的抑制,具有显著的生物学活性。
具体实施方式
本发明公开了植物作为宿主在表达Ibalizumab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Ibalizumab单克隆抗体。
本发明提供的植物作为宿主在表达Ibalizumab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
为了将外源蛋白在植物中的高效表达,将Ibalizumab抗体重链,轻链,(https://www.genome.jp/dbget-bin/www_bget?dr:D09575)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Ibalizumab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Ibalizumab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,分别获得pIbali-H,pIbali-L克隆载体(图1),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-Ibali-H,p35S-Ibali-L(图2)。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,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-Ibali-H以及p35S-Ibali-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Ibalizumab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以及50kDa条带(图3A),符合Ibalizumab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Ibalizumab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1.78mg/g。
实施例5 Ibalizumab对HIV的中和活性检测
采用HIV假病毒感染系统评价Ibalizumab的抗病毒活性。具体步骤为,将表达HIV毒株SF162或者HXB2的ENV蛋白的质粒转染293T细胞,假病毒分泌至上清中。收获大量的假病毒后,加入牛血清终浓度达到20%,分装冻存。将Ibalizumab按倍比稀释铺入96孔板中,终体积为50ul,其中用50ulDMEM培养基替代药物做阴性对照。加入HIV-1假病毒50ul,培养48h后,利用荧光素酶检测试剂测定每孔的相对荧光单位。半数中和剂量是能引起50%最大效应的剂量,用半数中和剂量(ND50)表示。结果如图4所示,Ibalizumab对SF162的中和活性为1.23ug/ml,对HIV病毒株HXB2的中和活性为5.72ug/ml,该结果说明Ibalizumab具有HIV中和活性。
本发明利用生菜来瞬时表达Ibalizumab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Ibalizumab单克隆抗体,可以大大缩短生产周期和生产成本
实施例6
对照组:利用动物细胞生产Ibalizumab抗体;
实验组1:本发明提供的植物生产Ibalizumab抗体;
实验组2:利用烟叶生产Ibalizumab抗体;
表1 Ibalizumab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与对照组的动物系统相比,本发明提供的生菜瞬时表达Ibalizumab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≤0.01)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的烟叶系统相比,生菜瞬时表达Ibalizumab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2与对照组相比,烟叶瞬时表达Ibalizumab抗体比动物系统显著(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Ibalizumab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> 王跃驹
<120> 植物作为宿主在表达HIV中和抗体中的应用
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
Claims (9)
1.植物作为宿主在表达HIV中和抗体中的应用;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。
2.一种表达载体,其特征在于,包括HIV中和抗体的重链序列或轻链序列以及载体。
3.根据权利要求2所述的表达载体,其特征在于,所述HIV中和抗体的重链序列或轻链序列为将HIV中和抗体的重链、HIV中和抗体的轻链的密码子优化为植物偏好的密码子,获得的优化HIV中和抗体的重链序列或优化的HIV中和抗体轻链序列。
4.根据权利要求3所述的表达载体,其特征在于,所述优化的HIV中和抗体的重链序列如SEQ ID No.2所示;所述优化的HIV中和抗体的重链的核苷酸序列如SEQ ID No.1所示;
所述优化的HIV中和抗体的轻链序列如SEQ ID No.4所示;所述优化的HIV中和抗体的轻链的核苷酸序列如SEQ ID No.3所示。
5.根据权利要求2至4任一项所述的表达载体,其特征在于,所述载体为双元植物载体。
6.根据权利要求2至5任一项所述的表达载体,其特征在于,其构建方法包括如下步骤:
步骤1:分别将HIV中和抗体重链、HIV中和抗体轻链的密码子优化为植物偏好的密码子,获得:
ⅰ.优化的HIV中和抗体的重链序列;
ⅱ.优化的HIV中和抗体的轻链序列;
步骤2:在所述优化的HIV中和抗体的重链序列的5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入Sac I位点;
在所述优化的HIV中和抗体的轻链序列的5’末端加入Xbal限制性酶切位点,在3’末端加入Sac I位点;
克隆到pUC57载体中,分别获得pIbali-H,pIbali-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-Ibali-H,p35S-Ibali-L。
7.根据权利要求2至6任一项所述的表达载体在表达HIV中和抗体中的应用。
8.一种植物作为宿主表达HIV中和抗体的方法,其特征在于,将如权利要求2至6任一项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HIV中和抗体。
9.根据权利要求8所述的方法,其特征在于,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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