CN110229847A - 生菜作为宿主在表达乙肝疫苗中的应用 - Google Patents
生菜作为宿主在表达乙肝疫苗中的应用 Download PDFInfo
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- CN110229847A CN110229847A CN201910549718.9A CN201910549718A CN110229847A CN 110229847 A CN110229847 A CN 110229847A CN 201910549718 A CN201910549718 A CN 201910549718A CN 110229847 A CN110229847 A CN 110229847A
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Abstract
本发明涉及生物技术领域,特别涉及利用植物来表达人乙型疫苗(即乙肝病毒(Hepatitis B virus,HBV)表面抗原蛋白)。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的真空渗透方法来表达人乙肝疫苗。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法确定重组人乙肝疫苗蛋白成功表达。表达的重组蛋白通过自组装成病毒样粒子后免疫新西兰白兔,获得的血清通过酶联免疫吸附剂测定法(ELISA)和假病毒颗粒中和试验证明植物表达的人乙肝疫苗具有生物学活性。
Description
技术领域
本发明涉及生物技术领域,特别涉及生菜作为宿主在表达乙肝疫苗中的应用。
背景技术
乙肝疫苗是一种预防乙型肝炎的疫苗。即从乙型肝炎病毒携带者血浆中分离乙肝表面抗原(HbsAg),经处理后而制成。乙肝疫苗接种后,可刺激免疫系统产生保护性抗体,这种抗体存在于人的体液之中,乙肝病毒一旦出现,抗体会立即作用,将其清除,阻止感染,并不会伤害肝脏,从而使人体具有了预防乙肝的免疫力。因此乙肝疫苗成为生活中不可缺少的一把保护伞。
乙型肝炎病毒(HBV)是一种很小的病毒,它属于嗜肝脱氧核糖核酸(DNA)病毒组中的一个成员。病毒颗粒由外膜和内核两部分组成,完整的HBV颗粒是直径42nm的球形颗粒,其外膜厚7nm,由蛋白质和膜脂质组成,称作乙型肝炎表面抗原(HBsAg)。由于其最早在澳大利亚发现,所以曾被称为“澳大利亚抗原”,简称“澳抗”。中心部分的直径约28nm,为病毒的核心,其中包括核心抗原(HBcAg)和e抗原(HBeAg),内核中心含有病毒基因(DNA)和DNA多聚酶。
HBV DNA的两链长短不一,长链(L)完整,为负链,长度恒定,约3200个核苷酸。短链(S)为正链,长度可变,约为长链长度的50~100%,链的增生按5′-3′顺序进行。在不同分子中短链3′端的位置是可变的,而短链和长链的5′端位置固定点为粘性末端,通过250~300个核苷酸碱基配对,以维持DNA分子的环状结构。在粘性末端两侧,两链5′端各有一个由11个碱基组成的直接重复序列(Direct repeat DR)-5′TTCACCTCTCC,该DR位于第1824个核苷酸者称DR1,位于第1590个核苷酸者称DR2,在病毒复制中起作用
乙型肝炎病毒侵入机体后,在肝细胞内进行复制繁殖。乙型肝炎病毒基因组DNA在肝细胞核内进行复制,转录,在合成核心颗粒后,被转运到肝细胞浆内,在通过内质网和细胞膜时合成其外壳部分,并以发芽的形式释放出肝细胞。
人体感染乙型肝炎病毒后,可引起细胞免疫及体液免疫应答,并激发自身免疫反应及免疫调节功能紊乱,致使病变的肝细胞产生或释放大量正常或异常的蛋白质,进一步促使免疫损害加重,使病情不断发展。
乙型肝炎疫苗的研制先后经历了血源性疫苗和基因工程疫苗阶段。目前基因工程乙肝疫苗技术已相当成熟,中国自行研制的疫苗经多年观察证明安全有效,亦已批准生产。乙肝疫苗的发展与应用,将对乙型肝炎的预防和控制起重要作用。
人类是乙型肝炎病毒的唯一宿主,当安全、有效、足量的乙型肝炎疫苗提供接种使用时,肯定将对控制乙型肝炎病毒的传播起到决定性的作用。中国已实施新生儿国家免疫规划,乙肝疫苗即是免费且强制性接种的疫苗之一。
目前使用的乙肝疫苗是基因工程疫苗--重组酵母乙肝疫苗和重组CHO乙肝疫苗,其主要成分是乙肝病毒的表面抗原,即一种乙肝病毒外衣壳蛋白,并非完整病毒。这种表面抗原不含有病毒遗传物质,不具备感染性和致病性,但保留了免疫原性,即刺激机体产生保护性抗体的能力。乙肝表面抗原曾经是从乙肝病毒携带者的血液,经纯化灭活等严格工序制备而来,即血源性疫苗,该疫苗由于安全、来源和成本等原因已被淘汰(如,有引起血源性疾病的嫌疑和浪费大量的血浆)。基因工程乙肝疫苗是采用基因工程技术生产的,即构建含有乙肝表面抗原基因的重组质粒,然后转染相应的宿主细胞,如酵母、CHO细胞,生产乙肝表面抗原蛋白。利用重组酵母生产的叫重组酵母乙肝疫苗,利用CHO细胞生产的叫重组CHO乙肝疫苗,剂量为每支5微克。它们都可以用于预防所有已知亚型的乙肝病毒感染,可以放心选用。
植物作为药物蛋白质的表达和生产系统,已经研究了近三十年。除了低成本和产量高等的优点外,基于植物的表达系统还降低了人和动物病原体从产生蛋白质的过程中传播到人类的风险。此外,植物真核蛋白内膜表达系统和分泌途径类似于哺乳动物细胞。植物表达系统可大量生产大分子量以及多亚基的药物蛋白质,且优于原核生物表达系统,比如大肠杆菌表达系统。如需要翻译后修饰的,糖基化的蛋白,以及需要组装的其它药物蛋白,其不能用原核系统实现。利用植物生产的药用蛋白作为生物试剂已经商业化。2012年批准的Elelyso是在美国上市的第一个也是迄今唯一一个转基因植物生产的新药。主要成分是葡糖脑苷脂酶,用于治疗1型戈谢病,而这种蛋白是利用胡萝卜生产的。在过去十年中,人们对药物蛋白质的需求急剧增加,所以FDA批准用于临床试验的植物药用蛋白质的数量也在不断增加。
植物瞬时表达系统可以大量生产重组蛋白用于临床研究或者应对突发性疾病。2014年,唯一用于有效抵抗埃博拉病毒爆发的抗体治疗药物,ZMappTM,就是农杆菌渗透方法在烟草叶片中生产。ZMapp的功效和安全性为推动植物制药业的行业开辟了道路。目前,烟草是最流行也是最成熟的瞬时表达蛋白的宿主植物,并且已经开发了各种载体和农杆菌渗透方法,用于在短时间内进行大规模生产。然而,烟草具有高纤维含量和潜在的有毒化合物,如生物碱尼古丁,显著增加了下游纯化过程中的成本,极大地阻碍了植物外源蛋白药物的进一步发展。与烟叶系统相比,生菜中含有更少的酚类和有毒化合物,因此,将生菜作为宿主在表达乙肝疫苗具有重要的现实意义。
发明内容
有鉴于此,本发明提供生菜作为宿主在表达乙型肝炎病毒疫苗中的应用。本发明利用生菜作为重组蛋白生产的有效平台,消除了植物生长周期,大大节省了前期培育植物的时间。本发明用生菜系统表达了乙肝疫苗,并且在温和的条件下成功分离出有活性的外源蛋白,证明生菜表达平台可以用来生产重组乙肝疫苗活性蛋白。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了生菜作为宿主在表达乙型肝炎病毒表面抗原中的应用;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。
本发明还提供了植物作为宿主在制备乙型肝炎病毒疫苗中的应用;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。
在本发明的一些具体实施方案中,所述乙型肝炎病毒疫苗为重组HBV表面抗原。
本发明还提供了一种表达载体,包括乙型肝炎病毒表面抗原的核苷酸序列和双元植物表达载体;具体包括人HBV病毒表面抗原的核苷酸序列和双元植物表达载体。
在本发明的一些具体实施方案中,所述表达载体中所述乙型肝炎病毒为HBV。
在本发明的一些具体实施方案中,所述乙型肝炎病毒的表面抗原大蛋白的核苷酸序列如SEQ ID No.1所示;所述乙型肝炎病毒的表面抗原大蛋白的氨基酸序列如SEQ IDNo.2所示;所述乙型肝炎病毒表面抗原小蛋白核苷酸序列如SEQ ID No.3所示;所述乙型肝炎病毒的表面抗原小蛋白的氨基酸序列如SEQ ID No.4所示。
在本发明的一些具体实施方案中,所述乙型肝炎病毒的表面抗原大蛋白的核苷酸序列如SEQ ID No.1所示;所述乙型肝炎病毒的表面抗原大蛋白的氨基酸序列如SEQ IDNo.2所示;所述乙型肝炎病毒的表面抗原小蛋白的核苷酸序列如SEQ ID No.3所示;所述乙型肝炎病毒的表面抗原小蛋白的氨基酸序列如SEQ ID No.4所示。
在本发明的一些具体实施方案中,所述表达载体的构建方法包括如下步骤:
步骤1:在乙型肝炎病毒(HBV)衣壳蛋白编码基因的核苷酸序列的5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入Sacl限制性酶切位点;
步骤2:取步骤1获得的修饰后的乙型肝炎病毒的核苷酸序列由金斯瑞公司克隆到载体pUC57载体中,分贝获得克隆载体pUC57-HBV-L和pUC57-HBV-S;
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LHBs(编码衣壳大蛋白)和SHBs(编码衣壳小蛋白),克隆至双元植物表达载体pCam35S,分别获得表达载体pCam35S-HBV-L和pCam35S-HBV-S。
具体的,本发明提供的表达载体的构建方法为:人乙肝病毒(NC_003977.2)基因由金斯瑞公司合成。在HBV序列5’末端加入XbaI限制性酶切位点,在3’末端加入SacI位点,并由金斯瑞公司克隆到pUC57载体中。人乙肝病毒(HBV)基因片段通过Xbal/Sacl从pUC57-HBVs中分离,并克隆到双元植物表达载体pCam35S,产生瞬时表达载体pCam35S-HBV-L和pCam35S-HBV-S。将植物表达构建体分别通过用Multiporator(Eppendorf,Hamburg,Germany)电穿孔转化到根癌农杆菌EHA105中。将所得菌株均匀地涂布在含有卡那霉素抗生素(50mg/L)的选择性YEP(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 MgSO4,15g/L琼脂,pH 7.2)平板上。在黑暗中28℃孵育48小时后,挑取单菌落接种到200mL YEB(不含有琼脂)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28℃孵育48小时以上,至OD600值达到1.5。然后收集培养液,离心(5000rpm)10min。将农杆菌细胞重悬在渗透培养基(10mM MES,10mM MgSO4)中至OD600为0.2。
所述克隆HBV基因片段(图1),并且构建两种基于双元植物表达载体p35S-HBV(图2)。
在本发明中,HBV表面抗原核苷酸序列(NC_003977.2,)如SEQ ID No.1所示;HBV表面抗原氨基酸序列如SEQ ID No.2(大蛋白)、SEQ ID No.3(小蛋白)所示。
在本发明中,所述乙型肝炎病毒的表面抗原大蛋白的核苷酸序列如SEQ ID No.1所示;所述乙型肝炎病毒的表面抗原大蛋白的氨基酸序列如SEQ ID No.2所示;所述乙型肝炎病毒的表面抗原小蛋白的核苷酸序列如SEQ ID No.3所示;所述乙型肝炎病毒的表面抗原小蛋白的氨基酸序列如SEQ ID No.4所示。
本发明还提供了所述表达载体在表达乙型肝炎病毒表面抗原的应用;具体的提供了所述表达载体在表达人乙型肝炎病毒表面抗原的应用。在本发明的一些具体实施方案中,所述乙型肝炎病毒为HBV。
本发明还提供了所述的表达载体在制备乙型肝炎病毒疫苗中的应用;具体的提供了所述表达载体在表达人乙型肝炎病毒疫苗中的应用。在本发明的一些具体实施方案中,所述乙型肝炎病毒为HBV。
本发明还提供了一种植物尤其是生菜作为宿主表达乙型肝炎病毒表面抗原的方法,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尤其是生菜组织后,提取、分离蛋白质,获得乙型肝炎病毒表面抗原。
本发明还提供了一种植物尤其是生菜作为宿主表达乙型肝炎病毒疫苗的方法,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尤其是生菜组织后,提取、分离蛋白质,获得乙型肝炎病毒疫苗。
在本发明的一些具体实施方案中,所述方法中所述乙型肝炎病毒为HBV。
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;
避光处理4d。
在本发明的一些具体实施方案中,所述农杆菌为根癌农杆菌EHA105。
具体的,农杆菌介导真空渗透的方法为:将制备好的农杆菌培养悬浮液置于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。然后将生菜组织从渗透液中轻轻取出,并用蒸馏水顺序冲洗三次,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。
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。为了增加浸入叶组织的土壤农杆菌的数量,使用剪刀将10%的生菜从头切掉使得生菜叶组织尽可能的浸润在渗透液中,和释放。与更长的真空暴露时间相比,该方法减少了叶片组织坏死。
在本发明的一些具体实施方案中,提取、分离蛋白质具体为:
经农杆菌真空渗透的生菜样品用搅拌器破碎,并用体积比为1:1比例的提取缓冲液(100mM KPi,pH7.8;5mM EDTA;10mM β-巯基乙醇)搅拌机中高速匀浆1~2min。将匀浆物调节至pH为8.0,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;2mM EDTA;10m β-巯基乙醇)中并在4℃下储存。
收集从农杆菌真空渗透生菜提取的纯化蛋白质,取样品(5uL)热变性(95℃)上样缓冲液(Biorad,Hercules,CA,USA)在12%Bis-Tris Plus SDS-凝胶(ThermoFisherScientific,Waltham,MA,USA)跑电泳,然后用考马斯亮蓝G250(Biorad)染色后对凝胶进行拍照。对于重组HBV蛋白的Western Blot蛋白印迹杂交,10ul的重组样品在15%Bis-Tris Plus聚丙烯酰胺凝胶上分离,并将其电泳转移到聚偏二氟乙烯(PVDF)膜上,用抗HBV的抗体(Abcam)进行免疫反应,稀释1:5000和辣根过氧化物酶(HRP)标记的山羊抗兔IgG(Beyotime),稀释度为1:10000,并使用ECL plus(Amersham Biosciences)显色,对显示图像进行拍照。
重组HBV疫苗蛋白的免疫原性通过酶联免疫吸附剂测定法(ELISA)检测。重组蛋白HBV病毒样蛋白颗粒利用HBV抗体进行检测。经过PBS缓冲液漂洗之后,1:10,000加入羊抗鼠或羊抗兔的二抗(IgG-HRP,Bio-Rad)室温孵育45分钟,然后加入2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(Roche).用ELISA酶标仪(Opsys MR,DynexTechnologies)读取405nm的吸光值。
植物来源的重组蛋白的下游加工通常较难并且昂贵,因为纤维素和细胞壁难以裂解以及较多的植物次生代谢产物。本发明用搅拌机搅拌匀浆,大大节省匀浆成本以及工艺。重组HBV表面抗原外壳蛋白经过SDS-PAGE分离我们在泳道中观察到估计分子量大约为46KD和24KD的条带(图3)。Western印迹分析也检测到相应的条带(图4),而阴性对照(NC)中检测不到相关信号。
通过ELISA法检测生菜表达的HBV重组蛋白免疫原活性。用HBV特异的抗体去滴定检测表达的HBV重组蛋白,可以明显,随着抗体稀释倍数增加,405nm处的吸光值线性下降,而用未免疫的空白血清则没有这种线性关系(图5)。这些结果表明,通过生菜系统瞬间表达的重组HBV蛋白具有生物学活性,生菜系统可能是生物活性重组药物蛋白质批量生产的合适的生物反应器。
用于真空农杆菌渗透的烟草植物的生长时间通常为4至6周。本发明利用生菜作为重组蛋白生产的有效平台,消除了植物生长周期长的影响,大大节省了前期培育植物的时间。本发明用生菜系统表达了人乙肝疫苗,并且在温和的条件下成功分离出有活性的外源蛋白,证明生菜表达平台可以用来生产人乙肝疫苗。
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示人乙肝病毒蛋白克隆载体(金斯瑞公司构建合成);
图2示植物瞬时表达载体p35S-HBVs构建流程;利用限制性内切酶(XbaI/SacI)双酶切,从图1克隆载体分别切下HBV片段,连接入pCam35S的XbaI/SacI位点,生成植物瞬时表达载体p35S-HBV;
其中,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示例用聚丙烯酰胺凝胶电泳(SDS-PAGE)检测纯化的重组HBV表面抗原蛋白;
图4示Western印记杂交检测纯化的重组人HBV表面抗原蛋白;
图5示重组蛋白免疫原性滴定检测结果;其中,图5(A)示LHBs免疫原性滴定检测结果;图5(B)示SHBs免疫原性滴定检测结果。
具体实施方式
本发明公开了生菜作为宿主在表达乙型肝炎病毒疫苗中的应用,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。
本发明研究表明,生菜系统可以是有效的表达平台,为快速,瞬时表达重组蛋白质提供了方法。真空农杆菌渗透方法简单,快速,减少叶片坏死,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。并且由于不需要特殊设备或液氮,成本效益更高。本发明证明该方法可以用于短时间内大规模生产HBV重组蛋白。
本发明提供了生菜作为宿主在表达乙型肝炎病毒疫苗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表达,本发明中HBV(NC_003977.2)由金斯瑞公司合成。在HBV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。人HBV基因片段通过Kpnl/Sacl从pUC57-INS中分离,并克隆到双元植物载体pCam35S,分别产生瞬时表达载体p35S-HBV。将植物表达构建体分别通过用Multiporator(Eppendorf,Hamburg,Germany)电穿孔转化到根癌农杆菌EHA105中。将所得菌株均匀地涂布于含有卡那霉素抗生素(50mg/L)的选择性YEP平板上。在黑暗中28℃孵育48小时后,挑取单菌落接种到200mL 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28℃孵育48小时以上。通过添加YEB培养基测量OD600值并调节至1.5。然后收集培养液,离心(5000rpm)10min。将农杆菌细胞重悬在渗透培养基(10mMMES,10mM MgSO4)中至O.D.600为0.2。
实施例2农杆菌介导的真空渗透
本发明优化了农杆菌真空渗透的方法。将制备好的农杆菌培养悬浮液置于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。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。
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。为了增加浸入叶组织的土壤农杆菌的数量,使用剪刀将10%的生菜从头切掉使得生菜叶组织尽可能的浸润在渗透液中,和释放。与更长的真空暴露时间相比,该方法减少了叶片组织坏死。
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-巯基乙醇)搅拌机中高速匀浆1~2min。将匀浆物调节至pH为8.0,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60分钟。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;2mM EDTA;10mM β-巯基乙醇)中并在4℃下储存。
实施例4 SDS-PAGE凝胶电泳以及Western Blot蛋白印迹杂交
收集从农杆菌真空渗透生菜提取的纯化蛋白质,取样品(5uL)热变性(95℃)加载缓冲液(Biorad,Hercules,CA,USA)在4~12%Bis-Tris Plus SDS-凝胶(ThermoFisher Scientific,Waltham,MA,USA)跑电泳,然后用考马斯亮蓝G250(Biorad)染色后对凝胶进行拍照。对于重组HBV蛋白的Western Blot蛋白印迹杂交,10ul的重组样品在15%Bis-Tris Plus聚丙烯酰胺凝胶上分离,并将其电泳转移到聚偏二氟乙烯(PVDF)膜上,用抗HBV的抗体分别进行免疫反应,稀释1:10000和辣根过氧化物酶(HRP)标记的山羊抗兔IgG(Beyotime),稀释度分别为1:10000,并使用ECL plus(AmershamBiosciences)显现,对显示图像进行拍照。
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HBV蛋白经过SDS-PAGE分离我们在泳道中观察到估计分子量对应的条带(图3),Western印迹分析也检测到大约46kD和24KD对应的条带(图4),对应分子量区域都能检测到信号,而阴性对照则检测不到任何信号(图4中的NC)。
对纯化所得乙肝表面抗原蛋白采用ELISA进行检测,如图5所示,随着抗体稀释倍数增加,405nm处的吸光值对应下降,而阴性对照只有很低的吸光值,表明没有相关功能蛋白。
实施例5重组乙肝疫苗的制备和疫苗效果检测
生菜表达的乙肝表面抗原纯化产物经检定合格后0.22μm除菌过滤。
甲醛处理,在过滤过的纯化产物中加入终浓度为200μg/ml的甲醛,置37℃保温72小时。
甲醛处理后的乙肝表面抗原经超滤、浓缩、除菌过滤后即为原液。
根据原液蛋白质含量进行稀释,是乙肝表面抗原含量为10μg/ml或20μg/ml。加入氢氧化铝佐剂吸附后,可加入硫柳汞作为防腐剂,即为半成品。
按照“生物制品分批规程”规定进行分批。
按照“生物制品分装和冻干规程”规定进行分装。
按照“生物制品包装规程”规定进行包装。
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人HBV疫苗蛋白。
实验组1:本发明提供的植物生产乙型肝炎病毒表面抗原;
对照组1:利用酵母细胞生产乙型肝炎病毒表面抗原;
实验组2:利用烟叶生产乙型肝炎病毒表面抗原;
表1乙型肝炎病毒表面抗原
*示与对照组1相比P≤0.05;**示与对照组1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1的动物系统相比,本发明提供的生菜瞬时表达乙型肝炎病毒表面抗原,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,生菜瞬时表达乙型肝炎病毒表面抗原,显著(P≤0.05)缩短了生产周期,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,烟叶瞬时表达乙型肝炎病毒表面抗原比动物系统显著(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乙型肝炎病毒表面抗原。
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> 生菜作为宿主在表达乙肝疫苗中的应用
<130> MP1900922
<160> 4
<170> SIPOSequenceListing 1.0
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<213> 乙型肝炎病毒表面抗原大外壳蛋白(surface antigen large coat proteinof Hepatitis B virus )
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tggccagacg ccaacaaggt aggagctgga gcattcgggc tgggtttcac cccaccgcac 180
ggaggccttt tggggtggag ccctcaggct cagggcatac tacaaacttt gccagcaaat 240
ccgcctcctg cctccaccaa tcgccagtca ggaaggcagc ctaccccgct gtctccacct 300
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caagatccca gagtgagagg cctgtatttc cctgctggtg gctccagttc aggaacagta 420
aaccctgttc tgactactgc ctctccctta tcgtcaatct tctcgaggat tggggaccct 480
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gggtttttct tgttgacaag aatcctcaca ataccgcaga gtctagactc gtggtggact 600
tctctcaatt ttctaggggg aactaccgtg tgtcttggcc aaaattcgca gtccccaacc 660
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ccatttgttc agtggttcgt agggctttcc cccactgttt ggctttcagt tatatggatg 1080
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atggagaaca tcacatcagg attcctagga ccccttctcg tgttacaggc ggggtttttc 60
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tgccggacct gcatgactac tgctcaagga acctctatgt atccctcctg ttgctgtacc 420
aaaccttcgg acggaaattg cacctgtatt cccatcccat catcctgggc tttcggaaaa 480
ttcctatggg agtgggcctc agcccgtttc tcctggctca gtttactagt gccatttgtt 540
cagtggttcg tagggctttc ccccactgtt tggctttcag ttatatggat gatgtggtat 600
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tgtctttggg tatacattta a 681
<210> 4
<211> 226
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<213> 乙型肝炎病毒表面抗原小外壳蛋白(surface antigen small coat proteinof Hepatitis B virus )
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Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala
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Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp
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Tyr Ile
225
Claims (10)
1.植物作为宿主在表达乙型肝炎病毒表面抗原中的应用;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.植物作为宿主在制备乙型肝炎病毒疫苗中的应用;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。
3.一种表达载体,其特征在于,包括乙型肝炎病毒表面抗原的核苷酸序列和双元植物表达载体。
4.如权利要求3所述的表达载体,其特征在于,所述乙型肝炎病毒的表面抗原大蛋白的核苷酸序列如SEQ ID No.1所示;所述乙型肝炎病毒的表面抗原大蛋白的氨基酸序列如SEQID No.2所示;所述乙型肝炎病毒的表面抗原小蛋白的核苷酸序列如SEQ ID No.3所示;所述乙型肝炎病毒的表面抗原小蛋白的氨基酸序列如SEQ ID No.4所示。
5.根据权利要求3或4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:在乙型肝炎病毒(HBV)的核苷酸序列的5’末端分别加入Xbal限制性酶切位点,在3’末端分别加入Sacl限制性酶切位点;
步骤2:取步骤1获得的修饰后的乙型肝炎病毒的核苷酸序列克隆到载体pUC57载体中,分别获得克隆载体pUC57-HBV-L和pUC57-HBV-S;
步骤3:通过Xbal/Sacl分别从步骤2所得的克隆载体pUC57-HBV-L和pUC57-HBV-S中获得基因片段LHBs和SHBs,克隆至双元植物载体pCam35S,分别获得表达载体pCam35S-HBV-L和pCam35S-HBV-S。
6.根据权利要求3至5任一项所述的表达载体在表达乙型肝炎病毒表面抗原中的应用。
7.根据权利要求3至5任一项所述的表达载体在制备乙型肝炎病毒疫苗中的应用。
8.植物作为宿主表达乙型肝炎病毒表面抗原的方法,其特征在于,将如权利要求3至5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,获得乙型肝炎病毒表面抗原。
9.植物作为宿主制备乙型肝炎病毒疫苗的方法,其特征在于,将如权利要求3至5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,获得乙型肝炎病毒疫苗。
10.根据权利要求8或9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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