CN111565747A - 与猪fc片段融合的抗原,以及包含该抗原的疫苗组合物 - Google Patents
与猪fc片段融合的抗原,以及包含该抗原的疫苗组合物 Download PDFInfo
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- CN111565747A CN111565747A CN201880085083.5A CN201880085083A CN111565747A CN 111565747 A CN111565747 A CN 111565747A CN 201880085083 A CN201880085083 A CN 201880085083A CN 111565747 A CN111565747 A CN 111565747A
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Abstract
本发明涉及与猪Fc片段融合的抗原、通过将Fc片段与各种抗原结合而具有自佐剂效应(self‑adjuvanting effect)的疫苗组合物以及产生该抗原的方法。
Description
技术领域
本发明涉及一种与猪Fc片段融合的抗原,包含该抗原的疫苗组合物等。
背景技术
预防传染病的疫苗能诱导针对病原体的特异性和有效的免疫反应,对预防各种传染病的疫苗需求持续增加。近年来,各种类型的疫苗如灭活疫苗、减毒活疫苗、自体疫苗等得到了发展。在这方面,由活细菌或减毒细菌生产的疫苗在预防方面非常有效,但由于存在退化(regression)风险、自我复制等问题,常常报告存在安全问题,使用死细菌的疫苗含有脂多糖(LPS)等有毒物质,或者有可能存在活细菌的风险。因此,为解决这些局限性而开发的DNA疫苗,通过插入宿主的基因组在致突变或致癌方面发挥作用的可能性也被提高。因此,利用纯化的抗原研制亚单位疫苗近年来得到了积极的发展。由于使用纯化的抗原,这种亚单位疫苗比其它疫苗更稳定,但免疫原性差,因此需要有效的佐剂来增强免疫原性。使用佐剂可以显著减少所使用的抗原量,因此,佐剂不仅可以在流行病蔓延等情况下对快速生产疫苗起决定性作用,而且还可以减少额外接种疫苗的次数等,从而显著降低成本和不便性。然而,很少有佐剂被批准用于疫苗。第一种佐剂“明矾”于1932年被批准用于人类疫苗,迄今为止应用最为广泛。从那时起,MF59、AS03等已开发并获得许可,但仍很少有佐剂获得用于疫苗的许可(韩国专利注册号10-0517114)。
因此,预期开发既能显著提高疫苗免疫原性,又稳定的佐剂将在疫苗领域的开发中发挥非常重要的作用。
发明内容
技术问题
本发明旨在解决上述问题,本发明的目的是提供一种与猪Fc片段融合的抗原、包括该抗原的疫苗组合物和制备该抗原的方法。
然而,本发明要解决的技术问题不限于上述技术问题,对于本领域技术人员来说,通过以下描述,其它未提及的技术问题将变得显而易见。
技术方案
本发明提供一种疫苗组合物,所述疫苗组合物包含作为活性成分的与由SEQ IDNO:4代表的猪Fc片段融合的抗原。
本发明还提供一种预防或治疗传染病的方法,其包括向个体施用包含与由SEQ IDNO:4代表的猪Fc片段融合的抗原作为活性成分的组合物。
本发明还提供一种组合物用于预防或治疗传染病的用途,所述组合物包含作为活性成分的与由SEQ ID NO:4代表的猪Fc片段融合的抗原。
在本发明的一个实施方式中,抗原可具有自佐剂效应,并通过与Fc片段融合而增加溶解度。融合可以是抗原通过肽键连接到Fc片段的N-末端和/或C-末端的形式,但只要是Fc片段和抗原可结合的形式,就不特别受限。
本发明还提供具有自佐剂效应的重组抗原的表达载体,所述表达载体包括编码由SEQ ID NO:4代表的猪Fc片段的多核苷酸和编码抗原的多核苷酸。
在本发明的一个实施方式中,在表达载体中,启动子基因、编码Fc片段的多核苷酸和编码抗原的多核苷酸;或者启动子基因、编码抗原的多核苷酸和编码Fc片段的多核苷酸可以以此顺序顺序地连接。
在本发明的另一实施方式中,启动子是来自花椰菜花叶病毒的35S启动子、来自花椰菜花叶病毒的19S RNA启动子、植物的肌动蛋白启动子、泛素蛋白启动子、巨细胞病毒(CMV)启动子、猿猴病毒40(SV40)启动子,呼吸合胞病毒(RSV)启动子、延伸因子-1α(EF-1α)启动子、pEMU启动子、MAS启动子、组蛋白启动子或Clp启动子,但本发明不限于此。
在本发明的另一个实施方式中,抗原蛋白可以是抗原、抗体、抗体片段、结构蛋白、调节蛋白、转录因子、毒素蛋白、激素、激素类似物、细胞因子、酶、酶片段、酶抑制剂、转运蛋白、受体、受体片段、生物防御诱导剂、贮藏蛋白、运动蛋白、开发蛋白或报告蛋白,但只要其能够通过在体内作为抗原诱导免疫应答,则不特别受限。
在本发明的另一实施方式中,表达载体可进一步包括编码伴侣结合蛋白(BiP)的多核苷酸、编码His-Asp-Glu-Leu(HDEL)肽的基因、M17的5'非翻译区(UTR)位点基因等。
在本发明的另一实施方式中,表达载体增加了融合Fc片段的靶抗原的表达量,并增加了靶抗原的溶解度。
本发明还提供了用所述表达载体转化的转基因生物体。
在本发明的一个实施方式中,转基因生物体可以是微生物,例如大肠杆菌(Escherichia coli)、芽孢杆菌(Bacillus)、沙门氏菌(Salmonella)、酵母等,昆虫细胞,包括人类细胞的动物细胞,动物,例如小鼠、大鼠、狗、猴子、猪、马、牛等,根癌农杆菌(Agrobacterium tumefaciens)、植物等,植物的例子包括粮食作物,包括水稻、小麦、大麦、玉米、豆类、土豆、红豆、燕麦和高粱;蔬菜作物包括拟南芥(Arabidopsis thaliana)、大白菜、白萝卜、辣椒、草莓、西红柿、西瓜、黄瓜、卷心菜、甜瓜(oriental melons)、南瓜、大葱(spring onions)、洋葱和胡萝卜;特殊用途作物包括人参、烟草、棉花、芝麻、甘蔗、甜菜、紫苏、花生和油菜;水果作物包括苹果树、梨树、枣树、桃子、葡萄、橘子、柿子、李子、杏和香蕉;花卉包括玫瑰、康乃馨、菊花、百合和郁金香,只要能够用本发明的表达载体进行转化的活体,就不特别受限。
此外,本发明还提供了一种产生重组抗原的方法,其包括:(a)培养转基因生物体;和(b)从转基因生物或培养液中分离和纯化与Fc片段融合的抗原。转基因生物体可以优选为细胞本身或含有细胞的培养物,并且培养液可以优选为通过培养细胞和去除细胞而获得的培养液,但本发明不限于此,可以使用包括本发明的重组抗原的任何培养液。
有益效果
根据本发明的与猪Fc片段融合的抗原具有自佐剂效应,因此可以在不使用额外佐剂的情况下增加抗原本身的免疫原性,并且可以增加抗原的溶解度和稳定性,从而促进抗原的分离和储存。此外,在根据本发明的表达载体中,可以通过将pFc与各种抗原融合来提高靶抗原的产量,因此预期该表达载体能够高效地生产疫苗。
附图说明
图1是示出根据本发明实施方式的pCAMBIA1300载体图谱的示意图。
图2是示出根据本发明实施方式的用于表达pFc融合VP1重组蛋白的基因的排列的图。
图3示出确认根据本发明实施方式的pFc融合VP1重组蛋白的表达量的蛋白质印迹法结果。
图4示出确认根据本发明实施方式的pFc融合VP1重组蛋白的稳定性的蛋白质印迹法结果。
图5示出确认根据本发明实施方式的pFc融合VP1重组蛋白的溶解度的蛋白质印迹法结果。
图6示出确认根据本发明实施方式的pFc融合GP5重组抗原的溶解度的蛋白质印迹法结果。
图7示出了确认根据本发明实施方式的pFc融合PCV2重组蛋白的溶解度和产量的蛋白质印迹法结果。
图8示出了确认根据本发明实施方式的pFc融合E2重组抗原的产量的结果。
图9示出了确认根据本发明实施方式的pFc融合的E2重组抗原的免疫原性的结果,其中该抗原给予一次。
图10示出根据本发明实施方式的pFc融合E2重组抗原的免疫原性的确认结果,其中该抗原给予两次。
具体实施方式
在本发明中,已证实当各种抗原与猪Fc片段结合时,该片段不仅增加了抗原的表达量和产量,而且增加了抗原的溶解度和稳定性,本发明的目的是提供一种与猪Fc片段融合的抗原,一种表达载体,所述表达载体包括编码猪Fc片段的多核苷酸和编码抗原的多核苷酸,以及使用所述表达载体生产重组抗原的方法。
如本文所使用的,术语“Fc片段”是指不具有抗原结合位点的Fc片段,当免疫球蛋白用木瓜蛋白酶消化时,其中只有重链(H链)部分通过S-S键连接,并且本发明的Fc片段优选为猪Fc片段,更优选由SEQ ID NO:4代表的猪Fc片段,但本发明不限于此,可以使用当与靶抗原融合时增加靶抗原的表达量和溶解度的任何Fc片段。另外,本发明的SEQ ID NO:4的Fc片段的变体在本发明的范围内。具体而言,该基因可包括与SEQ ID NO:4的碱基序列具有90%或更多、优选95%或更多、更优选98%或更多同源性的碱基序列。相对于多核苷酸的“序列同源性%”通过将最佳排列的序列与比较区域进行比较来确定,并且与参考序列(没有添加或删除)相比,相对于序列的最佳排列,比较区域中的多核苷酸序列的一部分可以包括添加或删除(即间隙)。
如本文所使用的,术语“抗原”统称为在体内诱导免疫反应的任何物质,且该抗原优选为病毒、化合物、细菌、花粉、癌细胞、虾或其一部分的肽或蛋白质,只要该物质能够在体内诱导免疫反应,就不特别受限。
如本文所使用的,术语“疫苗”是指生物制剂,其包括在体内引起免疫反应的抗原和通过注射或口服给人或动物而使活体获得免疫以预防传染病的免疫原。动物是指人或非人动物,非人动物是指猪、牛、马、狗、山羊、绵羊,但本发明不限于此。
在本文中,术语“靶蛋白”是指利用基因工程方法生成的蛋白质,优选地,靶蛋白是需要大量生产的市售抗原,更优选地,是抗原、抗体、抗体片段、结构蛋白、调节蛋白、转录因子、毒素蛋白、激素、激素类似物、细胞因子、酶、酶片段、酶抑制剂、转运蛋白、受体、受体片段、生物防御诱导剂、贮藏蛋白、运动蛋白、开发蛋白或报告蛋白,但本发明不限于此,可以使用能够用本发明的表达载体生产的任何蛋白质。
如本文所使用的,术语“表达载体”是指能够表达由插入载体中的异源核酸编码的肽或蛋白质的载体,并且优选地是指构建用于表达猪Fc片段融合靶抗原的载体。本文中使用的术语“载体”是指用于在体外、离体(ex vivo)或体内将碱基引入和/或转移到宿主细胞中的任何载体,并且可以是指可以附加另一个DNA片段以实现附加片段的复制的复制子。术语“复制子”是指作为体内DNA复制的自主单元,即能够通过自我调节进行复制的任何遗传单元(例如,质粒、噬菌体、粘粒(cosmid)、染色体、病毒等)。本发明的表达载体可以优选地包括作为RNA聚合酶结合的转录起始因子的启动子、用于调节转录的任意操纵子序列、编码合适的mRNA核糖体结合位点的序列、调节转录和翻译终止的序列,终止子等。更优选地,所述表达载体可进一步包括M17的5'UTR位点基因、用于将靶蛋白传送到囊泡的BiP基因、用于最小化蛋白质降解以使蛋白质能够稳定地保持在囊泡中的HDEL基因等。更优选地,所述表达载体还可以包括用于容易地分离重组蛋白的标签基因、用于选择抗生素抗性基因或选择转基因生物体等的标记基因,等。
可另外包括用于标签的基因以便于分离本发明的Fc片段之外的物质,其为标签蛋白,其示例可包括Avi标签、钙调素标签、多聚谷氨酸标签、E标签、FLAG标签、HA标签、His标签、Myc标签、S标签、SBP标签、IgG Fc标签、CTB标签、Softag 1标签、Softag 3标签、Strep标签、TC标签、V5标签、VSV标签、Xpress标签等。用于选择的标记基因的示例可包括抗除草剂(例如草甘膦和草铵膦)的基因、抗抗生素(如卡那霉素、G418、博来霉素、潮霉素和氯霉素)的基因、aadA基因等,启动子的示例可包括pEMU启动子、MAS启动子、组蛋白启动子、Clp启动子、源自花椰菜花叶病毒的35S启动子、源自花椰菜花叶病毒的19S RNA启动子、植物的肌动蛋白启动子、泛素蛋白启动子、巨细胞病毒(CMV)启动子、猿猴病毒40(SV40)启动子、呼吸道合胞病毒(RSV)启动子、延伸因子-1α(EF-1α)启动子等,终止子的示例可包括胭脂碱合酶(NOS)、水稻淀粉酶RAmyl A终止子、菜豆蛋白终止子、根癌农杆菌(Agrobacteriumtumefaciens)的章鱼碱(Octopine)基因终止子和大肠杆菌(E.coli)rrnB1/B2终止子,但这些仅仅是示例,本发明不限于此。
如本文所用,术语“融合抗原”是指由猪Fc片段与靶抗原融合产生的重组蛋白,优选地是指通过与Fc片段融合而增强溶解度的重组抗原,但本发明不限于此,通过与猪Fc片段结合产生的任何重组抗原都可以使用。
如本文所使用的,术语“转化”统称为通过注入DNA改变生物体的遗传特性,术语“转基因生物体”指通过使用分子遗传方法注入外部基因而产生的活生物体,优选地是指由本发明的重组载体转化的活生物体。只要其是活生物体,如微生物、真核细胞、昆虫、动物、植物等,则活生物体并不特别受限,其示例包括但不限于大肠杆菌、沙门氏菌、芽孢杆菌、酵母、动物细胞、小鼠、大鼠、狗、猴子、猪、马、牛、根癌农杆菌和植物。转基因生物体可以使用诸如转化、转染、农杆菌介导的转化方法、基因枪轰击、超声波、电穿孔和聚乙二醇(PEG)介导的转化方法等方法产生,但本发明不限于此,并且可以使用能够注入本发明的载体的任何方法。
如本文所使用的,术语“溶解度”是指靶蛋白或肽可溶解于适于施用于人体的溶剂中的程度。具体地说,溶解度可指示在特定温度下溶质相对于给定溶剂的饱和程度。溶解度可以通过测定溶质的饱和浓度来测量,例如,向溶剂中加入过量的溶质并搅拌和过滤该溶液,然后使用紫外光谱仪、高效液相色谱等测量其浓度,但本发明不限于此。高溶解度更适合于重组蛋白的分离和纯化,抑制重组蛋白的聚集,从而有效地维持重组蛋白的生理活性或药理活性。
如本文所使用的,术语“预防”是指通过施用根据本发明的疫苗组合物抑制或延迟疾病(例如传染病、癌症等)的发病的所有活动。
如本文所使用的,术语“治疗”是指通过施用根据本发明的疫苗组合物而改善或有益地改变传染病、癌症等的症状的所有活动。
如本文所使用的,术语“个体”是指可施用根据本发明的疫苗组合物的受试者,并且该受试者不受特别限制。
本发明的疫苗组合物可配制成口服制剂,例如粉末、颗粒、片剂、胶囊、悬浮液、乳剂、糖浆、气溶胶等,以及无菌注射溶液。可使用常用稀释剂或辅料(例如填充剂、增量剂(extenders)、粘合剂、润湿剂、崩解剂、表面活性剂等)来进行调配。口服给药的固体制剂包括片剂、丸剂、粉末、颗粒、胶囊等,这些固体制剂可通过将类卵磷脂乳化剂与至少一种辅料(例如淀粉、碳酸钙、蔗糖、乳糖、明胶等)混合来配制。除了简单的辅料外,还可以使用诸如硬脂酸镁和滑石粉之类的润滑剂。口服液体制剂可以包括悬浮液、内服液体、乳剂、糖浆等,并且这些液体制剂除了简单的常用稀释剂(例如水和液体石蜡)外,还可以包括各种类型的辅料,例如润湿剂、甜味剂、调味剂、防腐剂等。用于肠胃外给药的制剂包括无菌水溶液、非水溶剂、悬浮液、乳液和冻干制剂。非水溶剂和悬浮液的非限制性实例包括丙二醇、聚乙二醇、植物油(例如橄榄油)和可注射酯(例如油酸乙酯)。此外,疫苗组合物还可包括已知佐剂。尽管本领域已知的任何佐剂可以不受限制地用作佐剂,但疫苗组合物还可以包括例如弗氏完全佐剂或不完全佐剂以增加其免疫原性。
根据通用方法,本发明的疫苗组合物可以以口服制剂的形式配制,例如粉末、颗粒、片剂、胶囊、悬浮液、乳剂、糖浆、气溶胶等、外用制剂、栓剂和无菌注射溶液。
根据本发明的疫苗组合物的给药途径包括但不限于口服给药、静脉给药、肌肉给药、动脉内给药、髓内给药、硬膜内给药、心脏内给药、经皮给药、皮下给药、腹腔给药、鼻腔给药、肠道给药、局部给药、舌下给药、直肠给药。疫苗组合物可以口服或肠胃外给药。本文中使用的术语“肠胃外”意指包括皮下、皮内、静脉、肌肉、关节内、囊内(intrabursal)、胸骨内、硬膜内、病灶内和颅内注射或注射技术。本发明的疫苗组合物也可以直肠给药用栓剂的形式施用。
根据本发明的疫苗组合物的剂量是根据个体的年龄、体重、性别、身体状况等来选择的。根据存在的作为免疫原的重组蛋白和辅料,诱导个体免疫保护反应而不产生任何副作用所需的量可能有所不同。一般而言,相对于1毫升无菌溶液,每一剂量包括本发明的重组蛋白的量为0.1微克至1000微克,优选为0.1微克至100微克。如有需要,可在最初剂量的疫苗组合物后进行任意重复的抗原刺激。
如本文使用的,术语“佐剂”通常是指增强针对抗原的体液和/或细胞免疫应答的任何物质,并且术语“自佐剂反应”是指与现有抗原相比,重组抗原自身增强针对抗原的体液和/或细胞免疫应答的反应,并且优选地指通过将抗原结合到猪Fc片段来增加抗原的免疫原性。
以下,将描述示例性实施方式以帮助理解本发明。然而,提供以下实施例仅仅是为了便于理解本发明,而不是为了限制本发明的范围。
实施例
实施例1:pFc融合VP1重组蛋白表达载体的制备
为了制备表达载体,以通过增加靶蛋白的表达量和增强其溶解度来生产分离和纯化效率提高的重组蛋白,使用猪Fc片段(pFc)的pFc1(SEQ ID NO:1)、pFc2(SEQ ID NO:3),或pFc3(SEQ ID NO:5)构建表达载体。更具体地说,如图1和2所示,M17的5'非翻译区(UTR)位点基因(SEQ ID NO:7),编码伴侣结合蛋白(BiP)的多核苷酸(SEQ ID NO:8),口蹄疫病毒(FMDV)的VP1基因(SEQ ID NO:9),编码pFc的多核苷酸、编码His-Asp-Glu-Leu(HDEL)蛋白的多核苷酸按顺序克隆到pCAMBIA1300载体的CaMV 35S启动子基因和NOS终止子之间,从而完成表达载体的制备。对于pFc片段,通过插入pFc1、pFc2或pFc3制备不同的表达载体。
实施例2:pFc融合VP1重组蛋白表达实验
2.1确认pFc融合VP1重组蛋白表达量的实验
为了确认以与实施例1相同的方式制备的pFc融合VP1重组蛋白表达载体的蛋白表达量,通过PEG介导转化将该载体导入从拟南芥叶片中分离的原生质体中制备转基因生物体,然后收集培养的原生质体并进行裂解,从其表达重组蛋白的BiP:FMDV-VP1:pFc的表达模式使用pFc识别的抗猪二级抗体(1:5000,Abcam)进行蛋白质印迹法。更具体地说,将30μL细胞裂解产物与SDS样品缓冲液混合,然后加热。然后,在10%SDS-PAGE凝胶上按大小通过电泳分离蛋白质,将分离出的蛋白质转移到PVDF膜上,然后用5%脱脂牛奶封闭,然后将蛋白质与抗体结合并用制造商提供的方法用ECL溶液处理,以识别pFc融合的重组蛋白。其结果如图3所示。
如图3所示,证实在与各种pFc片段融合的重组蛋白中,pFc2融合重组蛋白的表达量最高。从以上结果可以证实,相同的免疫球蛋白片段并没有表现出相同的效果。
2.2pFc融合VP1重组蛋白的稳定性确认实验
为了确认以与实施例1相同的方式制备的pFc融合重组蛋白表达载体的蛋白质的稳定性,使用与实施例2.1相同的方法,通过蛋白质印迹法检测提取重组蛋白时的样品(0)和在4℃保存1小时后获得的样品(1)。其结果如图4所示。
如图4所示,已证实pFc2融合重组蛋白表现出最大的表达量和高稳定性。
2.3pFc2融合VP1重组蛋白的溶解度确认实验
为了确认以与实施例1相同的方式制备的pFc2融合重组蛋白表达载体的蛋白质的溶解度,将载体转化后的根癌农杆菌接种到本氏烟(Nicotiana benthamiana)的叶片中使用瞬态表达式方法表达pFc2融合重组蛋白(BiP:FMDV-VP1:pFc2),从植物叶片中提取蛋白质并离心,然后使用与实施例2.1中相同的方法对溶液中包含的可溶形式的蛋白质和颗粒部分(P)中存在的蛋白质进行蛋白质印迹法。作为对照,使用通过编码已知纤维素结合模块(CBM3)的多核苷酸(SEQ ID NO:13)而不是pFc片段融合产生的重组蛋白。其结果如图5所示。
如图5所示,已确认在颗粒部分中未观察到pFc2融合重组蛋白,而是包含在溶液中。然而,在CBM3融合重组蛋白的情况下,在颗粒部分观察到相当数量的重组蛋白。以上结果证实,pFc2融合重组蛋白由于靶蛋白与pFc2片段的结合,通过结构修饰,显示出增加的溶解度,从而证实pFc2融合重组蛋白在分离和纯化方面更为有效,由于抑制了重组蛋白的聚集,因此能够有效地维持其生理活性或药理活性。
实施例3:pFc2融合GP5重组抗原的溶解度确认实验
为了将pFc2片段与猪繁殖与呼吸综合征(PRRS)的GP5抗原蛋白融合,插入编码猪GP5抗原蛋白的多核苷酸(SEQ ID NO:11),而不是包括在实施例1的表达载体中的FMDV的VP1基因,以制备表达GP5:pFc2重组抗原的表达载体。然后将载体转化后的根癌农杆菌接种于本氏烟(Nicotiana benthamiana)的叶片中,采用瞬时表达法表达pFc2融合GP5的重组抗原(GP5:pFc2),从植株叶片中提取蛋白质并离心,然后用与实施例2.1相同的方法对溶液中包含的可溶形式的蛋白质和颗粒部分(P)中存在的蛋白质进行蛋白质印迹法。作为对照,使用与CBM3(SEQ ID NO:14)而不是pFc片段融合的GP5重组抗原,并且在CBM3融合GP5重组抗原的情况下,使用HA抗体进行实验用于蛋白质印迹法。其结果如图6所示。
如图6所示,已证实在pFc2融合GP5重组抗原的情况下,虽然在颗粒部分观察到一些蛋白质,但大多数蛋白质包含在溶液中。相反,在CBM3融合GP5重组抗原的情况下,在颗粒部分观察到相当数量的重组蛋白。上述结果证实,无论是哪种类型的蛋白,pFc2融合重组蛋白显示出提高的溶解度。
这些结果证实,通过将猪Fc片段,特别是含有由SEQ ID NO:4表示的氨基酸序列的pFc2片段与靶蛋白融合,提高了靶蛋白的表达量和溶解度,从而使靶蛋白能够稳定和容易地分离和储存。
实施例4:pFc2融合PCV2重组蛋白的产量和溶解度确认实验
为了将pFc2片段与猪圆环病毒2型(PCV2)蛋白融合,插入编码PCV2蛋白的多核苷酸(SEQ ID NO:15),而不是包含在实施例1的表达载体中的FMDV的VP1基因,以制备表达PCV2:pFc2重组蛋白的表达载体。然后,将载体转化后的根癌农杆菌接种于本氏烟(Nicotiana benthamiana)的叶片中,采用瞬时表达法表达pFc2融合的PCV2重组蛋白,从植株叶片中提取蛋白质并离心,然后用与实施例2.1相同的方法对溶液中包含的可溶形式的蛋白质和颗粒部分(P)中存在的蛋白质进行蛋白质印迹法。作为对照,使用与His标签而不是pFc片段融合的PCV2重组蛋白,并且对于His标签融合的PCV2重组蛋白,使用抗His抗体进行实验用于蛋白质印迹法。其结果如图7所示。
如图7所示,经证实,与His标签融合PCV2重组蛋白相比,pFc2融合PCV2重组蛋白主要包含在溶液中,并且显示出显著提高的产量。
实施例5:pFc2融合E2重组蛋白表达实验
5.1 pFc2融合抗原蛋白的分离
为了确认pFc2片段是否与抗原蛋白融合且可用,插入编码E2蛋白(其为猪瘟抗原)的多核苷酸(SEQ ID NO:17)而不是实施例1的表达载体中包含的FMDV的VP1基因,以制备表达BiP:E2:pFc2重组蛋白的表达载体。然后,利用所制备的表达载体,采用农杆菌介导的转化方法,对拟南芥进行转化,筛选出抗卡那霉素的拟南芥,最终获得经世代进化稳定表达pFc2融合E2重组蛋白的同源种子,从而完成转化植物的制备。然后,利用蛋白质提取中常用的蛋白质提取缓冲液从8g最终获得的转化植株中分离出蛋白质,并使用配备有蛋白质A-琼脂糖凝胶柱(Protein A-Sepharose column)的AKTA prime(通用电气医疗集团)分离pFc融合E2重组蛋白。然后,作为对照,使用通过CBD(SEQ ID NO:19)融合而是pFc片段融合产生的BiP:E2:CBD重组蛋白。用无定形纤维素(AMC)从5g转化植株中分离出CBD融合E2重组蛋白。然后,用磷酸盐缓冲液(PBS)透析分离出的重组蛋白,然后用离心过滤管浓缩。用SDS-PAGE和考马斯亮蓝染色法测定重组蛋白的量。此时,重组蛋白使用牛血清白蛋白(BSA)标准曲线进行定量。其结果如图8所示。
如图8所示,已证实,虽然CBD融合的E2重组抗原的产量约为每1g植物30微克,但pFc2融合的E2重组抗原的产量为每1g植物302微克。以上结果证实,使用pFc2片段可使靶抗原的表达量增加10倍或更多。
5.2 pFc2融合E2重组抗原的免疫原性及病毒中和能力确认实验
为证实pFc2融合E2重组抗原是否具有体内诱导抗体的免疫原性和病毒中和能力,采用6周龄雄性C57BL/6J小鼠进行实验。更具体地说,实验组小鼠一次(6周龄)或两次(6周龄和8周龄)给予1μg的pFc2融合E2重组抗原,阴性对照小鼠给予磷酸盐缓冲液。另外,作为阳性对照,在与实验组相同的剂量和相同的时间给小鼠给予CBD融合E2重组抗原。在给药每个抗原后,将每个抗原与等量的弗氏佐剂混合并给药,在一次给药组中施用完全佐剂,在两次给药组中,在给药完全佐剂后给药不完全佐剂。然后,分别于实验开始时和每个抗原给药后1周起每周采血,用猪瘟病毒临床诊断抗体试剂盒(CSFV-ab-ELISA试剂盒,MEDIANDIAGNOSTICS)检测,是否产生并维持针对给药抗原的特异性抗体。两次给药组在第二次给药结束后1周开始采血。每组用5只小鼠进行实验,实验结果如表1和图9和10所示。在表1中,S/P值在值为0.14或以上时确定为正值,在值小于0.14时确定为负值。
[表1]
如图9所示,经证实,在给予抗原一次的情况下,pFc2融合的E2重组抗原自给药后1周表现出高反应性,并且维持高活性超过8周。与之相反,证实CBD融合的E2重组抗原虽然显示正值,但其反应性较pFc2融合的E2重组抗原低。
如图10和表1所示,证实在两次给药抗原的情况下,CBD融合的E2重组抗原也表现出比一次给药更高的反应性,但与pFc2融合的E2重组抗原相比仍然具有较低的反应性。
同时,将同一小鼠血清样本送动植物检疫机关鉴定其病毒中和能力。其结果见下表2。
[表2]
如表2所示,经证实,虽然CBD融合的E2重组抗原在一些小鼠中在抗原给药一次时显示为阴性,但当给药一次和两次时,pFc2融合的E2重组抗原都显示出高效的病毒中和能力。
5.3 pFc2融合E2重组抗原的病毒中和能力确认实验
为证实pFc2融合的E2重组抗原在仔猪体内是否也表现出高效的病毒中和能力,选择4头猪瘟抗体阴性的仔猪,分两次给药pFc2融合的E2重组抗原,每隔2周取血清送动植物检疫机关鉴定其病毒中和能力。其结果见下表3。
[表3]
如表3所示,证实所有仔猪表现出与使用实施例3.2的小鼠进行的实验中的正值相同的值。
以上结果证实,将本发明的pFc2片段与疫苗用的各种多肽或蛋白质融合,不仅提高了靶抗原的产量,而且提高了靶抗原的溶解度和稳定性,抑制了靶抗原的聚集,因此,重组抗原可有效地维持其生理活性或药理活性。实验还证实,通过将抗原与pFc2片段融合制备重组抗原,pFc2片段表现出自佐剂效应(self-adjuvanting effect)和自佐剂作用(self-adjuvant effect),从而不仅可以显著提高靶抗原的免疫原性,而且可以显著提高靶抗原的产量和稳定性。
本发明的上述描述仅用于说明目的,本发明所属领域的普通技术人员应当理解,本发明可以容易地被修改为其它特定形式,而不改变本发明的技术精神或基本特征。因此,上述实施方式应被解释为仅用于说明目的而不是用于限制目的。
工业实用性
本发明涉及一种与猪Fc片段融合的抗原,更具体地说,涉及一种通过将Fc片段与各种抗原结合而具有自佐剂效应的疫苗组合物及其制备方法。由于与本发明的Fc片段融合的抗原具有自佐剂效应,它们可以显著提高疫苗组合物的预防和/或治疗效果,因此预期通过应用于各种疫苗组合物而被广泛使用。
<110> 巴伊沃爱普有限公司
<120> 与猪FC片段融合的抗原,以及包含该抗原的疫苗组合物
<130> MPO19-021CN
<150> KR 10-2018-0112443
<151> 2018-09-19
<160> 19
<170> KoPatentIn 3.0
<210> 1
<211> 690
<212> DNA
<213> 人工序列
<220>
<223> pFc1的DNA序列
<400> 1
ggaactaaga ctaagccacc ttgtcctatt tgtccagggt gcgaggtagc cggtcccagc 60
gtgtttattt ttccaccaaa accaaaggat actttgatga tatctcaaac accggaagtt 120
acttgcgttg tggtcgacgt ttcaaaagag catgccgaag ttcagttctc ttggtatgtg 180
gatggtgtgg aagtgcacac cgctgagaca cgtcctaaag aggaacagtt taactctact 240
tacagagtcg tgtccgtatt gcccattcag catcaagact ggcttaaggg aaaagaattt 300
aaatgtaagg taaataatgt tgatctgcca gcacctataa ctagaaccat ctcgaaagct 360
attggacaat ctagagaacc tcaagtttat acattgcctc ctccagctga ggaactttct 420
agaagtaaag tcactgttac atgcttagtt attggattct atccaccaga tatccatgtt 480
gaatggaaat caaatggtca gcccgaacct gagggcaact acagaacaac accaccacag 540
caagatgtag atggtacttt tttcctctac tcaaaactag ctgttgataa ggctaggtgg 600
gatcatggcg agacatttga gtgtgcagtc atgcacgaag cacttcataa tcactatacc 660
caaaagtcca taagtaagac gcaaggaaag 690
<210> 2
<211> 230
<212> PRT
<213> 人工序列
<220>
<223> pFc1的氨基酸序列
<400> 2
Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu Val
1 5 10 15
Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr
65 70 75 80
Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys
85 90 95
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala Pro
100 105 110
Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser Lys Val
130 135 140
Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His Val
145 150 155 160
Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr
165 170 175
Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser Lys
180 185 190
Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys
195 200 205
Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile
210 215 220
Ser Lys Thr Gln Gly Lys
225 230
<210> 3
<211> 681
<212> DNA
<213> 人工序列
<220>
<223> pFc2的DNA序列
<400> 3
gttggaagac catgccctat atgtcctgct tgtgaaggtc caggtccctc tgcttttata 60
ttcccaccaa agccgaagga taccttgatg atttcacgta caccacaagt tacttgtgtt 120
gttgtggatg tttcacaaga aaatcctgag gtacaattca gctggtatgt tgatggggta 180
gaagtgcaca ctgcacagac tcgaccaaag gaggcccagt ttaactcgac ttatagagtt 240
gtttctgttc tcccaatcca acacgaagat tggctgaagg gcaaggaatt tgaatgcaag 300
gttaacaata aagatctacc agcaccaatt accaggatta tttctaaggc aaaaggaccc 360
tccagagagc cccaagttta cacattgtct ccttctgctg aggagcttag tagaagtaaa 420
gtgagcatta cctgcttagt gacgggattc taccctccag acatcgacgt cgaatggaaa 480
tctaatggtc aacctgagcc agaaggtaac tataggacta ctccaccaca acaggacgtc 540
gatggcacat actttcttta ttcaaaactt gctgtcgata aggcaagttg gcaaagagga 600
gatccatttc agtgtgctgt aatgcatgag gctttgcata atcattatac acagaaatca 660
gtttctaaaa cacaagggaa a 681
<210> 4
<211> 227
<212> PRT
<213> 人工序列
<220>
<223> pFc2的氨基酸序列
<400> 4
Val Gly Arg Pro Cys Pro Ile Cys Pro Ala Cys Glu Gly Pro Gly Pro
1 5 10 15
Ser Ala Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
20 25 30
Arg Thr Pro Gln Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asn
35 40 45
Pro Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu Val His Thr
50 55 60
Ala Gln Thr Arg Pro Lys Glu Ala Gln Phe Asn Ser Thr Tyr Arg Val
65 70 75 80
Val Ser Val Leu Pro Ile Gln His Glu Asp Trp Leu Lys Gly Lys Glu
85 90 95
Phe Glu Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Thr Arg
100 105 110
Ile Ile Ser Lys Ala Lys Gly Pro Ser Arg Glu Pro Gln Val Tyr Thr
115 120 125
Leu Ser Pro Ser Ala Glu Glu Leu Ser Arg Ser Lys Val Ser Ile Thr
130 135 140
Cys Leu Val Thr Gly Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp Lys
145 150 155 160
Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro
165 170 175
Gln Gln Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val
180 185 190
Asp Lys Ala Ser Trp Gln Arg Gly Asp Pro Phe Gln Cys Ala Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Val Ser Lys Thr
210 215 220
Gln Gly Lys
225
<210> 5
<211> 702
<212> DNA
<213> 人工序列
<220>
<223> pFc3的DNA序列
<400> 5
attgagccac cgacacctat ttgtcctgaa atatgctctt gccctgcggc cgaagtttta 60
ggagcaccgt cggtctttct gtttccacct aaacctaagg acattttaat gatctctagg 120
acgcccaagg taacttgtgt tgttgttgat gtttctcaag aagaagctga ggttcaattc 180
tcctggtatg tagacggcgt tcaattgtac accgcacaga ctaggcctat ggaagaacag 240
tttaactcaa catacagagt agtgtccgtg ttgccgatcc aacatcaaga ttggttgaaa 300
ggtaaagagt ttaagtgtaa agtgaacaat aaggatctcc tttctcctat taccagaact 360
ataagtaaag ctaccggacc atctcgggtt ccacaggtct acactcttcc accagcttgg 420
gaggagctta gcaagtcaaa ggtaagcatc acttgtctcg taacgggatt ctatccacca 480
gatattgatg tggaatggca gagtaatggt caacaggaac ccgagggtaa ttaccgaaca 540
actcctcctc agcaggatgt tgacggtact tattttcttt attcaaagct agctgttgat 600
aaagtgagat ggcaacgtgg cgatttgttc cagtgcgcag tcatgcatga ggctcttcat 660
aatcactata cacaaaaatc aatttctaag acacaaggga ag 702
<210> 6
<211> 234
<212> PRT
<213> 人工序列
<220>
<223> pFc3的氨基酸序列
<400> 6
Ile Glu Pro Pro Thr Pro Ile Cys Pro Glu Ile Cys Ser Cys Pro Ala
1 5 10 15
Ala Glu Val Leu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Ile Leu Met Ile Ser Arg Thr Pro Lys Val Thr Cys Val Val
35 40 45
Val Asp Val Ser Gln Glu Glu Ala Glu Val Gln Phe Ser Trp Tyr Val
50 55 60
Asp Gly Val Gln Leu Tyr Thr Ala Gln Thr Arg Pro Met Glu Glu Gln
65 70 75 80
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln
85 90 95
Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
100 105 110
Leu Leu Ser Pro Ile Thr Arg Thr Ile Ser Lys Ala Thr Gly Pro Ser
115 120 125
Arg Val Pro Gln Val Tyr Thr Leu Pro Pro Ala Trp Glu Glu Leu Ser
130 135 140
Lys Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro
145 150 155 160
Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Gly
165 170 175
Asn Tyr Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe
180 185 190
Leu Tyr Ser Lys Leu Ala Val Asp Lys Val Arg Trp Gln Arg Gly Asp
195 200 205
Leu Phe Gln Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr
210 215 220
Gln Lys Ser Ile Ser Lys Thr Gln Gly Lys
225 230
<210> 7
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> M17基因的5'UTR
<400> 7
ggcgtgtgtg tgtgttaaag a 21
<210> 8
<211> 272
<212> DNA
<213> 人工序列
<220>
<223> 伴侣结合蛋白(BiP)的DNA序列
<400> 8
atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag 60
tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa 120
ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg 180
ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt 240
tgtcctctgc aatagaagag gctacgaagt ta 272
<210> 9
<211> 639
<212> DNA
<213> 人工序列
<220>
<223> FMDV-VP1的DNA序列
<400> 9
actacaagta ccggcgaatc tgctgatcca gttactgcta cagttgaaaa ttatggtgga 60
gaaacacaag tgcaaagaag acatcataca gatgtttctt ttatcctaga taggtttgtt 120
aaggtcactc ctaaggattc aattaatgtt ttggacctga tgcagactcc cccacataca 180
ttggttggcg ctctacttcg tactgcaact tattatttcg ctgatttaga ggtagccgtt 240
aaacacgaag gtgatttaac atgggttcct aatggagcac ctgaggctgc actcgataat 300
actactaatc caactgctta ccacaaagca ccactcacta gactcgcgct tccttacact 360
gccccgcata gggttcttgc tactgtttat aacgggaact gcaaatacgc aggtggttca 420
ttgcctaatg tacgaggaga tttgcaagta ttggctcaaa aagcagcatg gccattacct 480
acttctttta actatggagc tataaaggct acacgtgtga cggaacttct ttataggatg 540
aagagagctg agacatactg tcctagacca ttactggctg ttcatccatc cgccgcaaga 600
cacaaacaga aaattgtggc tcccgttaag cagagcctt 639
<210> 10
<211> 213
<212> PRT
<213> 人工序列
<220>
<223> FMDV-VP1的氨基酸序列
<400> 10
Thr Thr Ser Thr Gly Glu Ser Ala Asp Pro Val Thr Ala Thr Val Glu
1 5 10 15
Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val
20 25 30
Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Lys Asp Ser Ile
35 40 45
Asn Val Leu Asp Leu Met Gln Thr Pro Pro His Thr Leu Val Gly Ala
50 55 60
Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala Val
65 70 75 80
Lys His Glu Gly Asp Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala
85 90 95
Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His Lys Ala Pro Leu
100 105 110
Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr
115 120 125
Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val
130 135 140
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Trp Pro Leu Pro
145 150 155 160
Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu
165 170 175
Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu
180 185 190
Ala Val His Pro Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala Pro
195 200 205
Val Lys Gln Ser Leu
210
<210> 11
<211> 330
<212> DNA
<213> 人工序列
<220>
<223> GP5的DNA序列
<400> 11
aacggcaaca gctcgacata ccaatacata tataacttga cggtatgcga gctgaatggg 60
accgcctggt tgtctaccca cttttcttgg gcagtcgaga ccggaggcgg gggtagcaaa 120
aattgtatgg cttgccgcta cgcccgcacc cggttcacca acttcattgt agacgaccgg 180
gggaggattc atcggtggaa gtccccggtg gtggtggaga aatttggcaa agccgaaatt 240
ggcggcggtc ttgtcaccat caaacatgtc gtcctcgaag gggttaaagc tcaacccttg 300
acgaggactt cggctgagca atgggaagcc 330
<210> 12
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GP5的氨基酸序列
<400> 12
Asn Gly Asn Ser Ser Thr Tyr Gln Tyr Ile Tyr Asn Leu Thr Val Cys
1 5 10 15
Glu Leu Asn Gly Thr Ala Trp Leu Ser Thr His Phe Ser Trp Ala Val
20 25 30
Glu Thr Gly Gly Gly Gly Ser Lys Asn Cys Met Ala Cys Arg Tyr Ala
35 40 45
Arg Thr Arg Phe Thr Asn Phe Ile Val Asp Asp Arg Gly Arg Ile His
50 55 60
Arg Trp Lys Ser Pro Val Val Val Glu Lys Phe Gly Lys Ala Glu Ile
65 70 75 80
Gly Gly Gly Leu Val Thr Ile Lys His Val Val Leu Glu Gly Val Lys
85 90 95
Ala Gln Pro Leu Thr Arg Thr Ser Ala Glu Gln Trp Glu Ala
100 105 110
<210> 13
<211> 477
<212> DNA
<213> 人工序列
<220>
<223> CBM3的DNA序列
<400> 13
gtatcaggta accttaaggt ggagttttac aactcgaacc cttctgatac aactaactca 60
ataaacccac agttcaaagt tacaaacaca ggcagctctg cgatcgattt gtctaaatta 120
accctcagat actattatac ggttgatgga cagaaggacc agactttctg gtgtgatcat 180
gcagctatca ttggttctaa cggtagctac aacggaatta catcaaacgt gaagggcact 240
ttcgttaaga tgtcctctag cactaacaac gcagacacat atttggagat cagttttacg 300
gggggaaccc ttgaaccagg tgctcacgtc cagattcaag gaagattcgc taaaaacgac 360
tggtcgaact atacccaaag taatgattac agttttaaat ccgcctcaca atttgttgag 420
tgggatcagg tcactgctta cctgaacggg gttctagtgt ggggaaagga acctggt 477
<210> 14
<211> 159
<212> PRT
<213> 人工序列
<220>
<223> CBM3的氨基酸序列
<400> 14
Val Ser Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp
1 5 10 15
Thr Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser
20 25 30
Ser Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val
35 40 45
Asp Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile
50 55 60
Gly Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr
65 70 75 80
Phe Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu
85 90 95
Ile Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile
100 105 110
Gln Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn
115 120 125
Asp Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val
130 135 140
Thr Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro Gly
145 150 155
<210> 15
<211> 579
<212> DNA
<213> 人工序列
<220>
<223> PCV2的DNA序列
<400> 15
aaaaatggca ttttcaatac acgcctcagt cgaacttttg gatatactgt caagcgtact 60
acagtcacca cgccatcttg ggctgtggat atgatgagat ttaagttgga tgactttgtt 120
cctcctggag ggggaaccaa caaaatttct ataccgtttg agtactatag aatcagaaaa 180
gttaaggttg agttctggcc gtgttccccc ataactcagg gtgatagggg tgtgggttca 240
actgctgtta ttctagatga taacttcgta cctaaggcca acgcattgac ttatgacccc 300
tatgtaaact actcatctag acatacaatc ccacaacctt tctcctacca ctcgcgttat 360
tttacaccaa agcctgtttt agattctacc attgattatt tccaaccaaa taacaagagg 420
aatcagcttt ggttgagatt acaaacctca cggaacgtgg atcatgtcgg attgggtact 480
gcatttgaaa atagtaagta tgatcaggac tacaatatcc gtgtgacaat gtacgttcaa 540
tttagggaat ttaatcttaa agacccacca cttaatcca 579
<210> 16
<211> 193
<212> PRT
<213> 人工序列
<220>
<223> PCV2的氨基酸序列
<400> 16
Lys Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Phe Gly Tyr Thr
1 5 10 15
Val Lys Arg Thr Thr Val Thr Thr Pro Ser Trp Ala Val Asp Met Met
20 25 30
Arg Phe Lys Leu Asp Asp Phe Val Pro Pro Gly Gly Gly Thr Asn Lys
35 40 45
Ile Ser Ile Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu
50 55 60
Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser
65 70 75 80
Thr Ala Val Ile Leu Asp Asp Asn Phe Val Pro Lys Ala Asn Ala Leu
85 90 95
Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Pro Gln
100 105 110
Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp
115 120 125
Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp
130 135 140
Leu Arg Leu Gln Thr Ser Arg Asn Val Asp His Val Gly Leu Gly Thr
145 150 155 160
Ala Phe Glu Asn Ser Lys Tyr Asp Gln Asp Tyr Asn Ile Arg Val Thr
165 170 175
Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn
180 185 190
Pro
<210> 17
<211> 1026
<212> DNA
<213> 人工序列
<220>
<223> E2的DNA序列
<400> 17
aacggctagc ctgcaaggaa gattacaggt acgcaatatc atcaaccaat gagatagggc 60
tactcggggc cggaggtctc accaccacct ggaaagaata caaccacgat ttgcaactga 120
atgacgggac cgttaaggcc atttgcgtgg caggttcctt taaagtcaca gcacttaatg 180
tggtcagtag gaggtatttg gcatcattgc ataaggaggc tttacccact tccgtgacat 240
tcgagctcct gttcgacggg accaacccat caactgagga aatgggagat gacttcgggt 300
tcgggctgtg cccgtttgat acgagtcctg ttgtcaaagg aaagtacaat acaaccttgt 360
tgaacggtag tgctttctat cttgtctgtc caatagggtg gacgggtgtt atagagtgca 420
cagcagtgag cccaacaact ctgagaacag aagtggtaaa gaccttcagg agggacaagc 480
cctttccgca cagaatggat tgtgtgacca caacagtgga aaatgaagat ttattctact 540
gtaagttggg gggcaactgg acatgtgtga aaggtgaacc agtggtctac acgggggggc 600
tagtaaaaca atgcagatgg tgtggctttg acttcaatga gcctgacgga ctcccacact 660
accccatagg taagtgcatt ttggcaaatg agacaggtta cagaatagtg gattcaacag 720
actgtaacag agatggtgtt gtaatcagca cagaggggag tcatgagtgc ttgatcggta 780
acacgactgt caaggtgcat gcatcagatg aaagactggg ccccatgcca tgcagaccta 840
aagagatcgt ctctagtgca ggacctgtaa ggaaaacttc ctgtacattc aactacgcaa 900
aaactttgaa gaacaagtac tatgagccca gggacagcta cttccagcaa tatatgctta 960
agggcgagta tcagtactgg tttgacctgg acgtgactga ccgccactca gattacttcg 1020
cagaag 1026
<210> 18
<211> 341
<212> PRT
<213> 人工序列
<220>
<223> E2的氨基酸序列
<400> 18
Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asn
1 5 10 15
Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu
20 25 30
Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ile Cys
35 40 45
Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg
50 55 60
Tyr Leu Ala Ser Leu His Lys Glu Ala Leu Pro Thr Ser Val Thr Phe
65 70 75 80
Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp
85 90 95
Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys
100 105 110
Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val
115 120 125
Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro
130 135 140
Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro
145 150 155 160
Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu Asp
165 170 175
Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu
180 185 190
Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gln Cys Arg Trp Cys Gly
195 200 205
Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys
210 215 220
Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr Asp
225 230 235 240
Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys
245 250 255
Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg Leu
260 265 270
Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly Pro
275 280 285
Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys Asn
290 295 300
Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys
305 310 315 320
Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Val Thr Asp Arg His Ser
325 330 335
Asp Tyr Phe Ala Glu
340
<210> 19
<211> 429
<212> DNA
<213> 人工序列
<220>
<223> 纤维素结合域的DNA序列
<400> 19
tttcgaagtt caccagtgcc tgcacctggt gataacacaa gagacgcata ttctatcatt 60
caggccgagg attatgacag cagttatggt cccaaccttc aaatctttag cttaccaggt 120
ggtggcagcg ccattggcta tattgaaaat ggttattcca ctacctataa aaatattgat 180
tttggtgacg gcgcaacgtc cgtaacagca agagtagcta cccagaatgc tactaccatt 240
caggtaagat tgggaagtcc atcgggtaca ttacttggaa caatttacgt ggggtccaca 300
ggaagctttg atacttatag ggatgtatcc gctaccatta gtaatactgc gggtgtaaaa 360
gatattgttc ttgtattctc aggtcctgtt aatgttgact ggtttgtatt ctcaaaatca 420
ggaacttct 429
Claims (13)
1. 一种疫苗组合物,其包括作为活性成分的与由SEQ ID NO:4代表的猪Fc片段融合的抗原。
2.根据权利要求1所述的疫苗组合物,其中所述抗原通过与Fc片段融合具有自佐剂效应和增加的溶解度。
3. 一种具有自佐剂效应的重组抗原的表达载体,所述表达载体包括编码由SEQ IDNO:4表示的猪Fc片段的多核苷酸和编码抗原的多核苷酸。
4.根据权利要求3所述的表达载体,其中,在所述表达载体中,启动子基因、编码Fc片段的多核苷酸和编码抗原的多核苷酸顺序连接。
5. 根据权利要求4所述的表达载体,其中所述启动子包含选自由源自花椰菜花叶病毒的35S启动子、源自花椰菜花叶病毒的19S RNA启动子、植物的肌动蛋白启动子、泛素蛋白启动子、巨细胞病毒(CMV)启动子、猿猴病毒40(SV40)启动子、呼吸道合胞病毒(RSV)启动子、pEMU启动子、MAS启动子、组蛋白启动子、Clp启动子和延伸因子-1α(EF-1α)启动子组成的组中的一个或多个。
6.根据权利要求3所述的表达载体,其中所述表达载体进一步包含编码伴侣结合蛋白(BiP)的多核苷酸。
7.根据权利要求3所述的表达载体,其中所述表达载体进一步包含编码His-Asp-Glu-Leu(HDEL)肽的基因。
8.根据权利要求3所述的表达载体,其中所述表达载体进一步包含M17的5'非翻译区(UTR)位点基因。
9.根据权利要求3所述的表达载体,其中所述表达载体增加Fc片段融合抗原的表达量和溶解度。
10.由根据权利要求3至9中任一项所述的表达载体转化的转基因生物体。
11. 一种产生具有自佐剂效应的重组抗原的方法,所述方法包括:
(a) 培养根据权利要求10所述的转基因生物体;及
(b) 从转基因生物体或培养液中分离和纯化Fc片段融合抗原。
12. 一种预防或治疗传染病的方法,所述方法包括向个体施用包括作为活性成分的与由SEQ ID NO:4代表的猪Fc片段融合的抗原的组合物。
13. 一种组合物用于预防或治疗传染病的用途,所述组合物包括作为活性成分的与由SEQ ID NO:4代表的猪Fc片段融合的抗原。
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KR1020180112443A KR102200773B1 (ko) | 2018-09-19 | 2018-09-19 | 돼지의 Fc 단편과 융합된 항원 및 이를 포함하는 백신 조성물 |
PCT/KR2018/015399 WO2020059961A1 (ko) | 2018-09-19 | 2018-12-06 | 돼지의 fc 단편과 융합된 항원 및 이를 포함하는 백신 조성물 |
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