CN101307310B - 具有提高产率及免疫原性的重组蛋白质表达系统 - Google Patents
具有提高产率及免疫原性的重组蛋白质表达系统 Download PDFInfo
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Abstract
一种宿主细胞中重组目的蛋白质的表达系统，其包含主要由SEQ IDNO：1的蛋白质转导结构域(domain)的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞中表达重组目的蛋白质用的宿主-特异性转录及转译调控单元。本发明亦提供一种在宿主细胞中提高产率的生产重组目的蛋白质方法。
Description
技术领域
本发明涉及具有提高产率及免疫原性重组蛋白质的表达系统。
背景技术
由于越来越多诸如重组蛋白质的产品核准或即将核准用于人类，对于高效率生产治疗用生物制剂的需求在稳定地增加中。细菌发酵工艺自过去到现在仍是生产此等类型分子的主要工具。将生产工艺最佳化的主要目的是要以最低的成本、高产率地获得所需质量的产品，此通常由特异性表达构建体或系统的性质来决定。
据报J结构域的结构特征决定Hsp40蛋白质与其配体Hsp70s间交互作用的特异性[Hemmessy et al.，Protein Science(2005)，14:1697-1709]。咸信J结构域为Hsp40及类Hsp-40蛋白质与其配体Hsp70s间的交互作用的主要结合部位且为所需的最小区域[Walsh et al.，EMBO report(2004)，6：567-571]。据报通过DNA疫苗系统而与SV40病毒巨大T抗原的J结构域融合的肽可增强该肽的抗原性[Reimann and Schirmbeck，Immuno Rev.(2004)，199:54-67]。已知J结构域将DnaK接合至结合有DnaJ的受质，DnaK然后以其C端肽-结合性结构域结合[Greene et al.，Proc.Natl.Acad.Sci.USA(1998)，95:6108-6113]。
另一方面，蛋白质转导结构域(PTD)为小型肽，其可将比其大许多的分子运送至与典型胞饮作用无关的细胞中。该性质使得PTD成为将蛋白质及其它分子传送至研究用活细胞的理想工具[Beerens et al，Current GeneTherapy(2003)，3(5):486-494]。
然而，未曾暗示PTD或Hsp40J结构域的任一者可用于开发提高重组蛋白质产率及免疫原性的表达系统。
发明内容
依据本发明，提供一种在宿主细胞中重组目的蛋白质的表达系统，其包含主要由SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞中表达重组目的蛋白质用的宿主-特异性转录及翻译调控单元。
依据本发明，亦提供一种使用上述表达系统表达重组目的蛋白质的方法。本发明的方法包含：
构建表达载体，该表达载体包含主要由SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞-特异性转录及翻译调控单元；
在该宿主细胞中转入该表达载体；以及
从该宿主细胞中收集及分离该重组目的蛋白质。
因此，本发明亦提供通过使用上述表达系统所产生的重组蛋白质。结果，该重组蛋白质的免疫原性增加。
本发明亦提供一种包含上述重组蛋白质的疫苗。
本发明的其它目的及优点一部分记载于下述说明中，一部分从该说明显而易知，或者可通过实施本发明而知悉。本发明之目的及优点可通过权利要求所列特定的单件元及组合而实施及达成。
应了解前文的概述及下文的详细说明二者仅为例示性及阐释性的说明，而非如权利要求般限定本发明。
附图说明
为了说明本发明的目的，在附图中显示现阶段较佳的具体实施例。不过，应了解本发明非限于所示的具体实施例。
在附图中：
图1表示大肠杆菌的全细胞溶裂物的经考马斯蓝(
blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的pET22b-PTD-J-HSPA1A肽I构建体。第1行，标准分子量标记(示出对应于分子质量15kDa及20kDa的带)。第二行(0)，非被诱导的pET22b-PTD-J-HSPA1A肽I；第3行(1)，1小时后被诱导的pET22b-PTD-J-HSPA1A肽I；第4行(2)，2小时后被诱导的pET22b-PTD-J-HSPA1A肽I；第5行(3)，3小时后被诱导的pET22b-PTD-J-HSPA1A肽I；第6行(4)，4小时后被诱导的pET22b-PTD-J-HSPA1A肽I；第7行(5)，5小时后被诱导pET22b-PTD-J-HSPA1A肽I；第8行(6)，6小时后被诱导的pET22b-PTD-J-HSPA1A肽I；以及第9行(16)，16小时后被诱导的pET22b-PTD-J-HSPA1A肽I。
图2表示大肠杆菌的全细胞溶裂物的经考马斯蓝(
blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的pET22b-PTD-J-HSPA1A肽II构建体。第1行，标准分子量标记(示出对应于分子质量15kDa及20kDa的带)。第二行(0)，非被诱导之pET22b-PTD-J-HSPA1A肽II；第3行(1)，1小时后被诱导的pET22b-PTD-J-HSPA1A肽II；第4行(2)，2小时后被诱导pET22b-PTD-J-HSPA1A肽II；第5行(3)，3小时后被诱导pET22b-PTD-J-HSPA1A肽II；第6行(4)，4小时后被诱导的pET22b-PTD-J-HSPA1A肽II；第7行(5)，5小时后被诱导的pET22b-PTD-J-HSPA1A肽II；第8行(6)，6小时后被诱导的pET22b-PTD-J-HSPA1A肽II；以及第9行(16)，16小时后被诱导的pET22b-PTD-J-HSPA1A肽II。
图3表示大肠杆菌的全细胞溶裂物的经考马斯蓝(
blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的pET22b-PTD-J-HSPA1A肽III构建体。第1行，标准分子量标记(示出对应于分子质量15kDa及20kDa的带)。第二行(0)，非被诱导的pET22b-PTD-J-HSPA1A肽III；第3行(1)，1小时后被诱导的 pET22b-PTD-J-HSPA1A肽III；第4行(2)，2小时后被诱导的pET22b-PTD-J-HSPA1A肽III；第5行(3)，3小时后被诱导的pET22b-PTD-J-HSPA1A肽III；第6行(4)，4小时后被诱导的pET22b-PTD-J-HSPA1A肽III；第7行(5)，5小时后被诱导的pET22b-PTD-J-HSPA1A肽III；第8行(6)，6小时后被诱导的pET22b-PTD-J-HSPA1A肽III；以及第9行(16)，16小时后被诱导的pET22b-PTD-J-HSPA1A肽III。
图4表示大肠杆菌的全细胞溶裂物的经考马斯蓝(
blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的pET22b-PTD-J-鸡IGF-I构建体。第1行，标准分子量标记(示出对应于分子质量15kDa及20kDa的带)。第2行(0)，非被诱导的pET22b-PTD-J-鸡IGF-I；第3行(1)，1小时后被诱导的pET22b-PTD-J-鸡IGF-I；第4行(2)，2小时后被诱导之pET22b-PTD-J-鸡IGF-I；第5行(3)，3小时后被诱导的pET22b-PTD-J-鸡IGF-I；第6行(4)，4小时后被诱导的pET22b-PTD-J-鸡IGF-I；第7行(5)，5小时后被诱导的pET22b-PTD-J-鸡IGF-I；以及第8行(6)，6小时后被诱导的pET22b-PTD-J-鸡IGF-I。
图5表示大肠杆菌的全细胞溶裂物的经考马斯蓝(
blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的pET22b-PTD-J-HA RBD构建体。第1行，标准分子量标记(示出对应于分子质量25kDa及37kDa的带)。第2行(0)，非被诱导的pET22b-PTD-J-HARBD；第3行(1)，1小时后被诱导的pET22b-PTD-J-HA RBD；第4行(2)，2小时后被诱导的pET22b-PTD-J-HARBD；第5行(3)，3小时后被诱导的pET22b-PTD-J-HA RBD；第6行(4)，4小时后被诱导的pET22b-PTD-J-HARBD；第7行(5)，5小时后被诱导的pET22b-PTD-J-HA RBD；第8行(6)，6小时后被诱导的pET22b-PTD-J-HARBD；第9行(7)，7小时后被诱导的pET22b-PTD-J-HA RBD以及第10行(O/N)，16小时后被诱导的pET22b-PTD-J-HA RBD。
图6表示大肠杆菌的全细胞溶裂物的经考马斯蓝(blue)染色的SDS-PAGE。罗塞塔(Rosetta)菌株含有于IPTG存在下生长的 pET22b-PTD-J-HCV核心构建体。第1行，标准分子量标记(示出对应于分子质量25kDa及37kDa的带)。第2行(0)，非被诱导的pET22b-PTD-J-HCV核心；第3行(1)，1小时后被诱导的pET22b-PTD-J-HCV核心；第4行(2)，2小时后被诱导的pET22b-PTD-J-HCV核心；第5行(3)，3小时后被诱导的pET22b-PTD-J-HCV核心；第6行(5)，5小时后被诱导的pET22b-PTD-J-HCV核心；第7行(6)，6小时后被诱导的pET22b-PTD-J-HCV核心；以及第8行(16)，16小时后被诱导的pET22b-PTD-J-HCV核心。
图7a及图7b是检测血清中不同浓度的重组蛋白质PTD-J1-嗜中性肽-1(NP-1)、PTD-J1-核心及PTD-J1的印迹杂交图(dot blot)。
具体实施方式
总体言之，本发明涉及一种表达系统，其被引进宿主细胞以稳定且有效率地生产具有提高产率及免疫原性的重组目的蛋白质。
在本文中所用的术语「重组蛋白质」意指重组分子且通常为通过重组、化学或其它适当方法与蛋白质转导结构域及J结构域共价连接(即融合)的蛋白质或肽序列。该重组分子可视需要通过肽连接子(peptide linker)序列在1个或数个部位进行融合。该肽序列可包括1个或多个由宿主细胞所诱导的蛋白酶断裂的部位。
「多肽」指较佳主要由任何20个天然氨基酸组成不论其大小的任何聚合物。虽然术语「蛋白质」常被用于指称相对大型的蛋白质以及「肽」常被用于指称小型多肽，但在本领域这些术语的使用常重叠。
根据本发明，术语「免疫原性」指重组蛋白质诸如重组抗原性蛋白质激发免疫反应的能力。
根据本发明，提供一种在宿主细胞中重组目的蛋白质的表达系统，其包含主要由SEQ ID NO：1的蛋白质转导结构域(PTD)的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的 核酸片段，其中该核酸片段可运作地连接在宿主细胞中表达异源蛋白质用的宿主特异性转录及翻译调控单元。举例来说，编码PTD的核苷酸序列包含SEQ ID NO：2的核苷酸序列。SEQ ID NO：2的核苷酸序列可通过使用SEQ ID NO：5及SEQ ID NO：6的寡核苷酸而合成。SEQ ID NO：5及SEQ ID NO：6的寡核苷酸可在其5’端磷酰基化，以助该双股寡核苷酸插入由磷酸酯酶处理过的载体。依照另一实施例，HSP40J结构域可选自亚型A、B及C的HSP40J结构域所组成的族群。举例来说，该亚型A、B及C的HSP40J结构域可为人类基因组中41种DnaJ/HSP40蛋白质所含的J结构域或J-类似性结构域(Qiu et al.，Cell.Mol.Life Sci.63：2560-2570(2006))。依照本发明的一个特定实施例，该HSP40J结构域包含SEQ IDNO：4的核苷酸序列所编码的SEQ ID NO：3的肽。SEQ ID NO：4的核苷酸序列可通过使用SEQ ID NO：7至SEQ ID NO：16的寡核苷酸而合成。目的蛋白质可包括，但不限于，HSP肽、生长因子、病毒受体结合性结构域及病毒核心蛋白质。在本发明的实施例中，该重组蛋白质为在经免疫的宿主中显示免疫原性的抗原性多肽。在另一实施例中，本发明亦提供一种疫苗，其包含用上述表达系统所生产的重组蛋白质。
本发明可提供核酸序列，尤其是编码本发明重组蛋白质的DNA序列。在另一些实施例中，该DNA序列可由适于染色体外复制的载体所携带，该等载体诸如噬菌体、病毒、质体、噬质体(phagemid)、黏质体(cosmid)、酵母菌人造染色体YAC或附加体(episome)。更特定而言，编码所期望的重组蛋白质的DNA载体有助于本文所述的制备方法的实施及得到显著量的重组蛋白质。该DNA序列可被插入适当载体(即含有被插入的蛋白质-编码序列的转录及翻译所需的单元的载体)。各种宿主-载体系统可被用于表达蛋白质-编码序列。这些包括用病毒感染的哺乳动物细胞系统；用病毒感染的昆虫细胞系统；微生物诸如含酵母菌载体的酵母菌，或用噬菌体DNA、质体DNA或黏质体DNA转入的细菌。视所用的宿主-载体系统，可使用许多适当的转录及翻译单元之一。例如，该载体可含有启动子诸如细菌T7启动子及可诱导的操纵子(operator)诸如lac操纵子以调控转录。
其它载体及构建体包括染色体DNA序列、非染色体DNA序列及合成DNA序列，例如SV40的衍生物；细菌性质体；噬菌体DNA；杆状病毒；酵母菌质体；酵母菌人造染色体(YACs)；衍生自质体与噬菌体DNA的组合的载体；衍生自质体与病毒DNA的组合的穿梭载体(shuttlevectors)；病毒DNA诸如牛痘病毒(vaccinia)、腺病毒(adenovirus)、禽流感病毒(Avian influenza virus)及假性狂犬病病毒(pseudorabies)。不过，任何其它载体只要在所需宿主细胞中可以复制及存活，即可用于制备核酸表达构建体。该核酸序列可于侧面相接许多供分离及选殖入任何期望载体的限制核酸内切酶部位。亦加入蛋白质鉴定或纯化用标签诸如EE、6XHis、HA或MYC，以促进重组蛋白质接下来的纯化。此外，该核酸表达构建体亦可含有转录终止子。例如，该载体可含有终止核酸序列之转录用的T7终止子。
依据本发明的特定实施例，重组蛋白质可通过使用包含SEQ ID NO：2的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列的表达载体而产生。在另一实施例中，该表达载体可包括SEQID NO：2的核苷酸序列、SEQ ID NO：4的核苷酸序列及编码目的蛋白质的核苷酸序列，连同与该表达载体中的核苷酸序列，其可运作地连接在宿主特异性转录及翻译调控单元。
携带本发明的重组分子的载体可有效率地转导入目的细胞或这些细胞群中。转导效率可通过一种不同策略或不同策略的组合来监测及定量。
举例来说，涉及试管内检定的途径测量重组蛋白质被细胞摄入的量。该检定包括用例如放射活性原子、荧光、磷光或冷光标示物(例如荧光素、若单明、FITC)可侦测性地标记重组蛋白质，然后测量经标记的重组蛋白质的摄入量。另一选择为将该重组蛋白质用能形成可脱离标记的酶诸如山葵过氧化酶、β-半乳糖苷酶、氯霉素乙酰基转移酶或荧光素酶来标记。摄入量可通过数种公知方法来测量，这些公知方法诸如在标准细胞筛选器(例如FACS)中通过荧光显微镜检法或放射自显影法来定量经标记的细胞。
如上文所总述，宿主细胞可用于制备目的以增殖编码所期望的重组蛋白质的核酸。因此宿主细胞可为较高等的真核细胞诸如哺乳动物细胞或较低等的真核细胞诸如酵母菌细胞；或者宿主细胞可为原核细胞诸如细菌细胞。根据本发明的适当宿主细胞之代表例包括，但非限于，细菌细胞诸如大肠杆菌、链霉菌(Streptomyces)、伤寒沙门氏菌(Salmonella typhimurium)；真菌细胞，诸如酵母菌；昆虫细胞，诸如果蝇(Drosophila)S2及夜蛾(Spodoptera)Sf9；动物细胞，诸如MDCK、Hep-2、CHO或COS；人类细胞，诸如Jurkat或293细胞；腺病毒；植物细胞，或者任何已适应于在试管中增殖或原样再生之其它细胞。所属领域的技术人员可通过本文的教示明白适当宿主细胞的选择。
此外，编码所需重组蛋白质的核酸可通过转染细胞的标准技术引进宿主细胞。术语「转染」意指包含将核酸引进宿主细胞的所有公知技术，这些公知技术包括磷酸钙共沉淀法、由DEAE葡聚糖介导的转染、脂质转染法、电穿孔、显微注射、病毒转导及/或整合。
本发明还提供一种生产分离所需重组蛋白质的方法。在该方法中，将引入与调控序列可运作地连接的编码所需蛋白质的核酸的宿主细胞(例如酵母菌、真菌、昆虫、细菌或动物细胞)，在存在该重组蛋白质以刺激编码所需重组蛋白质的核苷酸序列转录下，在培养基中以生产的规模培养。接着，可将所需的重组蛋白质从所得的宿主细胞或从培养基中分离出。标准蛋白质纯化技术可被用于从培养基或从所得的细胞分离所需的蛋白质。特定而言，这些纯化技术可使用各种设备(包括滚瓶、旋转烧瓶、组织培养盘、生物反应器或发酵器)大规模(其量至少以毫克计)表达及纯化所需重组蛋白质。
本发明的重组蛋白质可通过已知技术的适当组合而分离及纯化。这些方法包括，例如，利用溶解度的方法诸如盐沉淀及溶剂沉淀、利用分子量差异的方法诸如透析、超过滤、凝胶过滤及SDS-聚丙烯酰胺凝胶电泳；利用电荷差异的方法诸如离子交换管柱层析法；利用特异性亲和性的方法诸如亲和性层析；利用疏水性差异的方法诸如逆向高效液相层析法；以及 利用等电点差异的方法诸如等电点聚焦电泳法、金属亲和性管柱诸如Ni-NTA。
因此，根据另一实施例，本发明提供一种在宿主细胞中生产具提高产率及免疫原性的重组目的蛋白质的方法。该方法包含：
构建包含表达载体，该表达载体包含主要由SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、编码Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞-特异性转录及翻译调控单元；
在该宿主细胞中转入该表达载体；以及
从该宿主细胞中收集及分离该重组目的蛋白质。
依据另一实施例，该方法尚包含用该重组蛋白质使生物免疫的步骤。该重组蛋白质的免疫原性可用经标记的抗体测定，该经标记的抗体可与转渍在受试膜上的重组蛋白质的抗原决定部位结合。再者，通过在试管内的抗原表达之分析以及测定活体内抗体反应的诱导及以抗原性多肽进行皮下激发免疫测试，来比较该重组蛋白质与其它多肽的免疫原性。
抗原性多肽可包括，但非限于，HSP肽、生长因子、病毒受体结合性结构域、病毒核心蛋白质或其组合。举例来说，能引起对宿主的免疫反应的其它蛋白质或肽亦可包含于本发明中。
现参照下列特别的非限定性实施例更详细地说明本发明。
实施例1：pET22b-PTD1-J1-Ag表达载体的构建
使用pET22b质体(Novagen，Madison，Wis.)建立pET22b-PTD-J1载体，其包括SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、SEQ ID NO：3的Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列，其中该核酸片段与在宿主细胞中表达重组目的蛋白质的宿主-特异性转录及翻译调控单元可运作地连接。编码大肠杆菌用的PTD的最适化密码子的寡核苷酸通过使用下表一所列的SEQ ID NO：5及SEQ ID NO：6的引物而化学合成。该二寡核苷酸的5’端在黏接(annealing)成双股形式之前通过聚 核苷酸激酶而被磷酰化，以插入已用NdeI及BamHI消化且通过牛肠碱性磷酸酯酶(CIAP)去磷酰化的pET22b质体，形成pET22b-PTD1。
表一
| PTD1f | T ATG TAT GGT CGT AAG AAA CGT CGT CAG CGTCGT CGT GG(SEQ ID NO：5) |
| PTD1r | (p)GATCCC ACG ACG ACG CTG ACG ACG TTT CTTACG ACC ATA CA(SEQ ID NO：6) |
HSP40基因的J结构域通过使用表二所示的SEQ ID NO：7至SEQ IDNO：16的寡核苷酸的集合式PCR而合成。这些寡核苷酸被设计用来扩增大肠杆菌用的最适化密码子。将PCR产物选殖入pGEM-T Easy载体(Promega)中以进行单一群落选择及DNA测序。具有编码DNA序列的正确J结构域的质体用BamHI及EcoRI一起消化，以从pGEM-T Easy载体除去0.2kb插入的DNA片段。然后将该DNA片段插入通过BamHI/EcoRI及CIAP处理的pET22b-PTD1载体，以建立pET22b-PTD1-J1表达载体。
表二
| J1nBF1 | tgg atc ctg ggt aaa gat tac tac cag act cac ggt ctc(SEQ IDNO：7) |
| nF2 | tac tac cag act cac ggt ctc gct cgt ggt gca tct gat gat gaaatc(SEQ ID NO：8) |
| F3 | ggt gca tct gat gat gaa atc aaa cgt gct tac cgt cgt cag gcactg(SEQ ID NO：9) |
| F4 | gct tac cgt cgt cag gca ctg cgt tac cat cca gac aaa aac aaagaa(SEQ ID NO：10) |
| F5 | tac cat cca gac aaa aac aaa gaa ccg ggtgca gaa gag aaattc(SEQ ID NO：11) |
| F6 | ccg ggt gca gaa gag aaa ttc aaa gag atc gca gaa gca tacgac gtt(SEQ ID NO：12) |
| EcoRI R1 | cga att cgc acc acc aga acc acc ttt cag acc ttc(SEQ IDNO：13) |
| R2 | aga acc acc ttt cag acc ttc ttc acc gta acg gtc gaa gat ttcacg(SEQ ID NO：14) |
| R3 | gta acg gtc gaa gat ttc acg ttt acg tgg atc gct cag aac gtcgta(SEQ ID NO：15) |
| R4 | tgg atc gct cag aac gtc gta tgc ttc tgc gat ctc(SEQ IDNO：16) |
通常将一种或多种由pET22b-PTD1-J1载体表达的重组多肽或抗原性(Ag)多肽转入成具有最适化大肠杆菌密码子的DNA。该一种或多种重组多肽编码区亦通过PCR扩增。在下列实施例中，这些编码多肽的DNA侧面与EcoRI及XhoI部位接合，以便于插入pET22b-PTD1-J1载体中。结果，构建成pET22b-PTD1-J1-Ag表达载体(以下简称为pET22b-PTD-J-Ag)。
组合式PCR
利用寡核苷酸套组合成密码子经最适化的cDNA。在PCR反应混合物中调整成0.5μM F1及R1以及0.05μM的F2、F3、R3、R2等等。反应条件为94℃历2分钟，继而20个循环的94℃历20秒/40℃历40秒/72℃历20秒以及于72℃进行延长历5分钟。将PCR产物选殖入供质体DNA分离及DNA测序用的pGEM-T Easy。
实施例2：PTD1-J1-Ag重组蛋白质的表达
将pET22b-PTD1-J1-Ag载体转入入大肠杆菌罗塞塔菌株(Novagen)胜任细胞。对于氨苄青霉素(ampicillin)及氯霉素具抗性的大肠杆菌群落在用1mM IPTG诱导之前，于TYD培养基(10g胰蛋白胨/20g酵母菌萃取物/5gNaCl/2g葡萄糖/公升，pH7.2)中培养及扩增至OD600＝0.3。依所列的时间表收集细胞样本。
将0.3OD600单位的大肠杆菌的全部蛋白质样本在30μL的2×SDS-PAGE加载缓冲液中溶裂。这些蛋白质样本以沸水浴处理5分钟后， 通过12.5％或15％SDS-PAGE而分离。该蛋白质带通过在SDS-PAGE凝胶上进行考马斯亮蓝R250染色而显色。
实施例3：免疫
小鼠通过皮下注射用TiterMAX金佐剂乳化的50μg多肽而免疫。每隔二个星期进行剂量追加。收集小鼠的血清以取得对抗该多肽的抗体。
印迹杂交法(Dot blotting)
将经系列稀释的重组蛋白质或合成多肽点加在PVDF膜上。将该膜用在TBSN(25mM Tris-HCl，pH7.4/150mM NaCl/0.02％Tween20)中的5％脱脂乳封阻1小时。用TBSN冲洗4次后，将该膜浸在经1000倍稀释的小鼠抗血清中进行整夜培养。未经结合的原发性抗体(Ab)通过用TBSN冲洗4次而移除，然后施用结合有HRP的山羊抗小鼠二次抗体(经2000倍稀释)历4小时。将经冲洗膜上的经标记蛋白质以化学发光套组(Pierce)按照厂商说明书所述进行测定。
实施例4：人类HSPA1A肽I的表达
将集合式PCR所用的寡核苷酸列于表三中。利用这些来合成SEQ IDNO：17的密码子经最适化的cDNA，该cDNASEQ ID NO：18的HAPA1A肽(SSSTQASLEI DSLFEGIDFY TSITRARFEE LCSDLFRSTL EPVEKALRDA KLDKAQI)。将该密码子经最适化的cDNA序列插入依照实施例1构建的表达载体pET22b-PTD1-J1中。
表三
| RI-I-f1 | G AAT TCT AGC AGC AGC ACC CAG GCG AGC CTGGAAATT GAT AGC CTG TTT(SEQ ID NO：19) |
| I-f2 | AGC CTG GAA ATT GAT AGC CTG TTT GAA GGCATT GAT TTT TAT ACC AGC(SEQ ID NO：20) |
| I-f3 | G TTT GAA GGC ATT GAT TTT TAT ACC AGC ATTACC CGT GCG CGT TTT GAA(SEQ ID NO：21) |
| I-r3 | G GGT GCT ACG AAA CAG ATC GCT GCA CAG TTC |
| TTC AAAACG CGC ACG GGT(SEQ ID NO：22) | |
| I-r2 | C ATC ACG CAG CGC TTT TTC CAC CGG TTC CAGGGT GCT ACG AAA CAG ATC(SEQ ID NO：23) |
| Xho-I-r1 | CTC GAG AAT CTG CGC TTT ATC CAG TTT CGCATC ACG CAG CGC TTT TTC(SEQ ID NO：24) |
将具有编码重组PTD-J-HSPA1A肽I的核酸片段的表达载体引进实施例2所述的大肠杆菌中。定量该重组PTD-J-HSPA1A肽I的表达并以重组PTD-J-HSPA1A肽I在所有蛋白质中所占的重量百分率来表示。参照图1，在最初6小时，该重组PTD-J-HSPA1A肽I的重量百分率在65％至92％的范围内。16小时后，该重组PTD-J-HSPA1A肽I的重量百分率为77％。
实施例5：人类HSPA1A肽II的表达
将集合式PCR所用的寡核苷酸列于表四中。利用这些来合成SEQ IDNO：25的密码子经最适化的cDNA，该cDNASEQ ID NO：26的HAPA1A肽II(NVTATDKSTG KANKITITND KGRLSKEEIE RMVQEAEKYK AEDEVQRERV SAKNALESYA F)。将密码子经最适的cDNA序列插入依照实施例1构建的表达载体中。
表四
| RI-II-fl | G AAT TCT AAC GTAACC GCT ACT GAC AAA TCCACT GGT AAA GCT AAC AAG(SEQ ID NO：27) |
| II-f2 | TCC ACT GGT AAA GCT AAC AAG ATC ACC ATCACC AAC GAC AAA GGT CGT C(SEQ ID NO：28) |
| II-f3 | ATC ACC AAC GAC AAA GGT CGT CTG TCC AAGGAA GAG ATC GAG CGTATG G(SEQ ID NO：29) |
| II-f4 | AAG GAA GAG ATC GAG CGT ATG GTT CAG GAAGCT GAA AAG TAC AAG(SEQ ID NO：30) |
| II-r3 | ACG CTG AAC TTC GTC TTC AGC CTT GTA CTTTTC AGC TTC CTG AAC C(SEQ ID NO：31) |
| II-r2 | AGC GTT CTT AGC GGA AAC ACG TTC ACG CTGAAC TTC GTC TTC AGC(SEQ ID NO：32) |
| Xho-II-r1 | CTC GAG GAA AGC GTA GGA TTC CAG AGC GTTCTT AGC GGA AAC ACG TTC A(SEQ ID NO：33) |
将具有编码重组PTD-J-HSPA1A肽II的核酸片段的表达载体如实施例2所述引进大肠杆菌中。定量该重组PTD-J-HSPA1A肽II的表达并以重组PTD-J-HSPA1A肽II在所有蛋白质中所占的重量百分率来表示。参照图2，在最初6小时，该重组PTD-J-HSPA1A肽II的重量百分率在27％至54％的范围内。16小时后，该重组PTD-J-HSPA1A肽II的重量百分率为10％。
实施例6：人类HSPA1A肽III的表达
将集合式PCR所用的寡核苷酸列于表五中。利用这些来合成SEQ IDNO：34的密码子经最适化的cDNA，该cDNASEQ ID NO：35的HAPA1A肽III(EDEGLKGKIS EADKKKVLDK CQEVISWLDA NTLAEKDEFE HKRKELEQVC NPIISGLYQG A)。将密码子经最适的cDNA序列插入依照实施例1构建的表达载体中。
表五
| RI-III-f1 | G AAT TCT GAA GAT GAA GGC CTG AAA GGCAAAATT AGC GAA GCG GAT(SEQ ID NO：36) |
| III-f2 | GGC AAAATT AGC GAA GCG GAT AAG AAAAAGGTG CTG GATAAA TGC CAG(SEQ ID NO：37) |
| III-f3 | G GTG CTG GAT AAA TGC CAG GAA GTG ATTAGC TGG CTG GAT GCGAAC ACC(SEQ ID NO：38) |
| III-f4 | G CTG GAT GCG AAC ACC CTG GCG GAA AAAGAT GAATTT GAA CAT AAA CGT(SEQ ID NO：39) |
| III-r3 | TTC CAG TTC TTT ACG TTT ATG TTC AAA TTCATC TTT TTC CGC CAG(SEQ ID NO：40) |
| III-r2 | CGG GTT GCA CAC CTG TTC CAG TTC TTT ACGTTTATG TTC AAATTC ATC(SEQ ID NO：41) |
| Xho-III-r1 | CTG GAG CGC GCC CTG ATA CAG GCC GCT AATAAT CGG GTT GCA CAC CTG(SEQ ID NO：42) |
将具有编码重组PTD-J-HSPA1A肽III的核酸片段的表达载体如实施例2所述引进大肠杆菌中。定量该重组PTD-J-HSPA1A肽III的表达并以重组PTD-J-HSPA1A肽III在所有蛋白质中所占的重量百分率来表示。参照图3，在最初5小时，该重组PTD-J-HSPA1A肽III的重量百分率在10％至33％的范围内。
实施例7：鸡IGF-I的表达
将集合式PCR所用的寡核苷酸列于表六中。使用这些寡核苷酸合成SEQ ID NO：43的密码子经最适化的cDNA，该cDNASEQ ID NO：44的鸡IGF-I(GPETLCGAEL VDALQFVCGD RGFYFSKPTG YGSSSAALHHKGIVDECCFQ SCDLRRLEMY CAPIKPPKSA)。将密码子经最适化的cDNA序列插入依照实施例1构建的表达载体中。
表六
| RI-F1new | GAATTCT GGT CCA GAA ACC CTG TGT GGT GCAGAA.CTG GTT GAT GCA CTG CAG TTC GTG(SEQ IDNO：45) |
| F2 | GAA.CTG GTT GAT GCA CTG CAG TTC GTG TGTGGT GAT CGT GGT TTC TAC(SEQ ID NO：46) |
| F3 | GGT GAT CGT GGT TTC TAC TTC AGC AAA CCG ACTGGT TAT GGT AGC TCT AGC(SEQ ID NO：47) |
| F4 | GGT TAT GGT AGC TCT AGC CGT CGT CTG CAT CACAAA GGT ATT GTG GAT G(SEQ ID NO：48) |
| F5 | CAC AAA GGT ATT GTG GAT GAA TGT TGC TTT CAGAGC TGT GAT CTG CGT CG(SEQ ID NO：49) |
| R2 | GAT TGG TGC ACA GTA CAT TTC CAG ACG ACGCAG ATC ACAGCT CTG(SEQ ID NO：50) |
| Xho-R1 | CTCGAG TGC GCT TTT CGG TGG TTT GAT TGG TGCACA GTA CAT TTC C(SEQ ID NO：51) |
将具有编码重组PTD-J-鸡IGF-I的核酸片段的表达载体如实施例2所述引进大肠杆菌中。定量该重组PTD-J-鸡IGF-I的表达并以重组PTD-J-鸡IGF-I在所有蛋白质中所占的重量百分率表示。参照图4，在最初6小时，该重组PTD-J-鸡IGF-I的重量百分率在74％至90％的范围内。
实施例8：禽流感病毒HA(H5)受体结合性结构域(RBD)的表达
将PCR所用的引物对列于表七中。利用这些引物合成SEQ ID NO：52的原生性病毒cDNA，该原生性病毒cDNASEQ ID NO：53的HA-RBD。将cDNA序列插入依照实施例1构建的表达载体中。
表七
| RIHA-RBD f | TG AAT TCG AAA CAC CTA TTG AGC AGA ATAAAC(SEQ ID NO：54) |
| XhoHA-RBD r | TCTCGAG AAT TGT TGA GTC CCC TTT CTT GAC(SEQ ID NO：55) |
将具有编码重组PTD-J-HA RBD的核酸片段的表达载体如实施例2所述引进大肠杆菌中。定量该重组PTD-J-HA RBD的表达及以重组PTD-J-HA RBD在所有蛋白质中所占的重量％表示。参照图5，在最初7小时，该重组PTD-J-HA RBD的重量百分率在12％至38％的范围内。16小时后重组PTD-J-HA RBD的重量百分率为32％。
实施例9：C型肝炎病毒(HCV)核心蛋白质的表达
将PCR所用的引物对列于表八中。利用这些引物合成SEQ ID NO：56的原生性病毒cDNA，该原生性病毒cDNA SEQ ID NO：57的HCV核心蛋白质。将该cDNA序列插入依照实施例1构建的表达载体中。
表八
| RI-Core f | TG AAT TCG ATG AGC ACA AAT CCT AAA CCT C(SEQ ID NO：58) |
| Xho-Core r | TCTCGAG AGC AGA GAC CGG AAC GGT GAT(SEQ ID NO：59) |
将具有编码重组PTD-J-HCV核心蛋白质的核酸片段的表达载体如实施例2所述引进大肠杆菌中。定量该重组PTD-J-HCV核心蛋白质的表达并以PTD-J-HCV核心蛋白质在所有蛋白质中所占的重量百分率表示。参照图5，在最初6小时，该重组PTD-J-HCV核心蛋白质的重量百分率的范围为11％至39％。
从上述实施例的结果显然可知使用该具有最适化密码子的pET22b-PTD-J表达载体所产生的重组蛋白质的重量百分率已证明为全部蛋白质的约90％。另一方面，若使用原生性cDNA，重组蛋白质在全部蛋白质中所占的重量百分率为约40％。因此本发明的表达载体可生产具有提高产率的重组蛋白质。
实施例10：增加嗜中性白血球肽-1(NP-1)的免疫原性
合成编码NP-1的密码子经最适化的cDNA序列并将其插入依照实施例1所述的方法构建的表达载体pET22b-PTD-J。将具有编码重组pET22b-PTD-J-NP-1的核酸片段的表达载体如实施例2所述引进大肠杆菌中。收集重组NP-1及将其从大肠杆菌中分离。
小鼠依照实施例3通过皮下注射重组NP-1而被免疫，以激发抗重组NP-1的抗体产生。然后从小鼠收集血清。使用实施例3所述的墨点检定法测定NP-1的免疫原性。
当使用重组NP-1做为抗原时，产生高滴度的血清。该血清可检测320pg的抗原，但不会与PTD1-J1-核心重组蛋白质交叉反应，此表示该血清会如图7a及图7b所示强力且特异性地对抗NP-1区域。
所属领域的技术人员当知道可在不偏离广义的本发明概念下修饰上述具体实施例。应了解本发明不局限于所揭示的特定具体实施例，而意欲涵盖如权利要求所界定的本发明的精神及范围内的修饰。
序列表
<110>康泰生医科技股份有限公司/财团法人台湾动物科技研究所
<120>具有提高产率及免疫原性的重组蛋白质表达系统
<130>7P04089-TW
<160>59
<170>PatentIn version 3.3
<210>1
<211>11
<212>PRT
<213>大肠杆菌
<400>1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210>2
<211>33
<212>DNA
<213>大肠杆菌
<400>2
tatggtcgta agaaacgtcg tcagcgtcgt cgt 33
<210>3
<211>72
<212>PRT
<213>人类(Homo sapiens)
<400>3
Gly Lys Asp Tyr Tyr Gln Thr His Gly Leu Ala Arg Gly Ala Ser Asp
1 5 10 15
Asp Glu Ile Lys Arg Ala Tyr Arg Arg Gln Ala Leu Arg Tyr His Pro
20 25 30
Asp Lys Asn Lys Glu Pro Arg Ala Glu Glu Lys Phe Lys Glu Ile Ala
35 40 45
Glu Ala Tyr Asp Val Leu Ser Asp Pro Arg Lys Arg Glu Ile Phe Asp
50 55 60
Arg Tyr Gly Glu Glu Gly Leu Lys
65 70
<210>4
<211>216
<212>DNA
<213>人类(Homo sapiens)
<400>4
ggtaaagatt actaccagac tcacggtctc gctcgtggtg catctgatga tgaaatcaaa 60
cgtgcttacc gtcgtcaggc actgcgttac catccagaca aaaacaaaga accgcgtgca 120
gaagagaaat tcaaagagat cgcagaagca tacgacgttc tgagcgatcc acgtaaacgt 180
gaaatcttcg accgttacgg tgaagaaggt ctgaaa 216
<210>5
<211>39
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>5
tatgtatggt cgtaagaaac gtcgtcagcg tcgtcgtgg 39
<210>6
<211>41
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>6
gatcccacga cgacgctgac gacgtttctt acgaccatac a 41
<210>7
<211>39
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>7
tggatcctgg gtaaagatta ctaccagact cacggtctc 39
<210>8
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>8
tactaccaga ctcacggtct cgctcgtggt gcatctgatg atgaaatc 48
<210>9
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>9
ggtgcatctg atgatgaaat caaacgtgct taccgtcgtc aggcactg 48
<210>10
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>10
gcttaccgtc gtcaggcact gcgttaccat ccagacaaaa acaaagaa 48
<210>11
<211>45
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>11
taccatccag acaaaaacaa agaaccgggt gcagaagaga aattc 45
<210>12
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>12
ccgggtgcag aagagaaatt caaagagatc gcagaagcat acgacgtt 48
<210>13
<211>36
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>13
cgaattcgca ccaccagaac cacctttcag accttc 36
<210>14
<211>48
<212>DNA
<213><213>人工合成
<220>
<223>寡核苷酸
<400>14
agaaccacct ttcagacctt cttcaccgta acggtcgaag atttcacg 48
<210>15
<211>48
<212>DNA
<213><213>人工合成
<220>
<223>寡核苷酸
<400>15
gtaacggtcg aagatttcac gtttacgtgg atcgctcaga acgtcgta 48
<210>16
<211>36
<212>DNA
<213><213>人工合成
<220>
<223>寡核苷酸
<400>16
tggatcgctc agaacgtcgt atgcttctgc gatctc 36
<210>17
<211>171
<212>DNA
<213>人类(Homo sapiens)
<400>17
agcagcagca cccaggcgag cctggaaatt gatagcctgt ttgaaggcat tgatttttat 60
accagcatta cccgtgcgcg ttttgaagaa ctgtgcagcg atctgtttcg tagcaccctg 120
gaaccggtgg aaaaagcgct gcgtgatgcg aaactggata aagcgcagat t 171
<210>18
<211>57
<212>PRT
<213>人类(Homo sapiens)
<400>18
Ser Ser Ser Thr Gln Ala Ser Leu Glu Ile Asp Ser Leu Phe Glu Gly
1 5 10 15
Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu Leu Cys
20 25 30
Ser Asp Leu Phe Arg Ser Thr Leu Glu Pro Val Glu Lys Ala Leu Arg
35 40 45
Asp Ala Lys Leu Asp Lys Ala Gln Ile
50 55
<210>19
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>19
gaattctagc agcagcaccc aggcgagcct ggaaattgat agcctgttt 49
<210>20
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>20
agcctggaaa ttgatagcct gtttgaaggc attgattttt ataccagc 48
<210>21
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>21
gtttgaaggc attgattttt ataccagcat tacccgtgcg cgttttgaa 49
<210>22
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>22
gggtgctacg aaacagatcg ctgcacagtt cttcaaaacg cgcacgggt 49
<210>23
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>23
catcacgcag cgctttttcc accggttcca gggtgctacg aaacagatc 49
<210>24
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>24
ctcgagaatc tgcgctttat ccagtttcgc atcacgcagc gctttttc 48
<210>25
<211>183
<212>DNA
<213>人类(Homo sapiens)
<400>25
aacgtaaccg ctactgacaa atccactggt aaagctaaca agatcaccat caccaacgac 60
aaaggtcgtc tgtccaagga agagatcgag cgtatggttc aggaagctga aaagtacaag 120
gctgaagacg aagttcagcg tgaacgtgtt tccgctaaga acgctctgga atcctacgct 180
ttc 183
<210>26
<211>61
<212>PRT
<213>人类(Homo sapiens)
<400>26
Asn Val Thr Ala Thr Asp Lys Ser Thr Gly Lys Ala Asn Lys Ile Thr
1 5 10 15
Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys Glu Glu Ile Glu Arg Met
20 25 30
Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu Asp Glu Val Gln Arg Glu
35 40 45
Arg Val Ser Ala Lys Asn Ala Leu Glu Ser Tyr Ala Phe
50 55 60
<210>27
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>27
gaattctaac gtaaccgcta ctgacaaatc cactggtaaa gctaacaag 49
<210>28
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>28
tccactggta aagctaacaa gatcaccatc accaacgaca aaggtcgtc 49
<210>29
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>29
atcaccaacg acaaaggtcg tctgtccaag gaagagatcg agcgtatgg 49
<210>30
<211>45
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>30
aaggaagaga tcgagcgtat ggttcaggaa gctgaaaagt acaag 45
<210>31
<211>46
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>31
acgctgaact tcgtcttcag ccttgtactt ttcagcttcc tgaacc 46
<210>32
<211>45
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>32
agcgttctta gcggaaacac gttcacgctg aacttcgtct tcagc 45
<210>33
<211>49
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>33
ctcgaggaaa gcgtaggatt ccagagcgtt cttagcggaa acacgttca 49
<210>34
<211>183
<212>DNA
<213>人类(Homo sapiens)
<400>34
gaagatgaag gcctgaaagg caaaattagc gaagcggata agaaaaaggt gctggataaa 60
tgccaggaag tgattagctg gctggatgcg aacaccctgg cggaaaaaga tgaatttgaa 120
cataaacgta aagaactgga acaggtgtgc aacccgatta ttagcggcct gtatcagggc 180
gcg 183
<210>35
<211>61
<212>PRT
<213>人类(Homo sapiens)
<400>35
Glu Asp Glu Gly Leu Lys Gly Lys Ile Ser Glu Ala Asp Lys Lys Lys
1 5 10 15
Val Leu Asp Lys Cys Gln Glu Val Ile Ser Trp Leu Asp Ala Asn Thr
20 25 30
Leu Ala Glu Lys Asp Glu Phe Glu His Lys Arg Lys Glu Leu Glu Gln
35 40 45
Val Cys Asn Pro Ile Ile Ser Gly Leu Tyr Gln Gly Ala
50 55 60
<210>36
<211>46
<212>DNA
<213>人工合成
<220>
<223>引物
<400>36
gaattctgaa gatgaaggcc tgaaaggcaa aattagcgaa gcggat 46
<210>37
<211>48
<212>DNA
<213>人工合成
<220>
<223>引物
<400>37
ggcaaaatta gcgaagcgga taagaaaaag gtgctggata aatgccag 48
<210>38
<211>49
<212>DNA
<213>人工合成
<220>
<223>引物
<400>38
ggtgctggat aaatgccagg aagtgattag ctggctggat gcgaacacc 49
<210>39
<211>49
<212>DNA
<213>人工合成
<220>
<223>引物
<400>39
gctggatgcg aacaccctgg cggaaaaaga tgaatttgaa cataaacgt 49
<210>40
<211>45
<212>DNA
<213>人工合成
<220>
<223>引物
<400>40
ttccagttct ttacgtttat gttcaaattc atctttttcc gccag 45
<210>41
<211>48
<212>DNA
<213>人工合成
<220>
<223>引物
<400>41
cgggttgcac acctgttcca gttctttacg tttatgttca aattcatc 48
<210>42
<211>48
<212>DNA
<213>人工合成
<220>
<223>引物
<400>42
ctcgagcgcg ccctgataca ggccgctaat aatcgggttg cacacctg 48
<210>43
<211>210
<212>DNA
<213>鸡(chicken)
<400>43
ggtccagaaa ccctgtgtgg tgcagaactg gttgatgcac tgcagttcgt gtgtggtgat 60
cgtggtttct acttcagcaa accgactggt tatggtagct ctagccgtcg tctgcatcac 120
aaaggtattg tggatgaatg ttgctttcag agctgtgatc tgcgtcgtct ggaaatgtac 180
tgtgcaccaa tcaaaccacc gaaaagcgca 210
<210>44
<211>70
<212>PRT
<213>鸡
<400>44
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe
1 5 10 15
Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Lys Pro Thr Gly Tyr Gly
20 25 30
Ser Ser Ser Ala Ala Leu His His Lys Gly Ile Val Asp Glu Cys Cys
35 40 45
Phe Gln Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Ile
50 55 60
Lys Pro Pro Lys Ser Ala
65 70
<210>45
<211>58
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>45
gaattctggt ccagaaaccc tgtgtggtgc agaactggtt gatgcactgc agttcgtg 58
<210>46
<211>48
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>46
gaactggttg atgcactgca gttcgtgtgt ggtgatcgtg gtttctac 48
<210>47
<211>51
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>47
ggtgatcgtg gtttctactt cagcaaaccg actggttatg gtagctctag c 51
<210>48
<211>49
<212>DNA
<220>
<213>人工合成
<220>
<223>寡核苷酸
<400>48
ggttatggta gctctagccg tcgtctgcat cacaaaggta ttgtggatg 49
<210>49
<211>50
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>49
cacaaaggta ttgtggatga atgttgcttt cagagctgtg atctgcgtcg 50
<210>50
<211>45
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>50
gattggtgca cagtacattt ccagacgacg cagatcacag ctctg 45
<210>51
<211>46
<212>DNA
<213>人工合成
<220>
<223>寡核苷酸
<400>51
ctcgagtgcg cttttcggtg gtttgattgg tgcacagtac atttcc 46
<210>52
<211>489
<212>DNA
<213>禽流感病毒(Avian influenza virus)
<400>52
aaacacctat tgagcagaat aaaccatttt gagaaaattc agatcatccc caaaagttct 60
tggtccagtc atgaagcctc attaggggtg agctcagcat gtccatacca gggaaagtcc 120
tcctttttca gaaatgtggt atggcttatc aaaaagaaca gtacataccc aacaataaag 180
aggagctaca ataataccaa ccaagaagat cttttggtac tgtgggggat tcaccatcct 240
aatgatgcgg cagagcagac aaagctctat caaaacccaa ccacctatat ttccgttggg 300
acatcaacac taaaccagag attggtacca agaatagcta ctagatccga agtaaacggg 360
caaagtggaa ggatggagtt cttctggaca attttaaaac cgaatgatgc aatcaacttc 420
gagagtaatg gaaatttcat tgctccagaa tatgcataca aaattgtcaa gaaaggggac 480
tcaacaatt 489
<210>53
<211>163
<212>PRT
<213>禽流感病毒(Avian influenza virus)
<400>53
Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile
1 5 10 15
Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser
20 25 30
Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp
35 40 45
Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser Tyr Asn
50 55 60
Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro
65 70 75 80
Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln Asn Pro Thr Thr Tyr
85 90 95
Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Arg Ile
100 105 110
Ala Thr Arg Ser Glu Val Asn Gly Gln Ser Gly Arg Met Glu Phe Phe
115 120 125
Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly
130 135 140
Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp
145 150 155 160
Ser Thr Ile
<210>54
<211>32
<212>DNA
<213>人工合成
<220>
<223>引物
<400>54
tgaattcgaa acacctattg agcagaataa ac 32
<210>55
<211>31
<212>DNA
<213>人工合成
<220>
<223>引物
<400>55
tctcgagaat tgttgagtcc cctttcttga c 31
<210>56
<211>573
<212>DNA
<213>c型肝炎病毒(Hepatitis c virus)
<400>56
atgagcacaa atcctaaacc tcaaagaaaa accaaaagaa acaccaaccg tcgcccagaa 60
gacgttaagt tcccgggcgg cggccagatc gttggcggag tatacttgtt gccgcgcagg 120
ggccccaggt tgggtgtgcg cacgacaagg aaaacttcgg agcggtccca gccacgtggg 180
agacgccagc ccatccccaa agatcggcgc tccactggca aggcctgggg aaaaccaggt 240
cgcccctggc ccctatatgg gaatgaggga ctcggctggg caggatggct cctgtccccc 300
cgaggctctc gcccctcctg gggccccact gacccccggc ataggtcgcg caacgtgggt 360
aaagtcatcg acaccctaac gtgtggcttt gccgacctca tggggtacat ccccgtcgta 420
ggcgccccgc ttagtggcgc cgccagagct gtcgcgcacg gcgtgagagt cctggaggac 480
ggggttaatt atgcaacagg gaacctaccc ggtttcccct tttctatctt cttgctggcc 540
ctgttgtcct gcatcaccgt tccggtctct gct 573
<210>57
<211>191
<212>PRT
<213>C型肝炎病毒(Hepatitis C virus)
<400>57
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Glu Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Thr
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys Ala Trp Gly Lys Pro Gly
65 70 75 80
Arg Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Val Val Gly Ala Pro Leu
130 135 140
Ser Gly Ala Ala Arg Ala Val Ala His Gly Val Arg Val Leu Leu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Phe Pro Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Ile Thr Val Pro Val Ser Ala
180 185 190
<210>58
<211>30
<212>DNA
<213>人工合成
<220>
<223>引物
<400>58
tgaattcgat gagcacaaat cctaaacctc 30
<210>59
<211>28
<212>DNA
<213>人工合成
<220>
<223>引物
<400>59
tctcgagagc agagaccgga acggtgat 28
Claims (12)
1.一种宿主细胞中重组目的蛋白质的表达系统，其特征在于包含由编码SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、编码SEQ ID NO：3的Hsp40 J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞中表达重组目的蛋白质用的宿主特异性转录及翻译调控单元。
2.根据权利要求1所述的表达系统，其特征在于该编码蛋白质转导结构域的核苷酸序列为SEQ ID NO：2的核苷酸序列。
3.根据权利要求1所述的表达系统，其特征在于该编码Hsp40 J结构域的核苷酸序列为SEQ ID NO：4的核苷酸序列。
4.根据权利要求1所述的表达系统，其特征在于该宿主细胞包括大肠杆菌。
5.根据权利要求1所述的表达系统，其特征在于该目的蛋白质包含Hsp肽、生长因子、病毒受体结合性结构域及病毒核心蛋白质中的至少一种。
6.一种重组蛋白质，其特征在于通过使用根据权利要求1所述的表达系统而生产，且该重组蛋白质具有增加的免疫原性。
7.一种疫苗，其特征在于包含权利要求6所述的重组蛋白质。
8.一种在宿主细胞中以增加的产率生产重组目的蛋白质的方法，其特征在于包含：
构建表达载体，该表达载体包含主要由SEQ ID NO：1的蛋白质转导结构域的核苷酸序列、编码SEQ ID NO：3的Hsp40-J结构域的核苷酸序列及编码目的蛋白质的核苷酸序列所组成的核酸片段，其中该核酸片段可运作地连接在宿主细胞特异性转录及翻译调控单元；
在宿主细胞中转入该表达载体；以及
从宿主细胞中收集及分离该重组目的蛋白质。
9.根据权利要求8所述的方法，其特征在于编码该蛋白质转导结构域的核苷酸序列为SEQ ID NO：2的核苷酸序列。
10.根据权利要求8所述的方法，其特征在于编码该Hsp 40 J结构域的核苷酸序列为SEQ ID NO：4的核苷酸序列。
11.根据权利要求8所述的方法，其特征在于该宿主细胞包括大肠杆菌。
12.根据权利要求8所述的方法，其特征在于该目的蛋白质包含Hsp肽、生长因子、病毒受体结合性结构域及病毒核心蛋白质中的至少一种。
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101079171A CN101307310B (zh) | 2007-05-15 | 2007-05-15 | 具有提高产率及免疫原性的重组蛋白质表达系统 |
| EP08748503.3A EP2154251B1 (en) | 2007-05-15 | 2008-05-15 | Expression system of recombinant proteins with enhanced yield and immunogenicity |
| AU2008250892A AU2008250892B2 (en) | 2007-05-15 | 2008-05-15 | Recombinant protein expression system with enhanced yield and immunogenicity |
| JP2010507780A JP5602012B2 (ja) | 2007-05-15 | 2008-05-15 | 収率及び免疫原性を高めた組換えタンパク質発現系 |
| PCT/CN2008/000948 WO2008138229A1 (fr) | 2007-05-15 | 2008-05-15 | Système d'expression de protéine recombinante à rendement et immunogénicité améliorés |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101079171A CN101307310B (zh) | 2007-05-15 | 2007-05-15 | 具有提高产率及免疫原性的重组蛋白质表达系统 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101307310A CN101307310A (zh) | 2008-11-19 |
| CN101307310B true CN101307310B (zh) | 2012-08-08 |
Family
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Family Applications (1)
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| CN2007101079171A Active CN101307310B (zh) | 2007-05-15 | 2007-05-15 | 具有提高产率及免疫原性的重组蛋白质表达系统 |
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| EP (1) | EP2154251B1 (zh) |
| JP (1) | JP5602012B2 (zh) |
| CN (1) | CN101307310B (zh) |
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| KR20180049238A (ko) * | 2012-12-05 | 2018-05-10 | 솔라 바이오사이언시즈 엘엘씨 | 단백질 발현 증진 폴리펩타이드 |
| TWI666218B (zh) * | 2016-02-05 | 2019-07-21 | 財團法人農業科技研究院 | 針對犬小病毒的重組蛋白疫苗 |
| CN113151296B (zh) * | 2021-03-22 | 2022-09-13 | 云南中烟工业有限责任公司 | 一种烟草热激蛋白相关的基因及其应用 |
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| CN1663613A (zh) * | 2004-12-29 | 2005-09-07 | 中国药科大学 | 能通过免疫防治ⅰ型糖尿病的重组蛋白和重组基因 |
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| US5976552A (en) * | 1995-04-28 | 1999-11-02 | Protein Sciences Corporation | Virus vaccines |
| EP1077263A1 (de) * | 1999-07-29 | 2001-02-21 | F.Hoffmann-La Roche Ag | Verfahren zur Herstellung von natürlich gefalteten und sekretierten Proteinen durch Co-Sekretion von Chaperonen |
| US6825005B2 (en) * | 2000-07-19 | 2004-11-30 | President And Fellows Of Harvard College | Methods and reagents to regulate apoptosis |
| US20020142299A1 (en) * | 2001-01-09 | 2002-10-03 | Davidson Beverly L. | PTD-modified proteins |
| KR20030074787A (ko) * | 2001-02-05 | 2003-09-19 | 스트레스젠 바이오테크놀러지스 코포레이션 | Ｂ형 간염 바이러스 치료 |
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| CN1663613A (zh) * | 2004-12-29 | 2005-09-07 | 中国药科大学 | 能通过免疫防治ⅰ型糖尿病的重组蛋白和重组基因 |
Non-Patent Citations (3)
| Title |
|---|
| Jorg Reimann et al..DNA vaccines expressing antigens with a stress protein-capturing domain display enhanced immunogenicity.Immunological Reviews.2004,19954-57. * |
| X.B.Qiu et al..The diversity of the DnaJ/Hsp40 family,the crucial partners for Hsp70 chaperones.Cell.Mol.Life Sci..2006,632563-2564. * |
| Yichen Lai et al..Selectively increasing inducible heat shock protein 70 via TAT-protein transduction protects neurons from nitrosative stress and excitotoxicity.Journal of Neurochemistry.2005,94361. * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2008250892B2 (en) | 2013-09-05 |
| JP5602012B2 (ja) | 2014-10-08 |
| EP2154251A1 (en) | 2010-02-17 |
| AU2008250892A1 (en) | 2008-11-20 |
| JP2010526544A (ja) | 2010-08-05 |
| EP2154251A4 (en) | 2010-08-04 |
| EP2154251B1 (en) | 2013-09-04 |
| CN101307310A (zh) | 2008-11-19 |
| WO2008138229A1 (fr) | 2008-11-20 |
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