CN100516220C - 双岐杆菌的丝氨酸蛋白酶抑制剂 - Google Patents
双岐杆菌的丝氨酸蛋白酶抑制剂 Download PDFInfo
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Abstract
本发明涉及双歧杆菌的新基因及此基因编码的多肽。特别地,本发明是涉及属于丝氨酸蛋白酶抑制剂超家族的基因以及此基因在细菌丝氨酸蛋白酶抑制剂生产中的应用。同样的,本发明提供了生产细菌丝氨酸蛋白酶抑制剂多核苷酸和/或多肽的载体、宿主细胞以及方法。
Description
本发明涉及双歧杆菌的新基因以及此基因编码的多肽。特别地,本发明是关于属于丝氨酸蛋白酶抑制剂(Serpin)超家族的一个基因及其在细菌丝氨酸蛋白酶抑制剂生产中的应用。同样提供生产细菌丝氨酸蛋白酶抑制剂多核苷酸和/或多肽的载体、宿主细胞、以及方法。
乳酸菌可用于长时间储存和制备食物原料,这主要得益于它的低pH及其发酵活性期内产生的产物的作用。此外,乳酸菌与多种食品产物的生产相关,例如奶酪或酸奶。
最近,乳酸菌特别是乳酸杆菌和双歧杆菌引起很大的关注,这是由于已经发现它们的一些菌株在人和动物的摄食过程中展现出一些很有价值的特性。这些菌株通常被认为是益生菌,发现它们能够在胃肠道中普遍存在的严苛环境条件下存活,并且能够至少在肠粘膜上短暂集群,在这里它们对掺入它们的生物的产生阳性反应。
在EP 0768375中公开了双歧杆菌属的这样一株益生菌株,此菌株可以移植入肠道菌群中。据报导,此双歧杆菌可以辅助宿主的免疫调节,并且可以竞争性地排除致病菌对肠细胞的粘附,从而可以支持个体健康的维持。
此外,在参考文献EP 0577903中使用了具有替代幽门螺杆菌能力的乳酸菌,而幽门螺旋菌是公认的溃疡发展原因。
并且,在WO 97/00078中公开的一个特有的乳酸杆菌菌株就是这样的益生菌,命名为乳酸杆菌GG(ATCC 53103)。此微生物可用来防止或治疗食物引起的过敏反应。
考虑到这些益生菌株可提供有价值的特性,特别希望能得到关于这些菌株生物学方面的详细信息,尤其是关于其与宿主的相互作用、它们能在肠道不同环境中生存的现象以及它们粘附肠粘膜的能力。特别地,其在免疫系统增强和防御病原体方面的作用引起了很大关注。
因此,本发明的一个问题就是提供关于展示出对人和/或动物有益的特性的细菌菌株资料并进行说明。
在研究益生菌双歧杆菌BL29菌株基因组过程中,本发明的发明者们惊讶地发现了一个展示出与丝氨酸蛋白酶抑制剂超家族基因具有中等程度的同源性的基因(丝氨酸蛋白酶抑制剂)。目前为止仅在更高等生物体的细胞中发现这种类型的基因,例如人和植物细胞,但在细菌中没有发现。
结果,本发明提供了一种核酸,此核酸为SEQ ID.NO.1或编码功能性多肽的其部分或变体,此变体与SEQ ID.NO.1具有约75%的同源性,优选80%,更优选85%,甚至更优选90%,甚至更优选95%。
依照一个替代性实施方案,本发明也涉及多肽SEQ ID.NO.2或其功能性部分或变体,此变体与SEQ ID.NO.2具有约75%的同源性,优选80%,更优选85%,甚至更优选90%,甚至更优选95%。
丝氨酸蛋白酶抑制剂(Serpins)包含不同的蛋白群,而此蛋白形成了包含100多个成员在内的超家族。多数丝氨酸蛋白酶抑制剂充当蛋白酶抑制剂并参与对一些蛋白酶活化的生理过程的调节,其对于个体非常重要,例如血液凝固、补体介导的溶解、免疫应答、肾小球肾炎、痛觉、炎症、胰腺炎、癌症、受精调节、细菌感染和病毒成熟。虽然丝氨酸蛋白酶抑制剂的主要功能体现在对丝氨酸蛋白酶活性的中和上,但发现这些多肽在细胞外基质重塑和细胞迁移过程中也起作用。
丝氨酸蛋白酶抑制剂的示例包括α1-抗胰蛋白酶、抗凝血酶III、纤溶酶原激活剂抑制剂1(PAI-1)或纤溶酶原激活剂抑制剂2。
到目前为止丝氨酸蛋白酶抑制剂是进行了大量研究的实验对象,它们都有一个共有的特征环,称为反应位点环(RSL),其从包含相关丝氨酸蛋白酶活性位点的识别序列的分子表面向外延伸。据认为,每个抑制剂的特异性主要由丝氨酸蛋白酶抑制剂的潜在切割位点氨基末端侧紧邻的氨基酸的身份所确定。已知这个氨基酸是Pi位点残基,据认为它与丝氨酸蛋白酶的活性位点的丝氨酸形成酰基键。
丝氨酸蛋白酶抑制剂似乎作为“自杀抑制剂”而与靶蛋白酶形成1∶1的化学数量复合物,从而阻断它们的活性。依照目前的资料显示抑制剂在反应中心被切割,并且复合物最有可能作为共价酰基-酶复合物被捕获。
由于丝氨酸蛋白酶抑制剂参与较高级的生物的复杂生物学过程,例如免疫系统的调节或炎症反应或甚至细胞外基质的重塑,所以就没有预想到它们出现在原核生物中。事实上,到目前为止没有源自于原核细胞的丝氨酸蛋白酶抑制剂的报道。
因此,本发明者第一次发现一些细菌细胞可能也包含丝氨酸蛋白酶抑制剂。不受任何理论的约束,目前假定衍生该抑制剂的双歧杆菌菌株的益生特性如免疫系统调节或已知的特性至少部分归因于丝氨酸蛋白酶抑制剂的存在。
在本申请的上下文中,本发明中的核酸指定为多核苷酸SEQ.ID.NO.1或可产生功能性多肽的其部分或变体。为了此目的,可在SEQ.ID.NO.1所示的核酸的末端进行一定程度的截短,其产生的多肽仍然能产生生物学功能。同样的,在所得到的多肽仍然保持生物学的功能的前提下,可通过一个或多个核苷酸的缺失、添加或取代而对核酸进行修饰。
同样的,此处描述的多肽也是如此。根据本发明,术语多肽指定为SEQ ID NO.2或其功能性部分或变体。因此,可在SEQ ID NO.2中所示的多肽的末端进行一定程度的截短,该多肽仍然能产生生物学功能。同样的,在所得到的多肽仍然保持生物学的功能的前提下,可通过一个或多个氨基酸的缺失、添加或取代而对多肽进行修饰。
根据一种实施方案,以上提及的核酸可插入到合适的宿主细胞并在其中表达。为了此目的,本发明的核酸可插入到允许其在所需宿主细胞中增殖和/或表达并允许其插入的合适载体中。此载体应包含一个能够稳定增殖的标记基因。
同样地,利用同源重组现象或其他仅允许核酸插入到宿主的染色体上的技术也可将本发明的核酸包含在宿主基因组中。这些技术在如EP 93105303.7中有描述,其内容在此处引用作为参考。
依赖于是否基因产物将过量表达,也即为了收集和纯化多肽,或表达至一定程度从而通过载体系统如微生物递送给个体,可将本发明的核酸置于内源或外源调节子如启动子控制之下。调节子如启动子优选为可调节的和/或可诱导的,并且可通过公认的且易操作的方法将其与编码分子进行可操作性连接。
然后,将这些重组的构建体导入以在合适的宿主细胞,如原核宿主细胞如大肠杆菌、乳酸杆菌、链球菌或双歧杆菌或真核宿主细胞如酿酒酵母、昆虫细胞、CHO或COS细胞中表达,并在允许异源基因表达的环境中培养转化或转导的宿主细胞。应该明白,本核发明中的多核苷酸的基因产物在真核表达系统中表达时会进行糖基化。
本发明另一个方面也包括至少包含本发明的核酸的一个拷贝的重组微生物。此核酸可包含在用来向个体递送靶物质的微生物中。在此方面,益生菌是适合的,因为它们能够通过个体的胃肠道并至少可短暂植入宿主的肠粘膜中。在这个位点上,它能够发挥衍生其的本发明的益生菌株BL29的生物学功能。不受任何理论的约束,目前认为此核酸的基因产物参与双歧杆菌BL29菌株所显示的抗炎活性。同样,任何已包含本发明的核酸的菌株都可作为宿主细胞,其中也可能包括额外的靶核酸拷贝。
宿主细胞可表达本发明中的核酸的基因产物。因而宿主细胞可以用来大量合成本发明的多肽。
本发明的“宿主细胞”可以通过重组方法得到,因为可将本发明的核酸插入到合适的细胞中。然而,为了增加含有此类多肽的双歧杆菌中的双歧杆菌-丝氨酸蛋白酶抑制剂量,双歧杆菌本身可经由普通突变筛选技术从而使得具有提高的相应基因产物量。
蛋白分离可通过已知的方法从宿主细胞或宿主细胞培养物上清中进行。Ausubel I.,Frederick M.,Current Protocols in Mol.Biol.(1992),John Wiley and Sons,New York中对这些方法进行了描述。可运用已知的包括免疫沉淀、凝胶过滤、离子交换层析、层析聚焦、等电聚焦、选择性沉淀、电泳等在内的蛋白纯化技术,通过亲和层析从重组产物中纯化多肽。
本发明进一步包括检测本发明的核酸或多肽的方法,此方法包括利用本发明中的任一核酸分子孵育样品如细胞裂解物或RNA样品的逆转录物,并确定该核酸分子在严格条件下与靶核酸分子的杂交从而确定核酸分子的存在,或利用抗体,优选抗本发明的多肽的单克隆抗体。也可利用PCR技术,优选应用通过如Roche Diagnostics GmbH,DE的LightCyclerTM进行的定量RT-PCR对基因进行定量检测。
由于丝氨酸蛋白酶抑制剂基因似乎与细菌菌株的益生活性有关,本发明中的核酸和/或多核苷酸同样可用于寻找可展示益生活性的额外菌株。
为了确定给定的细菌菌株是否合适,可确定与细菌菌株的一种或多种靶核酸进行杂交的核酸的大约量。可很容易通过如检测杂交作用的可视检查定性确定杂交的大约量。例如,如果利用凝胶解析可与样品中靶核酸杂交的标记核酸,生成的条带就可进行可视检测。同样的,FACS抗体应用可用于进行定量测量。
本发明的核酸或多肽可用于对可影响与丝氨酸蛋白酶活性相关的生物学过程的分子进行鉴定、表征和/或纯化。丝氨酸蛋白酶参与的生物学过程示例包括凝血、纤维蛋白溶解、免疫反应、补体激活、炎症反应、细胞外基质更新、细胞迁移和激素原激活、癌症转移。
同样的,本发明的核酸或多肽也可用于开发在治疗和/或诊断涉及丝氨酸蛋白酶活性的疾病状态中最终适用的分子。一旦了解了本发明中的丝氨酸蛋白酶抑制剂与靶蛋白酶的相互作用,就可设计蛋白酶的激动剂或拮抗剂以及丝氨酸蛋白酶抑制剂的激动剂或拮抗剂。如上所述,本发明考虑的疾病状态的非限制性示例为异常凝血、异常纤维蛋白溶解、免疫反应、补体激活、炎症反应、细胞外基质更新、细胞迁移和激素原激活、癌症转移。
多种模型系统的应用或结构研究能够开发用于调节本发明中的丝氨酸蛋白酶抑制剂和与之结合的蛋白酶的功能的激动剂或拮抗剂。可以想象,它们可为肽、突变的配体、可与本发明中的丝氨酸蛋白酶抑制剂相互作用的抗体或其他分子。
在适于表达多肽并收集和纯化多肽的条件下,通过在合适的宿主中表达本发明的核酸或包含此核酸的载体,本发明的核酸一般可用来合成用于大规模生产的多肽。
以下实施例例证了本发明,而没有对其进行限制。
实施例1
丝氨酸蛋白酶抑制剂基因的分离
在对插入的克隆进行荧光自动化序列分析和利用软件程序对这些核苷酸片段序列(插入序列)进行组装后,在利用定向测序法对长双歧杆菌BL29基因组序列进行分析的过程中发现了一个开放阅读框。为了完成这些操作,产生了基因组片段,连接到合适的用来扩增和繁殖的载体上,并对相应片段进行测序。通过合适的软件对片段的重叠和最后排序及其核苷酸序列进行评定。
利用以下算法评定了蛋白和/或核酸序列的同源性:
(1)BLASTP:将氨基酸询问序列与蛋白序列数据库进行比较
(2)BLASTN:将核苷酸询问序列与核苷酸序列数据库进行比较
(3)BLASTX:将在所有阅读框中翻译的核苷酸询问序列与蛋白序列数据库进行比较
(4)TBLASTN:将蛋白询问序列与在所有阅读框内动态翻译的核苷酸数据库进行比较
已经注意到与已知的鼠丝氨酸蛋白酶抑制剂α-2-抗纤溶酶具有约43%的中度总体同源性。而此多肽展示出与反应位点环(RSL)具有约63%的同源性。
实施例2
双歧杆菌丝氨酸蛋白酶抑制剂基因的克隆和多肽的分离
依照普通的技术,将编码从其信号肽上去除的推定的双歧杆菌丝氨酸蛋白酶抑制剂的核酸克隆到大肠杆菌表达载体pDEST 17(Invitrogen Life Technologies)上并在大肠杆菌中制备了相应的蛋白以作为6-His标记的融合蛋白(Janknecht R et al.(1991):Proc.Natl.Acad.Sci.USA:88(20)pp 8972-8976)。
按照Quiagen的QIA-Expressionist手册中的指导说明,在镍-氨三乙酸基质上利用金属亲和层析将6-His标记的融合蛋白纯化至均匀,并用于在兔中进行功能研究和多克隆抗体产生的研究。
实施例3
在BL29中表达的丝氨酸蛋白酶抑制剂样蛋白的潜在的抗炎活性
利用体外溶血测定法鉴定实施例2中分离得到的多肽作为丝氨酸蛋白酶抑制剂的活性。
为了此目的,将50μL连续双倍稀释的人AB血清(Sigma)与50μL 1.7%的绵羊抗体激活的绵羊红细胞悬液在96孔微量滴定板中混合。为了评估重组丝氨酸蛋白酶抑制剂的抗溶血活性,每孔加入1μg重组蛋白并且在37℃孵育混合物1小时。然后将滴定板离心,以沉淀完整的细胞沉淀并将细胞碎屑和上清转入另一新板中。溶血活性是通过测定在450nm释放血红蛋白的吸光度来测定。
将双歧杆菌丝氨酸蛋白酶抑制剂活性与人的丝氨酸蛋白酶抑制剂α1-抗胰蛋白酶(5μg/孔)进行了比较。以蒸馏水作为阳性对照获得了100%的红细胞溶解,人失活AB血清作为阴性对照(由于补体失活而无溶解)。
结果清楚地表明,从双歧杆菌获得的重组丝氨酸蛋白酶抑制剂与人α1-抗胰蛋白酶抑制红细胞溶解的程度相同,α1-抗胰蛋白酶是一种已知的丝氨酸蛋白酶抑制子。
序列表
<110>雀巢制品公司
<120>双歧杆菌的丝氨酸蛋白酶抑制剂
<130>NO 7204/WO
<140>WO 02060932
<141>2002-01-30
<150>EP 01102050.0
<151>2001-01-30
<160>2
<170>PatentIn version 3.1
<210>1
<211>1395
<212>DNA
<213>长双歧杆菌(Bifidobacterium longum)
<400>1
atgagcgagc aactgatgga acagtaccgg ttgcgcggac aacgcaaatg ccgtaacgct 60
tgtatcgccg ccatcgtgac agtagtgctt gtccttgccg tcgccggcgg cgtatggtgg 120
acggccggcg atggcagcgc attggttcgc aatatgttca agccgaaggc cacgcctgcc 180
acgcagccgg tagtcaacag caccgcaacc ttcgcctacc gcaccgcacc ggaattcctg 240
gcgatggaag ccggcgaccg aggcaccggc aatgtgaact actctcctgc ttcgatgtgg 300
atggcgttgg ccatcgccgc gcagggcgcc aatggcacga cccgctcgca actgaacgaa 360
ctgctgggct ccggttcgct gaccgatagc gactaccaat cgctgctaag ttcgatcaac 420
gggcaatatt cgggggcgaa atccgagatg agcgccgcga actcgctgtg gattgatgac 480
gactactctc ttgccagcga ttaccaatcc accgtcaaga agatgttcga ggccgaagtc 540
accacgttac cgttcgacga tcaggccgcc gccaagatgt ccgattggat tgccaagcat 600
acgaatggtt cgctcaagcc gaagatcacg ctgcgtgacc gtgaagtcct gtccatcatc 660
aacaccgtct atgcggatgg ccgctggaag gatccgttcg aagagcagtc caccggcaac 720
ggcaccttcc acggcgaagc cggagatgct caggtgccga tgatgcacca gaccttcagc 780
caaatggctt acggacatga tgagtacaac acttggcagc gggtggagat tccgttcgac 840
aacggcggca atctggccat cgtgctgccg gccgaagggc atttcgacga gttggccggc 900
gatgccgaga agctcagttg ggcgttcggt acatgctcga cggcatccct tggcgagggc 960
gcaatgggtt gcgccgcgga cagtatgccc ggctggggcg tctccgtcaa ctcggtcatg 1020
gtgaacgtca cgctaccgcg attcaccatc gacagcatgt tcgactcgga agccaccatc 1080
aaggcattcg aaaaactggg ggtgaccgat gcgttcagtg caggcgacgc cgacttcacc 1140
aagatgatcg acaccggttc gcacggcgag aacctgtata tcggctcgat tctgcaaggc 1200
acgcgcatcg aggtgaacga agccggcgcc aaggccatgt ccttcaccaa ggtcggcgca 1260
gactccgtta gcgcgccggt ggacaacgtc gagttcacgg tggatcgccc atttctgtat 1320
tcgtacgtca ccccggacgg cataccatta ttcatcggtg cggtgcgcaa cctcggcgga 1380
gtcggtggag aaaac 1395
<210>2
<211>465
<212>PRT
<213>长双歧杆菌(Bifidobacterium longum)
<400>2
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Cys Arg Asn Ala Cys Ile Ala Ala Ile Val Thr Val Val Leu Val Leu
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Ala Val Ala Gly Gly Val Trp Trp Thr Ala Gly Asp Gly Ser Ala Leu
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Val Arg Asn Met Phe Lys Pro Lys Ala Thr Pro Ala Thr Gln Pro Val
50 55 60
Val Asn Ser Thr Ala Thr Phe Ala Tyr Arg Thr Ala Pro Glu Phe Leu
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Ala Met Glu Ala Gly Asp Arg Gly Thr Gly Asn Val Asn Tyr Ser Pro
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Ala Ser Met Trp Met Ala Leu Ala Ile Ala Ala Gln Gly Ala Ash Gly
100 105 110
Thr Thr Arg Ser Gln Leu Asn Glu Leu Leu Gly Ser Gly Ser Leu Thr
115 120 125
Asp Ser Asp Tyr Gln Ser Leu Leu Ser Ser Ile Asn Gly Gln Tyr Ser
130 135 140
Gly Ala Lys Ser Glu Met Ser Ala Ala Asn Ser Leu Trp Ile Asp Asp
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Asp Tyr Ser Leu Ala Ser Asp Tyr Gln Ser Thr Val Lys Lys Met Phe
165 170 175
Glu Ala Glu Val Thr Thr Leu Pro Phe Asp Asp Gln Ala Ala Ala Lys
180 185 190
Met Ser Asp Trp Ile Ala Lys His Thr Asn Gly Ser Leu Lys Pro Lys
195 200 205
Ile Thr Leu Arg Asp Arg Glu Val Leu Ser Ile Ile Asn Thr Val Tyr
210 215 220
Ala Asp Gly Arg Trp Lys Asp Pro Phe Glu Glu Gln Ser Thr Gly Asn
225 230 235 240
Gly Thr Phe His Gly Glu Ala Gly Asp Ala Gln Val Pro Met Met His
245 250 255
Gln Thr Phe Ser Gln Met Ala Tyr Gly His Asp Glu Tyr Asn Thr Trp
260 265 270
Gln Arg Val Glu Ile Pro Phe Asp Asn Gly Gly Asn Leu Ala Ile Val
275 280 285
Leu Pro Ala Glu Gly His Phe Asp Glu Leu Ala Gly Asp Ala Glu Lys
290 295 300
Leu Ser Trp Ala Phe Gly Thr Cys Ser Thr Ala Ser Leu Gly Glu Gly
305 310 315 320
Ala Met Gly Cys Ala Ala Asp Ser Met Pro Gly Trp Gly Val Ser Val
325 330 335
Asn Ser Val Met Val Asn Val Thr Leu Pro Arg Phe Thr Ile Asp Ser
340 345 350
Met Phe Asp Ser Glu Ala Thr Ile Lys Ala Phe Glu Lys Leu Gly Val
355 360 365
Thr Asp Ala Phe Ser Ala Gly Asp Ala Asp Phe Thr Lys Met Ile Asp
370 375 380
Thr Gly Ser His Gly Glu Asn Leu Tyr Ile Gly Ser Ile Leu Gln Gly
385 390 395 400
Thr Arg Ile Glu Val Asn Glu Ala Gly Ala Lys Ala Met Ser Phe Thr
405 410 415
Lys Val Gly Ala Asp Ser Val Ser Ala Pro Val Asp Asn Val Glu Phe
420 425 430
Thr Val Asp Arg Pro Phe Leu Tyr Ser Tyr Val Thr Pro Asp Gly Ile
435 440 445
Pro Leu Phe Ile Gly Ala Val Arg Asn Leu Gly Gly Val Gly Gly Glu
450 455 460
Asn
465
Claims (15)
1.SEQ ID.NO.1所示核酸。
2.SEQ ID.NO.2所示多肽。
3.包含权利要求1中的核酸的载体。
4.包含权利要求1中的核酸的一个或多个拷贝或权利要求3中的载体的宿主细胞。
5.权利要求4中的宿主细胞,其中表达了权利要求2中的多肽。
6.权利要求1或2中的核酸或多肽在鉴定、表征和/或纯化可影响与丝氨酸蛋白酶活性相关的生物学过程的分子中的用途。
7.权利要求1或2中的核酸或多肽在开发用于治疗和诊断涉及丝氨酸蛋白酶活性的疾病状态的分子中的用途。
8.权利要求1中的核酸或权利要求2中的多肽在制备用于治疗和诊断涉及丝氨酸蛋白酶活性的疾病状态的载体中的用途。
9.权利要求8中的用途,其中载体是药物、食品或食品添加剂。
10.权利要求6到9中任一项的用途,其中生物学过程或疾病状态选自凝血、纤维蛋白溶解、免疫反应、补体激活、炎症反应、细胞外基质的更新、细胞迁移和激素原激活、癌症转移。
11.制备权利要求2中的多肽的方法,包括在合适的宿主中表达权利要求1中的核酸或权利要求3中的载体并对获得的多肽进行纯化。
12.用于检测益生菌菌株的方法,包括用权利要求1中的核酸或抗权利要求2中多肽的抗体筛选微生物中所述核酸或所述多肽的存在,并确定微生物中丝氨酸蛋白酶抑制剂的存在。
13.制备可表达或过量表达由权利要求1中的核酸编码的丝氨酸蛋白酶抑制剂的方法,包括用权利要求1的核酸转化微生物和表达编码丝氨酸蛋白酶抑制剂多肽的基因。
14.食品,其包含权利要求1中的核酸、权利要求2中的多肽、权利要求3中的载体或权利要求4或5中任一项的宿主细胞。
15.药品,其包含权利要求1中的核酸、权利要求2中的多肽、权利要求3中的载体或权利要求4或5中任一项的宿主细胞。
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