CN101012458A - 表达系统 - Google Patents
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- CN101012458A CN101012458A CNA2006101290967A CN200610129096A CN101012458A CN 101012458 A CN101012458 A CN 101012458A CN A2006101290967 A CNA2006101290967 A CN A2006101290967A CN 200610129096 A CN200610129096 A CN 200610129096A CN 101012458 A CN101012458 A CN 101012458A
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Abstract
表达系统。一种引起抗炭疽芽孢杆菌的保护性免疫应答的免疫原性试剂，所述试剂包含结合在一起代表炭疽芽孢杆菌全长保护性抗原(PA)的至多3个结构域或这些结构域的变异体的一种或多种多肽，并且所述结构域中的至少一个包含PA的结构域1或结构域4或其变异体。通过从大肠杆菌表达，生产所述免疫原性试剂的多肽以及全长PA。用该方法获得高产量的多肽。也描述并要求保护了在该方法中使用的细胞、载体和核酸。
Description
本申请是专利申请号为01812409.7、国际申请日为2001年7月6日(国际申请号为PCT/GB01/03065)、发明名称为“表达系统”的发明专利申请的分案申请。
发明领域
本发明涉及引起抵抗炭疽芽孢杆菌(Bacillus anthracis)感染的保护性免疫应答的多肽，涉及生产这些多肽的方法、可用于所述方法的重组大肠杆菌(Escherichia coli)细胞以及采用的核酸和转化载体。
背景技术
用于表达供疫苗系统用的保护性抗原(PA)的本发明系统，用蛋白酶缺陷型枯草杆菌(Bacillus subtilis)作为表达宿主。尽管这类系统就产物的量和纯度而论是可接受的，但仍有显著缺陷。首先，管理机构一般不熟悉这种宿主，批准决定可能因此而延迟。更重要的是，目前使用的枯草杆菌菌株产生热稳定孢子，因而需要使用专用的生产工厂。
WO00/02522具体描述了表达PA或某些免疫原性片段的VEE病毒复制子。
众所周知大肠杆菌是多种人用疫苗的表达系统。虽然容易使大肠杆菌发酵至非常高的细胞密度的能力，使得这种细菌成为表达许多蛋白质的理想宿主，但先前为用表达和从大肠杆菌细胞溶胶中纯化重组PA所作的尝试由于蛋白质收率低和蛋白酶降解而受到阻碍(Singh等，J.Biol.Chem.(1989)264；11099-11102，Vodkin等，Cell(1993)34；693-697以及Sharma等，Protein Expr.Purif.(1996)，7，33-38)。
在大肠杆菌细胞溶胶中过量表达作为稳定的可溶性蛋白的PA的策略最近已有描述(Willhite等，Protein and Peptide Letters，(1998)，5；273-278)。所用的策略是将亲和标志序列加至PA的N末端的一种策略，这提供了一种简单的纯化系统。
这种系统的一个问题是需要另一个下游的加工步骤，以便在可以使用PA之前除去所述标志。
密码子优化是现在众所周知并且用于合成基因设计的一种技术。遗传密码有一定程度的丰余性，因此大多数氨基酸由不止一个密码子序列编码。不同的生物优先利用这些不同密码子中的某一个。一般可预期，通过优化密码子，将提高特定蛋白的表达水平。
这一般是理想的，但PA除外，就PA而论，较高的表达水平会导致蛋白酶降解和/或细胞毒性。在这种情况下，提高表达水平可能是逆生产性的(counter-productive)，并且引起显著的细胞毒性。
然而意外的是，本申请人已经发现，在大肠杆菌中情况不是这样，在该系统中，无论在该菌株中是否存在蛋白酶，密码子优化均导致意想不到的高水平重组PA的表达。
此外，似乎PA保护性结构域的表达并不抑制大肠杆菌中的表达。
天然PA的晶体结构已经阐明(Petosa C.等Nature 385：833-838，1997)，表明PA由4个不同的功能独立的结构域组成：结构域1，分为1a(1-167号氨基酸)和1b(168-258号氨基酸)；结构域2，259-487号氨基酸；结构域3，488-595号氨基酸；和结构域4，596-735号氨基酸。
发明内容
本申请人已经确定，某些结构域当以分离形式、融合蛋白形式或者相互组合形式使用时，看来产生意想不到的良好保护性效应。
按照本发明，提供一种引起抗炭疽芽孢杆菌的保护性免疫应答的免疫原性试剂，所述试剂包含结合在一起代表炭疽芽孢杆菌全长保护性抗原(PA)的至多3个结构域或这些结构域的变异体的一种或多种多肽，并且所述结构域中的至少一个包含PA的结构域1或结构域4或其变异体。
具体地说，所述试剂将包含多肽的混合物或融合肽，其中所述各个多肽包含PA多个个别结构域中的一个。
具体地说，所述试剂包括非全长PA形式的包含PA结构域1或结构域4或其变异体的多肽。当存在时，结构域适为完整的，特别是结构域1全部存在。
本文使用的术语“多肽”包括蛋白质和肽。
用于本文时，表述“变异体”是指由于基础序列中一个或多个氨基酸缺失或取代为其它氨基酸而不同于所述序列、但仍引起抗炭疽芽孢杆菌的保护性免疫应答的氨基酸序列。当一种氨基酸被一种不同的、其性质大致相似的氨基酸取代时，所述氨基酸取代可以被称为“保守的”。当氨基酸被不同类型的氨基酸取代时，为非保守取代。广义上讲，较少的非保守取代在不影响所述多肽的生物活性的情况下是可行的。合适的变异体与所述PA序列将有至少60％相同，优选至少75％相同，更优选至少90％相同。
具体地说，特定变异体序列与所述PA序列的同一性可以采用Lipman和Pearson描述的多重比对法(Lipman D.J.和Pearson，W.R.(1985)Rapid and Sensitive Protein Similarity Searches，Science，vol 227，pp1435-1441)来评价。“优化”百分比分值可以用Lipman-Pearson算法的以下参数来计算：ktup＝1，gap penalty(空位罚分)＝4，和gap penaltylength(空位罚分长度)＝12。待评价相似性的序列应该用作“试验序列”，这意指用于比较的碱基序列(SEQ ID NO 1)应该首先进入该算法。
最好是，本发明的试剂包括具有野生型PA的结构域1序列和/或结构域4序列的多肽。
本发明的一个特别优选的实施方案包括炭疽芽孢杆菌PA的结构域4。
这些结构域包含以下表1中所示的以下序列。
表1
| 结构域 | 全长PA的氨基酸* |
| 41 | 596-7351-258 |
这些氨基酸编号是指在Welkos等Gene 69(1988)287-300中所示的序列，在下文中分别描述为SEQ ID NO 15(图4)和SEQ ID NO 3(图3)。
结构域1包括两个区，名为1a和1b。区1a包括氨基酸1-167，而区1b从氨基酸168至氨基酸258。看来区1a对于良好的保护性应答的产生是重要的，可能最好是全长的结构域。
在一个特别优选的实施方案中，用结构域1和结构域4或其保护性区的组合作为免疫原性试剂，该试剂引起抗炭疽芽孢杆菌的保护性免疫应答。这种组合例如可作为融合肽，可以用下文概述的本发明的表达系统来表达。
当使用结构域1时，适宜将其与所述PA序列的结构域2融合，可能最好将其与结构域2和结构域3融合。
这类组合及其在预防或治疗中的应用，构成了本发明的另一方面。
上述结构域适宜作为融合蛋白的部分，最好具有N末端谷胱甘肽-s-转移酶蛋白(GST)。GST不仅有助于所述蛋白的纯化，它也可以提供佐剂效应，可能是由于大小增加了。
本发明的多肽适宜用常规方法制备。例如，可以合成它们，或者可以用重组DNA技术制备它们。特别是，将编码所述结构域的核酸包括在表达载体中，用所述载体转化宿主细胞。然后可以采用常规方法进行宿主细胞的培养以及随后所需多肽的分离。这些方法中所用的核酸、载体和转化细胞构成了本发明的再一方面。
一般而言，所用的宿主细胞是常规用来制备PA的宿主细胞，例如枯草杆菌。
本申请人意外的发现，或者分离的或者组合的所述结构域可以在一定条件下在大肠杆菌中成功地表达。
因此，本发明还提供一种用于生产引起抗炭疽芽孢杆菌的保护性免疫应答的免疫原性多肽的方法，所述方法包括用或者(a)编码炭疽芽孢杆菌保护性抗原(PA)或可以引起保护性免疫应答的其变异体的核酸，或者(b)编码如上所述的炭疽芽孢杆菌保护性抗原(PA)的至少一个保护性结构域或可以引起保护性免疫应答的其变异体的核酸，转化大肠杆菌宿主；培养所述转化宿主，并且从中回收所述多肽，前提是：当所述多肽是炭疽芽孢杆菌保护性抗原(PA)或可以引起保护性免疫应答的其变异体时，所述核酸中胍和胞嘧啶残基的百分比超过35％。
应用这些选项，可以用优选的表达宿主获得高收率的产物。
在图1中显示了一个表，该表显示了在大肠杆菌和炭疽芽孢杆菌基因组中出现的密码子及其频率。显然，胍和胞嘧啶在大肠杆菌中出现的频率比在炭疽芽孢杆菌中出现的频率高得多。密码子选择含量的分析显示出以下结果：
| 物种 | 密码子第一个字母为GC | 密码子第二个字母为GC | 密码子第三个字母为GC | 总GC含量 |
| 大肠杆菌 | 58.50％ | 40.70％ | 54.90％ | 51.37％ |
| 炭疽芽孢杆菌 | 44.51％ | 31.07％ | 25.20％ | 33.59％ |
因此，看来大肠杆菌偏好的密码子是可能时包括胍或胞嘧啶的那些密码子。
通过在用来编码所述免疫原性蛋白的序列中将胍和胞嘧啶核苷酸的百分比提高至野生型炭疽芽孢杆菌基因中正常存在的百分比，密码子选择将使得在大肠杆菌中的表达得以改善。
在本发明中使用的编码核酸中胍和胞嘧啶残基的百分比至少在所述多肽为炭疽芽孢杆菌保护性抗原(PA)或可以引起保护性免疫应答的其变异体时，适宜超过40％，优选超过45％，最优选为50-52％。
保护性结构域的高水平表达可以采用编码这些单位的野生型炭疽芽孢杆菌序列来达到。然而，如上所述，通过提高所述核酸的GC％，可以进一步提高收率。
在一个具体实施方案中，所述方法包括表达炭疽芽孢杆菌的PA。
此外，按照本发明，提供一种已经用编码炭疽芽孢杆菌保护性抗原(PA)或可以引起保护性免疫应答的其变异体的核酸转化的重组大肠杆菌细胞，其中所述核酸中胍和胞嘧啶残基的百分比超过35％。
如上所述，所述编码核酸中胍和胞嘧啶残基的百分比适宜超过40％，优选超过45％，最优选为50-52％。
本发明的用来转化大肠杆菌细胞的核酸适宜为合成基因。特别是，所述核酸为图2中所示的SEQ ID NO 1的核酸或其修饰形式。
表述“修饰形式”是指编码PA或引起保护性免疫应答但利用某些不同密码子的片段或其变异体的其它核酸序列，条件是符合对按照本发明的GC百分含量的要求。合适的修饰形式是与SEQ ID NO 1至少80％相似，优选90％相似，最优选至少95％相似。特别是，所述核酸包含SEQ ID NO 1。
在一个替代实施方案中，本发明提供一种已经用编码炭疽芽孢杆菌保护性抗原(PA)的保护性结构域或可以引起保护性免疫应答的其变异体的核酸转化的重组大肠杆菌细胞。
所述核酸最好编码炭疽芽孢杆菌的结构域1或结构域4。
按照本发明，再提供一种生产免疫原性多肽的方法，所述多肽引起抗炭疽芽孢杆菌的保护性免疫应答，所述方法包括培养如上所述的细胞，并且从培养物中回收所需的多肽。这类方法是本领域众所周知的。
在再一方面，本发明提供一种大肠杆菌转化载体，所述载体包含编码炭疽芽孢杆菌保护性抗原(PA)或可以引起保护性免疫应答的其变异体的核酸，其中所述核酸中的胍和胞嘧啶残基的百分比超过35％。
本发明的又一方面包括一种大肠杆菌转化载体，所述载体包含编码炭疽芽孢杆菌保护性抗原(PA)的保护性结构域或可以引起保护性免疫应答的其变异体的核酸。
可供用于大肠杆菌转化的合适载体是本领域众所周知的。例如，T7表达系统提供良好的表达水平。然而，一种特别优选的载体包括可得自Avecia(UK)的pAG163。
SEQ ID NO 1的核酸或者编码PA并且GC含量至少为35％、优选至少40％、更优选至少45％、最优选为50-52％的其变异体，构成了本发明的另一方面。
如有必要，所述变异体的PA或结构域可以作为与另一蛋白的融合体表达，所述另一蛋白例如为提供一种不同免疫的蛋白、有助于所述产物纯化的蛋白或者用以确保翻译的良好起始的高表达蛋白(例如硫氧还蛋白，GST)。
可任选地加入其它系统例如T7溶菌酶至所述表达系统中，以改善对所述系统的阻抑，尽管就本发明而论，尚未记录到与细胞毒性相关的问题。
任何合适的大肠杆菌菌株都可以用于本发明的方法中。可得到许多蛋白酶缺陷型的菌株(例如Ion-、ompT-)，预期所述菌株会将蛋白水解减少至最低。然而，本发明人已经发现，不需要应用这类菌株来达到产物的良好收率，其它已知菌株例如K12可产生意想不到的高产物收率。
所述菌株的发酵一般在本领域知道的常规条件下进行。例如，发酵可以作为分批培养物进行，优选在大摇瓶中进行，使用含有抗生素以保持质粒并添加IPTG进行诱导的复合培养基。
适当地收获培养物，将细胞贮存于-20℃直至需要用于纯化。
对于大肠杆菌PA(或变异体或结构域)表达的合适的纯化方案可以根据在枯草杆菌表达中所用的方法进行改进。待使用的各个纯化步骤将取决于重组PA的物理特性。通常，在允许最大差别结合于所述离子交换柱条件下进行离子交换色谱分离，然后从窄梯度收集流分。在某些情况下，单个色谱分离步骤可能足以获得所需规格的产物。
可以根据需要用SDS PAGE或蛋白质印迹，分析流分中所述产物的存在。
正如下文说明的，已经达到了代表rPA的完整结构域或部分结构域的一组融合蛋白的成功克隆和表达。在A/J小鼠模型中评价了这些融合蛋白的免疫原性和抗STI孢子攻击的保护性功效。
所有所述rPA结构域蛋白在A/J小鼠中都有免疫原性，并且与GST对照免疫小鼠相比，至少赋予抗攻击的部分保护。连接于所述结构域蛋白N末端的载体蛋白GST，通过在免疫动物体内所激发的抗体应答表明，并不损害所述融合蛋白的体内免疫原性，或者不损害所述融合蛋白的体外免疫原性，因为用抗rPA抗血清在蛋白质印迹后可以检测到所述融合蛋白，这表明GST标志并不干扰rPA表位的识别。用较大的融合蛋白免疫，产生最高效价。具体地说，用全长GST 1-4融合蛋白免疫的小鼠，产生的平均血清抗rPA浓度大约是rPA免疫组的8倍(图5)。用切除所述GST的rPA结构域1-4免疫小鼠，产生的效价大约是用所述融合蛋白免疫所产生的效价的一半。这种融合蛋白的免疫原性高得多的原因尚不清楚。有可能这种蛋白大小的增加可能对免疫效应细胞有佐剂效应。它并未刺激这种应答达到在所述其它融合蛋白中的同一程度，并且GST标志的任何佐剂效应并不增强抗攻击的保护，因为经切割的蛋白的保护性与其融合蛋白对应物相似。
尽管用GST1、经切割的1、GST1b-2、GST1b-3和GST1-3免疫的组的抗rPA效价高，但在这些组中在102MLD的较低攻击水平下发生保护有一些被突破，并且与GST对照免疫小鼠相比，用这些蛋白质免疫并未延长那些小鼠的存活时间，这些小鼠的确死于攻击。这提示这些蛋白尚未恰当地引发所述免疫应答，以达到完全抗所述感染。正如小鼠和豚鼠的其它研究(Little S.F.等1986.Infect.Immun.52：509-512，Tumbull P.C.B.等，1986.Infect.Immun.52：356-363)所表明的，在抗PA的抗体效价与抗攻击的保护之间没有明确的关联。然而，保护需要一定阈值的抗体(Cohen S等，2000 Infect.Immun.68：4549-4558)，提示也需要免疫应答的细胞介导组分受刺激以提供保护(Williamson 1989)。
SDS Page和蛋白质印迹结果表明，GST1、GST1b-2和GST1-2是所产生的稳定性最低的融合蛋白，可能是由于所述蛋白在无结构域3的情况下对降解更为敏感，这种不稳定性可能导致了保护性表位的丧失。
所述蛋白质的结构构象对于刺激保护性免疫应答也可能是重要的。与其完整的对应物GST1-2和GST1-3相比时，从所述融合蛋白去除结构域1a，导致抗体效价降低和抵抗攻击的保护减弱。同样，仅用GST 1免疫的小鼠受到抵抗攻击的部分保护，但与结构域2联合时，如GST1-2融合蛋白，在102 MLD攻击水平下观测到完全的保护。然而，通过用GST1-2融合蛋白免疫激发的免疫应答不足以提供抗较高的103MLD攻击水平的完全保护，这又可能是由于该蛋白降解所致的保护性表位损失引起的。
用含结构域4的截短物免疫的所有组都受到完全的保护，抵抗103MLD的STI孢子的攻击(表1)，所述含结构域4的截短物包括单独的GST 4、单独的经切割的4、以及两种单独表达的结构域GST 1和GST4的混合物。Brossier等指出，用表达无结构域4的PA的炭疽芽孢杆菌突变型菌株免疫的小鼠的保护减弱(Brossier F.等2000.Infect.Immun.68：1781-1786)，并且这一点在该项研究中得以证实，在该项研究中，用GST 1-3免疫导致保护被突破，尽管有良好的抗体效价。这些数据表明，结构域4是PA的免疫显性亚单位。结构域4代表所述PA多肽羧基末端的139个氨基酸。它含有宿主细胞受体结合区(Little S.F.等，1996 Microbiology 142：707-715)，被鉴定为在位于氨基酸残基679-693之间的小环中或小环附近(Varughese M.等1999 Infect.Immun.67：1860-1865)。因此，它对于宿主细胞中毒是必不可少的，因为已经证明，已表达的在结构域4区中含突变(Varughese 1999出处同上)或缺失(Brossier 1999出处同上)的PA形式是无毒的。PA的晶体结构表明，结构域4、特别是该结构域的19个氨基酸的环(703-722)比相互之间紧密连接的其它3个结构域为暴露(Petosa 1997出处同上)。这一结构安排可能使得结构域4是对于免疫效应细胞的识别最为突出的表位，因此含结构域4的融合蛋白能够引发保护性最强的免疫应答。
这一研究进一步阐明了PA在保护性免疫应答激发中的作用，证明抗炭疽感染的保护作用可能归因于PA的单个结构域。
附图说明
现在将通过实施例并参考附图，具体描述本发明，在附图中：
图1是在大肠杆菌和炭疽芽孢杆菌中发现的密码子频率的表；
图2显示了按照本发明的核酸的序列，该核酸编码炭疽芽孢杆菌的PA，如Welkos等(出处同上)所发表的；和
图3显示了SEQ ID NO 3-14，它们是用来编码PA的各个结构域或结构域组合的氨基酸序列和DNA序列，如下文详述；
图4显示了SEQ ID NO 15-16，它们分别是PA结构域4的氨基酸序列和DNA序列；和
图5是一个表，显示了在初次免疫后37天，得自在第1天和第28天用10μg包含PA片段的融合蛋白肌内免疫的A/J小鼠的抗rPA IgG浓度；所示结果为取自每个治疗组5只小鼠的样品的平均数±sem。
具体实施方式
实施例1
在大肠杆菌中表达的研究
已经对rPA表达质粒pAG163::rPA进行了修饰，以用KmR标记取代原始的TcR基因。将该质粒转化到表达宿主大肠杆菌BLR(DE3)中，并且评价了表达水平和溶解性。该菌株的胞内蛋白酶La(Ion基因产物)和外膜蛋白酶OmpT有缺陷。
然而，表达研究并未表明与Ion+K12宿主菌株相比在该菌株内可溶性蛋白的积累有任何改善(即由于过度的蛋白水解，积累受到阻碍)。因此推断，rPA的任何胞内蛋白水解都不是La蛋白酶的作用所致。
实施例2
发酵分析
使用K12菌株UT5600(DE3)pAG163::rPA进行的发酵的进一步分析。
发现在该培养物中的rPA被分为可溶性部分不可溶性部分(估计有350mg/L不溶性部分，650mg/L全长可溶性部分)。所用的条件(37℃，1mM IPTG用于诱导)在摇瓶培养物中未产生任何可检测的可溶性rPA，也未得到以上实施例1中所述的结果，而存在大量的可溶性rPA是出乎意料的。然而，看来发酵的操作、诱导和收获点可能提供在大肠杆菌K12表达菌株中rPA的稳定积累。
实施例3
用最初作为不溶性内含体从UT5600(DE3)pAG163::rPA发酵分离出的物质，制备rPA样品。内含体用25mM Tris-HCl pH8洗涤2次，用同一缓冲液+2M尿素洗涤1次。然后将它们溶于缓冲液+8M尿素中，沉淀出碎片。通过在25mM Tris-HCl pH8中稀释并于4℃静置温育过夜，而去除尿素。将经稀释的样品上样至Q sepharose柱，用NaCl梯度洗脱蛋白质。合并含有最高纯度rPA的流分，将其分为等分样品并冻存于-70℃。用4-12％MES-SDS NuPAGE凝胶，与已知标准品对比测试该样品，表明其纯度高，并且内毒素污染少。
实施例4
所述产物的进一步表征
该产物的N末端测序表明，N末端序列的组成为
MEVKQENRLL(SEQ ID NO 2)
这证明该产物正如所预期的，且留有起始密码子甲硫氨酸。
发现该物质在蛋白质印迹中有反应；该样品的MALDI-MS表明，质量大约为82 700(与预期的质量为82 915相似)。已知分子量高并且与所用的质量标准(66KDa)有一定的距离，则认为这表明该物质没有显著地截短，而并未消除该样品内的微不均一性。
实施例5
PA各个结构域的测试
使用Pharmacia pGEX-6P-3表达系统，以与载体蛋白谷胱甘肽-s转移酶(GST)的融合蛋白在大肠杆菌中产生作为重组蛋白的PA的各个结构域。各个结构域的序列以及用来编码所述结构域的DNA序列如图3进行连接。相应的氨基酸序列和DNA序列在以下表2中提供。
用这些融合蛋白，以总共100μl体积、吸附于20％v/v铝胶的10μg相应融合蛋白，肌内免疫A/J小鼠(Harlan Olac)。
动物经两次免疫，通过用指定剂量水平的炭疽芽孢杆菌(STI菌株)孢子攻击，测定其保护性免疫的产生。下表显示了攻击后14天的存活者。
孢子的攻击水平/小鼠
| 结构域 | 氨基酸SEQID NO | DNASEQID NO | 5×104 | 9×104 | 9×105 | 1×106 | 5×106 |
| GST-1 | 3 | 4 | 4/4 | 3/5 | |||
| GST-1+2 | 5 | 6 | 4/4；5/5 | 4/5；5/5 | |||
| GST-1b+2 | 7 | 8 | 2/5 | 1/5 | |||
| GST-1b+2+3 | 9 | 10 | 2/5 | 3/5 | |||
| GST-1+2+3 | 11 | 12 | Nd | 4/5 | 3/5 | ||
| GST-1+2+3+4 | 13 | 14 | Nd | 5/5 | 5/5 | ||
| 1+2+3+4 | 13 | 14 | Nd | Nd | 5/5 | 5/5 |
所述数据表明，PA所有4个结构域的组合无论是否以与GST的融合蛋白存在，在直至高攻击水平下都是保护性的。去除结构域4，留下1+2+3，导致在所测试的最高攻击水平9×105下被突破。在9×104孢子下，结构域1+2的保护性与结构域1+2+3的组合一样。然而，去除结构域1a而留下GST与结构域1b+2的融合体，导致在所测试最高攻击水平(9×104)下保护被突破，加入结构域3仅有略微改善。
该数据表明，PA诱导的保护性免疫可以归因于单个结构域(完整的结构域1和结构域4)或者得自所有4个结构域作为全变物(permutation)的结构域组合。
结构域4的氨基酸序列和DNA编码序列示于图4，分别为SEQ IDNO 15和SEQ ID NO 16。
实施例6
结构域作为疫苗的进一步测试
从炭疽芽孢杆菌Sterne DNA，通过PCR扩增编码所述PA结构域-氨基酸1-259、168-488、1-488、168-596、1-596、260-735、489-735、597-735和1-735(分别为截短物GST1、GST1b-2、GST1-2、GST1b-3、GST1-3、GST2-4、GST3-4、GST4和GST1-4)的DNA，将其克隆到表达载体pGEX-6-P3(Amersham-Pharmacia)的XhoI/BamHI位点中，置于lac启动子下游并且与所述启动子符合读框。使用该系统产生的蛋白以具有N末端谷胱甘肽-s-转移酶蛋白(GST)的融合蛋白表示。然后，将带有编码所述PA结构域的DNA的重组质粒DNA转化到大肠杆菌BL21中，以供蛋白质表达研究用。
在含有50μg/ml氨苄青霉素、30μg/ml氯霉素和1％w/v葡萄糖的L-肉汤中，培养带有重组pGEX-6-P3质粒的大肠杆菌BL21。将培养物于30℃振荡(170rev/min)培养至A600nm为0.4，然后用0.5mM IPTG诱导。将培养物再孵育4小时，然后通过以10 000rpm离心15分钟收获培养物。
所述PA截短物-融合蛋白的初步提取表明，它们以内含体产生。将细胞沉淀重悬于磷酸缓冲盐溶液(PBS)中，在冰水浴中超声处理4×20秒。将悬浮液以15 000rpm离心15分钟，然后通过悬浮于8M尿素中于室温搅拌1小时，用尿素提取细胞沉淀。将悬浮液以15000rpm离心15分钟，上清液对含有400mM L-精氨酸和0.1mM EDTA的100mMTris pH8透析，然后在PBS中透析。
所述PA截短物-融合蛋白的成功重折叠，使其可以在谷胱甘肽Sepharose CL-4B亲和柱上纯化。将所有提取物(截短物GST1b-2-氨基酸残基168-487除外)上样到先前用PBS平衡的15ml谷胱甘肽Sepharose CL-4B柱(Amersham-Parmacia)，于4℃旋转温育过夜。柱子用PBS洗涤，用含150mM NaCl、1mM EDTA和20mM还原型谷胱甘肽的50mM Tris pH 7洗脱所述融合蛋白。合并通过SDS-PAGE分析鉴定的含PA截短物的流分，然后对PBS透析。用BCA(Perbio)测定蛋白浓度。
然而，用还原型谷胱甘肽未能从谷胱甘肽sepharose CL-4B亲和柱洗脱下截短物GST1b-2，因此用离子交换色谱纯化。具体地说，将截短物GST1b-2对20mM Tris pH8透析，然后上样至用同一缓冲液平衡的HiTrap Q柱(Amersham-Parmacia)。用20mM Tris pH8中的0-1M增加的NaCl梯度洗脱融合蛋白。合并含GST-蛋白的流分，浓缩，然后上样至先前用PBS平衡的HiLoad 26/60 Superdex 200凝胶过滤柱(Amersham-Parmacia)。合并含融合蛋白的流分，通过BCA(Perbio)测定蛋白浓度。产量在每升培养物1mg和43mg之间。
所述片段的分子量及其被抗PA抗体的识别用SDS PAGE和蛋白质印迹法来证实。通过SDS Page和蛋白质印迹法分析所述rPA截短物，显示出预期大小的蛋白条带。在所调查的所有所述rPA截短物中显然有一些降解，显示出与在枯草杆菌中表达的重组PA有相似性。rPA截短物GST1、GST1b-2和GST1-2在无结构域3的情况下对降解特别敏感。同样报道了结构域3中含有突变的rPA构建体不能从炭疽芽孢杆菌培养上清液中纯化(Brossier 1999)，表明结构域3可能使结构域1和结构域2稳定化。
在该项研究中使用雌性无特定病原体的A/J小鼠(Harlan UK)，因为它们是炭疽感染的稳定模型(Welkos 1986)。小鼠在年龄上相当，在该项研究开始时为7周龄。
在该项研究的第1天和第28天，用100μl总体积PBS中吸附于20％1.3％v/v铝胶(HCI Biosector，Denmark)的10μg融合蛋白为A/J小鼠免疫。也包括用得自枯草杆菌的rPA(Miller 1998)、重组GST对照蛋白或去除了GST标志的编码结构域1、4以及1-4的融合蛋白免疫的组。在后腿两个部位肌内给予免疫剂量。初次免疫后37天，取小鼠的血样，以供通过酶联免疫吸附测定(ELISA)进行血清抗体分析。
微量滴定板(Immulon 2，Dynex Technologies)用PBS中从枯草杆菌表达的(Miller 1998)5μg/ml rPA于4℃包被过夜，每块板的两列除外，这两列用5μg/ml抗小鼠Fab(Sigma，Poole，Dorset)包被。用含1％v/vTween 20的PBS(PBS-T)洗涤板，5％w/v脱脂奶粉的PBS溶液(blotto)于37℃封闭2小时。加入在1％blotto中两倍稀释的血清至所述rPA包被孔中，与加入至抗fab包被孔的鼠IgG标准(Sigma)一起，以双份重复进行分析，于4℃温育过夜。洗涤后，向所有孔中加入在PBS以1∶2000稀释的辣根过氧化物酶缀合的山羊抗小鼠IgG(SouthemBiotechnology Associates Inc.)，于37℃温育1小时。再次洗涤板，然后加入底物2，2’-Azinobis(3-乙基苯并噻唑啉磺酸)(1.09mM ABTS，Sigma)。于室温温育20分钟后，测定414nm下各孔的吸光度(TitertekMultiscan，ICN Flow)。用Titersoft version 3.1c软件计算标准曲线。效价以每ml血清μg IgG表示，计算组平均数±平均标准误差(sem)。结果示于图5。
所产生的所有rPA截短物都是免疫原性的，在A/J小鼠体内激发的平均血清抗rPA IgG浓度范围从GSTlb-2截短物免疫组的6μg/ml至GST 1-4截短物免疫组的1488μg/ml(图5)。GST对照免疫小鼠没有可检测的抗rPA抗体。
在免疫方案的第70天，用炭疽芽孢杆菌STI孢子攻击小鼠。从原液取出足够的STI孢子用于攻击，在无菌蒸馏水中洗涤，然后重悬于PBS中至1×107孢子/ml和1×106孢子/ml的浓度。分别以每只小鼠含1×106孢子和1×105孢子的0.1ml体积腹膜内攻击小鼠，在攻击后监测14天，以确定其保护状况。严格观测人道终点，使得表现出综合指示具有致死感染的大量临床体征的小鼠被剔除。攻击后存活14天的免疫小鼠数目示于表3。
表3
攻击水平MLD
结构域 存活者/受攻击的数目(％)
| 102MLD | 103MLD | |
| GST 1 | 3/5(60) | 1/5(20) |
| GST 1b-2 | 1/5(20) | nd |
| GST 1-2 | 5/5(100) | 3/5(60) |
| GST 1b-3 | 3/5(60) | nd |
| GST 1-3 | 4/5(80) | nd |
| GST 1-4 | nd | 5/5(100) |
| GST 2-4 | nd | 5/5(100) |
| GST 3-4 | nd | 5/5(100) |
| GST 4 | 5/5(100) | 5/5(100) |
| GST 1+GST 4 | nd | 5/5(100) |
| 经切割的1 | 1/5(20) | 2/5 |
| 经切割的4 | 5/5(100) | 5/5 |
| 经切割的1-4 | nd | 5/5 |
| rPA | nd | 4/4(100) |
| 对照 | 0/5(0) | 0/5(0) |
1 MLD＝约1×103STI孢子
nd＝未进行
用103MLD的STI孢子攻击的组都是完全受保护的，除了GST1、GST1-2和经切割的1免疫的组，在这些组中有一些保护被突破，仅用GST免疫的对照组全都死于感染，至死亡时的平均时间(MTTD)为2.4±0.2天。在102MLD的较低攻击水平下，GST1-2、GST4和经切割的4免疫的组都受到完全的保护，但在其它组中有一些保护被突破。在这些组中死亡小鼠的MTTD为4.5±0.2天，这与GST对照免疫组没有显著差异，GST对照免疫组全部死亡，MTTD为4±0.4天。
序 列 表
<110>英国国防部(The Secretary of State for Defence)
Williamson，Ethel D
Miller，Julie
Walker，Nieola J
Baillie，Leslie WJ
Holden，Paula T
Flick-Smith，Helen C
Bullifent，Helen L
Titball，Richard W
<120>表达系统
<130>CG/P/110/WOD
<140>PCT/GB01/03065
<141>2001-07-06
<150>GB 0016702.3
<151>2000-07-08
<160>16
<170>PatentIn Ver.2.1
<210>1
<211>2228
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：编码炭疽芽孢杆菌保护性抗原的核酸
<400>1
aagcttcata tggaagtaaa gcaagagaac cgtctgctga acgaatctga atccagctct 60
cagggcctgc ttggttacta tttctctgac ctgaacttcc aagcaccgat ggttgtaacc 120
agctctacca ctggcgatct gtccatcccg tctagtgaac ttgagaacat tccaagcgag 180
aaccagtatt tccagtctgc aatctggtcc ggttttatca aagtcaagaa atctgatgaa 240
tacacgtttg ccacctctgc tgataaccac gtaaccatgt gggttgacga tcaggaagtg 300
atcaacaaag catccaactc caacaaaatt cgtctggaaa aaggccgtct gtatcagatc 360
aagattcagt accaacgcga gaacccgact gaaaaaggcc tggactttaa actgtattgg 420
actgattctc agaacaagaa agaagtgatc agctctgaca atctgcaact gccggaattg 480
aaacagaaaa gctccaactc tcgtaagaaa cgttccacca gcgctggccc gaccgtacca 540
gatcgcgaca acgatggtat tccggactct ctggaagttg aaggctacac ggttgatgta 600
aagaacaaac gtaccttcct tagtccgtgg atctccaata ttcacgagaa gaaaggtctg 660
accaaataca aatccagtcc ggaaaaatgg tccactgcat ctgatccgta ctctgacttt 720
gagaaagtga ccggtcgtat cgacaagaac gtctctccgg aagcacgcca tccactggtt 780
gctgcgtatc cgatcgtaca tgttgacatg gaaaacatca ttttgtccaa gaacgaagac 840
cagtccactc agaacactga ctctgaaact cgtaccatct ccaagaacac ctccacgtct 900
cgtactcaca ccagtgaagt acatggtaac gctgaagtac acgcctcttt ctttgacatc 960
ggcggctctg ttagcgctgg cttctccaac tctaattctt ctactgttgc cattgatcac 1020
tctctgagtc tggctggcga acgtacctgg gcagagacca tgggtcttaa cactgctgat 1080
accgcgcgtc tgaatgctaa cattcgctac gtcaacactg gtacggcacc gatctacaac 1140
gtactgccaa ccaccagcct ggttctgggt aagaaccaga ctcttgcgac catcaaagcc 1200
aaagagaacc aactgtctca gattctggca ccgaataact actatccttc caagaacctg 1260
gctccgatcg cactgaacgc acaggatgac ttctcttcca ctccgatcac catgaactac 1320
aaccagttcc tggaacttga gaagaccaaa cagctgcgtc ttgacactga ccaagtgtac 1380
ggtaacatcg cgacctacaa ctttgagaac ggtcgcgtcc gcgttgacac aggctctaat 1440
tggtctgaag tactgcctca gattcaggaa accaccgctc gtatcatctt caacggtaaa 1500
gacctgaacc tggttgaacg tcgtattgct gctgtgaacc cgtctgatcc attagagacc 1560
accaaaccgg atatgactct gaaagaagcc ctgaagatcg cctttggctt caacgagccg 1620
aacggtaatc ttcagtacca aggtaaagac atcactgaat ttgacttcaa ctttgatcag 1680
cagacctctc agaatatcaa gaaccaactg gctgagctga acgcgaccaa tatctatacg 1740
gtactcgaca agatcaaact gaacgcgaaa atgaacattc tgattcgcga caaacgtttc 1800
cactacgatc gtaataacat cgctgttggc gctgatgaat ctgttgtgaa agaagcgcat 1860
cgcgaagtca tcaactccag caccgaaggc ctgcttctga acatcgacaa agacattcgt 1920
aagatcctgt ctggttacat tgttgagatc gaagacaccg aaggcctgaa agaagtgatc 1980
aatgatcgtt acgacatgct gaacatcagc tctctgcgtc aagatggtaa gacgttcatt 2040
gacttcaaga aatacaacga caaacttccg ctgtatatct ctaatccgaa ctacaaagtg 2100
aacgtttacg ctgttaccaa agagaacacc atcatcaatc catctgagaa cggcgatacc 2160
tctaccaacg gtatcaagaa gattctgatc ttctccaaga aaggttacga gatcggttaa 2220
taggatcc 2228
<210>2
<211>10
<212>PRT
<213>炭疽芽孢杆菌(Bacillus anthracis)
<400>2
Met Glu Val Lys Gln Glu Asn Arg Leu Leu
1 5 10
<210>3
<211>258
<212>PRT
<213>炭疽芽孢杆菌(Bacillus anthracis)
<400>3
Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser
1 5 10 15
Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro
20 25 30
Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser
35 40 45
Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile
50 55 60
Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala
65 70 75 80
Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val
85 90 95
Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
100 105 110
Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
115 120 125
Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu
130 135 140
Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser
145 150 155 160
Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro
165 170 175
Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
180 185 190
Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser
195 200 205
Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
210 215 220
Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr
225 230 235 240
Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val
245 250 255
Ala Ala
<210>4
<211>774
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID N0：3的DNA序列
<400>4
gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60
ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctc ttctactaca 120
ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180
caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240
acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300
tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360
caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420
aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaa acaaaaatct 480
tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540
gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600
acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660
tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720
ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agct 774
<210>5
<211>487
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：融合蛋白
<400>5
Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser
1 5 10 15
Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro
20 25 30
Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser
35 40 45
Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile
50 55 60
Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala
65 70 75 80
Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val
85 90 95
Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
100 105 110
Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
115 120 125
Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu
130 135 140
Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser
145 150 155 160
Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro
165 170 175
Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
180 185 190
Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser
195 200 205
Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
210 215 220
Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr
225 230 235 240
Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val
245 250 255
Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser
260 265 270
Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr
275 280 285
Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His
290 295 300
Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val
305 310 315 320
Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
325 330 335
Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu
340 345 350
Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn
355 360 365
Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val
370 375 380
Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln
385 390 395 400
Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu
405 410 415
Ala Pro Ile Ala Leu Ash Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile
420 425 430
Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu
435 440 445
Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn lle Ala Thr Tyr Asn Phe
450 455 460
Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val
465 470 475 480
Leu Pro Gln Ile Gln Glu Thr
485
<210>6
<211>1461
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID NO：5的DNA序列
<400>6
gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60
ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacttc ttctactaca 120
ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180
caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240
acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300
tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360
caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420
aataaaaaag aagtgatttc tagtgataac ttacaactgc cagaattaaa acaaaaatct 480
tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540
gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600
acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660
tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720
ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780
attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840
aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900
agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960
tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020
gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080
aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140
acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200
ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260
ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320
gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380
acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440
ttaccgcaaa ttcaagaaac a 1461
<210>7
<211>318
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：融合蛋白
<400>7
Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp
1 5 10 15
Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr
20 25 30
Phe Leu Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr
35 40 45
Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr
50 55 60
Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro
65 70 75 80
Glu Ala Arg His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp
85 90 95
Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn
100 105 110
Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg
115 120 125
Thr His Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe
130 135 140
Phe Asp Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser
145 150 155 160
Ser Thr Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr
165 170 175
Trp Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn
180 185 190
Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val
195 200 205
Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr
210 215 220
Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn
225 230 235 240
Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp
245 250 255
Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu
260 265 270
Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly
275 280 285
Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr
290 295 300
Gly Ser Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr
305 310 315
<210>8
<211>954
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID NO：7的DNA序列
<400>8
agtgctggac ctacggttcc agaccgtgac aatgatggaa tccctgattc attagaggta 60
gaaggatata cggttgatgt caaaaataaa agaacttttc tttcaccatg gatttctaat 120
attcatgaaa agaaaggatt aaccaaatat aaatcatctc ctgaaaaatg gagcacggct 180
tctgatccgt acagtgattt cgaaaaggtt acaggacgga ttgataagaa tgtatcacca 240
gaggcaagac acccccttgt ggcagcttat ccgattgtac atgtagatat ggagaatatt 300
attctctcaa aaaatgagga tcaatccaca cagaatactg atagtgaaac gagaacaata 360
agtaaaaata cttctacaag taggacacat actagtgaag tacatggaaa tgcagaagtg 420
catgcgtcgt tctttgatat tggtgggagt gtatctgcag gatttagtaa ttcgaattca 480
agtacggtcg caattgatca ttcactatct ctagcagggg aaagaacttg ggctgaaaca 540
atgggtttaa ataccgctga tacagcaaga ttaaatgcca atattagata tgtaaatact 600
gggacggctc caatctacaa cgtgttacca acgacttcgt tagtgttagg aaaaaatcaa 660
acactcgcga caattaaagc taaggaaaac caattaagtc aaatacttgc acctaataat 720
tattatcctt ctaaaaactt ggcgccaatc gcattaaatg cacaagacga tttcagttct 780
actccaatta caatgaatta caatcaattt cttgagttag aaaaaacgaa acaattaaga 840
ttagatacgg atcaagtata tgggaatata gcaacataca attttgaaaa tggaagagtg 900
agggtggata caggctcgaa ctggagtgaa gtgttaccgc aaattcaaga aaca 954
<210>9
<211>426
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：融合蛋白
<400>9
Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp
1 5 10 15
Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr
20 25 30
Phe Leu Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr
35 40 45
Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr
50 55 60
Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro
65 70 75 80
Glu Ala Arg His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp
85 90 95
Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn
100 105 110
Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg
115 120 125
Thr His Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe
130 135 140
Phe Asp Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser
145 150 155 160
Ser Thr Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr
165 170 175
Trp Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn
180 185 190
Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val
195 200 205
Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr
210 215 220
Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn
225 230 235 240
Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp
245 250 255
Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu
260 265 270
Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly
275 280 285
Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr
290 295 300
Gly Ser Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala
305 310 315 320
Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile
325 330 335
Ala Ala Val Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met
340 345 350
Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn
355 360 365
Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn
370 375 380
Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu
385 390 395 400
Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala
405 410 415
Lys Met Asn Ile Leu Ile Arg Asp Lys Arg
420 425
<210>10
<211>1278
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID NO：9的DNA序列
<400>10
agtgctggac ctacggttcc agaccgtgac aatgatggaa tccctgattc attagaggta 60
gaaggatata cggttgatgt caaaaataaa agaacttttc tttcaccatg gatttctaat 120
attcatgaaa agaaaggatt aaccaaatat aaatcatctc ctgaaaaatg gagcacggct 180
tctgatccgt acagtgattt cgaaaaggtt acaggacgga ttgataagaa tgtatcacca 240
gaggcaagac acccccttgt ggcagcttat ccgattgtac atgtagatat ggagaatatt 300
attctctcaa aaaatgagga tcaatccaca cagaatactg atagtgaaac gagaacaata 360
agtaaaaata cttctacaag taggacacat actagtgaag tacatggaaa tgcagaagtg 420
catgcgtcgt tctttgatat tggtgggagt gtatctgcag gatttagtaa ttcgaattca 480
agtacggtcg caattgatca ttcactatct ctagcagggg aaagaacttg ggctgaaaca 540
atgggtttaa ataccgctga tacagcaaga ttaaatgcca atattagata tgtaaatact 600
gggacggctc caatctacaa cgtgttacca acgacttcgt tagtgttagg aaaaaatcaa 660
acactcgcga caattaaagc taaggaaaac caattaagtc aaatacttgc acctaataat 720
tattatcctt ctaaaaactt ggcgccaatc gcattaaatg cacaagacga tttcagttct 780
actccaatta caatgaatta caatcaattt cttgagttag aaaaaacgaa acaattaaga 840
ttagatacgg atcaagtata tgggaatata gcaacataca attttgaaaa tggaagagtg 900
agggtggata caggctcgaa ctggagtgaa gtgttaccgc aaattcaaga aacaactgca 960
cgtatcattt ttaatggaaa agatttaaat ctggtagaaa ggcggatagc ggcggttaat 1020
cctagtgatc cattagaaac gactaaaccg gatatgacat taaaagaagc ccttaaaata 1080
gcatttggat ttaacgaacc gaatggaaac ttacaatatc aagggaaaga cataaccgaa 1140
tttgatttta atttcgatca acaaacatct caaaatatca agaatcagtt agcggaatta 1200
aacgcaacta acatatatac tgtattagat aaaatcaaat taaatgcaaa aatgaatatt 1260
ttaataagag ataaacgt 1278
<210>11
<211>595
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：融合蛋白
<400>11
Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser
1 5 10 15
Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro
20 25 30
Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser
35 40 45
Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile
50 55 60
Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala
65 70 75 80
Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val
85 90 95
Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
100 105 110
Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
115 120 125
Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu
130 135 140
Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser
145 150 155 160
Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro
165 170 175
Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
180 185 190
Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser
195 200 205
Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
210 215 220
Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr
225 230 235 240
Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val
245 250 255
Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser
260 265 270
Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr
275 280 285
Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His
290 295 300
Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val
305 310 315 320
Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
325 330 335
Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu
340 345 350
Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn
355 360 365
Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val
370 375 380
Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln
385 390 395 400
Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu
405 410 415
Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile
420 425 430
Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu
435 440 445
Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe
450 455 460
Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val
465 470 475 480
Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys
485 490 495
Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp
500 505 510
Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys
515 520 525
Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly
530 535 540
Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln
545 550 555 560
Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Ash Ile Tyr Thr
565 570 575
Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn lle Leu Ile Arg
580 585 590
Asp Lys Arg
595
<210>12
<211>1785
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID NO：11的DNA序列
<400>12
gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60
ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctc ttctactaca 120
ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180
caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240
acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300
tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360
caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420
aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaa acaaaaatct 480
tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540
gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600
acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660
tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720
ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780
attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840
aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900
agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960
tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020
gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080
aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140
acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200
ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260
ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320
gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380
acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440
ttaccgcaaa ttcaagaaac aactgcacgt atcattttta atggaaaaga tttaaatctg 1500
gtagaaaggc ggatagcggc ggttaatcct agtgatccat tagaaacgac taaaccggat 1560
atgacattaa aagaagccct taaaatagca tttggattta acgaaccgaa tggaaactta 1620
caatatcaag ggaaagacat aaccgaattt gattttaatt tcgatcaaca aacatctcaa 1680
aatatcaaga atcagttagc ggaattaaac gcaactaaca tatatactgt attagataaa 1740
atcaaattaa atgcaaaaat gaatatttta ataagagata aacgt 1785
<210>13
<211>735
<212>PRT
<213>人工序列
<220>
<223>人工序列的描述：融合蛋白
<400>13
Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser
1 5 10 15
Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro
20 25 30
Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser lle Pro Ser Ser
35 40 45
Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile
50 55 60
Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala
65 70 75 80
Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val
85 90 95
Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
100 105 110
Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
115 120 125
Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu
130 135 140
Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser
145 150 155 160
Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro
165 170 175
Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
180 185 190
Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser
195 200 205
Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
210 215 220
Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr
225 230 235 240
Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val
245 250 255
Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser
260 265 270
Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr
275 280 285
Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His
290 295 300
Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val
305 310 315 320
Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
325 330 335
Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu
340 345 350
Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn
355 360 365
Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val
370 375 380
Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln
385 390 395 400
Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu
405 410 415
Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile
420 425 430
Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu
435 440 445
Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe
450 455 460
Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val
465 470 475 480
Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys
485 490 495
Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp
500 505 510
Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys
515 520 525
Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly
530 535 540
Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln
545 550 555 560
Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr
565 570 575
Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg
580 585 590
Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp
595 600 605
Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr
610 615 620
Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser
625 630 635 640
Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile
645 650 655
Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly
660 665 670
Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr
675 680 685
Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu
690 695 700
Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly
705 710 715 720
Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly
725 730 735
<210>14
<211>2208
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：用来编码SEQ ID NO：13的DNA序列
<400>14
gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60
ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctc ttctactaca 120
ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180
caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240
acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300
tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360
caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420
aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaa acaaaaatct 480
tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540
gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600
acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660
tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720
ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780
attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840
aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900
agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960
tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020
gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080
aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140
acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200
ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260
ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320
gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380
acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440
ttaccgcaaa ttcaagaaac aactgcacgt atcattttta atggaaaaga tttaaatctg 1500
gtagaaaggc ggatagcggc ggttaatcct agtgatccat tagaaacgac taaaccggat 1560
atgacattaa aagaagccct taaaatagca tttggattta acgaaccgaa tggaaactta 1620
caatatcaag ggaaagacat aaccgaattt gattttaatt tcgatcaaca aacatctcaa 1680
aatatcaaga atcagttagc ggaattaaac gcaactaaca tatatactgt attagataaa 1740
atcaaattaa atgcaaaaat gaatatttta ataagagata aacgttttca ttatgataga 1800
aataacatag cagttggggc ggatgagtca gtagttaagg aggctcatag agaagtaatt 1860
aattcgtcaa cagagggatt attgttaaat attgataagg atataagaaa aatattatca 1920
ggttatattg tagaaattga agatactgaa gggcttaaag aagttataaa tgacagatat 1980
gatatgttga atatttctag tttacggcaa gatggaaaaa catttataga ttttaaaaaa 2040
tataatgata aattaccgtt atatataagt aatcccaatt ataaggtaaa tgtatatgct 2100
gttactaaag aaaacactat tattaatcct agtgagaatg gggatactag taccaacggg 2160
atcaagaaaa ttttaatctt ttctaaaaaa ggctatgaga taggataa 2208
<210>15
<211>140
<212>PRT
<213>炭疽芽孢杆菌(Bacillus anthracis)
<400>15
Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val
1 5 10 15
Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu
20 25 30
Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile
35 40 45
Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp Arg
50 55 60
Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe
65 70 75 80
Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn
85 90 95
Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile
100 105 110
Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys
115 120 125
Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly
130 135 140
<210>16
<211>423
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：结构域4的DNA编码序列
<400>16
tttcattatg atagaaataa catagcagtt ggggcggatg agtcagtagt taaggaggct 60
catagagaag taattaattc gtcaacagag ggattattgt taaatattga taaggatata 120
agaaaaatat tatcaggtta tattgtagaa attgaagata ctgaagggct taaagaagtt 180
ataaatgaca gatatgatat gttgaatatt tctagtttac ggcaagatgg aaaaacattt 240
atagatttta aaaaatataa tgataaatta ccgttatata taagtaatcc caattataag 300
gtaaatgtat atgctgttac taaagaaaac actattatta atcctagtga gaatggggat 360
actagtacca acgggatcaa gaaaatttta atcttttcta aaaaaggcta tgagatagga 420
taa 423
Claims (43)
1.一种用于生产引起抗炭疽芽孢杆菌(Bacillus anthracis)的保护性免疫应答的免疫原性多肽的方法，所述方法包括用编码炭疽芽孢杆菌保护性抗原(PA)或其变异体的核酸转化大肠杆菌宿主，所述变异体是通过删除和/或取代氨基酸序列中一个或多个氨基酸产生的并且其保护性免疫原性活性没有因此受损；培养所述转化宿主，并且从中回收所述多肽，其中核酸中至少部分野生型密码子被大肠杆菌偏好的密码子置换。
2.一种权利要求1的方法，其中所述核酸编码炭疽芽孢杆菌保护性抗原(PA)。
3.一种权利要求1或2的方法，其中所述核酸中胍和胞嘧啶残基的百分比超过35％。
4.一种权利要求3的方法，其中所述核酸中胍和胞嘧啶残基的百分比超过45％。
5.一种权利要求4的方法，其中所述核酸中胍和胞嘧啶残基的百分比为50-52％。
6.一种重组大肠杆菌(Escherichia coli)细胞，所述细胞已经用编码炭疽芽孢杆菌保护性抗原(PA)或其变异体的核酸转化，所述变异体是通过删除和/或取代氨基酸序列中一个或多个氨基酸产生的并且其保护性免疫原性活性没有因此受损，以及其中核酸中至少部分野生型密码子被大肠杆菌偏好的密码子置换。
7.一种权利要求6的重组大肠杆菌细胞，其中所述核酸中胍和胞嘧啶残基的百分比超过35％。
8.一种权利要求7的重组大肠杆菌细胞，其中所述核酸中胍和胞嘧啶残基的百分比超过45％。
9.一种权利要求8的重组大肠杆菌细胞，其中所述核酸中胍和胞嘧啶残基的百分比为50-52％。
10.一种权利要求6的重组大肠杆菌细胞，其中所述核酸是图2显示的SEQ ID NO 1或其修饰形式。
11.一种权利要求10的重组大肠杆菌细胞，其中所述核酸是SEQID NO 1。
12.一种用于生产引起抗炭疽芽孢杆菌的保护性免疫应答的多肽的方法，所述方法包括培养权利要求6-11中任一项的细胞，并且从培养物中回收所述保护性多肽。
13.一种大肠杆菌转化载体，所述载体包含编码炭疽芽孢杆菌保护性抗原(PA)或其变异体的核酸，所述变异体是通过删除和/或取代氨基酸序列中一个或多个氨基酸产生的并且其保护性免疫原性活性没有因此受损，以及其中核酸中至少部分野生型密码子被大肠杆菌偏好的密码子置换。
14.一种权利要求13的大肠杆菌转化载体，其中所述核酸中胍和胞嘧啶残基的百分比超过35％。
15.一种SEQ ID NO 1的核酸或其修饰形式，所述核酸或其修饰形式编码引起保护性免疫应答的PA或其变异体，以及其中核酸中至少部分野生型密码子被大肠杆菌偏好的密码子置换。
16.一种权利要求15的核酸，其具有至少35％GC含量。
17.一种权利要求15的核酸，其为SEQ ID NO 1。
18.一种权利要求15的核酸，其与SEQ ID NO 1至少90％相同。
19.一种权利要求15的核酸，其包含SEQ ID NO 1。
20.一种引起抗炭疽芽孢杆菌(Bacillus anthracis)的保护性免疫应答的免疫原性试剂，所述试剂包含一种或多种多肽，所述多肽结合在一起代表炭疽芽孢杆菌全长保护性抗原(PA)的至多3个结构域或这些结构域的变异体，并且所述结构域中的至少一个包含PA的结构域1或结构域4或其变异体。
21.一种权利要求20的免疫原性试剂，所述试剂包含炭疽芽孢杆菌PA的结构域4。
22.一种权利要求20或权利要求21的免疫原性试剂，所述试剂包含结构域1和4或它们的保护性区的组合。
23.一种权利要求22的免疫原性试剂，所述试剂包含野生型PA的结构域1和结构域4的序列。
24.一种权利要求23的免疫原性试剂，其中所述结构域以融合多肽的形式存在。
25.一种权利要求24的免疫原性试剂，所述试剂包含与所述PA序列的结构域2融合的结构域1。
26.一种权利要求25的免疫原性试剂，所述试剂与所述PA序列的结构域3融合。
27.一种权利要求23的免疫原性试剂，所述试剂包含多种多肽的混合物，其中一种多肽包含所述PA序列的结构域1，而其中一种多肽包含所述PA序列的结构域4。
28.一种权利要求20-27中任一项的免疫原性试剂，其中一种多肽与另一种多肽融合。
29.一种权利要求28的免疫原性试剂，其中所述另一种肽是谷胱甘肽-S-转移酶(GST)。
30.一种核酸，所述核酸编码权利要求20-27中任一项的免疫原性试剂的多肽。
31.一种表达载体，所述表达载体包含权利要求30的核酸。
32.一种用权利要求31的载体转化的细胞。
33.一种用于生产引起抗炭疽芽孢杆菌的保护性免疫应答的免疫原性多肽的方法，所述方法包括用编码炭疽芽孢杆菌保护性抗原(PA)的保护性结构域或其引起保护性免疫应答的变异体的核酸转化大肠杆菌宿主，培养所述转化宿主，并且从中回收所述多肽。
34.一种权利要求33的方法，其中核酸中至少部分野生型密码子被大肠杆菌偏好的密码子置换。
35.一种权利要求34的方法，其中所述核酸中胍和胞嘧啶残基的百分比超过45％。
36.一种权利要求35的方法，其中所述核酸中胍和胞嘧啶残基的百分比为50-52％。
37.一种权利要求33的方法，其中所述结构域是炭疽芽孢杆菌PA的结构域1和/或结构域4。
38.一种重组大肠杆菌细胞，所述细胞已经用编码炭疽芽孢杆菌保护性抗原(PA)的保护性结构域或其引起保护性免疫应答的变异体的核酸转化。
39.一种权利要求38的重组大肠杆菌细胞，其中所述核酸编码炭疽芽孢杆菌PA的结构域1或结构域4。
40.一种用于生产引起抗炭疽芽孢杆菌的保护性免疫应答的多肽的方法，所述方法包括培养权利要求38或39的细胞，并且从所述培养物中回收所述保护性多肽。
41.一种大肠杆菌转化载体，所述载体包含编码炭疽芽孢杆菌保护性抗原(PA)的保护性结构域或其可引起保护性免疫应答的变异体的核酸。
42.一种预防或治疗炭疽芽孢杆菌感染的方法，所述方法包括给予需要预防或治疗的哺乳动物足量的权利要求20-29中任一项的免疫原性试剂。
43.权利要求20-29中任一项的免疫原性试剂在制备用于预防或治疗炭疽芽孢杆菌感染的药物中的用途。
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| US7601351B1 (en) | 2002-06-26 | 2009-10-13 | Human Genome Sciences, Inc. | Antibodies against protective antigen |
| AU2003278776A1 (en) * | 2002-09-10 | 2004-04-30 | Vical Incorporated | Codon-optimized polynucleotide-based vaccines against bacillus anthracis infection |
| US9453251B2 (en) | 2002-10-08 | 2016-09-27 | Pfenex Inc. | Expression of mammalian proteins in Pseudomonas fluorescens |
| EP1572977B1 (en) | 2002-11-15 | 2010-03-03 | Gen-Probe Incorporated | Assay and compositions for detection of bacillus anthracis nucleic acid |
| EP1670505B1 (en) | 2003-09-18 | 2012-11-14 | GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Dna promoters and anthrax vaccines |
| FR2860005A1 (fr) * | 2003-09-23 | 2005-03-25 | Univ Aix Marseille Ii | Methode de detection d'agents bacteriens ou viraux de bioterrorisme |
| WO2005030991A1 (fr) * | 2003-09-23 | 2005-04-07 | Universite De La Mediterranee (Aix-Marseille Ii) | Methode de detection d’agents bacteriens ou viraux de bioterrorisme |
| GB0401036D0 (en) * | 2004-01-17 | 2004-02-18 | Royal Holloway University Of L | Improvements in or relating to vaccination |
| US8101735B2 (en) | 2004-06-16 | 2012-01-24 | Health Protection Agency | Preparation of protective antigen |
| US7355027B2 (en) * | 2004-06-16 | 2008-04-08 | Dynport Vaccine Company Llc | Bacillus anthracis protective antigen |
| AU2005269527B2 (en) | 2004-07-26 | 2011-12-01 | Pfenex Inc. | Process for improved protein expression by strain engineering |
| CN100336908C (zh) * | 2004-09-10 | 2007-09-12 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种制备重组炭疽保护性抗原的方法及其专用表达质粒 |
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| GB0504940D0 (en) * | 2005-03-10 | 2005-04-20 | Secr Defence | Vaccine formulation |
| EP1954307A4 (en) | 2005-11-14 | 2009-12-02 | Univ Maryland Biotech Inst | ORAL VACCINES AGAINST MILZBRAND ON SALMONELLA BASE |
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| US9580719B2 (en) | 2007-04-27 | 2017-02-28 | Pfenex, Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
| CA2685326A1 (en) | 2007-04-27 | 2008-11-06 | Dow Global Technologies Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
| US9616117B2 (en) | 2008-10-02 | 2017-04-11 | Pharmathene, Inc. | Anthrax vaccine formulation and uses thereof |
| US20110256172A1 (en) * | 2008-10-14 | 2011-10-20 | The Regents Of The University Of Michigan | Epitope-targeted anthrax vaccine |
| US20100183675A1 (en) * | 2009-01-22 | 2010-07-22 | Allan Watkinson | Stable vaccine compositions and methods of use |
| US8187611B2 (en) * | 2009-10-29 | 2012-05-29 | Albert Einstein College Of Medicine Of Yeshiva University | Anti-peptide antibodies that cross react with protective antigen of Bacillus anthracis and uses thereof |
| US20110110968A1 (en) * | 2009-11-12 | 2011-05-12 | Stanley Goldman | Human optimized Bacillus anthracis protective antigen |
| CN101798565B (zh) * | 2010-02-26 | 2013-08-07 | 中国人民解放军军事医学科学院生物工程研究所 | 能在芽孢表面展示pa20蛋白的炭疽芽孢杆菌及其应用 |
| RU2633504C1 (ru) * | 2016-12-27 | 2017-10-12 | Федеральное государственное учреждение "Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук (ФИЦ Биотехнологии РАН) | Штамм E.coli - продуцент полноразмерного протективного антигена Bacillus anthracis в форме вирусоподобных частиц |
| RU2633508C1 (ru) * | 2016-12-27 | 2017-10-12 | Федеральное государственное учреждение "Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук (ФИЦ Биотехнологии РАН) | Штамм E. coli - продуцент изолированного домена 1 протективного антигена Bacillus anthracis в форме вирусоподобных частиц |
| WO2020116686A1 (ko) * | 2018-12-06 | 2020-06-11 | 고려대학교 산학협력단 | 인간 항-antxr 키메라 항원 수용체 및 이의 용도 |
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| EP1808489B1 (en) | 2016-03-23 |
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| EP1301606A1 (en) | 2003-04-16 |
| US8313928B2 (en) | 2012-11-20 |
| JP2012010706A (ja) | 2012-01-19 |
| AU2007201553B2 (en) | 2010-04-22 |
| WO2002004646A1 (en) | 2002-01-17 |
| AU6930501A (en) | 2002-01-21 |
| CA2413045C (en) | 2016-06-21 |
| CA2413045A1 (en) | 2002-01-17 |
| CA2658217A1 (en) | 2002-01-17 |
| ZA200210206B (en) | 2004-03-17 |
| US20030170263A1 (en) | 2003-09-11 |
| AU2007201553A1 (en) | 2007-05-03 |
| CN1440459A (zh) | 2003-09-03 |
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