CN1084407A - 生产产气荚膜杆菌疫苗的方法 - Google Patents
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Abstract
本发明提供了生产新肽和疫苗的方法，包括给动
物使用所述新肽和疫苗后，它们能在动物体内诱导产
生直接抗产气荚膜杆菌α毒素(CPα)的抗体并由此
预防由产气荚膜杆菌和/或α毒素本身引起的感
染。本发明优选的肽含有产生气荚膜杆菌α毒素之
氨基酸247到370的氨基酸序列，但没有见于氨基
酸1到240之间的磷脂酶C和/或神经鞘髓磷脂水
解活性必需的抗原决定簇序列。另外还提供了产生
抗本发明产生之肽和疫苗的抗血清和抗体，特别是单
克隆抗体以及用于其生产目的之杂交瘤细胞系的方
法。
Description
本发明涉及生产能诱发人或动物中抗产气荚膜杆菌保护性免疫反应的新型多肽的方法;特别是能引发抗所述微生物α毒素的上述保护性反应的新肽，以及生产其抗体和抗血清的方法。
产气荚膜杆菌（C.Perfringens）在环境中广泛存在，并已在土壤中发现，使人和动物中的有机物质和类似肠菌丛的物质腐败。根据所产生的毒素的光谱（McDonel（1986）Pharmacology of Bacterial Toxins;F Dorner and J Drews（Editors）Pergamon Press，Oxford）可以将产气荚膜杆菌的不同菌株分成5个生物型（A-E）。生物型A菌株是人气性坏疽的特别重要的病原因子。该疾病在年纪较大者和糖尿病人群中，特别是经受了下肢手术的人群中有显著的增加（其中损害了向组织的血液供应可导致适于细菌繁殖的缺氧条件）。在经受过胃肠道手术的患者中，如果肠内容易污染了受损伤的组织后，则能够引起这种疾病，已经表明，在武装冲突期间会出现气性坏疽发病率的周期性增加。第一次世界大战期间，由于脏东西污染了伤员深部组织伤口，并且不能迅速治疗上述伤员从而导致了数万战士的死亡。
气性坏疽的发病机制在很大程度上归因于由细菌产生的强外毒素，其中α毒素作为引起疾病的主要因素受到注意。通过损坏并减少给组织的血液供应并因此促使感染扩散所需的条件，毒素可以在感染的起始病灶之表面上起作用。在疾病后期，毒素可影响全身引起死亡。早在1937年以前既已证明产气荚膜杆菌类毒素疫苗可提供抗实验诱导的气性坏疽保护作用（Penfold and Tolhurst（1937）Medical Journal of Australia PP604），并且随后的研究表明该疫苗的有效成份来自α毒素（Roberston and KePPie（1943），Lancet 2P311;Boyd et al（1972）J.Med.Microbiol 5，P467;Kameyama（1975）JaPanese Journal of Medicine，Science and Biology 25，P200）。
除这些进展外，还设有开发出适用于人的安全疫苗，而目前对气坏性坏疽的治疗通常是除去受影响的肢体或组织。
本发明之前已分离了编码α毒素的基因（Titball et al（1989）Infection and Immunity，Vol 57，P357-376）并检测了该蛋白质的结构一功能之间的关系（Titball and Rubidge 1990;Titball et al（1991）Infection and Immunity Vol 59.P1872-1874）。作为这些研究的一部分，确定了一些抗体抗原决定簇的位置（Logen et al（1992）Infection and Immunity Vol.59，P4338-4382）。
本发明的目的之一是提供生产新疫苗的方法，当给动物或人施用时，该疫苗可诱导生产直接抗产气荚膜杆菌α毒素（CP2）的抗体，并由此提供预防由产气荚膜杆菌和/或α毒素本身引起的感染。本发明的主要目的是提供相对安全并易于生产的上述疫苗。另外也提供生产抗体和抗血清的方法。
本发明的另一个目的是提供能诱导产生抗CPα抗体的疫苗，以便在研究气性坏疽发病机制中α毒素的作用时将其用作工具;上述疫苗没有其它产气荚膜杆菌类毒素活性。为了实现这些目的，本发明人已提供了生产该新肽的方法，在上述疫苗中可将该肽用作活性免疫剂或试剂。
因此，在其最主要的实施方案中，本发明提供了生产疫苗的方法，当给人或动物施用时，该疫苗能提供抗产气荚膜杆菌的保护性免疫反应，该方法包括将肽或肽结合物与药物可接受的载体混合，所述的肽或肽结合物包括产气荚膜杆菌α毒素氨基酸261到氨基酸300的抗原决定簇氨基酸序列，但没有磷脂酶C和/或见于α毒素氨基酸1到240之间的神经鞘髓磷脂水解活性所需的抗原决定簇，该方法最好包括掺入佐剂，如弗氏不完全或完全佐剂的步骤。
理论上，可以经本领域已知的肽化学合成法连接氨基酸来生产该肽或肽结合物;然后筛选上述产物活性。然而，可用本发明优选的方法生产能作为疫苗之活性剂的肽或肽结合物，以提供抗产气荚膜杆菌的保护性免疫反应，该方法包括将编码该肽的DNA序列插入到宿主微生物的DNA中，所述肽包括产气荚膜杆菌α毒素氨基酸261到300的抗原决定簇序列，但没有见于磷酸酶C和/或α毒素氨基酸1到240之间的神经鞘髓磷脂水解活性所需的抗原决定簇氨基酸序列，培养该生物体以使其表达所述的肽，并以纯化形式或作为非纯化部分分离该肽。
优选的方法是插入编码产气荚膜杆菌α毒素氨基酸261到氨基酸370，最好是编码247到370位氨基酸序列肽的DNA序列。
在最佳形式中，编码的肽仅由产气荚膜杆菌α毒素之氨基酸247到370的氨基酸序列组成或为该氨基酸序列与不是α毒素氨基酸1到氨基酸246的另一氨基酸序列之融合肽的形式或与如有其它所需效应之其他制剂所成之结合物形式。术语“其它氨基酸序列”是本领域专业人员已知的包括完整蛋白质以及适于使用者需要的相对短序列。例如也可以包括与前述提供其它免疫或标记目的的序列融合的非产气荚膜杆菌抗原蛋白质。
本发明的另一实施方案提供了生产含有适当剂量的前述肽或结合物的疫苗的方法，用其它制剂按需要选择性地补充这些肽或结合物以提供最佳保护。
本发明人和同事以前的工作已经确定，N末端（氨基酸1-249＝CPα249）或C末端（氨基酸250-370）区域均不能对其本身有致死性影响。据此，令人吃惊地发现，磷脂酶完全存在于N末端区域，而与抗原决定簇相关的已知神经鞘髓磷脂酶并不在C末端区域的序列内。
这些研究人员所做的其它实验已经表明，尽管针对这些N末端区域抗原决定簇的抗体可中和毒素的影响，但N末端区域本身并不能诱发保护性反应。因此，可以很容易地看出，有关的非活性C末端区域可引发保护性反应而有关的活性N末端则不能这一发现是完全令人惊奇的结果。
本发明人和其同事以前已确定了C末端抗原决定簇的位置，并发现其位于α毒素氨基酸序列的约273-275及295-297位。预期在不同分离物中这些抗原决定簇的位置或性质可轻微地变化而保持功能活性不变，因此，上述变化也包括在维持保护性反应的本发明范围内。
根据上述的公开内容，本领域专业人员会很清楚的了解C末端区域中的特定序列可比其它序列更有效地提供必需的免疫活性。这是因为保护性作用一般在一定程度上取决于肽在所需的抗原决定簇中的相互定向过程中的三维排列。这一点可由活性抗原决定簇支持N末端区域并不致死其本身的事实进一步证实，并说明C末端对于这些抗原决定簇的正确定位也是必须的。从本文所给的资料可很清楚地看出，本领域专业人员可以筛选用于所需活性的本发明的各种序列，并可用遗传工程技术如多聚酶链反应和基因克隆技术很容易地提供没有不需要的磷脂酶和神经鞘髓磷脂活性的各种序列。对“不需要的”区域在本发明作者及同事的文章中已有详细描述（Shuttleworth et al（1988）′Epitope mapping of Clostridium perfringens alpha-toxin′in F J Fehrenbach et al（Editors）bacterial Protein Toxins，Gustav Fischer Verlag，Stuttgart，p 65-66.Titball et al（1989）Infection and Immunity，Vol 57，p 357-376;Titball et al（1991）Infection and Immunity，Vol 59，5.p 1872-1874;Logan et al（1991）Infection Immunity，Vol 59，12.p 4338-4382）.
本发明还提供了生产编码本发明肽的重组DNA，含有上述DNA的质粒和细胞系，含有这些质粒或重组DNA本身的以便完成肽表达合适的大肠杆菌或酵母细胞。由PCR扩增编码所需序列如CPα247-370或CPα261-370的DNA来提供上述重组DNA，PCR使用的引物以天然产气荚膜杆菌DNA提供的双链序列的各自末端为目标，该引物形成双链末端的一半。将该扩增的DNA连接到适宜的载体中，可以适当与含有所需融合肽之其余部分的序列相邻接，并将该载体插入到适当的宿主细胞如大肠杆菌中。可以用已知的方法如Western印迹法筛选表达所需肽的细胞系。
应注意的是通过提供可裂解的相对较大的部分来选择特定融合肽，可利于分离该肽以便在开始纯化后得到与α毒素有关的肽。
本发明另一方面提供了用由本发明的肽生产抗该肽之抗血清及抗体的方法。此外，本发明还提供了抗本发明肽的单克隆抗体以及生产该抗体杂交瘤细胞。
用按本发明方法转化的微生物生产的肽注射宿主动物（如小鼠或兔），待到适宜的抗体产生期后，如14到28天从中分离血清，可以很容易地制备本发明提供的抗血清。用任何的已知方法，如使用本发明提供的固定化肽的亲和层析以保留抗体，即可从动物的血液及其血清中分离出抗体。可用已知方法生产表达抗本发明肽之抗体的杂交瘤细胞系，以很容易地制备相似的单克隆抗体。可使用将抗本发明肽的小鼠单克隆抗体轻链与人抗体重链掺在一起的已知方法，而使上述单克隆抗体人性化。
为了帮助本领域专业人员，现提供生产本发明肽和肽疫苗的有关附图及说明性实施例。这些非限定性实施例提供有关基本C末端区域肽之有效性的资料，据此本领域专业人员可以作出可能的改变也包括在本发明的范围内。
附图：
图1：显示在完整的α毒素序列（CPα1-370）＝A上、本发明的C末端区域肽（CPα247-370）＝B上及本发明融合肽（GST-CPα247-370）＝C上之主要抗原决定簇的相对位置。数字说明抗原决定簇的大致位置。
图2;显示用试验疫苗开始注射后（I.P.）V天log10抗体滴定度。用在X轴上的∧注释表明加强注射。上述滴定度对保护作用是非依赖性的。
序列目录1：显示按本发明优选的方法提供的细胞生产的优选α毒素剪短之肽的完整氨基酸序列。
序列目录2：显示天然产生的α毒素的完整氨基酸序列。
序列目录3：显示编码α毒素的完整DNA序列，其中本发明方法提供的优选肽的编码序列下面划了线。
实施例
产生气荚膜杆菌α毒素的C末端（CPα247-370）肽
在大肠杆菌中表达编码α毒素之C末端区域（氨基酸247-370＝CPα247-370）的α毒素基因片段，得到抗产气荚膜杆菌α毒素的候选疫苗肽。用多聚酶链反应（PCR）扩增α毒素基因序列之核苷酸823到1194间的区域，从而得到α毒素基因片断。将与核苷酸823起始区域同源的PCR引物掺入核苷酸尾部（GGGATG）以利于基因片段的克隆和表达。纯化PCR产物并将其与适当消化的pBluescriPt连接，并用常规方法（Maniatis et al 1989.Molecular Cloning：a Laboratory manual.Cold Spring Harbor Laboratory Press）进一步证实克隆之片的核苷酸序列可靠性。
从pBluescriPt克隆中分离CPα247-370编码片段（SmaI-HindⅢ片段），并将此片段与SmaI消化的PGEX-3X载体DNA（LRB-Pharmacia Biotechnology）连接，以实现表达。所得的重组质粒（PB3×13）表达了作为带有编码谷胱甘肽S转移酶的载体的融合蛋白（＝GST-CPα247-370）的CPα247-370。按制造商有关纯化GST的说明书纯化该蛋白质。
简单地说，在1升L-肉汤+氨苄青霉素中培养含PB3×13的大肠杆菌（37℃，150rpm），直到培养物的光密度（600nm）达到约0.3。加入IPTG直到终浓度为1mM，继续培养4小时，在30ml PBS+triton×100（1%）中重新悬浮细胞以制备 总细胞溶胞产物，并在冰上用Braun Labsonic超声处理器超声处理5×30秒。从谷胱甘肽-SePharose（LKB-Pharmacia）柱上选择性洗脱以纯化离心后得到的上清液。为了分离单独的CPα247-370片段，用因子（15U）将CPα247-370肽（在800μl中约10mg）室温消化过夜，通过谷胱甘肽-SePharose柱从GST分离出CPα247-370片段。
候选疫苗的毒性
通过腹腔注射将10μg量的疫苗经腹腔接种到一组6只小鼠体内，以确定疫苗融合肽的毒性。在这些剂量下候选疫苗是非致死性的，并且在直到接种后2星期小鼠都没有急性或慢性中毒症状。
对疫苗的抗体反应
通过腹腔注射，将疫苗与佐剂一起注入 6只小鼠体内，用ELISA监测抗α毒素抗体的出现。加强接种后，CPα247-370或GST-CPα247-370诱导了强烈的不断增强的抗α毒素抗体反应（图2）。CPα247-370与谷胱甘肽S转移酶融合并不影响抗该多肽的α毒素抗体反应。给予粗甲醛α类毒素后，观察到的抗CPα249（N末端区）CPα247-370或
GST-CPα247-370的抗体反应程度相似。
产生的抗疫苗抗体的性质
研究用CPα249，CPα247-370或GST-CPα247-370免疫之动物的血清体外中和与α毒素有关之生物活性的能力。全部都能有效地抑制毒素的磷脂酶C活性;而只有抗CPα247-370或GST-CPα247-370产生的抗血清抑制毒素的溶血活性。
抗毒性攻击保护作用
用5μg纯化的α毒素（约50LD50剂量）腹腔注射攻击单独用甲醛类毒素、CPα249、CPα247-370、GST-CPα247-370或GST免疫的动物。对照小鼠及用GST兔疫的小鼠在24小时内死亡，而用CPα249免疫的6只小鼠中的4只也在该期间死亡。用甲醛类毒素，CPα247-370或GST-CPα247-370免疫的小鼠则存活下来并且没有中毒迹象。
抗微生物攻击的保护作用
为了研究甲醛类毒素，CPα247-370或 GST-CPα247-370也诱导抗实验性气性坏疽之保护作用的可能性，用产气荚膜杆菌NCTC8237的1010个活细胞对已用这些候选疫苗免疫的每组6只小鼠进行肌内注射攻击。该攻击实验的被免疫动物全部存活，而未免疫的对照动物则在48小时内死亡。
结论
在该研究期间产生了用最容易的遗传工程方法加工的疫苗，CPα247-370代表毒素的C末端区。该蛋白质以至少高达10μg的剂量，在小鼠中未表现出毒性。用该蛋白质重复接种小鼠可诱导强烈的保护动物以对抗α毒素及对抗产气荚膜杆菌攻击的抗体反应。该疫苗可比甲醛类毒素提供数种潜在的优点：它更容易制备并且由于它没有甲醛、没有其它类毒素样产气荚膜杆菌毒素以及部分类毒素物质，所以该疫菌使用起来安全且反应原性较低。
表1：由候选疫苗免疫提供的抗α毒素攻击的保护作用。每组6只Balb/C小鼠在80天的时间内，接受与弗氏不完全佐剂混合的6×10μg抗原腹腔内注射。用10μg纯化的α毒素腹腔注射攻击这样小鼠并记录至24小时后的死亡数。
抗原 用α毒素攻击
剂量(μg) 存活者
无 10 0/6
甲醛类毒素 10 6/6
Cp α247-37010 6/6
GST-Cp α247-37010 6/6
Claims (18)
1、一种生产当给人或动物使用时能提供抗产气荚膜杆菌保护性免疫反应之疫苗的方法，该方法包括将肽或肽结合物与药物学上可接受的载体混合，所述的肽或肽结合物包括产气荚膜杆菌α毒素的从氨基酸261到氨基酸300的抗原决定簇氨基酸序列，但没有见于α毒素之氨基酸1到240之间的对磷脂酶C和/或神经鞘髓磷脂水解活性必需的抗原决定簇。
2、根据权利要求1的方法，其中还包括掺入佐剂的步骤。
3、一种生产能作为疫苗中活性剂的肽或肽结合物的方法，当给人或动物使用时，该疫苗提供抗产气荚膜杆菌的保护性免疫反应，该方法包括将编码 肽的DNA序列插入到宿主微生物的DNA中，培养该微生物以便表达该肽，并以纯化或非纯化形式分离该肽，所述肽包括产气荚膜杆菌α毒素的从氨基酸261到氨基酸300的抗原决定簇氨基酸序列，但没有见于在α毒素之氨基酸1到240间的对磷脂酶C和/或神经鞘髓磷脂水解活性必需的抗原决定簇。
4、根据权利要求3的方法，其中插入到微生物之DNA中的DNA序列编码产气荚膜杆菌α毒素的从氨基酸261到氨基酸370的氨基酸序列。
5、权利要求4要求的方法中，其中插入到微生物之DNA中的DNA序列编码产气荚膜杆菌α毒素的从氨基酸247到氨基酸370的氨基酸序列。
6、根据权利要求5的方法，其中插入到微生物之DNA中的DNA序列编码由产气荚膜杆菌α毒素的氨基酸247到氨基酸370的氨基酸序列或者以与另一不是α毒素之氨基酸1到氨基酸246的氨基酸序列所成之融合肽形式组成，或编码与具有其它所需作用的制剂所成之结合物形式的氨基酸247到370。
7、根据权利要求6的方法，其中肽是一种为进一步包括谷胱甘肽S转移酶之氨基酸序列的融合肽的肽结合物。
8、根据权利要求3至7中任一项的方法，其中将分离的纯化或未纯化的肽或肽结合物进一步与佐剂混合。
9、根据权利要求8的方法，其中佐剂是弗氏不完全佐剂。
10、根据要求3到9中任一项的方法，其中借助以天然产生的产气荚膜杆菌DNA作模板的多聚酶链反应提供编码肽或肽结合物的DNA。
11、根据权利要求10的方法，其中将产生的DNA插入到质粒中。
12、根据权利要求11的方法，其中通过将质粒包含于细胞内而将质粒插入到能支持肽表达的细胞中。
13、根据权利要求12的方法，其中细胞是大肠杆菌细胞或酵母。
14、生产能抑制人或动物中产气荚膜杆菌致死效应之抗血清的方法，该方法包括给动物使用按权利要求3到13中之任一项的方法生产的肽或肽结合物，并在抗体诱导期后从动物体内除去血清。
15、根据权利要求14的方法，其中动物是兔猪或小鼠。
16、生产能抑制人或动物中产气荚膜杆菌致死效应之抗体的方法，该方法包括用亲和层析法纯化按权利要求14或15的方法生产的抗血清。
17、根据权利要求16的方法，其中亲和层析使用按权利要求3到13中任一项的方法生产的固相化的肽或肽结合物以结合抗血清中的抗体，然后从固相化肽或肽结合物上洗脱这些抗体。
18、根据权利要求14的方法，其中抗体是抗体生成细胞与杂交瘤细胞融合所产生的单克隆抗体。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4877612A (en) * | 1985-05-20 | 1989-10-31 | Frank M. Berger | Immunological adjuvant and process for preparing the same, pharmaceutical compositions, and process |
| US5200318A (en) * | 1992-05-13 | 1993-04-06 | Miles Inc. | Diagnosis of IDDM with a panel of immunoreagents |
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1993
- 1993-05-20 US US08/341,538 patent/US5817317A/en not_active Expired - Lifetime
- 1993-05-20 ES ES93913200T patent/ES2185631T3/es not_active Expired - Lifetime
- 1993-05-20 NZ NZ253189A patent/NZ253189A/en not_active IP Right Cessation
- 1993-05-20 AT AT93913200T patent/ATE226635T1/de not_active IP Right Cessation
- 1993-05-20 PT PT93913200T patent/PT642581E/pt unknown
- 1993-05-20 WO PCT/GB1993/001039 patent/WO1993023543A1/en active IP Right Grant
- 1993-05-20 CN CN93107585A patent/CN1057533C/zh not_active Expired - Lifetime
- 1993-05-20 MX MX9302964A patent/MX9302964A/es unknown
- 1993-05-20 JP JP52002593A patent/JP3370672B2/ja not_active Expired - Fee Related
- 1993-05-20 GB GB9422512A patent/GB2283020B/en not_active Revoked
- 1993-05-20 IL IL10576393A patent/IL105763A/xx not_active IP Right Cessation
- 1993-05-20 DK DK93913200T patent/DK0642581T3/da active
- 1993-05-20 DE DE69332434T patent/DE69332434T2/de not_active Expired - Fee Related
- 1993-05-20 EP EP93913200A patent/EP0642581B1/en not_active Expired - Lifetime
- 1993-05-20 CA CA002136230A patent/CA2136230C/en not_active Expired - Fee Related
- 1993-05-20 AU AU43342/93A patent/AU671838B2/en not_active Ceased
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- 1996-10-04 US US08/725,518 patent/US5851827A/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101489584B (zh) * | 2006-04-17 | 2013-05-01 | 先灵-普劳有限公司 | 重组减毒梭状芽孢杆菌生物体和疫苗 |
| CN108904791A (zh) * | 2018-07-20 | 2018-11-30 | 中国兽医药品监察所 | 一种产气荚膜梭菌α毒素重组亚单位疫苗及其生产方法 |
| CN109602898A (zh) * | 2018-12-28 | 2019-04-12 | 江苏省农业科学院 | 一种产气荚膜梭菌α毒素基因工程疫苗及其制备方法 |
| CN109602898B (zh) * | 2018-12-28 | 2022-02-22 | 江苏省农业科学院 | 一种产气荚膜梭菌α毒素基因工程疫苗及其制备方法 |
| CN114621963A (zh) * | 2022-03-11 | 2022-06-14 | 牧原食品股份有限公司 | 一种猪魏氏梭菌a型的亚单位疫苗及其制备 |
Also Published As
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| DE69332434T2 (de) | 2003-06-26 |
| WO1993023543A1 (en) | 1993-11-25 |
| EP0642581B1 (en) | 2002-10-23 |
| CA2136230C (en) | 2008-11-18 |
| GB2283020B (en) | 1996-05-01 |
| IL105763A0 (en) | 1993-09-22 |
| CA2136230A1 (en) | 1993-11-25 |
| GB2283020A (en) | 1995-04-26 |
| AU671838B2 (en) | 1996-09-12 |
| JP3370672B2 (ja) | 2003-01-27 |
| DK0642581T3 (da) | 2003-02-17 |
| AU4334293A (en) | 1993-12-13 |
| GB9422512D0 (en) | 1995-01-04 |
| MX9302964A (es) | 1995-01-31 |
| DE69332434D1 (de) | 2002-11-28 |
| NZ253189A (en) | 1996-08-27 |
| CN1057533C (zh) | 2000-10-18 |
| US5851827A (en) | 1998-12-22 |
| IL105763A (en) | 2005-11-20 |
| JPH07506725A (ja) | 1995-07-27 |
| EP0642581A1 (en) | 1995-03-15 |
| ATE226635T1 (de) | 2002-11-15 |
| ES2185631T3 (es) | 2003-05-01 |
| US5817317A (en) | 1998-10-06 |
| PT642581E (pt) | 2003-03-31 |
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