CN1125174C - 重组噬菌体 - Google Patents
重组噬菌体 Download PDFInfo
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- CN1125174C CN1125174C CN97192116A CN97192116A CN1125174C CN 1125174 C CN1125174 C CN 1125174C CN 97192116 A CN97192116 A CN 97192116A CN 97192116 A CN97192116 A CN 97192116A CN 1125174 C CN1125174 C CN 1125174C
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- phage
- antibody
- component
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Classifications
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
本发明涉及一种用于治疗或预防细菌感染特别是诸如幽门螺旋杆菌感染的粘膜性细菌感染的噬菌体。特别地,它涉及用于上述目的的经过修饰的丝状噬菌体,如M13噬菌体。该噬菌体的表面包含有重组蛋白,其包含(i)由噬菌体表面蛋白衍生而来的组分1;(ii)包含有提供细菌抗原结合位点的抗体可变区序列的组分2,该组分2使前述噬菌体能结合到引起前述感染中涉及的细菌上并抑制其生长。
Description
本发明涉及用于治疗细菌感染,特别是如幽门螺旋杆菌(Helicobacter pylori)感染的粘膜性细菌感染的噬菌体。
噬菌体和抗生素抗性
抗生素抗性是医学和经济上日益严重的全球性问题。因而迫切需要有其它方法来消除细菌而避免发生这种抗性问题。
细菌噬菌体或噬菌体是一种特异性感染细菌的病毒。噬菌体首先与其宿主细菌结合并将编码不同噬菌体蛋白的基因转入到宿主中。噬菌体利用宿主细菌的蛋白质合成系统、氨基酸合成系统等以及由宿主产生的能量完成自身的复制(Maloy等编,微生物遗传学,(Microbial genetics),Jones and Bartlett出版社,1994)。
大多数噬菌体裂解宿主,或者通过其它机制来破坏特定的菌株。本发明基于这样的认识:噬菌体的遗传修饰,尤其是丝状噬菌体的遗传修饰,提供了一种构建新型的细菌专一性噬菌体用于消除如幽门螺旋杆菌等特定细菌的方法,此方法有望克服抗生素抗性的相关问题。
丝状噬菌体
具有头发状F菌毛的大肠杆菌(E.coli)是诸如M13、fd和f1等丝状噬菌体的宿主。这些Ff(丝状,F菌毛)噬菌体在序列和行为上十分相近(Rashed & Oberer(1986),微生物学综述(Microbiologicalreviews),50,401~427;Kornberg & Baker:DNA复制,557-570,W.H.Freeman and Co.,纽约,1992)。Ff噬菌体作为细菌病毒的一种,本身并不引起裂解性感染,但可以使被感染的宿主细胞产生并将噬菌体颗粒不通过裂解途径分泌出去。
噬菌体M13的单链基因组编码10种不同的蛋白。噬菌体DNA由一层蛋白质外壳包被,该外壳由大约2700个基因8蛋白质(g8p)组成。活的M13噬菌体在其顶端也表达5个43kDa的基因3蛋白质(g3p),该蛋白质负责使噬菌体粘附到大肠杆菌的菌毛上。该基因3蛋白质通过多肽链C端部分固定于病毒外壳上,多肽链的N-端球状区域是暴露的,调节噬菌体附着在宿主的F菌毛顶部。通过电子显微镜,可以看到附着到宿主上的粘附复合物的结构就象树干上的突结。在感染的过程中,g3p和g8p两个蛋白的先导序列引导着这些噬菌体蛋白质转运到细菌的内膜中。
Ff噬菌体作为克隆载体应用很广,因为它对被包装的DNA片段的长度没有限制,并且作为单链DNA也便于纯化。噬菌粒是一种包含M13(单链)和质粒(双链)的复制起点的载体。噬菌粒可以象质粒一样复制,也可在辅助噬菌体如M13K07的帮助下以重组M13噬菌体的形式被包装(Veria & Messing(1987),酶学方法(Methods in Enzymol.),153,3-11)。
重组抗体的生产
抗体分子包含有不连续的片段,它们可以用蛋白酶切开或由重组技术生产。其中的一个片段是Fv(可变区),仅由抗体的VL和VH区域组成。在美国专利4,946,778中描述了一种人工设计的单链Fv(ScFv),即重组的Fv片段,其中的两个可变区用中性接头人为连接后以单链多肽的形式表达。
Mc Cafferty及其合作者(Mc Cafferty(1990),自然(Nature),348,552-554;Winter & Milstein(1991),自然,349,293页)发展了一种重组抗体的生产技术。该技术依靠噬菌体展示系统,通过将VL(可变区轻链)和VH(可变区重链)基因克隆到噬菌体载体,从而使抗体片段以融合蛋白的形式展示在噬菌体表面。通过这种方式可以筛选到具有特定专一性和亲和力的抗体。有人建议将在原核体系中分离和制造的抗体称为“大肠杆菌克隆”抗体(Chiswell & McCafferty(1992),生物技术趋势,(Trends in Biotechnology)10,80-84)。
噬菌粒pCANTAB5是可购买到的,用于将抗体可变区基因克隆到M13基因3的先导序列和主体之间。该g3p先导序列将所产生的融合蛋白引导至大肠杆菌的内膜或周质中,再由g3p的主要结构域将融合蛋白附着到装配的噬菌体顶部。抗体-g3p基因的表达由噬菌粒上可诱导的lac启动子控制。
幽门螺旋杆菌感染
普遍认为,已知病例中84%的胃溃疡和95%的十二指肠溃疡主要是由于幽门螺旋杆菌感染造成的(Kuipers,E.L.,等(1995)营养药物治疗杂志(Aliment.Pharmacol.Ther.)9,(增刊2),59-69)。幽门螺旋杆菌生长于胃壁上,并由胃壁粘膜层保护避开胃中的酸性环境,同时其代谢过程自身也能分泌氨以中和酸。
传统的抗生素治疗对幽门螺旋杆菌几乎无效。这可能是因为:i)对于不直接暴露在血液循环中的微生物抗生素很难接近,ii)许多口服药在胃中快速通过,或者在胃中的酸性条件下降解。
本发明的目的在于提供一种新的治疗方法,该方法可消除细菌,尤其是消除造成粘膜性细菌感染如幽门螺旋杆菌的感染。特别是提供了经过遗传修饰的能专一性结合到另外的细菌宿主上的丝状噬菌体用于治疗过程。
基于重组噬菌体的治疗粘膜性细菌感染的方法优于传统的抗生素治疗,这是因为:
·它可消除具有传统抗生素抗性的细菌;
·重组噬菌体对特定细菌种类有高的专一性;
·噬菌体的繁殖有自限性;
·在由于幽门螺旋杆菌引起的感染中,可通过幽门螺旋杆菌的活动将噬菌体扩散到胃粘膜各处。
在本说明书和实施例中,“常规方法”或“常规操作”一词应理解为一般实验室操作中的方法和操作,如:Sambrook,J.,Fritsch,E.F和Maniatis,T.,(1989),分子克隆:实验室操作手册(第2版,冷泉港实验室,冷泉港,纽约)一书中所述。
第一方面,本发明提供了一种用于治疗和预防细菌感染的修饰过的噬菌体,该噬菌体表面存在的重组蛋白包含:
i)由噬菌体表面蛋白衍生而来的组分1;
ii)包含一种提供细菌抗原结合位点的抗体可变区序列的组分2,该组份2使得前述噬菌体能结合到引起前述感染中涉及的细菌上并抑制其生长。
前述修饰过的噬菌体可以是一种修饰过的丝状噬菌体,如经过修饰的M13噬菌体。
前述细菌感染可以是一种粘膜性细菌感染,如幽门螺旋杆菌感染。然而,本发明并不局限于能杀死幽门螺旋杆菌的噬菌体,而是包括经过改性的能用于杀死广泛范围细菌的噬菌体。这是因为基于本发明的公开,本领域技术人员可制备专一作用于任何一种细菌的本发明噬菌体。本发明的噬菌体适用于治疗任何与外界接触的粘膜细菌感染。这样的粘膜上皮组织的例子包括:鼻、肺、胃肠道、膀胱和阴道的粘膜。
可用本发明的噬菌体治疗的其它细菌感染的例子有:
·由大肠杆菌,腐生葡萄球菌(Staphylococcus saprophyticus),克雷伯氏菌(Klebsiella spp.),变形菌(Proteus spp)或绿脓杆菌(Pseudomonas aeruginosa)引起的尿道感染;
·由Clamycda引起的阴道感染;
·由链球菌(Streptococcus),葡萄球菌(Staphylococcus),流感嗜血菌(Haemophilus influence),肺炎球菌(Pneumococcus)或肺炎支原体(Mycoplasma pneumonic)引起的鼻,toncillar,肺部感染;
·由沙门氏菌(Salmonella),志贺氏菌(Shigella)耶尔森氏菌(Yersinia),肠弯曲杆菌(Campylobacter jejuni),大肠弯曲杆菌(Campylobacter coli),螺旋杆菌(Helicobacter),霍乱弧菌(Vibriocholera)或大肠杆菌(E.coli)引起的胃肠道感染。
上面提到的重组蛋白的组分1可以优选由负责未经修饰的该噬菌体吸附到细菌菌毛的蛋白质派生出来,如M13噬菌体中的g3p蛋白。
前述重组蛋白的组分2可以包含一个重组的单链Fv(ScFv)多肽。因此,前述重组蛋白可以是一个g3p-ScFv融合蛋白。
本发明的噬菌体的优选形式是一种用于治疗或预防幽门螺旋杆菌的噬菌体。其中前述重组多肽的抗体可变区序列是选自杂交瘤细胞系5F8(ECACC No.95121524),2H6(ECACC No.95121526)和5D8(ECACC No.95121527)单克隆抗体的单抗可变区序列。因而,本发明中的噬菌体可以是一种经过修饰的M13噬菌体,命名为B8,以编号NCIMB40779保藏在NCIMB,或者是它的一种保留有结合和感染幽门螺旋杆菌能力的衍生株。
具有预期特性的噬菌体可由下述任一方法获得:(a)筛选天然存在的噬菌体,或是从包含表达多种抗体片段的噬菌体文库中筛选。噬菌体文库可以从免疫细胞或大量的个体中获得。由于巨大的遗传多样性,该噬菌体文库应当包括预期的特定的噬菌体,这些噬菌体可针对个体先前接触过的多种细菌。
(b)对现存的噬菌体进行诱变。所有有生命的机体包括噬菌体均会产生突变。突变的频率可通过化学方法或者短波电磁辐照等方法加以提高。
(c)通过遗传修饰噬菌体结合区域中一个或多个氨基酸,或者改变其糖或脂的组成,从而改善天然或重组噬菌体的预期特性。这一方案在实验部分中有更进一步的描述。本发明的噬菌体可由下述方法生产:该方法包括(a)分离一种针对某细菌的抗体;(b)分离编码该抗体可变区重链和轻链的DNA;(c)将该DNA引入噬菌体DNA,使该抗体区域在噬菌体表面表达。
本发明另一方面,提供了修饰过的噬菌体用于制备治疗或预防由幽门螺旋杆菌引起的粘膜细菌感染的药物的用途,其中修饰的噬菌体是表面存在着包含下述成分的重组蛋白的M13噬菌体:(i)来自噬菌体表面蛋白的组分1;(ii)一种包含提供细菌抗原结合位点的抗体可变区序列的组分2,该组分2使得所述噬菌体能结合到细菌细胞表面,从而抑制参与所述细菌感染的病因的细菌细胞的生长;
其中,所述重组蛋白的组分1是来源于未经修饰的噬菌体中负责与细菌菌毛吸附的蛋白质,其由g3p蛋白衍生而来;
所述重组蛋白质的组分2包含有呈g3p-ScFv融合蛋白形式的ScFv多肽;
所述重组多肽中抗体可变区序列是选自杂交瘤细胞系5F8(ECACC No.9512524),2H6(ECACC No.95121526)和5D8(ECACCNo.95121527)的单克隆抗体的单克隆抗体可变区序列。
另外一方面,本发明提供了一种药物组合物,该组合物包含本发明的噬菌体和与之混合的药学上可接受的载体或赋形剂。
本发明的噬菌体适当的给药方法的例子包括:
·用于鼻、肺部的喷剂;
·用于治疗胃肠道粘膜时用奥美拉唑预治疗后再用碳酸氢盐缓冲液悬浮的噬菌体治疗;
·用于胃膜和阴道粘膜的粘膜附着凝胶混合物(如纤维素基凝胶,聚卡波非,泊洛沙姆等);
·用于膀胱的碳酸氢盐缓冲液。
服用的噬菌体数量可由技术人员决定。依照感染的类型,噬菌体的给药数量范围从104到1010个。
还有一方面,本发明提供了一种治疗哺乳动物细菌感染的方法,此方法包括服用本发明的噬菌体或药物组合物。所述细菌感染可以是象幽门螺旋杆菌感染那样的粘膜性细菌感染。本发明同样包括本发明的噬菌体用于制备一种用于治疗或预防粘膜性细菌感染如幽门螺旋杆菌感染的药物中的用途。
本发明的又一方面是选自5F8(ECACC No.95121524),2H6(ECACC No.95121526)和5D8(ECACC No.95121527)的杂交瘤,以及选自上述杂交瘤产生的单克隆抗体的单克隆抗体。杂交瘤技术是一种已知的先有技术。首先将免疫过的动物产生抗体的B细胞与骨髓瘤细胞融合,形成杂交瘤细胞系,再从中挑选能产生期望的抗体的杂交瘤细胞系。
实施例1:生产针对幽门螺旋杆菌的单克隆抗体
1.1抗原的制备
幽门螺旋杆菌17874,25,66,253和1139(来源于Astra Hassle,瑞典)用下面的培养条件培养:哥伦比亚琼脂中加入8.5%马血,10%马血清,1%isovitalex,微需氧环境下与Anaerocult C一起37℃培养。
幽门螺旋杆菌表面蛋白的制备按照Ma J-Y等(1994)(斯堪的纳维亚胃肠学杂志(Scand.J.Gastroenterol.)29,961-965页)中方法进行。简言之,总量为4克的5种幽门螺旋杆菌在100毫升0.2摩尔/升甘氨酸-盐酸缓冲液(pH2.2)中室温下培养15分钟。再用1M氢氧化钠中和。4℃下10,000转/分离心10分钟,丢弃沉淀,上清液对蒸馏水4℃下透析过夜,用作“幽门螺旋杆菌抗原制剂”或“幽门螺旋杆菌表面蛋白”。
1.2.生产单克隆抗体
免疫过程基本按照斯堪的纳维亚生理学报(Acta Physiol.Scand.),144,369-378页中Cabero,J.L等人叙述的方法进行。简言之,4℃下2mg/ml幽门螺旋杆菌表面蛋白与等体积弗氏完全佐剂一起乳化。两只雌性DBA/1鼠的后足垫分别注射抗原乳剂,每只剂量为50μl。11天后取腘淋巴结中的淋巴细胞与鼠骨髓瘤细胞(sp2系)在50%(w/w)聚乙二醇4000介导下融合。细胞融合悬液分别接种到5个微孔板中,用含有10%胎牛血清并加入50μg/ml庆大霉素的DEME培养基培养。
1.3.酶联免疫吸附测定(ELISA)
免疫板用50μl含有指定抗原(10μg/ml)的0.05M Na2CO3/NaHCO3缓冲液(pH9.8)包被,4℃孵育过夜。然后37℃下用含有0.05%吐温20的PBS(PBS-T)封闭未被结合的位点一小时。再加入一抗37℃培育1小时。用绵羊抗鼠IgG过氧化物酶偶联物作为二抗。结合的过氧化物酶用0.04%(w/v)OPD和14mM过氧化氢在0.1M柠檬酸/0.2MNaHPO4 pH5的条件下检测。加入2M H2SO4终止反应,492nm下检测。每次培育后均用PBS-T洗三次。
1.4初筛
用ELISA方法筛选融合的淋巴结细胞和骨髓瘤细胞,其中有45株杂交瘤克隆为幽门螺旋杆菌表面蛋白质阳性。通过免疫组化方法发现其中的8株明显可使琼脂平板上的幽门螺旋杆菌染色。命名为2H6(ECACCNo.95121526)、5D8(ECACC No.95121527)和5F8(ECACC No.95121524)的杂交瘤克隆与幽门螺旋杆菌的反应较其它菌株更强,故选作进一步研究。
实施例2:生产针对幽门螺旋杆菌的重组噬菌体M13
2.1材料
mRNA快速制备纯化试剂盒,鼠ScFv试剂盒,表达试剂盒,检测试剂盒,SfiI,NotI,T4DNA连接酶和绵羊抗M13抗体购自发马西亚生物技术公司(Pharmacia Biotech,Uppsala,瑞典)。dNTP混合物,10×PCR反应缓冲液和Ampli Taq DNA聚合酶购自Perkin Elmer公司。Bacto酵母提取物,Bacto蛋白胨,Bacto琼脂购自Difco实验室(Detroit,密执安,美国)。哥伦比亚琼脂板和布氏肉汤由林雪平大学微生物系提供(Linkoping,瑞典)。Anaerocult C由默克公司(Merck德国)提供。SlowfadeTM防衰试剂盒由分子探针公司(Molecular ProbesInc.美国)提供。
2.2.噬菌体抗体文库的构建
重组噬菌体抗体系统试剂盒(发马西亚生物技术公司)用于表达以融合蛋白形式展示在噬菌体表面的抗体片段。
使用mRNA快速制备纯化试剂盒(发马西亚生物技术公司)从杂交瘤细胞系(2H6,5D8和5F8)中用寡聚(dT)-纤维素介质的亲和层析法分离和纯化总mRNA。
用鼠SCFV试剂盒完成下列步骤:
·用试剂盒提供的反转录酶和引物混合物从杂交瘤mRNA中合成第一链cDNA;
·用不同的引物(VH和VL链引物,由试剂盒提供)用PCR反应扩增编码单抗中可变区轻链和重链的cDNA。用1.5%的琼脂糖凝胶电泳分析VH和VL链得到了正确大小的VH(340bp)和VL(325bp)的单带。
·1%琼脂糖凝胶分离并纯化扩增所得的VH和VL链并用试剂盒提供的DNA接头片段装配到单链FV(SCFV)基因中。接头片段的结构使其一头与重链的3′端退火,另一端则与轻链的5′端杂交。电泳后可看到正确大小的SCFV基因(750bp)单带。
·用一系列寡核苷酸引物(试剂盒提供)扩增组装好的抗体ScFvDNA片段,引入Not I和Sfi I限制性位点。用离心柱层析(试剂盒提供)除去接头、dNTP和Taq DNA聚合酶。SCFV片段用Not I和Sfi I切割后产生用于连接到载体pCANTAB5上的粘性末端。
下述步骤用表达试剂盒完成:
·将SCFV片段连接到事先用Not I和SfiI切割产生粘性末端的噬菌粒载体p CANTAB5(试剂盒提供)。用T4DNA将插入片段与相应的噬菌粒连接。于是将SCFV片段以正确的方向插入,邻近M13基因3并与之在同一读框中,从而可以表达SCFV-g3p融合基因。
·用大肠杆菌TG1(试剂盒提供)制备感受态细胞,用包含有lac启动子和氨苄青霉素抗性标记的重组噬菌粒转化感受态细胞。在含有葡萄糖和氨苄青霉素的培养基中30℃培养转化细胞,得到3.2×104个SCFV菌落。氨苄抗性菌落在培养基上划线培养以做为文库原种。
·氨苄抗性细胞由辅助噬菌体M13K07感染(试剂盒提供)。该噬菌体包含有卡那霉素抗性标记且能在含有卡那霉素和氨苄青霉素的葡萄糖缺乏培养基中生长。在没有葡萄糖的条件下,噬菌粒中的乳糖启动子不再受到抑制。于是产生了在外壳上带有重组抗体片段的噬菌体颗粒并释放到胞外。
·噬菌体展示的抗体通过与抗原的结合来挑选出其中可与幽门螺旋杆菌结合的抗体。一个培养瓶用5ml幽门螺旋杆菌表面蛋白(15μg/ml,50mM碳酸钠缓冲液,pH9.6)包被过夜。用PBS洗三次,含有10ml1%BSA(w/v)的PBS的培养瓶37℃培养1小时。PBS洗涤三次后将培养瓶在噬菌体上清液中(含有噬菌体的培养基)培养2小时。用含有0.1%(w/v)吐温20的PBS洗涤20次,再用PBS洗涤20次。结合的噬菌体颗粒通过加入1毫升100mM三乙胺轻摇10分钟,立即用0.5ml1M的TrisHCl pH7.4中和来洗脱。
用洗脱的噬菌体感染培养在SOBAG琼脂上处于对数生长期的大肠杆菌TG1。SOBAG琼脂包括2%Bacto-蛋白胨,0.5%Bacto-酵母提取物,0.05%NaCl,0.01M MgCl2,0.01%葡萄糖和0.01%氨苄青霉素。这些菌落再次用M13K07噬菌体挑选、转化,生长和复原一次。
第一轮筛选后的100个菌落,其中微孔板中再生的菌落中6%的菌落用酶联免疫吸附测定法鉴定为对幽门螺杆菌抗原制剂阳性。第三次筛选微孔板中的菌落中有95%的噬菌体抗体能与幽门螺旋杆菌抗原反应。
在用幽门螺旋杆菌抗原制剂作为抗原的噬菌体ELISA测定中,重组噬菌体B8的滴度比辅助噬菌体(野生噬菌体)要高10倍。选择噬菌体B8(NCIMB No.40779)作进一步分析。
实施例3:ELISA
使用检测试剂盒以酶联免疫吸附法(ELISA)检测和鉴别噬菌体展示的重组抗体。
用20μl幽门螺旋杆菌抗体(10μg/ml,50mM Na2CO3/NaHCO3pH9.6)包被96孔板,4℃下培养过夜。用含有0.05%吐温20的PBS溶液(PBS-T)洗涤孔板三次,再用含有1%BSA的300μl PBS37℃下封闭1小时。用等体积的1%BSA/PBS稀释重组噬菌体抗体,室温下培养20分钟。洗涤后,加入5×1010个噬菌体转导单位(200μl/孔),37℃培养2小时。用PBS-T洗涤孔板三次,再加入用1%BSA/PBS液稀释1∶5000倍后的HRP/Anti M13偶联物(由试剂盒提供),37℃培养1小时。用PBS-T洗涤孔板三次,加入2′2′-连氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐和过氧化氢作为过氧化物酶的底物,室温下培养30分钟。使用计算机化酶联免疫吸附测定读数仪405nm下测定吸光值。卵清蛋白(10μg/ml,溶于PBS)作为对照抗体。辅助噬菌体作为对照噬菌体。结果证实,重组噬菌体B8可特异性地与幽门螺旋杆菌表面抗原结合。
实施例4:免疫杂交
用聚丙烯酰胺凝胶电泳(SDS-PAGE)(10μg蛋白/孔)分离幽门螺旋杆菌抗原制剂的蛋白,将其用微型电泳转膜仪(伯乐公司,BioRad,Richmond,加州,美国)转移到硝酸纤维素膜上。用含有10%BSA,0.1%吐温-20的PBS封闭硝酸纤维素膜,室温下轻摇1小时。加入噬菌体B8(1011个转化单位/毫升)与硝酸纤维素膜一起4℃下轻摇培养过夜。用不加入一抗的过程作为阴性对照。用含有0.1%吐温20的PBS洗涤后,将硝酸纤维素膜与HRP/Anti-M13偶联物(用封闭液1∶5000稀释)一起轻摇1小时。用ECL检测试剂盒(Amersham,UK)检测结合。
硝酸纤维素膜用氨基黑染色,显示主带分别对应64KDa,36KDa,31KDa和27KDa的蛋白。收集的单克隆抗体(2H6,5D8和5F8,用于构建重组噬菌体的杂交瘤)可与32KDa和64KDa的条带反应。将噬菌体抗体B8用上述方法免疫杂交也有相似的染色结果。结果表明,对应于幽门螺旋杆菌特异性单克隆抗体的可变区重链和轻链的基因已在噬菌体上正确表达。
实施例5:重组噬菌体对细菌的作用效果
下列实验中,均用含有5%胎牛血清的布氏肉汤在10%CO2和5%O2的培养条件下37℃培养细菌。
当20μl细菌贮存液与10ml肉汤混合后实验开始。用PBS稀释培养基后等量地涂布在琼脂平板上,在一定时间后检定每毫升中菌落形成单位(CFU)。37℃下培养两天后计数每个平板上的菌落数。
5.1时间依赖效应
通过统计三天中有或无噬菌体时的CFU,研究重组噬菌体B8对幽门螺旋杆菌17874生长的时间依赖效应(见表1)。开始时每10毫升培养基中加入106个噬菌体(加噬菌体)。对照菌株中不加入噬菌体(无噬菌体)。
三天后,没有噬菌体加入的菌株中菌落数增加了5个数量级。在有噬菌体的培养基中,一天后菌落数下降了1个数量级,肉汤培养基外观从混浊变得不太混浊。
5.2.对不同菌株的作用效果
幽门螺旋杆菌17874,1139和244和金黄色葡萄球菌ATCC29213以及大肠杆菌TG1分别加入或不加入噬菌体(106个每10ml培养基),培养24小时。分析0时间和24小时的菌落形成单位(见表2)。三种幽门螺旋杆菌的菌落形成单位均被重组噬菌体降低,但是金黄色葡萄球菌和大肠杆菌不受影响。幽门螺旋杆菌17874也不受作为对照的辅助噬菌体M13K07的影响。
5.3.对幽门螺旋杆菌的作用效果
第二次实验(见表3)中,幽门螺旋杆菌17874,1139,253,25和66与链球菌(RafM87)一起进行测试。在没有噬菌体时,所有受试菌株的菌落形成单位在24小时培养过程中都增加。然而,在存在重组噬菌体(开始时106个噬菌体加至10ml培养基)的培养液中,幽门螺旋杆菌17874,1139和25的菌落形成单位减少而菌株253和66的生长速率与对照相比明显降低。因此,所有受试的幽门螺旋杆菌均可受到噬菌体的作用。
实施例6:幽门螺旋杆菌的免疫荧光染色
用PEG沉淀法浓缩噬菌体抗体B8。用培养基上的幽门螺旋杆菌(17874)进行免疫荧光染色。
用下列贮备试剂进行免疫荧光染色:
试剂A:用1%BSA/PBS 1∶1稀释的1012个转化单位/毫升的噬菌体抗体。
试剂B:用1%BSA/PBS稀释的绵羊抗M13抗体。
试剂C:用1%BSA/PBS 1∶100稀释的抗绵羊抗体异硫氰酸荧光素偶联物(抗绵羊IgFITC);
试剂D:1mg(w/v)的链霉蛋白酶溶于PBS。
培养三天后,幽门螺旋杆菌(5个不同菌株),金黄色葡萄球菌(ATCC29213)和链球菌(Raf M87)分别用含有1%BSA的PBS悬浮。分别滴加每种悬浮液20μl到载玻片上。干燥后用中性甲醛液固定2分钟。水洗载玻片6次后室温下晾干。与对照相比,用试剂D简单处理后可增加检测的信噪比。载玻片依次在30μl的试剂A,B和C中处理30分钟,两次处理之间均用PBS洗。晾干载玻片后在加盖玻片之前滴一滴Slow FadeTM。所有培养均在室温下进行。不加一抗的步骤作为阴性对照。
结果表明,幽门螺旋杆菌可被噬菌体抗体染色。这与培养实验中菌落形成单位的结果是十分一致的。因此,噬菌体B8的生物学作用的前提条件是细菌表面表达了特异性的抗原。
生物材料的保藏
下列杂交瘤已于1995年12月15日按照布达佩斯条约在欧洲动物细胞保藏中心(ECACC)(Salisbury,Wiltshire,英国)进行了保藏。
2H6(ECACC No.95121526)
5D8(ECACC No.95121527)
5F8(ECACC No.95121524)
重组噬菌体B8已于1995年12月20日按照布达佩斯条约在国家工业和海洋细菌保藏中心(NCIMB)(阿伯丁,苏格兰)进行了保藏。编号为NCIMB40779。
表1
无噬菌体 | 加噬菌体B8 | |
CFU/ml | ||
时间0 | 5×104 | 4×104 |
24小时 | 1.9×107 | 8×103 |
48小时 | 1.75×108 | 9.5×103 |
72小时 | 4×108 | 3×106 |
表2
幽门螺旋杆菌 | 金黄色葡萄球菌 | 大肠杆菌 | ||||
17874 | 1139 | 224 | ||||
CFU/ml | ||||||
无噬菌体 | 0小时 | 2.1×104 | 4.7×104 | 104 | 7.8×107 | 2.6×106 |
24小时 | 5.8×106 | 1.5×107 | 3.1×105 | 1010 | 4.4×108 | |
噬菌体B8 | 0小时 | 2.8×104 | 4.3×104 | 104 | 2.1×108 | 2.6×106 |
24小时 | 4×102 | 102 | 103 | 8×109 | 4.2×108 | |
噬菌体M13K07 | 0小时 | 3.9×104 | 未检测 | |||
24小时 | 4×106 | 未检测 |
表3
无噬菌体 | 加噬菌体B8 | |||
0小时 | 24小时 | 0小时 | 24小时 | |
17874 | 1.5×104 | 5×106 | 1.3×104 | 2×102 |
1139 | 104 | 7×106 | 8×103 | 1.1×103 |
253 | 1.1×104 | 1.7×107 | 1.3×104 | 4×105 |
25 | 1.4×104 | 9×107 | 7×103 | 3×102 |
66 | 1.4×104 | 2.1×107 | 1.4×104 | 4×104 |
链球菌 | 2.3×107 | 5.6×107 | 1.9×107 | 5.9×107 |
Claims (4)
1.修饰过的噬菌体用于制备治疗或预防由幽门螺旋杆菌引起的粘膜细菌感染的药物的用途,其中修饰的噬菌体是表面存在着包含下述成分的重组蛋白的M13噬菌体:
(i)来自噬菌体表面蛋白的组分1;
(ii)一种包含提供细菌抗原结合位点的抗体可变区序列的组分2,该组分2使得所述噬菌体能结合到细菌细胞表面,从而抑制参与所述细菌感染的病因的细菌细胞的生长;
其中,所述重组蛋白的组分1是来源于未经修饰的噬菌体中负责与细菌菌毛吸附的蛋白质,其由g3p蛋白衍生而来;
所述重组蛋白质的组分2包含有呈g3p-ScFv融合蛋白形式的ScFv多肽;
所述重组多肽中抗体可变区序列是选自杂交瘤细胞系5F8(ECACC No.9512524),2H6(ECACC No.95121526)和5D8(ECACCNo.95121527)的单克隆抗体的单克隆抗体可变区序列。
2.权利要求1中的噬菌体的用途,所述噬菌体称为B8,保藏在NCIMB,保藏号为NCIMB40779,或者其保留了结合和感染幽门螺旋杆菌能力的衍生株。
3.选自5F8(ECACC No.95121524),2H6(No.95121526)和5D8(ECACC No.95121527)的杂交瘤。
4.一种选自权利要求3的杂交瘤中所产生的单克隆抗体的单克隆抗体。
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CN97192116A Expired - Fee Related CN1125174C (zh) | 1996-02-06 | 1997-02-05 | 重组噬菌体 |
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Country | Link |
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EP (1) | EP0889955B1 (zh) |
JP (1) | JP4015195B2 (zh) |
KR (1) | KR100459638B1 (zh) |
CN (1) | CN1125174C (zh) |
AT (1) | ATE266091T1 (zh) |
AU (1) | AU712767B2 (zh) |
CA (1) | CA2244792A1 (zh) |
DE (1) | DE69728975T2 (zh) |
ES (1) | ES2221033T3 (zh) |
HU (1) | HUP9901242A3 (zh) |
IL (2) | IL125645A0 (zh) |
NO (1) | NO323359B1 (zh) |
NZ (1) | NZ331580A (zh) |
RU (1) | RU2215032C2 (zh) |
SE (1) | SE506771C2 (zh) |
WO (1) | WO1997029185A1 (zh) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002522029A (ja) | 1998-07-27 | 2002-07-23 | ジェネンテック・インコーポレーテッド | コートタンパク質の改変によるファージ提示における改良した形質転換効率 |
GB9908195D0 (en) * | 1999-04-09 | 1999-06-02 | Microbiological Res Authority | Treatment of intracellular infection |
JP2003502304A (ja) | 1999-06-14 | 2003-01-21 | ジェネンテック・インコーポレーテッド | ターンライブラリをファージ上に表示するための構造化ペプチド骨格 |
WO2002007742A2 (en) | 2000-07-25 | 2002-01-31 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Bacteriophage having multiple host range |
US6914123B2 (en) | 2001-04-17 | 2005-07-05 | Genentech, Inc. | Hairpin peptides with a novel structural motif and methods relating thereto |
KR20040036176A (ko) * | 2002-10-23 | 2004-04-30 | (주)엘피스바이오텍 | 단백질의 특이적 결합을 매개로 한 박테리오파아지의선택적 숙주감염을 이용한 단백질간의 상호작용을분석하는 방법 |
CN102296051B (zh) * | 2011-03-07 | 2014-09-17 | 江苏省农业科学院 | 一种宽宿主谱鸡白痢沙门氏菌噬菌体及其应用 |
RU2503716C1 (ru) * | 2012-09-27 | 2014-01-10 | Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) | ВИДОСПЕЦИФИЧЕСКИЙ ВИРУЛЕНТНЫЙ ШТАММ БАКТЕРИОФАГА, ОБЛАДАЮЩИЙ ЛИТИЧЕСКОЙ АКТИВНОСТЬЮ В ОТНОШЕНИИ Staphylococcus aureus, ВКЛЮЧАЯ МУЛЬТИРЕЗИСТЕНТНЫЕ ШТАММЫ |
GB201308745D0 (en) * | 2013-05-15 | 2013-06-26 | Imp Innovations | Bacteriophage |
CN106198523A (zh) * | 2016-07-06 | 2016-12-07 | 哈尔滨贝贝凯尔科技发展有限公司 | 微量元素检测试纸及其制备方法 |
KR20210027230A (ko) | 2017-10-04 | 2021-03-10 | 옵코 파마슈티칼스, 엘엘씨 | 암의 개인 맞춤형 치료에 관한 물품 및 방법 |
CN112739416A (zh) * | 2018-12-27 | 2021-04-30 | 国立大学法人东海国立大学机构 | 宿主细菌特异性纳米粒子 |
CN109679981A (zh) * | 2019-02-21 | 2019-04-26 | 武汉大学 | 一种甲酰基肽定向进化噬菌体及其制备方法与应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992001047A1 (en) * | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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SE9304060D0 (sv) * | 1993-12-06 | 1993-12-06 | Bioinvent Int Ab | Sätt att selektera specifika bakteriofager |
-
1996
- 1996-02-06 SE SE9600434A patent/SE506771C2/sv not_active IP Right Cessation
-
1997
- 1997-02-05 WO PCT/SE1997/000172 patent/WO1997029185A1/en active IP Right Grant
- 1997-02-05 AU AU16817/97A patent/AU712767B2/en not_active Ceased
- 1997-02-05 HU HU9901242A patent/HUP9901242A3/hu unknown
- 1997-02-05 IL IL12564597A patent/IL125645A0/xx active IP Right Grant
- 1997-02-05 KR KR10-1998-0705761A patent/KR100459638B1/ko not_active IP Right Cessation
- 1997-02-05 DE DE69728975T patent/DE69728975T2/de not_active Expired - Fee Related
- 1997-02-05 JP JP52844697A patent/JP4015195B2/ja not_active Expired - Fee Related
- 1997-02-05 EP EP97902815A patent/EP0889955B1/en not_active Expired - Lifetime
- 1997-02-05 RU RU98116471/13A patent/RU2215032C2/ru not_active IP Right Cessation
- 1997-02-05 NZ NZ331580A patent/NZ331580A/en unknown
- 1997-02-05 AT AT97902815T patent/ATE266091T1/de not_active IP Right Cessation
- 1997-02-05 CN CN97192116A patent/CN1125174C/zh not_active Expired - Fee Related
- 1997-02-05 ES ES97902815T patent/ES2221033T3/es not_active Expired - Lifetime
- 1997-02-05 CA CA002244792A patent/CA2244792A1/en not_active Abandoned
-
1998
- 1998-07-27 NO NO19983456A patent/NO323359B1/no unknown
- 1998-08-03 IL IL125645A patent/IL125645A/en not_active IP Right Cessation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992001047A1 (en) * | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
Also Published As
Publication number | Publication date |
---|---|
NO983456L (no) | 1998-10-06 |
JP4015195B2 (ja) | 2007-11-28 |
IL125645A (en) | 2006-08-20 |
RU2215032C2 (ru) | 2003-10-27 |
AU1681797A (en) | 1997-08-28 |
NO323359B1 (no) | 2007-04-10 |
HUP9901242A2 (hu) | 1999-07-28 |
EP0889955A1 (en) | 1999-01-13 |
NZ331580A (en) | 1999-09-29 |
DE69728975D1 (de) | 2004-06-09 |
NO983456D0 (no) | 1998-07-27 |
CN1210558A (zh) | 1999-03-10 |
HUP9901242A3 (en) | 2000-03-28 |
SE9600434D0 (sv) | 1996-02-06 |
ES2221033T3 (es) | 2004-12-16 |
DE69728975T2 (de) | 2005-05-04 |
WO1997029185A1 (en) | 1997-08-14 |
JP2000505648A (ja) | 2000-05-16 |
AU712767B2 (en) | 1999-11-18 |
CA2244792A1 (en) | 1997-08-14 |
ATE266091T1 (de) | 2004-05-15 |
KR100459638B1 (ko) | 2005-02-24 |
EP0889955B1 (en) | 2004-05-06 |
SE506771C2 (sv) | 1998-02-09 |
KR19990082041A (ko) | 1999-11-15 |
SE9600434L (sv) | 1997-08-07 |
IL125645A0 (en) | 1999-04-11 |
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