CN1479788A - 能够减小个体发展变态反应的倾向的乳酸菌 - Google Patents
能够减小个体发展变态反应的倾向的乳酸菌 Download PDFInfo
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Abstract
本发明涉及能够减小个体发展变态反应的倾向的新乳酸菌株。特别是,本发明涉及乳酸菌株的产生及其在减小个体发展变态反应的倾向中的用途,其中所述乳酸菌株表达含有耐受原肽的蛋白。本发明还涉及含所述微生物或其活性部分的食物或药物组合物。
Description
本发明涉及能够减小个体发展变态反应的倾向的新乳酸菌株。特别是,本发明涉及表达多肽的新乳酸菌株及其在减小个体发展变态反应的倾向中的用途,其中所述多肽含有耐受原肽。本发明还涉及含所述微生物或其活性部分的食物或药物组合物。
变态反应是免疫系统对各种物质(变应原)的不良反应。通常,个体对在环境中经常遇到的物质,如花粉或食物材料不能产生有价值的免疫反应,相信这种非-反应性主要是由于免疫系统自身的抑制机理。然而,在受损的情况下,免疫系统不能发挥其抑制活性(也被称为耐受性),产生对变应原的特异性免疫反应即变态反应。变态反应包括生物活性化合物,如炎性介质组胺,白三烯和酶从特定细胞,主要是肥大细胞和嗜碱性粒细胞到周围组织和血管结构中的释放。肥大细胞分布在个体的各处组织中,同时在血管系统内嗜碱性循环。在涉及IgE抗体的一系列事件中,所述化合物/介质是从在药理学反应中死亡的靶肥大细胞中释放的。
在过去,经历变态反应的个体数增加,其经常被归因于日益增长的,由废气导致的大气污染。且,据信蛋白质材料的持续消耗可促进所述发展,特别是可促进食物过敏事件的增长。此外,在发达国家遇到的微生物感染缺陷已经被暗示为特应性疾病增加的另一个可能性原因。
食物过敏,甚至是某些种类食物的不耐受已经变得十分普遍。人们发现绝大多数受影响的个体都对食物的某些成分不耐受,大约5%的群体甚至发展成对已摄取物质的反应,这种反应十分严重,因而须引起医生的注意。
近几年的证据已经确定了食物不耐受的主要原因在于机体吸收的物质常常被排斥,并被敏感个体的胃肠道吸收。据信这种不良-吸收源于排列在消化道中的组织的缺损(Peters等,(1988)Can,J.of Gastroenterol.2,127;Olaison等,(1990)Scand.J.of Gastroenterol.25,321;Hollander等,(1986)Ann.Of Int.Med.105,883)。最近确定的,排列于胃肠道的上皮细胞处理和描述抗原的能力也可解释导致炎症和变态反应的错误免疫反应的引发(Campbell等,(1999)Immunol.Rev.172,315)。
食物过敏的表现基本上可涉及机体中的任何组织。由于胃肠道较大的吸收面积,其很可能是攻击性物质的主要吸收位点。食物过敏的很多症状均是在消化道本身中表现的,但也可影响其它组织,包括皮肤和呼吸道。
在现有技术中,提出过使用不同的方法治疗变态反应,特别是食物过敏。
这种方法其中一个旨在改变变应原材料自身的来源,从而减小它的变应潜在性。这可通过分别限制或禁止食物或其组分而达到,而所述食物或其组分可能正是这种问题的原因。通常,所涉及的问题在于对各种食物材料中的特异性抗原物质(变应原)不太了解,因此在大多数情况下,不清楚应当选择性地除去哪个组分。
在US-5,480,660中报道过小麦过敏患者的变态反应发作可通过除去或减少面粉中,具有至多30,000 Da分子量的蛋白和50,000-70,000 Da分子量的蛋白而缓解。
JP 11 04 67 21中公开了一种减少含蛋白食物材料的变应原性的方法,其中所述食物材料与小麦粉混合,180℃烘焙该混合物超过3分钟。
US-4,293,571中描述了低变应原性组合物的制备,其中使蛋白质材料经过水解处理,剩余未水解的蛋白通过热处理而凝固,接着通过超滤步骤除去凝固的物质和可能构成变应原的巨肽。
US-5,039,532中公开了另一种制备低变应原食物材料的方法,其中使乳清产物经过酶水解。
然而,所有这些处理食物材料甚或将它从日常饮食中除去的方法最终证明难以实践,这是因为它们需要更改饮食,通常包括严格限量,而且这些方法很可能最终影响生活的质量和/或抑制个体预期的生长。
治疗食物过敏和食物不耐受的不同方法旨在恢复并维持肠的完整性,这样食物变应原基本上就不会通过。在这方面,US5,192,750描述了利用N-乙酰基葡糖胺使粘膜形成防止食物变应原传递所必需的屏障,并维持正常的功能。
根据另一个治疗变态反应的方法,建议个体相对于IgE分子进行接种,其可抑制肥大细胞和嗜碱细胞的引发。为此,WO97/31948建议接种特异性肽,所述特异性肽与IgE分子,即涉及介质释放的免疫球蛋白的三维构象部分类似,其中所述介质涉及变态反应和炎性反应的调节。人们认为个体自身的免疫系统将最终形成针对所述IgE分子的抗体,这样就可清除所述的IgE免疫球蛋白。然而,所述方法掩盖了所形成的抗体可能与其它免疫球蛋白种类交叉反应的缺点,这样会不利地影响所述个体的天然防御机理。
在本申请人所拥有的,至今仍未公开的专利申请EP 99200130.5中公开了另一种方法。其中提出一种低变应原性的组合物,其含有一种非-变应原性的,大量水解的蛋白材料和/或一种游离氨基酸,及至少一种各变应原蛋白的耐受原肽。虽然这种组合物可比现有技术提供更多的优点,但它仍难以获得耐受原肽,其每批不得不重新生产。
因此,现有技术需要提供治疗变态反应的改进方法。
因此,本发明的其中一个目的在于提供这样一种方法。
上述目的已经通过提供新的乳酸菌株而得到解决,所述乳酸菌株能够减小个体发展变态反应的倾向。所述新乳酸菌可表达一种含有耐受原肽的多肽,这样就可保留耐受原肽的特异性序列及可能的构型,保持稳定性,并因此可由个体的免疫系统处理并识别。
在附图中,
图1表示包含在β-乳球蛋白水解产物各部分中的肽的一级序列;直条代表二硫键;
图2表示在各实验中,使用耐受原肽/肽部分得到的结果。
乳酸菌,特别是乳杆菌和双岐杆菌已经显示可通过产生涉及免疫系统调节的信使(细胞因子)而调节免疫系统,且不会离开它们的腔栖息地。乳酸菌不能引发促炎细胞因子,如IL-8,TNF-α,MCP-1和GM-CSF的产生,但可促进炎症抑制细胞因子,如TGF-β的产生(Blum等,(1999)Antonie van Leeuwenhoek 76,199),其明显暗示了有关肠免疫内环境稳定和上皮屏障完整性的维持(Planchon等,(1999)J.Cell Physiol.181,55)。同样,乳杆菌匀浆的口服给药显示出对促细胞分裂剂-诱导的单核细胞增殖的抑制作用(Pessi等,(1999)Appl.Environ.Microbiol.65,4725)。
乳酸菌(LAB)可用于蛋白和肽的异源(超量-)产生,所述蛋白和肽具有允许基于染色体整合的外源基因或附加型质粒因子的表达的系统(见,例如Kuipers等,(1997)Tibtech 15,140)。此外,乳酸菌具有定居粘膜表面的潜力,并显示出一种内源性的佐剂活性,这种内源性的佐剂活性与肽聚糖降解所产生的胞壁酰肽有关。
此外,已经显示出某些乳杆菌菌株可促进抗原-特异性的免疫应答,特别是在IgA类(Majamaa等,(1995)J.Pediatr.Gastroenterol.Nutr.20,333)中。与抗原复合的人IgA不能激活补体,这对于粘膜表面完整性的维持比较重要。其可防止局部炎性反应,包括由于组织受损,粘膜渗透性提高导致的白细胞的流入和免疫效应子的释放。此外,通过这种免疫排除机理,IgA可限制旁观抗原的进一步吸收,并因此消除IgE和IgG同种型抗体介导的过敏反应。
此外,乳杆菌已经显示可通过分离的T细胞加强IFN-γ的产生(Halpern等,(1991)Int.J.Immunother.7,205)。最近,IFN-γ已经显示可抑制肠粘膜肥大细胞的TNF-α释放(Bissonnette等,(1995)Int.Arch.Allergy Immunol.107,156)。IFN-γ分泌的增加可防止TNF-α的有害作用(Hernandez-Pando等,(1994)Immunology 82,591),并因此消除与食物过敏有关的渗透性障碍。例如,变应性皮炎中,相对于抗原的摄取,肠屏障受损是对普通饮食和环境变应原的免疫应答增强的重要决定因素。
在通向本发明的广泛研究过程中,本发明人已经为此发明了将乳酸菌的有利特性与耐受原肽的潜在性结合,产生减小甚或使个体的变态反应倾向达到最小的方法。
本发明所用的乳酸菌株优选属于乳杆菌属或双岐杆菌属或乳球菌属,更优选嗜酸乳杆菌,约氏乳杆菌,加氏乳杆菌,干酪乳杆菌,类干酪乳杆菌,路氏乳杆菌,它们全部来源于人或动物源。
耐受原肽,其是一种来源于可引起个体变态反应的已知蛋白的肽,通常具有约200-6000 Da的大小,而且它是一种至少能够诱导对蛋白耐受性的肽,而其正来源于该蛋白。此外,除了由特异性耐受原肽所表示的耐受性外,还可诱导对具有相同抗原决定子的任何物质的耐受性。根据本发明,这种肽是通过重组方法插入到在乳酸菌内合成的多肽中的,多肽对于细菌可以是内源性的或外源性的。所述耐受原肽是以这种方式插入的,即它存在于所得重组多肽的周围,这样就可被免疫系统识别。
根据本领域熟知的方法,可在乳酸菌中表达所述多肽。例如,市售载体pNZ124(Platteuw等,(1994)Appl.Env.Microbiol.60,587),pGK12(Walke等,(1996)FEMS Microbiol.138,233)或pG+host9(Maguin等,(1996)J.Bacteriol 178,931)可用于附加型表达。然而,考虑到染色体整合较高的稳定性,这种做法优选用于编码各种多肽的重组基因。为了整合到染色体中,可应用同源重组,例如使用来源于乳酸菌的,含有耐受原肽并替换内源性基因的重组基因。然而,将重组基因引入到宿主染色体中的方法对于本领域技术人员是熟知的。
所述多肽可以是通常被表达为在细胞内停留的蛋白,但也可以是一种由乳酸菌分泌的多肽,或被插入到其细胞壁中的多肽,其具有含耐受原肽的部分,所述耐受原肽位于细胞外区域。本发明所用多肽的例子是pepN,pepX,乳酸脱氢酶或β-半乳糖苷酶,或bacteriosins类的成员或S-层蛋白或细胞壁固着蛋白酶。
多肽的性质并不重要,只要其中可插入耐受原肽,这样它就可进入免疫系统。然而,一旦已经鉴定了适宜的耐受原肽,那么本领域的普通技术人员就可在这个位置排列耐受原肽。为此,技术人员将考虑其中插入耐受原肽的多肽三维模型,并以这种信息为基础设计一种相应的重组蛋白。
在优选实施方案中,重组多肽是在细菌细胞壁中分泌或排列的,这样耐受原肽可持续存在于环境中。
所述耐受原肽来源于引起变态反应的食物材料。然而,它的氨基酸序列是否与在天然蛋白中发现的氨基酸序列相同并不重要,但具有基本相同的氨基酸序列的任何肽序列,和可能作为起始肽的三维构象,或从其衍生的,显示基本相同的结果,即,导致变态反应减少的多肽均适用于已知的目的。
作为耐受原肽的优选来源,可提到牛奶,大豆,花生,甲壳类动物,鱼,肉,芝麻或乳清,如β-乳球蛋白,牛血清白蛋白或酪蛋白。
为了鉴定耐受原肽,首先将目标变应原材料酶水解至约10-50%的程度,并通过热处理将蛋白混合物中的残留酶活性灭活。澄清所述蛋白水解液,如果需要,再进一步纯化。然后通过使溶液经过沉淀处理或使它通过色谱柱而分离由此获得的肽。
随后,通过在动物模型中进行试验而测定含不同肽部分的耐受原性。为此,给动物,如小鼠喂饲各种肽部分,接着利用所述变应原使其免疫,测量其反应,如外观的改变,或具体测量动物的IgE水平。结果,耐受原肽部分会减小动物的变态反应倾向,这样即可测定各种肽(部分)。
一旦耐受原肽及其氨基酸序列已经被证实,在乳酸菌中表达的多肽DNA-序列即通过基因工程而被插入到编码细菌蛋白载体的基因中。操纵微生物中DNA序列和表达蛋白的方法和技术在Sambrook等,ALaboratory Manual Cold Spring Harbor(1992)中描述。
本发明还涉及一种含有至少一种这种重组乳酸菌的食物和药物组合物。
所述菌株可以105-1012cfu(菌落形成单位)/g的量包含在组合物中。同样,乳酸菌培养物的上清液或其活性部分也可包含在所述组合物中。
所述食物组合物可以是牛奶,酸乳酪,凝乳,干酪,发酵牛奶,基于牛奶的发酵产品,冰淇淋,基于发酵谷物的产品,奶粉,婴儿代乳品或动物食品,如宠物食品。
所述药物组合物还可以是片剂,液体细菌混悬液或含细菌的一部分的制剂,如仅通过裂解细菌细胞并收集细胞壁成分获得的细胞壁部分,干燥的口服添加物,湿润的口服添加物,干燥的管式饲料或湿润的管式饲料。
药物制剂的给药途径是口服,但也可鼻饲。
本发明还涉及含本发明细菌或其一部分的疫苗。所述细菌可以是活的形式,或减弱的形式,即如果需要,减弱它们的生长潜力。可主要使用作为细胞一部分的,掩盖目标抗原的细胞壁。所述细胞壁可通过溶解细胞并分离其不同的成分而很容易地获得。
下列实施例用于进一步举例说明本发明,而不是对它的限制。
实施例1
耐受原肽的分离
a)肽的分离
将220gβ-乳球蛋白(Sigma)溶解在5%(w/w)浓度的双蒸馏水中。以1/100(w/w)的酶/底物(E/S)比,将TPCK处理过的胰蛋白酶加入到所得溶液中,并在40℃,pH7.6时,在不断搅拌下培养该溶液。1小时后,加入等量的酶,得到2/100的最终(E/S)比。继续培养4小时,并通过热处理(85℃,5分钟)使酶灭活,从而终止反应。随后冷冻干燥整个胰蛋白酶水解物,并通过阳离子树脂的制备型色谱分离所得的肽产物。
得到15个不同的肽部分,每个肽部分约有2-23个氨基酸(约240-2720 Da)。将各部分纳米过滤,膜渗滤,透析,再次冷冻干燥,并在室温干燥贮存直至使用。所述部分还可利用反相高效液相色谱(HPLC)描述它们的肽含量。收集全部15个部分(F1-F15),并列出所含的主要的肽(T)。
部分 | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 | F10 | F11 | F12 | F13 | F14 | F15 |
质量(g) | 52 | 13.9 | 11 | 11.8 | 2.57 | 1.94 | 4.37 | 13.1 | 5.01 | 6.53 | 1.05 | 3.46 | 1.9 | 2.67 | 28.8 |
富含的肽 | - | T6T17T18 | T23 | T23 | - | T7T9 | T20T21T24 | T9T12T24 | T12T21 | T10T21 | T11 | T10 | T10T11 | T10T11 | - |
将肽分离并测序。其序列在图1中表示。在不同的实验中,各种肽均被证实是耐受原性的,其分别在Recquet R,Bovetto L,Maynard F,FritschéR中详细描述,通过牛β-乳球蛋白的胰蛋白酶水解获得的肽可诱导小鼠特异性的口服耐受性,J.Allergy Clin Immunol(2000)105:514-21,其引入此处作为参考。
b)耐受原肽的合成
在上述参考文献中鉴定的三种β-乳球蛋白(BLG)的耐受原肽T6,T17和T6/T17和对照肽T13是利用BLG序列的侧翼残基,和外侧的Cys残基化学合成的,如下所列:T6 C F K
I D A L N E N K V S R
84 91T17 C N K
V L V L D T D Y K K Y S92 100T6/T17 C F K
I D A L N E N K V L V L D T D Y K K Y S84 100T13
L I V T Q T M K S R C1 8
c)动物试验
为了评估各种肽的耐受原性,使用Balb/c小鼠,这些小鼠是从IFFA-Crédo(L’Abresle France)获得的。所述动物全部是用没有牛奶的饮食饲养的。实验开始时,所述动物3周大。它们通过管饲法接受天然的β-乳球蛋白(5mg/g体重),各种量的消化β-乳球蛋白和各种量的,在上述a)中获得的不同肽。5天后,利用作为非相关抗原的卵清蛋白(V级,Sigma)和β-乳球蛋白,使接受这种饮食的所有小鼠免疫,以便评估免疫应答的特异性。延迟型过敏性的评估(DTH)是在系统攻击后21天,通过比较免疫前后左后足垫的厚度进行的。免疫24小时后,测量动物的足垫厚度(以Δ厚度(mm)表示)。随后,采集所有小鼠的血液,接着取得脾脏,按照治疗组将它们合并。对各组进行脾细胞特异性增殖测定。单独收集肠内含物。在-80℃时,单独冷冻血清和肠样品,直到进行各自的测定。测定血清和肠样品中的抗-β-乳球蛋白IgE和抗-卵清蛋白IgE的水平。
d)IgE抗体测定
稀释血清和肠流体,一式两份,利用ELISA测定抗-β-乳球蛋白和抗-卵清蛋白的IgE抗体。将20只未免疫小鼠的合并样品用作阴性对照。滴定度是通过计算样品的稀释度测定的,其产生两倍于阴性对照组的吸收度。所述滴定度是以稀释度倒数的log10表示的。
e)细胞培养物
在PBS中匀化脾细胞溶液并纯化。在β-乳球蛋白或植物凝集素A存在的情况下协同培养。在培养的最后6小时,加入含氚的胸腺嘧啶核苷([3H]-Thy(Amersham,Zürich),收集平板并通过闪烁计数分析。刺激指数是根据空白-扣除试验和对照水平的比计算的,所述对照水平是以通过三重培养掺入的[3H]-Thy的平均cpm表示的。
f)通过口服给予合成肽诱导的耐受性
在非肠道小鼠模型中,分析所述合成肽的耐受原能力。所得结果如下:
IgE滴定度(log)(试验/对照) | 脾淋巴细胞SI(试验/对照) | MLN淋巴细胞SI(试验/对照) | |
T-6 | 3.24/3.39 | 0.53 | 0.29 |
T-17 | 2.67/3.39 | 0.31 | 0.41 |
T-6/T-17 | 3.40/3.39 | 0.92 | 0.81 |
T-13 | 3.20/3.39 | 1.70 | 0.87 |
SI:刺激指数 MLN:肠系膜淋巴结
从上述可知,合成肽T-17既可减少IgE的产生又可减少T细胞的增殖,而肽T-6仅抑制T细胞的应答。相反,肽T-13,即对照肽,不能诱导对BLG的口服耐受性。
实施例2
重组多肽的构建
用细胞表面固着蛋白酶融合在实施例1中鉴定的,显示出耐受原性的肽T6和T17,使其在细菌表面显示。作为阴性对照肽T13,可使用β-乳球蛋白的N-末端构成部分。
所述细胞表面固着蛋白酶(上述)属于保加利亚乳杆菌,其序列在Gilbert等,(1996)J.Bacteriol,178,3059-3065中公开。此蛋白是由2000个氨基酸蛋白表示的,包括负责酶的细胞输出的33个氨基酸前导肽(前区),和一系列154个氨基酸的肽(前-区),其在裂解后可激活酶的蛋白分解活性,及活性位点的700-800个氨基酸。接下来的区(大约1000个氨基酸)可在所产生肽的细胞的裂解和转运特异性中起作用,并可跨越细胞壁。所述蛋白酶是通过其羧基末端与最后200个氨基酸细胞壁固着的,所述最后200个氨基酸负责与细胞壁多聚糖结构的特异性共价结合。
所述蛋白酶基因是通过使用下列两种引物,利用其启动子首先扩增的:
5’-TTTTGTGGATCCTTAACTTCATAGCACG-3’
(基因启动子的上游,携带BamHI位点)
5’-ATATTATCTAGAATTGAATAGATTGCC-3’
(所述基因的rho-依赖性终止子的下游,携带XbaI位点)
所述扩增产物是用BamHI和XbaI裂解的,并被克隆到乳酸菌载体pNZ124中,其已经用相同的限制酶消化,并最终通过电穿孔而被引入到没有质粒(β-半乳糖苷酶和蛋白酶阴性的)的乳酸乳球菌中。
用目标肽/多肽的序列替换克隆蛋白酶的活性位点区。为了达到目的,所述克隆蛋白酶是用NheI和PvuI裂解的,其中NheI位于前导肽裂解位点序列下游的50bp处,PvuI位于另一下游的800bp处。将编码目标肽的DNA序列插入到两个限制位点,如两个寡核苷酸之间,其被设计为一旦杂交,则在它们的末端产生两个限制位点。寡核苷酸的设计考虑到在与蛋白酶基因连接时,重组蛋白的读框保持开放。
较大多肽的插入是使用含两个限制位点的引物,通过DNA扩增进行的。用限制酶裂解扩增产物。在两种情况下,所述DNA片段与蛋白酶基因连接,并通过电穿孔而被引入到乳酸乳球菌和约氏乳杆菌中。
含肽T6和T17的重组质粒构建体已经按照布达佩斯条约,于2000年9月22日保藏在巴斯德研究所中,保藏号分别为CNCM I-2563和CNCM I-2564。
实施例3
乳酸乳球菌和约氏乳杆菌的转化
为了进行转化,使乳酸乳球菌菌株(MG 1363,没有质粒)和约氏乳杆菌La1(从Institute Pasteru获得,登记号为CNCM I-1225)在37℃的GasPak厌氧系统中的MRS肉汤培养基中过夜生长。将此培养物的等分试样用于接种其它含0.5M蔗糖的肉汤培养基(MRS)。以2%再次接种到200ml MRS+0.5M蔗糖中后,使所述培养物生长至OD595等于0.6。通过4℃时,以5000rpm离心10分钟来收集细胞,用1/2体积含1M蔗糖和2.5mM CaCl2的溶液洗一次,用1/4体积含1M蔗糖和2.5mM CaCl2的溶液洗一次,将离心后获得的颗粒在3.5ml的1M蔗糖,2.5mMCaCl2+0.459ml 87%甘油的溶液(最终浓度为10%)中重悬浮。所述细胞可直接用于转化或在-80℃冷冻。
为了进行电穿孔,将40μl细胞与10-100ng(<5μl体积)的DNA混合,并转移到冰冷的0.2cm电穿孔杯中。在冰冻的0.2cm电穿孔杯中应用200Ω,25μF,2.5kV的脉冲。将1ml的MRS+20mM MgCl2,2mMCaCl2加入到杯中,并在适宜的温度(对于乳酸乳球菌为30℃,对于约氏乳杆菌为37℃,当使用ts质粒(pG+host9)时,培养温度为30℃)培养所述混悬液2-3小时。在质粒DNA转化之后,分别将10μl和100μl等分试样平板接种在含适宜抗生素的MRS琼脂平板上,并在利用连接混合物转化之后,分别将100μl和500μl平板接种在含适宜抗生素的MRS琼脂平板上。所述平板在与上述相同的温度下,厌氧培养24-48小时。
作为一种选择性的介质,也可使用具有红霉素的MRS(2.5μg/ml)或具有卡那霉素的MRS(5μg/ml)。
实施例4
相对于耐受原性BLG肽产生的大鼠抗血清和单克隆抗体
将所述BLG肽T6,T17,T6/T17和T13(在实施例1中描述)与马来酰亚胺-激活的KLH偶联,并与Titermax佐剂一起,使用没有乳蛋白的饮食喂饲的大鼠免疫。在第一次免疫之后,以2周的时间间隔采集血液样品,如此一直进行115天,通过ELISA显示所采集的血液样品含有对BLG肽特异性的抗体。
抗-T6和抗-T13抗血清还与BLG的天然(ELISA)或变性(SDS-PAGE+蛋白质印迹)形式反应。抗-T6和抗-T13可特异性识别在重组乳酸乳球菌表面表达的BLG肽。结果在表I中显示。
表I
抗β-乳球蛋白肽抗血清的反应性
抗血清特异性 | T6 | T17 | T6/T17 | T13 | ||||||||
大鼠数 | A1 | A2 | A3 | B4 | B5 | B6 | C7 | C8 | C9 | D10 | D11 | D12 |
T6(ELISA) | +++ | +/- | + | - | NT | NT | - | NT | NT | - | NT | NT |
T17(ELISA) | - | NT | NT | + | - | - | +++ | NT | NT | - | NT | NT |
T6/17(ELISA) | +++ | NT | NT | + | NT | NT | +++ | + | - | - | NT | NT |
T13(ELISA) | - | NT | NT | - | NT | NT | - | NT | NT | + | + | +/- |
Nat.BLG(ELISA) | +++ | +/- | +++ | +/- | - | - | - | - | - | + | + | +/- |
Den.BLG(SDSPAGE+WB) | +++ | NT | ++ | - | NT | NT | - | NT | NT | +/- | +++ | ++ |
LI/T6(ELISA) | +/- | - | +++ | - | NT | NT | - | - | - | - | NT | NT |
LI/T17(ELISA) | - | NT | NT | + | - | - | - | - | - | - | NT | NT |
LI/T13(ELISA) | - | NT | NT | - | NT | NT | - | NT | NT | - | - | - |
ELISA/OD490nm(15’/37℃)
+++ >0.3
++ 0.2-0.3
+ 0.1-0.1
+/- 0.05-0.1
- <0.1
NT 没有检测到
nat.BLG 3x溶解在PBS中的结晶BLG(Sigma)
den.BLG 3x在SDS样品缓冲液中稀释并煮沸的结晶BLG(Sigma)
LI/T 具有插入BLG肽的保加利亚乳杆菌蛋白酶的重组乳酸乳球菌
为了产生杂交瘤分泌抗-BLG肽单克隆抗体,获得免疫大鼠的脾脏。按照标准技术用骨髓瘤细胞融合所述脾细胞,且第一轮筛选已经可鉴定产生抗-T6或抗-T17抗体的细胞群。这些细胞的克隆是使用细胞分选器进行的,从而确保每个孔中存在1个单一的细胞。
实施例5
利用抗体检测耐受原肽
为了进行测定,在某种意义上,不论所述乳酸菌是否表达耐受原肽,从而接近或被抗体识别,含重组基因的细菌都是在50ml培养基中生长的,并通过离心收集细胞。随后用50ml TBS洗该细胞两次,并最终在6ml TBS中悬浮该细胞。将75μl混悬液转移到孔中(半孔平底ELISA平板),并在37℃时,使平板在没有盖盖子的情况下保持24小时,从而使孔干燥。用TBS/PBS洗该平板3次,直到洗出未结合的细菌,随后在37℃时,用TBS-酪蛋白(1.5g/l)对细胞进行封闭处理。向孔中加入含针对特异性肽的抗体的大鼠抗血清。使用含0.1%吐温20的TBS-酪蛋白稀释液室温过夜培养该孔。接着,用TBS洗该孔3次,并加入发育抗体(HRP-结合的山羊抗-大鼠IgG),随后在37℃时,在含0.2%吐温20的TBS-酪蛋白稀释液中培养。用TBS洗该孔3次,并用OPD/H2O2展开,在490nm处阅读。
我们观察到乳酸乳球菌和约氏乳杆菌表现出一种阳性反应,表明耐受原肽可被抗体识别。
实施例6
兔子抗血清的制剂涉及保加利亚乳杆菌prtB蛋白酶的氨基酸残基472-659。
编码氨基酸残基472-659(氨基酸数1是起始甲硫氨酸)的prtB靶序列(bp 1414-1977)是使用pMD114作为模板,和
序列的上游引物(prtB5’-1414)
5‘-GCGGATCCGGCTTGGGCGGTGCAGATG-3’(含5’-BamHI位点),及
序列的下游引物(prtB5’-1977)
5’-CGCAAGCTTGTGCGAAGTGTTCATGGC-3’(含5’-HindIII位点)通过PCR扩增的。
所述特异性PCR产物是使用Taq DNA聚合酶(Perkin-Elmer),利用3次循环得到的,所述3次循环包括95℃时进行变性步骤30秒,56℃时进行引物退火步骤30秒,72℃时进行延伸步骤45秒,接着是进行退火步骤至60℃,循环27次。
在脱盐并除去引物和dNTPs(高纯度的PCR产物纯化试剂盒,Roche)后,用BamHI和HindIII消化该样品,然后经过琼脂糖凝胶电泳,从凝胶上回收目标DNA带(E.Z.N.A.凝胶提取试剂盒,Peqlab)。
所述纯化的扩增产物是在pQE-9大肠杆菌表达载体(Qiagen)的BamHI和HindIII位点之间连接的,其位于编码6-组氨酸标记物的序列下游。
大肠杆菌菌株M15[pREP4]是用这种载体转化的,并在1mM IPTG-介导的培养物的诱导后,获得异源蛋白的表达。
将1升培养物的细胞浓缩,并在变性条件(8M尿素)下溶解。通过离心获得澄清的溶解产物,并将其装到Ni2+-NTA琼脂糖珠(Qiagen)上,其在pH8.0时平衡,从而使6-组氨酸-标记的蛋白结合。pH6.3的清洗步骤后,在pH4.5时洗脱重组蛋白。
用100mM Tris-Cl(pH7.5)中和所述物质,通过二辛可宁酸蛋白测定法(Pierce)量化,并使用相同的缓冲液将其浓度调至1mg/ml。
用完全的Freund’s佐剂乳化所述抗原,并通过多次皮内注射将其给予两只兔子。注射是在第0,14,28和56天进行的。最终的出血发生在第80天。
所述抗血清的反应性,其出人意外的强,这是通过在重组Lc上进行ELISA,其中所述重组Lc在它们的细胞表面表达保加利亚乳杆菌prtB蛋白酶,并通过在表达蛋白酶分泌形式的重组Lc培养物上清液上进行免疫印迹而加以证实的。
申请人或代理人挡案号 | 国际申请号 |
关于微生物保藏的说明
(细则13之二)
A.对说明书第 页,第 行所述的微生物的说明 | |
B.保藏事项 | |
保藏单位名称 | 国立微生物保藏中心 |
保藏单位地址(包括邮政编码和国名)Institue Pasteur,28,rue du Dr Roux,74724 Paris Cedex 15,France | |
保藏日期 2002年9月22日 保藏编号 CNCM I-2563,CNCM I-2564 | |
C.补充说明(必要时) | |
D.本说明是为下列指定国作的(如果说明不是为所有指定国而作的) | |
E.补充说明(必要时) |
Claims (14)
1.一种乳酸菌类菌株,其表达含有耐受原肽的多肽,该耐受原肽与所述多肽是异源的,这样所述肽对于由胃/十二指肠酶所致的降解稳定,并保留了它的耐受原性,且所述肽是在多肽的周围显示的。
2.根据权利要求1所述的菌株,其选自乳杆菌属或双岐杆菌属。
3.根据权利要求1或权利要求2所述的菌株,其中所述多肽选自表面蛋白,细胞内蛋白或由细菌分泌的蛋白。
4.根据前述权利要求任何一项所述的菌株,其中所述耐受原肽得自食物材料。
5.一种含有前述权利要求任何一项所述的菌株或其上清液的食物组合物。
6.根据权利要求5所述的食物组合物,其选自牛奶,酸乳酪,凝乳、干酪,发酵牛奶,基于牛奶的发酵产品,冰淇淋,基于发酵谷物的产品,奶粉,婴儿代乳品或宠物食品。
7.根据权利要求5或6任何一项所述的食物材料,其中所述菌株是以107-1011cgu/剂量的用量包含在其中的。
8.一种含有权利要求1-4任何一项所述的菌株或其上清液的药物组合物。
9.根据权利要求8所述的药物组合物,其中所述菌株是以1010-1012cfu/剂量的用量形式包含在其中的。
10.根据权利要求1-4任何一项所述的菌株或其上清液用于制备治疗变态反应的可摄取载体的用途。
11.根据权利要求1-9任何一项所述的食物或药物组合物用于制备治疗变态反应的可摄取载体的用途。
12.根据权利要求11所述的用途,其中所述组合物经口腔或鼻途径给予。
13.一种含有权利要求1-4任何一项所述的菌株,或其一部分的疫苗。
14.根据权利要求13所述的疫苗,其中使用裂解细菌的细胞膜部分。
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EP1239032A1 (en) * | 2001-03-02 | 2002-09-11 | Société des Produits Nestlé S.A. | Lactic acid bacteria as agents for treating and preventing allergy |
EP1319410A1 (en) * | 2001-12-11 | 2003-06-18 | Société des Produits Nestlé S.A. | Use of micro-organisms for a directed delivery of substances to specific parts of the gut |
WO2004112773A1 (fr) * | 2003-04-24 | 2004-12-29 | Shin-Jen Shiao | Compositions pharmaceutiques utilisees pour le traitement de maladie immune et amelioration |
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KR20070089231A (ko) * | 2004-12-14 | 2007-08-30 | 알크-아벨로 에이/에스 | 이종 단백질성 화합물을 제시하는 박테리아 세포를포함하는 약제학적 조성물 |
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AR052472A1 (es) | 2005-05-31 | 2007-03-21 | Iams Company | Lactobacilos probioticos para felinos |
EP1885383B1 (en) | 2005-05-31 | 2016-09-21 | IAMS Europe B.V. | Feline probiotic bifidobacteria |
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WO2008093303A2 (en) | 2007-02-01 | 2008-08-07 | The Iams Company | Method for decreasing inflammation and stress in a mammal using glucose antimetaboltes, avocado or avocado extracts |
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US9232813B2 (en) * | 2008-07-07 | 2016-01-12 | The Iams Company | Probiotic supplement, process for making, and packaging |
BRPI1007274A2 (pt) * | 2009-01-27 | 2015-08-25 | Arla Foods Amba | Leite e produtos relacionados a leite de longo prazo de validade e um processo e instalação de processamento de leite para sua fabricação. |
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EP3338565A1 (en) * | 2010-06-04 | 2018-06-27 | N.V. Nutricia | Non-digestible oligosaccharides for oral induction of tolerance against dietary proteins |
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WO2013083140A1 (en) | 2011-12-07 | 2013-06-13 | N.V. Nutricia | Beta-lactoglobulin peptides for treating cow's milk protein allergy |
WO2016148562A1 (en) * | 2015-03-18 | 2016-09-22 | N.V. Nutricia | Method for inducing oral tolerance via administration of beta-lactoglobulin derived peptide in combination with probiotic |
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ZA200301244B (en) | 2004-05-14 |
KR20030034178A (ko) | 2003-05-01 |
CZ2003749A3 (cs) | 2003-06-18 |
HUP0302585A3 (en) | 2007-05-02 |
PL365246A1 (en) | 2004-12-27 |
NZ524527A (en) | 2005-05-27 |
JP2004514424A (ja) | 2004-05-20 |
SK3642003A3 (en) | 2003-08-05 |
AU2056102A (en) | 2002-04-02 |
CN1246456C (zh) | 2006-03-22 |
AU2002220561C1 (en) | 2002-04-02 |
BG107657A (bg) | 2003-12-31 |
IL154284A0 (en) | 2003-09-17 |
HUP0302585A2 (hu) | 2003-10-28 |
NO20031159L (no) | 2003-05-20 |
AR033574A1 (es) | 2003-12-26 |
BR0114105A (pt) | 2003-07-29 |
NO20031159D0 (no) | 2003-03-13 |
WO2002024883A2 (en) | 2002-03-28 |
US7498162B2 (en) | 2009-03-03 |
RU2294366C2 (ru) | 2007-02-27 |
EP1379635A2 (en) | 2004-01-14 |
WO2002024883A3 (en) | 2003-11-06 |
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