CN1505678A - 作为治疗和预防变态反应的药物的乳酸菌 - Google Patents
作为治疗和预防变态反应的药物的乳酸菌 Download PDFInfo
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Abstract
本发明涉及能降低个体对各种变应原产生变态反应倾向的乳酸菌。具体来讲，本发明涉及表达表面多肽的重组乳酸菌菌株，所述表面多肽包含可作为IgE分子Fc区的至少一部分的模拟物的肽或抗体片段。本发明也涉及含有所述微生物或其活性级分的食品或药物组合物。
Description
本发明涉及能降低个体产生变态反应倾向的新型乳酸菌菌株。具体来讲，本发明涉及表达表面多肽的重组乳酸菌菌株，所述表面多肽包括作为IgE分子Fc区的至少一部分的模拟物的小的和较大的肽。本发明也涉及含有所述微生物或其活性级分的食品或药用组合物。
免疫系统是一个复杂而多样性的防御系统，保护机体免受诸如病毒、细菌、寄生虫和真菌或仅仅较大的化学物质等任何生物或化学因子的攻击。虽然免疫系统是维护机体完整性不可缺少的部分，但在某些时候其自身也可能引起疾病，例如自身免疫病、炎症或变态反应。
变态反应是免疫系统对各种物质(变应原)的不当反应。一般来讲，个体不会对环境中经常遇到的物质(诸如花粉或食品)产生显著免疫反应，认为这种不反应性主要是因为免疫系统自身的抑制机制。然而，在受损条件下，免疫系统不实现所述抑制活性，产生对变应原的特异性免疫反应一变态反应。
普遍确立的一个变态反应机制涉及一系列事件，始于变应原摄取，此步需要变应原穿越上皮屏障、到达并激活位于紧密连接之下的固有层或上皮中的效应细胞。与变态反应相关的临床症状主要是早期特异性免疫应答和晚期炎症反应的结果。在早期，由宿主免疫系统产生针对变应性物质的免疫球蛋白E(IgE)，随之通过受体蛋白结合到诸如肥大细胞和嗜碱性粒细胞上。IgE分子结合并交联到细胞表面后，细胞释放组胺和细胞因子，继而通过募集炎性细胞到鼻和上呼吸道中，介导晚期反应。随后嗜酸性粒细胞、巨噬细胞、淋巴细胞、嗜中性粒细胞和血小板流入，开始了炎症期，放大初始的免疫应答，继而触发更多炎性细胞的释放。
过去，患有变态反应的个体数量一直在上升，这通常是归结于诸如排放废气引起的大气污染加重。人们也认为蛋白质类物质消费的增加造成所述，特别是造成食物过敏的发生日益增多。此外，在发达国家中遇到的微生物感染不足，也被认为是特应性疾病上升的另一可能原因。
因此，本领域存在着治疗变态反应的需求，至今为止也提出了各种不同的方法。
至于食物过敏的治疗，有些方法依赖于改进食品材料本身，以降低其变应原可能性。这可以通过改变其化学结构、或限制或禁止引起这一麻烦的食品材料或组分的方法来达到。但是所涉及的问题常常是，在相应的食品材料中具体变应原物质常常是未知的，所以多数情况下并不清楚该选择性去除或改变哪种组分。
一种不同的治疗食物过敏和食物不耐受的方法是恢复和维护肠道的完整性，使食物变应原基本不会穿过去。在这一方面，US 5,192,750描述了应用N-乙酰葡萄糖胺使粘膜能够对食物变应原的运输形成必要屏障，并维持正常功能。
一个最普遍的治疗变态反应的方法是一种免疫疗法，其中涉及重复注射变应原，持续几年的时间，来给患者对所述变应原脱敏。然而其过程耗费时间，需要多年的治疗，还常达不到给病人脱敏的目标。
根据最近的方法，建议给予个体接种针对IgE分子的疫苗，来抑制肥大细胞和嗜碱性粒细胞的触发。为此，WO 97/3 1948提供了供疫苗接种用的在三维构象上类似IgE分子部分的特异性多肽，IgE分子是与在变态反应和炎症反应调节中起作用的介质释放有关的免疫球蛋白。设想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，所述IgE免疫球蛋白会被清除掉。
然而，所述方法存在与正常疫苗接种程序所共有的缺点，因为所述生物学活性物质必须通过侵入性方式给药，例如采用静脉内注射，这种给药途径一般来讲是病人所不喜欢的。另一方面，当选择口服途径时，必须设计合适的盖仑制剂配方，以便让生物活性物质通过胃肠道而不受到破坏。在这一方法中所遇到的另一问题是，拟表位(mimotope)在多数情况下是短链肽，自身不能引发真正的免疫应答，所以在所述组合物中除了载体和赋形剂外，不得不包括佐剂。
因此，本领域需要提供治疗变态反应的改良方法。具体来讲，本发明的一个目的是提供能以有效、简便、成本合算的、最好不需要医师和不会带来与该治疗相关的负面影响的方式治疗变态反应的方法。
通过提供在其表面表达一种多肽的新型乳酸菌株，来达到以上目的，该多肽含有至少一种模拟IgE分子的构象表位的至少一部分(拟表位)的多肽序列。
在图中，
图1显示约氏乳杆菌(Lactobacillus johnsonii)重组体的蛋白质染色和免疫印迹分析。(A)野生型Lal的蛋白染色(第1道)，带有Lal的pMd112TT(LalTT，第2道)或pMD112ε4(Lalε4，第3道)。(B)在LalTT(第2道)和Lalε4(第3道)上的蛋白酶PrtB的表达。通过用1∶2000稀释度的抗PrtB血清进行免疫印迹分析，对约108个细菌进行分析。野生型Lal作为阴性对照上样(第1道)。用辣根过氧化物酶结合的山羊抗兔IgG(Fc)抗体检测结合抗体。(C)在LalTT表面破伤风拟表位的表达(第2道)。用1∶1000稀释度的抗TT血清进行免疫印迹分析，对约108个细菌/狭线进行分析。用Lal(第1道)和LalE4(第3道)作阴性对照。用辣根过氧化物酶结合的山羊抗兔IgG(Fc)抗体检测结合抗体。(D)在Lalε4表面ε4拟表位的表达(第3道)。用10μg/ml浓度的抗E4血清(SDS280)进行免疫印迹分析，对约108个细菌/狭线进行分析。用Lal(第1道)和LalTT(第2道)作阴性对照。用辣根过氧化物酶结合的山羊抗兔IgG(Fc)抗体检测结合抗体。箭头表示A-D中所看到的蛋白酶条带的高度。
图2显示了在ELISA中抗TT IgG与乳酸菌表面展示的ε拟表位的结合测定的结果。
图3显示了在ELISA中抗ε4 IgG与乳酸菌表面展示的ε拟表位的结合测定的结果。
在产生本发明的研究中已经发现，通过提供一种带有包含上述肽序列的重组表面多肽的乳酸菌，可以使个体获得对IgE的特异性免疫，从而使得所述个体对几乎所有抗原的变态反应都受到抑制。
虽然并不希望受任何理论的束缚，但目前的设想是，乳酸菌被摄食后在胃肠道会保持一段时间，能向个体免疫系统呈递所述抗原，使得所述个体发生有效免疫反应，从而产生抗IgE的抗体。这一事实更加出乎意料，因为不能预料到，包含这样一种表面蛋白的细菌，经过胃肠道途径实现给药后，是否最终向个体免疫系统呈递相应的抗原一拟表位一从而使免疫系统能识别该抗原并诱发对它的免疫应答。而且，很明显，所述拟表位呈递给免疫系统所处的生物学环境，使得应用佐剂诱发针对所述抗原的免疫应答来说，是不需要的。
按照优选实施方案，所述含有含拟表位表面多肽的乳酸菌属于乳杆菌群或双岐杆菌(Bifidobacterium)群或乳球菌(Lactococcus)群，更优选源自嗜酸乳杆菌(L.acidophilus)、约氏乳杆菌、加氏乳杆菌(L.gasseri)，干酪乳杆菌(L.casei)，类干酪乳杆菌(L.paracasei)或路氏乳杆菌(L.reuteri)菌群，所述这些菌群都来源于人类或动物。按照更优选的实施方案，所述乳酸菌是一种益生乳酸菌。作为益生菌，人们应理解为是能够以基本上有活力的活体形式穿越胃肠道和任选地也能够刺激宿主免疫系统的微生物。按照最优选的实施方案，所述乳酸菌是约氏乳杆菌。
只要可以插入所述“拟表位肽序列”使得它可以是免疫系统可及的，那么所述表面多肽的性质并不是关键。按照优选的实施方案，其中已插入模拟IgE免疫球蛋白的构象表位的序列的所述表面多肽/蛋白质，是保加利亚乳杆菌(Lactobacillus bulgaricus)的细胞表面锚定蛋白酶，其序列已发表在Gilbert等(1996)J.Bacteriol，178，3059-3065.。这种蛋白被鉴定是2000个氨基酸的蛋白，其构成为负责细胞输出所述酶的33个氨基酸的前导肽(前区)组成的、后接负责切割后激活所述酶的蛋白水解活性的一系列154个氨基酸(原区)和700-800个氨基酸的活性位点。有人提出，随后的区域(约1000个氨基酸)在切割特异性和所产生多肽的胞内转运方面起作用，并且也跨越细胞壁。所述蛋白酶以其羧基端锚定于细胞壁，最后200个氨基酸负责特异性共价连接到细胞壁的肽聚糖结构上。
可以按照本领域熟知的方法，让所述多肽在所述乳酸菌中表达。例如用市售载体pNZ124(Platteuw等，(1994)Appl.Env.Microbiol.60，587)、pGK12(Wakle等，(1996)FEMS Microbiol.138，233)或pG+host9(Maguin等，(1996)J.Bacteriol 178，193)可以用于附加型表达。目前，考虑到染色体整合的高度稳定性，这种方式对于编码相应多肽的重组基因可能是优选的。为了整合到染色体中，可以通过例如用来自包含所述耐受原性肽的乳酸菌的重组基因，取代内源基因，来应用同源重组。目前，将重组基因引入宿主染色体的方法完全在技术人员的技术范围内。
通过用用带有针对IgE的Fc部分的抗体筛选随机肽和人Fab抗体噬菌体展示文库，可以发现模拟IgE的构象表位的肽序列(Seq.Id.No.1-17)和抗独特型VH和VL序列(Seq.Id.No.18-19)。优选的拟表位和抗独特型Fab序列选自：
Ile-Asn-His-Arg-Gly-Tyr-Trp-Val (A) (Seq.Id.No.1)，
Arg-Asn-His-Arg-Gly-Tyr-Trp-Val (B) (Seq.Id.No.2)，
Arg-Ser-Arg-Ser-Gly-Gly-Tyr-Trp-Leu-Trp (C) (Seq.Id.No.3)，
Val-Asn-Leu-Thr-Trp-Ser-Arg-Ala-Ser-Gly (D) (Seq.Id.No.4)，
Val-Asn-Leu-Pro-Trp-Ser-Arg-Ala-Ser-Gly (E) (Seq.Id.No.5)，
Val-Asn-Leu-Thr-Trp-Ser-Phe-Gly-Leu-Glu (F) (Seq.Id.No.6)，
Val-Asn-Leu-Pro-Trp-Ser-Phe-Gly-Leu-Glu (G) (Seq.Id.No.7)，
Val-Asn-Arg-Pro-Trp-Ser-Phe-Gly-Leu-Glu (H) (Seq.Id.No.8)，
Val-Lys-Leu-Pro-Trp-Arg-Phe-Tyr-Gln-Val (I) (Seq.Id.No.9)，
Val-Trp-Thr-Ala-Cys-Gly-Tyr-Gly-Arg-Met (J) (Seq.Id.No.10)，
Gly-Thr-Val-Ser-Thr-Leu-Ser (K) (Seq.Id.No.11)，
Leu-Leu-Asp-Ser-Arg-Tyr-Trp (L) (Seq.Id.No.12)，
Gln-Pro-Ala-His-Ser-Leu-Gly (M) (Seq.Id.No.13)，
Leu-Trp-Gly-Met-Gln-Gly-Arg (N) (Seq.Id.No.14)，
Leu-Thr-Leu-Ser-His-Pro-His-Trp-Val-Leu-Asn-
His-Phe-Val-Ser (O) (Seq.Id.No.15)，
Ser-Met-Gly-Pro-Asp-Gln-Thr-Leu-Arg (P) (Seq.Id.No.16)，
Val-Asn-Leu-Thr-Trp-Ser (Q) (Seq.Id.No.17)，
Gln-Val-Lys-Leu-Leu-Glu-Ser-Gly-Pro-Gly-Leu-
Val-Lys-Pro-Ser-Glu-Thr-Leu-Ser-Leu-Thr-Cys-
Thr-Val-Ser-Gly-Gly-Ser-Ile-Ser-Ser-Gly-Gly-
Tyr-Tyr-Trp-Thr-Trp-Ile-Arg-Gln-Arg-Pro-Gly-
Lys-Gly-Leu-Glu-Trp-Ile-Gly-Tyr-Ile-Tyr-Tyr-
Ser-Gly-Ser-Thr-Ser-Tyr-Ash-Pro-Se -Leu-Lys-
Ser-Arg-Val-Thr-Met-Ser-Val-Asp-Thr-Ser-Lys-
Asn-Gln-Phe-Ser-Leu-Arg-Leu-Thr-Ser-Val-Thr-
Ala-Ala-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-Arg-
Glu-Arg-Gly-Glu-Thr-Gly-Leu-Tyr-Tyr-Pro-Tyr-
Tyr-Tyr-Ile-Asp-Val-Trp-Gly-Thr-Gly-Thr-Thr-
Val-Thr-Val-Ser-Ser (S) (Seq.Id.No.18)
Glu-Leu-Val-Val-Thr-Gln-Pro-Ala-Ser-Val-Ser-
Gly-Ser-Pro-Gly-Gln-Ser-Ile-Thr-Ile-Ser-Cys-
Thr-Gly-Thr-Arg-Ser-Asp-Val-Gly-Gly-Tyr-Asn-
Tyr-Val-Ser-Trp-Tyr-Gln-Gln-His-Pro-Gly-Lys-
Ala-Pro-Lys-Leu-Met-Ile-Tyr-Asp-Val-Ser-Asn-
Arg-Pro-Ser-Gly-Val-Ser-Asn-Arg-Phe-Ser-Gly-
Ser-Lys-Ser-Gly-Asn-Thr-Ala-Ser-Leu-Thr-Ile-
Ser-Gly-Leu-Gln-Ala-Glu-Asp-Glu-Ala-Asp-Tyr-
Tyr-Cys-Ser-Ser-Tyr-Thr-Ser-Ser-Ser-Thr-Leu-
Gly-Val-Phe-Gly-Gly-Gly-Thr-Lye-Leu-Thr-Val-
Leu-Gly (T) (Seq.Id.No 19)
关于肽的分离和/或制备以及模拟IgE的部分恒定区的抗独特型VH和VL序列的更详细的信息，参考WO97/31948，其中的内容通过引用结合到本文中。
本发明也涉及含有至少一种以上描述的这样的乳酸菌的食品和药物组合物，特别是疫苗。
所述菌株可以以从105到1012个cfu(菌落形成单位)/克材料的量包含在所述组合物中。所述食品组合物可以是乳、酸乳、凝乳、干酪、发酵乳、基于乳的发酵产品、冰淇淋、基于发酵谷物的产品、基于乳的粉末、婴儿配制食品，或对动物来说，宠物食品。所述药用组合物可以是片剂、液体细菌悬液、干口服补充剂、湿口服补充剂、干管饲剂(tube-feeding)或湿管饲剂的形式。
本发明所述乳酸菌和所述食品/药用组合物可以用于治疗与其中涉及IgE抗体的免疫反应的变态反应有关的任何疾病，例如鼻炎、特应性皮炎、红斑等。同样，人们会认识到，本发明所述细菌/组合物适合用作“接种疫苗用药物(vaccination agent)”，以普遍预防个体变态反应的发生。这只需让需要治疗变态反应的人进食按照本发明的食品或药用组合物，就可以容易地完成。所述细菌经摄取后，将在肠道内定居一段时间，因此，根据所述细菌的细胞数量和给予本发明组合物的时期，所述拟表位被呈递给个体，使之可以形成针对所述拟表位的免疫应答。人们将会认识到，除了含有本发明组合物所含的乳酸菌外，也可采用已知刺激免疫系统的药物，从而改善针对所述拟表位的免疫应答。
以下实施例进一步阐明了本发明，但不限于此。
实施例1
重组多肽的构建
将两种肽与保加利亚乳杆菌的细胞表面锚定蛋白酶融合，以展显在细菌Lal(CNCM I-1225)的表面，所述肽即肽ε4(Seq.Id.No.1)和另一破伤风毒素来源的肽(以下用TT表示；其序列见下)(用作对照)。
将所述拟表位(ε4)与保加利亚乳杆菌来源的细胞表面蛋白酶(PrtB)(Gilbert等，(1996)J.Bacteriol，178，3059-3065)符合读框地融合。
首先采用以下两种引物扩增所述蛋白酶基因和其启动子：
5`-TTITGTGGATCCTTAACTTCATAGCACG-3’
(所述基因启动子的上游，携带一个BamHI位点)
5`-ATATTATCTAGAATTGAATAGATTGCC-3’
(所述基因rho不依赖性终止子的下游，携带一个XbaI位点)
用BamHI和XbaI切割所述扩增产物，并克隆到用同样的限制性酶消化的乳酸菌载体pNZ124中，最终通过电穿孔引入到无质粒(β-半乳糖苷酶和蛋白酶阴性)乳酸乳球菌(Lactoccoccus lactis)中。
采用两端侧翼是两个半胱氨酸残基的所述TT肽和ε4肽的序列，取代所述已克隆的蛋白酶的活性位点区。加入所述半胱氨酸残基，是因为这两种肽从噬菌体展示文库中作为侧翼是两个半胱氨酸的环肽被分离出来的。因为这些肽并不代表天然的表位而是模拟天然表位，所以它们被称为拟表位：
9 18 27
5′TGC ATT AAT CAT AGA GGA TAT TGG GTT TGC 3′
--- --- --- --- --- --- --- --- --- --- ε4
Cys Ile Asn His Arg Gly Tyr Trp Val Cys
9 18 27
5′TGC ACA GAT CCT TCT GGA GCA TCT GCA CCT TGC 3′
--- --- --- --- --- --- --- --- --- --- --- TT
Cys Thr Asp Pro Ser Gly Ala Ser Ala Pro Cys
为达到此目的，用NheI在前导肽的切割位点的序列下游50bp处、以及用PvuI在更下游的800bp处，切割所述已克隆的蛋白酶。编码目标肽的DNA序列(参见上文)，作为两种寡聚核苷酸，插入到所述两个限制位点之间，这些设计是为了当它们杂交在其两端产生两个限制位点。所述寡聚核苷酸的设计考虑到但与所述蛋白酶连接时，所述重组蛋白的阅读框架仍然是可读框。用所述限制性酶切割所述扩增产物。在两种情况下，DNA片段与所述蛋白酶基因连接，并通过电穿孔将其导入约氏乳杆菌。
实施例2
约氏乳杆菌的转化
为了进行转化，将约氏乳杆菌Lal菌株(可得自the Institute Pasteur(巴斯德研究所)，保藏号CNCM I-1225)在MRS肉汤、37℃、缺氧条件下培养过夜。用一等份该培养物接种(1∶10)含0.5M蔗糖的另一培养肉汤(MRS)。在另外以2％在200ml MRS+0.5M蔗糖中再次接种后，让培养物生长至OD595为0.6。以5000rpm、4℃离心10分钟收集细胞，沉淀物用1/2体积的含1M蔗糖和2.5mM CaCl2的溶液洗涤两次，用1/4体积的含1M蔗糖、2.5mM CaCl2的溶液洗涤一次，离心后获得的沉淀物重新悬浮在3.5ml的含1M蔗糖、2.5mM CaCl2+0.459ml的87％甘油(终浓度10％)的溶液中。所述细胞或者直接用于转化或在-80℃冷冻。
对于电穿孔，将40μl细胞与10-100ng DNA(小于5μl的体积)混合，并转移至冰冷的0.2cm的电穿孔样品池中。在冰冷的0.2cm电穿孔样品池中，以200Ω、25μF、2.5kV施加脉冲。向该样品池中加入1ml的MRS+20mM MgCl2、2mM CaCl2，悬浮液保温于37℃2-3小时。分别用10μl、100μl等份在含合适抗生素的MRS琼脂平板上铺平板。这些平板在与前述相同的温度中、在缺氧条件下、温育20-48小时。采用含氯霉素(10μg/ml)的MRS作为选择性培养基。
实施例3
抗蛋白酶PrtB的抗血清的产生
为了产生针对PrtB的抗血清，用表达蛋白酶B(PrtB)的德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp bulgaricus)菌株ATCC11842皮下免疫兔子。让细菌在MRS肉汤中、在GasPak缺氧系统中42℃生长过夜。用一等份的培养物接种含0.5M蔗糖的另一MRS肉汤，让培养物于42℃生长5小时，直到OD595为0.6。以5000rpm、4℃离心收集所述细胞，沉淀物用10ml PBS洗涤两次，再重新悬浮于2ml PBS中。用1.5ml PBS重新悬浮的细胞免疫兔子3次，每次间隔2周，在最后一次给药后7天给兔子采血。用2×109约氏乳杆菌(Lal)纯化血清6次。
实施例4
针对所述TT和ε4拟表位的抗血清的产生
为了产生针对所述TT和ε4拟表位的抗血清，用聚肟-TT拟表位构建体或与匙孔血蓝蛋白(KLH)相结合的ε4拟表位来皮下免疫兔子。兔子免疫4次，每次间隔2周，最后一次注射后7天给动物采血。用与ε4拟表位偶联的CH-Sepharose 4B，对所述抗ε4血清进行免疫亲合层析。
实施例5
用抗体检测拟表位-PrtB融合蛋白
为了检测所述乳酸菌是否以为抗体可及或为抗体所识别的方式表达所述拟表位，将含有未经修饰的所述PrtB基因的细菌、或含有重组基因(分别编码所述ε4或所述TT拟表位)的细菌，培养于25ml含10μg/ml氯霉素的培养基中。在3000×g、4℃下离心15分钟收获细菌细胞，用5ml TBS洗涤，再离心。最后在450μl Tris缓冲盐溶液(TBS：25mM Tris/HCl，pH 7.0，0.8％NaCl，0.02％KCl)和150μl 4×非还原性样品缓冲液(80mM Tris/HCl pH 6.8，2.5％SDS，0.15％甘油，0.05％溴酚蓝)中重新悬浮细菌沉淀物。用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)(6％丙烯酰胺-Bis，0.5M Tris-HCl pH 8.8)，在25mM Tris、192mM甘氨酸缓冲液(pH 8.3)中，在100V下跑电泳60分钟，对20μl等份进行分离。该凝胶用BMFast染料(Boehringer Mannheim，德国)染色，或电转移到硝酸纤维素膜(Protran BA 83，Schleicher&Schuell，Dassel，德国)。转移后，用PBS/5％BSA在室温下对所述膜封闭2小时。用兔抗TT血清(1∶1000)或抗PrtB血清(1∶2000)在室温下对免疫印迹保温过夜，再在室温下用1∶1000稀释度的辣根过氧化物酶结合的山羊抗兔IgG保温3小时，用4-氯-1-萘酚处理2分钟，使免疫印迹显色。
如图1所示，可以观察到，用合适的抗体检测，重组约氏乳杆菌显示是特异性条带，表明用所述重组细菌可以产生正确构象的所述ε4和所述TT拟表位以及所述蛋白酶PrtB。
实施例6
ELISA检测约氏乳杆菌(Lal)的表面抗原表达
转化细菌在含有10μl/ml氯霉素的50ml培养基中生长过夜。以3000×g、4℃离心15分钟收获细菌细胞，用5ml TBS洗涤，再离心。最后该细菌沉淀物重新悬浮于900μl TBS和100μl 0.5M碳酸氢盐缓冲液(pH 9.6)中。Costar EIA/RIA半孔板(Costar，cambridge，MA)在37℃用每孔50μl细菌液(约108细菌)包被过夜。用10μl/ml浓度的TTd作为包被抗原，评估包被效率。用PBS/0.1％Tween-20彻底清洗板子，直到没有细菌残留。用PBS/5％BSA在37℃封闭各孔2小时，用50μl10μl/ml浓度的兔抗TT血清或亲和纯化的兔抗ε4血清IgG抗体，在37℃保温4小时。用PBS/0.1％Tween-20清洗六次后，将板子在37℃与1∶1000稀释度的辣根过氧化物酶结合的山羊抗兔IgG保温1.5小时。用PBS/0.1％Tween-20清洗六次，再用四甲基对二氨基联苯(TMB；Fluka Chemie AG，Buchs，瑞士)显色。用1M H2SO4终止反应，在450nm处用ELISA读出器(Molecular devices，Basel，瑞士)测量吸光度值。
如图2和3所示，抗TT和抗ε4抗体特异性识别表达TT和ε4拟表位的活重组Lal，表明这两种拟表位已表达并展示在所述细菌细胞表面。
序列表
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Claims (11)
1.一种乳酸菌群的细菌菌株，所述细菌菌株表达一种表面多肽，所述多肽含有模拟IgE分子构象表位的至少一部分的肽序列。
2.权利要求1所述的细菌菌株，所述细菌菌株选自乳杆菌(Lactobacillus)群或双岐杆菌(Bifidobacterium)群或乳球菌(Lactococcus)群，更优选源自嗜酸乳杆菌(L.acidophilus)、约氏乳杆菌(L.johnsonii)、加氏乳杆菌(L.gasseri)、干酪乳杆菌(L.casei)、类干酪乳杆菌(L.paracasei)或路氏乳杆菌(L.reuteri)菌群。
3.权利要求1或2所述的细菌菌株，其中所述表面多肽是保加利亚乳杆菌(L.bulgaricus)的细胞表面锚定蛋白酶。
4.任一前述权利要求所述的细菌菌株，其中所述肽序列来源于选自SEQ.ID.No.1-SEQ.ID.No.19的序列。
5.一种食品组合物，所述食品组合物含有任一前述权利要求的细菌菌株或其上清液。
6.权利要求5所述的食品组合物，所述组合物选自乳、酸乳、凝乳、干酪、发酵乳、基于乳的发酵产品、冰淇淋、基于发酵谷物的产品、基于乳的粉末、婴儿配制食品或宠物食品。
7.权利要求5或6所述的食品材料，其中所述细菌菌株的含量范围为107-1012cfu/剂型。
8.一种药用组合物，所述药用组合物含有权利要求1到4中任一项所述的细菌菌株。
9.权利要求8所述的药用组合物，其中所述细菌菌株的含量范围为1010-1012cfu/剂型。
10.权利要求1到4中任一项的细菌菌株在制备用于治疗变态反应和/或预防变态反应发生的可摄取载体中的应用。
11.权利要求6到9中任一项的食品组合物或药用组合物在制备用于治疗变态反应和/或预防变态反应发生的可摄取载体中的应用。
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| CN102028224A (zh) * | 2007-06-21 | 2011-04-27 | 东宇生物科技股份有限公司 | 抗过敏的乳酸菌 |
| CN111587118A (zh) * | 2018-01-12 | 2020-08-25 | Gi 医诺微新 | 包含益生菌和具有与IgE结合能力的多肽的组合物及其用途 |
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| US20050158294A1 (en) | 2003-12-19 | 2005-07-21 | The Procter & Gamble Company | Canine probiotic Bifidobacteria pseudolongum |
| US7955834B2 (en) | 2004-06-03 | 2011-06-07 | Biogaia Ab | Method for improved breast milk feeding to reduce the risk of allergy |
| US7862808B2 (en) * | 2004-07-01 | 2011-01-04 | Mead Johnson Nutrition Company | Method for preventing or treating respiratory infections and acute otitis media in infants using Lactobacillus rhamnosus LGG and Bifidobacterium lactis Bb-12 |
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| AU1120102A (en) * | 2000-11-02 | 2002-05-15 | Univ Singapore | AopB gene, protein, homologs, fragments and variants thereof, and their use for cell surface display |
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- 2002-03-04 KR KR1020087030377A patent/KR20080111172A/ko not_active Application Discontinuation
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- 2002-03-04 IL IL15746802A patent/IL157468A0/xx unknown
- 2002-03-04 RU RU2003129265/13A patent/RU2312892C2/ru not_active IP Right Cessation
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- 2002-03-04 BR BR0207749-3A patent/BR0207749A/pt not_active Withdrawn
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102028224A (zh) * | 2007-06-21 | 2011-04-27 | 东宇生物科技股份有限公司 | 抗过敏的乳酸菌 |
| CN102028224B (zh) * | 2007-06-21 | 2012-12-26 | 东宇生物科技股份有限公司 | 抗过敏的乳酸菌 |
| CN111587118A (zh) * | 2018-01-12 | 2020-08-25 | Gi 医诺微新 | 包含益生菌和具有与IgE结合能力的多肽的组合物及其用途 |
| TWI737955B (zh) * | 2018-01-12 | 2021-09-01 | 南韓商Ｇｉ創新股份有限公司 | 包含益生菌及IgE結合多肽的組成物及其用途 |
| CN111587118B (zh) * | 2018-01-12 | 2023-11-21 | Gi 医诺微新 | 包含益生菌和具有与IgE结合能力的多肽的组合物及其用途 |
Also Published As
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| AU2002256639B2 (en) | 2006-10-19 |
| IL157468A0 (en) | 2004-03-28 |
| WO2002086102A1 (en) | 2002-10-31 |
| PL367208A1 (en) | 2005-02-21 |
| MXPA03007881A (es) | 2003-12-04 |
| NO20033796D0 (no) | 2003-08-26 |
| US7235395B2 (en) | 2007-06-26 |
| JP2004528034A (ja) | 2004-09-16 |
| NZ527861A (en) | 2005-02-25 |
| BR0207749A (pt) | 2004-06-01 |
| RU2003129265A (ru) | 2005-04-10 |
| EP1370640A1 (en) | 2003-12-17 |
| AR035761A1 (es) | 2004-07-07 |
| US20040265290A1 (en) | 2004-12-30 |
| CA2438833A1 (en) | 2002-10-31 |
| ZA200307680B (en) | 2005-01-13 |
| RU2312892C2 (ru) | 2007-12-20 |
| EP1239032A1 (en) | 2002-09-11 |
| NO20033796L (no) | 2003-10-31 |
| KR20030082950A (ko) | 2003-10-23 |
| KR20080111172A (ko) | 2008-12-22 |
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