CN111363706A - 旱莲草内生细菌、旱莲草组合物及其应用 - Google Patents

旱莲草内生细菌、旱莲草组合物及其应用 Download PDF

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CN111363706A
CN111363706A CN202010283316.1A CN202010283316A CN111363706A CN 111363706 A CN111363706 A CN 111363706A CN 202010283316 A CN202010283316 A CN 202010283316A CN 111363706 A CN111363706 A CN 111363706A
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赵鑫
高秀梅
刘二伟
王亚静
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Abstract

本申请涉及旱莲草内生细菌及其组合物，它们的抑菌用途，本申请还涉及旱莲草组合物在调节受试者肠道菌群中的用途及方法。

Description

旱莲草内生细菌、旱莲草组合物及其应用
技术领域
本申请涉及旱莲草内生细菌及其组合物，以及它们的抑菌用途，本申请还涉及旱莲草组合物在调节受试者肠道菌群中的用途及方法。
背景技术
近年来，中医药对肠道菌群的调控越来越受到重视[1-3]。理论研究和实验研究都证实了一些单一的中药或化合物可以调节肠道微生态平衡[4,5]。一些中药可以直接抑制有害细菌，同时间接增加肠道菌群中有益细菌的丰度，从而产生抗菌化合物[6]。此外，中药促进的肠道微生物或植物性中药内生菌也可能产生抗菌药物，抑制病原微生物的生长，调节肠道菌群失调[7,8]。最近的研究表明，中医药在适当的临床剂量下，通过促进保加利亚乳杆菌的生长，在增加益生菌数量的同时减少致病菌数量，抑制致病菌金黄色葡萄球菌，优化肠道菌群结构，发挥其有益作用[9]。内生细菌及其代谢产物在农业、食品、医药、生态等领域发挥着重要作用[11,12]。近年来，植物内生菌的研究取得了一些进展，发现了许多药用菌株和化合物[10,13,14]。内生细菌具有广泛的抗菌活性，是抗菌物质的重要潜在来源[15]。植物内生细菌的抗菌物质具有较低的细胞毒性，大多是新奇的[16]。此外，内生菌可以调节植物次生代谢产物的生物合成[17]。中药植物内生微生物多为真菌，如shelby等在高羊茅内生真菌中发现的麦角生物碱等[18]。 Salituro等人从热带植物内生真菌假黑壳次生代谢神中获得小分子苯醌，作用类似胰岛素[19]；Yang等从西藏红豆杉内生真菌中提取具有抗菌活性的萘醌[20]；Wang等人从银杏叶中分离出一株内生次生孢子壳菌，并产生具有抗氧化活性的黄酮类化合物[21]。植物内生细菌也有报道，但中医药报道较少。内生细菌，包括芽孢杆菌、肠杆菌和节杆菌，普遍存在于植物的根和茎中[10]。植物内生细菌大多来源于根际，与土壤中的普通细菌相同[22]。产生抗生素或促进植物生长的生物量在生物控制、固氮和植物生长方面发挥着重要作用[23]。然而，从中草药或植物中筛选内生细菌，以及它们如何与植物相互作用以发挥其生物学功能仍有待研究。药用植物内生细菌能够经口进入肠道，影响肠道微生态平衡。因此，植物内生细菌的研究和应用具有广阔的前景。
旱莲草(Eclipta prostrata)(名称来源于全球菊科植物名录 http://www.theplantlist.org/tpl1.1/record/gcc-6746)是向日葵科的一种植物，在传统医案中被用于治疗子宫出血、牙齿松动、耳鸣和头晕[24]。我们先前的临床试验发现，含有旱莲草的配方有效地治疗了围绝经期症状[25]。药理研究表明，旱莲草具有保肝、止血、抗肿瘤、抗炎、抗高血压、降血糖和抗骨质疏松的作用[26-33]。现代中医学研究表明，中药的药效学与其所含化合物的种类和数量有关[17]。旱莲草含有多种活性成分，包括三萜、黄酮、噻吩[34-37]。有些是非微生物诱导和在草药中预先形成的抗菌物质；有些不是由草药本身产生的，而是由内生微生物产生的。
鉴于中药基于肠道菌群的药效作用，从中药中筛选和分析内生细菌是药效学研究的热点领域，具有重要的研究意义和应用前景。
发明内容
本研究从旱莲草野生植物中分离到多种内生细菌，其中EP01对金黄色葡萄球菌和大肠杆菌有明显的抑制作用。通过全基因组测序，对该细菌进行进一步鉴定，并对基因组中抗菌物质生物合成基因簇进行预测。最后通过动物实验验证了旱莲草提取物对肠道菌群的作用来自其内生细菌。
本发明的一个目的是提供一种旱莲草内生细菌：旱莲草内生细菌 EP01，其保藏于中国微生物菌种保藏管理委员会普通微生物中心，且具有保藏号CGMCC No.19512，保藏日期：2020年3月26日。
中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，位于北京市朝阳区北辰西路1号院3号。所述保藏的旱莲草内生细菌 EP01的建议分类命名为：格氏乳球菌Lactococcus garvieae。
本发明的另一个目的是提供一种组合物，其含有上述内生细菌 EP01。
在一些实施方案中，所述组合物为粉末或液体；优选地，所述组合物通过以下方式施用：口服使用、用于栓剂进行施用、用于皮下注射、用于静脉注射，或者作为用于吸入的液体。
本发明的第三个目的是提供上述的组合物在抑制细菌生长中的用途；所述细菌包括革兰氏阳性细菌和革兰氏阴性细菌。
在一些实施方案中，所述革兰氏阳性细菌包括但不限于：葡萄球菌属、链球菌属、梭菌属、放线菌属、奴卡菌属、红球菌属、棒状杆菌属、分支杆菌属、李氏杆菌属、肾杆菌属、炭疽杆菌属、破伤风杆菌属、抗酸菌属。
在一些实施方案中，所述革兰氏阴性细菌包括但不限于：淋球菌属、脑膜炎双球菌属、卡他菌属、耶尔森菌属、嗜肺军团菌属、志贺菌属、巴斯德菌属、霍乱弧菌属、副溶血性杆菌属、大肠杆菌属、沙门菌属、假单胞菌属、变形杆菌属、布氏杆菌属。
在一些优选的实施方案中，所述细菌为金黄色葡球菌和大肠杆菌。
本发明的第四个目的是提供旱莲草提取物在制备药物中的用途，所述药物用于调节受试者肠道微生物或治疗与肠道菌群紊乱相关疾病。
在一些实施方案中，所述旱莲草提取物是通过以下方法制备的：称取一定量旱莲草，经乙醇回流提取，优选70％乙醇回流提取，更优选8倍量70％乙醇回流提取两次，每次1.5h；合并滤液后，干燥得旱莲草提取物。
在一些实施方案中，所述旱莲草提取物调节受试者肠道微生物是通过增加肠道中细菌的数量，所述细菌选自：芽孢杆菌属，乳酸杆菌属，嗜臭菌属，乳球菌属，奇异菌属，普氏菌UCG-004属，或其任何组合。
在另一些实施方案中，所述旱莲草提取物调节受试者肠道微生物是通过抑制肠道中细菌的数量，所述细菌选自：瘤胃菌属9，厌氧棍状菌属，瘤胃菌属，普氏菌UCG-001属，不动杆菌，拟杆菌，副拟杆菌，或其任何组合。
在某些实施方案中，所述受试者是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩。在某些优选的实施方案中，所述受试者是人。
术语解释
如本文中所使用的，术语“16S rDNA序列”是指，编码16S rRNA 的DNA序列，其存在于所有细菌基因组中，一般由保守区和可变区组成。保守区在细菌间无显著差异，可用于构建所有生命的统一进化树。可变区在不同细菌中存在一定差异，对16S rDNA可变区进行测序，可对细菌菌群进行精细鉴定至种的级别。
如本文中所使用的，术语“有效剂量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本申请从旱莲草中分离到多株内生细菌，其中分离到的EP01对金黄色葡萄球菌和大肠杆菌有明显的抑制作用，证实旱莲草具有潜在的抑菌作用和价值。本申请还通过动物实验验证了旱莲草提取物对肠道菌群具有调节作用，并证实了其调节肠道菌群的作用来自其内生细菌。从旱莲草中筛选和分析内生细菌在中药药效学研究中，具有重要的研究意义和应用前景。
下面将结合实施例对本申请的实施方案进行详细描述，但是本领域技术人员将理解，下列实施例仅用于说明本申请，而不是对本申请的范围的限定。根据优选实施方案的下列详细描述，本申请的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了琼脂糖凝胶电泳法检测EP01-EP10菌株的16S rDNA 的结果图。
图2显示了旱莲草分离出的内生细菌的抗菌活性。指示菌株分别为大肠杆菌(A)和金黄色葡萄球菌(B)。NC，阴性对照盐水；PC，阳性对照0.4mg/mL庆大霉素。
图3显示了基于全基因组序列的EP01的系统进化树分析结果。
图4显示了旱莲草提取物对小鼠肠道菌群的影响。
关于生物材料保藏的说明
旱莲草内生细菌EP01保藏于中国微生物菌种保藏管理委员会普通微生物中心，且具有保藏号CGMCC No.19512，保藏日期：2020 年3月26日。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：旱莲草内生细菌的筛选与分离
将新鲜旱莲草的表面用无菌水清洗并在室温下干燥。无菌条件下，根、茎和叶分别剪碎取100mg于试管中。加入1毫升无菌水，样品以 4000转/分离心1分钟。每50μl上清液涂布于LB和MRS平板，37℃培养24小时，通过观察平板上菌落特征，从平板上筛选出10种不同表型的菌落进行抑菌试验，以进一步筛选。
实验结果：
从旱莲草中共筛选了10株细菌，其中，从根中筛选出5株细菌，茎中2株，叶中3株。将这10株细菌分别命名为EP01-EP10，从根和叶中筛选出的大多数菌落具有白色、圆形、表面光滑不透明、边缘整齐、革兰氏染色阳性等共同特征，具体的菌落特征见表1。通过形态学分析，这些细菌与最常见的根际细菌芽孢杆菌属相似。此外，从茎中筛选出的EP07菌落具有圆形、黄色、边缘整齐、低凸、光滑、革兰氏阴性等特性，此形态特征鲜有报道。
实施例2：分子生物学鉴定及抑菌试验
采用ctab/sds法提取EP01-EP10菌株的基因组DNA，并作为PCR 扩增的模板。用通用引物27f:5'-agagttttgatggctcag-3'(SEQ ID NO： 1)和1492r:5'-acggttatccttgttaccgatt-3'(SEQ ID NO：2)分别对10 个菌株的16s核糖体DNA序列进行扩增和测序。用聚合酶链式反应在 PCR热循环仪(MyCycler；Bio-Rad Laboratories Inc.，USA)中进行扩增。PCR扩增体系为25μl，包括0.2μl taq酶(0.5u/ml)、2.5μl 10×缓冲液、1.8μl mg2+、1μl dntps混合物、1μl模板DNA、0.5μl正向引物(10μM)、0.5μl反向引物(10μM)和17.5μl ddH2O，扩增条件： 95℃，3min；95℃，30s，55℃，60s，72℃，90s(30个循环)；72℃，5min；4℃终止反应。扩增产物经北京华大生物科技有限公司纯化测序。根据16S rDNA测序结果，结合芽孢杆菌属genbank中16S rDNA 序列。
以大肠杆菌(获自CMCC，保藏号为44103)和金黄色葡萄球菌 (获自ATCC，保藏号为6358)为指示菌，对筛选出的菌株进行抗菌活性检测。将10ml LB或MRS琼脂培养基倒入培养皿中，冷却备用。然后将干燥的牛津纸杯轻轻地放在培养基上，将10ml LB琼脂培养基与40-50℃下1μl活化的大肠杆菌或金黄色葡萄球菌培养物混合倒入培养板中。之后，取出牛津杯，每个孔中加入30μl的受试培养物。分别用等量灭菌水和0.4mg/ml庆大霉素作阴性对照和阳性对照。培养板在37℃下培养24小时，测量抗菌圈直径。每个实验重复3次。
实验结果：
图1为EP01-EP10 16S rDNA序列的凝胶电泳图。将16S rDNA 测序出的序列与genebank中细菌的序列进行比对(最低同源性为 98％)，最终鉴定出的最高同源性菌株结果如表1所示。结合形态学特征，从旱莲草中筛选出的10株细菌分别属于芽孢杆菌属(Bacillus)、乳球菌属(Lactococcus)、肠球菌属(Enterococcus)、微小杆菌属(Exiguobacterium)和泛菌属(Pantoea)。采用牛津杯法对EP01-EP10 进行了抑菌活性试验。实验结果如图2所示，EP01、EP03、EP05菌株对大肠杆菌和金黄色葡萄球菌具有较强的抗菌活性，具体的抑菌数据如表2所示。其他菌株对这两种病原菌均无抑菌作用。特别的是，EP01菌株抑制大肠杆菌的活性强于其他菌株。
表1.从旱莲草中筛选出内生细菌的菌落特征及同源性相似菌株
Figure BDA0002447525200000071
Figure BDA0002447525200000081
表2.旱莲草内生菌分离株抑菌圈测定
Figure BDA0002447525200000082
实施例3：基因组测序与抗菌药物挖掘
利用Illumina PE150测序技术对EP01内生细菌进行基因组测序，对质检合格的DNA样品构建插入片段为～400bp的片段，进行PE150 (pair-end)测序，即双端测序，单端测序读长150bp，每个样品提供不低于基因组100×覆盖深度的原始测序数据量。利用短序列组装软件SOAPdenovo2(http://soap.genomics.org.cn/)对二代测序后的优化序列进行多个Kmer参数的拼接，得到最优的contigs组装结果，然后把reads比对到contig上，根据reads的paired-end和overlap关系，对组装结果进行局部组装和优化，形成scaffolds，获得全基因组序列。 (基因组数据参见BioProject:PRJNA582067；BioSample: EP01:SAMN13164043)。在国家生物技术信息中心(ncbi)对基因组进行了注释。使用基于网络的基因组挖掘工具antiSmash (http://antismash.secondaryseconomicals.org)，对已知的和未知的抗菌化合物的基因簇进行基因组分析并收集基因组挖掘数据。
实验结果：
细菌EP01基因组由一个大小为2301882bp的环状染色体组成， G+C含量为39.14％。像将近80％的菌株一样，没有质粒。系统发育分析表明EP01与格式乳球菌(Lactococcus garvieae)[48,51]关系最为密切(图3)。EP01基因组的功能注释产生了2298个编码序列和 48个rRNAs和tRNAs。在预测的基因中，1676个(72.9％)与一般COG功能分类相关，1342个(58.4％)与基因本体相关。通过KEGG 注释，结果表明更多的基因分布在碳水化合物代谢和膜转运中。预计 EP01基因组中含有2个与抗菌化合物产生有关的基因簇，包括芳香多烯和细菌素。芳基多烯生物合成基因簇含有17个核心基因，分别编码酰基载体蛋白(acyl carrier protein)、3-酮酰基-ACP还原酶 (3-ketoacyl-ACP reductase)、6-磷酸-β-葡萄糖苷酶 (6-phospho-beta-glucosidase)、β-羟基酰基-ACP脱水酶 (beta-hydroxyacyl-ACP dehydratase)、3-酮基-L-古龙酸6-磷酸脱羧酶(3-keto-L-gulonate 6-phosphate)和胱硫醚γ合酶(cystathionine gamma-synthase)等，具体如表3所示。细菌素簇含有一个乳糖蛋白的结构基因(表3)。
EP01的基因组序列保存在GenBank中(BioProject: PRJNA582067；BioSample:EP01:SAMN13164043)。基于CAZy数据库(碳水化合物活性酶)的基因分类也确定了这两个微生物基因组的功能图谱。基因编码6种酶，大部分分布在糖苷水解酶(GHs)和糖基转移酶(GTs)中。
表3.细菌EP01抗菌物质基因簇的基因组探测结果
Figure BDA0002447525200000091
Figure BDA0002447525200000101
Figure BDA0002447525200000111
Figure BDA0002447525200000121
Figure BDA0002447525200000131
Figure BDA0002447525200000141
实施例4：旱莲草对C57小鼠肠道菌群的调节
实验动物为2月龄雌性健康小鼠C57BL/6J，由中国北京华富康有限公司提供。小鼠在中国医学科学院放射医学研究所(天津)饲养，室温(22±2℃)，相对湿度58-65％，光周期12h交替。在这项研究中，每组测试3只相同处理的小鼠。
旱莲草提取物的制备：称取2000g旱莲草，剪碎，8倍量70％乙醇回流提取两次，每次1.5h。合并滤液后，在60℃下旋转蒸发蒸馏至稠浸膏，随后60℃真空干燥得旱莲草提取物干膏200g。所得旱莲草提取物4℃保存备用。
如表4所示，实验共分三组，对照组(0周)、对照组(4周)和给药组(4周)，分别以三组实验小鼠的粪便作为检测样品。
对照组(0周)：计时0点健康小鼠。
给药组(4周)：旱莲草给药组剂量为4.8g·kg-1，相应的提取物剂量为0.48g·kg-1(表4)。按每10g体重灌服0.1mL旱莲草提取物计算，每只小鼠平均体重按40g计算。每天定时灌服。将上述提取物溶于水，小鼠灌胃给药连续4周。
对照组(4周)：与给药组给与旱莲草提取物相比，给予同体积的纯水。每天定期喂养每只小鼠，连续喂养4周。饲料满足SPF级饲养和营养需要。
表4.旱莲草动物实验给药分组及剂量
分组(给药/时间) 分组缩写 生药量 提取物剂量
对照组(0周) Control - -
对照组(4周) CO4 - -
旱莲草给药组 EP4 4.8g·kg-1 0.48g·kg-1
实验结果：
结果表明本研究的测序深度涵盖小鼠肠道菌群中所有物种(图 4a)。基于UniFrac的β多样性PCoA显示了每个治疗组的微生物群组成的明显聚类，旱莲草给药后菌群丰度和多样性均提高(图4b)。图4c显示了小鼠肠道菌群中最丰富的门，占有效序列的98.7％，在门的水平上，硬壁菌、拟杆菌和变形菌构成了所有样品中的三个优势门。与对照组小鼠肠道菌群相比，给药组的特点是硬壁菌增多，拟杆菌和变形菌减少，并且细菌种类丰富多样。热图显示了肠道菌群中含量排名前35个属的水平(图4d)。其中，如表5所示，对照组4周与0 周相比菌群存在变化，因此将给药组4周与对照组0周和4周均进行比较，将给药组处理后在肠道菌群所占比例高于对照组(0周和4周) 的菌群列于表5中。在旱莲草给药组小鼠的肠道菌群中，芽孢杆菌、乳杆菌、嗜臭菌、乳球菌、奇异菌、普氏菌UCG-004等菌属所占比例均高于对照组(0周和4周)；瘤胃菌属、厌氧棍状菌、未鉴定的瘤胃球菌科、瘤胃梭菌、普氏菌UCG-001、不动杆菌、拟杆菌、副拟杆菌等菌属所占比例均低于对照组(0周和4周)。其中，旱莲草给药组的乳球菌和芽孢杆菌丰度均高于对照组。而在实施例2中已经证实，旱莲草内生菌中富含芽孢杆菌属和乳球菌属。尤其，具有抑菌作用的内生细菌EP01为乳球菌属。因此，以上实验结果证明了旱莲草提取物调节肠道菌群的作用与旱莲草的内生菌相关。尤其是，本发明分离鉴定出的内生细菌EP01更是起到了显著调节肠道菌群的作用，使肠道菌群中的益生菌比例增加，更接近健康的肠道菌群比例。例如，芽孢杆菌、乳杆菌、乳球菌均为已报道的肠道益生菌所在菌属，有益于健康【49-51】。
表5.旱莲草施用后菌属在肠道菌群所占比例高于对照组(0周和4周) 的菌属
Figure BDA0002447525200000161
表6.旱莲草施用后菌属在肠道菌群所占比例低于对照组(0周和4周) 的菌属
Figure BDA0002447525200000162
Figure BDA0002447525200000171
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
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序列表
<110> 天津中医药大学
<120> 旱莲草内生细菌、旱莲草组合物及其应用
<130> IDC190472
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<170> PatentIn version 3.5
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Met Lys Val Ala Ile Val Thr Gly Gly Thr Arg Gly Ile Gly Arg Ser
1 5 10 15
Ile Ser Ile Glu Leu Tyr Lys Glu Gly Tyr Lys Val Ile Ala Ile Tyr
20 25 30
Gln Gly Asn Lys Ala Ala Ala Gln Ser Leu Ala Asp Glu Tyr Pro Asp
35 40 45
Ile Asp Val Gln Thr Cys Asp Ile Ser Arg Glu Lys Asp Val Ser Lys
50 55 60
Ile Ile Lys Thr Ile Tyr Glu Thr Tyr Gly Arg Ile Asp Cys Leu Ile
65 70 75 80
Asn Asn Ala Gly Ile Thr Arg Asp Gly Tyr Phe Leu Met Met Ser Thr
85 90 95
Glu Lys Trp Gly Thr Val Leu Asn Val Asn Ile Leu Gly Ala Val Asn
100 105 110
Ile Ser Lys Ser Val Leu Arg Tyr Met Lys Val Lys Lys Asn Gly Gly
115 120 125
Lys Val Val Asn Ile Ser Ser Thr Ser Gly Val Thr Gly Gln Ile Gly
130 135 140
Gln Ala Asn Tyr Ser Ala Thr Lys Gly Ala Ile Ile Ser Leu Thr Lys
145 150 155 160
Ser Trp Ala Lys Glu Phe Ala Ala Asp Asn Ile Asn Val Asn Cys Val
165 170 175
Ser Pro Gly Phe Ile Asn Thr Asp Met Thr Ser Asn Leu Lys Asn Lys
180 185 190
Ala Glu Ile Ile Glu Asn Leu Ile Pro Leu Gln Arg Phe Gly Asp Pro
195 200 205
Gln Glu Val Ala Trp Leu Val Ala Phe Leu Ser Ser Asp Lys Ala Asn
210 215 220
Tyr Ile Thr Gly Lys Asn Phe Val Ile Asp Gly Gly Met Ile Asn Asp
225 230 235 240
<210> 5
<211> 486
<212> PRT
<213> Lactococcus garvieae
<400> 5
Met Lys Lys Phe Glu Gln Thr Phe Pro Glu Asn Phe Leu Trp Gly Gly
1 5 10 15
Ala Ile Ala Ala Cys Gln Ser Glu Gly Ala Phe Asp Leu Asp Gly Lys
20 25 30
Gly Leu Thr Val Ala Asp Ile Ala Val Lys His Asp Lys Thr Val Pro
35 40 45
Arg Ser Val Arg Lys Leu Ile Thr Ala Glu Arg Leu Glu Lys Val Leu
50 55 60
Gln Glu Thr Asp Asp Arg Lys Phe Pro Lys Arg His Gly Ile Asp Phe
65 70 75 80
Tyr His Arg Phe Lys Glu Asp Ile Ala Trp Cys Ala Glu Met Gly Phe
85 90 95
Lys Val Phe Arg Phe Ser Ile Ala Trp Gly Arg Ile Phe Pro Asn Gly
100 105 110
Asp Asp Ala Lys Ala Asn Met Lys Gly Leu Ala Phe Tyr Asp Gln Val
115 120 125
Ile Ala Glu Val Ile Lys His Gly Met Glu Pro Leu Ile Thr Ile Asn
130 135 140
His Phe Asp Met Pro Met Asn Leu Val Thr Lys Tyr Gly Gly Trp Lys
145 150 155 160
Asn Arg Gln Leu Ile Asp Phe Tyr Val Thr Tyr Ala Lys Thr Leu Phe
165 170 175
Glu Arg Tyr Gly Glu Lys Val Asn Tyr Trp Ile Ala Phe Asn Glu Ile
180 185 190
Asn Gly Ala Arg Phe Asn Val Phe Tyr Ser Thr Gly Ile Val Lys Asp
195 200 205
Asp Glu Ala Thr Tyr Leu Gln Asp Cys Tyr Gln Ala Ala His His Gln
210 215 220
Phe Val Ala Ser Ala Leu Ala Lys Lys Ala Leu Ser Lys Val Ser Lys
225 230 235 240
Glu Ala Met Leu Gly Cys Met Val Ala Lys Phe Thr Thr Tyr Pro Ala
245 250 255
Thr Cys Lys Pro Glu Asp Ala Leu Glu Ala Gln Lys Asn Glu Gln Met
260 265 270
Asp Asn Tyr Tyr Phe Thr Asp Thr Ile Ile Arg Gly Glu Tyr Pro Pro
275 280 285
Tyr Ala Ala Arg Phe Trp His Glu Asn Gly Ile Glu Leu Gln Ile Phe
290 295 300
Glu Glu Asp Glu Ala Val Leu Lys Arg Tyr Pro Ala Asp Phe Leu Ala
305 310 315 320
Phe Ser Tyr Tyr Met Ser Ser Ile Ser Ala Ala Asp Lys Glu Asn Phe
325 330 335
Glu Glu Thr Asp Gly Asn Leu Lys Asn Thr Leu Lys Asn Pro His Leu
340 345 350
Lys Ala Ser Glu Trp Gly Trp Gln Ile Asp Pro Ile Gly Leu Arg Tyr
355 360 365
Thr Leu Asn Asp Leu Tyr Asn Arg Tyr Ser Ile Pro Leu Phe Ile Val
370 375 380
Glu Asn Gly Ile Gly Ala Glu Asp Val Leu Glu Asp Gly Lys Ile Asp
385 390 395 400
Asp Asp Tyr Arg Ile Asp Tyr Leu Ser Ala His Ile Lys Gln Ile Glu
405 410 415
Glu Ala Ile Arg Asp Gly Val Gln Leu Ile Gly Tyr Thr Thr Trp Ser
420 425 430
Ala Ile Asp Ile Val Ser Ser Gly Thr Ser Glu Met Ala Lys Arg Tyr
435 440 445
Gly Phe Ile Tyr Val Asp Gln Asp Asp Leu Gly His Gly Thr Leu Lys
450 455 460
Arg Ile Pro Lys Lys Ser Phe His Trp Tyr Lys Ala Val Ile Ala Ala
465 470 475 480
Asn Gly Leu Gly Leu Asp
485
<210> 6
<211> 555
<212> PRT
<213> Lactococcus garvieae
<400> 6
Met Thr Asp Lys Lys Phe Gly Ser Asp Leu Val Val Asp Ser Leu Ile
1 5 10 15
Asn His Asn Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp
20 25 30
Arg Val Phe Asp Arg Leu Glu His Asp Ser Lys Ala Pro Lys Leu Ile
35 40 45
Val Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly
50 55 60
Arg Ile Thr Gly Glu Pro Gly Val Val Ala Val Thr Ser Gly Pro Gly
65 70 75 80
Val Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Gly Asp
85 90 95
Ala Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys
100 105 110
Arg Ala His Gln Ser Met Asp Asn Ala Gln Met Met Ala Pro Ala Ser
115 120 125
Lys Tyr Ser Ser Glu Ile Leu Asp Pro Ser Thr Ile Ser Glu Thr Ile
130 135 140
Ala Asn Ala Tyr Arg Ile Ala Lys Ala Gly Arg Ala Gly Ser Ser Phe
145 150 155 160
Leu Ser Ile Pro Gln Asp Val Ile Asp Ser Glu Val Ser Val Asn Ala
165 170 175
Ile Lys Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp
180 185 190
Ile Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile
195 200 205
Leu Val Gly Ala Gly Gly Ser Asp Glu Lys Val Ala Lys Ala Leu Arg
210 215 220
Asn Leu Leu Ser His Val Ser Ile Pro Val Val Glu Thr Phe Gln Gly
225 230 235 240
Ala Gly Val Ile Ser Arg Asp Leu Glu His Thr Phe Phe Gly Arg Ile
245 250 255
Gly Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp
260 265 270
Leu Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn
275 280 285
Trp Asn Ala Glu Ile Asp Ser Arg Ile Val Val Ile Asp Asn Ala Ile
290 295 300
Ala Glu Ile Asp Thr Tyr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp
305 310 315 320
Ile Ala Gln Thr Leu Glu Asn Leu Leu Pro Ala Val Arg Gly Tyr Asn
325 330 335
Leu Pro Glu Gly Ser Val Glu Tyr Leu Lys Gly Leu Arg Asp Val Ile
340 345 350
Asn Gln His Glu Phe Asp Ile Glu Ala Ala Glu Glu Gly Arg Leu His
355 360 365
Pro Leu Asp Leu Val Asn Thr Phe Gln Glu Val Val Lys Asp Asp Glu
370 375 380
Thr Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg Tyr
385 390 395 400
Phe Arg Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln
405 410 415
Thr Leu Gly Ile Ala Leu Pro Trp Gly Ile Thr Ala Ala Leu Leu Arg
420 425 430
Pro Gly His Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe
435 440 445
Thr Gly Gln Glu Leu Glu Val Ala Val Arg Leu Asn Leu Pro Leu Ile
450 455 460
His Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu
465 470 475 480
Glu Lys Lys Tyr Gly Arg Ala Ala Gly Val Asn Phe Gly Ser Val Asp
485 490 495
Tyr Val Lys Tyr Ala Glu Ala Met Gly Ala Thr Gly His Arg Val His
500 505 510
Ser Ala Lys Glu Leu Lys Glu Leu Leu Glu Ser Ile Pro Asn Thr Thr
515 520 525
Gly Pro Val Val Ile Asp Val Pro Ile Asp Tyr Ser Asp Asn Ile Lys
530 535 540
Leu Ala Asp Thr Leu Leu Pro Glu Glu Phe Tyr
545 550 555
<210> 7
<211> 240
<212> PRT
<213> Lactococcus garvieae
<400> 7
Met Lys Val Ala Ile Val Thr Gly Gly Thr Arg Gly Ile Gly Arg Ser
1 5 10 15
Ile Ser Ile Glu Leu Tyr Lys Glu Gly Tyr Lys Val Ile Ala Ile Tyr
20 25 30
Gln Gly Asn Lys Ala Ala Ala Gln Ser Leu Ala Asp Glu Tyr Pro Asp
35 40 45
Ile Asp Val Gln Thr Cys Asp Ile Ser Arg Glu Lys Asp Val Ser Lys
50 55 60
Ile Ile Lys Thr Ile Tyr Glu Thr Tyr Gly Arg Ile Asp Cys Leu Ile
65 70 75 80
Asn Asn Ala Gly Ile Thr Arg Asp Gly Tyr Phe Leu Met Met Ser Thr
85 90 95
Glu Lys Trp Gly Thr Val Leu Asn Val Asn Ile Leu Gly Ala Val Asn
100 105 110
Ile Ser Lys Ser Val Leu Arg Tyr Met Lys Val Lys Lys Asn Gly Gly
115 120 125
Lys Val Val Asn Ile Ser Ser Thr Ser Gly Val Thr Gly Gln Ile Gly
130 135 140
Gln Ala Asn Tyr Ser Ala Thr Lys Gly Ala Ile Ile Ser Leu Thr Lys
145 150 155 160
Ser Trp Ala Lys Glu Phe Ala Ala Asp Asn Ile Asn Val Asn Cys Val
165 170 175
Ser Pro Gly Phe Ile Asn Thr Asp Met Thr Ser Asn Leu Lys Asn Lys
180 185 190
Ala Glu Ile Ile Glu Asn Leu Ile Pro Leu Gln Arg Phe Gly Asp Pro
195 200 205
Gln Glu Val Ala Trp Leu Val Ala Phe Leu Ser Ser Asp Lys Ala Asn
210 215 220
Tyr Ile Thr Gly Lys Asn Phe Val Ile Asp Gly Gly Met Ile Asn Asp
225 230 235 240
<210> 8
<211> 161
<212> PRT
<213> Lactococcus garvieae
<400> 8
Met Lys Lys Leu Asn Ile Thr Gln Ile Met Ala Leu Leu Pro His Thr
1 5 10 15
Tyr Pro Phe Leu Leu Ile Asp Ser Val Glu Asn Tyr Ser Gly Gly Glu
20 25 30
Ile Leu Cys Arg Lys Asn Val Thr Ile Asn Glu Pro Phe Phe Gln Gly
35 40 45
His Phe Pro Lys Gln Pro Ile Met Pro Gly Val Leu Leu Ile Glu Cys
50 55 60
Gly Ala Gln Ala Ala Ala Leu Met Tyr Ile Leu Asp Ser Leu Ser Asp
65 70 75 80
Gln Val Asn Ile Ser Gly Thr Asp Phe Glu Lys Leu Cys Leu Glu Glu
85 90 95
Asn Leu Ser Asp Lys Val Gly Tyr Leu Ala Ser Ile Lys Asn Phe Lys
100 105 110
Leu Lys Thr Leu Val Ser Pro Gly Asp Thr Leu Ser Ile Arg Cys Arg
115 120 125
Ser Phe Ile Lys Leu Gly Lys Leu Ser Glu Ile Glu Val Arg Ile Thr
130 135 140
Asn Asp Ser Arg Lys Glu Val Ala Ser Gly Arg Ile Leu Val Ser Gln
145 150 155 160
Lys
<210> 9
<211> 735
<212> PRT
<213> Lactococcus garvieae
<400> 9
Met Gly Asn Gln Val Asn Ile Thr Gly Val Gly Val Val Ser Ser Leu
1 5 10 15
Gly Lys Asn Leu Glu Glu His Lys Lys Asn Leu Phe Ser Gln Thr Glu
20 25 30
Gly Ile Lys Gln Lys Arg Phe Ile Asn Gly Glu His Glu Leu Asn Ser
35 40 45
Tyr Val Gly Ala Val Glu Ser Glu Leu Glu Val Pro Glu Lys Tyr Gln
50 55 60
Gln Glu Thr Arg Asn Phe Lys Phe Ala Phe Leu Ala Phe Glu Glu Ala
65 70 75 80
Ile Ser Ser Ala Asn Leu Gln Leu Glu Glu Glu Lys Asp Lys Lys Ile
85 90 95
Ala Ile Cys Leu Gly Thr Ser Leu Gly Gly Lys Val Leu Gly Gln Glu
100 105 110
Leu Leu Tyr Ser Phe Glu Asp Asn Arg Leu Asp Met Ser Asn Asp Leu
115 120 125
Phe Lys Lys Arg Ser Leu His Tyr Ile Ala Asp Glu Leu Ile Ala Tyr
130 135 140
His Gly Ile Lys Ala Ser Tyr Tyr Val Ile Ser Thr Ala Cys Ser Ala
145 150 155 160
Ser Asn Asn Ala Val Ile Leu Gly Thr Gln Leu Leu Gln Asp Gly Lys
165 170 175
Cys Asp Ile Ala Ile Cys Gly Gly Cys Asp Glu Leu Ser Asp Ile Ser
180 185 190
Leu Ala Gly Phe Thr Ser Leu Gly Ala Ile Asn Lys Glu Ser Pro Cys
195 200 205
Gln Pro Tyr Ser Thr Gly Ser Gly Ile Ser Leu Gly Glu Gly Ala Gly
210 215 220
Phe Ile Val Leu Glu Lys Asn Lys Val Gly Lys Tyr Gly Lys Val Leu
225 230 235 240
Gly Gly Ser Ile Thr Ser Asp Ala Tyr His Ile Thr Ala Pro Lys Ala
245 250 255
Thr Gly Glu Gly Ala Gly Gln Ile Ala Leu Asn Leu Ser Glu Gln Ser
260 265 270
Gly Val Pro Leu Asn Lys Val Asp Tyr Ile Asn Gly His Gly Thr Gly
275 280 285
Thr Arg Ala Asn Asp Gly Met Glu Lys Ser Met Ile Gly Lys Asn Phe
290 295 300
Pro Leu Thr Val Arg Val Ser Ser Thr Lys Gly Gln Thr Gly His Thr
305 310 315 320
Leu Gly Ala Ala Gly Ile Ile Glu Thr Ile Asn Cys Ile Leu Ala Ile
325 330 335
Glu Glu Lys Thr Val Pro Ala Thr Lys Thr Leu Asp Ser Tyr Gln Glu
340 345 350
Asp Cys Phe Val Arg Asn Gln Asn Ile Pro Lys Asp Ile Ser Tyr Ala
355 360 365
Leu Asn Phe Ser Phe Ala Phe Gly Gly Asn Asn Ser Gly Val Leu Leu
370 375 380
Ala Ser Lys Asp Thr Pro Glu Ala Thr Phe Leu Ala Ser Glu Asp Ile
385 390 395 400
His Met Asp Ile Ile Ser His Ala Ser Thr Leu Glu Ser Leu Glu Asp
405 410 415
Lys Ser Ser Arg Tyr Glu Asn Ile Ser Lys Ser Phe Ser Lys Ile Arg
420 425 430
Gly Leu Arg Tyr Thr Asp Phe Ser Pro Ser Pro Lys Ile Asn Pro Ala
435 440 445
Gln Tyr Arg Arg Met Asp Asn Phe Ser Lys Met Ile Ala Gly Thr Val
450 455 460
Ala Lys Ala Ile Glu Arg Ser Ser Leu Asp Phe Lys Lys Ile Asp Leu
465 470 475 480
Asn Lys Val Gly Ile Leu Phe Thr Thr Pro Ser Gly Pro Leu Glu Val
485 490 495
Val Glu Asp Ile Glu Lys Gln Ile Arg Val Glu Gly Tyr His Gln Val
500 505 510
Ser Ala Ser Lys Phe Pro Phe Thr Val Met Asn Ala Ala Ala Gly Met
515 520 525
Leu Ser Ile Leu Phe Lys Ile Lys Gly Pro Val Ser Val Ile Ser Ser
530 535 540
Asn Val Gly Phe Ala Asp Gly Ile Ile Tyr Ala Glu Glu Met Met Lys
545 550 555 560
Asn Glu Lys Leu Ser His Val Leu Ile Val Ser Ala Ser Gln Trp Thr
565 570 575
Asp Phe Ser Leu Phe Ala Trp Glu Lys Leu Gly Tyr Asp Pro Glu Lys
580 585 590
Phe Ser Pro Ser Asp Tyr Cys Ser Thr Leu Ile Val Gly Asn Ser Ser
595 600 605
Glu Lys Arg Glu Ala Arg Ile Leu Gly Ala Glu Gln Ile Lys Tyr Asp
610 615 620
Asn Lys Val Arg Thr Asn Glu Glu Ile Lys His Asp Leu Ile Ser Leu
625 630 635 640
Ile Glu Ala Glu Leu Ser Ile Lys Gly Ile Pro Phe Asp Thr Leu Lys
645 650 655
Gly Val Val Trp Asn Ala Asn Lys Lys Thr Glu Glu Lys Glu Tyr Asp
660 665 670
Val Leu Arg Asp Trp Leu Ala Ala Leu Asp Ile Pro Val Tyr Ser Leu
675 680 685
Asp Glu Leu Asp Phe Ser Ser Asp Gly Ala Gly Glu Glu Leu Asp Tyr
690 695 700
Val Leu Asn Lys Glu Asp Leu Lys Leu Gly Asp Tyr Leu Ile Ile Ser
705 710 715 720
Tyr Ser Arg Phe Gly Gly Leu Ser Thr Val Leu Leu Glu Lys Ile
725 730 735
<210> 10
<211> 385
<212> PRT
<213> Lactococcus garvieae
<400> 10
Met Asn Leu Gly Glu Ser Gln Arg Leu Ser Gln Ile Ala Ser Ala Leu
1 5 10 15
His Gln Ala Gly His Glu Ile His Ile Leu Gly Glu Val Phe Tyr Pro
20 25 30
Phe Leu Phe Gln His Asp Ala Tyr Ile Phe His Gln Cys Pro Phe Asp
35 40 45
Thr Gln Val Tyr Cys Arg Glu Arg Tyr Gln Glu Phe Phe Ser Phe Asp
50 55 60
Met Asp Phe Asn Phe Leu Thr Asp Glu Glu Ile Arg Asn Met Cys Asp
65 70 75 80
Tyr Glu Arg Glu Leu Leu Val Asn Gln Gln Phe Asp Ala Val Phe Thr
85 90 95
Gly Tyr Arg Leu Ser Ile Val Val Ser Cys Lys Ile Glu Lys Ile Pro
100 105 110
Leu Val Trp Ile Ile Ser Gly Ser Val Gln Ile Asp Glu Ile Ile His
115 120 125
Asn Ile Asp Gly Ile Ile Pro Gly His Met Ser Phe Asp Lys Glu Asn
130 135 140
Glu Lys Ala Lys Lys Ser Ile Lys Arg Leu Val Leu Ser Tyr Ser Lys
145 150 155 160
Ser Val Glu Ser Trp Asn Lys Tyr Leu Ile Ser Glu Gly Ala Leu Pro
165 170 175
Met Lys Ser Gly Leu Glu Leu Phe Ile Gly Asn Leu Asn Leu Ile Ala
180 185 190
Asp Tyr Ser Lys Phe Tyr Ser Phe Asp Ser Glu Ser Asn Tyr Lys Ile
195 200 205
Val Gly Pro Ile Leu Phe Ser Ser Lys Gln Ser Asp Phe Arg Arg Leu
210 215 220
Lys Gly Glu Asn Arg Ile Leu Leu Ser Phe Gly Thr Ser Phe Asn Lys
225 230 235 240
Glu Trp Val Ile Asp Phe Val Ser His Leu Pro Lys Gly Pro Ser Tyr
245 250 255
Ile Leu Thr Thr Ser Gly Glu Glu Phe Tyr Ile Asp Arg Lys Asp Ile
260 265 270
Glu Leu Val Asp Phe Val Asp Phe Gly Ser Leu Arg Ala Lys Ile Ser
275 280 285
Phe Ala Ile Ile His Gly Gly Gln Gly Thr Val Tyr Ala Met Ala Gln
290 295 300
Gln Gly Ile Pro Phe Ile Gly Ile Pro Phe Phe Asn Glu Gln Leu Trp
305 310 315 320
Asn Ile Lys Lys Phe Ser Asp Tyr Gly Ala Ala Met Leu Leu Ser Lys
325 330 335
Gly Glu Lys Ile Ser Glu Lys Met Ser Met Phe Gln Ser Glu Ile Glu
340 345 350
Ala Tyr Ser Glu Lys Met Ser Glu Ile Asn Arg Gly Ile Phe Glu Glu
355 360 365
Ser Gln His Ser Leu Thr Lys Val Ser Glu Ile Leu Glu Ser Phe Leu
370 375 380
Glu
385
<210> 11
<211> 363
<212> PRT
<213> Lactococcus garvieae
<400> 11
Met Ala Asn Ile Asn Glu Val Thr Arg Glu Ser Trp Ile Leu Asn Thr
1 5 10 15
Phe Pro Glu Trp Gly Thr Trp Leu Asn Glu Glu Ile Ala Ala Thr Val
20 25 30
Val Pro Glu Glu Asn Phe Ser Met Trp Trp Leu Gly Asn Cys Gly Leu
35 40 45
Trp Ile Lys Thr Pro Ala Ala Ala Asn Ile Val Met Asp Leu Trp Ser
50 55 60
Ser Arg Gly Lys Ser Ser Lys Lys Ile Lys Asn Met Val Arg Gly His
65 70 75 80
Gln Met Ala Asn Met Ala Gly Ala Arg Gln Leu Gln Pro Asn Leu Arg
85 90 95
Val Gln Pro Met Val Ile Asp Pro Phe Ala Ile Glu Glu Leu Asp Tyr
100 105 110
Tyr Leu Val Ser His Phe His Ser Asp His Ile Asp Leu Ser Thr Ala
115 120 125
Ala Ala Ile Val Asn Asn Pro Lys Leu Lys His Val Lys Leu Val Gly
130 135 140
Pro Tyr Glu Cys Gly Glu Ile Trp Lys Lys Trp Gly Val Pro Glu Lys
145 150 155 160
Arg Ile Ile Ile Val Lys Pro Gly Glu Ala Phe Thr Val Lys Asp Val
165 170 175
Thr Val His Ala Val Glu Ser Phe Asp Arg Thr Cys Leu Val Thr Leu
180 185 190
Pro Val Glu Gly Ala Glu Ser Gln Gly Gly Glu Leu Ser Gly Leu Thr
195 200 205
Ile Thr Asp Ala Glu Met Ala Arg Lys Ala Val Asn Tyr Ile Phe Glu
210 215 220
Thr Pro Gly Gly Thr Ile Tyr His Gly Ala Asp Ser His Phe Ser Asn
225 230 235 240
Tyr Phe Ala Lys His Gly Lys Asp Phe Ser Ile Asp Val Ala Leu Asn
245 250 255
Asn Tyr Gly Glu Asn Pro Arg Gly Ile Gln Asp Lys Met Thr Ser Val
260 265 270
Asp Leu Leu Arg Met Ala Glu Asn Leu Gln Ala Lys Val Ile Ile Pro
275 280 285
Val His Tyr Asp Ile Trp Ser Asn Phe Met Ala Ser Thr Ala Glu Ile
290 295 300
Leu Glu Leu Trp Lys Met Arg Lys Glu Arg Leu Gln Tyr Asp Phe His
305 310 315 320
Pro Phe Ile Trp Glu Val Gly Gly Lys Tyr Thr Tyr Pro Leu Asp Lys
325 330 335
Glu Lys Ile Glu Tyr His His Pro Arg Gly Phe Glu Asp Cys Phe Glu
340 345 350
Glu Asp Ser Asn Ile Gln Phe Lys Ala Leu Leu
355 360
<210> 12
<211> 224
<212> PRT
<213> Lactococcus garvieae
<400> 12
Met Met Lys Met Asn Lys Lys Leu Pro Asn Leu Gln Val Ala Leu Asp
1 5 10 15
His Ser Glu Leu His Ser Ala Leu Lys Ala Ala Val Ser Val Gly His
20 25 30
Glu Val Asp Ile Ile Glu Ala Gly Thr Val Cys Leu Leu Gln Val Gly
35 40 45
Ser Gln Leu Val Glu Ile Leu Arg Gly Leu Phe Pro Asp Lys Thr Leu
50 55 60
Val Ala Asp Thr Lys Cys Ala Asp Ala Gly Glu Thr Val Ala Lys Asn
65 70 75 80
Asn Ala Leu Cys Gly Ala Asn Trp Met Thr Cys Ile Cys Ser Ala Thr
85 90 95
Leu Pro Thr Met Lys Ala Ala Arg Lys Ala Ile Gln Glu Ile Asn Asp
100 105 110
Lys Asn Gly Glu Ile Gln Val Glu Leu Tyr Gly Asp Trp Thr Tyr Glu
115 120 125
Gln Ala Lys Thr Trp Leu Asp Ala Gly Ile Ser Gln Val Ile Tyr His
130 135 140
Gln Ser Arg Asp Ala Leu Leu Glu Gly Lys Thr Trp Ser Glu Lys Asp
145 150 155 160
Leu Asp Lys Ile Lys Lys Leu Ile Asn Met Gly Phe Arg Val Ser Val
165 170 175
Thr Gly Gly Leu Ala Thr Glu Asn Leu Glu Leu Phe Arg Gly Leu Asp
180 185 190
Ile Phe Thr Phe Ile Val Gly Arg Gly Ile Thr Glu Ala Pro Lys Pro
195 200 205
Ala Glu Ala Ala Gln Glu Phe Lys Ala Glu Ile Leu Arg Ile Trp Gly
210 215 220
<210> 13
<211> 287
<212> PRT
<213> Lactococcus garvieae
<400> 13
Met Thr Arg Pro Ile Gly Ile Tyr Glu Lys Ala Met Pro Arg His Phe
1 5 10 15
Ser Trp Lys Glu Arg Leu Ala Phe Ala Lys Glu Leu Gly Phe Asp Phe
20 25 30
Val Glu Met Ser Val Asp Glu Asp Asp Ser Arg Leu Ala Arg Leu Asn
35 40 45
Trp Thr Lys Glu Glu Arg Leu Glu Val Val Gln Ala Ile Tyr Glu Thr
50 55 60
Gly Ile Arg Ile Gln Ser Leu Cys Phe Ser Ala His Arg Arg Phe Pro
65 70 75 80
Leu Gly Ser Asn Asn Pro Glu Thr Glu Ala Lys Ser Leu Lys Ile Met
85 90 95
Glu Gln Cys Ile Glu Leu Ala Gln Asp Leu Gly Ile Arg Val Ile Gln
100 105 110
Val Ala Gly Tyr Asp Val Tyr Tyr Glu Glu Lys Ser Thr Glu Thr Arg
115 120 125
Ala Arg Tyr Ile Lys Asn Leu Arg Lys Val Cys Asn Trp Ala Glu Gln
130 135 140
Thr Gln Val Ile Leu Ala Val Glu Thr Met Asp Asp Pro Phe Ile Asn
145 150 155 160
Ser Val Gln Lys Tyr Leu Thr Ile Glu Lys Glu Val Ala Ser Pro Tyr
165 170 175
Leu Phe Val Tyr Pro Asp Thr Gly Asn Ile Ser Ala Trp His Asn Asp
180 185 190
Ile Tyr Ser Glu Leu Phe Leu Gly His Leu Ser Ile Ala Gly Leu His
195 200 205
Leu Lys Asp Thr Tyr Ala Val Thr Glu His Ser Lys Gly Gln Phe Arg
210 215 220
Asp Val Pro Phe Gly Lys Gly Ser Val Asp Trp Glu Ile Met Phe Asn
225 230 235 240
Ile Leu Lys Glu Thr Asn Tyr Gln Gly Pro Phe Leu Ile Glu Met Trp
245 250 255
Ser Glu Asn Cys Asp Thr Val Glu Glu Thr Arg Ala Ala Ile Arg Glu
260 265 270
Ala Gln Thr Phe Leu Tyr Pro Leu Ile Glu Lys Ala Gly Leu Lys
275 280 285
<210> 14
<211> 232
<212> PRT
<213> Lactococcus garvieae
<400> 14
Met Ile Leu Glu Glu Leu Arg Lys Arg Val Tyr Ala Ala Asn Val Ser
1 5 10 15
Leu Pro Glu Tyr Gly Leu Val Lys Phe Thr Trp Gly Asn Val Ser Ala
20 25 30
Ile Asp Arg Glu Leu Gly Tyr Val Val Ile Lys Pro Ser Gly Val Glu
35 40 45
Tyr His Asn Leu Arg Pro Asp Asn Met Val Ile Thr Asp Leu Glu Gly
50 55 60
Lys Val Ile Glu Gly Glu Leu Asn Pro Ser Ser Asp Leu Met Thr His
65 70 75 80
Leu Glu Leu Tyr Lys Ala Tyr Pro Glu Ile Gly Gly Val Val His Thr
85 90 95
His Ser Thr Glu Ala Val Gly Trp Ala Gln Ala Gly Arg Asp Ile Pro
100 105 110
Phe Tyr Gly Thr Thr His Ala Asp Tyr Phe Tyr Gly Gly Ile Pro Val
115 120 125
Val Arg Ser Leu Thr Ala Gly Glu Ile His Ser Asp Tyr Glu Arg Lys
130 135 140
Thr Gly Thr Val Ile Ile Glu Thr Leu Arg Glu Arg Gly Ser Ser Tyr
145 150 155 160
Lys Val Val Pro Gly Val Leu Val Arg His His Gly Pro Phe Thr Trp
165 170 175
Gly Lys Thr Pro Glu Glu Ala Val Tyr His Ser Val Val Leu Glu Glu
180 185 190
Val Ala Arg Met Ala Arg Leu Thr Glu Gln Leu Asn Pro Lys Ala Glu
195 200 205
Glu Ala Pro Thr Tyr Leu Leu Asp Lys His Tyr Leu Arg Lys His Gly
210 215 220
Ser Ala Ala Tyr Tyr Gly Gln Lys
225 230
<210> 15
<211> 236
<212> PRT
<213> Lactococcus garvieae
<400> 15
Met Lys Phe Asn Ile Glu Ile Tyr Ser Asp Gly Ala Val Ile Ser Asp
1 5 10 15
Met Ile Ala Met Lys Glu Ala Gly Leu Ile Ser Gly Phe Thr Thr Asn
20 25 30
Pro Ser Leu Met Lys Gln Ala Gly Val Thr Asp Tyr Met Ser Phe Val
35 40 45
Gln Glu Ala Leu Gly Lys Ile Glu Gly Met Pro Ile Ser Phe Glu Val
50 55 60
Phe Gly Asp Asp Phe Ala Thr Met Glu Lys Glu Ala Leu Lys Leu Ala
65 70 75 80
Ala Leu Gly Asn Asn Val Phe Val Lys Ile Pro Ile Met Asn Thr Arg
85 90 95
Gly Glu Ser Ser Ile Pro Leu Ile Lys Lys Leu Ser Asp Leu Gly Leu
100 105 110
Lys Leu Asn Ile Thr Ala Leu Met Thr Glu Lys Gln Val Gln Asp Thr
115 120 125
Val Asp Ala Leu Thr Thr Gly Val Glu Ser Ile Val Ser Val Phe Ala
130 135 140
Gly Arg Ile Ala Asp Thr Gly Val His Pro Glu Pro Phe Val Lys Lys
145 150 155 160
Ser Val Glu Ile Cys His Arg Lys Glu Gly Val Lys Leu Leu Trp Ala
165 170 175
Ser Thr Arg Glu Val Leu Asn Ile Val Gln Ala Gln Asp Leu Gly Val
180 185 190
Asp Ile Ile Thr Val Pro Pro Ala Leu Leu Asn Lys Leu Ser Met Tyr
195 200 205
His Lys Asp Leu Asn Thr Leu Ser Leu Glu Thr Val Gln Thr Phe Asn
210 215 220
Lys Asp Ile Lys Ala Leu Gly Phe Thr Ile Leu Asp
225 230 235
<210> 16
<211> 695
<212> PRT
<213> Lactococcus garvieae
<220>
<221> misc_feature
<222> (71)..(71)
<223> Xaa can be any naturally occurring amino acid
<400> 16
Met Asp Asn Arg His Ile Glu His Met Lys His Asp Arg Glu Glu Met
1 5 10 15
Glu His His Gly Glu His Asp Met Met Asn His Gly Gly His Met Met
20 25 30
His Met Gly Asn Met Ser Lys Lys Leu Lys Val Ser Ile Ala Leu Met
35 40 45
Phe Pro Leu Leu Ala Ile Ser Asn Ile Ala Gly Phe Gln Leu Phe His
50 55 60
Phe Ser Gly Asp Ser Leu Xaa Leu Ala Ile Ser Asn Ile Ala Gly Phe
65 70 75 80
Gln Leu Phe His Phe Ser Gly Asp Ser Leu Ile Arg Leu Ile Leu Gly
85 90 95
Ser Ile Ile Phe Phe Tyr Gly Gly Thr Pro Phe Phe Ser Gly Ala Arg
100 105 110
Gly Glu Leu Gln Ala Lys Lys Pro Ala Met Met Met Leu Ile Thr Met
115 120 125
Gly Ile Val Val Ala Tyr Leu Tyr Ser Val Tyr Ala Thr Val Val Val
130 135 140
Trp Ala Gly Gly Glu Ala Met Asp Phe Trp Phe Glu Leu Ser Thr Leu
145 150 155 160
Ile Val Ile Met Leu Ala Gly His Ile Ile Glu Met Arg Ala Val His
165 170 175
Arg Ala Gly Asp Ala Leu Lys Asp Leu Ala Ser Leu Ile Pro Lys Lys
180 185 190
Ala His Leu Lys Asp Gly Lys Asp Val Asp Ile Ser Asp Leu Lys Val
195 200 205
Asp Asp Ile Leu Leu Val Lys Gly Asn Glu Arg Ile Pro Ala Asp Gly
210 215 220
Ile Val Ile Ser Glu Thr Ala His Leu Ser Val Asp Glu Ser Met Ile
225 230 235 240
Thr Gly Glu Ser Arg Pro Val Pro Lys Glu Ile Gly Gly Thr Val Tyr
245 250 255
Gly Gly Ser Leu Asn Gln Asn Leu Pro Phe Glu Met Lys Val Thr Ser
260 265 270
Leu Gly Lys Asp Ser Leu Leu Ser Gln Ile Ala Gln Leu Val Glu Lys
275 280 285
Ala Gln Lys Gln Lys Ser Ala Ser Glu Asn Leu Ala Asp Lys Val Ala
290 295 300
Gly Trp Leu Phe Trp Thr Ala Leu Ile Val Gly Val Ala Ser Phe Ile
305 310 315 320
Ile Trp Thr Met Thr Ser Thr Leu Ser Phe Ala Leu Met Thr Ala Val
325 330 335
Ser Val Phe Val Ile Ala Cys Pro His Ala Leu Gly Leu Ala Val Pro
340 345 350
Leu Val Val Ala Arg Leu Thr Asn Ile Ser Ala Lys Asn Gly Leu Leu
355 360 365
Ile Gln Asn Arg Thr Ala Leu Glu Ser Ile His Lys Val Arg Tyr Ala
370 375 380
Leu Met Asp Lys Thr Gly Thr Leu Thr Asp Gly Lys Phe Thr Val Arg
385 390 395 400
Gln Val Val Lys Val Asp Asp Glu Glu Asp Ile Asp Ile Leu Gln Val
405 410 415
Met Ala Ala Leu Glu Asn Gly Ser Thr His Pro Ile Ala Leu Ser Ile
420 425 430
Val His Ala Val Glu Asp Leu Gln Leu Glu Ala Arg Asn Ile Glu Ser
435 440 445
Ile Pro Gly Val Gly Leu Glu Gly Ser Val Gln Gly Lys Asn Tyr Ser
450 455 460
Val Val Ser Tyr Ala Tyr Leu Gln Glu His Ser Ile Ala Val Asp Gln
465 470 475 480
Asp Glu Ile Gln Lys Val Leu Ala Gln Gly Leu Thr Val Ser Phe Leu
485 490 495
Ile Ser Asp Arg Lys Leu His Gly Phe Val Ala Leu Gly Asp Ser Leu
500 505 510
Lys Asp Asp Ala Lys Phe Phe Ile Thr Glu Leu Lys Lys Arg Gly Ile
515 520 525
Leu Pro Ile Met Leu Thr Gly Asp Asn Lys Ala Thr Ala Ala Lys Ile
530 535 540
Ala Lys Asn Leu Asp Met Thr Glu Phe Tyr Ala Glu Leu Lys Pro Glu
545 550 555 560
Asp Lys Ala Arg Ile Val Lys Thr Tyr Gln Glu Lys Gly Asn Val Leu
565 570 575
Phe Ile Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Ala Thr Ser Asn
580 585 590
Leu Gly Ile Ala Ile Gly Gly Gly Thr Ser Val Ala Ile Asn Thr Ala
595 600 605
Asp Val Val Leu Val Asn Ser Asn Pro Ser Asp Val Leu Ala Leu Ile
610 615 620
Glu Ile Ser Lys Arg Ser Asn Arg Lys Met Lys Gln Asn Leu Trp Phe
625 630 635 640
Gly Ala Gly Tyr Asn Ile Val Ala Ile Pro Val Ala Ala Gly Ile Phe
645 650 655
Tyr Pro Thr Phe Gly Ile Ser Ile Asn Pro Leu Ala Ala Ala Val Leu
660 665 670
Met Ser Ile Ser Thr Val Val Val Ser Ile Asn Ala Met Gly Leu Lys
675 680 685
Tyr Glu Lys Ala Gln Glu Lys
690 695
<210> 17
<211> 279
<212> PRT
<213> Lactococcus garvieae
<400> 17
Met Asp Leu Asn Phe Ile Glu Leu Leu Lys Ile Ile Phe Leu Gly Ile
1 5 10 15
Val Glu Gly Ile Thr Glu Trp Leu Pro Ile Ser Ser Thr Gly His Met
20 25 30
Leu Leu Val Asp Ala Phe Ile His Thr Asp Met Thr Gln Ala Phe Lys
35 40 45
Asp Met Phe Phe Val Val Ile Gln Leu Gly Ala Ile Ile Ala Val Ile
50 55 60
Val Glu Phe Trp Ser Lys Met Asn Pro Phe Val Asn Ile Glu Gly Lys
65 70 75 80
Gly Val Arg Ile Lys Arg Ser Val Phe Asn Met Trp Leu Lys Val Ile
85 90 95
Val Ala Phe Leu Pro Thr Ala Val Phe Gly Leu Phe Leu Asn Asp Phe
100 105 110
Leu Glu Glu Asn Phe Gly Thr Pro Leu Thr Ile Ala Ile Met Leu Ile
115 120 125
Ile Tyr Gly Ile Ala Phe Ile Val Val Glu Arg Trp Asn Lys Thr Arg
130 135 140
Gln Pro Lys Ile Thr Ser Ile Lys Gly Leu Thr Tyr Gln Ala Ala Leu
145 150 155 160
Ile Ile Gly Met Phe Gln Val Leu Ser Met Ile Pro Gly Thr Ser Arg
165 170 175
Ser Gly Ala Thr Ile Val Gly Ala Leu Leu Ile Gly Val Ser Arg Val
180 185 190
Val Ala Ala Glu Phe Thr Phe Phe Leu Ala Val Pro Thr Met Leu Gly
195 200 205
Ala Ser Ala Phe Lys Leu Leu Lys Phe Gly Leu His Phe Ser Leu Ser
210 215 220
Glu Val Ile Ala Leu Ile Val Gly Met Ala Val Ala Tyr Ile Val Ser
225 230 235 240
Val Phe Val Ile Lys Phe Leu Met Ser Tyr Ile Lys Arg His Asp Phe
245 250 255
Gln Val Phe Gly Trp Tyr Arg Ile Ala Leu Gly Val Leu Val Ile Ile
260 265 270
Phe Ile Leu Phe Gly Val Ile
275
<210> 18
<211> 452
<212> PRT
<213> Lactococcus garvieae
<400> 18
Met Glu Arg Lys Ala Val Lys Asn Leu Ile Ile Gly Phe Gly Lys Ala
1 5 10 15
Gly Lys Thr Leu Ala Lys Ser Leu Ala Arg Lys Gly Glu Ala Val Ala
20 25 30
Ile Val Glu Gln Ser Pro Arg Met Tyr Gly Gly Thr Cys Ile Asn Ile
35 40 45
Gly Cys Ile Pro Ser Lys Ser Leu Ile Val Asn Gly Gln Lys Gly Glu
50 55 60
Lys Phe Ala Asp Ala Ala Leu Gln Lys Val Asn Leu Val Glu Lys Leu
65 70 75 80
Asn Arg Lys Asn Tyr His Met Ile Ala Asp Glu Pro Thr Ala Thr Val
85 90 95
Ile Asp Gly Lys Ala Arg Phe Leu Ser Asp His Glu Val Glu Ile Ser
100 105 110
Arg Glu Gly Gln Pro Ala Met Leu Val Glu Ala Glu Arg Ile Phe Ile
115 120 125
Asn Thr Gly Ala Arg Ala Ile Leu Pro Ser Ile Leu Gly Met Ser Glu
130 135 140
Cys Lys Asn Leu Val Thr Ser Thr Gly Leu Met Asp Leu Gln Lys Arg
145 150 155 160
Pro Asp His Leu Val Ile Ile Gly Ser Gly Tyr Ile Gly Leu Glu Phe
165 170 175
Ser Ser Met Phe Leu Ser Tyr Gly Ser Arg Val Thr Val Ile Asp Ser
180 185 190
His Arg Thr Phe Leu Pro Arg Gln Asp Arg Asp Ile Ala Ala Arg Val
195 200 205
Lys Lys Asp Leu Glu Glu Ala Gly Ala Val Phe Lys Leu Gly Tyr Glu
210 215 220
Ile Asn Gly Val Phe Asp Arg Asp Glu Lys Ser Ile Leu Asn Ile Thr
225 230 235 240
Phe Asp Gly Gln Ala Glu Glu Val Glu Ala Asp Lys Ile Leu Leu Ala
245 250 255
Thr Gly Arg Lys Pro Asn Ile Ser Gly Leu Gly Leu Glu Asn Thr Ser
260 265 270
Val Lys Ile Gly Glu Asp Gly His Ile Leu Val Asn Glu Lys Leu Glu
275 280 285
Thr Ser Ala Lys Asn Ile Trp Ala Leu Gly Asp Val His Gly Gly Pro
290 295 300
Gln Phe Thr Tyr Thr Ser Leu Asp Asp Tyr Arg Ile Ile Glu Ser Gln
305 310 315 320
Leu Tyr Gly Asp Lys Gly Arg Thr Leu Asp Asn Arg Gly Glu Leu Ala
325 330 335
Ser Ser Val Phe Ile Thr Pro Thr Leu Ser Asn Val Gly Leu Thr Glu
340 345 350
Thr Glu Ala Gln Ala Gln Arg Lys Asn Tyr Arg Leu Phe Arg Leu Glu
355 360 365
Ala Thr Ala Ile Pro Lys Ser Ala Val Leu Lys Gln Ser Lys Gly Leu
370 375 380
Leu Lys Ala Leu Val Asp Pro Glu Thr Glu Glu Ile Leu Gly Ala Thr
385 390 395 400
Leu Tyr Ala Glu Asp Ser His Glu Val Ile Asn Ile Leu Ser Leu Ala
405 410 415
Met Lys Ala Lys Leu Pro Tyr Thr Met Ile Arg Asp His Ile Phe Thr
420 425 430
His Pro Thr Met Ser Glu Ala Leu Asn Asp Leu Phe Ser Asn Ala Asn
435 440 445
Glu Val Lys Lys
450
<210> 19
<211> 381
<212> PRT
<213> Lactococcus garvieae
<400> 19
Met Lys Ile Arg Thr Lys Met Ile His Gly Gly Ile Ser Glu Asp Ala
1 5 10 15
Thr Ser Gly Ala Val Ser Val Pro Ile Tyr Gln Val Ser Thr Tyr Lys
20 25 30
Gln Glu Gly Val Gly Gln Pro Lys Ala Tyr Glu Tyr Ser Arg Ser Gly
35 40 45
Asn Pro Thr Arg His Ala Leu Glu Ser Leu Ile Ala Asp Leu Glu Gly
50 55 60
Gly Val Arg Gly Phe Ala Phe Gly Ser Gly Leu Ala Gly Ile His Ala
65 70 75 80
Val Leu Ser Ile Phe Lys Ala Gly Asp His Ile Ile Met Gly Asn Asp
85 90 95
Val Tyr Gly Gly Thr Phe Arg Leu Met Asp Lys Val Met Gln Asn Phe
100 105 110
Gly Leu Glu Tyr Asp Leu Val Asp Leu Thr Asp Phe Ser Ala Leu Asn
115 120 125
Lys Ala Ile Lys Pro Thr Thr Thr Ala Leu Tyr Phe Glu Thr Pro Ser
130 135 140
Asn Pro Leu Met Lys Ile Leu Asp Ile Arg Gln Leu Thr Glu Val Ala
145 150 155 160
Gln Glu Asn Asn Leu Ile Thr Ile Val Asp Asn Thr Phe Ala Thr Pro
165 170 175
Tyr Phe Gln Arg Pro Leu Ser Leu Gly Ala Asp Ile Val Leu His Ser
180 185 190
Gly Thr Lys Tyr Leu Gly Gly His Ser Asp Val Val Ser Gly Leu Val
195 200 205
Thr Thr Asn Asp Thr Asn Leu Ala Glu Arg Ile Gly Phe Met Gln Asn
210 215 220
Ser Ile Gly Gly Val Leu Gly Pro Gln Asp Ser Trp Leu Val Gln Arg
225 230 235 240
Gly Ile Lys Thr Leu Ala Leu Arg Met Glu Ala His Ala Thr Asn Ala
245 250 255
Gln Lys Ile Ala Glu Phe Leu Glu Lys Ser Pro Gln Val Ser Lys Val
260 265 270
Tyr Tyr Pro Gly Leu Ser Thr Asp Ser Gly Tyr Glu Ile Ala Ser Lys
275 280 285
Gln Met Ser Gly Phe Gly Gly Met Leu Ser Phe Glu Leu Gln Asp Glu
290 295 300
Ser Lys Val Lys Tyr Phe Val Glu Ser Leu Lys Tyr Phe Thr Leu Ala
305 310 315 320
Glu Ser Leu Gly Gly Val Glu Ser Leu Ile Glu Val Pro Ala Val Met
325 330 335
Thr His Ala Ser Ile Pro Lys His Leu Arg Glu Ala Val Gly Ile Lys
340 345 350
Asp Gly Leu Ile Arg Leu Ser Val Gly Ile Glu Asp Ile His Asp Leu
355 360 365
Leu Glu Asp Leu Gln Thr Ala Phe Glu Ile Ile Arg Asn
370 375 380
<210> 20
<211> 131
<212> PRT
<213> Lactococcus garvieae
<400> 20
Met Lys Lys Ile Val Leu Leu Leu Ala Val Leu Ala Leu Gly Thr Val
1 5 10 15
Thr Phe Glu Lys Leu Gly Ile Ala Asp Leu Glu Gln Thr Ser Gly Gly
20 25 30
Leu Thr Ala Glu Gln Leu Glu Lys Arg Gly Glu Val Gln Thr Arg Lys
35 40 45
Val Ala Asn Val Gly Gly Gly Thr Trp Tyr Tyr Met Val Asn Val Asn
50 55 60
Gly Arg Thr Ile Ser Glu Tyr Asn His Arg Asp Arg Lys His Ser Ala
65 70 75 80
Thr Val Ser Arg Thr Asn Gly Val Phe Asn Lys Ser Val Arg Pro Ala
85 90 95
Gly Gln Thr Ala Arg Ala Ser Leu Pro Tyr Lys Ser Gly Ala Asn Ser
100 105 110
Val Tyr Trp Asn Asn Ser Pro Lys Glu Ala Val Gly Asn Tyr Asn Gly
115 120 125
Ala Lys Phe
130

Claims (9)

1.一种旱莲草内生细菌EP01，其保藏于中国微生物菌种保藏管理委员会普通微生物中心，且具有保藏号CGMCC No.19512。
2.一种组合物，其含有权利要求1所述的内生菌。
3.权利要求2所述的组合物，所述组合物为粉末或液体；优选地，所述组合物通过以下方式施用：口服使用、用于栓剂进行施用、用于皮下注射、用于静脉注射，或者作为用于吸入的液体。
4.权利要求2或3所述的组合物在抑制细菌生长中的用途，其中所述细菌包括革兰氏阳性细菌和革兰氏阴性细菌；
优选地，所述革兰氏阳性细菌包括但不限于：葡萄球菌属、链球菌属、梭菌属、放线菌属、奴卡菌属、红球菌属、棒状杆菌属、分支杆菌属、李氏杆菌属、肾杆菌属、炭疽杆菌属、破伤风杆菌属、抗酸菌属；
优选地，所述革兰氏阴性细菌包括但不限于：淋球菌属、脑膜炎双球菌属、卡他菌属、耶尔森菌属、嗜肺军团菌属、志贺菌属、巴斯德菌属、霍乱弧菌属、副溶血性杆菌属、大肠杆菌属、沙门菌属、假单胞菌属、变形杆菌属、布氏杆菌属；
更优选地，所述细菌为金黄色葡球菌和大肠杆菌。
5.旱莲草提取物在制备药物中的用途，所述药物用于调节受试者肠道微生物或治疗与肠道菌群紊乱相关疾病。
6.权利要求5所述的用途，其中，所述旱莲草提取物是通过以下方法制备的：称取一定量旱莲草，经乙醇回流提取，优选70％乙醇回流提取，更优选8倍量70％乙醇回流提取两次，每次1.5h；合并滤液后，干燥得旱莲草提取物。
7.权利要求5或6所述的用途，其中，所述旱莲草提取物调节受试者肠道微生物是通过增加肠道中细菌的数量，所述细菌选自：芽孢杆菌属，乳酸杆菌属，嗜臭菌属，乳球菌属，奇异菌属，普氏菌UCG-004属，或其任何组合。
8.权利要求5或6所述的用途，其中，所述旱莲草提取物调节受试者肠道微生物是通过抑制肠道中细菌的数量，所述细菌选自：瘤胃菌属9，厌氧棍状菌属，瘤胃菌属，普氏菌UCG-001属，不动杆菌，拟杆菌，副拟杆菌，或其任何组合。
9.权利要求4-8任一项所述的用途，所述受试者是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩；优选地，受试者是人。
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