CN112083169A - 斑马鱼感染爱德华氏菌的体液小分子代谢标记物及其应用 - Google Patents
斑马鱼感染爱德华氏菌的体液小分子代谢标记物及其应用 Download PDFInfo
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Abstract
本发明公开了斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,为精氨酸,脯氨酸,缬氨酸,亮氨酸,异亮氨酸,Cadaverine中的至少一种,优选脯氨酸和缬氨酸。本发明还公开了斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在提高斑马鱼应答迟钝爱德华氏菌感染的免疫力中的应用,在调控斑马鱼精氨酸‑脯氨酸代谢通路、缬氨酸‑亮氨酸‑异亮氨酸生物合成代谢通路中的应用。本发明通过实验发现,脯氨酸、缬氨酸在斑马鱼响应迟钝爱德华氏菌感染时起着重要的作用,在斑马鱼先天免疫反应中起着极其重要的功能,可以用于代谢组功能的研究。本发明对斑马鱼响应迟钝爱德华氏菌感染免疫应答相关的代谢组学的研究具有重要的参考价值。
Description
技术领域
本发明涉及斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物及其应用,,属于代谢组学技术领域。
背景技术
代谢组学(metabonomics/metabolomics)是效仿基因组学和蛋白质组学的研究思想,对生物体内所有代谢物进行定量分析,并寻找代谢物与生理病理变化的相对关系的研究方式,是系统生物学的组成部分。其研究对象大都是相对分子质量1000以内的小分子物质。代谢组是指某一生物或细胞在一特定生理时期内所有的低分子量代谢产物,代谢组学则是对某一生物或细胞在一特定生理时期内所有低分子量代谢产物同时进行定性和定量分析的一门新学科,它是以组群指标分析为基础,以高通量检测和数据处理为手段,以信息建模与系统整合为目标的系统生物学的一个分支。先进分析检测技术结合模式识别和专家系统等计算分析方法是代谢组学研究的基本方法。代谢组学是研究代谢组在某一时刻细胞内所有代谢物的集合的一门学科。基因与蛋白质的表达紧密相连,而代谢物则更多地反映了细胞所处的环境,这又与细胞的营养状态,药物和环境污染物的作用,以及其它外界因素的影响密切相关。因此有人认为,“基因组学和蛋白质组学告诉你什么可能会发生,而代谢组学则告诉你什么确实发生了”。
斑马鱼是一种常见的热带鱼。斑马鱼体型纤细,成体长3~4cm,对水质要求不高。孵出后约4个月达到性成熟,成熟鱼每隔几天可产卵一次。卵子体外受精,体外发育,胚胎发育同步且速度快,胚体透明。发育温度要求在25~31℃之间。斑马鱼由于个体小,养殖花费少,能大规模繁育,且具许多优点,吸引了众多研究者的注意。经过30多年的研究应用和系统发展,已有约20个斑马鱼品系,斑马鱼基因数据库里有相关的资料可供查询和下载,方便了研究。斑马鱼的细胞标记技术、组织移植技术、突变技术、单倍体育种技术、转基因技术、基因活性抑制技术等已经成熟,且有数以千计的斑马鱼胚胎突变体,是研究胚胎发育分子机制的优良资源,有的还可做为人类疾病模型。斑马鱼已经成为最受重视的脊椎动物发育生物学模式之一,在其它学科上的利用也显示很大的潜力。由于斑马鱼基因与人类基因的相似度达到87%,这意味着在其身上做药物实验所得到的结果在多数情况下也适用于人体,因此它受到生物学家的重视。因为斑马鱼的胚胎是透明的,所以生物学家很容易观察到药物对其体内器官的影响。此外,雌性斑马鱼可产卵200枚,胚胎在24小时内就可发育成形,这使得生物学家可以在同一代鱼身上进行不同的实验,进而研究病理演化过程并找到病因。
迟钝爱德华氏菌(Edwardsiella tarda,Et)又称迟缓爱德华氏菌或缓慢爱德华氏菌,属于肠杆菌科的爱德华菌属(Edwardsiella),是爱德华氏菌属细菌家族中较早发现的成员之一,该菌是爱德华氏菌属的模式种,其模式为ATCC15947(DSM 30052),DNA中G+Cmol%为55~58(Tm、Bd)。迟钝爱德华氏菌是一种典型的胞内寄生细菌,是目前水产养殖中危害极大的病原菌,它可引起鱼类产生爱德华菌病,使鱼类腹部积水肿胀、体表出血、肠内出现黏液,造成鱼类的大量死亡,严重危害了鱼类的养殖,带来了巨大的经济损失。迟钝爱德华氏菌的感染范围包括鳗鲡,斜带石斑鱼、鲇鱼、鲻鱼、真鲷、牙鲆、大马哈鱼、大菱鲆、鲶鱼、罗非鱼和豹蟾鱼等。使用抗生素可以有效抑制爱德华菌的感染,但抗生素的广泛使用可能会使其产生抗药性,规范与限制使用抗生素可降低毒性反应(Gootz et al.2010)。
目前,调节斑马鱼响应迟钝爱德华氏菌感染免疫应答相关的代谢组在很大程度上尚不清楚。
发明内容
针对上述现有技术,本发明提供了斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,及其应用。
本发明是通过以下技术方案实现的:
斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,为精氨酸,脯氨酸,缬氨酸,亮氨酸,异亮氨酸,Cadaverine中的至少一种,优选脯氨酸和缬氨酸。
所述斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在检测斑马鱼感染迟钝爱德华氏菌中的应用(非诊断目的)。
所述斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在制备用于检测斑马鱼感染迟钝爱德华氏菌的检测试剂盒中的应用。
所述斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在制备治疗斑马鱼感染迟钝爱德华氏菌的药物中的应用。
脯氨酸或/和缬氨酸在作为斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物中的应用。
斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在提高斑马鱼应答迟钝爱德华氏菌感染的免疫力中的应用,在制备用于提高斑马鱼应答迟钝爱德华氏菌感染的免疫力的药物或保健品中的应用。
斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在调控斑马鱼精氨酸-脯氨酸代谢通路中的应用。
斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在调控斑马鱼缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路中的应用。
本发明通过实验发现,脯氨酸、缬氨酸在斑马鱼响应迟钝爱德华氏菌感染时起着重要的作用,在斑马鱼先天免疫反应中起着极其重要的功能,可以用于代谢组功能的研究、斑马鱼感染迟钝爱德华氏菌的检测、治疗等。本发明对斑马鱼响应迟钝爱德华氏菌感染免疫应答相关的代谢组学的研究具有重要的参考价值。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。提及的术语和短语如有与公知含义不一致的,以本发明所表述的含义为准。
附图说明
图1:群体斑马鱼感染EIB202结果,其中,A:对照组;B:垂死组;C:生存组。
图2:群体斑马鱼感染EIB202的存活曲线。
图3:斑马鱼体液代谢物的分类示意图。
图4:代谢组平台技术操作可重复性分析。
图5:斑马鱼感染EIB202体液的代谢组学,其中,A:68种代谢物的非监督模式聚类图;B:3个样本聚类图;C,D,E:3个样本Z值图。
图6:EIB202感染斑马鱼后体液代谢物质变化,其中,A:EIB202感染后发生极显著差异变化的44种代谢物聚类图;B:垂死组与对照组37种代谢物Z值图;C:生存组与对照组33种代谢物Z值图。
图7:斑马鱼感染EIB202显著变化差异的代谢物统计分析,其中,A:垂死组;B:生存组。
图8:PCA模式识别EIB202感染斑马鱼并鉴定重要的生物标志物。
图9:脯氨酸、缬氨酸的原始峰值。
图10:缬氨酸和脯氨酸含量。
图11:EIB202感染斑马鱼代谢通路变化比较,其中,A:生存组;B:垂死组。
图12:EIB202感染斑马鱼代谢通路变化机制。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实验1斑马鱼应答迟钝爱德华氏菌EIB202感染的代谢组学研究
1.1EIB202半数致死剂量感染斑马鱼体液标本的获取
1.1.1半数致死剂量的测定
为了研究群体斑马鱼应对EIB202感染的普遍水平,特别测定了斑马鱼感染后死亡率为50%的EIB202剂量,即半数致死剂量(lethal dose,LD50)。设置不同梯度的EIB202菌数注射斑马鱼,约72h后统计死亡率,结果显示,EIB202注射剂量为2.5×103CFU时,10条斑马鱼的死亡率为30%,剂量为5×103CFU时死亡率为50%,剂量为1×104CFU时死亡率为60%,剂量为5×104CFU时死亡率为80%。
正式试验注射感染42条斑马鱼,攻毒剂量为5×105CFU/mL即2.5×103CFU。同时设置生理盐水对照组,注射同等体积(5L)的生理盐水,对照组注射20条斑马鱼。
1.1.2EIB202半数致死剂量感染对斑马鱼生存的影响
为了研究斑马鱼感染半数致死剂量EIB202时受伤和健康斑马鱼体液小分子代谢物的变化情况,用2.5×103CFU剂量注射感染42条斑马鱼,试验用斑马鱼大小均一,约3cm长(图1)。注射后观察斑马鱼生存情况。注射后约36h开始有斑马鱼出现受伤垂死症状,伤口开始红肿、感染化脓、鱼体发黑(图1B),游向水体的上层。对照组和生存组斑马鱼体表无异常(图1A和C)。72h后逐步稳定不再死亡。存活曲线见图2。注射感染42条斑马鱼,其中死亡22条,死亡率为52.3%,基本为半数死亡。受伤的斑马鱼一旦出现垂死症状,立即取样,收取其体液样品。注射72h后群体斑马鱼稳定下来不再死亡,将对照组和生存组斑马鱼同时切段浸提体液。
1.2数据的可靠性分析和标准化
收集斑马鱼感染LD50剂量EIB202的三组(对照组,垂死组,生存组)体液样品后,运用软件X Calibur software(Thermo fisher 2.1)从总离子色谱图(total ionschromatograms,TIC)标出每一个化合物的单离子峰,共106个,并提取峰面积,每个单离子峰逐一运用NIST MS search 2.0软件从NIST(National Institute of Standards andTechnology)数据库中得到鉴定,并将出峰时间保留。删除硅烷化试剂,具有相同名字的代谢物进行合并,共得到68种小分子代谢物(图3)。其中碳代谢相关代谢物28种,氮代谢相关代谢物21种,脂类代谢相关代谢物9种,核苷酸代谢相关代谢物10种。
为了说明EIB202半数致死剂量感染斑马鱼后的3组数据技术操作的可重复性,分别计算出各组两个技术重复的相关系数,相关系数范围是0.934-0.9997,以相关系数为0.934这组数据作散点图,见图4,由图知,散点图呈线性,相关性好,技术操作是可重复的。
1.3斑马鱼应答EIB202感染的代谢组学分析
1.3.1斑马鱼应答EIB202感染的代谢组
标准化后的数据先用来等级聚类(Hierarchical Clustering),聚类是在R软件的平台上完成的(图5A,B),对照组12个样品,垂死组12个样品,生存组12个样品。图5A是三个组所有代谢物的聚类图,图5B为样本聚类图,三个组的12个样品很好的聚成三支。Z值也称为标准得分,它计算多少代谢物的面积标准差偏离平均值,用来确定对照组样品中每种代谢物的平均值和标准差。对于对照样品,每一代谢物的值减去对照组该代谢物的平均值再除以标准偏差。对于垂死组和生存组来说,Z值即是代谢物的面积减去对照组的平均值再除以标准偏差,这样,处理组和对照组之间的偏差就被精确计算了(图5C)。由图5C可知,垂死组和生存组相对对照组而言有很大的变化,垂死组Z值变化范围为-21.2至64.1,存活组Z值变化范围为-13.6至224。
1.3.2斑马鱼应答EIB202感染的差异代谢组
将68种代谢物四分位标准化后的数据进行SPSS检验,即代谢物之间的差异进行Mann-Whitney检验(Wilcoxon rank sum test)和Kruskal-Wallis(KW)检验。双尾检验p值小于0.05表明差异显著,p值小于0.01表明差异极显著,两种参数检验都是假设数据是非对称分布的。经过SPSS检验,我们从68种代谢物中筛选出37种垂死组差异极显著、33种生存组差异极显著的化合物。做等级聚类热图(图6A)和Z值图(图6B,C)。
垂死组显著差异变化37种差异代谢物中17种为碳代谢相关,其中8种上调9种下调;10种氮代谢相关,1种上调9种下调;5种脂类代谢相关,全部上调;5种核苷酸代谢相关,3种上调2种下调。这表明EIB202感染斑马鱼后垂死组斑马鱼脂类代谢和核苷酸代谢以上调为主,碳代谢和氮代谢均以下调为主(图7A)。生存组显著差异变化的33种代谢物中,16种与碳代谢相关,其中12种上调4种下调;9种与氮代谢相关,其中4种上调5种下调;5种与脂肪酸代谢相关,全部上调;3种与核苷酸代谢相关,全部上调。这个结果表明EIB202感染斑马鱼后生存组的碳代谢、脂类代谢及核苷酸代谢以上调为主,氮代谢以下调为主(图7B)。
将垂死组斑马鱼37种显著差异变化的代谢物运用在线软件MetPA进行通路富集,共得到45个通路,其中差异极显著(p值小于0.01)的有5个,但其中3个Impact值为0;差异显著(p值小于0.05)的通路有5个。表1列出了差异显著但Impact值不为0的7个通路。
表1斑马鱼感染EIB202垂死组差异代谢物通路富集结果
为了研究生存组斑马鱼体液小分子代谢物的变化,将存活生存组和对照组的68种代谢物运用SPSS进行两两比较,生存组显著差异变化的物质共33个,其中L-Pipecolicacid是KEGG检测不到的化合物。
生存组与垂死组相比,碳代谢是相反的趋势,以上调为主,这是二者最大的差异。氮代谢不再大部分下调,但仍过半数下调,是相似的趋势,脂类代谢和核苷酸代谢也是相同的变化趋势。
将生存组斑马鱼37种显著差异变化的代谢物运用在线软件MetPA进行通路富集,共得到31个通路.,其中差异极显著(p值小于0.01)的有1个,但Impact值为0;差异显著(p值小于0.05)的通路有3个,但其中1个Impact值为0,表2列出了差异显著同时Impact值不为0的两个通路。
表2斑马鱼感染EIB202生存组代谢物通路富集结果
如上所述,EIB202半数致死剂量感染斑马鱼后垂死组和生存组与对照组相比差异显著的代谢通路均有Arginine(精氨酸)and proline metabolism(脯氨酸)和Valine(缬氨酸),leucine(亮氨酸)and isoleucine biosynthesis(异亮氨酸)。
1.3.3s-plot模式识别
通过SPSS两两比较的方法分别得出EIB202半数致死剂量感染斑马鱼的垂死组与对照组、生存组与对照组的差异显著物质,用这些差异显著代谢物分别富集代谢通路,得到两个共同差异显著的代谢通路:精氨酸-脯氨酸代谢通路和缬氨酸-亮氨酸-异亮氨酸代谢通路。
现在进一步从代谢物出发,运用多重比较的方法得出EIB202半数致死剂量感染斑马鱼的垂死组、生存组、对照组的差异显著物质,进行三个组的模式识别和模式识别里重要的代谢物分析。
将EIB202感染斑马鱼的3个组标准化的值进行SPSS多重比较,从68种物质筛选得到显著差异变化的45种物质,我们运用PCA(princical component analysis)模式识别的方法进行研究,它是一种非监督模式的分析方法(Wang et al.2012b),包括PCA,PLS-DA(偏最小二乘法投影关联分析)和OPLS-DA(正交偏最小二乘投影判别分析)集成研分析究斑马鱼感染EIB202后小分子代谢物的变化。OPLS-DA分析将包括对照组在内的3个组的小分子代谢物分开在4个象限,区分明确(图8A)。为了鉴别可以进行模式识别的重要代谢物,S-plot里用p(1)0.05和p(corr)(1)0.5双重界定,用p(1)和p(corr)(1)绝对值的高或低来代表各代谢物在3个组别模式识别中的相关性,绝对值越大,则越相关。我们从S-plot分析中得到L-Proline、L-Valine、Cadaverine(图8B)。这说明这3种代谢物在区分三个组时最相关,是斑马鱼应对EIB202感染反应中起着极其重要的功能的代谢物。
L-Proline是精氨酸-脯氨酸代谢通路中重要的差异代谢物,而L-Valine是缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路中重要的差异代谢物,且二者的变化趋势是一致的,垂死组下调而生存组上调,而Cadaverine的变化趋势却相反,垂死组上调而生存组下调。
EIB202感染斑马鱼的三个组的缬氨酸、脯氨酸的峰图的截图如图9,标准化后的缬氨酸、脯氨酸峰面积值散点图如图10,垂死组含量下调而生存组上调。
1.3.4生存组及垂死组差异代谢通路的比较
在精氨酸-脯氨酸代谢通路中,脯氨酸在垂死组中显著下调而在生存组中显著上调;在缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路中,缬氨酸在垂死组中显著下调而在生存组中显著上调(图11)。图中灰色的小分子代谢物未检测到,绿色是下调的代谢物,红色是上调的代谢物。
EIB202感染斑马鱼后,宿主斑马鱼体内Arginine and proline metabolism和Valine,leucine and isoleucine biosynthesis两条代谢通路变化最为显著,其中,脯氨酸和缬氨酸是最重要的差异物质。脯氨酸是一种非必需氨基酸,可通过外界摄取,也可体内合成,脯氨酸经由谷氨酸合成酮戊二酸,至此进入三羧酸循环。而缬氨酸只能从外界摄取,经琥珀酰辅酶A进入三羧酸循环(图12)。脯氨酸和缬氨酸含量的升高可能都会导致宿主三羧酸循环的增强,从而促进呼吸代谢,获得更多的能量供生命活动需要,最终在细菌感染后生存下来。
1.4缬氨酸、脯氨酸对斑马鱼的免疫保护
选择L-脯氨酸、L-缬氨酸作外源添加,添加后再进行EIB202攻毒,统计死亡率,并检验有无显著差异。结果见表3,添加脯氨酸、缬氨酸后死亡率显著降低,说明脯氨酸和缬氨酸可以帮助斑马鱼增强对EIB202感染的抵抗能力。
表3斑马鱼添加脯氨酸、缬氨酸后后应对EIB202感染的相对免疫保护率
*p小于0.05,差异显著
1.5讨论
本发明通过研究感染迟钝爱德华氏菌后垂死和生存状态的斑马鱼个体体内小分子代谢物的变化,寻求能抵御细菌感染的小分子代谢物,以有效减少抗生素的使用。
本发明运用基于GC-MS的代谢组学研究方法,用迟钝爱德华氏菌EIB202半数致死剂量感染斑马鱼,分析感染后垂死斑马鱼和存活斑马鱼体内小分子代谢物的变化,统计显著差异变化的小分子代谢物的分类,结果发现垂死组斑马鱼碳代谢以上调为主氮代谢以下调为主,这可能说明垂死组斑马鱼的能量代谢急剧增加,宿主需要能量,而氮代谢下调从而氨基酸合成减弱,这将会导致免疫蛋白合成的减弱,从而造成斑马鱼免疫力下降,宿主不可避免随感染时间加长逐步死亡;进一步用差异显著的小分子代谢物作通路富集,结果富集到两个同时差异显著的通路:精氨酸-脯氨酸代谢通路和缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路。在精氨酸-脯氨酸代谢通路中,垂死组和生存组中天冬氨酸均下调而尿素上调,唯有脯氨酸在垂死组中显著下调而在生存组中显著上调;在缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路中,垂死组和生存组中异亮氨酸均下调,但缬氨酸在垂死组中显著下调而在生存组中显著上调。
接下来运用PCA对迟钝爱德华氏菌EIB202半数致死剂量感染斑马鱼体液代谢数据进行了模式识别,并确定了模式识别中重要的代谢物:L-Proline、L-Valine、Cadaverine。脯氨酸和缬氨酸与通路富集中富集到的重要代谢物是一致的,属于正调控。
将脯氨酸和缬氨酸外源添加至斑马鱼体内后,再进行EIB202攻毒,结果发现死亡率显著下降,说明脯氨酸和缬氨酸可以帮助斑马鱼抵御EIB202感染。
Antunes等(Antunes et al.2011)以沙门氏菌(salmonella)为病原菌以小鼠作为感染对象,研究了感染沙门氏菌的小鼠粪便和肝脏的代谢组学变化,结果发现,粪便中共检测到924种代谢物,其中58%的物质发生了2倍甚至更高倍数的变化,显示沙门氏菌感染后的小鼠粪便生化成分有极大的变化。除了在宿主肠道增殖,沙门氏菌可移植至多个器官从而导致小鼠全身感染,尤其是肝脏和脾脏。因此进一步分析了感染后肝提取物化学成分的变化,结果共检测到1149种物质,其中高达64%发生了显著变化。这些代谢产物涉及到类固醇、花生酸类物质、胆汁酸、碳水化合物和卟啉等等。变化最显著的是固醇类花生酸的激素。这说明宿主在受沙门氏菌感染后代谢通路发生了显著变化。其中类花生酸代谢和类固醇代谢被扰乱,类花生酸合成酶和类固醇合成酶的转录水平受到影响。
Lin等(Lin et al.2010)研究了流感病毒感染两个细胞株(A549和AGS)基于GC-MS的代谢组学。感染后分别得到12种和10种差异显著的代谢物,A549中8种代谢物病毒感染后下调,33.3%上调而AGS细胞系中病毒感染后仅有1个代谢物(10%)表达下调。将代谢组数据进行PCA和OPLS-DA分析,结果发现代谢组的数据靶定在脂肪酸生物合成和胆固醇新陈代谢,说明该流感病毒依靠宿主代谢网络的改变获取能量,另外,在分化程度降低的细胞株A549中病毒复制引起的代谢变化更为显著。
细菌和病毒等病原微生物感染宿主后,引起宿主一系列的代谢变化,通过生物学分析软件,可以找出变化最显著的代谢物,与该病原菌感染最相关,并靶定出重要的代谢通路。
1.6小结
通过分析迟钝爱德华氏菌EIB202半数致死剂量感染斑马鱼的代谢组学数据得出两个重要的通路:精氨酸-脯氨酸代谢通路和缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路,并进一步分析得出通路中两种与EIB202感染相关的物质:脯氨酸和缬氨酸;将脯氨酸和缬氨酸外源添加至斑马鱼,显著降低了EIB202感染的死亡率。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (10)
1.斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,其特征在于:为精氨酸,脯氨酸,缬氨酸,亮氨酸,异亮氨酸,Cadaverine中的至少一种。
2.根据权利要求1所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,其特征在于:为脯氨酸和缬氨酸。
3.根据权利要求1所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物,其特征在于:为脯氨酸、缬氨酸和Cadaverine。
4.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在检测斑马鱼感染迟钝爱德华氏菌中的应用。
5.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在制备用于检测斑马鱼感染迟钝爱德华氏菌的检测试剂盒中的应用。
6.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在制备治疗斑马鱼感染迟钝爱德华氏菌的药物中的应用。
7.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在提高斑马鱼应答迟钝爱德华氏菌感染的免疫力中的应用,在制备用于提高斑马鱼应答迟钝爱德华氏菌感染的免疫力的药物或保健品中的应用。
8.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在调控斑马鱼精氨酸-脯氨酸代谢通路中的应用。
9.权利要求1或2或3所述的斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物在调控斑马鱼缬氨酸-亮氨酸-异亮氨酸生物合成代谢通路中的应用。
10.脯氨酸或/和缬氨酸在作为斑马鱼感染迟钝爱德华氏菌的体液小分子代谢标记物中的应用。
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