CN117106041A - Kdo糖蛋白缀合物及其应用 - Google Patents
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Abstract
本发明提供Kdo糖蛋白缀合物及其应用,Kdo糖蛋白缀合物由Kdo衍生物、连接子和载体蛋白三部分构成。Kdo单糖是众多耐药菌表面重要的保守结构,将设计和合成的Kdo单糖衍生物通过连接子与载体蛋白偶联构建非天然糖蛋白缀合物。本发明还涉及将该糖蛋白缀合物作为一种抗原激发多价抗血清和特异性识别Kdo的单克隆抗体,应用于金黄色葡萄球菌、大肠杆菌等耐药菌疫苗和诊断工具的开发。
Description
技术领域
本发明涉及药物领域,特别涉及一种非天然Kdo糖蛋白缀合物及其应用。
背景技术
面对抗生素的挑战,细菌可以迅速演变出耐药性,甚至演变成具有多重耐药性的超级细菌。尤其是感染率很高的严重耐药致病菌之一的金黄色葡萄球菌,会引起骨髓炎、肺炎、脑膜炎和中毒性休克综合征等严重甚至致命性疾病。随着耐药细菌在世界范围内的泛滥以及感染病例的不断上升,人类有可能重新面临没有抗生素可用的危险局面。目前已经上市的脑膜炎奈瑟菌疫苗,b型流感嗜血杆菌疫苗,肺炎链球菌疫苗、伤寒杆菌疫苗等的抗菌范围较窄,对耐药菌预防和诊断的方法仍然具有一定的局限性。因此,需要研发针对不同耐药菌的疫苗和诊断工具,让人类获得更多对抗耐药细菌感染的工具。
3-脱氧-D-甘露-2-辛酮糖酸(Kdo)属于存在于耐药菌细胞壁表面的一种特异性的高度保守结构,通常参与维持细胞膜的完整性,又可以刺激免疫系统产生特异性抗体,具有优秀的免疫原性,对于开发针对耐药菌感染的疫苗和诊断工具有重要的潜在应用价值。
由于从天然耐药菌表面提取的脂多糖中难以获得结构单一的Kdo单元,从而限制了后续单克隆抗体制备及其在细菌检测方面的应用,因此有必要通过化学合成获得结构单一的纯净Kdo衍生物(Gold(I)-catalyzed Synthesis ofβ-Kdo Glycosides Using Kdoortho-Hexynylbenzoate as Donor.Xuemeng Mi,Qixin Lou,Wenjing Fan,Liqin Zhuang,YouYang*.Carbohydr.Res.2017,448,161–165.)。为了进一步探索Kdo衍生物在抗耐药菌疫苗和细菌检测及诊断中的应用,需要将其与适当的载体蛋白偶联后形成糖蛋白缀合物,引起T细胞依赖性的免疫应答,有效增强糖类物质的免疫原性,这是开发糖类疫苗的一个重要策略。
发明内容
本发明的目的在于,提供一种结构简单且构型明确的Kdo糖蛋白缀合物,对金黄色葡萄球菌和大肠杆菌有着特殊的识别和结合能力,小剂量即能够在体内作为抗原激发出大量抗体。
本发明的第二个目的在于,提供Kdo糖蛋白缀合物在制备金黄色葡萄球菌或大肠杆菌抗菌疫苗中的应用。
本发明的第三个目的在于,提供Kdo糖蛋白缀合物在制备耐药菌检测和诊断试剂中的应用。
为了实现上述目的,本发明提供了一种Kdo糖蛋白缀合物,由Kdo衍生物、连接子和载体蛋白三部分构成,所述Kdo蛋白缀合物具有通式I所示的结构:
其中,
L基团为-(CH2)m-NH2,-(CH2)m-N3,或者-(CH2)m-SH;m为1-10中的自然数,通过糖苷化反应连接于Kdo的C-2位;
C基团为载体蛋白,选自CRM197,HSA,TT,DT,或者OMPC中的一种。
本发明中含3-脱氧-D-甘露-2-辛酮糖酸(Kdo)结构的单糖衍生物具有通式II所示的结构:
所述单糖的L基团为-(CH2)m-NH2,-(CH2)m-N3,或者-(CH2)m-SH;m为1-10,连接子连接至所述单糖的C-2位。
通式中的n是指载体蛋白平均连接kdo衍生物的数量。
作为一个优选方案,所述L基团为-(CH2)m-NH2且m为5。
本发明优选实施例中,所述连接子为二(N-羟基琥珀酰亚胺)戊二酸酯(DSG)。
作为一个优选方案,所述Kdo糖蛋白缀合物结构式如CRM197-1或HSA-1所示:
在下述实施例中,过量的化学交联剂双琥珀酰亚胺戊二酸酯与Kdo单糖C-2位衍生的氨基反应形成酰胺键,转化为相应的活化单酯,通过离心萃取法有效除去有机溶剂后,与载体蛋白CRM197或HSA共价偶联,并使用超滤离心法进行蛋白的浓缩和纯化获得Kdo糖蛋白缀合物。利用10%十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-4800plus)分析并计算平均每个载体蛋白偶联上的单糖数量。
为了实现上述第二个目的,本发明提供了Kdo糖蛋白缀合物在制备金黄色葡萄球菌或大肠杆菌抗菌疫苗中的应用。
本发明还提供了一种用于预防和治疗金黄色葡萄球菌或大肠杆菌感染的疫苗,所述疫苗利用所述Kdo糖蛋白缀合物作为抗原。
为了实现上述第三个目的,本发明提供了Kdo糖蛋白缀合物在制备耐药菌检测和诊断试剂中的应用,所述耐药菌是指耐金黄色葡萄球菌或耐大肠杆菌。
本发明还提供了一种耐药菌的检测和诊断试剂,所述检测和诊断试剂利用所述Kdo糖蛋白缀合物作为抗原诱导产生抗体与耐药菌结合,所述耐药菌是指耐金黄色葡萄球菌或耐大肠杆菌。
根据本发明的另一方面,还提供Kdo糖蛋白缀合物作为抗原的抗体制备方法。
在本发明优选实施例中,Kdo糖蛋白缀合物与弗氏佐剂(FA)混合形成乳剂,按照0-14-28天的免疫程序腹股沟皮下免疫雌性C57BL/6J小鼠,混合乳剂诱导产生更多的交叉反应抗体,并于最后一次免疫7天后取眼眶血分离得到抗血清,通过酶联免疫吸附实验结果发现小鼠血清中IgG抗体滴度水平显著提高,免疫应答强烈。尤其是IgG1、IgG2b、IgG3抗体滴度水平也有明显的升高,证明该糖蛋白缀合物能诱导机体产生较强的T细胞依赖性保护性免疫反应,可以作为开发抗耐药菌疫苗的潜在抗原。
在优选实施例中,Kdo糖蛋白缀合物与弗氏佐剂(FA)混合形成乳剂,按照0-14-28-42天的免疫程序皮下多点免疫雌性BALB/c小鼠,并于最后一次免疫7天后取眼眶血选择抗血清效价达到8K稀释度的小鼠。按照B淋巴细胞能够产生抗体,瘤细胞体外无限传代的原理,提取小鼠脾脏和淋巴组织与对数生长期并预先用8-AG处理的骨髓瘤SP2/0细胞融合形成既可以无限增殖又可以产生抗体的杂交瘤细胞,并进行多次细胞亚克隆,直至得到Kdo抗原识别率达到100%的细胞株。继续将挑选出的细胞株腹腔注射10周龄左右的BALB/c小鼠体内,一周后采集腹水得到针对Kdo的大量腹水单抗。
在优选实施例中,选取含有或不含有Kdo结构的几种灭活耐药菌首先与单抗腹水抗体共孵育,基于AF488的山羊抗小鼠IgG抗体(绿色)染色标记,可以抽取约10%的细菌通过流式细胞仪进行荧光分析,并可利用共聚焦激光显微镜(LeicaTCS SP8)观察灭活细菌和腹水抗体之间的结合情况。实验结果表明Kdo存在于细菌表面不同位置时,细菌与单克隆腹水抗体的结合结果差异明显,因此大量制备的单克隆抗体可以作为含有Kdo结构的耐药细菌的诊断工具,并有潜力应用于抗菌疫苗的开发。
本发明的优点在于,本发明利用结构简单且构型明确的Kdo糖蛋白缀合物作为抗原,小剂量即能够在体内激发出大量抗体,通过单克隆技术制备的单克隆腹水抗体表现出与Kdo存在于细菌表面不同部位的耐药菌,如金黄色葡萄球菌、大肠杆菌等,有着不同的识别和结合能力,有潜力应用于抗耐药菌疫苗和诊断工具的开发。
附图说明
图1A和图1B所示的是本发明实施例1中获得的糖蛋白缀合物CRM197-1的表征结果;
图2A和图2B所示的是本发明实施例1中获得的糖蛋白缀合物HSA-1的表征结果;
图3所示的是本发明所述糖蛋白缀合物CRM197-1在C57BL/6J小鼠体内的免疫反应结果;
图4A-4D所示的分别是灭活的致病菌(肺炎链球菌19F、金黄色葡萄球菌、大肠杆菌K12、产碱普罗威登斯菌O36)与单克隆腹水抗体(2E6/3N11/2G20)结合的免疫荧光和流式细胞检测的实验结果。
具体实施方式
以下,结合具体实施方式对本发明的技术进行详细描述。应当知道的是,以下具体实施方式仅用于帮助本领域技术人员理解本发明,而非对本发明的限制。
实施例1.Kdo糖蛋白缀合物
在本实施例中,提供一种Kdo糖蛋白缀合物,使用的Kdo单糖衍生物由本课题组合成研究的同学提供,也可以根据Gold(I)-catalyzed Synthesis ofβ-Kdo GlycosidesUsing Kdo ortho-Hexynylbenzoate as Donor.Xuemeng Mi,Qixin Lou,Wenjing Fan,Liqin Zhuang,You Yang*.Carbohydr.Res.2017,448,161–165.中公开的方法进行合成。该糖蛋白缀合物由Kdo衍生物与载体蛋白CRM197或HSA通过连接子二(N-羟基琥珀酰亚胺)戊二酸酯(DSG)连接而成,分别具有结构式CRM197-1和结构式HSA-1所示的结构:
在本实施例中,以活化酯法将Kdo衍生物和连接子二(N-羟基琥珀酰亚胺)戊二酸酯偶联。然后,将活化后的Kdo衍生物连接到载体蛋白CRM197或HSA上,得到所述糖蛋白缀合物CRM197-1或糖蛋白缀合物HSA-1。
在本实施例中,所述糖蛋白缀合物CRM197-1的合成路线为:
所述糖蛋白缀合物CRM197-1的制备方法具体为:
向双琥珀酰亚胺戊二酸酯(15mg,46μmol)和三乙胺(10μL,0.07mmol)的DMSO溶液(200μL)中加入Kdo衍生物1(1.5mg,4.6μmol)的DMSO溶液(80μL)。室温搅拌反应2小时后,反应液中加入PBS(100mM,pH=7.4,640μL),再加入5mL氯仿离心萃取(2min,1800g)。上层水相移至1.5mL EP管中离心(1min,14500g),至观察溶液无分层时取上层水相。取1mg CRM197(17.3nmol,Creative BioMart)溶于1mLPBS(100mM,pH=7.4),加入上述水相室温搅拌18小时。反应完全后转移至超滤管(30kDaMWCO,Millipore)加入超纯水和PBS分别离心3次(4000rpm,15min,4℃),获得所述糖蛋白缀合物CRM197-1。
以10%SDS-PAGE(120V,90min)和MALDI-TOF-MS检测获得的所述糖蛋白缀合物CRM197-1的平均分子量,获得如图1A和图1B所示的结果。结果表明,所述糖蛋白缀合物CRM197-1中每个CRM197蛋白平均连接约9.2个kdo衍生物。
在本实施例中,所述糖蛋白缀合物HSA-1的合成路线为:
所述糖蛋白缀合物HSA-1的制备方法具体为:
向双琥珀酰亚胺戊二酸酯(10mg,30μmol)和三乙胺(10μL,0.07mmol)的DMSO溶液(200μL)中加入Kdo衍生物1(1mg,3μmol)的DMSO溶液(80μL)。室温搅拌反应2小时后,反应液中加入PBS(100mM,pH=7.4,640μL),再加入5mL氯仿离心萃取(2min,1800g)。上层水相移至1.5mL EP管中离心(1min,14500g),至观察溶液无分层时取上层水相。取1mg HSA(15nmol)溶于1mL PBS(100mM,pH=7.4),加入上述水相室温搅拌18小时。反应完全后转移至超滤管(30kDa MWCO,Millipore)加入超纯水和PBS分别离心3次(4000rpm,15min,4℃),获得所述糖蛋白缀合物HSA-1。
以10%SDS-PAGE(120V,90min)和MALDI-TOF-MS检测获得的所述糖蛋白缀合物HSA-1的平均分子量,获得如图2A和图2B所示的结果。结果表明,所述糖蛋白缀合物HSA-1中每个HSA蛋白平均连接约8.2个Kdo衍生物。
实施例2.糖蛋白缀合物CRM197-1的免疫活性测试
在本实施例中,提供实施例1获得的糖蛋白缀合物CRM197-1的免疫活性测试结果,通过酶联免疫吸附实验的免疫结果表明该糖蛋白缀合物CRM197-1能诱导机体产生较强的免疫应答。产生高滴度的IgG1、IgG2b和IgG3抗体,特别是IgG1和IgG3抗体的产生,表明CRM197-1糖缀合物可以诱导更强有力的T细胞依赖的保护性免疫反应,这对于预防性疫苗是非常理想的。
在本实施例中,所述糖蛋白缀合物CRM197-1的免疫活性测试具体包括以下几个具体步骤。
1.糖蛋白缀合物CRM197-1免疫C57BL/6J小鼠
用6-8周雌性C57BL/6J小鼠(每组4-6只)皮下免疫CRM197-1糖缀合物抗原,给药组每只小鼠在第0天注射含0.4μg糖抗原的糖蛋白缀合物CRM197-1,第14和28天加强免疫含0.8μg糖抗原的糖蛋白缀合物CRM197-1,每只小鼠注射100μL糖蛋白缀合物CRM197-1与弗氏佐剂1:1(V/V)混合制成的乳剂。对照组只注射PBS或PBS加佐剂,给药组只注射糖缀合物抗原或糖缀合物加佐剂。初次免疫加弗氏完全佐剂,加强免疫加弗氏不完全佐剂。在第0,14,21,35天眼眶取血,提取血清。
2.免疫后C57BL/6J小鼠多抗血清酶联免疫吸附实验(ELISA)
使用高结合力96孔酶标板(康宁)进行ELISA试验。将HSA-1(10μg/mL)溶于碳酸盐缓冲液(0.05M,pH=9.6)中,4℃下静置20小时包被酶标板。第二天,酶标板用PBS-T(含0.1%Tween-20的PBS)洗3次,用2%BSA-PBS在37℃下封闭1小时。PBS-T洗3次后,加入1%BSA-PBS倍比稀释的抗血清,37℃下孵育2小时。PBS-T洗3次,分别加入HRP标记的IgG、IgG1、IgG2a、IgG2b、IgG3山羊抗小鼠二抗,37℃下避光孵育1小时。用PBS-T洗3次,加入TMB底物在37℃下孵育20分钟至显色。加入2%硫酸终止反应并用Synergy2多功能酶标仪读取450nm下吸光度,获得如图3所示的检测结果。
结果表明,免疫35天后,小鼠血清中IgG抗体滴度水平显著提高,免疫应答强烈。免疫后IgG1、IgG2b、IgG3抗体滴度水平也有明显的升高。该糖蛋白缀合物能诱导机体产生较强的T细胞依赖性保护性免疫反应。
实施例3.糖蛋白缀合物CRM197-1的单克隆腹水抗体的制备
在本实施例中,提供实施例1获得的糖蛋白缀合物CRM197-1作为抗原制备单克隆腹水抗体的过程。通过免疫BALB/c小鼠制备的几种腹水的免疫荧光实验和流式细胞术实验结果表明:CRM197-1作为抗原诱导小鼠所产生的单克隆腹水抗体可与几种耐药菌产生不同的荧光结合,尤其是与金黄色葡萄球菌和大肠杆菌发生显著结合,表明糖蛋白缀合物CRM197-1有较大潜力应用于抗金黄色葡萄球菌感染和大肠杆菌感染的疫苗开发,也可应用于针对含有Kdo结构的耐药菌的检测和诊断工具的开发。
在本实施例中,所述糖蛋白缀合物CRM197-1作为抗原制备单克隆腹水抗体具体包括以下几个关键步骤。
免疫:用8-12周雌性BALB/c小鼠3只,皮下免疫CRM197-1糖缀合物抗原,采用皮下多点免疫的方法进行四次常规免疫,免疫程序为0-14-28-42天,每次免疫计量为每剂含200μg蛋白的CRM197-1糖缀合物,最后一次加强免疫后一周取眼眶血检测抗血清的ELISA效价(HSA-1抗原包被96孔板,ELISA操作同实施例2中2),分别检测小鼠抗血清效价大于1:8000(8K稀释度下阳性组大于阴性组的二倍)时进入融合阶段。
融合:选择免疫后BALB/c小鼠效价达到8K稀释度的小鼠,在5min内将小鼠的脾脏和淋巴组织取出剪碎于培养皿中,加不完全培养基15mL,将200目筛网置于培养皿上,继续研磨组织至无块状,再加入15mL培养基冲洗,一起收集到50mL离心管中洗涤(1500rpm,5min,4℃),弃掉上清并轻敲管底打散组织,加入1mL红细胞裂解液,避光静置1.5min。之后加入20mL RDF不完全培养基,1500rpm离心5min,弃上清,重复两遍,去除块状组织,将培养基弃净。
将处于对数生长期并预先用8-AG处理的SP2/0细胞和刚制取的脾细胞按3:1或5:1体积混合后离心(1700rpm,7min,4℃),弃掉上清后轻拍管底拍散细胞。在1min内加入37℃预温的1mL45%PEG(分子量4000),边加边震摇离心管,静置1.5min。之后在2.5min内加入5mL RDF不完全培养基后静置5min,再于1min内加完10mL RDF不完全培养基后离心(1000rpm,5min,4℃),弃上清之后加入HAT完全培养基调整细胞浓度后铺满384孔板。
亚克隆:融合完成后,稀释细胞并铺满384孔板,细胞培养10-14天。取每个孔里的上清液做ELISA检测针对抗原的反应,进行亲和力排序,亲和力高的挑取亚克隆。将挑选出的亲和力高的孔中细胞铺满96孔板,ELISA检测每个孔中上清针对抗原的反应,蛋白抗原包被浓度1μg/mL,抗体稀释倍数1:3.125K/6.25K/12.5K/25K/50K/100K,亲和力高的进入下一轮亚克隆。重复上述步骤直到孔中细胞株针对Kdo的识别率达到100%,此时认为细胞可以成功建株。
腹水制备:将选取的2E6、3N11、2G20三种杂交瘤细胞(3N11杂交瘤细胞保藏于中国典型培养物保藏中心,保藏地址:湖北省武汉市武昌区八一路299号,保藏日期为2022年09月18日。所述菌株命名为Kdo单克隆杂交瘤细胞株3N11,保藏编号为CCTCC NO:C2022211。)首先培养至生长旺盛,形态良好,处于对数生长期的状态。将弗氏不完全佐剂1mL/只腹腔注射10周龄的BALB/c小鼠体内,防止实体瘤的形成。一周之后,调整对数生长期的杂交瘤细胞数量为2×106个/mL,1mL/只腹腔注射到预处理的BALB/c小鼠体内,一周后处死小鼠,用7号针头刺入腹腔,即有腹水流出,采集腹水于15mL离心管后离心(3000rpm,10min,4℃),去除最上层油脂和最下层细胞,上清液加入50%甘油冻存于-20℃冰箱保存,得到单克隆腹水抗体2E6、3N11、2G20。
实施例4.糖蛋白缀合物CRM197-1的单克隆腹水抗体的免疫荧光识别应用
1.紫外灭活致病细菌与单抗腹水结合的免疫荧光实验
由北京北纳创联生物技术研究院提供的湿热灭活(121℃,30min)的肺炎链球菌19F(Streptococcus Pneumoniae 19F ATCC49619)、金黄色葡萄球菌(Staphylococcusaureus ATCC6538)、大肠杆菌K12(E.coli serotype K12ATCC25404)、产碱普罗威登斯菌O36(Providencia alcalifaciens O36ATCC9886)加入PBS制成1×107cfu/mL的细菌悬液。将细菌解冻,离心收集(10000rpm,5min,rt),用PBS-T洗3次。加入3%BSA-PBS在37℃下封闭1小时。后用PBS-T洗3次,分别加入在0.5%BSA-PBS中1:10稀释的单克隆腹水抗体,4℃过夜孵育。第二天,PBS-T洗3次,加入AF488标记的山羊抗小鼠IgG抗体,37℃下避光孵育1小时。用PBS-T洗3次之后,用激光扫描共聚焦显微镜观察细菌,用Leica Application Suite X图像分析软件进行分析处理。
免疫荧光结果显示单克隆腹水抗体与Kdo存在于胞外多糖(EPS)的金黄色葡萄球菌和Kdo存在于荚膜多糖(CPS)的大肠杆菌K12的结合能力最强,与Kdo存在于O抗原的产碱普罗威登斯菌O36的结合能力稍弱,而与不含Kdo结构的革兰氏阳性肺炎链球菌19F基本不发生结合。
2.紫外灭活致病细菌与单抗腹水结合的流式细胞术实验
将上述各细菌解冻,离心收集(10000rpm,5min,rt),用PBS-T洗3次。加入3%BSA-PBS在37℃下封闭1小时。用PBS-T洗3次后,分别加入在0.5%BSA-PBS中1:10稀释的抗血清和非免疫血清,4℃过夜孵育。第二天,PBS-T洗3次,加入AF488标记的山羊抗小鼠IgG抗体,37℃下避光孵育1小时。用PBS-T洗3次,再用Lytoflex LX流式细胞仪分析。
流式细胞术实验发现,含有Kdo结构的致病细菌金黄色葡萄球菌、大肠杆菌K12、产碱普罗威登斯菌O36与单克隆腹水抗体结合后均产生荧光信号增强现象,而单克隆腹水抗体与不含Kdo的革兰氏阳性肺炎链球菌19F基本不产生荧光信号。该结果与免疫荧光实验结果同时说明糖蛋白缀合物CRM197-1作为抗原在小鼠体内激发产生的单克隆腹水抗体能够与含有Kdo结构的致病细菌产生不同程度的结合,单克隆腹水抗体与四种致病菌的结合结果如图4A-4D所示。
上述检测结果表明,本发明所述的糖蛋白缀合物CRM197-1的免疫原性很强,在较低的剂量下可以诱发高滴度的抗体应答。基于Kdo衍生物构建的糖蛋白缀合物产生的免疫响应主要是IgG型T细胞依赖性免疫应答,且经单克隆技术诱导出的单克隆腹水抗体可与含有Kdo结构的致病细菌产生不同的结合,尤其是与金黄色葡萄球菌和大肠杆菌的结合效果显著。因此,本发明所述的糖蛋白缀合物可被进一步应用于金黄色葡萄球菌感染和大肠杆菌感染的抗菌疫苗的开发,并且大量制备的单克隆腹水抗体可作为不同耐药菌的诊断工具。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种Kdo糖蛋白缀合物,其特征在于,由Kdo衍生物、连接子和载体蛋白三部分构成,所述Kdo蛋白缀合物具有通式I所示的结构:
其中,
L基团为-(CH2)m-NH2,-(CH2)m-N3,或者-(CH2)m-SH;m为1-10中的自然数,通过糖苷化反应连接于Kdo的C-2位;
C基团为载体蛋白,选自CRM197,HSA,TT,DT,或者OMPC中的一种。
2.根据权利要求1所述的一种Kdo糖蛋白缀合物,其特征在于,所述L基团为-(CH2)m-NH2,且m为5。
3.根据权利要求1所述的一种Kdo糖蛋白缀合物,其特征在于,所述Kdo糖蛋白缀合物结构式如CRM197-1或HSA-1所示:
4.权利要求1所示的Kdo糖蛋白缀合物在制备金黄色葡萄球菌或大肠杆菌抗菌疫苗中的应用。
5.权利要求1所示的Kdo糖蛋白缀合物在制备耐药菌检测和诊断试剂中的应用,其特征在于,所述耐药菌是指耐金黄色葡萄球菌或耐大肠杆菌。
6.一种用于预防和治疗金黄色葡萄球菌或大肠杆菌感染的疫苗,其特征在于,所述疫苗利用权利要求1所述的Kdo糖蛋白缀合物作为抗原。
7.一种耐药菌的检测和诊断试剂,其特征在于,所述检测和诊断试剂利用权利要求1所述的Kdo糖蛋白缀合物作为抗原诱导产生抗体与耐药菌结合,所述耐药菌是指耐金黄色葡萄球菌或耐大肠杆菌。
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