CN117462664A - 一种多杀性巴氏杆菌外膜囊泡的制备方法及其应用 - Google Patents
一种多杀性巴氏杆菌外膜囊泡的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开一种多杀性巴氏杆菌外膜囊泡的制备方法及其应用,包括下述步骤:将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上进行培养;取预培养菌液的上清液,离心,过滤,将滤液收集到超滤器中浓缩,浓缩液超速离心,弃上清液,沉淀用PBS缓冲液重悬,于‑80℃条件下冻存;先制备氨基修饰的介孔二氧化硅纳米粒使其带正电;将二氧化硅分散液与石斛多糖在室温下剧烈搅拌后,离心处理水洗,即得介孔二氧化硅载石斛多糖;用微型挤出机对介孔二氧化硅载石斛多糖和外膜囊泡行挤压,然后灭菌,得到多杀性巴氏杆菌外膜囊泡。本发明制得的外膜囊泡包裹介孔二氧化硅载石斛多糖可以产生强大而持久的抗菌免疫力。
Description
技术领域
本发明生物制品技术领域,特别涉及一种多杀性巴氏杆菌外膜囊泡的制备方法及其应用。
背景技术
多杀性巴氏杆菌(Pasteurella multocida,P.multocida)作为一种人畜共患的病原体,它可在多种动物物种中引起疾病,并且是许多具有重要经济意义疾病的病原体,包括禽霍乱、牛出血性败血症、地方性动物患性肺炎和猪萎缩性鼻炎;人类接触患多杀性巴氏杆菌病动物的唾液时,软组织的机会性感染相对常见,其他严重并发症,如肺炎、脑膜炎和感染引起的败血症,也可能发生在老年人等免疫力低下的个体上。多杀性巴氏杆菌作为人类疾病病原体的重要性渐渐得到了关注。多杀性巴氏杆菌的主要表面成分包括衣壳蛋白、脂多糖和粘附素等,这些成分在多杀性巴氏杆菌逃避宿主先天免疫机制的过程中起主要作用,也是主要的毒力决定因素和免疫原性结构。现有的多杀性巴氏杆菌疫苗保护效率不高,在接种疫苗的动物中也存在很多感染该病菌的现象。由于缺乏有效和安全的疫苗,通过免疫预防多杀性巴氏杆菌病受到阻碍。因此,在预防多杀性巴氏杆菌病方面需要安全有效的疫苗。
纳米技术作为近些年的研究热点,已被广泛用于疫苗研发。纳米疫苗的优点主要包括:避免抗原的快速降解,提高疫苗的稳定性、具有佐剂的增强特异性免疫应答的作用、可以作为载体负载或充当佐剂来刺激免疫细胞成熟。细菌膜泡(MVs)最初被发现是通过挤压革兰氏阴性细菌外膜的起泡而产生的,因此通常被称为外膜囊泡(OMVs)。OMVs可以使细菌与外界环境相互作用,具有介导细菌发病机制、使细菌在应激条件下存活以及调节细菌群落内的微生物相互作用等功能。OMVs的形态通常是直径在20-250nm范围内的球形双层膜结构,主要由脂质、蛋白质和脂多糖、脂蛋白、肽聚糖、核酸等各种病原体相关分子模式(PAMPs)组成,其结构和组成是OMVs发挥功能的基础。细菌抗原和具有免疫刺激特性的多个PAMPs的共存以及蛋白质-脂质复合体的纳米结构使OMV有望成为预防和治疗细菌感染的候选疫苗。然而,OMVs不可在体内过量输入,且稳定性有待改善。
发明内容
本发明提供一种多杀性巴氏杆菌外膜囊泡的制备方法及其应用,本发明为了增强其诱导免疫应答的功能,选用石斛多糖介孔二氧化硅纳米粒(DP-MSN)为佐剂,将OMVs与DP-MSN有效结合。
为解决上述技术问题,本发明所采取的技术方案是:
本发明提供一种多杀性巴氏杆菌外膜囊泡的制备方法,包括下述步骤:
(1)外膜囊泡的制备
将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上进行培养,然后挑取单菌落接种于马丁肉汤培养基中进行培养,扩大培养后得预培养菌液;取预培养菌液的上清液,超速离心,过滤,将滤液收集到超滤器中浓缩,浓缩液超速离心,弃上清液,沉淀用PBS缓冲液重悬,于-80℃条件下冻存;
(2)介孔二氧化硅载石斛多糖纳米颗粒的合成
将介孔二氧化硅分散于甲苯中,超声中加入APTES反应,离心收集沉淀,沉淀用乙醇和ddH2O分别洗涤数次后,分散于ddH2O中,得到氨基修饰的介孔二氧化硅纳米粒使其带正电;将二氧化硅分散液与石斛多糖在室温下剧烈搅拌后,离心处理,水洗两次,即得介孔二氧化硅载石斛多糖;
(3)外膜囊泡包裹介孔二氧化硅载石斛多糖的合成:
用装有0.2μm聚碳酸酯薄膜过滤膜的微型挤出机对介孔二氧化硅载石斛多糖和外膜囊泡行多次挤压挤压数次进行结合,得到多杀性巴氏杆菌外膜囊泡DP-MSN-OMV。
其中,步骤(1)中,将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上进行培养的条件:37℃,培养时间为24h,单菌落接种于马丁肉汤培养基中进行培养的条件:37℃、200r/min摇床培养18h;按体积比1∶100扩大培养。
其中,步骤(1)中,培养菌液上清液的超速离心具体为:在4℃条件下,以5×103g离心10min,取上清液再次以4℃,5×103g的条件离心10min;过滤时分别经0.45μm和0.22μm的滤膜过滤以确保没有活菌和细胞碎片残留;采用具有100kDa滤膜的超滤器浓缩;浓缩液超速离心的条件:1×105g超速离心1-3h。
其中,步骤(1)中,介孔二氧化硅的制备方法如下:将十六烷基三甲基甲苯磺酸铵、三乙醇胺、ddH2O混合后加热至80℃,搅拌1h至完全溶解,快速加入正硅酸四乙酯,在80℃的条件下反应2h,然后离心收集沉淀,沉淀使用ddH2O和乙醇交替洗3次后分散于乙醇中,即得二氧化硅;将分散于乙醇中的二氧化硅,加入到乙醇和盐酸溶液中,70℃反应12h,离心收集沉淀,使用ddH2O和乙醇交替洗3次后,甲苯洗2次后沉淀分散于甲苯中,得到介孔二氧化硅。
其中,乙醇和盐酸溶液中乙醇和盐酸的体积比为:(8-12):1。
其中,步骤(2)中,加入APTES反应条件为:在N2保护下,80℃反应12h。
其中,步骤(3)中,介质化硅载石斛多糖和外膜囊泡混悬液的体积比为1:1。
上述制备方法制得的多杀性巴氏杆菌外膜囊泡在预防多杀性巴氏杆菌病中的应用。
石斛多糖(Dendrobium polysaccharides,DP)是从铁皮石斛中分离出来的主要生物活性成分,具有抗氧化、抗肿瘤、抗肥胖、抗高血压和有效增强免疫的药理作用,与大多数多糖相似,已在临床上被用作刺激和改善免疫反应的免疫佐剂,具有良好的生物活性,但其应用时往往需要大剂量且在体内代谢很快,难以发挥药效,因此需要选择一种合适的剂型。介孔二氧化硅纳米粒(MSN)作为一种药物传递载体,粒径可在50~300nm之间调节,粒子形状稳定且规整,具有多孔性、比表面积大、便于修饰、毒性低等特点,具有极大开发前景。纳米载体可增加药物的稳定性和安全性,有效地控制药物负载量,还能改善药物释放速度,从而提高药物的治疗活性。
巨噬细胞是先天免疫系统中具有重要作用的免疫细胞,具有吞噬、迁移、产生炎症反应、分泌细胞因子以及向T细胞呈递抗原的功能,可以表达许多天然免疫受体,包括如Toll样受体、炎症体和凝集素样受体,这些受体位于细胞膜、细胞质和内膜室。巨噬细胞可以通过表面暴露、囊泡或细胞质模式识别受体(PRRs)感知病原体相关分子模式,从而向炎症和促炎细胞因子发出信号,随后介导吞噬作用和炎症反应。
将多杀性巴氏杆菌OMVs作为亲本病原体的一种疫苗候选抗原首先需要了解其免疫原性。在本发明中,我们从多杀性巴氏杆菌培养物中将OMVs进行提取和表征,以研究其形态学特征,然后通过蛋白质谱分析其蛋白组成以及其在发病机制和免疫中的作用,然后构建了一种以DP-MSN为佐剂的新型亚单位疫苗DP-MSN-OMV,并对该亚单位疫苗进行表征,分析了其对巨噬细胞RAW264.7增殖、吞噬、细胞因子分泌的影响,获得DP-MSN-OMV对巨噬细胞的免疫调节作用和结果。以期为基于OMV的多杀性巴氏杆菌疫苗设计提供基础信息和潜在策略。最后在小鼠体内对DP-MSN-OMV的免疫功能进行研究。结果表明,本发明制得的外膜囊泡包裹介孔二氧化硅载石斛多糖DP-MSN-OMV可以产生强大而持久的抗菌免疫力。本研究旨在为开发安全高效的多杀性巴氏杆菌亚单位疫苗提供科学依据。
附图说明
图1为MSN、DP-MSN、OMV、DP-MSN-OMV透射电镜图。
图2为OMV以及DP-MSN-OMV的粒径(A)、电势(B)、OMVCFU单位产量(C)以及浓度(D)和稳定性(E)结果图。
图3为多杀性巴氏杆菌OMV和多杀性巴氏杆菌裂解蛋白进行SDS-PAGE分析图。
图4为OMV、DP-MSN、MSN-OMV和DP-MSN-OMVSDS-PAGE分析图。
图5为OMV亚细胞定位分析图。
图6为OMV的KEEG分析图。
图7为OMV的COG分析结果图。
图8为OMV的GO分析结果图。
图9为RAW264.7巨噬细胞被不同蛋白浓度的OMV、MSN-OMV和DP-MSN-OMV刺激后增殖情况结果图。
图10为OMV、MSN-OMV和DP-MSN-OMV被RAW264.7巨噬细胞摄取情况的共聚焦激光扫描显微镜观察图。
图11为OMV、MSN-OMV和DP-MSN-OMV被RAW264.7巨噬细胞摄取情况的流式细胞结果图。
图12为RAW264.7巨噬细胞被OMV、MSN-OMV和DP-MSN-OMV刺激后TNF-α(A)、IL-1β(B)、IL-10(C)和TGF-β1(D)细胞因子释放定量检测结果图。
图13为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠血清中OMV特异性IgG(A)、IgG1(B)和IgG2a(C)的水平。
图14为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠血清中P.multocida特异性IgG(A)、IgG1(B)和IgG2a(C)的水平。
图15为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠淋巴结中树突状细胞的CD80+CD11c+(A、B)和MHC-II+CD11c+(C、D)的表达水平。
图16为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠脾脏中CD4/CD8T(A)和CD19+B(B)细胞的激活和分化水平。
图17为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠脾脏中CD4T细胞中Th1(A)、Th2(B)和Th17(C)细胞分化水平。
图18为OMV、MSN-OMV和DP-MSN-OMV免疫后小鼠脾脏中CD4(A)和CD8(B)中TCM和TEM细胞的分化水平。
具体实施方式
下面将结合本发明具体实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本实施例提供一种多杀性巴氏杆菌外膜囊泡的制备方法,包括下述步骤:
(1)外膜囊泡OMV的制备
将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上,于37℃条件下培养24h,挑取单菌落接种于10mL马丁肉汤培养基中,于37℃、200r/min摇床培养18h后,按体积比1∶100扩大培养1000mL菌液。采用稀释涂布分离计数法测定细菌培养中菌落形成单位(Colony-Forming Units,CFU)的数量。将菌液倍比稀释,取1×106、1×107和1×108稀释度的菌液各100μL涂布于TSA平板并计数,每个稀释度重复3次并进行活菌数测定。
将培养至对数生长末期的1000mL菌液计数后,在4℃条件下,以5×103g离心10min,取上清液再次以4℃,5×103g的条件离心10min;收集上清液,分别经0.45μm和0.22μm的滤膜过滤以确保没有活菌和细胞碎片残留,将滤液收集到具有100kDa滤膜的超滤器中浓缩,将含OMV的浓缩液以1×105g超速离心2h,弃上清液;沉淀用PBS缓冲液重悬,于-80℃条件下冻存。
(2)介孔二氧化硅载石斛多糖纳米颗粒(DP-MSN)的合成
首先制备介孔二氧化硅纳米粒,将CTATos、TEAH3、ddH2O混合后加热至80℃,搅拌1h至完全溶解,快速加入TEOS,在80℃的条件下反应2h,然后离心收集沉淀,沉淀使用ddH2O和乙醇交替洗3次后分散于乙醇中,即得二氧化硅;将分散于乙醇中的二氧化硅,加入到乙醇和盐酸溶液(V乙醇:V盐酸=10:1)中,70℃反应12h,离心收集沉淀,使用ddH2O和乙醇交替洗3次后,甲苯洗2次后沉淀分散于甲苯中,得到介孔二氧化硅;将介孔二氧化硅分散于甲苯中,超声中加入APTES,在N2保护下,80℃反应12h,离心收集沉淀,沉淀用乙醇和ddH2O分别洗涤数次后,分散于ddH2O中,得到氨基修饰的介孔二氧化硅纳米粒使其带正电。将二氧化硅分散液与石斛多糖在室温下剧烈搅拌12h后,离心处理,水洗两次,即得介孔二氧化硅载石斛多糖,测试上清液中石斛多糖的含量。
(3)外膜囊泡OMV包裹介孔二氧化硅载石斛多糖纳米颗粒(DP-MSN-OMV)的合成
用用装有0.2μm聚碳酸酯薄膜过滤膜的Avanti微型挤出机对混合物以1:1的体积进行10次挤压结合,得到DP-MSN-OMV。
本实施例所使用的仪器设备如下:
Model 550酶标仪检测仪(SpectraMaxM5,Molecular Devices);100kDa超滤管(Millipore);-80℃冰箱(Thermo702);高速冷冻离心机(High-speed RefrigerateCentrifuge,HITACHI);恒温培养箱(GNP-9270,上海精宏实验设备有限公司);摇床(HZ-9210K、DHZ-CA,华利达);电泳仪(Bio-Rad);电泳槽(Bio-Rad);透射电子显微镜(TecnaiTMG2Spirit BioTWIN,FEI);纳米颗粒跟踪分析仪(ZetaView PMX 110,ParticleMetrix);用ZetaView 8.04.02 SP2软件进行纳米颗粒追踪分析;超声波细胞粉碎机(JY92-IIDN,宁波新芝生物科技股份有限公司);Orbitrap Exploris 480质谱仪(Thermo);ASY-nLCTM1200纳升级UHPLC(Thermo Fisher/LC140);低温离心机(Scilogex/D3024R);冷冻干燥机(Labogene/ScanSpeed40);用Proteome Discoverer(PD,Thermo)搜库;用Interproscan软件进行GO功能注释;用GraphPad Prism 8.0进行统计分析和作图。
本实施例所采用的菌株及主要试剂如下:
多杀性巴氏杆菌(CVCC500)购自国家兽医微生物菌(毒)种保藏中心。胰蛋白胨大豆琼脂(Tryptone Soya Agar,TSA)、马丁肉汤培养基(Martin Broth,MB)、胰蛋白胨(tryptone)购自美国赛默飞世尔科技有限公司;BCA Protein Assay Kit购自北京索莱宝科技有限公司;Tris-乙二胺四乙酸缓冲液(Tris-ethylenediaminetetraacetic acid,TE)购自生工生物工程(上海)股份有限公司;一步法凝胶制备试剂盒(12%)购自杭州弗德生物科技有限公司;Protein Marker(10-180kDa)购自北京全式金生物技术股份有限公司;细胞膜红色荧光探针(DiD Perchlorate/DiIC18(5))、溶酶体绿色荧光探针(LysoTrackerGreen DND-26)购自翌圣生物科技(上海)股份有限公司;Hoechst 33342和CCK-8购自美国APExBIO生物科技有限公;脂多糖(LPS)购自Sigma-Aldrich;DMEM、RPMI-1640 10%、胎牛血清(FBS)、青霉素(50U/ml)、链霉素(50μg/ml)、0.25%胰蛋白酶均购自Gibco;小鼠细胞因子IL-1β、IL-10、TNF-α和TGF-β1ELISA试剂盒购自联科生物有限公司(中国浙江);HRP标记的山羊抗小鼠IgG、IgG1和IgG2a购自Abcam公司。RAW264.7小鼠巨噬细胞购自中国科学院细胞库。
实验方法
1.OMV以及DP-MSN-OMV的理化性质分析方法
1.1形态学观察
用透射电镜(Transmission Electron Microscopy,TEM)对DP-MSN-OMV、MSN-OMV、MSN、OMV、DP-MSN进行形态学观察。分别吸取样品滴加于200目铜网上,使用醋酸双氧铀进行染色后常温干燥。使用TecnaiTMG2 Spirit BioTWIN于80kv的条件下进行电镜检测成像。
1.2纳米颗粒跟踪检测
选择纳米颗粒跟踪检测(Nanoparticle Tracking Analysis,NTA)的方法,使用Zetaview(Particle Metrix,Germany)仪器,分别对DP-MSN-OMV、MSN-OMV、MSN、OMV、DP-MSN进行粒径-浓度和电位-浓度的检测,使用无菌PBS缓冲液将样品按1:5000进行稀释,将每帧平均计数粒子保持在100-200个左右,记录并分析11个位置的NTA测量结果。同时将OMV与DP-MSN-OMV置于4℃储存,间隔一周进行一次NTA检测,以观察其稳定性情况。
1.3蛋白含量的测定与分析
使用BCA蛋白浓度测定试剂盒检测OMV蛋白含量。按照试剂盒说明书进行操作。以BSA作为蛋白标准品,制备标准曲线。将OMV样品用PBS缓冲液作适当稀释,加20μL到样品孔中;每孔再加入200μL含Cu+的BCA工作液,37℃放置15-30min;用酶标仪测定562nm波长下的吸光度,根据标准曲线计算出OMV蛋白浓度。将OMV以及多杀性巴氏杆菌裂解蛋白、DP-MSN、MSN-OMV和DP-MSN-OMV进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)并用考马斯蓝(CBB R-250)染料处理凝胶进行染色。
接下来取60μL OMV样品加入15μL 5×Loading Buffer,于100℃金属浴中煮沸15min,以备上样。采用一步法凝胶制备试剂盒(12%)制备蛋白预制胶,按照试剂盒说明书将蛋白预制胶制备好后,入电泳槽,倒入1×Tris-甘氨酸电泳缓冲液(Tris-GlycineRunning Buffer)依次加入分子量范围为10kDa-180kDa的分子标志物和OMV样品,各蛋白样品设置3个重复。在120V电压下电泳1h。
通过蛋白组学定性分析进一步确定OMV中的蛋白成分,由北京诺禾致源科技股份有限公司对蛋白质含量为50μg的多杀性巴氏杆菌OMV样品进行蛋白组学分析鉴定。
收集OMV样品并与DB蛋白溶液(8M尿素、100mM双硼酸三乙胺[TEAB])、胰蛋白酶和100mM TEAB缓冲液混合,在37℃下消化4小时。将消化后的OMV样品与甲酸混合,装入C18脱盐柱,用洗涤缓冲液(0.1%甲酸,3%乙腈)洗涤,然后加入洗脱缓冲液(0.1%甲酸,70%乙腈)。收集洗脱液并冻干,用于LC-MS/MS检测。
胰肽在EASY-nLCTM 1200超高效液相色谱系统上分离。从OMV样品中洗脱出的肽段使用Orbitrap Exploris 480质谱仪进行分析,质谱仪采用数据依赖模式,离子源为Nanospray FlexTM。概览扫描范围为m/z 350至1500,分辨率为60000(m/z 200)。全扫描根据丰度从高到低选择前体,然后通过高能碰撞解离(HCD)进行破碎。这一过程的分辨率为15000(在m/z 200处),归一化碰撞能量设定为30%。
使用Proteome Discoverer,根据UniProt Pasteurel-la_multocida数据库(1632170-uniprot-Pasteurella_multocida.fasta(23309个序列))分别搜索得到的光谱。搜索时最多有两次漏切,将氨基甲酰基作为固定修饰,将蛋氨酸(M)的氧化作为动态修饰,将N端的蛋氨酸作为损失。前体离子的质量容限为10ppm,生成离子的质量容限为0.02Da。检索结果经P D2.5分析,肽谱匹配度超过99%。鉴定出的蛋白质至少含有一个独特的肽段。经鉴定的肽谱和蛋白质都被保留下来并进行分析,FDR不超过1.0%。
使用Cell-mPLOC 2.0预测亚细胞定位。使用Interproscan(程序与Pfam数据库(http://pfam.xfam.org/)进行基因本体(GO)功能分析。鉴定出的蛋白质与在线京都基因组百科全书(KEGG)数据库(http://www.genome.jp/kegg/)和同源群(COG)数据库(http://www.ncbi.nlm.nih.gov/COG/)进行了比对(blastp,evalue≤1e-4),选择得分最高的比对结果来注释蛋白质家族和通路。2对免疫细胞的免疫效果研究
2.1 CCK-8法检测巨噬细胞的增殖
以5×103mL-1(100μl)的密度将细胞接种于96孔细胞培养板中,于含有10%胎牛血清、50U/ml青霉素和50μg/ml链霉素的DMEM培养液中,在37℃、5%CO2中孵育24小时待细胞贴壁后,弃去上层营养液。各孔分别加入OMV、MSN-OMV和DP-MSN-OMV(OMV终浓度为25、5、1、0.2和0.04μg/mL,DP-MSN和MSN终浓度为50、25、5、1和0.2μg/mL)的培养液100μL,每个药物浓度重复6孔。同时设置空白对照、细胞对照以及LPS对照。5%CO2、37℃条件下培养48h后,以10μL/孔的体积加入CCK-8试剂。待显色后,在酶标仪上通过测量450nm处的OD值来观察细胞的生长增殖情况。
2.2巨噬细胞的摄取
用DiD(λex/em644/663nm)与DP-MSN-OMV、MSN-OMV和OMV在37℃孵育30min进行荧光标记,以检测RAW264.7对DP-MSN-OMV、MSN-OMV和OMV的摄取。将RAW264.7细胞按5×105个/孔的密度接种在8孔玻片中,然后在5%CO2、37℃条件下孵育24小时。用DiD标记的对DP-MSN-OMV、MSN-OMV和OMV分别处理1、2、4小时后,然后用PBS洗涤3次。之后,选择Lyso TrackerGreen DND-26(λex/em504/511nm)对溶酶体染色1h,使用Hoechst 33342(λex/em350/461nm)对细胞核染色,最后用4%多聚甲醛固定细胞,再用PBS冲洗。在Leica TCS-SP5共聚焦激光扫描显微镜下观察载玻片以确定DP-MSN-OMV、MSN-OMV和OMV的在细胞内的分布情况。为了进一步获得细胞摄取的更多定量和动态变化,用流式细胞分析仪来评估RAW264.7细胞与DiD标记的DP-MSN-OMV、MSN-OMV和OMV(OMV终浓度为5μg/mL,DP-MSN和MSN终浓度为10μg/mL)作用1、2小时和4小时后的细胞摄取。
2.3巨噬细胞体外细胞因子的分泌
将巨噬细胞按上述方法培养,将含有预定浓度的DP-MSN-OMV、MSN-OM和OMV(OMV蛋白终浓度为62.5、12.5、2.5和0.5μg/mL,DP-MSN和MSN终浓度为100、20、4和0.8μg/mL)与细胞在5%CO2、37℃条件下培养共同培养24小时后,收集细胞上清液,通过ELISA Kit检测试剂盒对TNF-α、IL-1β、IL-10和TGF-β1这4种细胞因子进行定量检测。检测步骤按照试剂盒的说明书进行。
3对小鼠的免疫效果研究
3.1动物免疫
选择6~8周龄的BALB/c雌性小鼠为免疫对象,随机分为5组,每组5只,分别为DP-MSN-OMV免疫组、MSN-OMV免疫组、Alum-OMV免疫组、OMV免疫组以及空白对照组。免疫组每次每只小鼠皮下注射0.2ml相应的疫苗,每只小鼠的用药量为OMV2μg,DP-MSN或MSN200μg;空白对照组皮下注射0.2mlPBS。第0天皮下注射第一次免疫后,于第7天和第14天进行强化免疫。
3.2小鼠血清IgG抗体及其亚型检测
分别在三免14、28、42天后,每组随机选取5只小鼠摘眼球采血,用离心法提取血清,用ELISA法检测血清中OMV和P.multocida特异性IgG水平。在三免28天后,每组随机选取5只小鼠摘眼球采血,用离心法提取血清,用ELISA法检测血清中OMV和P.multocida特异性IgG1,IgG2a水平。将P.multocida破碎后的上清蛋白和OMV用BCA蛋白浓度试剂盒检测蛋白浓度后,用PH9.6的碳酸盐缓冲包被液分别调整浓度至3μg/ml,按每孔100μl加至96孔板,37℃孵育2小时后4℃保存过夜。次日用PBST洗涤,重复三次后将96孔板拍干。每孔加入300μl的5%脱脂乳,在37℃孵育2h,用PBST洗涤三次。将小鼠血清用脱脂乳10000倍稀释,按每孔100μl加至96孔板,同时设立阴性和空白对照,37℃孵育1h后用PBST洗涤三次。将HRP偶连山羊抗小鼠IgG、IgG1、IgG2a抗体(Abcam)分别用脱脂乳5000倍稀释,按每孔100μl加至96孔板,37℃孵育1h,用PBST洗涤三次后按每孔100μl加入TMB显色液,置37℃温箱避光孵育10min,每孔加入100μl 0.5M H2SO4终止反应,以空白孔调零,用酶标仪测量450nm波长下的OD值。
3.3淋巴结分析
通过检测共刺激分子的表达以分析纳米疫苗是否具有激活和扩增淋巴结中树突细胞的作用。在小鼠三免7天后,随机取6只,在超净台中用镊子将处死小鼠的淋巴结取出,在含IV型胶原酶(1mg/mL)和脱氧核糖核酸酶I(0.01mg/mL)的RPMI 1640培养液中于37℃消化60min,终止消化后,经200目细胞筛过滤至离心管内,离心(1800r/min,5min),后用PBS洗涤一次,获得淋巴单细胞悬液。分别用混合的抗鼠抗体(FITC-CD11c、APC-CD80、PE-CD86、PE-Cy7-MHC-I、PerCP-Cy5.5-MHC-II)分别在4℃避光的条件下将细胞染色30min后,抗体均来自联科生物,通过流式细胞仪进行检测并用FlowJo软件进行分析。
3.4脾脏分析
为了分析免疫后小鼠脾脏B和T淋巴细胞亚群的激活和分化情况。在小鼠三免后42天,随机取5只,按上述方法取小鼠的脾脏制成单细胞悬液后,用混合的抗鼠抗体(FITC-CD3、PE-CD4、APC-CD8和PE-Cy7-CD19)在4℃下避光染色15分钟后,通过流式细胞仪进行检测并用Flow Jo软件进一步分析活化淋巴细胞(CD19+)、CD4 T细胞(CD3+CD4+)和CD8 T细胞(CD3+CD8+)的百分比。
通过流式细胞术对免疫后小鼠脾脏中T细胞分化的情况进行分析。小鼠三免7天后,随机取5只,无菌取出小鼠脾脏,去掉脾脏上黏连的白色胰腺,然后将脾脏放到含RPMI1640培养液的研磨管中,通过全自动样品快速研磨仪60Hz振荡30s,经200目细胞筛过滤将多于组织及杂物等去除,经红细胞裂解液作用5分钟后离心(1800r/min,5min),用PBS洗涤一次,获得脾淋巴细胞悬液。用混合抗鼠抗体(FITC-CD3ε、PerCP-Cy5.5-CD4、PE-IFN-γ、APC-IL-4、PE-IL-17)在4℃下避光染色15分钟后,通过流式细胞仪进行检测并用Flow Jo软件进行分析。
最后通过流式细胞术检测免疫后小鼠脾脏中心记忆T细胞(TCM)和效应记忆T细胞(TEM)的分化情况。在小鼠三免后42天,随机取5只,按上述方法取小鼠的脾脏制成单细胞悬液后,用混合的抗鼠抗体(PE-CD4、APC-CD8、PE-Cy5-CD44和FITC-CD62L)在4℃下避光染色15分钟后,通过流式细胞仪进行检测并用FlowJo软件进行分析。
实验结果
1.OMV以及DP-MSN-OMV的理化性质分析
1.1OMV以及DP-MSN-OMV的TEM、NTA和Zeta电位的结果本研究通过超滤浓缩法制备了多杀性巴氏杆菌OMV,由TEM(图1)和NTA结果结果可知,纯化的多杀性巴氏杆菌OMV表现出均匀的球形形态,直径大小在20-300nm之间,且具有典型的双层膜囊泡状结构,OMV平均直径约128.5nm,其中粒径在10-50nm的OMV数量较少,观察到的多数OMV粒径在100-200nm之间,Zeta电位为-26.94Mv,且通过此方法制得的OMV可达每毫升3×1012个囊泡。MSN是基于表面活性剂模板法合成的,制备的MSN为纳米球形结构,尺寸均匀且具有均匀的内孔结构,平均直径在96.9nm左右,负载DP后的MSN仍为球型纳米颗粒,平均直径增加到119.3nm,Zeta电位为-23.47mV,通过物理挤压将OMV包覆在DP-MSN表面,形成稳定的纳米粒子DP-MSN-OMV,结合后的DP-MSN-OMV呈明显的核-壳结构,与单独OMV和DP-MSN相比平均直径增大到143.6nm,Zeta电位液升高至-35.57mV(图2A、B),表明OMV与DP-MSN结合成功。NTA还提供了有关计算OMV/CFU的颗粒计数的信息,据估计大约每1个细菌释放2.7个OMV(图2C)。并且在30天内,OMV的粒径从135.5nm增加到210.9nm,增加了75.5nm,而DP-MSN-OMV的粒径仅增加了9.7nm(图2E),表明DP-MSN-OMV体系具有良好的稳定性。
1.2 OMV中的蛋白质成分分析
将提取的OMV进行SDS-PAGE和LC-MS/MS分析。首先对多杀性巴氏杆菌OMV和多杀性巴氏杆菌裂解蛋白进行SDS-PAGE分析。从图3中可知:OMV蛋白和多杀性巴氏杆菌裂解蛋白两者的蛋白质谱不同,但OMV中的主要蛋白质条带都在多杀性巴氏杆菌裂解蛋白质谱中可见(图3),证明此方法制得的OMV没有杂菌污染,并保留了多种免疫原性蛋白质。接下来对制备的OMV、DP-MSN、MSN-OMV和DP-MSN-OMV进行SDS-PAGE分析。从图4可知:多杀性巴氏杆菌OMV的膜蛋白组成在整个制备过程中主要蛋白组成未发生变化,且在DP-MSN纳米颗粒中未检测到蛋白。
经质谱分析,多杀性巴氏杆菌OMV中鉴定到的肽段数为1500个,共鉴定出429种蛋白质。鉴定出的多数蛋白质都与毒力有关,包括丝状血凝素(FHA)、神经氨酸酶、柔毛组装蛋白、结构蛋白、结合蛋白和转运蛋白。此外,还发现了转运糖、氨基酸、离子的前导蛋白和跨膜通道蛋白。(表1)。
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其中参与细胞壁/膜生物发生的蛋白质,如OmpA、OmpW、Tol-Pal和mltC是囊泡形成的主要调节因子,可能与OMV的生物发生有关;TolC家族蛋白、OmpA与促进细菌的生存有关;核糖体蛋白(rpsG、rplQ、rplB)、与外膜相关的延伸因子(TufB、fusA)、DNA导向的RNA聚合酶(RpoA、RpoB、RpoC)以及代谢酶(aceE、SucB和fabG)推测与OMV可以促进遗传物质以及参与翻译和代谢酶的蛋白质转移到其他细菌的功能有关;丝状血凝素蛋白、OmpA和OmpW可能参与OMV与宿主细胞特异地相互作用;OmpA可以介导激活宿主免疫反应。
根据亚细胞定位分析,OMVs中的蛋白质来自细菌内不同的亚细胞区。大多数蛋白质(46.54%)被归类为细胞质蛋白质,其次是周质体蛋白质,占20.74%,外膜蛋白占17.51%(图5)。其中,细胞质蛋白所占比例最大。。为了确定所涉及的代谢和信号通路,对OMV蛋白进行了KEGG通路分析。在本研究鉴定的蛋白质中,3.4%参与了细胞过程,9.8%参与了环境信息处理,30.5%参与了遗传信息获取并促进了新陈代谢。参与代谢途径的蛋白质最多,这可能与OMV的生物遗传机制有关(图6)。
在COG分析中发现的大多数囊泡蛋白可能参与了多杀菌球菌的OMVs,主要与翻译、核糖体结构和生物发生;细胞壁/膜/包膜生物发生;碳水化合物转运和代谢;氨基酸转运和代谢;无机离子转运和代谢;细胞内贩运、分泌和囊泡转运;以及防御机制有关(图7)。
从GO分析结果可以看出,OMV蛋白的生物过程富集于氨基酸、脂肪和碳水化合物的运输、氧化还原、代谢和翻译等过程。从细胞组成上看,OMV的蛋白富集于核糖体、细胞膜、细胞外膜、细胞质和细胞周质等,OMV蛋白的分子功能富集于核糖体和核苷酸的结和组成等(图8)。
综上所述,多杀性巴氏杆菌OMV的蛋白质可能主要与其生物发生、促进细菌的生存、促进遗传物质和蛋白质向其他细菌的转移、调节宿主细胞和宿主免疫反应有关。本研究制备的多杀性巴氏杆菌OMV包含多种与毒力和感染机制有关的蛋白质和脂蛋白,这些蛋白质和脂蛋白触发宿主的先天免疫,并引发细胞和体液适应性免疫反应。是作为新型疫苗候选抗原的基础条件之一。
2免疫效果研究
2.1对免疫细胞的免疫效果研究
2.1.1对巨噬细胞增殖的影响
与OMV、MSN-OMV和DP-MSN-OMV(OMV终浓度为25、5、1、0.2和0.04μg/mL,DP-MSN和MSN终浓度为50、25、5、1和0.2μg/mL)作用后的巨噬细胞增殖水平均高于空白对照组(图9),说明它们均能有效促进巨噬细胞的增殖。比较各免疫组增殖水平发现,DP-MSN-OMV显著或极显著高于同浓度下的OMV组,且在OMV蛋白浓度为0.2μg/mL时,DP-MSN-OMV组增殖水平显著高于同浓度下的MSN-OMV组,促进巨噬细胞的增殖效果最明显。
2.1.2对巨噬细胞摄取的影响
激光共聚焦扫描显微镜图像显示OMV、MSN-OMV和DP-MSN-OMV都被成功内化,且主要富集在巨噬细胞内的溶酶体上,并可以看出孵育1h后,仅有少量在细胞内内化,在2h和4h后,巨噬细胞内OMV、MSN-OMV和DP-MSN-OMV的内化逐渐增加。且巨噬细胞对OMV、MSN-OMV和DP-MSN-OMV的摄取速度较快,几乎在4h后即可吞噬所有药物(图10)。
流式细胞术进一步检测RAW264.7细胞对DiD荧光标记的OMV、MSN-OMV和DP-MSN-OMV的摄取结果显示,在孵育1h后,RAW264.7细胞对OMV、MSN-OMV和DP-MSN-OMV的摄取量分别达69.3%、70.8%、82.2%;2h时摄取量略有增长,可达74.4%、88.0%和92.5%;在孵育4h后摄取量为95.5%、96.6%和98.4%,OMV几乎完全被摄取。DP-MSN-OMV处理后巨噬细胞的吞噬作用显著高于显示OMV和MSN-OMV组(图11)。因此,DP-MSN-OMV比起单独的OMV可以显著增强巨噬细胞的吞噬功能。且RAW264.7细胞对OMV、MSN-OMV和DP-MSN-OMV的摄取具有时间依赖性。
2.1.3对对巨噬细胞细胞因子分泌的影响
巨噬细胞可以通过分泌多种具有免疫增强作用的细胞因子来促进免疫应答,IL-1β、TNF-α是M1型巨噬细胞表达的促炎细胞因子,促进Th1免疫应答,TGF-β1、IL-10是M2型巨噬细胞表达的炎症趋化因子,M2型巨噬细胞通常起免疫调节作用,促进Th2免疫应答。结果表明,OMV、MSN-OMV和DP-MSN-OMV都能够显著促进巨噬细胞分泌细胞因子,其中与单独的OMV相比,DP-MSN-OMV能显著或极显著促进巨噬细胞产生TNF-α、IL-1β、IL-10、TGF-β1这四种细胞因子(图12A-D),与MSN-OMV相比,DP-MSN-OMV在OMV蛋白浓度为12.5μg/mL时能够显著促进巨噬细胞产生细胞因子IL-1β和TGF-β1(图12B、D)。且巨噬细胞分泌的这5种细胞因子呈剂量依赖性。可见OMV、MSN-OMV和DP-MSN-OMV可以同时促进巨噬细胞诱导Th1和Th2免疫应答。
2.2 DP-MSN-OMV对小鼠的免疫效果研究
2.2.1免疫后小鼠的抗体反应
小鼠最后一次免疫后42天内,DP-MSN-OMV组、MSN-OMV免疫组和Alum-OMV和OMV组均诱导了小鼠血清中高水平的OMV特异性IgG(图13A),这证明各实验组均能提高动物体特异性IgG抗体水平。在免后第14、28天DP-MSN-OMV组相较于Alum-OMV组无显著差异,在免后第42天DP-MSN-OMV组的OMV特异性IgG水平明显高于Alum-OMV组,在免后第14、28、42天DP-MSN-OMV组相较于MSN-OMV组均显著的提高了动物体特异性IgG抗体水平。可见将DP-MSN作为佐剂的多杀性巴氏杆菌OMV能有效刺激动物体内抗原特异性体液免疫应答。在小鼠体内,Th1型免疫应答会产生IgG2a型抗体,Th2型免疫应答产生IgG1型抗体。免后第28天的小鼠血清经倍比稀释后,血清中IgG1和IgG2a均得到了显著提高,其中以DP-MSN-OMV效果最好(图13B、C)。说明小鼠Th1和Th2型免疫应答均得到了显著增强,免疫对机体粘膜免疫也有显著增强的作用。
为进一步确认免疫后小鼠对P.multocida的免疫效果,我们进一步检测了血清中Pm.特异性的IgG、IgG1和IgG2a的水平。如图所示,DP-MSN-OMV组、MSN-OMV免疫组和Alum-OMV和OMV组分别免疫后P.multocida特异性IgG水平得到了显著提高,同时在免后第14、28、42天DP-MSN-OMV组相较于MSN-OMV组、Alum-OMV组和OMV组P.multocida特异性IgG水平均显著提高,且免后第28天的小鼠血清中P.multocida特异性IgG1水平得到了显著提高,其中DP-MSN-OMV效果显著优于MSN-OMV组和Alum-OMV组。而IgG2a水平DP-MSN-OMV组较MSN-OMV组和Alum-OMV组增加并不显著。(图14)。
2.2.2淋巴结中树突状细胞的活化和成熟
树突状细胞的激活是发展细胞免疫反应的第一步,LN中DC细胞首先对抗原进行摄取进而诱导T细胞反应,本研究通过分析LN中DC细胞激活标志物的表达,对OMV及DP-MSN-OMV在促进LN中DC细胞的活化和成熟从而产生免疫反应方面的作用进行评估。对小鼠淋巴结的树突细胞进行荧光标记的抗体染色,通过流式细胞术分析后,结果显示,以DP-MSN为佐剂的OMV疫苗组的小鼠LN中CD80+CD11c+、CD86+CD11c+、MHC-I+CD11c+和MHC-II+CD11c+的水平均高于单独免疫OMV的小鼠。且以DP-MSN为佐剂的OMV疫苗组CD80+CD11c+和MHC-II+CD11c+的表达水平高于以铝为传统佐剂的OMV疫苗组。同时由于OMV固有的抗原特性,与未免疫小鼠相比,OMV组和MSN-OMV组共刺激表达分子的百分比也有所增加(图15)。这些结果表明,纳米疫苗可以通过MHC-II途径促进抗原呈递,并通过MHC-I通路促进抗原的交叉呈递,且疫苗刺激后的LN中DC细胞的共刺激分子CD80/CD86的表达上调,即疫苗可以显著促进LN中DC细胞的活化和成熟,显著增强细胞免疫应答。
2.2.3脾脏中淋巴细胞的激活和分化
T细胞和B细胞是两个主要的淋巴细胞亚群,它们可以介导获得性免疫的产生。T淋巴细胞主要负责建立细胞介导的免疫反应,而B淋巴细胞则负责体液免疫反应。本研究对疫苗免疫后的小鼠脾脏内T细胞亚群和B细胞的百分比进行了定量分析。将免疫后小鼠的脾脏制成单细胞悬液,用荧光标记的抗体染色,通过流式细胞术分析后,结果如图所示,DP-MSN-OMV组的CD4/CD8T细胞比例显著高于MSN-OMV组、以铝为佐剂的OMV组和OMV组(图16A),DP-MSN-OMV组的CD19+B细胞比例著高于和OMV组和未免疫组,而与以铝为佐剂的OMV组相比,增加不显著(图16B)。这些结果提示,DP-MSN-OMV纳米疫苗比铝为佐剂的OMV和OMV疫苗能进一步提高CD4/CD8T和CD19+B细胞的比例。
2.2.4脾脏中T细胞亚群分化
在各种细胞因子的存在下,APC通过MHC-II类分子将多肽抗原递送给原始T细胞,原始T细胞被激活后产生不同的效应T辅助细胞亚群,包括Th1、Th2和Th17。其中Th1细胞分泌IFN-γ和TNF-α等细胞因子,主要负责细胞介导的免疫反应,IL-4可抑制Th1细胞分化,继而诱导另Th2细胞亚群,Th2细胞分泌IL-4、IL-5和IL-13等细胞因子,主要负责体液免疫;Th17细胞则分泌IL-17,对于宿主抵御细菌和真菌感染至关重要,可能是疫苗诱导免疫的有用靶点。本研究进一步评估了DP-MSN-OMV和OMV对小鼠脾脏的原始CD4T细胞分化的影响,通过流式细胞术检测了免疫后小鼠脾脏中Th1(CD4IFN-γT细胞)、Th2(CD4IL-4T细胞)和Th17(CD4IL-17T细胞)的百分比。结果发现,DP-MSN-OMV免疫组小鼠脾脏的Th1、Th2和Th17细胞亚群分化的比例均显著或极显著高于以铝为佐剂的OMV疫苗组和单独的OMV组,且DP-MSN-OMV免疫组小鼠脾脏的Th1细胞亚群的分化的比例显著高于MSN-OMV免疫组(图17A),Th2和Th17细胞亚群的分化的比例相较于MSN-OMV免疫组也有增加但并不显著(图17B、C),同时OMV免疫组的Th1、Th2和Th17细胞亚群分化的比例均高于未免疫组。这些结果表明,DP-MSN作为OMV疫苗的佐剂比起传统的铝佐剂和单独的OMV疫苗可以进一步增加CD4T细胞中Th1、Th2和Th17细胞的比例,并产生相应的细胞因子,诱导有效的混合Th1/Th2/Th17免疫反应。
2.2.5脾脏中记忆细胞的分化
记忆细胞在产生免疫记忆和发挥保护性免疫应答方面有着重要作用。经抗原刺激后的初始T细胞增殖并分化为效应T细胞和记忆T细胞。根据记忆T细胞的功能可分为中央记忆T细胞(TCM)和T效应记忆T细胞(TEM)。TCM存在于淋巴器官中,不能立即被激活,当再次受到抗原刺激,可以快速增殖和分化,为周围部位的效应性T细胞提供补给;TEM主要存在于周围组织和炎症部位,具有快速效应功能,可快速产生效应细胞因子,进行免疫保护,防止感染部位的再次感染。为了进一步确认DP-MSN-OMV在适应性免疫应答中的诱导作用,将免疫后小鼠的脾脏制成单细胞悬液,用荧光标记的抗体染色,通过流式细胞术对CD4和CD8TCM(CD44+CD62L+)和TEM(CD44+CD62L-)细胞进行分析,结果如图所示,用DP-MSN-OMV免疫的小鼠脾脏中的TCM(CD44+CD62L+)和TEM(CD44+CD62L-)细胞均显著高于OMV免疫组,且DP-MSN-OMV免疫组TCM(CD44+CD62L+)细胞显著高于以铝为佐剂的OMV免疫组(图18A)。CD8TCM(CD44+CD62L+)和TEM(CD44+CD62L-)细胞的结果与CD4相似(图18B)。结果表明,DP-MSN-OMV可以有效诱导记忆T细胞,产生强大的免疫记忆。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (8)
1.一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于包括下述步骤:
(1)外膜囊泡的制备
将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上进行培养,然后挑取单菌落接种于马丁肉汤培养基中进行培养,扩大培养后得预培养菌液;取预培养菌液的上清液,超速离心,过滤,将滤液收集到超滤器中浓缩,浓缩液超速离心,弃上清液,沉淀用PBS缓冲液重悬,于-80℃条件下冻存;
(2)介孔二氧化硅载石斛多糖纳米颗粒的合成
将介孔二氧化硅分散于甲苯中,超声中加入APTES反应,离心收集沉淀,沉淀用乙醇和ddH2O分别洗涤数次后,分散于ddH2O中,得到氨基修饰的介孔二氧化硅纳米粒使其带正电;将二氧化硅分散液与石斛多糖在室温下剧烈搅拌后,离心处理,水洗两次,即得介孔二氧化硅载石斛多糖;
(3)外膜囊泡包裹介孔二氧化硅载石斛多糖的合成:
用装有0.2μm聚碳酸酯薄膜过滤膜的微型挤出机对介孔二氧化硅载石斛多糖和外膜囊泡挤压数次进行结合,得到多杀性巴氏杆菌外膜囊泡DP-MSN-OMV。
2.根据权利要求1中所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:步骤(1)中,将多杀性巴氏杆菌划线接种于马丁肉汤固体培养基平板上进行培养的条件:37℃,培养时间为24h,单菌落接种于马丁肉汤培养基中进行培养的条件:37℃、200r/min摇床培养18h;按体积比1∶100扩大培养。
3.根据权利要求1中所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:步骤(1)中,培养菌液上清液的超速离心具体为:在4℃条件下,以5×103g离心10min,取上清液再次以4℃,5×103g的条件离心10min;过滤时分别经0.45μm和0.22μm的滤膜过滤以确保没有活菌和细胞碎片残留;采用具有100kDa滤膜的超滤器浓缩;浓缩液超速离心的条件:1×105g超速离心1-3h。
4.根据权利要求1中所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:步骤(1)中,介孔二氧化硅的制备方法如下:将十六烷基三甲基甲苯磺酸铵、三乙醇胺、ddH2O混合后加热至80℃,搅拌1h至完全溶解,快速加入正硅酸四乙酯,在80℃的条件下反应2h,然后离心收集沉淀,沉淀使用ddH2O和乙醇交替洗3次后分散于乙醇中,即得二氧化硅;将分散于乙醇中的二氧化硅,加入到乙醇和盐酸溶液中,70℃反应12h,离心收集沉淀,使用ddH2O和乙醇交替洗3次后,甲苯洗2次后沉淀分散于甲苯中,得到介孔二氧化硅。
5.根据权利要求4所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:乙醇和盐酸溶液中乙醇和盐酸的体积比为:(8-12):1。
6.据权利要求1中所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:步骤(2)中,加入APTES反应条件为:在N2保护下,80℃反应12h。
7.据权利要求1中所述的一种多杀性巴氏杆菌外膜囊泡的制备方法,其特征在于:步骤(3)中,介孔二氧化硅载石斛多糖和外膜囊泡混悬液的体积比为1:1。
8.权利要求1-7任一项制备方法制得的多杀性巴氏杆菌外膜囊泡在预防多杀性巴氏杆菌病中的应用。
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