CN116747295A - 一种IgM功能化的仿生纳米类毒素疫苗及其制备方法和应用 - Google Patents
一种IgM功能化的仿生纳米类毒素疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及疫苗技术领域,具体是一种IgM功能化的仿生纳米类毒素疫苗及其制备方法和应用。本发明的红细胞膜‑杂合脂质体类毒素疫苗可以装载细菌成孔毒素,无须灭活蛋白毒素,使其活性极大增强,最大限度保留免疫原性,进一步以叶酸对纳米疫苗进行修饰,在体内可吸附天然IgM于纳米载体表面,显著提高抗原递呈效率,增强免疫应答,免疫接种后产生高滴度的特异性抗体预防细菌感染。
Description
技术领域
本发明涉及疫苗技术领域,具体地说,是一种IgM功能化的仿生纳米类毒素疫苗及其制备方法和应用。
背景技术
细菌感染是世界范围内的一类高发病率和高致死率疾病,而细菌耐药给抗细菌感染治疗带来了严峻挑战,至2050年耐药细菌感染将导致全球上千万人死亡,累计花费上百万亿美元。创伤后细菌感染治疗中,脓毒症、多器官功能障碍综合症严重影响患者生存率。创伤后感染的主要病原体极大比例为多重耐药(MDR)菌,严重创伤后脓毒症患者MDR菌感染占29.37%,死亡患者中因MDR菌感染占28%。而抗生素耐药问题是抗细菌感染治疗中面临的最为严峻的问题,在临床的使用而广泛发生。因此,亟需新的给药策略以改善细菌乃至耐药菌感染的预防和治疗效果。
一些感染包括创伤后感染常见的细菌有葡萄球菌属、链球菌属、破伤风梭菌等,均可产生外毒素。成孔毒素是外毒素的一种,是多种细菌感染的主要毒力因子,通过破坏上皮屏障和免疫系统来协助病原菌的生长、侵袭和定殖,是抗细菌感染的主要靶点之一。通过抑制成孔毒素来治疗耐药细菌感染,可以非直接的方式杀灭病原菌从而避免产生耐药性。抗毒素疫苗是指将抗毒素策略与疫苗治疗进行结合,尽可能地减少细菌耐药现象的产生。传统的抗毒素疫苗(类毒素疫苗)通过变性蛋白的方法来减少毒素抗原的毒性,存在的主要问题是难以同时保证灭活毒素的安全性和免疫原性。因此根据成孔毒素的生物特性,结合仿生纳米策略构建新的疫苗载体系统很有意义。
游离成孔毒素可与细胞膜表面的一些特异性受体结合,后续折叠组装成多聚体,识别、组装插入细胞膜表面的特定区域,如红细胞、巨噬细胞、内皮细胞、上皮细胞等。红细胞是众多成孔毒素发挥毒性的主要靶细胞之一,因此可以作为毒素吸附的介质。文献(Nanoparticle-detained toxins for safe and effective vaccination.NatNanotechnol 2013,8(12),933-938)将红细胞膜包载于聚合物纳米粒子表面,可以吸附并中和金黄葡萄球菌α溶血素。但是该方法需要借助聚合物纳米粒子内核,制备方法较为复杂。文献(Erythroliposomes:Integrated hybrid nanovesicles composed oferythrocyte membranes and artificial lipid membranes for pore-forming toxinclearance.ACS Nano.2019,13,4148-4159;Broad-spectrum and powerfulneutralization of bacterial toxins by erythroliposomes with the help ofmacrophage uptake and degradation.Acta Pharmaceutica Sinica.B,2022,12(11):4235-4248)和中国专利文献CN113041224 A;CN 113041346 A;CN 113041345A描述了一种红细胞膜与脂质体融合后制备的纳米载体吸附金黄葡萄球菌a溶血毒素。以上方法仅是利用红细胞膜和脂质体的杂合载体作为纳米佐剂发挥作用,无其他增强免疫应答的机制。如何提高纳米佐剂的抗原递呈效果是一个重要问题。
纳米载体应用于机体后,吸附各种血浆蛋白使其维持特定的内源性蛋白功能,产生更多生物学效应。例如以叶酸修饰的脂质体入血后,可大量吸附天然IgM,天然IgM为叶酸结合蛋白,在调控叶酸修饰的纳米药物体内性能中发挥一定作用。而血液中的免疫球蛋白IgM可以作为天然佐剂,增加抗原递呈,增强纳米疫苗的体内免疫应答。脂质体吸附IgM后,脾脏B细胞分布显著增加。小鼠经免疫刺激后,疫苗产生的特异性抗体滴度能长期维持较高水平。但是目前尚未有藉由IgM功能化增强疫苗免疫应答作用用于细菌毒素疫苗的报道。
发明内容
本发明的目的在于提供一种新型的由IgM功能化作用增强抗原递呈从而增强免疫应答的仿生纳米类毒素疫苗,主要涉及脂质材料与细胞膜形成杂合载体,而后装载细菌成孔毒素形成类毒素疫苗、载体表面修饰叶酸分子形成类毒素疫苗。
为了实现上述目的,本发明采用以下技术方案:
1、叶酸修饰脂质体的制备
称取一定量的蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400加入二氯甲烷溶解,40℃条件下在旋转蒸发仪上除去二氯甲烷形成薄膜。
2、红细胞膜的制备
BLAB/c雌性6-8周龄小鼠用3%异氟烷麻醉后经眼球取血。在4℃条件下,提取小鼠纯红细胞。将上述纯红细胞膜加入等渗液重悬,而后加入低渗液使红细胞溶胀,4℃条件下20000g离心重复洗涤三次,得到红细胞膜。
3、叶酸修饰的红细胞膜杂合脂质体的制备
薄膜水化法制备脂质体后,水化过程中加入红细胞膜,水化后冰浴超声5min,脂质体挤出器连续挤出后制备杂合载体。
4、叶酸修饰的细胞膜杂合脂质体疫苗的制备
将上述载体制备完成后与细菌成孔毒素在37℃条件下共孵育1h,即得到叶酸修饰的细胞膜杂合脂质体疫苗。
5、对叶酸修饰的细胞膜杂合脂质体疫苗的理化性质进行表征
采用动态光散射法测定其粒径,Zeta电位,多分散系数。采用透射电镜观察其形态和大小。
6、通过皮下注射类毒素疫苗免疫小鼠,测定其淋巴结中生发中心成熟B淋巴细胞比例,测定其血清抗体滴度及抗体中和毒素的效果,考察类毒素疫苗的免疫激活情况。
7、通过树突细胞对类毒素疫苗的摄取实验,考察类毒素疫苗的抗原摄取及提呈效果。
8、通过皮下注射类毒素疫苗免疫小鼠,腹腔注射细菌成孔毒素(如创伤弧菌溶血毒素A(VvhA)),考察疫苗接种预防细菌成孔毒素感染的效果。
9、通过皮下注射类毒素疫苗免疫小鼠,腹腔注射耐药细菌,考察疫苗接种预防耐药细菌感染的效果。
构建红细胞膜-脂质体杂合载体,将叶酸修饰于载体表面,一方面由红细胞膜对毒素的高吸附能力,脂质体对毒素吸附空间的提高,可以吸附多种细菌产生的致孔毒素作为类毒素蛋白抗原模型,将毒素以多聚体的形式装载于纳米载体上,中和毒素毒性的同时,最大限度的保留其抗原活性。同时,将叶酸分子作为结合IgM的靶分子修饰于红细胞膜杂合脂质体表面,在体内吸附IgM,利用其天然佐剂的作用增强类毒素疫苗的机体免疫应答,制备一种用于细菌感染防治的类毒素疫苗。目前尚未有以IgM功能化增强疫苗免疫应答作用用于细菌类毒素疫苗的报道,有望极大的增强细菌类毒素疫苗的免疫效果。
基于上述技术方案,本发明的第一方面,提供一种IgM功能化的仿生纳米类毒素疫苗的制备方法,包括以下步骤:
(A)叶酸修饰的脂质体(FA-Lips)的制备:
称取一定量的蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400加入二氯甲烷溶解,40℃条件下在旋转蒸发仪上除去二氯甲烷形成薄膜;
所述的叶酸修饰的脂质体由蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400组成,表示为FA-Lips。胆固醇是脂质膜的重要组成部分,对脂质体吸附毒素的效果影响较大。所述的蛋黄卵磷脂与胆固醇的摩尔比为4:1~4:4,优选4:3和4:4。DSPE-mPEG2000、FA-DSPE-mPEG3400对脂质体疫苗的稳定性有重要影响,所述的蛋黄卵磷脂与DSPE-mPEG2000、FA-DSPE-mPEG3400的摩尔比为4:(0.8~0.4):(0.6~0.2),优选4:0.8:0.2。
进一步的,所述的蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400的摩尔比为4:(1~4):(0.8~0.4):(0.6~0.2)。优选为4:4:0.8:0.2。
(B)红细胞膜的制备:
BLAB/c雌性6-8周龄小鼠用3%异氟烷麻醉后经眼球取血;在4℃条件下,提取小鼠纯红细胞;将上述纯红细胞加入等渗液重悬,而后加入低渗液使红细胞溶胀,4℃条件下20000g离心重复洗涤三次,得到红细胞膜;
(C)叶酸修饰的红细胞膜杂合脂质体的制备:
薄膜水化法制备脂质体后,水化过程中加入红细胞膜,水化后冰浴超声5min,脂质体挤出器连续挤出后制备杂合载体;
(D)叶酸修饰的细胞膜杂合脂质体类毒素疫苗的制备:
将步骤(C)制备得到的杂合载体与细菌成孔毒素在37℃条件下共孵育1h,得到叶酸修饰的细胞膜杂合脂质体疫苗(RCM-FA-Lips),即为所述的IgM功能化的仿生纳米类毒素疫苗。
进一步的,所述的步骤(B)中,在4℃条件下,700g离心5min,用等渗液洗涤两次,而后3200g离心5min,用等渗液洗涤三次,提取小鼠纯红细胞,用等渗液1:1的比例将红细胞重悬,加入950μL低渗液,再加入50uL PBS,4℃条件20000g离心10min,用低渗液洗涤三次,得到类白色沉淀红细胞膜,置于-80℃条件下备用。
进一步的,所述的步骤(C)中,脂质体与红细胞膜(膜蛋白定量)的质量比为(9~36):1,优选18:1。
进一步的,所述的步骤(C)中,叶酸在红细胞膜-杂合脂质体上的修饰质量比为5-15%。
进一步的,所述的步骤(C)中,脂质体挤出器连续挤出通过400nm、200nm、100nm聚碳酸酯膜各20次制备杂合载体。
进一步的,所述的细菌成孔毒素为创伤弧菌溶细胞素VvhA,金黄色葡糖球菌α毒素,铜绿假单胞菌外毒素A,李斯特菌溶血素O,大肠杆菌α溶血素,链球菌溶血素O,破伤风溶血素。
进一步的,所述的步骤(D)中,红细胞膜融合叶酸修饰脂质体与细菌成孔毒素的质量比为15.8:1~3.9:1,优选7.9:1。
在本发明的一个具体实施方式中,所述的细菌成孔毒素为创伤弧菌溶血毒素A(VvhA)。
本发明的纳米载体疫苗技术可以应用于多种细菌的致孔毒素装载,创伤感染常见的细菌有葡萄球菌属、链球菌属、假单胞菌、破伤风梭菌、海洋创伤弧菌等,这些细菌均可产生致孔毒素(创伤弧菌溶细胞素VvhA,金黄色葡糖球菌α毒素,铜绿假单胞菌外毒素A,李斯特菌溶血素O,大肠杆菌α溶血素,链球菌溶血素O,破伤风溶血素)。利用致孔毒素在细胞膜(红细胞、巨噬细胞等)表面聚集打孔机制,可将此类毒素吸附于本发明构建的杂合载体上,降低毒性的同时完整保留外毒素蛋白的免疫原性。通过Fa修饰,并吸附IgM作为纳米佐剂可以提高抗原递呈细胞的摄取及抗原提呈效率,增加体液免疫应答,用于复杂细菌感染的防治,发挥高效、广谱的作用。
本发明的第二方面,提供一种采用如上所述的制备方法制备得到的IgM功能化的仿生纳米类毒素疫苗。
本发明的第三方面,提供一种如上所述的IgM功能化的仿生纳米类毒素疫苗在制备细菌感染防治产品中的应用。
本发明优点在于:
本发明的仿生纳米类毒素疫苗一方面无须灭活蛋白毒素,使其活性极大增强,提高免疫原性,进一步利用叶酸配体的修饰从而吸附天然IgM于纳米载体表面,显著提高抗原递呈效率,增强免疫应答,免疫接种后产生高滴度的特异性抗体预防细菌感染。
附图说明
图1.载成孔毒素脂质体(RCM-FA-Lips)的理化性质表征。其中:A为(RCM-FA-Lips)的平均粒径,B为(RCM-FA-Lips)的分散指数,C为(RCM-FA-Lips)的Zeta电位,D为(RCM-FA-Lips)负染后的透射电镜结果。
图2.RCM-FA-Lips的吸附毒素能力评价。其中:A、B:VvhA的溶血量效关系(n=3);C、D:RCM-FA-Lips吸附VvhA毒素蛋白的量效关系(n=3);E、F:RCMs、FA-Lips和RCM-FA-Lips与VvhA孵育后对红细胞的溶血百分比(n=3);***P<0.001。
图3.RCM-FA-Lips的细胞解毒能力评价。NCM460细胞毒性测定结果(n=3);***P<0.001。
图4.皮肤解毒实验考察RCM-FA-Lips的体内解毒能力。其中:A为小鼠皮肤损伤模型,B为小鼠皮肤组织的H&E染色结果。
图5.RCM-FA-Lips的抗原摄取及提呈效果评价。
图6.疫苗免疫后小鼠淋巴结生发中心B细胞免疫荧光染色。其中:A为PBS组的生发中心B细胞免疫荧光染色,B为RCM-Lips(VvhA)组的生发中心B细胞免疫荧光染色,C为RM-FA-Lips组的生发中心B细胞免疫荧光染色。
图7.疫苗免疫后小鼠淋巴结中生发中心B细胞的流式结果。其中:A为PBS组的生发中心B细胞流式结果,B为RCM-Lips(VvhA)组的生发中心B细胞流式结果,C为RM-FA-Lips组的生发中心B细胞流式结果。
图8.疫苗免疫后小鼠抗体滴度。****P<0.0001
图9.疫苗免疫后小鼠生存率。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1:叶酸修饰脂质体的制备
称取摩尔比为4:4:0.8:0.2的蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400加入二氯甲烷溶解,40℃条件下在旋转蒸发仪上除去二氯甲烷形成薄膜。脂质体由蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400组成,表示为FA-Lips。
实施例2:红细胞膜的制备
BLAB/c雌性6-8周龄小鼠用3%异氟烷麻醉后经眼球取血。在4℃条件下,700g离心5min,用等渗液洗涤两次,而后3200g离心5min,用等渗液洗涤三次,提取小鼠纯红细胞,用等渗液1:1的比例将红细胞重悬,加入950μL低渗液,再加入50uL PBS,4℃条件20000g离心10min,用低渗液洗涤三次,得到类白色沉淀红细胞膜,置于-80℃条件下备用。
实施例3:叶酸修饰的红细胞膜杂合脂质体的制备
薄膜水化法制备脂质体后,水化过程中加入一定量的红细胞膜(膜蛋白定量),脂质体与红细胞膜的质量比为18:1,水化后冰浴超声5min,脂质体挤出器连续挤出通过400nm、200nm、100nm聚碳酸酯膜各20次制备杂合载体。
实施例4:叶酸修饰的细胞膜杂合脂质体疫苗的制备
将实施例3制备好的叶酸修饰的细胞膜杂合脂质体与创伤弧菌溶血素A(VvhA)37℃条件下共孵育1h,制备叶酸修饰的细胞膜杂合脂质体疫苗(RCM-FA-Lips)。红细胞膜融合叶酸修饰脂质体与VvhA的质量比为7.9:1。
实施例5:对叶酸修饰的细胞膜杂合脂质体疫苗的理化性质进行表征
使用马尔文粒径仪测定实施例4的红细胞膜融合脂质体疫苗的粒径和Zeta电位,并与叶酸修饰的脂质体(FA-Lips),红细胞囊泡(RCMVs)进行对比。采用负染法进行样品制备,将一定浓度的RCM-FA-Lips醋酸铀进行负染,TEM观察。
结果如图1所示,RCMVs平均粒径为120.6nm,FA-Lips的平均粒径为93.9nm,RCM-FA-Lips的平均粒径为101.1nm(图1A),RCMVs、FA-Lips和RCM-FA-Lips的分布均较窄(图1B)。RCM-FA-Lips Zeta电位值(-22.9mV)介于RCMVs(-10.4mV)与FA-Lips(-37.7mV)之间(图1C)。TEM结果显示,RM-FA-Lips呈规则类球形,大小均一,粒径约100nm(图1D)。以上结果表明,处方量毒素负载对融合脂质体的理化性质无显著影响。
实施例6:
通过体外溶血试验测定RCM-FA-Lips对毒素的负载量。具体操作:将100μg VvhA与不同量的RCM-FA-Lips室温下1h,然后与适量2.5%红细胞膜在37℃下振荡孵育1h,2000g离心5min,测定上清在540nm的吸光度值并按下述公式计算溶血百分数。其中阴性对照为生理盐水,阳性对照为1%TritonX-100。根据溶血百分数为0时RCM-FA-Lips的用量计算RCM-FA-Lips对毒素的负载量。
溶血率(%)=[(A样品-A阴性对照)/(A阳性对照-A阴性对照)]×100%
结果显示,40μg的创伤弧菌溶血毒素A(VvhA)可以使1mL 2.5%红细胞悬液完全溶血;314μg的RCM-FA-Lips几乎完全中和40μg的VvhA毒素蛋白,抗溶血率达到98.78±0.08%。而FA-Lips和RCMVs组均产生溶血现象,无中和毒素能力(图2),显示出RCM-FA-Lips良好的安全性。
实施例7:
文献表明创伤弧菌溶血毒素A(VvhA)对上皮细胞有一定的细胞毒性。本发明采用NCM460为细胞模型,采用结肠上皮细胞(NCM460)为细胞模型,CCK-8法测定细胞活力,以研究RCM-FA-Lips的细胞水平解毒能力。NCM460按5000个/孔接种于96孔板,37℃、5% CO2培养过夜。将50μL RCM-FA-Lips、FA-Lips、红细胞膜(RCMs)和生理盐水分别与不同浓度的创伤弧菌溶血毒素A(VvhA)(10、50、100、200和400μg/mL)室温孵育1h。而后加入96孔板中,与NCM460在37℃下共孵育12h。孵育完毕后每孔加入10μL CCK-8溶液,继续37℃下孵育1h,酶标仪测量450nm处OD值,计算细胞存活率。结果显示,细胞活力与创伤弧菌溶血毒素A(VvhA)呈剂量依赖性,与对照组相比RCM-FA-Lips组显著提高细胞活力,具有一定的毒性抑制作用,而FA-Lips组和RCMs组也具有一定的毒素吸附能力,但随着毒素浓度的增加,这种毒素中和能力显著降低(图3)。上述结果提示RCM-FA-Lips具有较好的体外安全性。
实施例8:
取BALB/c雌性8-10周龄小鼠,颈椎脱臼法处死,取小鼠骨髓细胞。用含20ng/mLGM-CSF,20ng/mL IL-4的1640培养基诱导骨髓细胞分化成树突状细胞(DCs)。
将蛋黄卵磷脂、胆固醇、DSPE-mPEG2000以4:4:1的摩尔比制备成脂质体,加入一定量的DiI染料稀释液,薄膜分散法成膜后,避光水化半小时,然后4℃条件下,14000g离心15min,去除游离的染料,洗涤三次,加水重悬,而后与红细胞膜以18:1的质量比融合成红细胞膜杂合脂质体(RCM-Lips),再与VvhA以7.9:1的质量比制备成脂质体疫苗(RCM-Lips(VvhA)),作为对照组。将蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400以4:4:0.8:0.2的摩尔比制备成脂质体,加入一定量的DiI染料稀释液,薄膜分散法成膜后,避光水化半小时,然后4℃条件下,14000g离心15min,去除游离的染料,洗涤三次,加水重悬,而后与红细胞膜以18:1的质量比融合成红细胞膜杂合脂质体(RCM-FA-Lips),再与VvhA以7.9:1的质量比制备成脂质体疫苗(RCM-FA-Lips)。
取BALB/c雌性8-10周龄小鼠血清,将上述两组类毒素疫苗按1:1的比例分别与血清共孵育1h,使类毒素疫苗能够充分吸附血清中的IgM。
将DC细胞密度调整为5×105个/mL,取1mL接种于共聚焦专用皿,37℃孵育12h。吸弃培养基,取一定量的上述两组与血清共孵育的类毒素疫苗分别加入到对应的皿中,37℃避光孵育1h,吸弃上清,PBS洗涤2次,4%多聚甲醛固定15分钟,PBS洗涤3次,加入0.1%Triton X-100作用1分钟,PBS洗涤,加入1mL 20μg/mL DAPI染色10min,PBS洗涤2次,加入1mL PBS,置于激光共聚焦下观察DC对于类毒素疫苗的摄取情况,激发波长为549nm。
结果显示图5,DCs对RCM-FA-Lips(VvhA)的摄取更显著。结果表明叶酸修饰的脂质体能够起到更好的抗原提呈效率。
实施例9:
取BALB/c雌性8-10周龄小鼠,随机分为三组。分别每7天颈背部皮下注射200μLSaline、RCM-Lips(VvhA)、RCM-FA-Lips(VvhA),其中VvhA剂量均为100μg。参考文献方法,疫苗接种共三次,第一次为初始免疫,第二次与第三次为加强免疫。
于第三次免疫接种完成后第7天,将小鼠脱颈处死,取小鼠腋窝淋巴结O.C.T包埋后,10μm厚度制备冰冻切片,进行免疫荧光染色(IF),首先用1×PBS将组织切片洗涤三次后滴加封闭液(10% BSA+0.1% TritonX-100),室温孵育30min,完成后用封闭液稀释的荧光标记抗体(Pacific Blue标记抗小鼠B220抗体、Alexa Fluor 488标记抗小鼠IgD抗体、Alexa Flour 647标记抗小鼠GL-7抗体)4℃避光条件下染色过夜,甘油明胶进行封片。共聚焦显微镜进行观察并拍摄荧光照片。
同样于第三次免疫接种完成后第7天,将小鼠脱颈处死,取小鼠腋窝淋巴结组织研磨均匀,4℃条件下700g离心5min,弃去上清加培养基重悬,得到淋巴结细胞。然后加入含1% BSA的1×PBS溶液,置于4℃条件下封闭10min。加入上述荧光标记抗体,4℃条件下孵育30min免疫荧光染色后流式细胞仪分析B细胞的比例。
结果如图6,绿色为B细胞,蓝色为未成熟B细胞,红色为生发中心B细胞。从结果中可以看出,RCM-Lips(VvhA)和RCM-FA-Lips(VvhA)组均显示出了生发中心的存在,而阴性对照组(生理盐水组)无。然而与RCM-Lips(VvhA)组比较,RCM-FA-Lips(VvhA)显示出面积更大的GC区域。
通过流式细胞术对淋巴结进行了定量分析。将淋巴结消化后,同样用前述荧光标记抗体对细胞悬液进行染色,再用流式细胞仪进行分选。结果显示(图7),RCM-FA-Lips(VvhA)能够显著提高GC B细胞的比例,约为RCM-Lips(VvhA)组的1.3倍。说明叶酸修饰的纳米类毒素疫苗有更强的抗原递送效率,增强了免疫激活能力。
实施例10:
取实施例9小鼠第三次接种完成后第7天的全血,收集血清,于-80℃保存,待测。
采用间接酶联免疫吸附测定(ELISA)法检测小鼠血中的VvhA特异性抗体。
从图8可以看出,Saline组无VvhA特异性抗体产生。而RCM-Lips(VvhA)组和RCM-FA-Lips(VvhA)组能刺激机体产生更高的血清抗体滴度。RCM-FA-Lips(VvhA)抗体的表达水平约为RCM-Lips(VvhA)的2倍(P<0.0001)。上述结果表明,RCM-FA-Lips(VvhA)能诱导机体产生较强的免疫反应。
实施例11:
取BALB/c雌性8-10周龄小鼠,随机分为三组,每组10只。分别每7天颈背部皮下注射200μL Saline、甲醛脱毒VvhA、RCM-Lips(VvhA)、RCM-FA-Lips(VvhA),其中VvhA剂量均为100μg。疫苗接种共三次,第一次为初始免疫,第二次与第三次为加强免疫。第三次免疫完成后,小鼠腹腔注射1×106CFU创伤弧菌后(100μL),给药结束后立即开始计时,观察小鼠精神状态、进食情况,并记录小鼠生存情况。
生理盐水组小鼠在给予创伤弧菌后立即出现精神萎靡、被毛竖立、食欲减退、呼吸困难等症状,在注射创伤弧菌后72h内陆续死亡。甲醛脱毒VvhA免疫组24h内死亡2只,72h内共死亡4只,小鼠生存率为60%。而RCM-Lips(VvhA)组小鼠在给予创伤弧菌后48小时内死亡1只,72小时内共死亡2只,其余全部存活,存活率为80%,RCM-FA-Lips(VvhA)组无死亡小鼠(图9)。上述结果表明RCM-FA-Lips(VvhA)免疫后能够产生更好的免疫保护作用。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.一种IgM功能化的仿生纳米类毒素疫苗的制备方法,其特征在于,包括以下步骤:
(A)叶酸修饰的脂质体FA-Lips的制备:
称取蛋黄卵磷脂、胆固醇、DSPE-mPEG2000、FA-DSPE-mPEG3400加入二氯甲烷溶解,40℃条件下在旋转蒸发仪上除去二氯甲烷形成薄膜;所述的蛋黄卵磷脂、胆固醇、DSPE-mPEG、FA-DSPE-mPEG的摩尔比为4:(1~4):(0.8~0.4):(0.6~0.2);
(B)红细胞膜的制备:
BLAB/c雌性6-8周龄小鼠取血,在4℃条件下,提取小鼠纯红细胞,将上述纯红细胞加入等渗液重悬,而后加入低渗液使红细胞溶胀,4℃条件下20000g离心重复洗涤三次,得到红细胞膜;
(C)叶酸修饰的红细胞膜杂合脂质体的制备:
薄膜水化法制备叶酸修饰的脂质体FA-Lips后,水化过程中加入红细胞膜,水化后冰浴超声5min,脂质体挤出器连续挤出后制备杂合载体;
(D)叶酸修饰的细胞膜杂合脂质体疫苗的制备:
将步骤(C)制备得到的杂合载体与细菌成孔毒素在37℃条件下共孵育1小时,得到叶酸修饰的细胞膜杂合脂质体疫苗RCM-FA-Lips,即为所述的IgM功能化的仿生纳米类毒素疫苗。
2.根据权利要求1所述的制备方法,其特征在于,所述的步骤(B)中,在4℃条件下,700g离心5min,用等渗液洗涤两次,而后3200g离心5min,用等渗液洗涤三次,提取小鼠纯红细胞,用等渗液1:1的比例将红细胞重悬,加入950μL低渗液,再加入50uL PBS,4℃条件20000g离心10min,用低渗液洗涤三次,得到类白色沉淀红细胞膜,置于-80℃条件下备用。
3.根据权利要求1所述的制备方法,其特征在于,所述的步骤(C)中,脂质体与红细胞膜的质量比为(9~36):1。
4.根据权利要求1所述的制备方法,其特征在于,所述的步骤(C)中,叶酸在红细胞膜-杂合脂质体上的修饰质量比为5-15%。
5.根据权利要求1所述的制备方法,其特征在于,所述的步骤(C)中,脂质体挤出器连续挤出通过400nm、200nm、100nm聚碳酸酯膜各20次制备杂合载体。
6.根据权利要求1所述的制备方法,其特征在于,所述的细菌成孔毒素为创伤弧菌溶细胞素VvhA,金黄色葡糖球菌α毒素,铜绿假单胞菌外毒素A,李斯特菌溶血素O,大肠杆菌α溶血素,链球菌溶血素O,破伤风溶血素。
7.根据权利要求1所述的制备方法,其特征在于,所述的步骤(D)中,红细胞膜融合叶酸修饰脂质体与细菌成孔毒素的质量比为15.8:1~3.9:1。
8.一种采用如权利要求1-7任一所述的制备方法制备得到的IgM功能化的仿生纳米类毒素疫苗。
9.一种如权利要求8所述的IgM功能化的仿生纳米类毒素疫苗在制备细菌感染防治产品中的应用。
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