CN110294797A - 旋毛虫副肌球蛋白H-2d限制性Th表位P5、其组合物及应用 - Google Patents
旋毛虫副肌球蛋白H-2d限制性Th表位P5、其组合物及应用 Download PDFInfo
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Abstract
本发明属于免疫生物学领域,涉及一种旋毛虫副肌球蛋白H‑2d限制性Th表位P5、其组合物及应用。具体地,所述的旋毛虫副肌球蛋白Th2表位,其氨基酸序列如SEQ ID NO:5所示。本发明的表位能够刺激/促进脾淋巴细胞显著增殖,刺激/促进小鼠脾细胞CD4+T细胞增高,刺激/促进Th0细胞向Th2分化,刺激/促进Th1型细胞因子IFN‑γ、IL‑2以及Th2型细胞因子IL‑4、IL‑5升高;刺激/促进Th0细胞向Th2细胞分化,免疫小鼠后均可诱导产生Th2免疫反应,具有应用于制备疫苗例如多表位疫苗的潜力。
Description
本发明是申请号为201510012293.X的母案的分案申请,该母案的申请日为2015年01月09日,发明名称为“旋毛虫副肌球蛋白H-2d限制性Th表位P2、其组合物及应用”。
技术领域
本发明属于免疫生物学领域,涉及一种旋毛虫副肌球蛋白H-2d限制性Th表位P5。本发明还涉及其组合物及应用。
背景技术
旋毛虫病是一种呈全球性分布的人兽共患寄生虫病。人的感染主要来源于高致病性的旋毛形线虫(Pozio E.World distribution of Trichinella spp.infections inanimals and humans.[J].Vet Parasitol,2007,149(1-2):3-21.)。由于旋毛虫病临床症状复杂,包括腹泻,发热,肌痛,水肿等,令其难以正确诊断,对及时的药物治疗造成了一定困难且药物治疗不能解决该虫的反复感染问题(Gottstein B,Pozio E,NocklerK.Epidemiology,diagnosis,treatment,and control of trichinellosis.[J].ClinMicrobiol Rev,2009,22(1):127-145.)。旋毛虫病的流行,不仅直接威胁着人类的身体健康,而且对畜牧业、肉食品业以及外贸出口等造成重大经济损失,是我国“十一五”期间重点攻克的食源性寄生虫病之一。
副肌球蛋白(paramyosin,Pmy)是多种无脊椎动物主要的结构蛋白,在寄生性蠕虫的不同发育阶段呈持续性表达,是参与肌肉收缩的粗肌丝的主要成分(Epstein H F,Miller D R,Ortiz I,et al.Myosin and paramyosin are organized about a newlyidentified core structure[J].J Cell Biol,1985,100(3):904-915.)。在某些寄生蠕虫的非肌肉部位,如体表、消化道和生殖道也有副肌球蛋白的分布(Zhao Q P,Moon S U,Na BK,et al.Paragonimus westermani:biochemical and immunologicalcharacterizations of paramyosin.[J].Exp Parasitol,2007,115(1):9-18.MatsumotoY,Perry G,Levine R J,et al.Paramyosin and actin in schistosomal teguments[J].Nature,1988,333(6168):76-78.)。副肌球蛋白同时也是免疫调节分子,在寄生性蠕虫与宿主的相互作用中发挥重要功能(Loukas A,Jones M K,King L T,et al.Receptor forFc on the surfaces of schistosomes.[J].Infect Immun,2001,69(6):3646-3651.DengJ,Gold D,Loverde P T,et al.Inhibition of the complement membrane attackcomplex by Schistosoma mansoni paramyosin.[J].Infect Immun,2003,71(11):6402-6410.)。旋毛虫的副肌球蛋白基因(Ts-Pmy GenBank accession No.:EF429310)全长为2996bp,其开放读码框架(ORF)2655bp,编码885个氨基酸,理论分子量为102kDa。
近年来免疫学的进展使人们认识到蛋白质抗原并非通过其完整分子发挥功能,而是由抗原分子中具有特异性的特殊化学基团,即表位(epitope)来完成的。
此外,一种新型疫苗,即表位疫苗的出现为病原体疫苗研制提供了新的思路。现代免疫理论认为,有效的保护性免疫源于一组表位的合理组合与搭配(吴玉章,免疫识别、分子设计与抗原工程.第三军医大学学报,2000(10):917-918)。理想的疫苗分子中应同时包含目的抗原B细胞表位和自身或外源T细胞表位,可诱导出高度特异性的体液或细胞免疫反应,并由此赋予较广的保护性。表位疫苗恰好可以满足这些条件。它不仅能够组合具有高度抗原性的表位,避开天然蛋白的空间位阻,而且可选择能够被大多数个体识别的保守区,从而增加了疫苗的安全性(Rosa,D.S.,S.P.Ribeiro and E.Cunha-Neto,CD4+T cellepitope discovery and rational vaccine design.Arch Immunol Ther Exp(Warsz),2010.58(2):p.121-130)。
现有研究证实,表位疫苗能够诱导产生与全蛋白相当或者更强的免疫反应。表位疫苗最常用的组合方式是优势B表位、Th表位和CTL表位基因或者表位肽串联连接。在构建表位疫苗时,考虑到CD4+T细胞在适应性免疫功能中所起的作用,含有适合的Th细胞表位对于疫苗的有效性是十分必要的(Rosa,D.S.,S.P.Ribeiro and E.Cunha-Neto,CD4+T cellepitope discovery and rational vaccine design.Arch Immunol Ther Exp(Warsz),2010.58(2):p.121-130)。在体液免疫中,CD4+T细胞中的Th2细胞可促进B细胞分化成浆细胞产生中和抗体,以及促使辅助记忆性B细胞对再感染迅速唤起回忆反应。研究表明,在构建表位疫苗时加入适合的CD4+T细胞表位,有助于提高疫苗的保护效果。
Th细胞即辅助性T细胞(helper T cell,Th)。Th细胞主要分为Th1细胞与Th2细胞两种,其中,Th1细胞参与细胞免疫和迟发性超敏性炎症反应;Th2可辅助B细胞分化为抗体分泌细胞,参与体液免疫应答。
Th细胞是根据功能分类的一个T细胞亚群。据其分泌的细胞因子的不同可分为Th0、Th1、Th2和Th3四个亚型。Th1细胞主要分泌白细胞介素(interleukin,IL)-2,干扰素(interferon,IFN)-γ,IFN-a,肿瘤坏死因子(tumor necrosis factor,TNF)-β等,主要介导细胞毒和局部炎症有关的免疫应答,辅助抗体生成,参与细胞免疫及迟发型超敏性炎症的发生。Th2细胞主要分泌IL-4,IL-5,IL-6和IL-10等,主要功能为刺激B细胞增殖并产生免疫球蛋白G(immunoglobulinG,IgG)1和免疫球蛋白E(immunoglobulinE,IgE)抗体,与体液免疫有关。TH2辅助细胞主要为对抗细胞外多细胞寄生虫的免疫反应,其主要为白介素4(IL-4)所驱动诱发,其主要的执行的细胞因子是IL-4,IL-5&IL-13,其最重要的执行细胞为肥大细胞(Mast cell)嗜酸细胞(Eosinophil)及嗜碱细胞(Basophil)另外还有产生IgE的B细胞以及分泌IL-4/IL-5的CD4 T细胞等、其主要的转录因子为STAT6另外还有GATA等等。CD4 T细胞分泌的IL-5会活化嗜酸细胞,使其能够攻击细胞外寄生虫,另外IL-4&IgE会活化肥大细胞而放出组织胺(histamine)血清素(serotonin)等造成气管收缩腹泻及肠蠕动而排出寄生虫。
目前一般认为:在旋毛虫感染过程中,宿主的免疫保护作用主要基于Th2细胞介导的体液免疫反应。研究发现,排虫数目较多的小鼠,其Th2细胞因子占优势,特异性抗体IgG1呈高水平;而排虫数目较少的小鼠,其Th1细胞因子占优势,抗体IgG2a升高。如果寻找到Ts-Pmy的Th2表位,并将其与保护性B细胞表位构建成表位肽嵌合疫苗,诱导机体向Th2型免疫应答定向发展。不仅可以避免全蛋白分子中那些不利表位的影响,而且应该能够增强B细胞表位的免疫保护效果。
故此,需要筛选获得Ts-Pmy的Th2表位,并可将其与前期筛选获得的具有保护性的Ts-Pmy的B细胞表位构建成表位肽嵌合疫苗,以期对旋毛虫感染及其所致疾病进行有效地防治。
发明内容
本发明人经过深入的研究和创造性的劳动,得到了一种分离的多肽。所述多肽为Ts-Pmy的抗原表位,特别是Ts-Pmy的Th2表位。本发明人惊奇地发现,该多肽能够刺激Th0细胞向Th2细胞分化,免疫小鼠后均可诱导产生Th2免疫反应,具有应用于制备疫苗例如表位疫苗特别是多表位疫苗的潜力。由此提供了下述发明:
本发明的一个方面涉及一种分离的多肽,其为或者包含SEQ ID NO:2-5中任一项所示的氨基酸序列。SEQ ID NO:2-5依次命名为P2、P3、P4以及P5。
P2:Q F E I D R L A A A L A D A E(SEQ ID NO:2)
P3:K K Q A D Q L R A L Q S S F E(SEQ ID NO:3)
P4:E A T Q R E L R A T Q A E L Q(SEQ ID NO:4)
P5:A I A Q R K L S A L S A E L E(SEQ ID NO:5)
所述多肽为Ts-Pmy的抗原表位,特别是Ts-Pmy的Th表位,具体为Ts-Pmy的Th2表位。
本发明的另一方面涉及一种分离的多核苷酸,其编码本发明任一项所述的多肽。
本发明的再一方面涉及一种抗原表位偶联物,包括抗原表位和偶联部分,其中,所述抗原表位为本发明任一项所述的任一多肽,所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白和生物素中的一种或多种;具体地,所述偶联部分为载体蛋白BSA或KLH。
本发明的再一方面涉及一种组合物,其包含一种或多种(例如选自SEQ ID NO:2、3、4和/或5所示的多肽)本发明任一项所述的多肽,和/或一种或多种本发明所述的抗原表位偶联物,以及任选的药学上可接受的辅料例如佐剂;具体地,所述组合物为疫苗制剂;具体地,所述疫苗制剂为表位疫苗制剂;更具体地,所述表位疫苗制剂还包含一个或多个“Ts-Pmy的B细胞表位”;特别具体地,所述Ts-Pmy的B细胞表位为选自SEQ ID NO:13-17中任一项所示的氨基酸序列。
ALSTPTFSTLPA(SEQ ID NO:13)
LPWHFKSRHRYQ(SEQ ID NO:14)
SHWNSHSTPARA(SEQ ID NO:15)
LSTPYSKSQAST(SEQ ID NO:16)。
上述Ts-Pmy的B细胞表位SEQ ID NO:13-16可以参考中国专利ZL201110448420.2(公开号为CN 102558306A)。
EEAEGTTDAQIDANRKRESE(SEQ ID NO:17)
上述Ts-Pmy的B细胞表位SEQ ID NO:17可以参考Junfei Wei,Yuan Gu,JingYang,Yaping Yang,Shaohua Wang,Shijuan Cui,Xinping Zhu,Identification andcharacterization of protective epitope of Trichinella spiralis paramyosin,Vaccine 29(2011)3162–3168。
本发明的再一方面涉及任意一种或者几种本发明所述的多肽或者本发明所述的抗原表位偶联物在制备治疗和/或预防旋毛虫感染的药物中的用途。
本发明的再一方面涉及一种在体外抗旋毛虫的方法,包括使用有效量的任意一种或者几种本发明任一项所述的多肽或者本发明所述的抗原表位偶联物的步骤。在本发明的一个实施方案中,所述方法是非治疗目的的。
本发明的再一方面涉及任意一种或者几种本发明所述的多肽或者本发明所述的抗原表位偶联物在制备促进或刺激脾淋巴细胞分泌IL-4、IL-5、IFN-γ和/或IL-2的药物或试剂,或者在制备刺激/促进Th0细胞向Th2细胞分化的药物或试剂中的用途。
本发明的再一方面涉及一种抗体,其能够特异性地结合本发明任一项所述的多肽或者本发明所述的抗原表位偶联物。
在本发明中,术语“特异性结合”具有免疫学的一般含义,例如抗原抗体间的结合。
本发明的再一方面涉及一种抗体偶联物,包括抗体部分和偶联部分,其中,所述抗体部分为本发明任一项所述的抗体,所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白和生物素中的一种或多种。
本发明的再一方面涉及一种药物组合物,其包含本发明任一项所述的抗体或者本发明所述的抗体偶联物;可选地,所述药物组合物还包含药学上可接受的载体或辅料。
本发明中,术语“Th2免疫反应”,如果没有特别说明,是指:即体液免疫(humoralimmunity),即以B细胞产生抗体来达到保护目的的免疫机制。指B细胞在T细胞辅助下,接受抗原刺激后形成效应B细胞和记忆细胞。效应B细胞产生的具有专一性的抗体与相应抗原特异性结合后完成的免疫反应。体液免疫的关键过程是产生高效而短命的效应B细胞,由效应B细胞分泌抗体清除抗原。产生寿命长的记忆细胞,在血液和淋巴中循环,随时“监察”,如有同样抗原再度入侵,立即发生免疫反应以消灭之(二次反应)。
术语“Th0细胞”,如果没有特别说明,是指:未接触抗原的T细胞被称为Th细胞前体(Thcell precursor)通过接触天然免疫细胞摄取的抗原而分化成一种不确定的状态,称为Th0细胞。Th1,Th2细胞均由前体细胞Th0分化而来。Th0细胞既分泌Th1型细胞因子IL-2和IFN-γ,又分泌Th2型细胞因子IL-4,可在不同信号的刺激下分化为Th1或Th2细胞。
发明的有益效果
本发明的多肽(抗原表位)能够刺激/促进脾淋巴细胞显著增殖,刺激/促进小鼠脾细胞CD4+T细胞增高,刺激/促进Th0细胞向Th2分化,刺激/促进Th1型细胞因子IFN-γ、IL-2以及Th2型细胞因子IL-4、IL-5升高;刺激/促进Th0细胞向Th2细胞分化,免疫小鼠后均可诱导产生Th2免疫反应,具有应用于制备疫苗例如多表位疫苗的潜力。
附图说明
图1:脾淋巴细胞增殖实验(表位肽体外鉴定)。小鼠脾淋巴细胞分别以候选表位肽及rTs-Pmy进行刺激,结果显示候选Th表位肽P2、P3、P4、P5及P12可刺激脾淋巴细胞显著增殖(SI>2)。
图2:ELISPOT IL-4(表位肽体外鉴定)。
图3:ELISPOT IL-5(表位肽体外鉴定)。
图4:ELISPOT IFN-γ(表位肽体外鉴定)。
图5:ELISPOT IL-2(表位肽体外鉴定)。
ELISPOT结果显示:与培养基阴性对照孔相比,候选Th表位肽P2、P3、P4、P5及P12均可刺激脾淋巴细胞分泌Th2型细胞因子IL-4及IL-5增高(图2,3),而Th1型细胞因子IFN-γ及IL-2分泌水平接近培养基阴性对照孔(图4,5)。
图6:脾淋巴细胞增殖实验(表位肽体内鉴定)。候选Th表位肽P2、P3、P4、P5及P12免疫小鼠的脾淋巴细胞分别以各自免疫原候选表位肽进行刺激,结果显示,除P12外,其余表位肽均可刺激脾淋巴细胞显著增殖(SI>2)。
图7:流式细胞仪检测表位肽免疫小鼠CD4+T细胞的相对数量变化(表位肽体内鉴定)。与PBS对照组相比,Th表位肽P2、P3、P4、P5、P12均刺激小鼠脾细胞CD4+T细胞增高。
图8:ELISPOT检测IL-4(表位肽体内鉴定)。
图9:ELISPOT检测IL-5(表位肽体内鉴定)。
图10:ELISPOT检测IFN-γ(表位肽体内鉴定)。
图11:ELISPOT检测IL-2(表位肽体内鉴定)。
ELISPOT结果显示:与培养基阴性对照孔相比,候选Th表位肽P2、P3、P4、P5及P12均可刺激脾淋巴细胞分泌Th2型细胞因子IL-4(图8),P2、P3、P4及P5可刺激Th2型细胞因子IL-5(图9)及Th1型细胞因子IFN-γ(图10)及IL-2增高(图11)。
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。本领域技术人员将会理解,下面的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技术或条件者,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。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:rTs-Pmy蛋白的制备
Ts-Pmy质粒菌液为转化有含有副肌球蛋白基因(Ts-Pmy GenBank accessionNo.:EF429310)读码框全长质粒的大肠杆菌,等同于公开号为CN100999737A(申请号为200710000018.1)的专利申请中实施例2制备的转化有副肌球蛋白基因(Ts86 cDNA)的大肠杆菌BL21,其制备方法亦可以参考该专利申请。参考该专利申请制得重组Ts-Pmy蛋白(rTs-Pmy蛋白)。
实施例2:多肽序列的合成
根据重组Ts-Pmy蛋白(rTs-Pmy蛋白)的氨基酸序列合成12条多肽序列,如下面的表1。其中位置是指在rTs-Pmy蛋白上的氨基酸位置。
委托北京奥维亚生物技术有限公司合成。
表1:多肽序列
实施例3:多肽的体外实验鉴定
1.实验样品:
实施例1制备的重组Ts-Pmy蛋白(rTs-Pmy)。
实施例2合成的多肽P1-P12。
2.实验动物及分组:
6-8周龄BALB/c雌性小鼠随机分为2组,分别为重组蛋白组和PBS对照组,每组6只。
3.实验方法
(1)免疫动物:以每只小鼠rTs-Pmy 50μg(溶于50μl PBS)或PBS 50μl加等体积佐剂MONTANIDETM ISA 50 V2(Seppic公司),皮下多点免疫小鼠,于2周及4周后以同样剂量加强免疫两次。
(2)脾淋巴细胞增殖分离:
末次免疫一周后,处死小鼠并分离脾淋巴细胞(按照达科为生物技术有限公司小鼠淋巴细胞分离液说明书操作)。具体步骤如下:
A.断颈处死小鼠,浸泡于75%的乙醇中;
B.在超净台中取出小鼠脾脏。注意无菌操作;
C.在35mm培养皿中放入4-5mL EZ-SepTMMouse 1×淋巴细胞分离液(取用前恢复至室温并摇匀);
D.把悬有脾脏细胞的分离液立即转移到15mL离心管中,覆盖200-500μL的1640培养基(保持液面分界明显);
E.室温,800g离心30min。设置较慢的加速度和减速度,如果有十档,设为第三档。离心结束后细胞分层;
F.吸出淋巴细胞层,再加入10mL 1640培养基,颠倒洗涤。室温,250g离心10min收集细胞;
G.倾倒上清液,用无血清培养基或其他培养液重悬细胞,细胞计数。
(3)脾淋巴细胞增殖实验:
选用CellTiterAqueous单溶液细胞增殖检测试剂盒(Promega)测试。
A.计数完毕后,用含有细胞刺激剂的完全培养基(1640培养基,含10%胎牛血清)稀释脾淋巴细胞(多肽工作浓度2.5μg/ml,rTs-Pmy工作浓度15μg/ml),使细胞的终浓度为5×106个/ml。每组同时设立阴性对照(不含有刺激物的完全培养基,其余同实验组)整块细胞培养板中还要设立1组空白对照(不加细胞,只加入培养基和所有检测试剂);
B.37℃5%CO2温箱孵育48h;
C.融化CellTiterAQueous One Solution Reagent。室温静止90分钟或37℃水浴10分钟;
D.在96孔板中,每孔100μl培养基加20μl CellTiterAQueous One SolutionReagent;
E.在37℃,5%CO2的环境下孵育1-4个小时;
F.490nm读取吸光度值;
G.刺激指数(Stimulation index,SI)计算:SI=(实验组OD值-空白对照组OD值)/(阴性对照组OD值-空白对照组OD值)。
(4)ELISPOT检测小鼠脾细胞分泌的细胞因子:
为分析筛选出的多肽诱导CD4+T淋巴细胞的极化方向,首先检测Th1型细胞因子IFN-γ及Th2型细胞因子IL-4。筛选获得的Th2型表位肽进一步检测Th1型细胞因子IL-2及Th2型细胞因子IL-5。具体步骤如下:
A.包被抗体:用无菌PBS将包被抗体按比例稀释(IFN-γ1:250,IL-2,IL-4和IL-5为1:200),每孔100μl加至ELISPOT板,4℃包被过夜;
B.封闭:弃去包被液,用封闭液(RPMI 1640,含1%的L-谷氨酸盐、10%灭活胎牛血清、青霉素104U/ml+链霉素104μg/ml)200μl/孔洗涤一次,加封闭液200μl/孔,室温孵育2h;
C.对照组的设立:每组细胞因子的检测中都设立使用刀豆蛋白A(Con A)作为刺激物的阳性对照,每孔加1μg的Con A。同时还要设立1组阴性对照(加细胞,只加入完全培养基和所有检测试剂);
D.准备脾淋巴细胞悬液:用1640培养基稀释淋巴细胞,100μl/孔,使每个孔中脾细胞数量约为1×107个/ml(检测IL-2,IL-4和IL-5)或5×106/ml(检测IFN-γ)。每个样本设2个复孔;
E.盖上板盖,37℃5%CO2温箱孵育48h(注意:碰撞会引起细胞移位,造成斑点模糊、拖尾。在整个培养过程中应避免移动、碰撞培养板,并尽量减少开关培养箱的次数);
上述5步需在无菌状态下进行操作,下面步骤不再需要无菌操作。可以在安全柜外操作。
A.加检测抗体:培养完毕后,弃去细胞悬液,加入ddH2O,200μl/孔洗板,孵育5min/次,洗2次。随后加入PBST(PBS,含0.05%Tween-20pH 7.2)洗板,孵育5min/次,洗3次;按1:200用稀释液(PBS,含10%灭活胎牛血清,pH 7.2)稀释检测抗体,100μl/孔;盖上板盖,室温孵育2h(移液器吸取液体时应注意不要划伤膜);
B.加入酶标记物Streptacidin-HRP:弃去检测抗体,以洗涤液Ⅰ洗板3次,2min/次;按照说明书要求稀释酶标记物Streptacidin–HRP(1:100),100μl/孔;盖上板盖,室温孵育1h;
C.加入显色剂:弃去酶标记物Streptacidin-HRP,用洗涤液Ⅰ洗板4次,2min/次;用洗涤液Ⅱ(PBS,pH 7.2)洗板2次,2min/次。按照AEC显色试剂盒操作说明配制和加入显色剂,70μl/孔。每块板需配制7ml;
D.终止反应:IFN-γ和IL-2显色时间10s-1min,IL-4,IL-6和IL-10显色时间2-3min。可根据实际显色情况灵活掌握时间,不要过度显色。待斑点生长至适合的大小之后,用ddH2O洗涤2遍,终止显色过程。将板倒扣在吸水纸上,拍干细小的水珠,室温静置2-3h,让ELISPOT板底部的结合膜自然风干。注意此处禁止将板放到烤箱内,否则会导致膜发脆、破裂;
E.斑点计数:将ELISPOT板置于CTL ELISPOT自动读板仪内,调整适合的参数,斑点计数,并记录斑点的各种参数,做统计分析。
4.实验结果
(1)脾淋巴细胞增殖实验:小鼠脾淋巴细胞分别以候选多肽及rTs-Pmy进行刺激,结果显示,P2、P3、P4、P5及P12可刺激脾淋巴细胞显著增殖(SI>2,图1)。
(2)ELISPOT检测细胞因子:ELISPOT结果显示:与培养基阴性对照孔相比,候选Th表位肽P2、P3、P4、P5及P12均可刺激脾淋巴细胞分泌Th2型细胞因子IL-4及IL-5增高(图2,3),而Th1型细胞因子IFN-γ及IL-2分泌水平接近培养基阴性对照孔(图4,5)。此结果表明,P2、P3、P4、P5及P12能刺激Th0细胞向Th2分化,为Ts-Pmy蛋白的Th2表位。
实施例4:表位肽的体内实验鉴定
1.实验样品:
单个Th表位肽P2、P3、P4、P5、P12。
2.实验动物及分组:
6-8周龄BALB/c雌性小鼠随机分为6组,分别为Th表位肽组和PBS对照组,每组6只。
3.实验方法
(1)免疫动物:
以每只小鼠50μg单个Th表位肽P2、P3、P4、P5、P12(溶于50μl PBS)或PBS 50μl加等体积佐剂MONTANIDETM ISA 50 V2(Seppic公司),皮下多点免疫小鼠,于2周及4周后以同样剂量加强免疫两次。
(2)脾淋巴细胞增殖实验:末次免疫一周后,处死小鼠并分离脾淋巴细胞,分别以候选表位肽进行刺激,选用CellTiterAqueous单溶液细胞增殖检测试剂盒(Promega)测试,计算淋巴细胞刺激指数(Stimulation index,SI)。
(3)表位肽免疫小鼠CD4+T细胞的相对数量检测:
末次免疫后10天,分离小鼠脾细胞,以PBS洗涤2遍,调整细胞浓度为1×107个/mL,分别取100μL细胞悬液置于两个1.5ml EP管中,实验管加入PE标记的抗小鼠CD4抗体、FITC标记的抗小鼠CD3抗体,对照管加入PE及FITC标记的同型抗体,冰上避光孵育30min染色,洗涤2次,最后用500μL PBS重悬,流式细胞仪检测表位肽免疫小鼠CD4+T细胞的相对数量变化。
(4)ELISPOT检测脾细胞分泌细胞因子:
为分析筛选出的表位肽免疫小鼠后诱导CD4+T淋巴细胞的极化方向,末次免疫后10天,取小鼠脾细胞,以表位肽为刺激物,检测Th1型细胞因子IFN-γ、IL-2及Th2型细胞因子IL-4、IL-5。
4.实验结果
(1)脾淋巴细胞增殖实验:Th表位肽免疫小鼠脾淋巴细胞分别以各种免疫候选表位肽进行刺激,结果显示P2、P3、P4及P5可刺激脾淋巴细胞显著增殖(SI>2,图6)。
(2)表位肽免疫小鼠CD4+T细胞的相对数量检测:
结果显示:Th表位肽P2、P3、P4、P5、P12均刺激小鼠脾细胞CD4+T细胞增高,提示表位肽免疫后能激发CD4+T淋巴细胞应答(图7)。
(3)ELISPOT检测脾细胞分泌细胞因子:经表位肽体内鉴定试验,获得了4个可在体内有效呈递的Th2表位肽P2、P3、P4及P5。这4个肽均可刺激Th2型细胞因子IL-4(图8)、IL-5(图9)增高。此外,这4个肽还均可刺激Th1型细胞因子IFN-γ(图10)、IL-2(图11)增高。
综上,通过以上实验,本发明人获得了Ts-Pmy的4个Th2表位,这些表位多肽免疫小鼠后均可诱导产生Th2免疫反应。以上结果提示:这些表位可作为候选疫苗组份,从而为制备抗旋毛虫病的多表位疫苗奠定了基础。
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,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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<110> 首都医科大学
<120> 旋毛虫副肌球蛋白H-2d 限制性Th表位P5、其组合物及应用
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Claims (8)
1.一种分离的多肽,其氨基酸序列如SEQ ID NO:5所示。
2.一种分离的多核苷酸,其编码权利要求1所述的多肽。
3.一种抗原表位偶联物,包括抗原表位和偶联部分,其中,所述抗原表位为权利要求1所述的多肽,所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白和生物素中的一种或多种;优选地,所述偶联部分为载体蛋白BSA或KLH。
4.一种组合物,其包含权利要求1所述的多肽,和/或权利要求3所述的抗原表位偶联物,以及任选的药学上可接受的辅料例如佐剂;优选地,所述组合物为疫苗制剂;优选地,所述疫苗制剂为表位疫苗制剂;更优选地,所述表位疫苗制剂还包含一个或多个Ts-Pmy的B细胞表位;特别优选地,所述Ts-Pmy的B细胞表位的氨基酸序列如SEQ ID NO:13-17中任一序列所示。
5.根据权利要求4所述的组合物,其还包含一种或多种多肽,并且所述多肽的氨基酸序列如SEQ ID NOs:3-4中任一序列所示。
6.权利要求1所述的多肽、权利要求1所述的多肽与选自氨基酸序列如SEQ ID NOs:3-4中任一序列所示多肽的一种或多种的组合或者权利要求3所述的抗原表位偶联物在制备治疗和/或预防旋毛虫感染的药物中的用途。
7.一种在体外抗旋毛虫的方法,包括使用有效量的权利要求1所述的多肽、权利要求1所述的多肽与选自氨基酸序列如SEQ ID NOs:3-4中任一序列所示多肽的一种或多种的组合或者权利要求3所述的抗原表位偶联物的步骤。
8.权利要求1所述的多肽、权利要求1所述的多肽与选自氨基酸序列如SEQ ID NOs:3-4中任一序列所示多肽的一种或多种的组合或者权利要求3所述的抗原表位偶联物在制备促进或刺激脾淋巴细胞分泌IL-4、IL-5、IFN-γ和/或IL-2的药物或试剂,或者在制备刺激/促进Th0细胞向Th2细胞分化的药物或试剂中的用途。
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