CN101921325A - 一种增加CD4+CD25+Foxp3+调节性T细胞的抗原及其应用 - Google Patents
一种增加CD4+CD25+Foxp3+调节性T细胞的抗原及其应用 Download PDFInfo
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Abstract
本发明属于免疫学领域,具体涉及一种可以增加CD4+CD25+Foxp3+调节性T细胞的血吸虫来源的蛋白抗原分子-日本血吸虫热休克蛋白60KDa(SjHSP60)及其应用。SjHSP60全长氨基酸序列如SEQ.ID.NO.1所示,与感染宿主的HSP60具有一系列相同或高度相似的交叉性T细胞表位。用SjHSP60体内免疫小鼠或体外刺激小鼠脾、***细胞,均可显著增加CD4+CD25+Foxp3+Tregs。在实际应用上,SjHSP60可有效地减轻关节炎所致的炎性症状和免疫病理反应,因此在诱导免疫抑制,应用于治疗免疫性疾病方面具有广阔的前景。
Description
技术领域
本发明属于免疫学领域,具体涉及一种增加CD4+CD25+Foxp3+调节性T细胞(Tregs)的血吸虫来源的抗原分子-日本血吸虫热休克蛋白60KDa(Schistosoma japonicum heatshock protein 60KDa,SjHSP60)及其应用。
背景技术
上个世纪九十年代前后,西方发达国家提出来“卫生假说”,认为在卫生环境大大改善的发达国家,一些过敏性疾病和自身免疫病的发病率大为增加,这是与发达国家卫生条件改善,人群发生病原体感染的机会减少有关(Wills-Karp,et al,Nat Rev Immunol,2001,1:69-75)。其后的若干年,科学家们通过一些过敏性疾病和自身免疫病的动物模型研究证明,一些病原体的慢性感染确实可以预防这些免疫性疾病的发生或者减轻免疫病理损伤(Dunne,Nat Rev Immunol,2005,5:420-426):如曼氏血吸虫感染可以预防或减轻I型糖尿病,实验性脑炎,甲状腺炎;绦虫感染会减少实验性结肠炎的发病;链球菌感染可以抑制胶原诱导型关节炎的发生发展,等等。从进化史的角度看,慢性感染是病原体和宿主在长期共同进化彼此选择后,最终所形成的对双方都有利的一种平衡状态。在这种状态下,病原体不会被宿主完全清除,可长期存在于宿主;宿主既可维持对病原体有一定抵抗力,也不会因为过强的免疫反应而产生免疫病理损伤,而且还预防了自身免疫病的发生。
随着免疫学的发展,在免疫***中发现了一群天然存在的CD4+CD25+调节性T细胞(Tregs)(Sakaguchi,et al.,J Immunol,1995,155:1151-64),这群细胞主要由胸腺产生,参与维持外周自身免疫耐受和免疫内环境稳定;后来进一步发现,一些病原体的感染(病毒,细菌,寄生虫)也可以在外周从CD4+T细胞中诱导产生CD4+CD25+Tregs(Bluestone and Abbas,Nat Rev Immunol,2003,3:253-7)。如丙型肝炎病毒(HCV)感染人体后,可以诱导特异性的CD4+CD25+Tregs(Bolacchi,et al.,Clin Exp Immunol,2006,144:188-96);小鼠百日咳杆菌感染模型中研究发现,CD4+CD25+Tregs发生增多(McGuirk,et al.,J Exp Med,2002,195:221-31);寄生虫类感染中,曼氏血吸虫感染后4周,发现肠系膜***中CD4+CD25+Tregs发生了扩增,然后聚集到肝脏和脾脏中发挥免疫抑制效应(Wilson et al,.unpublished observaton)。进一步探究导致慢性感染的病原体是如何诱导调节性T细胞,从而抑制免疫攻击,对于提高宿主抗感染力和治疗免疫病意义都非常重大。寄生虫中的蠕虫类,是一类具有复杂生物学功能的多细胞真核生物,能够很好的逃避宿主免疫攻击,长期存在于宿主机体中(Maizels,et al.,J ExpMed,2009,206:2059-66)。蠕虫类中血吸虫感染是一种非常典型的慢性感染,在其感染模型中研究发现,其虫卵抗原是刺激诱导CD4+CD25+Tregs产生的关键因素(Baumgart,et al.,JImmunol,2006,176:5374-87);此外,本课题组也已发现,日本血吸虫虫卵抗原也可以刺激诱导CD4+CD25+Tregs从而减轻哮喘所导致的气道炎症反应(Yang,et al.,Immunology,2006,120:8-18)。
关于特异性抗原在外周诱导免疫负调控的研究,在自身免疫研究领域已有一些报道,有些自身抗原的自身反应性在逃避了胸腺中枢的阴性选择后,它们还可以在外周诱导自身反应性调节性T细胞,从而参与免疫调控。这些自身抗原包括:一些组织抗原(谷氨酸脱羧酶GAD,少突胶质细胞蛋白MOG,肌球蛋白等);免疫调控分子(泛素,白介素,肌动蛋白等);应激蛋白(HSP40,HSP60,HSP70)(Merbl,et al.,J Clin Invest,2007,117:712-8)。实际上,免疫***天然就存在这些抗原的抗体或反应性T细胞,或其特定的受体(配体),它们会时刻监视着机体免疫***,维持免疫内环境稳定(Cohen,Nat Rev Immunol,2007,7:569-74)。早些年就有研究发现,人自身HSP60或其多肽可预防或减轻I型糖尿病(Elias and Cohen,Diabetes,1996,45:1168-72),且于两年前已经开始应用于临床III期实验(Eldor,et al.,DiabetesMetab Res Rev,2009,25:316-20);通过大鼠佐剂性关节炎模型研究发现,鼠自身HSP60也可减轻关节炎症病理反应(Durai,et al.,J Autoimmun,2009,33:208-13)。而且非常特别的是,HSP60属于进化上最为保守的分子家族-热休克蛋白家族,在所有真核和原核生物中高度同源。因此,病原体很可能会利用这个分子去模拟宿主HSP60的作用,诱导免疫调控,抑制宿主免疫攻击,维持慢性感染。如果能够鉴定和验证出这些病原体来源的“模拟分子”,不仅可以找到打破慢性感染的靶标,还可应用于预防或治疗免疫性疾病。
对于过敏性疾病和自身免疫病的治疗,临床上很长时间以来一直在用一些直接抑制免疫应答行为的化学药物,如激素类,或者联合应用硫唑嘌呤(抗胸腺球蛋白类药物),环孢素A等(Bougneres,et al.,Diabetes,1990,39:1264-72)。但是其中有些药物不仅价格比较贵,会让患者产生难以忍受的副作用,而且一旦停止用药,自身免疫反应会立即反弹,疾病会卷土重来。而廉价的纯化蛋白分子,不仅安全没有毒副作用,更重要的是,它是通过免疫学途径去启动其自身的调控机制,从根本上解决了问题;相比较于同类的短肽疫苗,氨基酸序列较长而不易被降解,且具有多个表位,可以联合发挥作用,更易被机体免疫***所识别;此外,蛋白分子的制备,一般都是通过生物表达***(原核或真核表达***),而不是生物合成,这有助于形成蛋白质的高级空间结构,能更有效地形成免疫学表位。
已公开的日本血吸虫热休克蛋白60KDa(Schistosoma japonicum heat shock protein60KDa,SjHSP60)基因在Genebank中序列号为AY813151。
总之,利用慢性感染中病原体来源的抗原,寻找诱导调节性T细胞的特异分子,不仅有利于鉴定出导致慢性感染主要因素,从而可以针对性地提高宿主抗感染力,而且在进一步研究应用于治疗过敏性疾病或自身免疫病方面具有更广阔的前景。
发明内容
本发明旨在提供一种增加CD4+CD25+Foxp3+调节性T细胞的蛋白。
本发明所说的增加CD4+CD25+Foxp3+调节性T细胞的蛋白,是日本血吸虫热休克蛋白60KDa(SjHSP60),其全长氨基酸序列SEQ.ID.NO.1所示,由572个氨基酸构成。
本发明所说的增加CD4+CD25+Foxp3+调节性T细胞的蛋白,还包括缺失了一段多肽的SjHSP60,即SjHSP60(D),SjHSP60(D)的序列如SEQ.ID.NO.3所示,由548个氨基酸组成。
本发明SEQ.ID.NO.1所示的蛋白与Genebank中SjHSP60基因(序列号为:AY813151)的蛋白质序列并不完全相同(一致性为87%)。但是SEQ.ID.NO.1分别与其它种属同源蛋白中曼氏血吸虫HSP60(SmHSP60,Genebank号:AF310263)和人类HSP60(HomoHSP60,Genebank号:BC003030)的一致性更高,分别为92%和72%,而AY813151的蛋白质序列与SmHSP60和HomoHSP60的一致性分别为81%和65%。这是单克隆测序与大规模测序的差异所在,本发明是经过“Primer walking”测序拼接后翻译出来的序列,所以更精确。
本发明所说的蛋白SjHSP60,主要通过以下两个途径增加CD4+CD25+Foxp3+Tregs,从而增强机体免疫抑制力:
1.上述所说蛋白诱导CD4+CD25-T细胞转化为CD4+CD25+Foxp3+Tregs;
2.上述所说蛋白直接使CD4+CD25+Tregs发生扩增;
本发明公开了SjHSP60和SjHSP60(D)作为增强CD4+CD25+Foxp3+调节性T细胞抑制效应的免疫抑制剂的应用。
本发明公开了SjHSP60和SjHSP60(D)在制备治疗免疫反应引起的炎症性疾病药物中的应用。特别是在制备防治关节炎所致的炎性症状和免疫病理药物中的应用。
本发明所说的SjHSP60和SjHSP60(D)蛋白使扩增后的CD4+CD25+Tregs的免疫抑制力增强。
本发明所说的蛋白应用于实验性炎症动物模型中有抑制炎性症状和免疫病理反应的作用,在治疗免疫性疾病方面具有比较广阔的应用前景。
附图说明
图1SjHSP60序列同源性比对分析,天然表达分析。
A SjHSP60的全长氨基酸序列,与宿主(人,小鼠)的HSP60进行序列比对;
B SjHSP60在虫体中的天然表达情况;
表1SjHSP60与宿主(人,小鼠)自身HSP60的交叉性T细胞表位分析。
图2SjHSP60增加CD4+CD25+Foxp3+Tregs。
A用SjHSP60体内免疫小鼠,小鼠脾和***中CD4+CD25+Foxp3+Tregs比例显著增加;
B用SjHSP60体内免疫小鼠,检测小鼠脾和***中CD4+CD25+Foxp3+Tregs比例的流式细胞术检测图;
C用SjHSP60体外处理小鼠脾和***细胞,CD4+CD25+Foxp3+Tregs比例均发生增加;
D用SjHSP60体外处理小鼠脾和***细胞,检测CD4+CD25+Foxp3+Tregs比例的流式细胞术检测图;
图3SjHSP60减轻CIA(胶原性关节炎)小鼠炎性症状和病理损伤。
A SjHSP60体内免疫可减轻CIA小鼠关节炎性症状;
B SjHSP60体内免疫可减轻CIA小鼠踝关节肿胀度;
C SjHSP60体内免疫可减轻CIA小鼠关节周围软组织肿胀和骨关节损害;
D SjHSP60体内免疫可减轻CIA小鼠关节及其周围软组织的炎症病理反应;
图4SjHSP60增加CIA小鼠CD4+CD25+Foxp3+Tregs比例,抑制炎症抗原(CollagenII,CII)的特异性体液和细胞免疫应答。
A SjHSP60体内免疫CIA小鼠,小鼠脾和***中CD4+CD25+Foxp3+Tregs比例显著增加;
B SjHSP60体内免疫CIA小鼠,检测小鼠脾和***中CD4+CD25+Foxp3+Tregs比例的流式细胞术检测图;
C SjHSP60体内免疫后,CIA小鼠体内针对炎症抗原C II的特异性体液免疫应答水平明显降低;
D SjHSP60体内免疫后,CIA小鼠体内针对炎症抗原CII的特异性细胞免疫应答水平明显降低:
图5HSP60增加CD4+CD25+Foxp3+Tregs可通过诱导CD4+CD25-T转化为CD4+CD25+Foxp3+Tregs。
A用SjHSP60处理小鼠CD4+T,CD4+CD25-T细胞,SjHSP60诱导CD4+CD25-T转化为CD4+CD25+Foxp3+Tregs;
B用SjHSP60处理小鼠CD4+T,CD4+CD25-T细胞,SjHSP60诱导CD4+CD25-T转化为CD4+CD25+Foxp3+Tregs的流式细胞术检测图;
图6SjHSP60增加CD4+CD25+Foxp3+Tregs可通过直接扩增CD4+CD25+Tregs,且扩增后的CD4+CD25+Tregs免疫抑制力更强。
A SjHSP60直接扩增CD4+CD25+Tregs;
B扩增后的CD4+CD25+Tregs具有更强的免疫抑制力;
图7:SjHSP60(D)仍可显著增加CD4+CD25+Foxp3+Tregs比例。
A SjHSP60的蛋白质空间结构和表面功能残基(蛋白质-蛋白质作用位点)的预测和分析;
B通过两次PCR得到了接头部分重叠且缺失了SJMHE1的两个PCR产物;
C通过第三次PCR得到了缺失SJMHE1的SjHSP60(D);
D用SjHSP60(D)体内免疫小鼠,小鼠脾和***中CD4+CD25+Foxp3+Tregs比例显著增加;
E用SjHSP60(D)体内免疫小鼠,检测小鼠脾和***中CD4+CD25+Foxp3+Tregs比例的流式细胞术检测图;
F用SjHSP60(D)体外处理小鼠脾和***细胞,CD4+CD25+Foxp3+Tregs比例显著增加;
G用SjHSP60(D)体外处理小鼠脾和***细胞,检测CD4+CD25+Foxp3+Tregs比例的流式细胞术检测图;
图8:去除SjHSP60全长序列中一段多肽(第435到第458个氨基酸,SJMHE1)的引物设计法和三步PCR示意图。
具体实施方式:
在本发明中所使用的术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体的制备实施例和应用实施例,并参照数据进一步详细地描述本发明。应理解为:这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下的实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。所用试剂的来源、商品名以及有必要列出其组成成分者,均在首次出现时标明,其后所用相同试剂如无特殊说明,均与首次标明的内容相同。
本发明实施例中所提及的表达载体pGEX-6p-1可从GE Healthcare公司购买(货号:28-9546-48)。
日本血吸虫总cDNA的获取:日本血吸虫感染性钉螺(阳性钉螺)购自江苏省血吸虫病防治研究所。感染性钉螺可以释放出具有感染性的尾蚴(日本血吸虫发育阶段之一,此阶段具有感染性)从而用来攻击感染实验性小鼠。然后尾蚴在小鼠体内可以继续发育为童虫,最终发育为日本血吸虫成虫,寄居在小鼠的门静脉***。剖杀小鼠后,可以通过灌注小鼠门静脉***取出成虫,从而可以用来抽提日本血吸虫总RNA,最后再逆转录成日本血吸虫总cDNA。
实施例一:SjHSP60同源性的序列比对分析,交叉性表位分析,及其天然表达的检测。
1、根据Genebank中SjHSP60基因(序列号为:AY813151)全长cDNA序列,先行设计引物:上游引物为5’-cgcggatcccaaccggtgacaatgttacgag-3’(含BamHI酶切位点及起始密码字ATG),下游引物为5’-ccgctcgagattaagagcaggcagtgtttac-3’(含XholI酶切位点及终止密码子TAA),由上海英潍捷基贸易有限公司(Invitrogen)合成;通过PCR方法从日本血吸虫总cDNA中扩增SjHSP60ORF全长序列,送生物技术公司(上海英潍捷基贸易有限公司,Invitrogen)测序,并回收PCR产物;测序结果如SEQ.ID.NO.2所示,再通过在线生物学工具Biology WorkBench(http://workbench.sdsc.edu/)将其翻译成氨基酸序列;并与人和小鼠的HSP60(Genebank中基因序列号分别为:人HSP60-BC003030,小鼠HSP60-NM010477)进行序列比对。
SjHSP60开放阅读框ORF的全长序列,如SEQ.ID.NO.2所示,共1719bp。
SjHSP60开放阅读框ORF的全长序列翻译后的氨基酸序列为如SEQ.ID.NO.1所示,共572个氨基酸。
2、综合应用在线表位预测软件:IEDB(http://tools.immuneepitope.org/main/index.html),SYFPEITHI(http://www.syfpeithi.de/home.htm)分析SjHSP60分别与人HSP60,小鼠HSP60的交叉性T细胞表位。
3、用BamHI(NEB,#R3136V)和XholI(NEB,#R0146V)将质粒pGEX-6P-1和此上(1)中回收的PCR产物进行双酶切;酶切产物经琼脂糖凝胶电泳进行分离鉴定后,再分别割胶回收质粒和PCR片断的双酶切产物;通过T4DNA连接酶(NEB,#M0202S)进行载体和PCR产物的连接,构建重组表达载体pGEX-SjHSP60;转化E.coli BL21感受态细胞,37℃培养过夜后,挑取单克隆菌落,接种于含Amp的LB培养液中,37℃振摇培养过夜后,抽提质粒后行双酶切鉴定,进一步送测序公司鉴定。鉴定成功后,将菌液进行1∶100扩大培养,至OD值达0.6-0.8时,加入IPTG使其终浓度为0.05mmol/L,37℃诱导表达5h;收集菌液,4℃高速离心20min,用1×PBS重悬细菌沉淀;通过超声破碎仪和反复冻融法裂菌;再次4℃高速离心20min,分别取上清和沉淀,以及未裂解的总蛋白进行SDS-PAGE电泳分析蛋白的表达量及分布情况;鉴定为可溶性表达后,通过融合蛋白GST亲和层析法,用Glutathione Sepharose 4B(GE Healthcare,17-0751-01)进行融合蛋白GST-SjHSP60的纯化;并用蛋白酶PreScissionTMProtease(GE Healthcare,270843)对融合蛋白进行酶切,最后得到纯化的SjHSP60;经过SDS-PAGE电泳分析纯化结果,纯度>95%。纯化的SjHSP60交由上海中科院英沐生物科技有限公司制备抗SjHSP60的单克隆抗体(anti-SjHSP60mAb)。制备可溶性的成虫(SWA)和虫卵抗原(SEA),通过Weatern Blot实验检测anti-SjHSP60mAb的特异性和抗体效价;验证成功后,再用anti-SjHSP60mAb,通过免疫组织化学方法,检测日本血吸虫成虫(雌虫和雄虫)和虫卵中SjHSP60的天然表达和分布情况。
结果如图1和表1所示,SjHSP60与人、小鼠HSP60序列上具有高度的同源性,序列的一致性分别达到72%和70%;通过T细胞表位预测后,发现SjHSP60,与人HSP60,与小鼠HSP60,均具有一系列相同或高度相似的MHCI和MHCII类交叉性T细胞表位。
表1
实施例二:SjHSP60体内免疫小鼠或体外刺激小鼠淋巴细胞,观察SjHSP60对CD4+CD25+Foxp3+Tregs的影响。
1.将实施例一(3)中纯化的SjHSP60,滤菌并通过Polymyxin B-Agarose(Sigma,P1411)去除其中的类毒素及其相关分子,然后再通过Limulus amebocyte lysate(LAL)检测试剂盒E-TOXATE Kits(Sigma,ET0200),验证纯化蛋白的内毒素去除效果,<0.001FU/ml(0.1pg/ml),最后用DC Protein Assay(BIA-RAD,500-0111)进行蛋白定量。将SjHSP60与不完全弗氏佐剂IFA(Sigma,F5501)等体积混合,使SjHSP60浓度为250ug/ml,反复涡悬振荡混匀。对BALB/c小鼠(SPF级,6-8周龄,雌性,购自上海斯莱克实验动物有限责任公司)进行腹股沟皮下免疫,剂量为25ug/100ul/只,首次免疫后,隔两周再同样免疫一次,设PBS免疫组为对照组。整个过程小鼠饲养于SPF级动物房(南京医科大学实验动物中心)。末次免疫结束后一周,剖杀小鼠,分离各组小鼠的脾和***,研磨过滤后,制备成单细胞悬液;再经红细胞裂解液去除红细胞,制备成单淋巴细胞悬液;进行细胞计数后,对各组每只小鼠取1×106个细胞,按MouseRegulatory T Cell Staining Kit(eBioscience,88-8111-40)操作说明书进行淋巴细胞的染色标记,再经流式细胞仪FACSCalibur检测CD4+CD25+Foxp3+Tregs比例。
2.分离正常BALB/c小鼠的脾和***,按此上(1)中方法,制备成单淋巴细胞悬液;细胞计数后,分别转移至96孔圆底培养板,5×105个细胞/孔;再分三组进行刺激,分别是SjHSP60组(0.1ug/ml)/PBS组/无刺激(Medium)组,每组设4孔;在37℃,5%CO2细胞孵箱培养3天后,收集各组细胞并进行计数,每组取1×106个细胞,同此上(1)中方法检测CD4+CD25+Foxp3+Tregs比例。
结果如图2所示,体内外实验均证明,SjHSP60可以增加CD4+CD25+Foxp3+Tregs比例。
实施例三:探究SjHSP60能否在炎症模型中发挥抑制炎症及免疫病理反应的作用。
1.按实施例二(1)中方法,分两组:SjHSP60组/PBS组,体内免疫DBA1小鼠(南京大学国家遗传工程小鼠资源库,6-8周龄,雌性)。建立胶原诱导型关节炎(CIA)小鼠模型:混合牛源II型胶原(CII)Bovine Collagen Type II(BD,354257)和Mycobacteriumtuberculosis H37RA(Difco,3114),用1×PBS稀释,使两者浓度分别为2mg/ml,3mg/ml;在混合体系中再加入等体积的不完全弗氏佐剂CFA,涡悬振荡仪上混匀2min,反复6-8次,每隔30min一次;于首次免疫SjHSP60/PBS后一周,在两组小鼠尾部皮下免疫混合抗原诱导CIA,100ul/只。诱导后一周,对两组小鼠再分别免疫SjHSP60/PBS一次。诱导后4周,开始动态观察两组小鼠关节炎症表现,隔天一次;按如下标准进行炎性症状严重程度评分:
0分:正常,无症状;
1分:红斑,只有踝关节轻微肿胀;
2分:红斑,从踝关节到跖骨、掌指关节轻微肿胀;
3分:红斑,从踝关节到掌指关节中度肿胀;
4分:红斑,从踝关节到指尖严重肿胀;
每侧肢体最高分为4分,最低分为0分;每只小鼠总得分最高为16分,最低为0分。
2.与此上(1)中观察时间点同步,使用电子数显游标卡尺(购自温州三和量具仪器有限公司)测量各组小鼠左后肢踝关节直径。
3.诱导后7周剖杀各组小鼠,取各组小鼠前爪固定于含10%***的1×PBS中,送标本至上海瑞金医院伤骨科研究所进行X光检测。
4.X光检测后,将标本继续固定于含10%***的1×PBS中,交由江苏省人民医院病理科进行标本脱钙、石蜡包埋、切片及病理HE染色;显微镜下观察HE病理结果。
结果如图3所示,SjHSP60免疫组的CIA小鼠,其关节的炎性症状,关节肿胀度,关节损害程度以及病理炎症反应都显著减轻。
实验例四:观察SjHSP60能否增加CIA小鼠CD4+CD25+Foxp3+Tregs,从而发挥免疫抑制作用。
1.分离CIA诱导后3W和7W各组(HSP60组/PBS组)小鼠脾和***,按实施例二(1)中方法,检测各组小鼠CD4+CD25+Foxp3+Tregs比例。
2.在剖杀此上(1)中各组小鼠前,对各组小鼠采血,并分离出血清;再经ELISA检测各组血清中CII特异性抗体(IgG,IgG2a,IgG1),具体操作是:用高亲和力酶标板预包被CII,5ug/ml,4℃包被过夜;弃去包被液,PBS-T洗板2-3遍;5%脱脂牛奶进行封闭,200ul/孔,37℃孵育2小时;弃去封闭液,PBS-T洗板2-3遍;1∶20稀释各组血清后,加入酶标板,100ul/孔,4℃孵育过夜;弃去血清,PBS-T洗板3-4遍;加入稀释后的二抗:HRP anti-mouse IgG(Boster,BA1050)1∶10000稀释,HRP anti-mouse IgG2a(BD,553391)和HRP anti-mouse IgG1(BD,559626)均1∶1000稀释,100ul/孔,37℃孵育1小时;弃去二抗,PBS-T洗板4-5遍;加入TMB显色液(eBioscience,00-4201-56),室温避光放置10-15min后,加入2N H2SO4终止反应,100ul/孔;最后在酶标仪上进行读值。
3.取此上(1)中分离后得到的各组每只小鼠的淋巴细胞,分别转移至96孔圆底细胞培养板,4×105个/孔,每只小鼠设9个孔,再各分为三组处理:Medium组(不刺激)/ConA刺激组(2ug/ml)/CII刺激组(5ug/ml),3孔/组;按实施例四(5)中方法,通过3H掺入法检测各组淋巴细胞增殖情况:培养至56小时,加入氚胸腺嘧啶(3H-thymidine,3H-TDR),0.5μCi/孔;继续培养16小时(总计培养3天)后,经Beckman液体闪烁计数仪检测细胞的3H-TDR掺入情况,从而反映出CII特异性的细胞免疫应答水平。
结果如图4所示,SjHSP60使CIA模型小鼠体内CD4+CD25+Foxp3+Tregs比例显著增加;进一步的实验也证明,针对C II特异性的体液和细胞免疫应答都有明显的抑制。
实施例五:SjHSP60体外分别刺激小鼠CD4+T细胞,CD4+CD25-T细胞,进一步观察SjHSP60如何增加CD4+CD25+Foxp3+Tregs。
分离正常BALB/c小鼠脾和***,制备成单淋巴细胞悬液;用试剂盒CD4+T cellIsolation Kit(MACS,130-090-860)分选出CD4+T细胞;剩余的CD4-淋巴细胞经50ug/ml丝裂霉素,37℃,5%CO2处理30min后,作为抗原递呈细胞(APC)使用。再取部分CD4+T细胞,进一步再用CD4+CD25+Regulatory T Cell Isolation Kit(MACS,130-091-041)去除CD4+CD25+Tregs,从而分离出CD4+CD25-T细胞。具体分选步骤均参照产品说明书。通过流式细胞术检测CD4+T细胞和CD4+CD25-T细胞纯度,纯度分别>90%和>95%。将两种细胞分别转移96孔圆底培养板,2×105个细胞/孔,再加入APC,2×105个/孔;CD4+T细胞和CD4+CD25-T细胞再各自分两组处理,刺激组(SjHSP60,0.1ug/ml)/不处理组(Medium);在37℃,5%CO2细胞孵箱培养3天后,收集各组细胞并计数;每组取1×106个细胞,流式细胞术检测CD4+CD25+Foxp3+Tregs比例。此实例中相关具体方法可参考实施例二(1)。
结果如图5所示,SjHSP60增加CD4+CD25+Foxp3+Tregs可通过诱导CD4+CD25-T细胞转化而来。
实施例六:观察SjHSP60增加CD4+CD25+Foxp3+Tregs是否可通过直接扩增CD4+CD25+Tregs,扩增后的CD4+CD25+Tregs免疫抑制力如何。
1.按实施例二(2)中方法分离正常BALB/c小鼠的脾和***,制备成单淋巴细胞悬液;用CD4+CD25+Regulatory T Cell Isolation Kit按照说明书步骤分选出CD4+CD25+Tregs。参照实施例五中方法,流式细胞术检测CD4+CD25+Tregs的细胞纯度,纯度>95%。转移CD4+CD25+Tregs至96孔圆底细胞培养板,2×105个细胞/孔;再分三组处理:anti-CD3(BD,553057)刺激组(1ug/ml)/SjHSP60刺激组(0.5ug/ml)/SjHSP60(0.5ug/ml)+anti-CD3(1ug/ml)刺激组,均为5孔/组。按实施例四(3)中方法,通过3H掺入法检测各组CD4+CD25+Tregs的增殖情况。
2.按此上(1)中具体方法分选并刺激处理CD4+CD25+Tregs,培养3天后,收集并计数;转移至96孔圆底细胞培养板,1×105个/孔,4孔/组。再按实施例五中方法分选正常BALB/C小鼠CD4-细胞(作为APC使用)和CD4+CD25-T细胞,分别加入每孔CD4+CD25+Tregs,1×105个/孔。共培养3天后,同样按实施例四(3)中方法,通过3H掺入法检测各组CD4+CD25-T细胞的增殖情况,进而反映各组CD4+CD25+Tregs的抑制力。
结果如图6所示,SjHSP60还可通过直接扩增CD4+CD25+Tregs,增加CD4+CD25+Foxp3+Tregs;且扩增后的CD4+CD25+Tregs具有更强的免疫抑制力。
实施例七:去除SjHSP60全长序列中第435到第458氨基酸的多肽(SJMHE1)后,得到SjHSP60(D),观察SjHSP60(D)是否还能增加小鼠CD4+CD25+Foxp3+Tregs比例。
1.应用在线蛋白质结构和功能预测软件Phyre server(http://www.sbg.bio.ic.ac.uk/phyre/),预测SjHSP60蛋白分子的空间结构以及表面功能残基(蛋白-蛋白作用位点)。
2.按附图中图8所示设计引物:
P1(5’-3’):cgcggatcccaaccggtgacaatgttacgag
P2(5’-3’):cacacctgttcgctgtatgccttcctcaattgct
P3(5’-3’):attgaggaaggcatacagcgaacaggtgtgcg
P4(5’-3’):ccgctcgagattaagagcaggcagtgttta
以实施例一(3)中构建的重组表达载体pGEX-SjHSP60为模板:以P1/P2为上下游引物进行PCR I;以P3/P4为上下游引物进行PCRII;两次PCR后,得到了缺失SJMHE1且有部分重叠的两个PCR产物。
3.等摩尔混合此上(2)中PCR I和PCRII产物(质量比大约为3.5∶1),以此为模板,以P1/P4为上下游引物,进行PCRIII,得到了缺失SJMHE1的SjHSP60(D),共1816bp。其全长cDNA序列和经过翻译出来的氨基酸序列分别为SEQ.ID.NO.4和SEQ.ID.NO.3所示。
4.回收此上(3)中PCR III产物;按实施例一(3)中方法,用回收的PCR III产物和pGEX-6P-1构建重组表达载体pGEX-SjHSP60(D);诱导表达和纯化SjHSP60(D);再进行内毒素的去除和定量验证,最后进行蛋白定量。按实施例二(1)中具体方法,用SjHSP60(D)以及多肽SJMHE1(由上海生工公司合成,纯度>99%,序列如SEQ.ID.NO.5所示)体内免疫正常BALB/c小鼠,设PBS免疫组为对照组,检测各组小鼠脾和***中CD4+CD25+Foxp3+Tregs比例。
5.按实施例二(2)中方法,用SjHSP60(D)/多肽SJMHE1/PBS体外刺激正常BALB/c小鼠的脾和***细胞,检测各组小鼠脾和***中CD4+CD25+Foxp3+Tregs比例。
结果如图7所示,经过软件预测发现,SjHSP60的三维空间结构表面,除了SJMHE1,还存在其它一些功能性残基(蛋白-蛋白作用位点);进一步体内外实验证明,缺失SJMHE1的SjHSP60(D)仍可增加小鼠脾和***中CD4+CD25+Foxp3+Tregs比例。结合图2分析,可以发现:与SJMHE1相比较,SjHSP60和SjHSP60(D)体内免疫小鼠(in vivo)增加CD4+CD25+Foxp3+Tregs比例的能力更强一些;而体外刺激淋巴细胞(in vitro),SJMHE1,SjHSP60和SjHSP60(D)三者增加CD4+CD25+Foxp3+Tregs比例的能力无明显差异。
本发明不限于这些公开的实例方案,本发明将覆盖在专利书中所描述的范围,以及权利要求范围的各种变型和等效变化。
SEQUENCE LISTING
<110>南京医科大学
<120>一种增加CD4+CD25+Foxp3+调节性T细胞的抗原及其应用
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atgttacgag ctctaagttc acttcgtggt agtttggttc cagctagaca aaaagttgtt 60
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ggtgttatgc ttgctgttga tgctgttgtc aaagagctca aatctttttc gaagcaaata 480
tccacaccag aagagatagc acaagttgcg acaatatccg ctaacggtga caaggctatt 540
ggcgacctaa tcgcttcagc tatgaagagg gttgggaatg atggcacgat aactgttaag 600
gatggcaaaa ctcttcacga tgagttggag tttatcgaag gtatgaaatt cgaccgtggt 660
tatatctctc catattttat caacactgag aaaggagccc gatgtgaatt ccaagatgcc 720
ttcatattgt tttctgaaaa aaagataaac agcattcaga cattattacc agctctcgaa 780
ctatgccacc aacaaaaacg gccgctgctg attatcgccg aggatgttga aggtgaagct 840
cttacagcac ttgtactaaa tcgcttaaaa ctgggcttgc aggtctgtgc agtcaaagca 900
ccaggttttg gggataatcg caaaaataca ttaaaagata tggctgtagc aacaggtggt 960
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agcgagtatg aaaaagaaaa aatgcacgaa cgcttggcca agttgtcaaa cggcgtcgca 1200
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acaggtgtgc ggattgttct acgtgcactt tcaactccgt gctataccat tgcacataac 1440
gcaggtgtca acgcctctgt tgtcgttgaa aaagtcaaag gtatgagcca gaatatgggt 1500
tacgatgctc aaaatgatgt ttacgttgat atgatcgagg ctggaattat tgacccaact 1560
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Leu Glu Phe Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile Ser Pro
210 215 220
Tyr Phe Ile Asn Thr Glu Lys Gly Ala Arg Cys Glu Phe Gln Asp Ala
225 230 235 240
Phe Ile Leu Phe Ser Glu Lys Lys Ile Asn Ser Ile Gln Thr Leu Leu
245 250 255
Pro Ala Leu Glu Leu Cys His Gln Gln Lys Arg Pro Leu Leu Ile Ile
260 265 270
Ala Glu Asp Val Glu Gly Glu Ala Leu Thr Ala Leu Val Leu Asn Arg
275 280 285
Leu Lys Leu Gly Leu Gln Val Cys Ala Val Lys Ala Pro Gly Phe Gly
290 295 300
Asp Asn Arg Lys Asn Thr Leu Lys Asp Met Ala Val Ala Thr Gly Gly
305 310 315 320
Ile Val Phe Gly Asp Glu Ala Asp Met Tyr Lys Leu Glu Asp Val Gln
325 330 335
Leu Gln Asp Leu Gly Arg Val Ser Glu Ala Val Ile Thr Lys Asp Asp
340 345 350
Cys Leu Leu Met Arg Gly Arg Gly Asn Lys Met Asp Ile Asp Lys Arg
355 360 365
Ile Ala Gln Ile Lys Asp Glu Met Glu Ala Ser Asn Ser Glu Tyr Glu
370 375 380
Lys Glu Lys Met His Glu Arg Leu Ala Lys Leu Ser Asn Gly Val Ala
385 390 395 400
Val Ile Lys Val Gly Gly Ser Ser Glu Val Glu Val Ser Glu Lys Lys
405 410 415
Asp Arg Tyr Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Ile Glu Glu
420 425 430
Gly Ile Gln Arg Thr Gly Val Arg Ile Val Leu Arg Ala Leu Ser Thr
435 440 445
Pro Cys Tyr Thr Ile Ala His Asn Ala Gly Val Asn Ala Ser Val Val
450 455 460
Val Glu Lys Val Lys Gly Met Ser Gln Asn Met Gly Tyr Asp Ala Gln
465 470 475 480
Asn Asp Val Tyr Val Asp Met Ile Glu Ala Gly Ile Ile Asp Pro Thr
485 490 495
Lys Val Val Arg Thr Ala Leu Val Asp Ala Ala Gly Val Ala Ser Leu
500 505 510
Leu Thr Thr Ala Glu Thr Val Val Thr Asp Leu Pro Lys Glu Glu Thr
515 520 525
Gly Gly Asn Ala Ala Gly Met Gly Gly Met Gly Gly Met Gly Gly Met
530 535 540
Gly Gly Met Met
545
<210>4
<211>1647
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<213>Schistosoma japonicum
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atgttacgag ctctaagttc acttcgtggt agtttggttc cagctagaca aaaagttgtt 60
caaaggtttt atgcaaagga agtgaagttc ggggctgacg caagatcagc tatgttgatt 120
ggtgttgatg tactggcgga tgctgttgca gtcaccatgg gccctaaagg tcgtaacgta 180
ataatagaaa gctcgtggaa atctccaaaa atcacaaaag atggggttac tgttgccaaa 240
ggtattgagc tgaaggataa gttccagaat attggggcta agttagtaca ggacgttgct 300
aacaacacca acgaagaggc tggggatggt actacaacag ccactgtttt agcgcgagct 360
atagctaaag agggttttga aaagataagt aaaggcgcta atcctatcga gttccgaaga 420
ggtgttatgc ttgctgttga tgctgttgtc aaagagctca aatctttttc gaagcaaata 480
tccacaccag aagagatagc acaagttgcg acaatatccg ctaacggtga caaggctatt 540
ggcgacctaa tcgcttcagc tatgaagagg gttgggaatg atggcacgat aactgttaag 600
gatggcaaaa ctcttcacga tgagttggag tttatcgaag gtatgaaatt cgaccgtggt 660
tatatctctc catattttat caacactgag aaaggagccc gatgtgaatt ccaagatgcc 720
ttcatattgt tttctgaaaa aaagataaac agcattcaga cattattacc agctctcgaa 780
ctatgccacc aacaaaaacg gccgctgctg attatcgccg aggatgttga aggtgaagct 840
cttacagcac ttgtactaaa tcgcttaaaa ctgggcttgc aggtctgtgc agtcaaagca 900
ccaggttttg gggataatcg caaaaataca ttaaaagata tggctgtagc aacaggtggt 960
atagtgttcg gtgacgaagc agatatgtac aagttagagg atgtacaact gcaagatctg 1020
ggacgtgtgt cagaagctgt aataacaaaa gacgattgtt tactaatgcg aggtcgtgga 1080
aacaaaatgg acatcgacaa acgtatcgca caaattaaag atgaaatgga agcctccaac 1140
agcgagtatg aaaaagaaaa aatgcacgaa cgcttggcca agttgtcaaa cggcgtcgca 1200
gtaattaagg ttggtggaag tagtgaagta gaagttagtg agaaaaagga tcgatatact 1260
gatgcactca atgcaacgag agcagcaatt gaggaaggca tacagcgaac aggtgtgcgg 1320
attgttctac gtgcactttc aactccgtgc tataccattg cacataacgc aggtgtcaac 1380
gcctctgttg tcgttgaaaa agtcaaaggt atgagccaga atatgggtta cgatgctcaa 1440
aatgatgttt acgttgatat gatcgaggct ggaattattg acccaactaa ggtcgttaga 1500
actgcattgg ttgatgcagc tggtgttgct tccttactca caactgcaga aaccgtggtg 1560
acggatttac caaaagaaga gaccggaggc aacgcagcag gaatgggtgg gatgggaggt 1620
atgggtggaa tgggaggcat gatgtaa 1647
<210>5
<211>24
<212>PRT
<213>人工序列
<400>5
Val Pro Gly Gly Gly Thr Ala Leu Leu Arg Cys Ile Pro Val Leu Asp
1 5 10 15
Thr Leu Ser Thr Lys Asn Glu Asp
20
Claims (8)
1.一种增加CD4+CD25+Foxp3+调节性T细胞的抗原,是日本血吸虫热休克蛋白60KDa,它的全长氨基酸序列如SEQ.ID.NO.1所示。
2.权利要求1所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原作为增强CD4+CD25+Foxp3+调节性T细胞抑制效应的免疫抑制剂的应用。
3.权利要求1所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原在制备治疗免疫反应引起的炎症性疾病药物中的应用。
4.权利要求1中所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原在制备防治关节炎所致的炎性症状和免疫病理药物中的应用。
5.一种增加CD4+CD25+Foxp3+调节性T细胞的抗原,是缺失了第435至第458个氨基酸的日本血吸虫热休克蛋白60KDa,它的氨基酸序列如SEQ.ID.NO.3所示。
6.权利要求5所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原作为增强CD4+CD25+Foxp3+调节性T细胞抑制效应的免疫抑制剂的应用。
7.权利要求5所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原在制备治疗免疫反应引起的炎症性疾病药物中的应用。
8.权利要求5中所说的增加CD4+CD25+Foxp3+调节性T细胞的抗原在制备防治关节炎所致的炎性症状和免疫病理药物中的应用。
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PCT/CN2011/071738 WO2011116655A1 (zh) | 2010-03-25 | 2011-03-11 | 一种增加cd4+cd25+foxp3+调节性t细胞的抗原及其应用 |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011116655A1 (zh) * | 2010-03-25 | 2011-09-29 | 南京医科大学 | 一种增加cd4+cd25+foxp3+调节性t细胞的抗原及其应用 |
CN103063846A (zh) * | 2012-12-20 | 2013-04-24 | 南京医科大学 | SjHSP60在制备监测血吸虫病患者综合肝脏病理损伤程度的诊断试剂中的应用 |
CN103372214A (zh) * | 2012-04-13 | 2013-10-30 | 北京艾棣维欣生物技术有限公司 | 治疗和/或预防ⅰ型糖尿病的药物组合物及其应用 |
CN110818787A (zh) * | 2019-12-04 | 2020-02-21 | 南京医科大学 | 一种增加Ly6C+巨噬细胞的抗原及其应用 |
CN112707959A (zh) * | 2020-11-11 | 2021-04-27 | 南京医科大学 | 一种多肽、制备方法及应用 |
Families Citing this family (1)
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WO2018031871A1 (en) * | 2016-08-12 | 2018-02-15 | Translate Bio Ma, Inc. | Ex vivo modulation of foxp3 expression |
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CN1952140A (zh) * | 2005-10-20 | 2007-04-25 | 中国疾病预防控制中心寄生虫病预防控制所 | 日本血吸虫疫苗抗原基因的克隆、表达和应用 |
CN101002948A (zh) * | 2006-12-26 | 2007-07-25 | 南京医科大学 | 基于t细胞表位的抗日本血吸虫感染的肽-dna双疫苗 |
CN101624422A (zh) * | 2008-07-09 | 2010-01-13 | 中国农业科学院上海兽医研究所 | 日本血吸虫重组多表位抗原及其表达、纯化方法与应用 |
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CN101671689B (zh) * | 2009-07-29 | 2013-05-01 | 中国农业科学院上海兽医研究所 | 含日本血吸虫基因的重组表达载体及其应用 |
CN101921325B (zh) * | 2010-03-25 | 2012-06-27 | 南京医科大学 | 一种增加CD4+CD25+Foxp3+调节性T细胞的抗原及其应用 |
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CN1952140A (zh) * | 2005-10-20 | 2007-04-25 | 中国疾病预防控制中心寄生虫病预防控制所 | 日本血吸虫疫苗抗原基因的克隆、表达和应用 |
CN101002948A (zh) * | 2006-12-26 | 2007-07-25 | 南京医科大学 | 基于t细胞表位的抗日本血吸虫感染的肽-dna双疫苗 |
CN101624422A (zh) * | 2008-07-09 | 2010-01-13 | 中国农业科学院上海兽医研究所 | 日本血吸虫重组多表位抗原及其表达、纯化方法与应用 |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011116655A1 (zh) * | 2010-03-25 | 2011-09-29 | 南京医科大学 | 一种增加cd4+cd25+foxp3+调节性t细胞的抗原及其应用 |
CN103372214A (zh) * | 2012-04-13 | 2013-10-30 | 北京艾棣维欣生物技术有限公司 | 治疗和/或预防ⅰ型糖尿病的药物组合物及其应用 |
CN103372214B (zh) * | 2012-04-13 | 2017-09-29 | 北京艾棣维欣生物技术有限公司 | 治疗和/或预防ⅰ型糖尿病的药物组合物及其应用 |
CN103063846A (zh) * | 2012-12-20 | 2013-04-24 | 南京医科大学 | SjHSP60在制备监测血吸虫病患者综合肝脏病理损伤程度的诊断试剂中的应用 |
CN110818787A (zh) * | 2019-12-04 | 2020-02-21 | 南京医科大学 | 一种增加Ly6C+巨噬细胞的抗原及其应用 |
CN110818787B (zh) * | 2019-12-04 | 2023-08-04 | 南京医科大学 | 一种增加Ly6C+巨噬细胞的抗原及其应用 |
CN112707959A (zh) * | 2020-11-11 | 2021-04-27 | 南京医科大学 | 一种多肽、制备方法及应用 |
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WO2011116655A1 (zh) | 2011-09-29 |
CN101921325B (zh) | 2012-06-27 |
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