CN106383227B - 尿液中hiv相关蛋白的检测 - Google Patents
尿液中hiv相关蛋白的检测 Download PDFInfo
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- CN106383227B CN106383227B CN201610701916.9A CN201610701916A CN106383227B CN 106383227 B CN106383227 B CN 106383227B CN 201610701916 A CN201610701916 A CN 201610701916A CN 106383227 B CN106383227 B CN 106383227B
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Abstract
公开了检测哺乳动物中HIV感染的方法。该方法包括从哺乳动物的尿液样品中分离外泌体和检测所述分离的外泌体中HIV特异性生物标志物的存在的步骤。还公开了诊断患有HIV相关疾病、特别是患有HIV相关肾病的哺乳动物的方法。
Description
本申请要求2009年10月2日提交的美国专利申请流水第12/572,652号的优先权,该美国专利申请要求2008年10月6日提交的美国临时申请流水第61/102,941号的优先权。通过引用将所有上述申请的全文并入本文。
发明领域
本发明通常涉及诊断方法,并且具体而言,涉及使用尿液中的生物标志物来检测HIV感染、诊断HIV或HIV相关疾病的方法。
背景技术
通常对血清或血浆进行HIV测试。HIV抗体的检测是HIV-1感染的推定证据,并且通常通过Western印迹方法进行验证。通过p24抗原测定进行的病毒检测或通过RT-PCR进行的病毒RNA检测也用于确定循环中病毒的量。CD4/CD8T细胞比和其他免疫功能测试也常用于监测免疫状态和向AIDS的进展。最近,还开发了使用唾液或口腔中上皮细胞的HIV测试。然而,目前没有可用的检测尿液中抗原或抗体的测试。尿液中HIV蛋白的检测可以提供检测HIV感染或疾病进展,特别是肾脏并发症的更快速的方法。
HIV相关肾病(HIVAN)是尤其困扰非洲人种的肾病,其特征为肾脏肥大和快速进展以及阶段性肾病。HIVAN是由HIV-1病毒直接感染肾脏细胞所引起并且通过病毒基因产物而导致肾脏损伤。HIVAN也能够由HIV感染期间细胞因子释放的改变所引起。HIVAN的病因学仍然未知。据估算,90%的HIVAN患者是非洲人种,这表明该疾病具有遗传倾向(Wyatt,CM.,Klotman,P.E.HIV-Associated Nephropathy in the Era of Antiretroviral Therapy(逆转录病毒疗法时代的HIV相关肾病).American Journal of Medicine Review 2007)。
当患者的肾脏活组织切片检查表现出局灶性节段性肾小球硬化,伴随肾小管扩张和炎症、微囊型肾小管、退变的肾小球毛细血管,并联合明显的蛋白尿时,可诊断为HIVAN(同上)。然而,该方法是创伤性操作并且因为会造成创伤有时会被患者拒绝。因此,需要对于HIV或HIV相关疾病的可靠、快速、合算和较低创伤性的诊断测试。
发明概述
本发明一方面涉及检测哺乳动物中HIV感染的方法。在一个实施方案中,该方法包括以下步骤:从所述哺乳动物的尿液样品中分离外泌体,以及检测来自所述分离的外泌体的HIV相关生物标志物。
在相关实施方案中,该方法还包括以下步骤:根据所述分离的外泌体中HIV相关生物标志物是否存在来确定该哺乳动物是否被HIV感染。
在相关的实施方案中,通过离心分离外泌体。
在另一相关的实施方案中,通过过滤分离外泌体。
在另一相关实施方案中,通过一种或多种选自以下的技术检测外泌体:电泳、Western印迹、HPLC、FPLC、MS和蛋白质测序。
在另一相关实施方案中,通过SELDI-TOF-MS和LC-MS/MS检测外泌体。
在另一相关实施方案中,HIV相关生物标志物选自Nef、HIV包膜gp120、HIV蛋白酶、Vif、Gag-Pol、Gag、p24、Rev、逆转录酶(RT)、Tat、p1、p17、Vpu、Vpr、gp41和DNA聚合酶。
在另一相关实施方案中,HIV相关生物标志物是Nef、HIV蛋白酶、Vif、Pol或Gag。
在另一相关实施方案中,HIV相关生物标志物是Nef。
在一个相关实施方案中,哺乳动物是人、猴、大猩猩或狒狒。
在另一相关实施方案中,哺乳动物是人。
本发明的另一方面涉及诊断哺乳动物中HIV相关疾病的方法。在一个实施方案中,该方法包括以下步骤:从所述哺乳动物的尿液样品中分离外泌体,以及检测来自所述分离的外泌体的HIV相关生物标志物。
在另一相关实施方案中,所述HIV相关疾病是HIV相关肾病。
在另一相关实施方案中,该方法还包括以下步骤:根据所述分离的外泌体中所述HIV相关生物标志物是否存在来确定该哺乳动物是否患有HIV相关肾病。
在一个相关实施方案中,哺乳动物是人、猴、大猩猩或狒狒。
在另一相关实施方案中,哺乳动物是人。
本发明的另一方面涉及监测哺乳动物中HIV感染进展的方法。在一个实施方案中,该方法包括以下步骤:从所述哺乳动物的尿液样品中分离外泌体,以及检测来自所述分离的外泌体的所述HIV相关生物标志物。
在另一相关实施方案中,该方法还包括以下步骤:根据所述分离的外泌体中所述HIV相关生物标志物是否存在来确定所述哺乳动物中HIV感染进展。
本发明的另一方面涉及监测哺乳动物中HIV相关肾病进展的方法。在一个实施方案中,该方法包括以下步骤:从所述哺乳动物的尿液样品中分离外泌体,以及检测来自所述分离的外泌体的所述HIV相关生物标志物。
在另一相关实施方案中,该方法还包括以下步骤:根据所述分离的外泌体中所述HIV相关生物标志物是否存在来确定所述哺乳动物中HIV相关肾病进展。
本发明的另一方面涉及监测用抗HIV药剂治疗哺乳动物的功效的方法。该方法包括下述步骤:在给予药剂之前,测定获自哺乳动物的尿液样品中的尿液外泌体中HIV相关生物标志物谱;测定所述哺乳动物的一个或多个给药后的尿液样品中的尿液外泌体中HIV相关生物标志物谱;将给药前样品中HIV相关生物标志物谱与给药后样品中HIV相关生物标志物谱进行比较;以及确定所述药剂的功效。
在相关实施方案中,该方法还包括以下步骤:改变所述药剂对所述哺乳动物的给药。
本发明的另一方面涉及用于检测哺乳动物中HIV感染或监测哺乳动物中HIV感染进展的试剂盒。所述试剂盒包含用于制备检测用外泌体样品的一种或多种试剂和至少一种作为标准的HIV相关生物标志物。
在另一相关的实施方案中,通过Western印迹检测外泌体。
在另一相关实施方案中,试剂盒还包含标签。
在另一相关实施方案中,试剂盒还包含带有说明书的标签。
本发明的另一方面涉及用于诊断哺乳动物中HIV相关疾病或监测哺乳动物中HIV相关疾病进展的试剂盒。所述试剂盒包含用于制备检测用外泌体样品的一种或多种试剂和至少一种作为标准的HIV相关生物标志物。
在另一相关实施方案中,所述HIV相关疾病是HIV相关肾病。
在一个相关实施方案中,HIV相关生物标志物选自Nef、HIV包膜gp120、HIV蛋白酶、Vif、Gag-Pol、Gag、p24、Rev、逆转录酶(RT)、Tat、p1、p17、Vpu、Vpr、gp41和DNA聚合酶。
在另一相关实施方案中,HIV相关生物标志物是Nef、HIV蛋白酶、Vif、Pol或Gag。
在另一相关实施方案中,HIV相关生物标志物是Nef。
附图简要说明
图1是流程图,其示出了使用来自哺乳动物的尿液样品检测哺乳动物中HIV感染或监测哺乳动物中HIV感染进展的方法的实施方案。
图2A-2C是来自HIVAN组中患者的尿液外泌体的样品SELDI-TOF-MS谱的组图。
图3A-3D是来自AA HIV+组中患者的尿液外泌体的样品SELDI-TOF-MS谱的组图。
图4A-4C是来自HIV白种人组中患者的尿液外泌体的样品SELDI-TOF-MS谱的组图。
图5A-5E是来自FSGS组中患者的尿液外泌体的样品SELDI-TOF-MS谱的组图。
图6A-6C是来自正常对照组中患者的尿液外泌体的样品SELDI-TOF-MS谱的组图。
图7A-7E是分离自HIVAN组(图A)、FSGS组(图B)、非裔美国人(AA)HIV+组(图C)、白种人HIV+组(图D)和正常对照组(图E)中患者的尿液外泌体的透射电镜(TEM)图像的组图。
图8是显示来自HIV+患者和对照的尿液囊泡的Western印迹分析图像的组图。通过超滤从尿液分离囊泡并且分析囊泡中HIV Nef或其他HIV蛋白的存在。上排图使用抗HIVNef单克隆抗体,而下排图使用合并的HIV+患者血清作为一抗。患者27、28、30、41和104是AA。患者108、103、86和48是HIV+白种人患者。最后是三个HIV阴性个体、重组HIV Nef和p24的对照图。
发明详述
除非另外指明,下文详细描述的实施方案的实践使用本领域技术内的诊断学、分子生物学、细胞生物学、生物化学和免疫学的常规方法。这些技术在文献中有详细的解释。本文引用的所有出版物、专利和专利申请,不论上文还是下文的,都通过引用整体而并入本文。
本发明一方面涉及检测哺乳动物中HIV感染的方法。如图1所示,方法100的实施方案包括以下步骤:从哺乳动物的尿液样品中分离(110)外泌体,以及检测(120)来自所述分离的外泌体的HIV相关生物标志物的存在。
在一个实施方案中,该方法还包括以下步骤:根据所述分离的外泌体中HIV相关生物标志物是否存在来确定(130)该哺乳动物是否被HIV感染。
外泌体是由多种哺乳动物细胞类型分泌的50-90nm的囊泡。外泌体首先发现于成熟中的哺乳动物网织红细胞,被证实为许多质膜蛋白的选择性移除的机制。当细胞膜的部分自发内陷并且被内吞时,细胞内产生外泌体。内化的部分破裂成较小的囊泡,该囊泡随后从细胞排出。上述后一阶段是在包含许多小囊泡的晚期内体与细胞膜融合时发生,这触发囊泡从细胞释放。囊泡(一旦释放后被称为外泌体)由嵌入原始细胞膜共有的配体的脂筏组成。
尽管外泌体的蛋白组成根据细胞来源而不同,但大多数外泌体包含可溶性蛋白Hsc 70。诸如树突细胞和B细胞的某些免疫细胞分泌外泌体,许多科学家相信外泌体在介导对病原体和肿瘤的适应性免疫反应中起功能作用。据报道,未成熟的树突细胞来源的外泌体可以介导HIV转染(Wiley RD et al.,Proc Natl Acad Sci U.S.A.2006年1月17日;103(3):738-743)。
可以通过离心或过滤完成分离步骤110。在一个实施方案中,通过离心使尿液样品中的外泌体沉淀。洗涤沉淀的外泌体并以合适的浓度进行重悬用于进一步分析。在某些实施方案中,将尿液样品以100,000g或更高速离心10-120分钟以沉淀外泌体。在一个实施方案中,将尿液样品以100,000g离心60-120分钟以沉淀外泌体。
在某些其他实施方案中,通过两步离心法将尿液样品中的外泌体沉淀,所述两步离心法包括用低速离心去除尿液中的细胞和其他大颗粒和用高速离心沉淀外泌体。在一个实施方案中,首先将尿液样品以5,000-25,000g离心5-30分钟。然后将上清转移到另一个管中,以100,000g或更高速再次离心30-120分钟以沉淀外泌体。在优选实施方案中,首先将尿液样品以20,000-22,000g离心10-20分钟。然后将上清转移到另一管中,以100,000g再次离心30-90分钟以沉淀外泌体。然后将沉淀的外泌体重悬于液体介质中用于进一步分析。
液体介质可以是等渗的、低渗的或高渗的。在某些实施方案中,液体介质含有缓冲剂和/或至少一种盐或盐的组合。缓冲剂可以将pH保持在特定范围内,例如,1-12,并且缓冲剂也被称为pH稳定剂。更通常地,pH为约pH 5.0至约pH 12.0。pH稳定剂的具体实例是两性离子。pH稳定剂的具体非限制性实例包括三(羟甲基)氨基甲烷盐酸盐(TRIS)、N-(2-羟乙基)哌嗪-N'-2-乙磺酸(HEPES)、3-(N-吗啡啉)丙磺酸(MOPS)、2-(N-吗啡啉)乙磺酸(MES)、N-三(羟甲基)甲基-2-氨基乙磺酸(TES)、N-[羧甲基]-2-氨基乙磺酸(ACES)、N-(2-乙酰氨基)-2-亚氨基二乙酸(ADA)、N,N-二(2-羟乙基)-2-氨基乙磺酸(BES)、N-(2-羟乙基)哌嗪-N'-(2-羟基丙磺酸)(HEPPSO)、N-三(羟甲基)甲基甘氨酸(TRICINE)、N,N-二(2-羟乙基)甘氨酸(BICINE)、4-(环己胺基)-l-丁磺酸(CABS)、3-(环己胺基)-1-丙磺酸(CAPS)、3-(环己胺基-2-羟基-1-丙磺酸(CAPSO)、2-(环己胺基)乙磺酸(CHES)、N-(2-羟乙基)哌嗪-N'-(3-丙磺酸)(EPPS)、哌嗪-N,N'-二(2-乙磺酸)(PIPES)、[(2-羟基-l,l-二[羟甲基]乙基)氨基]-l-丙磺酸(TAPS)、N-三(羟甲基)甲基-4-氨基丁磺酸(TABS)、2-氨基-2-甲基-1-丙醇(AMP)、3-[(l,l-二甲基-2-羟乙基)氨基]-2-羟基丙磺酸(AMPSO)、乙醇胺和3-氨基-l-丙磺酸。pH稳定剂的其他具体非限制性实例包括氯化钾、柠檬酸、邻苯二甲酸氢钾、硼酸、磷酸二氢钾、二乙醇胺、柠檬酸钠、磷酸二氢钠、醋酸钠、碳酸钠、四硼酸钠、二甲胂酸、咪唑、2-氨基-2-甲基-1-丙二醇、tricine、甘氨酸-甘氨酸、bicine和磷酸盐缓冲液(例如,磷酸钠或磷酸钠-钾等)。
使用的缓冲剂或pH稳定剂通常为约0.1mM至约500mM、约0.5mM至约100mM、约0.5mM至约50mM、约1mM至约25mM或约1mM至约10mM。更具体地,缓冲剂的浓度可以为约(即10%以内)1mM、2mM、5mM、10mM、15mM、20mM、25mM、30mM、40mM或50mM。
液体介质还可以含有螯合剂。螯合剂通常与金属离子形成多重键,并且是可以多价螯合金属的多齿配体。金属螯合作用可以相应地降低或阻止微生物的生长或生物分子(例如,肽或核酸)的降解,从而可以提高对结合至底物上的生物分子的保护作用。螯合剂的具体非限制性实例包括EDTA(乙二胺四乙酸)、EGTA(乙二醇-O,O'-二(2-氨乙基)-N,N,N',N'-四乙酸)、GEDTA(乙二醇醚二胺四乙酸)、HEDTA(N-(2-羟乙基)乙二胺-N,N',N'-三乙酸)、NTA(次氮基三乙酸)、水杨酸、三乙醇胺和卟吩。螯合剂的通常浓度为约0.1mM至约100mM、约0.5mM至约50mM或约1mM至约10mM。
液体介质还可以含有变性剂。变性剂和去垢剂通常在疏水环境和亲水环境之间形成化学桥,所述化学桥破坏或减小保持天然蛋白结构所需的疏水力。变性剂和去垢剂的具体非限制性化学种类包括阴离子表面活性剂、非离子表面活性剂、阳离子表面活性剂和两性电解质表面活性剂。去垢剂的具体非限制性实例包括硫氰酸胍、SDS、十二烷基硫酸钠、NP40、曲拉通X-100、吐温、胆酸钠、脱氧胆酸钠、氯化苄乙氧铵、CTAB(十六烷基三甲基溴化铵(Cetyltrimethylammonium bromide))、十六烷基三甲基溴化铵(Hexadecyltrimethylammonium bromide)和N,N-二甲基癸烷基-N-氧化胺。
液体介质还可以含有变性剂。还原剂和抗氧化剂通常抑制微生物的生长并将生物分子的氧化进行还原。这类试剂的具体非限制性种类包括自由基清除剂。还原剂和抗氧化剂的具体非限制性实例包括DTT(二硫苏糖醇)、二硫赤藓糖醇、尿素、尿酸、2-巯基乙醇、半胱氨酸、维生素E、维生素C、连二亚硫酸盐、硫代乙醇酸和焦亚硫酸盐。
液体介质还可以含有防腐剂或稳定剂。如果需要抑制或推迟HIV相关生物标志物的降解,可以使用防腐剂或稳定剂。防腐剂和稳定剂的具体非限制性实例包括叠氮化钠和聚乙二醇(PEG)。防腐剂和稳定剂的通常浓度为约0.05%至约1%。
液体介质还可以含有蛋白酶抑制剂。蛋白酶抑制剂抑制肽的降解。蛋白酶抑制剂的具体非限制性种类包括底物(例如,肽)与蛋白酶结合的可逆抑制剂或不可逆抑制剂。蛋白酶抑制剂的具体非限制性种类包括丝氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂。蛋白酶抑制剂的具体非限制性实例包括:PMSF、PMSF Plus、APMSF、抗凝血酶III、氨肽酶抑制剂、抗痛素、抑肽酶、苯丁抑制素、苯甲脒、胰凝乳蛋白酶抑制剂、钙蛋白酶抑制剂I和II、E-64、3,4-二氯异香豆素、DFP、弹性蛋白酶抑制剂、亮抑肽酶、胃蛋白酶抑制剂、1,10-邻二氮杂菲、磷酰二肽、TIMP-2、TLCK、TPCK、胰蛋白酶抑制剂(大豆或鸡卵白蛋白)、hirustasin、α-2-巨球蛋白、4-(2-氨乙基)-苯磺酰氟盐酸盐(AEBSF)和Kunitz型蛋白酶抑制剂。
在另一实施方案中,通过使尿液样品通过滤器来收集外泌体,所述滤器的孔径小于外泌体的平均大小。然后将外泌体从滤器移出,并以合适浓度重悬用于进一步分析。在某些实施方案中,使用分子量截留值为500kd-50kd的离心滤器来收集尿液样品中的外泌体。在一个实施方案中,使用分子量截留值为100kd的离心滤器来收集尿液样品中的外泌体。
可以使用能够鉴定HIV相关生物标志物的任何技术进行检测步骤120。如本文所用的术语“HIV相关生物标志物”是指与HIV感染、HIV感染进展和诸如HIV相关肾病的HIV相关疾病有关的蛋白或蛋白片段。HIV相关生物标志物的实例包括但不限于HIV蛋白,例如HIV包膜gp120/gp41、HIV蛋白酶、Nef、Vif、Gag-Pol、Gag、p24、Rev、逆转录酶、Tat、p1、p17、Vpr、Vpu和DNA聚合酶。
多种技术可被用于鉴定HIV相关生物标志物。这些技术的实例包括,但不限于,诸如单向和双向凝胶分析的电泳、Western印迹、ELISA、HPLC、FPLC、质谱、蛋白质测序、抗体阵列及以上的组合。在一个实施方案中,通过Western印迹鉴定生物标志物,在另一实施方案中,通过表面增强激光解吸/电离飞行时间质谱(SELDI-TOF-MS)和LC-MS/MS鉴定生物标志物。
通过将尿液样品中HIV相关生物标志物谱与数据库中存储的HIV相关生物标志物谱进行比较来进行确定步骤。基于比较的结果作出诊断。HIV相关生物标志物谱可以包含零至多个HIV相关生物标志物。例如,健康哺乳动物的HIV相关生物标志物谱可以不包含HIV相关生物标志物。另一方面,患有HIVAN的患者的HIV相关生物标志物谱可以包含多个HIV相关生物标志物。
在一个实施方案中,方法100是用于监测哺乳动物中HIV感染的进展。在该实施方案中,来自所述哺乳动物的医疗记录的相关信息也可用于确定步骤130中以作出诊断。
在另一实施方案中,方法100用于诊断哺乳动物中HIV相关肾病。在该实施方案中,来自所述哺乳动物的医疗记录的遗传背景和相关信息也可用于确定步骤130中以作出诊断。在另一实施方案中,方法100也可用于监测哺乳动物中HIV相关肾病的进展。
在另一实施方案中,方法100用于监测HIV治疗的临床试验中的效果。在这类临床试验中,尿液外泌体中HIV相关生物标志物谱可被用做指示哺乳动物对治疗的生理反应的读出信息。
在一个相关实施方案中,哺乳动物是人、猴、大猩猩或狒狒。
在优选相关实施方案中,哺乳动物是人。
本发明的另一方面提供了监测用抗HIV药剂治疗哺乳动物的功效的方法。在一个实施方案中,该方法包括下述步骤:在给予药剂之前检测获自哺乳动物的样品中的尿液外泌体中的HIV相关生物标志物谱;检测获自所述哺乳动物的一个或多个给药后的样品中的尿液外泌体中的HIV相关生物标志物谱;将给药前样品中HIV相关生物标志物谱与给药后样品中HIV相关生物标志物谱进行比较。
在另一实施方案中,所述方法还包括以下步骤:确定所述药剂的功效和任选地改变所述药剂对所述哺乳动物的给药。根据这样的实施方案,HIV相关生物标志物谱可被用做药剂功效的指示,即使没有可观察到的表型反应。
本发明的另一方面涉及用于检测哺乳动物中HIV感染或监测哺乳动物中HIV感染进展的试剂盒。所述试剂盒包含用于制备检测用外泌体样品的一种或多种试剂和至少一种作为标准的HIV相关生物标志物。
在另一相关的实施方案中,通过Western印迹检测外泌体。
本发明的另一方面涉及用于诊断哺乳动物中HIV相关疾病或监测哺乳动物中HIV相关疾病进展的试剂盒。所述试剂盒包含用于制备检测用外泌体样品的一种或多种试剂和至少一种作为标准的HIV相关生物标志物。
在另一相关实施方案中,HIV相关疾病是HIV相关肾病。
在另一相关实施方案中,HIV相关生物标志物选自Nef、HIV包膜gp120、HIV蛋白酶、Vif、Gag-Pol、Gag、p24、Rev、逆转录酶(RT)、Tat、p1、p17、Vpu、Vpr、gp41和DNA聚合酶。
上述的试剂盒通常带有包括组分或使用说明描述的标签或包装插页。示例性的说明包括收集尿液样品、从尿液中收获外泌体和检测HIV相关生物标志物的说明。上述的试剂盒还可以包含适于重悬从尿液样品中分离的外泌体的液体。上述的试剂盒可以包含用于收集尿液样品的容器和/或用于从尿液样品中分离外泌体的离心滤器。
通过随后的实施例进一步说明本发明,这些实施例不应解释为是对本发明的限制。将本申请各处引用的所有参考文献、专利和公开专利申请的内容以及图和表格通过引用的方式并入本文。
实施例1:材料和方法
患者
从亚特兰大大都会地区的四个临床单位征募了处于疾病不同阶段的HIV+患者来参与本研究。本研究仅排除了进行透析的患者。所有样品都是根据莫尔豪斯医学院的机构伦理审查委员会和人体试验委员会批准的操作程序进行收集,并且根据机构伦理审查委员会创建的指导,从所有患者和健康志愿者获得知情同意书。将患者分为五组:患有HIV的非裔美国人患者(AA HIV+)、患有HIV的白种人患者(白种人HIV+)、患有HIVAN的患者(HIVAN)、未患HIV但患有FSGS的非裔美国人患者和健康对照。还从患者的医疗记录中收集了相关信息。
样品收集和保存
在常规就诊期间从患者收集尿液样品。从患者的医疗记录获得临床数据。将尿液收集在无菌容器中并运回实验室。使用Multistix 10SG试剂带(Bayer Corporation,Elkhart,IN)对每个样本进行尿检并且利用Siemens Clinitek Microalbumin计量棒(Bayer Corp.)测定白蛋白/肌酸比值。在Siemens Clinitek Status仪器(Bayer Corp.)上读取所述试剂带。将样品以2,000g离心10分钟以去除全细胞和沉淀。将剩余的尿液样品以4ml体积分装并在-80℃保存直至分析。
外泌体的分离
评估了以下两种分离外泌体的方法:高速超速离心法或使用分子量截留滤器的超滤法。对于超速离心法,将4ml尿液转移到聚碳酸酯离心管中并以21,000g离心15分钟。弃上清,并以100,000g再次离心60分钟以沉淀外泌体。将多余的尿液倒出,并将小团在100μl磷酸盐缓冲盐溶液(PBS)中复原并在4℃保存。对于超滤法,将4ml尿液加到Amicon Ultra离心过滤装置(Ultracel,截留值100k,Millipore,Inc.)中并在吊桶式转子中以4,000g离心20分钟。使用100μl PBS冲洗滤器并稀释滞留物。使用二辛可宁酸蛋白检测(Pierce)测定蛋白浓度。
表面增强激光解吸/电离飞行时间质谱
将通过亲水性和带电残基结合蛋白的正常相(normal phase)芯片(蛋白芯片NP20;Ciphergen Biosystems,Fremont,CA)用于分析。将5μl囊泡制备物一式两份地加到芯片上并在恒湿箱中孵育30分钟。用5μl高效液相色谱(HPLC)级的水洗涤芯片3次并风干10分钟。根据厂商说明书,制备50%乙腈/0.5%三氟乙酸中的饱和芥子酸(SPA,CiphergenBiosystems,CA)。每个点添加1μl基质溶液(SPA)并风干,随后在ProteinChip Reader II(Ciphergen Biosystems)上使用以下设定进行读取:激光强度250;检测器敏感度10;高质量300Kda,优化为3Kda至50Kda。将数据获得方法设定为自动激光调整并自动鉴定3Kda至50Kda的峰。
LC-MS/MS
使用LTQ质谱仪(ThermoFinnigan)通过LC/MS分析收集的外泌体。首先,用分析当天新鲜配制的2D凝胶上样缓冲液(Q-biosciences)提取成团的外泌体。然后,使用4倍体积的冰冷(-20℃)丙酮沉淀溶解的小团并在-20℃孵育过夜。以19,200g离心收集沉淀物。将小团干燥并重新溶解在50mM碳酸氢铵(AmBIC)中。首先,用2μl的500mM DTT贮液(Qbiosciences,单次使用)将蛋白溶液在56℃还原30分钟。然后,通过添加2μl的1M碘乙酸(iodacetic acid)贮液(IAA;Q-biosciences,单次使用)将溶液烷基化并在室温避光孵育30分钟。用50mM AmBic将新鲜小管胰蛋白酶(Promega Gold质谱级)从8μl稀释到312μl并保持在冰上。然后将10μl稀释的胰蛋白酶添加到反应中并将其在37℃振荡孵育4小时。然后加入50μl的0.5%甲酸,将混合物直接分析或在-20℃保存用于分析。使用自动上样器以每分钟10μl的流速将10μl样品上样到captrap(Michrom)C18肽阱(peptide trap)。10分钟后,液体被转换到0.5mm×50μm的C18柱(Microm)。使用线性梯度的5-40%乙腈水溶液洗脱肽超过50分钟。使用微喷雾电离装置(microspray ionization)(Michrom Advance)以约每分钟3μl的流速将洗脱的肽直接引入到LTQ质谱仪中。使用Excalibur 2.2软件套装分析样品,从而以数据依赖扫描模式分析离子。母体扫描后,进行三个丰度最高的离子的数据依赖扫描。使用Bio Works 3.1(ThermoFinnigan)将文件与包括人和HIV蛋白的NR数据库子集进行检索比对。DTA生成阀值设为200,肽的公差设为0.5Da,蛋白的公差设为1.0Da。使用>10.0的一致性评分和>1.0的Xcorr评分产生初始蛋白鉴定列表。
电泳和Western印迹
SDS PAGE电泳。将样品在Tris-甘氨酸SDS样品还原缓冲液中85℃加热2分钟,并上样到4-12%的Criterion XT Bis-Tris预制丙烯酰胺凝胶(BioRad,Hercules,CA)。每孔上样约200ng样品。对照为重组HIV NeF(获赠于Andrea Raymond博士)和HIV重组p24(Immunodiagnostics,Inc.),每孔上样30至40ng。使用Gel Code Blue(Pierce,Inc.)对凝胶进行染色,或将蛋白转移到PVDF膜(Immobilon-P,Millipore Corp,Billerica,MA)上用于Western印迹分析。SNAP ID系统(Millipore,Corp)被用于HIV Nef或HIV蛋白是否存在的Western印迹分析。使用单克隆鼠抗HIV Nef单克隆抗体(1:1500,Chemicon hit.,CA)和二抗羊抗鼠IgG(H+L)过氧化物酶偶联抗体(1:15,000,Jackson Immunoresearch,WestGrove,PA)进行HIV Nef鉴定。使用合并的人HIV+血清(1:15,000)作为一抗和羊抗人IgG(H+L)过氧化物酶偶联抗体(1:15,000,Jackson Immunoresearch)检测HIV蛋白。将膜与化学发光底物(SuperSignal West Femto Maximum,Pierce,Inc.)孵育并且在X射线胶片(CL-Xposure,Kodak)上曝光和冲洗。
透射电镜
将样品在0.1M二甲胂酸盐缓冲液中的2.5%戊二醛中4℃固定2小时,然后用0.1M二甲胂酸盐缓冲液洗涤2次,每次5分钟。将样品用0.1M二甲胂酸盐缓冲液中的1%四氧化锇在4℃固定1小时,然后用二甲胂酸盐缓冲液洗涤2次,用去离子水洗涤3次,每次5分钟。切薄片,用0.5%水性乙酸双氧铀在室温染色2小时,用JEOL 1200EX透射电镜观察。
实施例2:尿液囊泡的分离、超速离心法与超滤法比较:
通过超速离心法或超滤法从6个不同HIV+患者的尿液中分离囊泡,以确定两种方法中的哪种得到最大量的蛋白。超滤法分离的蛋白(中位数2930μg)都多于超速离心法(中位数591μg)。
实施例3:来自患者的尿液外泌体的SELDI-TOF-MS谱
通过SELDI-TOF-MS分析了来自不同组患者的尿液外泌体。通过LC-MS/MS验证结果。来自代表性患者的SELDI-TOF-MS谱在图2-6中示出。表1总结了不同测试组中通过SELDI-MS检测的并通过LC-MS/MS验证的蛋白。
表1:通过SELDI-MS在尿液样品中检测的蛋白
患者 | MW | 蛋白 |
HIVAN | 10,585 | HIV包膜gp;HIV蛋白酶 |
23,546 | HIV包膜gp;HIV Nef;HIV Vif | |
33,464 | HIV蛋白gp;mu A03009B12Rik蛋白 | |
45,632 | HIV包膜gp;HIV pol蛋白 | |
66,587 | HIV包膜gp;HIV Nef;PgD合酶 | |
78,942 | 未知 | |
AA HIV | 23,684 | HIV包膜gp;HIV Nef;PgD合酶 |
83,256 | 未知 | |
FSGS | 66,533 | 未知 |
白种人HIV | 23,935 |
表2总结了个体患者中的尿液蛋白谱。
表2:个体患者中的尿液蛋白谱
透射电镜(TEM)
利用TEM使患者来自尿液的囊泡可视化。从4ml尿液中分离外泌体,进行固定和包埋用于TEM。图中示出了囊泡在以下中的分布:A)HIVAN;B)局灶性节段性肾小球硬化;C)AAHIV+;和D)白种人HIV+;E)AA HIV阴性。与白种人HIV+患者和AA正常患者相比,HIVAN、FSGS和AA HIV+患者明显具有更多的囊泡群。
Ingenuity通路分析(Ingenuity Pathways Analysis)
如实施例1和2所示,AA HIV+患者的SELDI-TOF-MS峰表现出与HIVAN患者的SELDI-TOF-MS峰极度相似和与FSGS患者的SELDI-TOF-MS峰轻微相似的蛋白模式,这表明峰与HIVAN相似的的AA HIV+患者可能先倾向于发展为HIVAN。FSGS患者和AA HIV+患者的基线蛋白值(30-2000mg/dl)在相同的范围内。与HIVAN的蛋白值相似而不同于FSGS患者,AA HIV+患者中检测到的蛋白与HIVAN患者中检测到的蛋白类似。这强调了以下重要性:即,无论是否存在肾病,HIV感染仍然是HIVAN发展的主要原因;并且HIV感染之前肾脏机能不全的在先条件不是HIVAN发展的必要先决条件。
与AA HIV+患者不同,白种人HIV+患者的蛋白谱与HIVAN患者的蛋白谱完全不同。这表明,除了肾组织中肾细胞的简单感染或感染的免疫细胞的浸润之外的其它因素可能介导肾病的表达。具有使用LC-MS/MS可检测到的Nef的AA HIV+和HIVAN患者数(12/15)支持了关于表达Nef的转基因小鼠与HIVAN患者的肾脏病理学之间的相似性的早期结论,这提示Nef可能参与引起对HIV患者的肾脏损伤。这可以解释表达Nef的转基因小鼠和HIVAN患者的肾脏病理学之间的相似性与AA HIV+和HIVAN患者的蛋白谱中Nef表达之间的相似性的关系,如果存在关系的话。这还为Nef在HIVAN的病理学中所起的作用提供了更多的启示。通过LC-MS/MS还在HIVAN和AA HIV+患者中检测到HIV包膜gp。尽管肾脏的局部HIV感染的暗示可能超出了HIVAN发展,但由于肾脏作为潜在的病毒贮库,所以可以推断该贮库中这些病毒的一部分通过一定途径进入尿液。
透射电镜(图7)表明AA HIV+、FSGS和HIVAN患者的尿液中具有明显的囊泡,而在白种人HIV+和AA正常患者的尿液中却不明显。由于HIVAN外泌体溶液的最初的图像显示出极密的外泌体群而难以视觉分辨,所以将其稀释了10倍,这表明HIVAN患者产生囊泡的速度可能高于所有其他患者组。HIV相关的肾脏损伤可能是AA HIV+和HIVAN患者中外泌体分泌明显增加的原因。
实施例4:Western印迹分析,HIV Nef和其他HIV蛋白存在的验证
使用超滤法分离来自14名HIV+AA患者和9名HIV+白种人患者的尿液囊泡样品,并使用Western印迹分析来分析HIV Nef和其他HIV蛋白的存在。通过Western印迹,所有HIV+AA样品是HIV Nef阳性的,尽管没有通过质谱在样品41中检测到HIV Nef(图8)。该差异可能是由于质谱分析所利用的分离方法造成的,所用的分离方法为超速离心法,得到较少蛋白。仅在4名HIV+白种人患者中鉴定出HIV Nef,而质谱鉴定出3个没有HIV Nef的样品。所有HIV+患者都具有通过Western印迹可检测出的HIV蛋白,但是HIV蛋白的种类和量不同(图8)。
以上描述是为了教导本领域普通技术人员如何实践本发明,并不意欲详述本发明的所有显而易见的修改和变化,这些修改和变化在本领域技术人员阅读本说明书后会变得明显。然而,意欲将所有显而易见的修改和变化包含在由随后的权利要求所定义的本发明的范围内。除非文中具体指出相反,实施方案意欲涵盖按照任何顺序的组分和步骤,只要能有效实现所要实现的目的。
Claims (20)
1.通过检测外泌体中的HIV相关生物标志物蛋白来检测哺乳动物中HIV感染或监测哺乳动物中HIV感染进展的试剂盒,包含:
用于制备检测用外泌体的一种或多种试剂,其中所述外泌体分离自所述哺乳动物的尿液样品;
用于从所述尿液样品中分离外泌体的离心滤器,所述滤器具有小于外泌体的大小的孔径,其中所述外泌体为50-90nm的囊泡;和
至少一种标准品,
其中所述标准品是HIV相关生物标志物蛋白。
2.如权利要求1所述的试剂盒,其中所述HIV相关生物标志物蛋白选自Nef、HIV包膜gp120、HIV蛋白酶、Vif、Gag-Pol、Gag、p24、Rev、逆转录酶、Tat、p1、p17、Vpu、Vpr、gp41和DNA聚合酶。
3.如权利要求1所述的试剂盒,其中所述HIV相关生物标志物蛋白是Nef、HIV蛋白酶、Vif、Pol或Gag。
4.如权利要求1所述的试剂盒,其中所述HIV相关生物标志物蛋白是Nef。
5.如权利要求1所述的试剂盒,其中所述哺乳动物是人、猴、大猩猩或狒狒。
6.如权利要求5所述的试剂盒,其中所述哺乳动物是人。
7.如权利要求1所述的试剂盒,还包含用于通过一种或多种选自以下的技术来检测所述外泌体样品中HIV相关生物标志物蛋白的一种或多种试剂:电泳、Western印迹、HPLC、FPLC、质谱和蛋白质测序。
8.如权利要求1所述的试剂盒,还包含用于通过Western印迹来检测所述外泌体样品中HIV相关生物标志物蛋白的一种或多种试剂。
9.如权利要求1所述的试剂盒,还包含用于通过SELDI-TOF-MS或LC-MS/MS来检测所述外泌体样品中HIV相关生物标志物蛋白的一种或多种试剂。
10.如权利要求1所述的试剂盒,其中所述离心滤器的分子量截留值为500kd至50kd。
11.如权利要求1所述的试剂盒,其中所述离心滤器的分子量截留值为100kd。
12.如权利要求1所述的试剂盒,还包含标签、说明书或二者。
13.如权利要求1所述的试剂盒,还包含外泌体重悬溶液。
14.如权利要求13所述的试剂盒,其中所述重悬溶液包含缓冲剂或蛋白酶。
15.如权利要求1所述的试剂盒,其中所述分离为离心分离。
16.如权利要求15所述的试剂盒,其中所述离心分离为将所述尿液样品以100,000g或更高速离心10-120分钟。
17.如权利要求15所述的试剂盒,其中所述离心分离为将所述尿液样品以100,000g离心60-120分钟。
18.如权利要求15所述的试剂盒,其中所述离心分离为将所述尿液样品以100,000g离心60分钟。
19.如权利要求15所述的试剂盒,其中所述离心分离为将所述尿液样品以5,000-25,000g离心5-30分钟,将上清转移至另一管,将转移的上清以100,000g或更高速离心30-120分钟。
20.如权利要求15所述的试剂盒,其中所述离心分离为将所述尿液样品以20,000-22,000g离心15分钟,将上清转移至另一管,将转移的上清以100,000g离心30-90分钟。
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CA2738666A1 (en) | 2010-04-15 |
EP2331969A2 (en) | 2011-06-15 |
CA2738666C (en) | 2018-05-15 |
EP2331969A4 (en) | 2012-05-16 |
WO2010042425A2 (en) | 2010-04-15 |
CN102171574B (zh) | 2016-09-21 |
CN102171574A (zh) | 2011-08-31 |
AU2009302612B2 (en) | 2016-07-14 |
US9581596B2 (en) | 2017-02-28 |
AU2016244203B2 (en) | 2018-06-21 |
US20100086956A1 (en) | 2010-04-08 |
CN106383227A (zh) | 2017-02-08 |
EP2331969B1 (en) | 2014-11-26 |
US20120214151A1 (en) | 2012-08-23 |
US20120208175A1 (en) | 2012-08-16 |
WO2010042425A3 (en) | 2010-07-22 |
AU2016244203A1 (en) | 2016-11-03 |
US9541554B2 (en) | 2017-01-10 |
AU2009302612A1 (en) | 2010-04-15 |
AP2011005665A0 (en) | 2011-04-30 |
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