CN1243568C - 预防人类沙眼衣原体感染的重组蛋白及其用途 - Google Patents
预防人类沙眼衣原体感染的重组蛋白及其用途 Download PDFInfo
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Abstract
本发明提供了一种重组蛋白疫苗，它是由卡介苗热休克蛋白65和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位相连接而形成的融合蛋白；编码这种重组蛋白疫苗的氨基酸序列和核苷酸序列；含有该核苷酸序列的表达载体；含有该表达载体的宿主细胞，以及该重组蛋白疫苗的制备方法。本发明的融合蛋白在应用于人体后可有效地预防沙眼衣原体感染。
Description
技术领域
本发明涉及一种基因工程领域，具体地涉及基因工程重组蛋白疫苗(下文有时也称为“基因工程重组蛋白”)，特别是涉及一种预防人类沙眼衣原体感染，尤其是预防泌尿生殖系统感染的重组蛋白疫苗；编码这种重组蛋白疫苗的核苷酸序列(下文有时也称为“基因”)；含有该核苷酸序列的表达载体；含有该表达载体的宿主细胞，以及该重组蛋白疫苗的制备方法；本发明还涉及该基因工程重组蛋白在制备用于预防人类沙眼衣原体感染的疫苗制品中的用途以及含有该基因工程重组蛋白的疫苗制品。
背景技术
衣原体是不运动的专营细胞内寄生的微生物，它有独特的生活周期：有原生小体(elementary body，下文有时简称为“EB”)和原始小体(initial body或网状体reticulate body，下文有时简称为“RB”)两个发育阶段。EB具有感染能力，吸附于宿主细胞，被宿主细胞膜吞噬入胞浆内。大约感染10小时后，EB开始分化，增大变为RB，RB是衣原体的增殖型，具有增殖能力。RB行二分裂法增殖，经反复分裂，形成包涵体，包涵体内充满了EB，RB和中间体。大约2-4天，RB停止分裂，包涵体内充满了以EB为主的衣原体小集落，包涵体膜和宿主细胞膜先后破裂，EB被释放到寄生宿主外，再感染其他细胞，开始新的生活周期。
沙眼衣原体(Chlamydia Trachomatis，下文有时简称为“CT”)根据感染宿主的不同可分为小鼠生物型和人生物型，小鼠生物型可致鼠肺炎而与人类无关，而人生物型有15个血清型A、B、Ba、C、D、E、F、G、H、I、J、K、L1、L2、L3，其都是以人类作为唯一宿主的病原体。
A、B、Ba和C的感染可导致沙眼，人类可通过反复感染沙眼衣原体，出现严重的角膜血管翳和形成瘢痕，这种损伤可引起睑内翻倒睫，角膜浑浊加重导致失明。沙眼是世界上致人类失明的最主要的原因。
D～K可导致包涵体性结膜炎、泌尿生殖系统疾病，其泌尿生殖道感染是性传播疾病，流行病学调查显示，美国每年有300-400万受感染人群。由于衣原体本身具有不易感染鳞状上皮细胞而易侵犯柱状上皮细胞的特性，宫颈表皮为柱状上皮细胞，因而宫颈为CT感染的常见部位，进而CT沿柱状上皮上行，导致子宫内膜炎，输卵管炎，盆腔炎，并可造成输卵管性不孕及输卵管异位妊娠，严重威胁妇女健康。
孕期对CT易感性的升高导致孕妇感染率为20-30％，而CT通过垂直传播又可导致胎儿感染。有学者报道，胎儿通过感染的宫颈，约50-70％成为CT感染新生儿，患有CT包涵体性结膜炎和肺炎。
性病淋巴肉芽肿(LVG)主要也是通过性接触传播。男性以腹股沟横痃为常见，女性以肛门直肠淋巴结肿多见，病变逐步扩及其他淋巴结，有的淋巴结肿胀化脓、穿孔形成瘘管、晚期病变为外生殖器橡皮肿及直肠狭窄。因此，CT感染的危害性是相当大的，正因为如此，沙眼衣原体疫苗的研制显得尤为重要和迫切。
经过几十年的研究，一直无理想的疫苗问世，其部分原因是由于介导生殖道粘膜抗衣原体感染的宿主的免疫机制尚不完全明了。但根据基因敲除鼠及T细胞克隆的一些实验提供证据表明CD4+T细胞在对衣原体生殖道感染的保护中起主要作用，而CD8+T细胞和体液免疫的作用则次要一些。上述观点在这一研究领域已取得共识。
目前沙眼衣原体疫苗的研究热点集中在以下几个方面：
DNA疫苗(DNA vaccine)，沙眼衣原体DNA疫苗目前还存在很多问题如：仅能诱导部分保护，Th1免疫反应和抗体反应均较弱。
减毒活疫苗(live attenuated chlamydia vaccine)，Hua Su等(Hua Su，Qonald Messer，William Whitmire等，雌性小鼠生殖道的亚临床衣原体感染产生强烈的保护免疫反应：减毒活疫苗菌株开发的结论(Subclinical Chlamydial Infection of the Female MouseGenital Tract Generates a Potent Protective Immune Response：Implications forDevelopment of Live Attenuated Chlamydial Vaccation Strains)，感染与免疫(Infectionand Immunity)，January 2000年，1月，P.192-196，vol.68.)用沙眼衣原体感染后用少量土霉素干涉治疗，使小鼠处于轻度感染的亚临床状态，以达到类似于减毒活疫苗的效果，其实验结果表明与自然感染相比，这种轻度感染后机体产生良好的保护性免疫，由此推断减毒活疫苗可能具有非常好的前景。但事实上，人们对沙眼衣原体的基因系统尚不完全明了，无法实现减毒突变(attenuating mutaion)，因此目前这种疫苗可能会导致严重的自身免疫或免疫病理反应。
DC(Dendritic Cells)细胞的回输(chlamydia-pulsed DC)，Hua Su等用热灭活的衣原体-脉冲DC(Heat-killed chlamydia-pulse DC)自体回输的办法，发现产生非常好的免疫效果(Hua su，Ronald Messer，William，等，1998.用使用非活性的衣原体体外装载的树状细胞免疫后的抗衣原体腔道感染的接种(Vaccination againstChlamydial Tract Infection after Immunization with Dendritic Cells Pulsed Ex Vivo withNonviable Chlamydiae).实验医学杂志(J.Exp.Med.)Volume 188，Number 5，September7，1998809-818)。就目前来讲，用这种方法来实现免疫保护是不现实的，但它提示非活菌感染的免疫一样可以实现免疫保护。
表位疫苗(Epitopes vaccine)，主要外膜蛋白(MOMP)是沙眼衣原体的最主要的一种外膜蛋白，很多实验表明沙眼衣原体的MOMP能产生一定的保护性的免疫。在MOMP中已发现了20多个具有免疫原性的表位(Linette Ortiz，Karen P.Demick，JeanW.Petersen等，1996.激活来自感染的人的II型HLA-限制性T细胞的沙眼衣原体主要外膜蛋白(Chlamydia trachomatis Major Outer Membrane Protein(MOMP)EpitopesThat Activate HLA Class II-Restricted T Cells from infected Humans)，免疫学杂志(TheJournal of Immunology)，1996，157：4554-4567.)，从而为表位疫苗的设计奠定了一定的基础。
目前的工作核心是如何提高疫苗的免疫原性和保护能力，延长免疫保护的时间。将热休克蛋白65与已发现的MOMP上的一些表位相连为达到上述目的提供了一种可能性。
热休克蛋白(heat shock protein，下文有时简称为“HSP”)是存在于多种生物体内的一个分子伴侣蛋白质家族。在免疫应答的过程中，热休克蛋白可表现如下三种基本的功能：1、协助抗原性物质进入包括树突状细胞在内的抗原提呈细胞；2、在抗原提呈细胞中和加工处理的抗原物质相互作用使之进入MHC I类抗原提呈途径，进而激活抗原特异性CTL；3、刺激树突状细胞表达协同刺激分子(如B7等)和分泌细胞因子等(Suzue K，Zhou X，Eisen HN，Young RA.Heat shock fusion proteins asvehicles for antigen delivery into the major histocompatibility complex class I presentationpathway.Proc.Natl.Acad.Sci.U.S.A.1997 Nov 25；94(24)：13146-13151)。由于具有上述特点，将热休克蛋白65与已发现的MOMP上的一些表位相连形成融合蛋白以达到提高免疫原性和保护能力，以及延长免疫保护的时间的目的。
发明内容
本发明要解决的技术问题是提供一种由卡介苗热休克蛋白65和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位的多肽融合形成的基因工程重组蛋白疫苗，它们对人沙眼衣原体感染有预防作用。
本发明之二是提供一种编码这种重组蛋白的氨基酸序列。
本发明之三是提供一种编码这种重组蛋白的核苷酸序列。
本发明之四是提供一种含有该核苷酸序列的表达载体。
本发明之五是提供一种含有该表达载体的宿主细胞。
本发明之六是提供一种制备该重组蛋白疫苗的方法。
本发明之七涉及该基因工程重组蛋白在制备用于预防人类沙眼衣原体感染的疫苗制品中的用途。
本发明之八涉及含有该基因工程重组蛋白的疫苗制品。
本发明提供了一种重组蛋白疫苗，它是由卡介苗热休克蛋白65和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位相连接而形成的融合蛋白，其中，所述的能激活人T细胞的沙眼衣原体主要外膜蛋白的表位的多肽具有SEQ ID No：2所示的氨基酸序列。在本发明的重组蛋白疫苗中，卡介苗热休克蛋白65可以位于该融合蛋白的氨基端，能激活人T细胞的沙眼衣原体主要外膜蛋白的表位位于该融合蛋白的羧基端。
本发明提供了编码重组蛋白疫苗的氨基酸序列，其可以具有选自如下的任一氨基酸序列：
1)SEQ ID NO：4所示的氨基酸序列；和
2)由在严紧的杂交条件下与编码1)的氨基酸序列的核苷酸序列杂交的核苷酸序列所编码的氨基酸序列。
本发明还提供了编码本发明的重组蛋白疫苗的核苷酸序列，该核苷酸序列可以具有选自如下的任一序列：
1)SEQ ID NO：3所示的核苷酸序列；和
2)由在严紧的杂交条件下与1)的核苷酸序列杂交的核苷酸序列。
能激活人T细胞的沙眼衣原体主要外膜蛋白的表位多肽的核苷酸序列如SEQ IDNO：1所示或其功能等价物。
本发明还提供了包含本发明的重组蛋白的核苷酸序列的表达载体及其宿主细胞。还提供了本发明的重组蛋白疫苗的制备方法、疫苗制品及本发明的重组蛋白疫苗在预防人类沙眼衣原体感染中的用途。
因此，正是本发明人经过长期的大量的研究，解决了现有的技术难题，首次开拓性地将卡介苗热休克蛋白65与沙眼衣原体MOMP上的一些表位相连形成了全新的基因工程重组蛋白，从而提高了抗衣原体感染的疫苗的免疫原性和保护能力，并延长了其免疫保护的时间。
另外，需要指出的是，在本申请的上下文的公开内容的基础上，本发明的其它具有实质性特点的方面和创造性的有益效果对本领域的普通技术人员来说是显而易见的。
附图说明
图1是HSP65-Chlamy构建于PET28(a+)的模式图。
图2是三轮PCR结果琼脂糖凝胶电泳照片，Line 1：DL2000 DNA marker 2：第一轮PCR 3：第二轮PCR 4：第三轮PCR。
图3是pMD18T-chlamy重组质粒用EcolRI和HindIII酶切鉴定的琼脂糖凝胶电泳照片，Line1：DL2000DNA marker 2：重组质粒的酶切鉴定。
图4是HSP65编码基因克隆入pET28(a+)的重组质粒用NcoLI和EcoRI酶切鉴定琼脂糖凝胶电泳照片：Line1重组质粒的酶切鉴定2 DL2000 DNA marker。
图5是pET28(a+)-HSP65-chlamy重组质粒用EcolRI和HindIII酶切鉴定琼脂糖凝胶电泳照片：Line1：DL2000 DNA marker 2：重组质粒的酶切鉴定。
图6是Hela单层细胞上接种CT-D 48～72小时后可见大量包涵体生长(20x)照片，白色箭头所指为沙眼衣原体包涵体。
图7是重组蛋白疫苗的表达及纯化SDS照片：1为蛋白质marker，2为目的蛋白的表达，3、4、5为目的蛋白的纯化。
图8是沙眼衣原体感染疫苗免疫后小鼠阴道组织轻度炎症照片：白色线圈内示固有层血管扩张充血，散在淋巴细胞浸润，纤维组织增生。
图9是沙眼衣原体感染后小鼠阴道组织中度炎症照片：白色线圈内示固有层血管有较多淋巴细胞浸润。
图10是沙眼衣原体感染后小鼠阴道组织3度炎症：白色线圈内示固有层血管有大量淋巴细胞浸润。
图11是证明疫苗免疫组的抗体效价明显高于PBS免疫组的用ELISA法检测的C57小鼠抗体效价的直方图：1：空白对照；2：PBS免疫；3：疫苗免疫；标本1-10。
具体实施方式
在本发明的上下文中，所使用的术语除非另外说明，一般具有本领域的普通技术人员通常理解的含义。特别地，下列术语具有如下的含义：
“重组蛋白疫苗”是指疫苗来源于通过基因工程的方法在原核或真核细胞中表达的蛋白质。
“卡介苗热休克蛋白65”是指来源于卡介苗的分子量为65kDal的热休克蛋白，其氨基酸序列如SEQ ID NO：4中氨基酸残基1-546所示。
“能激活人T细胞的沙眼衣原体主要外膜蛋白的表位多肽”是指来源于沙眼衣原体主要外膜蛋白的9个表位相连接而形成的多肽，其氨基酸序列如SEQ ID NO：2所示。
“人沙眼衣原体感染”是指沙眼衣原体通过某种途径进入人体并感染人的多种器官(如泌尿生殖系统、呼吸系统、眼睑结膜等)的状态，包括急性感染和慢性感染以及无症状携带状态。
“严紧的杂交条件”是指为避免反应体系中非同源性或部分同源性的核酸序列形成杂交复合物而采用的杂交条件，如较高的反应温度和低离子强度。
″治疗″或″预防″包括：
(1)预防疾病，也就是使疾病的临床症状不会在哺乳动物中发展，所述的哺乳动物可能与该疾病的病原体接触或易患有该疾病但不曾经历或显现出疾病的该症状，
(2)抑制疾病，也就是阻止或减轻该疾病或其临床症状的发展，或
(3)缓解疾病，也就是引起疾病或其临床症状的退化。
本发明提供了一种重组蛋白疫苗，它是由卡介苗热休克蛋白65和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位相连接而形成的融合蛋白，其中，所述的能激活人T细胞的沙眼衣原体主要外膜蛋白表位的多肽可为任何能激活人T细胞的主要外膜蛋白表位的氨基酸序列，优选地具有SEQ ID NO：2所示的氨基酸序列或其功能等价物。在本发明的重组蛋白疫苗中，卡介苗热休克蛋白65可为来源于卡介苗的分子量为65kDal的热休克蛋白，其氨基酸序列优选地为SEQ ID NO：4所示氨基酸残基1-546，或其功能等价物。其可以位于该融合蛋白的氨基端，能激活人T细胞的沙眼衣原体主要外膜蛋白的表位位于该融合蛋白的羧基端。
本发明的重组蛋白疫苗具有选自如下的任一氨基酸序列：
1)SEQ ID NO：4所示的氨基酸序列；和
2)由在严紧的杂交条件下与编码1)的氨基酸序列的核苷酸序列杂交的核苷酸序列所编码的氨基酸序列。
本发明还提供了编码本发明的重组蛋白疫苗的核苷酸序列，该核苷酸序列可以具有选自如下的任一序列：
1)SEQ ID NO：3所示的核苷酸序列；和
2)由在严紧的杂交条件下与1)的核苷酸序列杂交的核苷酸序列。
能激活人T细胞的沙眼衣原体主要外膜蛋白的表位多肽的核苷酸序列如SEQ IDNO：1所示或其功能等价物。
卡介苗热休克蛋白65是一种来源于卡介苗的蛋白质，它和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位多肽相连融合形成的融合蛋白(序列如SEQ ID NO：4所示)在进入人体后均可刺激人体DC细胞上调MHC(I类和H类)和共刺激分子(B7.2)的水平，分泌细胞因子，卡介苗热休克蛋白65还有一独特之处，它可以协助与它形成融合蛋白表位多肽进入包括树突状细胞在内的抗原提呈细胞，并进入MHC I类途径加工提呈，进而激活抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)对病原菌进行特异性攻击和杀伤，而且诱导这种CTL无须借助于外源佐剂或CD4+细胞的参与。
本发明的重组蛋白疫苗可通过本领域已知的方法，例如按照Molecular Cloning一书(J.Sambrook，Cold Spring Harbor Laboratory Press，Molecular cloning，1989)所述进行生产，以下的实施例详细地例举了一种生产本发明的重组蛋白疫苗的方法。
因此，本发明还提供了含有上述编码本发明的重组蛋白疫苗的核苷酸序列的表达载体；含有该表达载体的宿主细胞，其可以为本领域常规的各种原核细胞，真核细胞或哺乳动物细胞；以及该重组蛋白疫苗的基因工程制备方法。
另外，本发明还涉及该基因工程重组蛋白在制备用于预防人类沙眼衣原体感染的疫苗制品中的用途以及含有该基因工程重组蛋白的疫苗制品。本领域技术人员可以理解的是，这些疫苗制品可用本领域周知的各种常规方法制备。
本发明的重组蛋白疫苗可通过皮下注射的方式给人接种，接种的剂量为100-500μg。为了加强效果，可进行2-3次的加强免疫。时间间隔可为2周-2月。
下面结合具体的制备实施例和生物学效果实施例，并参照附图进一步详细地描述本发明。应理解，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。
实施例
在如下实施例中，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，例如Molecular Cloning一书(J.Sambrook，Cold Spring HarborLaboratory Press，Molecular cloning，1989)所述的方法。
在如下实施例中，所用试剂的来源、商品名和/或有必要列出其组成成分者，均只标明一次。在其后所用相同试剂如无特殊说明，不在赘述上述内容。
实施例1 表位基因的人工构建
将主要外膜蛋白上所选定的表位基因串连在一起，并在氨基端构建EcoRI的酶切位点，在羧基端构建HindIII的酶切位点，获取可激活人T细胞的沙眼衣原体主要外膜蛋白表位的编码基因(以下称之为“chlamy”)。
具体方法是：通过3轮PCR循环对所选定的表位基因进行人工构建。引物3与引物4互为模板及引物，进行第一轮PCR；再以第一轮PCR产物为模板，以引物2及引物5为引物，进行第二轮PCR；再以第二轮PCR产物为模板，以引物1及引物6为引物，进行第三轮PCR。
引物序列如下：
primer1(chlamy-1)
5’GAATTCCCGGCATACGGTCGTCATATGCAGGATGCTGAGATGTTCACCAACGCTGCTTGCATGGCT3’
primer2(chlamy-2)
5’AACGCTGCTTGCATGGCTCTCAACATTTGGGACGAGCTCAACGTACTGTGCAACGCTGCTGAGTTT 3’
primer3(chlamy-3)
5’TGCAACGCTGCTGAGTTTACCATTAACAAGCCGAAAGGTTACGTAGGCAAAGAATTTCCGCTGGCTCTG 3’
primer4(an-chlamy-3)
5’CTGCCATTCGTGGTAGTCGATAGATGCGTCCTTAGTACCGGTAGCTGCGTCCAGAGCCAGCGGAAATTC 3’
primer5(an-chlamy-2)
5’GATGTACGGGGTAAACATGTTCAGACGATAAGACAGTGCCAGAGAAGCCTGCCATTCGTGGTAGTC 3’
primer6(an-chlamy-1)
5’AAGCTTGTAGGTGTCTGCGTCGAAAGAAGCACGAGACCATTTAACACCGATGTACGGGGTAAACAT 3’
由引物3和引物4互为模板和引物进行第一轮PCR反应如下：
在一个500ul微量离心管中加入下列试剂：
引物32.5ul(10u mol/L)
引物42.5ul(10u mol/L)
10×PCR缓冲液(TakaRa公司，成分见产品说明)5μl
dNTPs(10mmol/L)1μl
Taq DNA聚合酶(TakaRa公司)(5u/μl)0.5μl
加去离子水至终体积50μl
混合后加入矿物油3滴
反应条件：94℃，1min；55℃，1min；72℃，1min，30个循环周期后，72℃延伸10min。形成第一轮PCR产物(产物1)如下：(未写出其互补链)
TGCAACGCTGCTGAGTTTACCATTAACAAGCCGAAAGGTTACGTAGGCAAAGAATTTCCGCT
GGCTCTGGACGCAGCTACCGGTACTAAGGACGCATCTATCGACTACCACGAATGGCAG
第二轮PCR循环以产物1为模板，分别以引物2和引物5为5′端引物和3′端引物，反应体系如下：
产物1 1ul
10(PCR缓冲液(含氯化镁)5μl
dNTPs(10mmol/L)1μl
引物2和引物5各2.5μl
Taq DNA聚合酶(5u/μl)0.3μl
加去离子水至终体积50μl
混合后加入矿物油3滴
反应条件：94℃，1′；55℃，1′；72℃，1′，30个循环周期后，72℃延伸10min。形成第二轮PCR产物(产物2)如下：(未写出其互补链)
AACGCTGCTTGCATGGCTCTCAACATTTGGGACGAGCTCAACGTACTGTGCAACGCTGCTGA
GTTTACCATTAACAAGCCGAAAGGTTACGTAGGCAAAGAATTTCCGCTGGCTCTGGACGCAG
CTACCGGTACTAAGGACGCATCTATCGACTACCACGAATGGCAGGCTTCTCTGGCACTGTCTT
ATCGTCTGAACATGTTTACCCCGTACATC
第三轮PCR循环以产物2为模板，分别以引物1和引物6为5′端引物和3′端引物，反应体系如下：
产物2 1ul
10×PCR缓冲液(含氯化镁)5μl
dNTPs(10mmol/L)1μl
引物1和引物6各2.5μl
Taq DNA聚合酶(5u/μl)0.3μl
加去离子水至终体积50μl
混合后加入矿物油3滴
反应条件：94℃，1min；55℃，1min；72℃，1min，30个循环周期后，72℃延伸10min。形成第三轮PCR产物(终产物)SEQ ID No：1。
三轮PCR产物的琼脂糖凝胶电泳结果见附图2。
实施例2 表位基因的TA克隆
采用TA克隆方法(J.Sambrook，Cold Spring Harbor Laboratory Press，Molecular cloning，1989)克隆PCR产物，TA克隆的载体为PMD 18-T Vector(TakaRa公司)。具体方法如下：
一DNA的回收：
1.将第三轮PCR产物用2％琼脂糖凝胶电泳(1×TAE，150-200mA，0.5小时)；
2.从琼脂糖凝胶上切下含DNA片段的凝胶，放入一离心管中；
3.加入3倍体积的溶胶液(北京鼎国公司)，45-55℃水浴5-10min使胶完全融化；
4.加入10ul玻璃奶(北京鼎国公司)，轻弹管底混匀，然后在45-55℃水浴5-10min，期间每2-3min混匀一次；
5. 5000g离心60sec，弃上清；
6.加400ul漂洗液，轻弹管底混匀玻璃奶，然后同上离心，弃上清；
7.再加入400ul漂洗液，轻弹管底混匀玻璃奶，然后同上离心弃上清，并用加样器尽量除净漂洗液；然后室温晾干玻璃奶；
8.加10-30ul无菌双蒸水将玻璃奶悬浮起来，45-55℃水浴5-10min；
9. 10000g离心2min，回收上清备用。
二连接反应：
1.取3ul上清液电泳，并在紫外灯下观察电泳带，与marker相比较，估计回收DNA浓度；
2.连接反应体系：
Insert DNA(序列参见SEQ ID NO：1)0.05-0.3pmol
pMD18-T Vect(TakaRa公司) 0.5-1ul
Solution I(TakaRa公司) 5ul
DH2O up to 10ul
16℃反应1h
三转化：感受态细胞的制备方法是：将大肠杆菌JM109(Novagen公司)在LB琼脂培养基上划线，37℃培养12-16h；次日从琼脂平板上取一单菌落于2ml LB培养基中，37℃以225r/min速度震荡培养12-16h；取1ml上述培养物接种于100ml LB培养基中，37℃以225r/min的速度震荡培养直至OD值为0.5左右(大约3h)；将菌液冰浴2h，然后2,500g，4℃离心20min收集菌体；加入100ml冰冷的Trituration缓冲液(100mmol/L CaCl2，70mmol/L MgCl2，40mmol/L醋酸钠，pH5.5)，混匀，置冰上45min；1,800g，4℃离心10min，弃上清，加入10ml冰冷的Trituration缓冲液悬浮细胞；按每份200ul分装，4℃可保存1-2周。若需长期保存，可加甘油至终浓度为15％，置-70℃备用。
转化的方法是：将200ul感受态细胞置冰上融化，然后加入3ul DMSO或β-巯基乙醇，混合后，加入10μl连接反应液(含重组质粒)，温和混匀，置冰上30min；42℃45sec，然后迅速放回冰中1-2min；加入2ml LB培养液，37℃以225r/min的速度振荡培养1h；4,000g离心10sec，弃上清，用200ul LB培养液重悬菌体；将菌液铺于含有适当抗生素的LB琼脂培养板上，涂匀，室温放置20-30min，倒置于37℃孵箱中培养12-16h。
四鉴定：用限制性内切酶消化的方法初步鉴定重组克隆。并由北京鼎国生物技术发展中心进行DNA的测序。测序结果无误(具体序列参见SEQ ID No.1)。重组的pMD-18-chlamy质粒用EcoRI和HindIII酶切后释放出chlamy(300bp)片段见附图3。
五菌种及保存：重组质粒冰冻保存于-20℃。含重组质粒的菌株在含20％甘油培养液中保存于-20℃或-70℃。
实施例3
获取结核分枝杆菌65KD热休克蛋白(BCG HSP65)的编码基因并将其克隆入pET28(+)表达载体
卡介苗结核分枝杆菌来源于长春生物制品研究所。采用苏通马铃薯培养基(Starch Potato Code NO C250-1北京鼎国生物)培养卡介苗结核分枝杆菌，培养的温度为37-39℃，生长出的卡介苗结核分枝杆菌呈现干皱浅黄色的菌膜。收集菌膜，从中提取卡介苗结核分枝杆菌基因组DNA。
提取结核分枝杆菌基因组DNA的方法参照Molecular Cloning一书(J.Sambrook，从哺乳动物分离高分子量DNA(Isolation of high-molecular-weight DNA from mammalian cells)，9.16-9.22，Cold Spring HarborLaboratory Press，Molecular cloning，1989)。
通过PCR自结核分枝杆菌分离热休克蛋白65(HSP65)结构基因。5′端引物序列为5′CCATG GCC AAG ACA ATT GCG3′，3′端引物序列为5′GAA TTC GCT AGC CAT ATG GAA ATC CAT GCC ACC CAT 3′。
所述PCR操作程序是：在一个500μl微量离心管中加入下列试剂：模板cDNA 5μl(mmol/L)
10×PCR缓冲液(含氯化镁)5μl
dNTPs(10mmol/L)1μl
5′端和3′端引物(0.01mmol/L)各0.5μl
Taq DNA聚合酶(5u/μl)0.25μl
加去离子水至终体积50μl
混合后加入矿物油3滴
反应条件：94℃，30sec；55℃，1min；72℃，2min，30个循环周期后，72℃延伸10min。
采用TA克隆方法克隆PCR产物，DNA的提取、连接及转化方法见实施例2
用限制性内切酶NcoI(TakaRa公司)和EcoRI(TakaRa公司)消化含HSP65的重组pMD18-T载体和pET28a(+)(Novagen公司)，反应体系如下：
7μl质粒DNA
1μl 10×缓冲液(TakaRa公司)
1ul 0.1％BSA(TakaRa公司)
0.5μl限制性内切酶NcoI(10单位/μl)
0.5μl限制性内切酶EcoRI(10单位/μl)
37℃4h
提取HSP65DNA插入片段和酶切后的pET28a(+)线性载体(方法见实施例2。在电泳后紫外灯下估算这两种DNA的浓度。建立连接反应体系如下：
pET28a(+)线性质粒DNA 5ul
HSP65DNA插入片段3ul
10×T4DNA连接酶缓冲液1ul
T4DNA连接酶1ul 16℃12h
连接后的转化方法见实施例2。具体序列参见SEQ ID No：3(1-1638bp)。酶切鉴定结果见附图4。
实施例4 构建卡介苗热休克蛋白65-可激活人T细胞的沙眼衣原体主要外膜蛋白表位多肽融合蛋白的基因
提取插入了HSP65基因的表达载体pET28a(+)质粒(见实施例3)，并用限制性内切酶EcoRI和HindIII(TakaRa公司)消化该重组质粒和插入了可激活人T细胞的沙眼衣原体主要外膜蛋白表位基因(chlamy)的pMD18-T(见实施例2)，反应体系如下：
8μg质粒(重组HSP65的pET28a或重组chlamy的pMD 18-T)
1μl 10×缓冲液(TakaRa公司)
0.5μl限制性内切酶EcoRI(10单位/μl)
0.5μl限制性内切酶HindIII(10单位/μl)
混合后37℃温育30-120min。
如上述，用同样的方法提取酶切后的线性质粒pET28a(+)和chlamy插入片段。并将二者进行连接：
连接反应：
质粒DNA(0.5μg/μl)3μl
DNA插入片段(序列参见SEQ ID NO.2)(300ng/μl)5μl
10×T4DNA酶连接缓冲液1μl
T4DNA连接酶1μl
混合后置14-16℃水浴6-12h。
将该重组pET-28a(+)质粒转化JM109大肠杆菌。方法同前。
卡介苗热休克蛋白65-可激活人T细胞的沙眼衣原体主要外膜蛋白表位多肽融合蛋白的基因的测序由北京鼎国生物技术发展中心承担，结果表明，所得到的卡介苗热休克蛋白65可激活人T细胞的沙眼衣原体主要外膜蛋白表位多肽融合蛋白的基因和设计的完全一致，具体序列参见SEQID NO：3所示。重组的pET-28a(+)-HSP65-chlamy质粒用EcoRI和HindIII酶切后释放出chlamy(300bp)片段见附图5。
实施例5
卡介苗热休克蛋白65可激活人T细胞的沙眼衣原体主要外膜蛋白表位多肽融合蛋白的基因的表达
将在实施例4中最终获得的重组质粒转化入表达菌BL21(DE3)(Novagen公司)中，单菌落的细菌接种于50ml LB培养基中，于250ml三角烧瓶中，37℃水浴震荡培养至OD600为0.6。加入IPTG使其终浓度为01mM，37℃水浴震荡培养2-3h。置三角烧瓶于冰上5min，4℃离心5min(5000g)。吸弃上清，收集细菌，立即使用或冻存。
实施例6
卡介苗热休克蛋白65可激活人T细胞的沙眼衣原体主要外膜蛋白表位多肽融合蛋白的纯化。
样品的准备
1.将细菌重悬于细胞裂解液中；
细胞裂解液：20mM Tris，0.5M NaCl
5mM imidazole 0.1mM PMSF调pH至7.9
2.加入10mM MgSO4和20ug/ml Dnase I(GIBCO公司)于细胞裂解液中，在冰上孵育30min除去DNA；
3.12000r/min离心15min收集沉淀物，然后用细胞裂解液离心洗一次；
4.将沉淀物溶解在结合缓冲液中，冰上孵育1-2h。
结合缓冲液：20mM Tris，0.5M Nacl
5mM imidazole 6M urea调pH至7.9
5.12000r/min离心15min除去絮状物。
柱的准备
1.将层析柱较准到理想的容量，用防水笔作好标记；
2.用磷酸盐缓冲液装柱，然后封住出口；
磷酸盐缓冲液：20mM phosphate，pH 7.21M NaCl
配制方法：溶液A Na2HPO4.12H2O 35.8g双馏水加到100ml
溶液B NaH2PO4.H2O 13.8g 双馏水加到100ml
溶液C将溶液B逐渐加到溶液A中，一直pH为7.2为止
3.开始加入层析介质(Ni2+-Saphrose-4-B)
4.让柱开始流动并加入更多的介质，一直到介质水平达到标记的柱水平；
5.打开出口，加磷酸盐缓冲液，弹击柱边使介质下沉，持续弹击一直到柱床高度恒定为止；
6.加更多的介质，重复第1-6步，当弹击不在改变柱床的体积时，关闭柱的出口。
7.加入磷酸盐缓冲液来充满柱，盖上柱的上盖，打开出口；
8.让柱开始以最小100个柱床体积/h流动，最短流动30min；
9.如果柱中介质的容积需要调整，关闭柱的出口，打开柱的上盖，加入更多的介质，然后应该再以最大流速冲洗柱至少30min。
用金属离子给介质充电
在螯合介质能够被用于纯化过程以前，必须用所选择的金属离子为其充电。
1.用至少5个柱床体积的双蒸水洗柱
2.将3-5个柱床体积的20mM金属离子溶液加到柱中；
50mM镍离子溶液的配制 NiSO4.6H2O 13.1g双馏水加到1000ml。
3.用5个柱床体积的洗脱缓冲液洗柱
洗脱缓冲液：20mM Tris，pH 7.9
0.5M Nacl
1M imidazole
6M urea
4.用10个柱床体积的结合缓冲液洗柱。
结合缓冲液：20mM Tris，pH7.9
0.5M NaCl
5mM imidazole
6M urea
吸附
1.将样品加到金属螯合的柱中，并以预先确定的最佳流速让其流过柱床；
2.用含16mM咪唑的结合缓冲液洗柱，一直到流出物的吸收值回到基线水平。
洗脱 用洗脱液洗脱时，会出现一个峰值，继续洗脱直到吸收值不在下降为止，用容器接取不同时段的流出液，并进行SDS电泳，判定纯化效果纯度可达98％以上。纯化后的SDS-PAGE见附图7。
咪唑洗脱液：20mM Tris，pH 7.9，0.5M NaCl，
1M imidazole，6M urea。
实施例7 衣原体培养纯化
一培养
1.在24孔板中加入Hela细胞(吉林大学新民校区基础医学院免疫教研室)1-2×105个/孔，1-2d融合成致密的单层。
培养液用IMDM(Iscove′s modified Dulbecco′s medium，GIBCO)+10％的胎牛血清(天津TBD生物技术发展中心)100ug庆大霉素/ml。
2.弃取培养液，将菌种(北京协和医院，D型沙眼衣原体)接种于细胞单层上，室温下3000r/min/h，移去用于保存菌种的蔗糖-磷酸盐运送液(简称SPG：Sucrose蔗糖75g，KH2PO4 0.52g，Na2HPO4 1.22g，glutamic acid 0.72g，H2O to 1 liter，pH7.4-7.6)。
3.加入衣原体生长液1ml IMDM+10％的胎牛血清100ug庆大霉素/ml 1ug/ml的放线菌酮。48-72h后倒置显微镜下可看到超过90％的细胞中长出衣原体，移去生长液，加入少许冷的SPG，用吸管反复吹吸将细胞移下。胞悬液存于-70℃冰箱中。
纯化(密度剃度离心法)
1.细胞悬液用超声波处理(20秒)
2.悬液离心500g 15min 4℃取上清
3.上清被平铺在8ml 35％(vol/vol)肾造影剂(76％，上海淮海制药厂)上。
35％肾造影剂65％0.01M HEPES含0.1M NaCl
4. 4℃离心43000g 1h
5.沉淀物被SPG重新悬起，集中在一起混匀。
6.混匀物被铺在不连续的密度的造影剂上。
13ml 40％，8ml 44％，5ml 52％，4℃离心43000g 1h，EB位于44/52％的造影剂界面上。
7.收集3倍体积SPG稀释，离心30000g 30min
8.沉淀物用SPG重悬，储存于-70℃。
Hela单层细胞接种沙眼衣原体48～72h后可见包涵体生长见附图6。
实施例8疫苗免疫后，小鼠生殖道衣原体感染的保护性实验
一材料
1.C57雌性小鼠(中国军事医学科学院)6-8周龄
2.D型衣原体
3.抗原HSP65及可激活人T细胞的沙眼衣原体主要外膜蛋白表位的融合蛋白(以下称”HSP65chlamy”)、PBS
二方法
1.将20只小鼠随机分两组，一组为疫苗免疫组，一组为PBS免疫组，疫苗组于0、14、21d每只鼠在四个腋窝处注射共注射50ug。而PBS组用同样的方法等体积注射PBS。
2.在第21d每只鼠注射5mg黄体酮
3.在第28d阴道接种衣原体1.5×104IFU
4.在第35d，将20只鼠的阴道、子宫、输卵管取出，进行组织病理学检查。比较疫苗免疫组与PBS免疫组的病理情况。
三病理分级标准：
0正常
1+最小反应(少量的炎症细胞)
2+轻度反应(炎症细胞增加，伴有轻度间质增厚，并扩散到周围脂肪组织)
3+中等反应(炎症细胞显著存在，伴有临近脂肪组织的清除)
4+严重反应(中度到严重的反应广泛存在，伴受影响组织的清除，伴有临近脂肪组织的多病灶坏死)。
三实验结果及结论
PBS免疫组：10例生殖系统标本卵巢和子宫内膜均正常；
阴道的炎症反应：7例为3+，3例为2+
疫苗免疫组：10例生殖系统标本卵巢和子宫内膜均正常；
阴道的炎症反应：2例为2+，8例为1+
结论：免疫了该疫苗后，小鼠的生殖系统的炎症减轻，疫苗产生了一定的保护作用。
各级炎症反应的病理切片见附图8、9、10。
实施例9 疫苗免疫后，小鼠足底实验
一材料
1.C57雄性小鼠(中国军事医学科学院)，6-8周龄
2.D型衣原体
3.抗原HSP65及可激活人T细胞的沙眼衣原体主要外膜蛋白表位的融合蛋白(以下称“HSP65chlamy”)、PBS。
二方法
1.将20只小鼠随机分两组，一组为疫苗免疫组，一组为PBS免疫组，疫苗组于0、14、21d每只鼠在四个腋窝处注射共注射50ug。而PB 组用同样的方法等体积注射P B S。
2.在第28d每只小鼠左后脚掌足底皮下注射25ulPBS，右后脚掌皮下注射25ul 2×104IFU热灭活的CT(56℃30min)。
3.在72h测量脚掌的厚度。
三实验结果及结论
1.PBS组：左脚平均厚度2.8±0.2cm，右脚平均厚度3.1±0.4
2.疫苗组：左脚平均厚度2.95±0.3，右脚平均厚度6.7±0.2
实验结果表明：疫苗免疫组的小鼠的右脚注射了CT-D后，其肿胀的程度要明显高于注射了PBS的左脚，也明显高于PBS免疫组。
实施例10 疫苗免疫后，CTL杀伤实验
一材料
1.C57雄性小鼠，6-8周龄
2.D型衣原体
3.抗原HSP65及可激活人T细胞的沙眼衣原体主要外膜蛋白表位的融合蛋白(以下称“HSP65chlamy”)、PBS
二方法
1.将20只小鼠随机分两组，一组为疫苗免疫组，一组为PBS免疫组，疫苗组于0、14、21d每只鼠在四个腋窝处注射共注射50ug。而PBS组用同样的方法等体积注射PBS。
2.条件培养基(condition medium)的制备：取一只正常小鼠，断颈处死后，酒精消毒腹部，用经过消毒处理的器具取出脾脏，放入无菌平皿中，转移入超净台，用毛玻璃研碎，用完全培养液悬浮脾细胞，纱布过滤，2500r/min，5min，除去上清，用0.83％氯化铵1ml重悬，冰上2min，2500r/min，5min，除去上清，用完全培养液悬浮脾细胞，将其移入培养瓶中，并加入25ug刀豆蛋白A(ConA)，再加入完全培养液至总体积为5ml。培养37℃24h，2500r/min，5min，收集上清即为条件培养基。
3.靶细胞的抗原装载提呈及Na2 51CrO4的标记
1)将已在100ml培养瓶中长成稀疏单层约2×106个细胞时，倒出培养液，加入无血清IMDM+10％条件培养基，培养16-24h。
2)1ml 5×106IFU热灭活的CT EBs中加入5ul的lipofectin，混匀，5min，然后加入1)中，37℃4h。
3)倒出瓶中液体，用0.25％胰酶(华美生物工程公司)消化，加入5ml完全培养液，计数，1200r/min，10min，重悬于100ul完全培养液中，转移至无菌塑料管中，并加入100ul Na2 51CrO4(含200uCi)(PerkinElmer公司)，震荡培养1h。
4)完全培养液洗3-4次。
5)用完全培养液将细胞的浓度调成1×105个细胞/ml。
4.将疫苗免疫和PBS免疫鼠的脾和淋巴节取出，研磨，脾细胞的处理如上述，并将脾和淋巴节细胞计数，调成2×107个细胞/ml。向96孔板中加入脾和淋巴细胞100ul，均设3个浓度梯度分别为2×107，1×107，5×106，设3复孔。
5.将标记好的B16细胞100ul加入含有效应细胞的孔中，并另设3复孔的自发释放(100ul的靶细胞+100ul完全培养液)和最大释放(100ul的靶细胞+100ul 2mol盐酸)。
6. 37℃ 5％CO2，12-16h。
7.将96孔培养板以150g离心5min。
8.取100ul上清，将其移至塑料管(用于γ计数)
9.用γ计数仪测定各管的cpm值。
10.杀伤率的计算：
杀伤率＝(实验孔值-自发释放孔值/最大释放孔值-自发释放孔值)*100％
三实验结果及结论
实验结果表明：疫苗免疫组的脾细胞和淋巴细胞对靶细胞具有一定的杀伤作用，在效靶比为100∶1的情况下，杀伤率平均为50-60％。而对照组的杀伤率平均为5-10％。
实施例11 ELISA检测免疫疫苗后C57小鼠抗体效价
一材料
C57小鼠(雄性6-8周龄)、衣原体CT-D、PBS、包被缓冲液1％BSA、羊抗鼠IgG-HRP(北京鼎国公司)、OPD-底物缓冲液(0.2M Na2HPO425.7ml；0.1M柠檬酸24.3ml)
二方法
1.将纯化后的衣原体置于56℃水浴中，30min。经预实验后，1∶80用包被缓冲液(Na2CO3 1.59g，NaHCO3 2.93g加水至1000ml)稀释。
2.包被：加入96孔板，100ul/孔。4℃，过夜。
3.洗涤：洗液洗板3次，5min/次。
4.封闭：加1％BSA封闭，200ul/孔。40℃，1h。
5.洗涤：洗液洗板3次，5min/次。
6.加样：将PBS免疫及疫苗免疫鼠(免疫方法同前)各10只的血清用样品稀释液做100倍稀释，100ul/孔。设复孔。40℃，30min。
7.加酶标抗体：将羊抗鼠IgG-HRP(北京鼎国生物，0.1ml，1∶1000)用洗液稀释500倍，100ul/孔。40℃，30min。
8.洗涤：洗液洗板三次，5min/次。
9.显色：新鲜配制OPD-底物缓冲液，100ul/孔。室温避光反应5-10min。
10.终止：加2mol/L H2SO4，50ul/孔。
11.测A490。
| 标本 | 空白对照 | PBS组 | 疫苗免疫组 | |||||||||
| 1 | 0.004 | 0 | 0.005 | 0.003±0.003 | 0.166 | 0.082 | 0.123 | 0.124±0.042 | 0.293 | 0.467 | 0.358 | 0.382±0.087 |
| 2 | 0.077 | 0.143 | 0.201 | 0.140±0.062 | 0.327 | 0.542 | 0.410 | 0.426±0.11 | ||||
| 3 | 0.131 | 0.122 | 0.178 | 0.144±0.03 | 0.410 | 0.412 | 0.378 | 0.4±0.019 | ||||
| 4 | 0.83 | 0.120 | 0.141 | 0.115±0.029 | 0.418 | 0.322 | 0.438 | 0.393±0.062 | ||||
| 5 | 0.119 | 0.142 | 0.09 | 0.117±0.026 | 0.342 | 0.198 | 0.201 | 0.247±0.082 | ||||
| 6 | 0.100 | 0.123 | 0.175 | 0.133±0.038 | 0.211 | 0.105 | 0.156 | 0.157±0.053 | ||||
| 7 | 0.092 | 0.108 | 0.087 | 0.096±0.01 | 0.478 | 0.412 | 0.388 | 0.426±0.047 | ||||
| 8 | 0.115 | 0.141 | 0.176 | 0.144±0.031 | 0.378 | 0.422 | 0.398 | 0.399±0.022 | ||||
| 9 | 0.092 | 0.126 | 0.242 | 0.153±0.079 | 0.412 | 0.402 | 0.423 | 0.412±0.011 | ||||
| 10 | 0.087 | 0.125 | 0.092 | 0.101±0.017 | 0.510 | 0.378 | 0.469 | 0.452±0.068 | ||||
实验结果及结论
1空白对照：0.003±0.0008；
2PBS免疫组：0.1038±0.0095；
3疫苗免疫组：0.435±0.012。
实验结果表明：疫苗免疫组的抗体效价明显高于PBS免疫组
序列表
<110>北京迪威华宇生物技术有限公司
<120>预防人类沙眼衣原体感染的重组蛋白疫苗及其用途
<130>
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Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro
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Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile
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Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro
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Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr
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Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln
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Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro
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Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu
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Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala
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gcc acc gca gcg att tcg gcg ggt gac cag tcc atc ggt gac ctg atc 480
Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile
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Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu
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Cys Asn Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly
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Lys Glu Phe Pro Leu Ala Leu Asp Ala Ala Thr Gly Thr Lys Asp Ala
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Ser Ile Asp Tyr His Glu Trp Gln Ala Ser Leu Ala Leu Ser Tyr Arg
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Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln
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Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg
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Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly
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Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala
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Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln
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Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly
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His His His
Claims (20)
1.一种重组蛋白疫苗，其特征在于，它是由卡介苗热休克蛋白65和能激活人T细胞的沙眼衣原体主要外膜蛋白的表位相连接而形成的融合蛋白，其中所述的能激活人T细胞的沙眼衣原体主要外膜蛋白的表位的多肽具有SEQ ID NO：2所示的氨基酸序列。
2.按照权利要求1所述的重组蛋白疫苗，其具有SEQ ID NO：4所示的氨基酸序列。
3.按照权利要求1所述的重组蛋白疫苗，其中，卡介苗热休克蛋白65位于该融合蛋白的氨基端，能激活人T细胞的沙眼衣原体主要外膜蛋白的表位位于该融合蛋白的羧基端。
4.编码权利要求1-3任一项所述的重组蛋白疫苗的氨基酸序列。
5.按照权利要求4所述的氨基酸序列，其具有SEQ ID NO：4所示的氨基酸序列。
6.按照权利要求4所述的氨基酸序列，其中所述的能激活人T细胞的沙眼衣原体主要外膜蛋白的表位的多肽具有SEQ ID NO：2所示的氨基酸序列。
7.按照权利要求4所述的氨基酸序列，其中所述的卡介苗热休克蛋白65具有SEQ ID NO：4所示的氨基酸序列残基1-546。
8.编码权利要求1至3中任意一项所述的重组蛋白疫苗的核苷酸序列。
9.按照权利要求8所述的核苷酸序列，其具有SEQ ID NO：3所示的核苷酸序列。
10.按照权利要求8所述的核苷酸序列，其中，所述的能激活人T细胞的沙眼衣原体主要外膜蛋白的表位的多肽具有SEQ ID NO：1所示的核苷酸序列。
11.按照权利要求8所述的核苷酸序列，其中所述的卡介苗热休克蛋白65具有SEQ ID NO：3所示的核苷酸序列1-1638bp。
12.含有权利要求8-11中任一项的核苷酸序列的表达载体。
13.按照权利要求12所述的表达载体，其是质粒。
14.含有权利要求12-13任一项所述的表达载体的宿主细胞。
15.按照权利要求14所述的宿主细胞，其为原核细胞，真核细胞或哺乳动物细胞。
16.一种制备权利要求1-3中任一项的重组蛋白疫苗的方法，包括培养如权利要求14或15所述的宿主细胞。
17.权利要求1-3中任一项所述的重组蛋白疫苗在制备用于预防人类沙眼衣原体感染的疫苗制品中的用途。
18.按照权利要求17所述的用途，其中所述的人类沙眼衣原体感染是泌尿生殖系统感染。
19.一种用于预防人类沙眼衣原体感染的疫苗制品，含有权利要求1-3中任一项所述的重组蛋白疫苗和载体或赋形剂。
20.按照权利要求19所述的疫苗制品，其中所述的人类沙眼衣原体感染是泌尿生殖系统感染。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021419779A CN1243568C (zh) | 2002-08-29 | 2002-08-29 | 预防人类沙眼衣原体感染的重组蛋白及其用途 |
| PCT/CN2003/000430 WO2004020471A1 (fr) | 2002-08-29 | 2003-06-03 | Proteine recombinee de prevention de chlamydia trachomatis humain et ses utilisations |
| AU2003246103A AU2003246103A1 (en) | 2002-08-29 | 2003-06-03 | Recombinant protein for preventing human chlamydia trachomatis and the uses thereof |
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| CNB021419779A CN1243568C (zh) | 2002-08-29 | 2002-08-29 | 预防人类沙眼衣原体感染的重组蛋白及其用途 |
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| US6225443B1 (en) * | 1999-05-19 | 2001-05-01 | Wisconsin Alumni Research Foundation | Cytotoxic T lymphocyte epitopes of the major outer membrane protein of chlamydia trachomatis |
| CN100400099C (zh) * | 2001-01-04 | 2008-07-09 | 北京迪威华宇生物技术有限公司 | 预防和治疗人前列腺癌的重组蛋白疫苗 |
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| WO2004020471A1 (fr) | 2004-03-11 |
| AU2003246103A1 (en) | 2004-03-19 |
| CN1478549A (zh) | 2004-03-03 |
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