CN108949783A

CN108949783A - 一种重组卡介苗及其应用

Info

Publication number: CN108949783A
Application number: CN201710355603.7A
Authority: CN
Inventors: 刘军; 张鹭
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2017-05-19
Filing date: 2017-05-19
Publication date: 2018-12-07
Anticipated expiration: 2037-05-19
Also published as: CN108949783B

Abstract

本发明涉及基因工程领域和结核病疫苗技术领域。本发明提供了一类包含结核分枝杆菌基因组RD4区相关编码基因的重组卡介苗。它是将含有编码结核分枝杆菌基因组RD4区蛋白基因的重组大肠杆菌‑分枝杆菌穿梭质粒转化入卡介苗中形成的重组卡介苗。所述重组卡介苗实现了RD4区基因、蛋白的表达。免疫动物后，包涵完整RD4区的重组BCG菌株安全性不会降低，包涵部分RD4区(Rv1501‑Rv1508c)的重组BCG菌株安全性明显上升。包涵完整/部分RD4区基因的重组BCG菌株展示出更好的抗感染保护效果。该重组卡介苗可用于结核病的预防或治疗。

Description

一种重组卡介苗及其应用

技术领域

本发明属于基因工程疫苗和新型结核病疫苗领域。具体地说，本发明提供了一种新型的抗致病性分枝杆菌的重组卡介苗及其在预防和/或治疗由致病性分枝杆菌引起的感染中的应用。

背景技术

结核病是由结核分枝杆菌(Mycobacterium tuberculosis，Mtb)引起的呼吸道疾病，是世界范围内单病因导致的死亡率最高的传染病。据WHO统计，全球有三分之一(约20亿人)感染了结核菌，代表着活动性结核的巨大隐患。2015年全球140万人死于结核病，1040万人新增感染结核菌(其中近63％出现结核症状)。中国是全球22个结核病高危国家地区之一，结核病患者的人数在全球排名第二(仅次于印度)，耐药结核病人数为全球第一。

在全球范围内,结核病的有效控制依然面临许多困难和挑战，包括缺乏快速准确的诊断技术，缺乏有效的抗结核疫苗，和过长的结核病药物疗程(9到12个月)。而结核病和艾滋病的合并感染，以及越来越多的耐多药(MDR-TB)和广泛耐药结核(XDR-TB)的传播更加剧了结核病防控工作的严峻性。因此，急需开发安全有效的抗结核感染的新型疫苗，预计这类疫苗每年可以减少800-1000万以上新发结核病例。

卡介苗(Mycobacterium bovis BCG，BCG)是迄今为止唯一被批准使用的抗结核疫苗，1974年被世界卫生组织纳入扩大免疫接种计划(EPI，Expanded Program onImmunization)，在全球(包括中国)广泛使用。每年有超过一亿的儿童接种卡介苗，总共接种卡介苗的人数超过40亿，使其成为人类历史上最广泛使用的疫苗。尽管已经问世使用了近100年，卡介苗有两个主要的缺陷：一是卡介苗虽然对儿童的粟粒性肺结核和结核性脑膜炎有一定的保护作用，对成人肺结核的保护效果却非常有限，临床试验的结果参差不齐(0-80％)；二是卡介苗在免疫能力低下人群中的安全性问题。基于这些因素，BCG只能在特定人群、特定时期提供保护，显然已经不是一株理想的疫苗，新一代的抗结核疫苗必须比现有的卡介苗有更好的保护效果和更优的安全性，使其能在所有的人群(包括HIV感染人群)中广泛使用。

在研的重组卡介苗主要有两种，一是由UCLA的Marcus Horwitz教授实验室所构建的rBCG30，是在卡介苗中过表达抗原85B(Ag85B)的重组卡介苗。二是由德国马普研究院的Stefan Kaufmman教授构建的rBCG::ΔureC-llo+，是在卡介苗中表达李斯特菌(Listeriamonocytogenes)中的溶血素(listeriolysin O)使其在巨噬细胞中能够穿跃吞噬体(phagosome),进入细胞质(cytosol)，以增加抗原被T细胞识别的机会。前一种重组卡介苗(rBCG30)在2004年在美国完成临床一期实验后没有进一步的深入研究，学术界对于其只通过过表达一种抗原是否能起到很好的保护效果持怀疑态度。后一种重组卡介苗(rBCG::ΔureC-llo+)进入临床二期实验，但因为加入的融血素listeriolysin是李斯特菌的外毒素(toxin)，研究界对于其安全性和可推广性有疑虑。AERAS公司采用相似的策略在卡介苗中表达产气夹膜杆菌(Clostridium perfringens)的溶血素(perfringolysin O)而构建的重组卡介苗[AERAS-422:BCG(ΔureC::pfoA Rv3407+fbpB+fbpA)]在最近的临床实验中出现安全性有问题，已经停止进一步的研究工作。这些最新的研究表明，尽管这几种重组卡介苗的研发工作开展得比较早，但并不意味着它们能成功成为新一代疫苗的组分。重组卡介苗的构建仍然需要新的思路和策略。

结核分枝杆菌H37Rv的RD4区存在于MTBC的大多数成员，例如M.tuberculosis，M.africanum，M.canettii和M.microti中，是一个包含了至少11个基因(Rv1506c-1516c)的12.6kbp大小的片段。但在从阿根廷、荷兰、英国和西班牙的牛群以及人群中分离出来的常见的典型M.bovis菌株中并不存在。源自M.bovis的所有BCG菌株都不存在RD4区。申请者前期研究发现，与MTBC密切相关的鱼型分枝杆菌M.marinum的基因组中含有扩展的RD4区域，该区域包括了至少40个基因，参与脂多糖(LOS)的生物合成。因此，似乎有这样一个趋势，RD4区基因簇随着M.marinum,M.tuberculosis和M.bovis(包括BCG菌株)这样的顺序在分枝杆菌基因组中逐渐消失，可能在病原菌-宿主相互作用中发挥作用。

目前，编码RD4区的大多数蛋白的功能还是未知的，但是有研究表明，这些蛋白参与了含海藻糖的糖脂类生物合成。在M.marinum扩展的RD4区中的很多基因参与到了糖基化酰基海藻糖LOSs的生物合成。Rv1511和Rv1512分别被预测为核苷糖脱水酶和差向异构酶；Rv1516c很有可能是糖基转移酶。M.marinum中的MMAR_2327是Rv1508c的同源蛋白，参与到M.marinum中LOSs的生物合成过程。M.tb H37Rv并不合成LOSs，其细胞内也没有LOSs这种脂质，然而，用转座子突变失活Rv1503c和Rv1506c基因，破坏了M.tb中2,3-二-O-乙酰基海藻糖(2,3-di-O-aceyltrehalose)的脂质合成。Rv1503c和Rv1506c失活的M.tb突变体无法诱导吞噬细胞内吞噬泡的成熟，导致其弱毒。相反，在斑马鱼胚胎感染模型中，M.marinum体内LOSs生物合成途径中的基因被破坏，增强了M.marinum毒性。因此，RD4区在分枝杆菌毒力中的作用是不同的，而这种不同似乎具有分枝杆菌物种相关性，反映出宿主与病原体之间相互作用的复杂性。推测RD4区在BCG中的表达/过表达可能改变BCG的细胞表面的特征，促进与LOS类脂类相关的PE-PGRS蛋白的分泌，从而提高疫苗抗原的呈递和保护效果。

经检索，未见将结核分枝杆菌RD4相关区域重组到卡介苗中，构建表达或过表达相关基因的新型重组卡介苗的报道和专利申请。

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发明内容

针对现有技术的空白，本发明将包含结核分枝杆菌全部/部分RD4区的基因片段(Rv1501-1516c/Rv1501-1508c)克隆入卡介苗，在卡介苗中表达或过表达相关抗原蛋白，形成重组卡介苗。特别涉及相应的免疫原性成分、疫苗或治疗成分，用来预防和/或治疗由致病性分枝杆菌，如：结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌、溃疡分枝杆菌、海洋分枝杆菌等引起的感染。免疫原性成分或治疗成分由结核分枝杆菌Rv1501-1516c区全部或部分基因簇蛋白或其对应的编码核酸组成。

具体技术方案如下：

本发明提供一种编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列：

(1)所述核苷酸序列与SEQ ID NO.1具有至少70％，至少80％，至少90％，至少95％，至少98％，至少99％或100％同一性；或

(2)所述核苷酸序列与SEQ ID NO.2具有至少70％，至少80％，至少90％，至少95％，至少98％，至少99％或100％同一性。

本发明提供一种重组质粒，所述重组质粒是将编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列插入到大肠杆菌-分枝杆菌穿梭质粒中构成的重组质粒。

编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列可来源于结核分枝杆菌的不同菌株，例如(但不局限于)：Mycobacterium tuberculosis H37Ra，Mycobacteriumtuberculosis strain F1，Mycobacterium tuberculosis str.Erdman，Mycobacteriumtuberculosis CDC1551等等；所述核苷酸序列可通过PCR扩增或人工合成获得。

本发明提供一种重组卡介苗菌株，将包涵结核分枝杆菌全部/部分RD4区的基因片段克隆入卡介苗，在卡介苗中表达或过表达相关抗原蛋白，形成重组卡介苗菌株；可通过将上述重组质粒转化至卡介苗菌株实现。

本发明提供一种重组卡介苗菌株，所述重组卡介苗菌株名称为rBCG08c。

本发明提供另一种重组卡介苗菌株，所述重组卡介苗菌株名称为rBCG16c。

本发明提供一种重组卡介苗菌株的制备方法，包括以下步骤：

(1)扩增或人工合成编码含有结核杆菌RD4区全长或部分基因的片段；

(2)将步骤(1)获得的目的基因序列插入到大肠杆菌-分枝杆菌穿梭质粒的序列中，构建含目标基因的重组大肠杆菌-分枝杆菌穿梭质粒；

(3)将步骤(2)获得的含有目标基因的重组大肠杆菌-分枝杆菌穿梭质粒转化入卡介苗中，即得到重组卡介苗菌株。

本发明提供一种重组卡介苗疫苗，所述疫苗包含本发明所述重组卡介苗菌株以及药学上可接受的佐剂或缓冲液体系。

本发明提供一种编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列、包含上述核苷酸序列的重组质粒、和/或包含上述重组质粒的菌株在制备抗致病性分枝杆菌感染的疫苗中的应用。

本发明提供一种使用上述重组卡介苗菌株或重组卡介苗疫苗预防和/或治疗致病性分枝杆菌感染的方法。

本发明提供一组多肽，所述多肽由权利要求1所述核苷酸序列编码产生：

(1)所述多肽包含Rv1501(SEQ ID NO.17)、Rv1502(SEQ ID NO.18)、Rv1503(SEQID NO.19)、Rv1504c(SEQ ID NO.20)、Rv1505(SEQ ID NO.21)、Rv1506c(SEQ ID NO.22)、Rv1507c(SEQ ID NO.23)、Rv1507A(SEQ ID NO.24)、Rv1508c(SEQ ID NO.25)；或，

(2)所述多肽包含Rv1501(SEQ ID NO.17)、Rv1502(SEQ ID NO.18)、Rv1503(SEQID NO.19)、Rv1504c(SEQ ID NO.20)、Rv1505(SEQ ID NO.21)、Rv1506c(SEQ ID NO.22)、Rv1507c(SEQ ID NO.23)、Rv1507A(SEQ ID NO.24)、Rv1508c(SEQ ID NO.25)、Rv1508A(SEQID NO.26)、Rv1509(SEQ ID NO.27)、Rv1510(SEQ ID NO.28)、gmdA(SEQ ID NO.29)、epiA(SEQ ID NO.30)、Rv1513(SEQ ID NO.31)、Rv1514c(SEQ ID NO.32)、Rv1515c(SEQ IDNO.33)、Rv1516c(SEQ ID NO.34)。

本发明提供一种包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列或上述核苷酸序列编码的多肽在制备抗致病性分枝杆菌感染的基因工程亚单位疫苗中的应用。

本发明中的编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列随机插入到载体，如腺病毒或牛痘病毒载体中，以DNA疫苗形式直接用于人类或其他哺乳动物或动物，在体内表达抗原，导致机体对致病性分枝杆菌引起的结核感染的抗性。因此，本发明中的多肽和核酸可以构成治疗性成分，应用于人类或其他哺乳动物或动物中，来预防和/或治疗结核分枝杆菌感染。

本发明所述编码包含结核分枝杆菌RD4区基因的核苷酸序列包含自身启动子序列、编码序列和调节序列，可指导目的基因在BCG菌株中的表达；不受大肠杆菌-分枝杆菌穿梭质粒携带的启动子和调节序列的影响；同时表达的蛋白的性质和原始结核分枝杆菌自身表达的蛋白的性质完全一致。

本发明的一个实施例中，将编码结核杆菌RD4区基因(Rv1501-1516c(SEQ IDNO.2)、Rv1501-1508c(SEQ ID NO.1))的序列扩增后，插入到大肠杆菌-分枝杆菌穿梭质粒pMV306中，Rv1501-1516c片段插入位置为pMV306中SalI和NheI酶切位点之间，Rv1501-1508c片段插入位置为pMV306中XbaI和HindIII酶切位点之间。

本发明所述RD4区及相关基因的基因序列，源于美国NIH GenBank公开数据库(参考基因组在NCBI中编号NC_000962.3)。Rv1501-1508c片段位于H37Rv基因组1691890..1699894,complement之间，编码9个蛋白。Rv1501-1516c片段位于H37Rv基因组1691890..1708539,complement之间，编码18个蛋白。上述目标片段可以通过人工基因合成的方式获得，也可以通过PCR反应扩增获得。在本发明的一个实施例中，目标基因片段是从结核杆菌H37Rv菌株的基因组扩增获得。

本发明所述重组质粒制备过程中，所采用的大肠杆菌-分枝杆菌穿梭质粒可选自pSMT3，pMV206，pMV261，pMV306，pMV361，pCherry中的一种，但不限于此。大肠杆菌-分枝杆菌穿梭质粒的作用是携带外源的目的基因片段进入到卡介苗中，进一步利用大肠杆菌-分枝杆菌穿梭质粒可在卡介苗复制的能力，最终有利于编码的目标抗原在卡介苗中的表达。在本发明的一个实施例中，采用的大肠杆菌-分枝杆菌穿梭质粒是pMV306。

本发明用于制备重组卡介苗菌株的起始菌株，可以是现用于临床免疫接种的任何卡介苗菌株，如卡介苗瑞典株、卡介苗巴斯德株，卡介苗丹麦株，卡介苗哥本哈根株，卡介苗日本株，卡介苗中国株、卡介苗巴西株、卡介苗Tice株，卡介苗俄罗斯株，等等，但并不局限于上述菌株。

本发明所述致病性分枝杆菌可为结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌、溃疡分枝杆菌、海洋分枝杆菌中的一种或几种的组合，但并不局限于上述菌株。

本发明所述疫苗可用于人类、哺乳动物、或其他动物免疫。

本发明所述疫苗可包含佐剂(但不局限于所列佐剂)：DDA、TDB、Novasome、gp96、MF59等

本发明所述疫苗可使用如下缓冲体系(但不局限于所列缓冲体系)：柠檬酸缓冲液、磷酸缓冲盐溶液等。

实验结果表明，本发明提供的重组卡介苗实现了目标蛋白在BCG菌株中的表达，免疫动物后，重组卡介苗不影响或提高疫苗安全性，并显著提高疫苗的抗感染保护效果。

本发明重组卡介苗具有以下优势：

1.重组表达了所有BCG疫苗株缺失的RD4区抗原，改善了BCG缺失重要保护性抗原的缺陷。

2.重组表达的RD4区蛋白的性质不变。RD4区蛋白在重组卡介苗的表达策略是通过利用其自身的启动子和调节序列，从而不需要额外的温度或其他特殊条件的诱导表达，充分实现重组表达的RD4区蛋白与结核分枝杆菌自身的RD4区蛋白在结构和功能上的完全一致。

3.重组卡介苗安全性不变。用本发明提供的重组表达RD4区蛋白的重组卡介苗rBCG::RD4，感染T、B细胞联合免疫缺陷SCID小鼠。同时将卡介苗rBCG::306(BCG含空质粒pMV306)和PBS分别作为阳性对照和阴性对照。结果显示，新型重组卡介苗rBCG::RD4具有与现有卡介苗一致或更高的安全性。

4.重组卡介苗有利于人体应用。RD4区蛋白在重组卡介苗的表达策略是通过利用其自身的启动子和调节序列，从而不需要额外的温度或其他特殊条件的诱导表达，有利于重组卡介苗的人体应用。

5.重组卡介苗保护效果强。过表达全长或部分RD4区蛋白的新型的重组卡介苗rBCG::RD4，其免疫保护性均显著强于原始母体卡介苗。这种增强的保护性经证实，是与卡介苗重组表达RD4区蛋白密切相关。

附图说明

图1：重组卡介苗菌株的分子水平验证

图1A：重组BCG菌株基因组中目标基因的PCR验证。对分别重组了pMV306-Rv1501-1508c或pMV306-Rv1501-1516c的重组BCG菌株(BCG-Japan和BCG-China)进行PCR分析验证。两组PCR特异性引物Rv1507a(针对Rv1501-1508c)和Rv1515c(针对Rv1501-1516c)分别用来验证两个重组菌株。Rv1507a在所有重组RD4区的菌株中都可检测到。Rv1515c只在重组了RD4全长(Rv1501-1516c)的菌株中检测到。重组了pMV306的菌株作为对照组。

图1B：在重组BCG和重组M.marinum菌株中，用RT-PCR分析Rv1501、Rv1507c和Rv1516c基因的表达。

图2：利用Rv1505c多抗血清对重组卡介苗rBCG::RD4进行Western blot验证。

检测重组菌株细胞裂解液中Rv1505c蛋白的表达。1，2分别代表重组菌株的不同克隆。每个图的最下面一条是考马斯亮蓝染色，作为上样对照。

图3：重组卡介苗rBCG::RD4的安全性分析

感染了重组BCG-China菌株的SCID小鼠的生存曲线。每组SCID小鼠(n＝20)分别尾静脉感染10⁷CFU的BCG菌株，观察SCID小鼠死亡情况，并绘制其生存曲线。Log-rank test(Mantel-Cox)用来分析统计学差异。*，P<0.05；**，P<0.01。

图4：重组卡介苗rBCG::RD4免疫对感染动物存活能力的保护性分析。

免疫重组卡介苗rBCG::RD4的斑马鱼在M.marinum 535攻击下的生存曲线。每组成年斑马鱼(n＝20)分别腹腔免疫10⁴CFU的BCG菌株或PBS。免疫30天之后，进行10CFU的M.marinum 535的攻击。观察斑马鱼死亡情况，并绘制其生存曲线。*，P<0.05；**，P<0.01。

图5：重组卡介苗rBCG::RD4免疫对感染动物体内载菌量的影响

免疫重组卡介苗rBCG::RD4的斑马鱼在M.marinum 535攻击下，体内荷菌数比较。每组成年斑马鱼(n＝15)分别腹腔免疫10⁴CFU的BCG菌株或PBS。斑马鱼在免疫30天之后，进行10CFU的M.marinum 535的攻击。攻击30天后，选择6条幸存的斑马鱼进行荷菌计数。每条鱼内的荷菌数就是一个数据。统计学分析采用非参数检验(Kruskal-Wallis test)，然后进行邓恩(Dunn)的多重比较检验。*，P<0.05；**，P<0.01。两个重组BCG组的斑马鱼体内的细菌数明显低于PBS组。

具体实施方式

以下结合具体实施例对上述方案做进一步说明，并不作为对本发明的限制。

实施例1：重组质粒pMV306-Rv1501-Rv1508c和pMV306-Rv1501-Rv1516c的构建和鉴定

整合型载体pMV306用于分子克隆。分子生物学技术按照常规进行：

(1)首先构建pMV306-Rv1501-Rv1502

Rv1501-Rv1502基因利用PCR技术从结核分枝杆菌H37Rv基因组中扩增，(上游引物5'-CACTGGTCGACAATGTCACTTCATTTAGCAAC-3'(SEQ ID NO.3)；下游引物5'-CATGAAAGCTTCGAATCATTGGAACAGCGG-3'(SEQ ID NO.4))，扩增条件分别为：98℃5min，[98℃10s,(Tm-5)℃10s，72℃1min/kbp]30个循环，72℃10min。PCR产物用AxyPrep PCR产物回收试剂盒回收(Axygen)。用SalI和HindIII酶切基因片段和pMV306质粒，AxyPrep DNA凝胶回收试剂盒回收酶切片段并连接，形成重组质粒pMV306-Rv1501-Rv1502。

(2)构建pMV306-Rv1501-Rv1508c重组质粒，其步骤如下：

Rv1503-Rv1508c基因利用PCR技术从结核分枝杆菌H37Rv基因组中扩增，(引物5'CCTCGAAGCTTTCATGATACCGGTTCCATAGGTCCAATC-3'(SEQ ID NO.5),5'-TTGGCTAGCAACCGCGCGAGGTCCTC-3'(SEQ ID NO.6))，获得的片段进行HindIII和NheI双酶切，用HindIII和XbaI酶切pMV306-Rv1501-Rv1502质粒，分别回收酶切片段后连接，构建pMV306-Rv1501-Rv1508c。进行测序验证。

(3)构建pMV306-Rv1501-Rv1516c重组质粒，其步骤如下：

文献报道中的细菌人工染色体Rv264(含Rv1501-Rv1516c等区域的人工染色体)进行三酶切(HindIII、NheI和BglII)，将切下来的10K左右的片段(Rv1503-Rv1516c)进行胶回收，然后将其连接到用HindIII和XbaI酶切过的pMV306-Rv1501-Rv1502载体中去，其连接产物为pMV306-Rv1501-Rv1516c。

上述每一步都通过酶切鉴定和序列分析验证。证明构建的重组表达质粒完全正确。测序结果表明，克隆的Rv1501-Rv1516c基因与美国NIH GenBanK中分别公布的结核分枝杆菌H37Rv全基因组序列中对应的基因的编码序列完全一致。重组质粒pMV306-Rv1501-Rv1508c中的插入片段对应SEQ ID NO.1；重组质粒pMV306-Rv1501-Rv1516c中的插入片段对应SEQ ID NO.2。重组BCG菌株基因组中目标基因的PCR验证、RT-PCR验证结果见附图1。

实施例2：重组卡介苗rBCG::RD4的建立与验证

(1)卡介苗感受态细胞制备：

取对数生长期的BCG-China、BCG-Japan菌株lml，无菌接种于50ml添加了10％ADC(DIFCO，Bection-Dickinson)、0.2％甘油和0.05％Tween80的Middlebrook 7H9液体培养基(DifcoTM)中，37℃静止培养至OD600＝0.8-1.0。4℃离心收集细菌。用10％甘油重悬后，用原培养体积的1/2，1/10和1/50的甘油洗涤3次，最后用1ml预冷甘油重悬，分装为100μL每管，-80℃保存备用。

(2)卡介苗的电转化：

在标记好的0.2cm的Bio-Rad电转杯中，分别加入5μL高浓度质粒(如pMV306、pMV306-Rv1501-Rv1508c、pMV306-Rv1501-Rv1516c)和200μLBCG感受态细胞，轻柔吹吸混匀，用Bio-rad GenePulser电穿孔仪电穿。电穿参数为：电压2.5KV、电阻1000Ω、电容25μF脉冲波进行电转化，时间常数介于15-20ms。

(3)重组菌株筛选：

转化完成后，立即将细菌转移入10ml的7H9液体培养基中，37℃振荡过夜培养；次日离心收集细菌，涂布接种于添加10％OADC(DIFCO，Bection-Dickinson)和0.5％甘油的Middlebrook 7H11琼脂培养基(DifcoTM)(含25μg/ml卡那霉素)，37℃培养4周。挑取抗性生长克隆，接种于7H9液体培养基(含25μg/ml卡那霉素)37℃扩大培养4周后，分别离心收集细菌和上清，针对重组基因进行PCR、RT-PCR和Western Blotting鉴定。

(4)重组菌株验证：

提取重组卡介苗rBCG::RD4细菌基因组或者直接选取1μL裂解液作为模板，利用基因Rv1507A(针对Rv1501-1508c)和Rv1515c(针对Rv1501-1516c)的特异性引物进行PCR，结果验证了对应基因片段成功整合进入重组卡介苗rBCG::RD4基因组。含有空质粒pMV306的重组卡介苗(rBCG::306)同上制备，并作为实验对照，无目标基因扩增条带。见附图1A。对应引物如下(5’to 3’)：

Rv1507a：

Forward：TGTGCTAGC ATGCAATCAGGTCAAAATATCCTCGCC(SEQ ID NO.7)

Reverse：TGTGAGCTC TCAACCCGCTAGAAGGCCGGTG(SEQ ID NO.8)

Rv1515c：

Forward：GTTGCTAGC ATGTCGACAAACCCAGGACCAGCC(SEQ ID NO.9)

Reverse：TGGGAGCTC TCACCGGGTCTTGATACCGATGAAGG(SEQ ID NO.10)

离心收集分枝杆菌培养物(5ml，OD600＝1.0)，重悬在800μL Trizol中。细菌用磁珠震荡破碎。上清先用氯仿-异戊醇(24:1)抽提，再用异丙醇沉淀。RNA粗提取物样本用gDNAEraser和PrimeScript^TMRT reagent Kit(Takara)按照操作手册处理。合成后的cDNA作为模版，针对Rv1501，Rv1507c和Rv1516c进行了RT-PCR验证，结果表明Rv1501和Rv1507c的转录子可以在全部的重组菌株中检测到，同时Rv1516c的转录仅可以在完整重组RD4片段的菌株中被检测到。含有空质粒pMV306的重组卡介苗(rBCG::306)同上制备，并作为实验对照，无目标基因表达。见附图1B。对应引物如下(5’to 3’)：

Rv1501：

Forward：GGCGCTAGCATGATTCCTGTAAAGGTTGAAAACAATAC(SEQ ID NO.11)

Reverse：TTTCAAGAAAGGTAAAGAAATGAGGGTCATAC(SEQ ID NO.12)

Rv1507c：

Forward：TGTGCTAGCTTGAAGAAAGTCGCGATTGTTCAATC(SEQ ID NO.13)

Reverse：CGTGTGCTGTTCTTCGAGGTAAATCGGCGCG(SEQ ID NO.14)

Rv1516c：

Forward：TATAAGCTTTCCGAATCCCTTGTGAAGTAGTAATGTGCGAGC(SEQ ID NO.15)

Reverse：CGATCCAGTAGTCGTCCGCCTCGCACAACGC(SEQ ID NO.16)

抗Rv1505c鼠多克隆抗体由本实验室制备，化学发光法进行显色。结果证实重组卡介苗rBCG::RD4细菌裂解液中有分子量约25kDa的特异性蛋白表达，含有空质粒pMV306的重组卡介苗(rBCG::306)同上制备，并作为实验对照，无目标蛋白表达，参见附图2。

实施例3：重组卡介苗rBCG::RD4的安全性评价

6周龄的雌性SCID小鼠随机分组，每组20只。每只小鼠尾静脉注射100μL(1×107CFU)重组卡介苗rBCG::RD4和rBCG::306，以PBS作为阴性对照。感染后的第二天，每组处死2只小鼠，无菌分离肺脏和脾脏并计数细菌负荷数，以确定感染剂量。剩余的小鼠进行长期的观察，记录其体重变化、死亡情况，绘制小鼠生存曲线。

统计发现，野生型BCG(即BCG::pMV306)组、BCG::Rv1501-1508c组和BCG::Rv1501-1516c组的半数死亡时间分别为63、77和67.5天(附图3)。Log-rank统计分析表明BCG::Rv1501-1508c组的小鼠的生存期明显大于对应的BCG::pMV306组(P<0.01)和BCG::Rv1501-1516c组(P<0.01)。然而对照组BCG::pMV306组和BCG::Rv1501-1516c组之间并没有显著性差异。结果表明重组完整RD4区对BCG本身的安全性并没有造成显著影响，重组部分RD4区(Rv1501-1508c)则显著提高重组BCG的安全性。

实施例4：重组卡介苗rBCG::RD4免疫对感染动物存活能力的保护性分析

利用斑马鱼-M.marinum感染模型。成年斑马鱼随机分为4组(每组20条)：PBS(阴性对照组)和三组实验组(BCG::pMV306、BCG::Rv1501-Rv1508c(即rBCG08c)和BCG::Rv1501-Rv1516c(即rBCG16c))。实验组分别用10⁴CFU的BCG菌株进行腹腔注射免疫。免疫30天之后，用10CFU M.marinum 535通过腹腔注射感染，每天记录斑马鱼的生存情况。生存曲线通过Log-rank统计分析表明，未免疫PBS组、BCG::pMV306免疫组、BCG::Rv1501-Rv1508c免疫组和BCG::Rv1501-Rv1516c免疫组的半数死亡时间分别为27.5、30、45.5和54天(附图4)。在M.marinum 535的攻击下，免疫BCG::Rv1501-Rv1508c组的斑马鱼和BCG::Rv1501-Rv1516c组的斑马鱼的生存时间明显比BCG::pMV306组长很多，Log-rank统计学分析显示均存在显著性差异(*，P<0.05或**，P<0.01)。作为对照组，BCG::pMV306免疫组生存曲线与PBS组存在显著性差异(*，P<0.05)。

实施例5：重组卡介苗rBCG::RD4免疫对感染动物体内载菌量的影响

重组卡介苗免疫及M.marinum 535感染实验同实施例4。感染30天后，每组选取6条斑马鱼处死并计数鱼体内的细菌载量。结果显示：BCG::Rv1501-1508c(即rBCG08c)免疫组和BCG::Rv1501-1516c(即rBCG16c)免疫组的斑马鱼体内的M.marinum 535数量比BCG::pMV306免疫对照组分别低1.73和2.25log10CFU(附图5)。BCG::Rv1501-1516c组与BCG::pMV306对照组存在显著性差异(*,P<0.05)。BCG::Rv1501-1508c组和BCG::Rv1501-1516c组的斑马鱼体内的M.marinum 535数量显著低于PBS组(附图5)。图4、5表明，重组RD4区基因的确提高了重组BCG菌株抗感染保护效果。统计学分析方法采用非参数检验(Kruskal-Wallistest)，然后进行Dunn多重比较检验(Dunn's Multiple Comparison test)。

SEQUENCE LISTING

<110> 复旦大学

<120> 一种重组卡介苗及其应用

<160> 34

<170> PatentIn version 3.3

<210> 1

<211> 8005

<212> DNA

<213> Rv1501-Rv1508c核苷酸序列

<400> 1

atgattcctg taaaggttga aaacaatact tcgctcgatc aggtgcaaga cgctcttaat 60

tgcgtcgggt acgcggttgt agaagatgtg cttgatgagg cgtcactggc agcgacccgt 120

gatcgcatgt atcgtgtaca ggagcggatt cttaccgaga ttggcaaaga gcggctggca 180

agggccggtg agctcggtgt tcttcgactc atgatgaagt atgaccctca tttctttacc 240

tttcttgaaa tacccgaagt cctaagcatc gttgatcgtg tgctatctga aacggccatc 300

ttacatctgc agaatggctt tatccttccg tccttcccgc ccttctccac gccggacgtt 360

tttcagaatg cgttccacca agactttccc agggttctgt ccggttacat tgcctccgtc 420

aatattatgt tcgccatcga tccctttaca cgagacaccg gcgcaacgct cgtagtgccg 480

gggagccacc agcgcataga gaaaccggac catacctacc tcgcgcgcaa tgccgttccc 540

gttcaatgcg cggcgggctc gttgttcgtt tttgactcta cgctttggca tgcggctggc 600

cgaaacacct ccggcaaaga ccgcttggcc ataaatcatc agtttacgcg ctcgtttttc 660

aagcagcaga tcgactacgt ccgcgcgctg ggcgacgccg tggttctgga gcagcctgcg 720

cgtactcagc aactgctcgg atggtacagt cgagtggtta ccaatctgga cgagtattac 780

cagccgccgg acaagcgatt gtatcggaag gggcaaggct agttttgcga gaattccgtt 840

gcgcctattt gaaagcccga catgaaacga tcgcttttaa gcgcatatgt ctgttctgca 900

aaaatgtcta atttttccga taaaggttgg tgggaaagct cgatgcgtgc cgtgttttgt 960

aggtggccgg atgatccact tagacaggcc gtggaagcag aatttgcgcg tcccgatggc 1020

gttgcggtgg cgtaatggcc tggcgaaagc tcgggagaat ttttgctccg tcgggcgaac 1080

tcgactggtc gcgaagtcat gctgcgctac cggttcctga atggatcgag ggtgatattt 1140

tccgcatcta tttcagcggc cgcgatggtc agaatcgttc cagtatcggt agcgtgatcg 1200

tcgatctcgc cgtgggcggc aagattctgg acattccggc ggagccgatt ttgcgccccg 1260

gcgctcgagg aatgtttgac gactgtgggg tgtcaatcgg atcgattgtg cgtgccggcg 1320

atacgcgact tttgtactac acgggctgga atctcgctgt caccgtgccc tggaaaaaca 1380

ccataggcgt ggcgattagc gaagcaggtg caccattcga gcgatggtct acttttcccg 1440

tcgttgcgct ggacgagcgt gatccattct cgctttctta tccctgggtc atccaagatg 1500

gagggacata ccgtatgtgg tatggctcaa atctaggctg gggagagggc accgacgaga 1560

tacctcacgt gatcaggtat gcgcaatcaa gggacggtgt ccactgggaa aagcaggatc 1620

gcgtgcatat cgacacaagc ggatccgaca atagcgcggc ctgtaggccg tacgtcgtcc 1680

gcgatgcggg agtatacaga atgtggtttt gcgctcgcgg tgcgaaatat cggatttact 1740

gcgctacatc ggaggatggt ttgacttggc ggcaactcgg caaagatgag ggcatcgacg 1800

tttcgccaga tagctgggac tcggatatga tcgagtatcc ttgcgtgttc gatcacaggg 1860

gacagcgctt tatgctttat tcgggcgatg gctacggtcg caccgggttc ggtttggcgg 1920

tgctggagaa ctgatcaggg ctgacaatag atgtttagcg gctgatgatg cgcttcccgc 1980

tcgaataggc tgagaccatt attgccgcgg tagcgatgat ttcccggatt atcgtcgtcg 2040

ccgcgatcac tcactgctcg tcgaggccct ttaagggctt cattgtatcc ttcgcactgc 2100

ttatcttcat gcgcgcaacg tcaggatgcg cgtgagcgcc tcgacaacgc ggctctgatc 2160

tacctcctga agtccaaccc acatcggcag acggattagg cgggaagcca cgtcgttggt 2220

gacggtcagg ttgccattgg tgcggccgta gcgacgcccg gccggcgaat cgtgaagcgg 2280

cacgtaatga aagaccgcgc ctataccttc gctcgtcaga cgcgccagca cctcctcccg 2340

atcggcgctg ggcgctagta acacgtagta catgtgggcg ttgtgagagc agccctgtgg 2400

gatgatcgga cggcgcagga gcccccgctg ttccaatgat tcgaagcttt catgataccg 2460

gttccatagg tccaatcgga tacgcgtgat ccgctcggct tcctcgaact gagcccatag 2520

aaaggcagcg actaattcgc tgggcaaata ggaagaccct ttgtcctgcc acgtatattt 2580

gtcgacctcg ttgcgaagga agcggctgcg attggtgccc ttttccctga gaatctctgc 2640

ccggagcagg aagtcttatg agttgacaag cagggcgccg ccttcgccgg aaatcacatt 2700

cttggtctcg tgaaatgaga gcgctcccag gtcgccgatg ctgccgagcg cccgcccacg 2760

atacgacgcc atcgcgcctt gggccgcgtc ttcgaccacc gccaggttgt ggtgcgtggc 2820

gatcttcatg atcgcgtcca tctcgcaggc cacgccggca tagtgaacgg ggacgatggc 2880

cttggttcgc ggggtgatgg cgtctacgat gcgagtttca tcaatgttga gcgtgtcggg 2940

ccgaatatcg acaaagactg gcacaccacc gcgcaacacg aaggcgttgg cggtagagac 3000

aaaggtgtat gacggcagta tgacttcgtc cccctcctct atgtccagaa gcagcgccat 3060

catttccagc gcggcggtgc atgagggggt gagtagtgcc ttgcgacaac cggtctgctg 3120

ttcgagccat gcatggctac gccgggtgaa gggaccatcg ccggccaggt ggccgcaaga 3180

atgcgcttcg gcgatgtacg cgagctcccg gccggtcatg tacggccgat tgaatggaac 3240

tttgtgatct gacactcgac gccaacttct caaatcatcg aacagggcgc tgaagtgttc 3300

ggtgatcggg gtcgaacatc caccagaatt ctccttgtgg ccggcggatc cctagccttt 3360

tcaggtatcc caacatgcct tcactatttc ttcatatctt ccgcaactcc gtgctgggca 3420

ccggacggcg ctccgtcttg gttcctatat agacaccatc cgcgtcagcg tcgccaagga 3480

gtagggcgcc cgctccgacc acacaccgtg aaccgatggt gatatggtcg cgtagcgttg 3540

cattgacgcc aatgaaagat tgctcctcta ttaccacgcc accggatacg acgatatgag 3600

acgctagaaa acagtgatcg tgaatcgtcg agtgatggcc gatatgattg ccgctccaca 3660

atgtgacgtt gttgccaatc gatacgaatg gctggatagt gttgtcttca agcaggaaga 3720

cattttcacc gatccgccca tcgttcaaga cggtagcgtg ggagctcaca tagctggcga 3780

gttcgtagcc gagagcctta gcggcaagat atttttcctt ccgcacaccg ttcagtttgg 3840

cgtaggccag cgccacgaac atcgcgtggg actccggcgg aaagcgttgt gcgacctcgt 3900

cgaaggccac taaaggcagg ccgcaaaact cggacacgct tgcatagtct cggtcgactg 3960

tgaacgcgac gacctcatat tccgaatccc ttgtgaagta gtaatgtgcg agctgagcga 4020

tgtcgccgct cccaaaaatt accaatggtt tggtcatgac gccttcctaa ccagaattgt 4080

gaattcatac aagccgtagt cgtgcagaag cgcaacactc ttggagtacc tgcgcttgca 4140

gagatcaaat agggcgcatg ggtcagcata gtacaggtcg tcgcgcatct ttgatgcatc 4200

ggaataagat gtcaggcaat taaaagagaa gccacggcga ctcgcggcat tcagcatgtc 4260

gagcgtcgct tcgatgtgag cgcaccattc cgtgtccaac gatttcagac gaacattgaa 4320

tattccactc gcgacgctat agtccgcctc ccgatctatg cgcgccgcgc agatgaagtc 4380

tgcgttcgcc cgaccttcga aacgtagtgc ggccgcgcgc accatttcgg gggagacgtc 4440

gatgccggtg taatcagttt tgaagccacg cgcatctagg tagtccagta gagccccata 4500

gccacagcct agatcgttga tcgaaaatgg gtccgccgca ttgacaatgc gcaccagctg 4560

gtcaaagcgc aacgcctgcc cggcttcgcc gttccaatcg acgccgcgcg ggtgccgtgt 4620

gcttcgagtt tcgatgcgta gtaacgggcc acgtcagcga gcatggtcgt tgcgtcttcc 4680

gccatgaagc tgcctcacga tttgtgtgtg tgggcgtcgg tgcgtgggtc cgagactata 4740

ccttcaacag ttgcatgccg aggctgcggc gggcaatgac ccaaaaaccc gccggcacgg 4800

ttcgccgagc aaggaagcgt ggagacgata gataatttca ctggcgacag tacctcaaat 4860

agtccggagc ctcggctccg acgttaaaga gcagatccag aatcgacacg gcgggctcga 4920

accctcccca caattgctta taatcgcggt agccgtcata atcgaaccaa gttacccgga 4980

tgctaagttc gtcgaacacg cgctcatcga catacgaacg ggctgagggg ccagagacat 5040

attcggtcgc tgcggcctgt tggcagaggt tggccagtct ctcggtcttg ccgtcggcta 5100

attcgtagtc ccacgaattt gccagtcgcg tgctgatacc gagataactg caaatcgcat 5160

tcaatagacg cctgttgagt aaggaaagat tcgtgtgctg ttcttcgagg taaatcggcg 5220

cgagccagtc agcgatctcc gcaaaatgag cggccgcgct gtagttgaat tctagtgccc 5280

gccagtgcgc tttcgcccaa tcggtgccgt cgatcagcgt ctcacgtatc ttttgatgga 5340

aacgtccctt cacctggacg ggaacagtta tccactgtaa cccctggctc gttttgatcc 5400

gatttctgtt tcgccaatca cgcttggtat attgcatgtc atcatagatg atgaattcat 5460

cgacgaatgc aatcaggtca aaatatcctc gccaaggtat gtaatttgat tgaacaatcg 5520

cgactttctt caacgcggtg tctccaattt agaataacaa atacgtcgcg cccgcgacag 5580

ctccgctgga gcgagttcaa gcgattctgc gacatattca atatggtgct cgggaaggcc 5640

aggatgggcc gcgacccggg gcgtccggtg cgcgatgaac gtcgcatcgt ctcctgtgag 5700

ataattgcat ccgatcatat agggctggct gcggctaggt tgctggcaaa aagatatcgc 5760

ggccgatccg tttctggttt tgtcttgatg atcaaatccg cttccgttca cgagatcgat 5820

tcctggtctt cccccagcgt cgcgatgtcg ataggtgtcg cgctttgttc gtacccgcac 5880

tacgcggcgg cgagaacctc gccaccgaat cgggattggg gggaggatac cactcggtcg 5940

aggcccgtca ccggccttct agcgggttga ccatcagtgt ttgcagggcc ctatcccggt 6000

atggcgcacc acgggatcgg cagcgttccg gttgctggcg tggtacctcg ttgtggcgcc 6060

gtggtccatg tcgattgagt gcgtggatca gtgtaaaccg ttgcgcgcca tgttctgtag 6120

gcactggttc gggttgtggt taggctgcac ggttggcagg ttaccaacca ctgagcccct 6180

gggcggatgt gagctcggac tccgcctatg gggtgtaatt ttggcagatt gggccgggtc 6240

cccgtggtga ggactcctca accggattgg gtaagcatga ggtggtgctg gcagcggtgt 6300

cctggtcgct ctcccgagta ggcccgttgt gactgtcatg tgggcgagcg ggtttgcgcg 6360

cgtaggagac gatgattact acgcacgtga ccaaccacaa gaacggtgcc catgtcaccg 6420

tggtgaaaac gagtggcgtg gtaccgacta cccctttggc tcccagctgt ccatagagcg 6480

gcacgtagaa cggctggccc gggaccgcga cgttgacgat gctcagcgcc acggccaaac 6540

tcacgcagac gccgaccgcg cggcggcggt ctccatgggc tgcgagttgg tcgaatatcc 6600

cagcaccagg aggcccgttg gggtctcggg ctaccagtgc agcgattggc aagacgaaaa 6660

cgagatagta gaaggcgacg tccgcggggg agaaggtggc ggtggcgagc aacacaatcc 6720

ccaccatgac aggcgggata cggcgtccga gcgccagcac ggcgaccacg actatgacta 6780

ggacagcaaa cccgatctgc gttcgcggac cagtgaggaa accctctggg atcttgcccg 6840

attgatagtt cttgatgcta tcggggatca gcaggagtgc cttgccaaag gacacgttcc 6900

gcgggtctcg aagccctccg aacgaactat tgaacttgat gatgccgtgg atcgactgtg 6960

cgatcgtccc cgggaagcct cgtggccaca acagaaaggc tgcgatattg gacaccacca 7020

cgccggtgat cccgatacca gcccaccgcc attgtcgagc cgccaacaac accacgccga 7080

gaacgacgaa ctgcggcttt accaggacgg ccaagatcac cgtgatggtg gcgaggcccc 7140

accgctgtcg ggacaacgcc acgaagtaag ccagcgcgat cggtaccacg aaccctgtcg 7200

agttgcctcg atcgatgacc ccccacgccg ggatggccgc ggcgcccagt gtcacgaaga 7260

tgaccactcg ctccagacca cgtgcccccc gggccgccca gatggcggga gatatgaccg 7320

ccatcgttag ggcgaccagg taacagatca gccccaagcg cggcgcaccc agccaatggc 7380

tgggtagtcc gaaaatcgca tacggtatgc gggcgggggc ccatgcagca accgcggtcg 7440

gctggtaatc ggcgggtagc gagatcaggt agtccgcggg attgggttga atcccggcgg 7500

cggcgaccat ggcgtagtcg ctgaagcagt gccgaccgat attcatgccc caatcaagcc 7560

aacagtcccc agggactacc aaaagagtgg aaaagacgtc gaccgcgtac cactgactga 7620

gggcgtacgc cgtcgccgcc gaaatcaccg acgccagcag gatggtgccg agcatgaggg 7680

tgcgctcgga ttgggagccg atcgcccaga gccgctcccg gctcgcggtc acggcaccgc 7740

gcaacacctc cgggggtcgc ttcatctgga ttctcctcgg ttctgcgcga aacggtagca 7800

gagcgccatg gttgccaacg cggtcgccgg gcagtctaga ccggatcttc ctcgtggcaa 7860

ccgacaacag gacgtcgttg ccgaaagggc gctgggcacc gacatctagg atgaacccac 7920

agccacgccc cgacgttatg ccatggcgaa gagcgaccgg caggagcggg aacccagtga 7980

agcgagcgct catcaccgga atcac 8005

<210> 2

<211> 16650

<212> DNA

<213> Rv1501-Rv1516c核苷酸序列

<400> 2

atgattcctg taaaggttga aaacaatact tcgctcgatc aggtgcaaga cgctcttaat 60

tgcgtcgggt acgcggttgt agaagatgtg cttgatgagg cgtcactggc agcgacccgt 120

gatcgcatgt atcgtgtaca ggagcggatt cttaccgaga ttggcaaaga gcggctggca 180

agggccggtg agctcggtgt tcttcgactc atgatgaagt atgaccctca tttctttacc 240

tttcttgaaa tacccgaagt cctaagcatc gttgatcgtg tgctatctga aacggccatc 300

ttacatctgc agaatggctt tatccttccg tccttcccgc ccttctccac gccggacgtt 360

tttcagaatg cgttccacca agactttccc agggttctgt ccggttacat tgcctccgtc 420

aatattatgt tcgccatcga tccctttaca cgagacaccg gcgcaacgct cgtagtgccg 480

gggagccacc agcgcataga gaaaccggac catacctacc tcgcgcgcaa tgccgttccc 540

gttcaatgcg cggcgggctc gttgttcgtt tttgactcta cgctttggca tgcggctggc 600

cgaaacacct ccggcaaaga ccgcttggcc ataaatcatc agtttacgcg ctcgtttttc 660

aagcagcaga tcgactacgt ccgcgcgctg ggcgacgccg tggttctgga gcagcctgcg 720

cgtactcagc aactgctcgg atggtacagt cgagtggtta ccaatctgga cgagtattac 780

cagccgccgg acaagcgatt gtatcggaag gggcaaggct agttttgcga gaattccgtt 840

gcgcctattt gaaagcccga catgaaacga tcgcttttaa gcgcatatgt ctgttctgca 900

aaaatgtcta atttttccga taaaggttgg tgggaaagct cgatgcgtgc cgtgttttgt 960

aggtggccgg atgatccact tagacaggcc gtggaagcag aatttgcgcg tcccgatggc 1020

gttgcggtgg cgtaatggcc tggcgaaagc tcgggagaat ttttgctccg tcgggcgaac 1080

tcgactggtc gcgaagtcat gctgcgctac cggttcctga atggatcgag ggtgatattt 1140

tccgcatcta tttcagcggc cgcgatggtc agaatcgttc cagtatcggt agcgtgatcg 1200

tcgatctcgc cgtgggcggc aagattctgg acattccggc ggagccgatt ttgcgccccg 1260

gcgctcgagg aatgtttgac gactgtgggg tgtcaatcgg atcgattgtg cgtgccggcg 1320

atacgcgact tttgtactac acgggctgga atctcgctgt caccgtgccc tggaaaaaca 1380

ccataggcgt ggcgattagc gaagcaggtg caccattcga gcgatggtct acttttcccg 1440

tcgttgcgct ggacgagcgt gatccattct cgctttctta tccctgggtc atccaagatg 1500

gagggacata ccgtatgtgg tatggctcaa atctaggctg gggagagggc accgacgaga 1560

tacctcacgt gatcaggtat gcgcaatcaa gggacggtgt ccactgggaa aagcaggatc 1620

gcgtgcatat cgacacaagc ggatccgaca atagcgcggc ctgtaggccg tacgtcgtcc 1680

gcgatgcggg agtatacaga atgtggtttt gcgctcgcgg tgcgaaatat cggatttact 1740

gcgctacatc ggaggatggt ttgacttggc ggcaactcgg caaagatgag ggcatcgacg 1800

tttcgccaga tagctgggac tcggatatga tcgagtatcc ttgcgtgttc gatcacaggg 1860

gacagcgctt tatgctttat tcgggcgatg gctacggtcg caccgggttc ggtttggcgg 1920

tgctggagaa ctgatcaggg ctgacaatag atgtttagcg gctgatgatg cgcttcccgc 1980

tcgaataggc tgagaccatt attgccgcgg tagcgatgat ttcccggatt atcgtcgtcg 2040

ccgcgatcac tcactgctcg tcgaggccct ttaagggctt cattgtatcc ttcgcactgc 2100

ttatcttcat gcgcgcaacg tcaggatgcg cgtgagcgcc tcgacaacgc ggctctgatc 2160

tacctcctga agtccaaccc acatcggcag acggattagg cgggaagcca cgtcgttggt 2220

gacggtcagg ttgccattgg tgcggccgta gcgacgcccg gccggcgaat cgtgaagcgg 2280

cacgtaatga aagaccgcgc ctataccttc gctcgtcaga cgcgccagca cctcctcccg 2340

atcggcgctg ggcgctagta acacgtagta catgtgggcg ttgtgagagc agccctgtgg 2400

gatgatcgga cggcgcagga gcccccgctg ttccaatgat tcgaagcttt catgataccg 2460

gttccatagg tccaatcgga tacgcgtgat ccgctcggct tcctcgaact gagcccatag 2520

aaaggcagcg actaattcgc tgggcaaata ggaagaccct ttgtcctgcc acgtatattt 2580

gtcgacctcg ttgcgaagga agcggctgcg attggtgccc ttttccctga gaatctctgc 2640

ccggagcagg aagtcttatg agttgacaag cagggcgccg ccttcgccgg aaatcacatt 2700

cttggtctcg tgaaatgaga gcgctcccag gtcgccgatg ctgccgagcg cccgcccacg 2760

atacgacgcc atcgcgcctt gggccgcgtc ttcgaccacc gccaggttgt ggtgcgtggc 2820

gatcttcatg atcgcgtcca tctcgcaggc cacgccggca tagtgaacgg ggacgatggc 2880

cttggttcgc ggggtgatgg cgtctacgat gcgagtttca tcaatgttga gcgtgtcggg 2940

ccgaatatcg acaaagactg gcacaccacc gcgcaacacg aaggcgttgg cggtagagac 3000

aaaggtgtat gacggcagta tgacttcgtc cccctcctct atgtccagaa gcagcgccat 3060

catttccagc gcggcggtgc atgagggggt gagtagtgcc ttgcgacaac cggtctgctg 3120

ttcgagccat gcatggctac gccgggtgaa gggaccatcg ccggccaggt ggccgcaaga 3180

atgcgcttcg gcgatgtacg cgagctcccg gccggtcatg tacggccgat tgaatggaac 3240

tttgtgatct gacactcgac gccaacttct caaatcatcg aacagggcgc tgaagtgttc 3300

ggtgatcggg gtcgaacatc caccagaatt ctccttgtgg ccggcggatc cctagccttt 3360

tcaggtatcc caacatgcct tcactatttc ttcatatctt ccgcaactcc gtgctgggca 3420

ccggacggcg ctccgtcttg gttcctatat agacaccatc cgcgtcagcg tcgccaagga 3480

gtagggcgcc cgctccgacc acacaccgtg aaccgatggt gatatggtcg cgtagcgttg 3540

cattgacgcc aatgaaagat tgctcctcta ttaccacgcc accggatacg acgatatgag 3600

acgctagaaa acagtgatcg tgaatcgtcg agtgatggcc gatatgattg ccgctccaca 3660

atgtgacgtt gttgccaatc gatacgaatg gctggatagt gttgtcttca agcaggaaga 3720

cattttcacc gatccgccca tcgttcaaga cggtagcgtg ggagctcaca tagctggcga 3780

gttcgtagcc gagagcctta gcggcaagat atttttcctt ccgcacaccg ttcagtttgg 3840

cgtaggccag cgccacgaac atcgcgtggg actccggcgg aaagcgttgt gcgacctcgt 3900

cgaaggccac taaaggcagg ccgcaaaact cggacacgct tgcatagtct cggtcgactg 3960

tgaacgcgac gacctcatat tccgaatccc ttgtgaagta gtaatgtgcg agctgagcga 4020

tgtcgccgct cccaaaaatt accaatggtt tggtcatgac gccttcctaa ccagaattgt 4080

gaattcatac aagccgtagt cgtgcagaag cgcaacactc ttggagtacc tgcgcttgca 4140

gagatcaaat agggcgcatg ggtcagcata gtacaggtcg tcgcgcatct ttgatgcatc 4200

ggaataagat gtcaggcaat taaaagagaa gccacggcga ctcgcggcat tcagcatgtc 4260

gagcgtcgct tcgatgtgag cgcaccattc cgtgtccaac gatttcagac gaacattgaa 4320

tattccactc gcgacgctat agtccgcctc ccgatctatg cgcgccgcgc agatgaagtc 4380

tgcgttcgcc cgaccttcga aacgtagtgc ggccgcgcgc accatttcgg gggagacgtc 4440

gatgccggtg taatcagttt tgaagccacg cgcatctagg tagtccagta gagccccata 4500

gccacagcct agatcgttga tcgaaaatgg gtccgccgca ttgacaatgc gcaccagctg 4560

gtcaaagcgc aacgcctgcc cggcttcgcc gttccaatcg acgccgcgcg ggtgccgtgt 4620

gcttcgagtt tcgatgcgta gtaacgggcc acgtcagcga gcatggtcgt tgcgtcttcc 4680

gccatgaagc tgcctcacga tttgtgtgtg tgggcgtcgg tgcgtgggtc cgagactata 4740

ccttcaacag ttgcatgccg aggctgcggc gggcaatgac ccaaaaaccc gccggcacgg 4800

ttcgccgagc aaggaagcgt ggagacgata gataatttca ctggcgacag tacctcaaat 4860

agtccggagc ctcggctccg acgttaaaga gcagatccag aatcgacacg gcgggctcga 4920

accctcccca caattgctta taatcgcggt agccgtcata atcgaaccaa gttacccgga 4980

tgctaagttc gtcgaacacg cgctcatcga catacgaacg ggctgagggg ccagagacat 5040

attcggtcgc tgcggcctgt tggcagaggt tggccagtct ctcggtcttg ccgtcggcta 5100

attcgtagtc ccacgaattt gccagtcgcg tgctgatacc gagataactg caaatcgcat 5160

tcaatagacg cctgttgagt aaggaaagat tcgtgtgctg ttcttcgagg taaatcggcg 5220

cgagccagtc agcgatctcc gcaaaatgag cggccgcgct gtagttgaat tctagtgccc 5280

gccagtgcgc tttcgcccaa tcggtgccgt cgatcagcgt ctcacgtatc ttttgatgga 5340

aacgtccctt cacctggacg ggaacagtta tccactgtaa cccctggctc gttttgatcc 5400

gatttctgtt tcgccaatca cgcttggtat attgcatgtc atcatagatg atgaattcat 5460

cgacgaatgc aatcaggtca aaatatcctc gccaaggtat gtaatttgat tgaacaatcg 5520

cgactttctt caacgcggtg tctccaattt agaataacaa atacgtcgcg cccgcgacag 5580

ctccgctgga gcgagttcaa gcgattctgc gacatattca atatggtgct cgggaaggcc 5640

aggatgggcc gcgacccggg gcgtccggtg cgcgatgaac gtcgcatcgt ctcctgtgag 5700

ataattgcat ccgatcatat agggctggct gcggctaggt tgctggcaaa aagatatcgc 5760

ggccgatccg tttctggttt tgtcttgatg atcaaatccg cttccgttca cgagatcgat 5820

tcctggtctt cccccagcgt cgcgatgtcg ataggtgtcg cgctttgttc gtacccgcac 5880

tacgcggcgg cgagaacctc gccaccgaat cgggattggg gggaggatac cactcggtcg 5940

aggcccgtca ccggccttct agcgggttga ccatcagtgt ttgcagggcc ctatcccggt 6000

atggcgcacc acgggatcgg cagcgttccg gttgctggcg tggtacctcg ttgtggcgcc 6060

gtggtccatg tcgattgagt gcgtggatca gtgtaaaccg ttgcgcgcca tgttctgtag 6120

gcactggttc gggttgtggt taggctgcac ggttggcagg ttaccaacca ctgagcccct 6180

gggcggatgt gagctcggac tccgcctatg gggtgtaatt ttggcagatt gggccgggtc 6240

cccgtggtga ggactcctca accggattgg gtaagcatga ggtggtgctg gcagcggtgt 6300

cctggtcgct ctcccgagta ggcccgttgt gactgtcatg tgggcgagcg ggtttgcgcg 6360

cgtaggagac gatgattact acgcacgtga ccaaccacaa gaacggtgcc catgtcaccg 6420

tggtgaaaac gagtggcgtg gtaccgacta cccctttggc tcccagctgt ccatagagcg 6480

gcacgtagaa cggctggccc gggaccgcga cgttgacgat gctcagcgcc acggccaaac 6540

tcacgcagac gccgaccgcg cggcggcggt ctccatgggc tgcgagttgg tcgaatatcc 6600

cagcaccagg aggcccgttg gggtctcggg ctaccagtgc agcgattggc aagacgaaaa 6660

cgagatagta gaaggcgacg tccgcggggg agaaggtggc ggtggcgagc aacacaatcc 6720

ccaccatgac aggcgggata cggcgtccga gcgccagcac ggcgaccacg actatgacta 6780

ggacagcaaa cccgatctgc gttcgcggac cagtgaggaa accctctggg atcttgcccg 6840

attgatagtt cttgatgcta tcggggatca gcaggagtgc cttgccaaag gacacgttcc 6900

gcgggtctcg aagccctccg aacgaactat tgaacttgat gatgccgtgg atcgactgtg 6960

cgatcgtccc cgggaagcct cgtggccaca acagaaaggc tgcgatattg gacaccacca 7020

cgccggtgat cccgatacca gcccaccgcc attgtcgagc cgccaacaac accacgccga 7080

gaacgacgaa ctgcggcttt accaggacgg ccaagatcac cgtgatggtg gcgaggcccc 7140

accgctgtcg ggacaacgcc acgaagtaag ccagcgcgat cggtaccacg aaccctgtcg 7200

agttgcctcg atcgatgacc ccccacgccg ggatggccgc ggcgcccagt gtcacgaaga 7260

tgaccactcg ctccagacca cgtgcccccc gggccgccca gatggcggga gatatgaccg 7320

ccatcgttag ggcgaccagg taacagatca gccccaagcg cggcgcaccc agccaatggc 7380

tgggtagtcc gaaaatcgca tacggtatgc gggcgggggc ccatgcagca accgcggtcg 7440

gctggtaatc ggcgggtagc gagatcaggt agtccgcggg attgggttga atcccggcgg 7500

cggcgaccat ggcgtagtcg ctgaagcagt gccgaccgat attcatgccc caatcaagcc 7560

aacagtcccc agggactacc aaaagagtgg aaaagacgtc gaccgcgtac cactgactga 7620

gggcgtacgc cgtcgccgcc gaaatcaccg acgccagcag gatggtgccg agcatgaggg 7680

tgcgctcgga ttgggagccg atcgcccaga gccgctcccg gctcgcggtc acggcaccgc 7740

gcaacacctc cgggggtcgc ttcatctgga ttctcctcgg ttctgcgcga aacggtagca 7800

gagcgccatg gttgccaacg cggtcgccgg gcagtctaga ccggatcttc ctcgtggcaa 7860

ccgacaacag gacgtcgttg ccgaaagggc gctgggcacc gacatctagg atgaacccac 7920

agccacgccc cgacgttatg ccatggcgaa gagcgaccgg caggagcggg aacccagtga 7980

agcgagcgct catcaccgga atcacaggac cggacggctc gtatctcgct aagctcccgc 8040

tgaagggata tgtggccgct ggtagcccgg ccgaggtcta tttctgctgg gcgacacgga 8100

attatcgcga attgtatggg ttgctcgcgg tcaacagcat ctggttcaat cacgaatcac 8160

cgcgtcacgg cgagacattc atgactcgta atcctgcacc atatcgcggt cggcaacgag 8220

gcgctgatcg atgcgcagac gctgatgcgc cggcccaccc ggataggtat cagtattggg 8280

gcgttccggc cagcgtacga ggcgtgatcg accgcgcaat gggtgtttgc gttgagtaat 8340

aatctgaacc gtgtgaacgc atgcatggat ggattccttg cccgtatccg ctcacatgtt 8400

gatgcgcacg cgccagaatt gcgttcactg ttcgatacga tggcggccga ggcccgattt 8460

gcacgcgact ggctgtccga ggacctcgcg cggttgcctg tcggtgcagc attgctggaa 8520

gtgggcgggg gggtacttct gctcagctgt caactggcgg cggagggatt tgacatcacc 8580

gccatcgagc cgacgggtga aggttttggc aagttcagac agcttggcga catcgtgctg 8640

gaattggctg cagcacgacc caccatcgcg ccatgcaagg cggaagactt tatttccgag 8700

aagcggttcg acttcgcctt ctcgctgaat gtgatggagc acatcgacct tccggatgag 8760

gcagtcaggc gggtatcgga agtgctgaaa ccgggggcca gttaccactt cctgtgcccg 8820

aattacgtat tcccgtacga accgcatttc aatatcccaa cattcttcac caaagagctg 8880

acatgccggg tgatgcgaca tcgcatcgag ggcaatacgg gcatggatga cccgaaggga 8940

gtctggcgtt cgctcaactg gattacggtt cccaaggtga aacgctttgc ggcgaaggat 9000

gcgacgctga ccttgcgctt ccaccgtgca atgttggtat ggatgctgga acgcgcgctg 9060

acggataagg aattcgctgg tcgccgggca caatggatgg tcgctgctat tcgctcggcg 9120

gtgaaattgc gtgtgcatca tctggcaggc tatgttcccg ctacgctgca gcccatcatg 9180

gatgtgcggc taacgaagag gtaatgacat ggcgcaagcg acatcgggca ttcgcgcggc 9240

actttcgcaa cctgctgtgt atgaggcgta tcagcggatt gcgggcgcta aaagcgggct 9300

tgcgtggatc acaaccgacc ccatccagtc gttgccaggc atgcgtactc tcgacctcgg 9360

ttgctggcca gcggtgatac acagctcccc gccagtggac gtgacatgta cgagagacgg 9420

catgagcgcg gaatgtgcga ccgtgccgtc gagatgaccg acgtcggcgc tacggcagcc 9480

cccaccggac ctatcgcgcg gggcagcgtc gctcgggtcg gcgcggcgac cgcgttggcc 9540

gttgcctgcg tctacacggt catctatctg gcggcccgcg acctaccccc ggcttgtttt 9600

tcgatattcg cggtgttttg gggggcgctc ggcattgcca ccggcgccac ccacggcctc 9660

ctgcaagaaa cgacccgcga ggtccgctgg gtgcgctcca cccaaatagt tgcgggccat 9720

cgtacccatc cgctgcgggt ggccgggatg attggcaccg tcgcggccgt cgtaattgcg 9780

ggtagctcac cgctgtggag ccgacagcta ttcgtcgagg ggcgctggct gtccgtgggg 9840

ctactcagcg ttggggtggc cgggttctgc gcgcaggcga ccctgctggg cgcgctggcc 9900

ggcgtcgacc ggtggacaca gtacgggtca ctgatggtga ccgacgcggt catccggttg 9960

gcggtcgccg cggcagcggt tgtgatcgga tggggtctgg ccgggtactt gtgggccgcc 10020

accgcgggag cggtggcgtg gctgctcatg ctgatggcct cgcccaccgc gcgcagcgcg 10080

gccagcctgc tgacgcccgg gggaatcgcc acgttcgtgc gcggtgccgc tcattcgata 10140

accgccgcgg gtgccagcgc gattctggta atgggtttcc cagtgttgct caaagtgacc 10200

tccgaccagt taggggcaaa gggcggagcg gtcatcctgg ctgtgacctt gacgcgtgcg 10260

ccgcttctgg tcccactgag cgcgatgcaa ggcaacctga tcgcgcattt cgtcgaccgg 10320

cgcacccaac ggcttcgggc gctgatcgca ccggcgctgg tcgtcggcgg catcggtgcg 10380

gtcgggatgt tggccgcagg gcttaccggt ccctggttgc tgcgtgttgg attcggcccc 10440

gactaccaaa ctggcggggc gttgctggcc tggttgacgg cagcggcggt agctatcgcc 10500

atgctgacgc tgaccggcgc cgccgcggtc gcggccgcac tgcaccgggc gtatttgctg 10560

ggctgggtca gcgcgacggt ggcgtcgacg ctgttgctgc tgctgccgat gccgctggag 10620

acgcgcaccg tgatcgcgct gttgttcggt ccaacggtgg gaatcgccat ccatgtggcc 10680

gcgttggcgc ggcgacccga ctgatttgtg ccccaggtcg acaaatcacg ccgtctcgtc 10740

agtgagcact ccgtcctcgg gtccgatcct tccaggagac gttgcaacct gatttggctc 10800

aaattggtgc gcaccgaggg tcgggcacat cgtagggtcg caacagtcac atgtgtcact 10860

gcaccgggcg acacccgatg tcccggctct cagcgacagc tgtctgacct gtggttttgt 10920

tcccaagttg gtcgtggctg tgcgggattg gaggtggcgt gggggtcgcg tcgtatggat 10980

tctcctcctc ggttccgcgc gaaacggccg caggcgcaat ggtcaccaac ttggccgcgg 11040

tggagtctag cctcacattt tcctggtcgc ccccgacaac caggaggtcg ctgcagaacg 11100

ggcgttccct acccacatct actatgaagc gacagcggcg ccccgctgtg atggctgagc 11160

atgaccgaca gaggcgggaa gacagtgaag cgagcgctca tcaccggaat caccggccag 11220

gacggctcgt atctcgccga actgctgctg gccaaggggt atgaggttca cgggctcatc 11280

cggcgcgctt cgacgttcaa cacctcgcgg atcgatcacc tctacgtcga cccgcaccaa 11340

ccgggcgcgc ggctgtttct gcactatggt gacctgatcg acggaacccg gttggtgacc 11400

ctgctgagca ccatcgaacc cgacgaggtg tacaacctgg cggcgcagtc acacgtgcgg 11460

gtgagcttcg acgaacccgt gcacaccggt gacaccaccg gcatgggatc catgcgactg 11520

ctggaagccg ttcggctctc tcgggtgcac tgccgcttct atcaggcgtc ctcgtcggag 11580

atgttcggcg cctcgccgcc accgcagaac gagctgacgc cgttctaccc gcggtcaccg 11640

tatggcgccg ccaaggtcta ttcgtactgg gcgacccgca attatcgcga agcgtacgga 11700

ttgttcgccg ttaacggcat cttgttcaat cacgaatcac cgcggcgcgg tgagacgttc 11760

gtgacccgaa agatcaccag ggccgtggca cgcatcaagg ccggtatcca gtccgaggtc 11820

tatatgggca atctggatgc ggtccgcgac tgggggtacg cgcccgaata cgtcgaaggc 11880

atgtggcgga tgctgcagac cgacgagccc gacgacttcg ttttggcgac cgggcgcggt 11940

ttcaccgtgc gtgagttcgc gcgggccgcg ttcgagcatg ccggtttgga ctggcagcag 12000

tacgtgaaat tcgaccaacg ctatctgcgg cccaccgagg tggattcgct gatcggcgac 12060

gcgaccaagg ctgccgaatt gctgggctgg agggcttcgg tgcacactga cgagttggct 12120

cggatcatgg tcgacgcgga catggcggcg ctggagtgcg aaggcaagcc gtggatcgac 12180

aagccgatga tcgccggccg gacatgaacg cgcacacctc ggtcggcccg cttgaccgcg 12240

cggcccgggt ctacatcgcc gggcatcgcg gcctggtcgg gtccgcgctg ctacgcacgt 12300

ttgcgggcgc ggggttcacc aacctgctgg tgcggtcacg cgccgagctt gatctgacgg 12360

atcgggccgc gacgttcgac ttcgttctcg agtcgaggcc gcaggtcgtc atcgacgcgg 12420

cggcccgggt cggcggcatc ctggccaacg acacctaccc ggccgatttc ctgtcggaaa 12480

acctccagat ccaggtcaac ctgctggatg ccgccgtggc ggcgcgggtg ccgcggctgc 12540

tgttcctggg ctcgtcgtgc atctacccga aactcgcccc gcagccgatc ccggagagcg 12600

cgctgctcac cggtccgttg gagccgacca acgacgcgta cgcgatcgcc aaaatcgccg 12660

gcatccttgc ggtccaggcg gtgcgccgcc aacatggcct gccgtggatc tcggcgatgc 12720

ccaccaacct gtacgggcca ggcgacaact tttcgccgtc cggctcgcat ctgctgccgg 12780

cactcatccg ccgctatgac gaggccaaag ccagtggcgc gcccaacgtg accaactggg 12840

gcaccggcac gccccgacgg gagttgctgc acgtcgacga cctggcgagc gcatgcctgt 12900

atctgctgga acatttcgac gggccgaccc atgtcaacgt gggaaccggc atcgaccaca 12960

ccatcggcga gatcgccgag atggtcgcct cggcggtagg ctatagcggc gaaacccgct 13020

gggatccaag caaaccggac ggaacaccac gcaaactgct ggatgtttcg gtgctacggg 13080

aggcgggatg gcggccttcg atcgcgctgc gcgacggcat cgaggcgacg gtggcgtggt 13140

atcgcgagca cgcgggaacg gttcggcaat gaggctggcc cgtcgcgctc ggaacatctt 13200

gcgtcgcaac ggcatcgagg tgtcgcgcta ctttgccgaa ctggactggg aacgcaattt 13260

cttgcgccaa ctgcaatcgc atcgggtcag tgccgtgctc gatgtcgggg ccaattcggg 13320

gcagtacgcc aggggtctgc gcggcgcggg cttcgcgggc cgcatcgtct cgttcgagcc 13380

gctgcccggg ccctttgccg tcttgcagcg cagcgcctcc acggacccgt tgtgggaatg 13440

ccggcgctgt gcgctgggcg atgtcgatgg aaccatctcg atcaacgtcg ccggcaacga 13500

gggcgccagc agttccgtct tgccgatgtt gaaacgacat caggacgcct ttccaccagc 13560

caactacgtg ggcgcccaac gggtgccgat acatcgactc gattccgtgg ctgcagacgt 13620

tctgcggccc aacgatattg cgttcttgaa gatcgacgtt caaggattcg agaagcaggt 13680

gatcgcgggt ggcgattcaa cggtgcacga ccgatgcgtc ggcatgcagc tcgagctgtc 13740

tttccagccg ttgtacgagg gtggcatgct catccgcgag gcgctcgatc tcgtggattc 13800

gttgggcttt acgctctcgg gattgcaacc cggtttcacc gacccccgca acggtcgaat 13860

gctgcaggcc gatggcatct tcttccgggg cagcgattga cgcgccggcg cgtcaatcta 13920

tttcgacatt cgcgtgaaga cgttttccca gaatcgactg ttgtaggcgt agaactcccg 13980

gccgcgtagg taggcatgtg atattcgcct tcccccgaac gggtagcggc gatgaaggtc 14040

gcccatgcgg cgcagatcac cgaagaccgc gcttggttcc cggtgcgagc cgacgcccgt 14100

ggtgtcgaac tcgcacagca cacaccgaat cgtgaccggc tcgcatacca gcgcggcccg 14160

caatatgaat tcctggtcgg cggcgatccc gaaatcaagg tcgtagccac cgatcttggc 14220

caccagcgat gatccgaaga acgatgcttg atgcggaaca acctgcttgc cggccaggaa 14280

tttgcgcagg ctgaaaggta tcgggccgcg cacccgatcg agcccgacga gacgatccat 14340

cccgaagccc cacaattcgg acaccggtcc cttgccggat agcgcctcca cggcctgggc 14400

taccacgtcg ggcccggaaa aacgatcggc ggagtgcaag aaccacaaca gatcacccga 14460

tgcgtgcgcg atgccctggt tcatcgcgtc gtaccgcccg ccgtcgggct cggactgcca 14520

atacgcgaag cctggttcac acccggacag gtatgccacc acgtcgtcgc cgctgccacc 14580

gtcgattacg atgtgctcga tgcgtccccg gtagcgttgc gcccgcacac ttttcaccgt 14640

gcgctgcaac ccgtcgaggt cgttgaacga gatcgttatc accgagacgg tcggagcaga 14700

cgtcaccgag ttcccctagg ttgctggcgg cgattgtgga tcaccgggtc ttgataccga 14760

tgaaggtgcc tcgaagattc gccgcatagg aacctccgag caacgactcg gcgatgcttg 14820

gttccaagtt gtcgtactcc tccatcacca ggtcgacgcc gacgtctttg atggcctgaa 14880

gtaggtgctc gcgttgaatc cagaatgacc ggcgattgtc ccaggacgcc cattttgcgg 14940

tgtcgcgctg gccaaacgag cggtcgtcgg aaaactcggt aaaccaccta ccgggaagtc 15000

cctcatgttc ggtgggcgcc gagagcatga acttcaccgg cgccggccgc cgcagcaacc 15060

gatcggtcaa ttgtcgtgcc gtcgtgggca accggagcca tttatcgctc cggttgatga 15120

tcgagaagtg cgtctggaga atcagcagct tgttcgttac cgacgagagg gtttccaggt 15180

attgcttcgg attctccagg tggtagaaga ggccgcagca gaagacggta tcgaagagcc 15240

cgtggttggc gatgttgagg gcgttgtcgt ggacgaaccg gagattcggc aggttggtct 15300

tcgatttgat gtagttgcag gccgccatgt tcagctcgcg aacctcgatc ccgaggacct 15360

gaaatcccat gcgcgcgaac ccgaccgcgt acccgccttc caagcagccg acatcggcca 15420

ggcgtaggtg gctcttgtcc ccgggaaaga cggtttccag aatcccgcgc gccgagatga 15480

accaggacga ttcgtctaac gtgcgcgagg actccggtat cgtcaaggtt ccgtcgtcga 15540

ggcgaacgtt gtgggcggtg aattgtaccg cgccggccga atgttcctgt gccatcactt 15600

ggttagcccc ttcggctggt cctgggtttg tcgacatggt caggctcgac agccgcgtcg 15660

gagccgggag ggccacacat ccacgagccc cctgcggctc ggcgtcgcgg cggcgagctt 15720

gcgccactgg gtcttgagcc gccgcgcggg tgtcgccccg cggtgctgca gcgccagcat 15780

ggcgatccgg ggatggcgcg cgatggtttc ctgcagcgcg gcgcgcccct ccgggcctgg 15840

aacgttggcg atctggcgaa ggatccagtc ggccatgacg gcgatgagct cctcgcgcgc 15900

ggggtctccc gggaacaggt cgagcatcgc gtcaaacgtc gccgcatgcc ccggaccctg 15960

cgtcaaccag aactttggcg ggtccaccac ctggttgtgc cacatgcctt gggcgtggcg 16020

gcgatacacg gccatggtgt cgggcaacat ggcgatgtcg ccatgcaccg cgtgccggac 16080

gtgcagatac cagtccaggg gcatgacgtc ggcaggaatg tcgtcgtagc gctcgaggcg 16140

acggtacacg gccgagttgg tctggatgaa gttcatcaag atcaacgcat ccaggctcaa 16200

gttgccccgc acccgaaccg gggggaactt cgagtccttg gcatggccgt cctcccatat 16260

cactcggacg ggatggaagc acaccgtcgt cttggggtgc cggtcgagga atgcgacctg 16320

tttgcttagc ttcagcggat cgatccagta gtcgtccgcc tcgcacaacg cgacgtactc 16380

gccgcgagcg gccgacaggg cgccggtcag gttcccattg aggccgaggt tttcggtcct 16440

gaagatcggc cggaacacgt gcgggtaccg ctcggcgtac tcacggatga tcgccggggt 16500

ggcatcggtc gacgcgtcgt cggcgacgat gatctccacc gggaagtcgg tttgctggtc 16560

gagaaagctg tcgaaggcct gacgggcgta gcccgcctgg ttgtgagtgg tcgagacgat 16620

gctcaccttg gggcaaagct ggggactcac 16650

<210> 3

<211> 32

<212> DNA

<213> 人工序列

<400> 3

cactggtcga caatgtcact tcatttagca ac 32

<210> 4

<211> 30

<212> DNA

<213> 人工序列

<400> 4

catgaaagct tcgaatcatt ggaacagcgg 30

<210> 5

<211> 39

<212> DNA

<213> 人工序列

<400> 5

cctcgaagct ttcatgatac cggttccata ggtccaatc 39

<210> 6

<211> 26

<212> DNA

<213> 人工序列

<400> 6

ttggctagca accgcgcgag gtcctc 26

<210> 7

<211> 36

<212> DNA

<213> 人工序列

<400> 7

tgtgctagca tgcaatcagg tcaaaatatc ctcgcc 36

<210> 8

<211> 31

<212> DNA

<213> 人工序列

<400> 8

tgtgagctct caacccgcta gaaggccggt g 31

<210> 9

<211> 33

<212> DNA

<213> 人工序列

<400> 9

gttgctagca tgtcgacaaa cccaggacca gcc 33

<210> 10

<211> 35

<212> DNA

<213> 人工序列

<400> 10

tgggagctct caccgggtct tgataccgat gaagg 35

<210> 11

<211> 38

<212> DNA

<213> 人工序列

<400> 11

ggcgctagca tgattcctgt aaaggttgaa aacaatac 38

<210> 12

<211> 32

<212> DNA

<213> 人工序列

<400> 12

tttcaagaaa ggtaaagaaa tgagggtcat ac 32

<210> 13

<211> 35

<212> DNA

<213> 人工序列

<400> 13

tgtgctagct tgaagaaagt cgcgattgtt caatc 35

<210> 14

<211> 31

<212> DNA

<213> 人工序列

<400> 14

cgtgtgctgt tcttcgaggt aaatcggcgc g 31

<210> 15

<211> 42

<212> DNA

<213> 人工序列

<400> 15

tataagcttt ccgaatccct tgtgaagtag taatgtgcga gc 42

<210> 16

<211> 31

<212> DNA

<213> 人工序列

<400> 16

cgatccagta gtcgtccgcc tcgcacaacg c 31

<210> 17

<211> 273

<212> PRT

<213> Rv1501氨基酸序列

<400> 17

Met Ile Pro Val Lys Val Glu Asn Asn Thr Ser Leu Asp Gln Val Gln

1 5 10 15

Asp Ala Leu Asn Cys Val Gly Tyr Ala Val Val Glu Asp Val Leu Asp

20 25 30

Glu Ala Ser Leu Ala Ala Thr Arg Asp Arg Met Tyr Arg Val Gln Glu

35 40 45

Arg Ile Leu Thr Glu Ile Gly Lys Glu Arg Leu Ala Arg Ala Gly Glu

50 55 60

Leu Gly Val Leu Arg Leu Met Met Lys Tyr Asp Pro His Phe Phe Thr

65 70 75 80

Phe Leu Glu Ile Pro Glu Val Leu Ser Ile Val Asp Arg Val Leu Ser

85 90 95

Glu Thr Ala Ile Leu His Leu Gln Asn Gly Phe Ile Leu Pro Ser Phe

100 105 110

Pro Pro Phe Ser Thr Pro Asp Val Phe Gln Asn Ala Phe His Gln Asp

115 120 125

Phe Pro Arg Val Leu Ser Gly Tyr Ile Ala Ser Val Asn Ile Met Phe

130 135 140

Ala Ile Asp Pro Phe Thr Arg Asp Thr Gly Ala Thr Leu Val Val Pro

145 150 155 160

Gly Ser His Gln Arg Ile Glu Lys Pro Asp His Thr Tyr Leu Ala Arg

165 170 175

Asn Ala Val Pro Val Gln Cys Ala Ala Gly Ser Leu Phe Val Phe Asp

180 185 190

Ser Thr Leu Trp His Ala Ala Gly Arg Asn Thr Ser Gly Lys Asp Arg

195 200 205

Leu Ala Ile Asn His Gln Phe Thr Arg Ser Phe Phe Lys Gln Gln Ile

210 215 220

Asp Tyr Val Arg Ala Leu Gly Asp Ala Val Val Leu Glu Gln Pro Ala

225 230 235 240

Arg Thr Gln Gln Leu Leu Gly Trp Tyr Ser Arg Val Val Thr Asn Leu

245 250 255

Asp Glu Tyr Tyr Gln Pro Pro Asp Lys Arg Leu Tyr Arg Lys Gly Gln

260 265 270

Gly

<210> 18

<211> 299

<212> PRT

<213> Rv1502氨基酸序列

<400> 18

Met Ala Trp Arg Lys Leu Gly Arg Ile Phe Ala Pro Ser Gly Glu Leu

1 5 10 15

Asp Trp Ser Arg Ser His Ala Ala Leu Pro Val Pro Glu Trp Ile Glu

20 25 30

Gly Asp Ile Phe Arg Ile Tyr Phe Ser Gly Arg Asp Gly Gln Asn Arg

35 40 45

Ser Ser Ile Gly Ser Val Ile Val Asp Leu Ala Val Gly Gly Lys Ile

50 55 60

Leu Asp Ile Pro Ala Glu Pro Ile Leu Arg Pro Gly Ala Arg Gly Met

65 70 75 80

Phe Asp Asp Cys Gly Val Ser Ile Gly Ser Ile Val Arg Ala Gly Asp

85 90 95

Thr Arg Leu Leu Tyr Tyr Thr Gly Trp Asn Leu Ala Val Thr Val Pro

100 105 110

Trp Lys Asn Thr Ile Gly Val Ala Ile Ser Glu Ala Gly Ala Pro Phe

115 120 125

Glu Arg Trp Ser Thr Phe Pro Val Val Ala Leu Asp Glu Arg Asp Pro

130 135 140

Phe Ser Leu Ser Tyr Pro Trp Val Ile Gln Asp Gly Gly Thr Tyr Arg

145 150 155 160

Met Trp Tyr Gly Ser Asn Leu Gly Trp Gly Glu Gly Thr Asp Glu Ile

165 170 175

Pro His Val Ile Arg Tyr Ala Gln Ser Arg Asp Gly Val His Trp Glu

180 185 190

Lys Gln Asp Arg Val His Ile Asp Thr Ser Gly Ser Asp Asn Ser Ala

195 200 205

Ala Cys Arg Pro Tyr Val Val Arg Asp Ala Gly Val Tyr Arg Met Trp

210 215 220

Phe Cys Ala Arg Gly Ala Lys Tyr Arg Ile Tyr Cys Ala Thr Ser Glu

225 230 235 240

Asp Gly Leu Thr Trp Arg Gln Leu Gly Lys Asp Glu Gly Ile Asp Val

245 250 255

Ser Pro Asp Ser Trp Asp Ser Asp Met Ile Glu Tyr Pro Cys Val Phe

260 265 270

Asp His Arg Gly Gln Arg Phe Met Leu Tyr Ser Gly Asp Gly Tyr Gly

275 280 285

Arg Thr Gly Phe Gly Leu Ala Val Leu Glu Asn

290 295

<210> 19

<211> 182

<212> PRT

<213> Rv1503氨基酸序列

<400> 19

Asp Phe Leu Leu Arg Ala Glu Ile Leu Arg Glu Lys Gly Thr Asn Arg

1 5 10 15

Ser Arg Phe Leu Arg Asn Glu Val Asp Lys Tyr Thr Trp Gln Asp Lys

20 25 30

Gly Ser Ser Tyr Leu Pro Ser Glu Leu Val Ala Ala Phe Leu Trp Ala

35 40 45

Gln Phe Glu Glu Ala Glu Arg Ile Thr Arg Ile Arg Leu Asp Leu Trp

50 55 60

Asn Arg Tyr His Glu Ser Phe Glu Ser Leu Glu Gln Arg Gly Leu Leu

65 70 75 80

Arg Arg Pro Ile Ile Pro Gln Gly Cys Ser His Asn Ala His Met Tyr

85 90 95

Tyr Val Leu Leu Ala Pro Ser Ala Asp Arg Glu Glu Val Leu Ala Arg

100 105 110

Leu Thr Ser Glu Gly Ile Gly Ala Val Phe His Tyr Val Pro Leu His

115 120 125

Asp Ser Pro Ala Gly Arg Arg Tyr Gly Arg Thr Asn Gly Asn Leu Thr

130 135 140

Val Thr Asn Asp Val Ala Ser Arg Leu Ile Arg Leu Pro Met Trp Val

145 150 155 160

Gly Leu Gln Glu Val Asp Gln Ser Arg Val Val Glu Ala Leu Thr Arg

165 170 175

Ile Leu Thr Leu Arg Ala

180

<210> 20

<211> 199

<212> PRT

<213> Rv1504c氨基酸序列

<400> 20

Met Ser Asp His Lys Val Pro Phe Asn Arg Pro Tyr Met Thr Gly Arg

1 5 10 15

Glu Leu Ala Tyr Ile Ala Glu Ala His Ser Cys Gly His Leu Ala Gly

20 25 30

Asp Gly Pro Phe Thr Arg Arg Ser His Ala Trp Leu Glu Gln Gln Thr

35 40 45

Gly Cys Arg Lys Ala Leu Leu Thr Pro Ser Cys Thr Ala Ala Leu Glu

50 55 60

Met Met Ala Leu Leu Leu Asp Ile Glu Glu Gly Asp Glu Val Ile Leu

65 70 75 80

Pro Ser Tyr Thr Phe Val Ser Thr Ala Asn Ala Phe Val Leu Arg Gly

85 90 95

Gly Val Pro Val Phe Val Asp Ile Arg Pro Asp Thr Leu Asn Ile Asp

100 105 110

Glu Thr Arg Ile Val Asp Ala Ile Thr Pro Arg Thr Lys Ala Ile Val

115 120 125

Pro Val His Tyr Ala Gly Val Ala Cys Glu Met Asp Ala Ile Met Lys

130 135 140

Ile Ala Thr His His Asn Leu Ala Val Val Glu Asp Ala Ala Gln Gly

145 150 155 160

Ala Met Ala Ser Tyr Arg Gly Arg Ala Leu Gly Ser Ile Gly Asp Leu

165 170 175

Gly Ala Leu Ser Phe His Glu Thr Lys Asn Val Ile Ser Gly Glu Gly

180 185 190

Gly Ala Leu Leu Val Asn Ser

195

<210> 21

<211> 221

<212> PRT

<213> Rv1505氨基酸序列

<400> 21

Met Thr Lys Pro Leu Val Ile Phe Gly Ser Gly Asp Ile Ala Gln Leu

1 5 10 15

Ala His Tyr Tyr Phe Thr Arg Asp Ser Glu Tyr Glu Val Val Ala Phe

20 25 30

Thr Val Asp Arg Asp Tyr Ala Ser Val Ser Glu Phe Cys Gly Leu Pro

35 40 45

Leu Val Ala Phe Asp Glu Val Ala Gln Arg Phe Pro Pro Glu Ser His

50 55 60

Ala Met Phe Val Ala Leu Ala Tyr Ala Lys Leu Asn Gly Val Arg Lys

65 70 75 80

Glu Lys Tyr Leu Ala Ala Lys Ala Leu Gly Tyr Glu Leu Ala Ser Tyr

85 90 95

Val Ser Ser His Ala Thr Val Leu Asn Asp Gly Arg Ile Gly Glu Asn

100 105 110

Val Phe Leu Leu Glu Asp Asn Thr Ile Gln Pro Phe Val Ser Ile Gly

115 120 125

Asn Asn Val Thr Leu Trp Ser Gly Asn His Ile Gly His His Ser Thr

130 135 140

Ile His Asp His Cys Phe Leu Ala Ser His Ile Val Val Ser Gly Gly

145 150 155 160

Val Val Ile Glu Glu Gln Ser Phe Ile Gly Val Asn Ala Thr Leu Arg

165 170 175

Asp His Ile Thr Ile Gly Ser Arg Cys Val Val Gly Ala Gly Ala Leu

180 185 190

Leu Leu Gly Asp Ala Asp Ala Asp Gly Val Tyr Ile Gly Thr Lys Thr

195 200 205

Glu Arg Arg Pro Val Pro Ser Thr Glu Leu Arg Lys Ile

210 215 220

<210> 22

<211> 166

<212> PRT

<213> Rv1506c氨基酸序列

<400> 22

Met Arg Ile Val Asn Ala Ala Asp Pro Phe Ser Ile Asn Asp Leu Gly

1 5 10 15

Cys Gly Tyr Gly Ala Leu Leu Asp Tyr Leu Asp Ala Arg Gly Phe Lys

20 25 30

Thr Asp Tyr Thr Gly Ile Asp Val Ser Pro Glu Met Val Arg Ala Ala

35 40 45

Ala Leu Arg Phe Glu Gly Arg Ala Asn Ala Asp Phe Ile Cys Ala Ala

50 55 60

Arg Ile Asp Arg Glu Ala Asp Tyr Ser Val Ala Ser Gly Ile Phe Asn

65 70 75 80

Val Arg Leu Lys Ser Leu Asp Thr Glu Trp Cys Ala His Ile Glu Ala

85 90 95

Thr Leu Asp Met Leu Asn Ala Ala Ser Arg Arg Gly Phe Ser Phe Asn

100 105 110

Cys Leu Thr Ser Tyr Ser Asp Ala Ser Lys Met Arg Asp Asp Leu Tyr

115 120 125

Tyr Ala Asp Pro Cys Ala Leu Phe Asp Leu Cys Lys Arg Arg Tyr Ser

130 135 140

Lys Ser Val Ala Leu Leu His Asp Tyr Gly Leu Tyr Glu Phe Thr Ile

145 150 155 160

Leu Val Arg Lys Ala Ser

165

<210> 23

<211> 231

<212> PRT

<213> Rv1507c氨基酸序列

<400> 23

Met Lys Lys Val Ala Ile Val Gln Ser Asn Tyr Ile Pro Trp Arg Gly

1 5 10 15

Tyr Phe Asp Leu Ile Ala Phe Val Asp Glu Phe Ile Ile Tyr Asp Asp

20 25 30

Met Gln Tyr Thr Lys Arg Asp Trp Arg Asn Arg Asn Arg Ile Lys Thr

35 40 45

Ser Gln Gly Leu Gln Trp Ile Thr Val Pro Val Gln Val Lys Gly Arg

50 55 60

Phe His Gln Lys Ile Arg Glu Thr Leu Ile Asp Gly Thr Asp Trp Ala

65 70 75 80

Lys Ala His Trp Arg Ala Leu Glu Phe Asn Tyr Ser Ala Ala Ala His

85 90 95

Phe Ala Glu Ile Ala Asp Trp Leu Ala Pro Ile Tyr Leu Glu Glu Gln

100 105 110

His Thr Asn Leu Ser Leu Leu Asn Arg Arg Leu Leu Asn Ala Ile Cys

115 120 125

Ser Tyr Leu Gly Ile Ser Thr Arg Leu Ala Asn Ser Trp Asp Tyr Glu

130 135 140

Leu Ala Asp Gly Lys Thr Glu Arg Leu Ala Asn Leu Cys Gln Gln Ala

145 150 155 160

Ala Ala Thr Glu Tyr Val Ser Gly Pro Ser Ala Arg Ser Tyr Val Asp

165 170 175

Glu Arg Val Phe Asp Glu Leu Ser Ile Arg Val Thr Trp Phe Asp Tyr

180 185 190

Asp Gly Tyr Arg Asp Tyr Lys Gln Leu Trp Gly Gly Phe Glu Pro Ala

195 200 205

Val Ser Ile Leu Asp Leu Leu Phe Asn Val Gly Ala Glu Ala Pro Asp

210 215 220

Tyr Leu Arg Tyr Cys Arg Gln

225 230

<210> 24

<211> 167

<212> PRT

<213> Rv1507A氨基酸序列

<400> 24

Met Gln Ser Gly Gln Asn Ile Leu Ala Lys Val Cys Asn Leu Ile Glu

1 5 10 15

Gln Ser Arg Leu Ser Ser Thr Arg Cys Leu Gln Phe Arg Ile Thr Asn

20 25 30

Thr Ser Arg Pro Arg Gln Leu Arg Trp Ser Glu Phe Lys Arg Phe Cys

35 40 45

Asp Ile Phe Asn Met Val Leu Gly Lys Ala Arg Met Gly Arg Asp Pro

50 55 60

Gly Arg Pro Val Arg Asp Glu Arg Arg Ile Val Ser Cys Glu Ile Ile

65 70 75 80

Ala Ser Asp His Ile Gly Leu Ala Ala Ala Arg Leu Leu Ala Lys Arg

85 90 95

Tyr Arg Gly Arg Ser Val Ser Gly Phe Val Leu Met Ile Lys Ser Ala

100 105 110

Ser Val His Glu Ile Asp Ser Trp Ser Ser Pro Ser Val Ala Met Ser

115 120 125

Ile Gly Val Ala Leu Cys Ser Tyr Pro His Tyr Ala Ala Ala Arg Thr

130 135 140

Ser Pro Pro Asn Arg Asp Trp Gly Glu Asp Thr Thr Arg Ser Arg Pro

145 150 155 160

Val Thr Gly Leu Leu Ala Gly

165

<210> 25

<211> 599

<212> PRT

<213> Rv1508c氨基酸序列

<400> 25

Met Ile Pro Val Met Ser Ala Arg Phe Thr Gly Phe Pro Leu Leu Pro

1 5 10 15

Val Ala Leu Arg His Gly Ile Thr Ser Gly Arg Gly Cys Gly Phe Ile

20 25 30

Leu Asp Val Gly Ala Gln Arg Pro Phe Gly Asn Asp Val Leu Leu Ser

35 40 45

Val Ala Thr Arg Lys Ile Arg Ser Arg Leu Pro Gly Asp Arg Val Gly

50 55 60

Asn His Gly Ala Leu Leu Pro Phe Arg Ala Glu Pro Arg Arg Ile Gln

65 70 75 80

Met Lys Arg Pro Pro Glu Val Leu Arg Gly Ala Val Thr Ala Ser Arg

85 90 95

Glu Arg Leu Trp Ala Ile Gly Ser Gln Ser Glu Arg Thr Leu Met Leu

100 105 110

Gly Thr Ile Leu Leu Ala Ser Val Ile Ser Ala Ala Thr Ala Tyr Ala

115 120 125

Leu Ser Gln Trp Tyr Ala Val Asp Val Phe Ser Thr Leu Leu Val Val

130 135 140

Pro Gly Asp Cys Trp Leu Asp Trp Gly Met Asn Ile Gly Arg His Cys

145 150 155 160

Phe Ser Asp Tyr Ala Met Val Ala Ala Ala Gly Ile Gln Pro Asn Pro

165 170 175

Ala Asp Tyr Leu Ile Ser Leu Pro Ala Asp Tyr Gln Pro Thr Ala Val

180 185 190

Ala Ala Trp Ala Pro Ala Arg Ile Pro Tyr Ala Ile Phe Gly Leu Pro

195 200 205

Ser His Trp Leu Gly Ala Pro Arg Leu Gly Leu Ile Cys Tyr Leu Val

210 215 220

Ala Leu Thr Met Ala Val Ile Ser Pro Ala Ile Trp Ala Ala Arg Gly

225 230 235 240

Ala Arg Gly Leu Glu Arg Val Val Ile Phe Val Thr Leu Gly Ala Ala

245 250 255

Ala Ile Pro Ala Trp Gly Val Ile Asp Arg Gly Asn Ser Thr Gly Phe

260 265 270

Val Val Pro Ile Ala Leu Ala Tyr Phe Val Ala Leu Ser Arg Gln Arg

275 280 285

Trp Gly Leu Ala Thr Ile Thr Val Ile Leu Ala Val Leu Val Lys Pro

290 295 300

Gln Phe Val Val Leu Gly Val Val Leu Leu Ala Ala Arg Gln Trp Arg

305 310 315 320

Trp Ala Gly Ile Gly Ile Thr Gly Val Val Val Ser Asn Ile Ala Ala

325 330 335

Phe Leu Leu Trp Pro Arg Gly Phe Pro Gly Thr Ile Ala Gln Ser Ile

340 345 350

His Gly Ile Ile Lys Phe Asn Ser Ser Phe Gly Gly Leu Arg Asp Pro

355 360 365

Arg Asn Val Ser Phe Gly Lys Ala Leu Leu Leu Ile Pro Asp Ser Ile

370 375 380

Lys Asn Tyr Gln Ser Gly Lys Ile Pro Glu Gly Phe Leu Thr Gly Pro

385 390 395 400

Arg Thr Gln Ile Gly Phe Ala Val Leu Val Ile Val Val Val Ala Val

405 410 415

Leu Ala Leu Gly Arg Arg Ile Pro Pro Val Met Val Gly Ile Val Leu

420 425 430

Leu Ala Thr Ala Thr Phe Ser Pro Ala Asp Val Ala Phe Tyr Tyr Leu

435 440 445

Val Phe Val Leu Pro Ile Ala Ala Leu Val Ala Arg Asp Pro Asn Gly

450 455 460

Pro Pro Gly Ala Gly Ile Phe Asp Gln Leu Ala Ala His Gly Asp Arg

465 470 475 480

Arg Arg Ala Val Gly Val Cys Val Ser Leu Ala Val Ala Leu Ser Ile

485 490 495

Val Asn Val Ala Val Pro Gly Gln Pro Phe Tyr Val Pro Leu Tyr Gly

500 505 510

Gln Leu Gly Ala Lys Gly Val Val Gly Thr Thr Pro Leu Val Phe Thr

515 520 525

Thr Val Thr Trp Ala Pro Phe Leu Trp Leu Val Thr Cys Val Val Ile

530 535 540

Ile Val Ser Tyr Ala Arg Lys Pro Ala Arg Pro His Asp Ser His Asn

545 550 555 560

Gly Pro Thr Arg Glu Ser Asp Gln Asp Thr Ala Ala Ser Thr Thr Ser

565 570 575

Cys Leu Pro Asn Pro Val Glu Glu Ser Ser Pro Arg Gly Pro Gly Pro

580 585 590

Ile Cys Gln Asn Tyr Thr Pro

595

<210> 26

<211> 120

<212> PRT

<213> Rv1508A氨基酸序列

<400> 26

Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Pro Asp Gly Ser Tyr

1 5 10 15

Leu Ala Lys Leu Pro Leu Lys Gly Tyr Val Ala Ala Gly Ser Pro Ala

20 25 30

Glu Val Tyr Phe Cys Trp Ala Thr Arg Asn Tyr Arg Glu Leu Tyr Gly

35 40 45

Leu Leu Ala Val Asn Ser Ile Trp Phe Asn His Glu Ser Pro Arg His

50 55 60

Gly Glu Thr Phe Met Thr Arg Asn Pro Ala Pro Tyr Arg Gly Arg Gln

65 70 75 80

Arg Gly Ala Asp Arg Cys Ala Asp Ala Asp Ala Pro Ala His Pro Asp

85 90 95

Arg Tyr Gln Tyr Trp Gly Val Pro Ala Ser Val Arg Gly Val Ile Asp

100 105 110

Arg Ala Met Gly Val Cys Val Glu

115 120

<210> 27

<211> 293

<212> PRT

<213> Rv1509氨基酸序列

<400> 27

Met Phe Ala Leu Ser Asn Asn Leu Asn Arg Val Asn Ala Cys Met Asp

1 5 10 15

Gly Phe Leu Ala Arg Ile Arg Ser His Val Asp Ala His Ala Pro Glu

20 25 30

Leu Arg Ser Leu Phe Asp Thr Met Ala Ala Glu Ala Arg Phe Ala Arg

35 40 45

Asp Trp Leu Ser Glu Asp Leu Ala Arg Leu Pro Val Gly Ala Ala Leu

50 55 60

Leu Glu Val Gly Gly Gly Val Leu Leu Leu Ser Cys Gln Leu Ala Ala

65 70 75 80

Glu Gly Phe Asp Ile Thr Ala Ile Glu Pro Thr Gly Glu Gly Phe Gly

85 90 95

Lys Phe Arg Gln Leu Gly Asp Ile Val Leu Glu Leu Ala Ala Ala Arg

100 105 110

Pro Thr Ile Ala Pro Cys Lys Ala Glu Asp Phe Ile Ser Glu Lys Arg

115 120 125

Phe Asp Phe Ala Phe Ser Leu Asn Val Met Glu His Ile Asp Leu Pro

130 135 140

Asp Glu Ala Val Arg Arg Val Ser Glu Val Leu Lys Pro Gly Ala Ser

145 150 155 160

Tyr His Phe Leu Cys Pro Asn Tyr Val Phe Pro Tyr Glu Pro His Phe

165 170 175

Asn Ile Pro Thr Phe Phe Thr Lys Glu Leu Thr Cys Arg Val Met Arg

180 185 190

His Arg Ile Glu Gly Asn Thr Gly Met Asp Asp Pro Lys Gly Val Trp

195 200 205

Arg Ser Leu Asn Trp Ile Thr Val Pro Lys Val Lys Arg Phe Ala Ala

210 215 220

Lys Asp Ala Thr Leu Thr Leu Arg Phe His Arg Ala Met Leu Val Trp

225 230 235 240

Met Leu Glu Arg Ala Leu Thr Asp Lys Glu Phe Ala Gly Arg Arg Ala

245 250 255

Gln Trp Met Val Ala Ala Ile Arg Ser Ala Val Lys Leu Arg Val His

260 265 270

His Leu Ala Gly Tyr Val Pro Ala Thr Leu Gln Pro Ile Met Asp Val

275 280 285

Arg Leu Thr Lys Arg

290

<210> 28

<211> 432

<212> PRT

<213> Rv1510氨基酸序列

<400> 28

Met Tyr Glu Arg Arg His Glu Arg Gly Met Cys Asp Arg Ala Val Glu

1 5 10 15

Met Thr Asp Val Gly Ala Thr Ala Ala Pro Thr Gly Pro Ile Ala Arg

20 25 30

Gly Ser Val Ala Arg Val Gly Ala Ala Thr Ala Leu Ala Val Ala Cys

35 40 45

Val Tyr Thr Val Ile Tyr Leu Ala Ala Arg Asp Leu Pro Pro Ala Cys

50 55 60

Phe Ser Ile Phe Ala Val Phe Trp Gly Ala Leu Gly Ile Ala Thr Gly

65 70 75 80

Ala Thr His Gly Leu Leu Gln Glu Thr Thr Arg Glu Val Arg Trp Val

85 90 95

Arg Ser Thr Gln Ile Val Ala Gly His Arg Thr His Pro Leu Arg Val

100 105 110

Ala Gly Met Ile Gly Thr Val Ala Ala Val Val Ile Ala Gly Ser Ser

115 120 125

Pro Leu Trp Ser Arg Gln Leu Phe Val Glu Gly Arg Trp Leu Ser Val

130 135 140

Gly Leu Leu Ser Val Gly Val Ala Gly Phe Cys Ala Gln Ala Thr Leu

145 150 155 160

Leu Gly Ala Leu Ala Gly Val Asp Arg Trp Thr Gln Tyr Gly Ser Leu

165 170 175

Met Val Thr Asp Ala Val Ile Arg Leu Ala Val Ala Ala Ala Ala Val

180 185 190

Val Ile Gly Trp Gly Leu Ala Gly Tyr Leu Trp Ala Ala Thr Ala Gly

195 200 205

Ala Val Ala Trp Leu Leu Met Leu Met Ala Ser Pro Thr Ala Arg Ser

210 215 220

Ala Ala Ser Leu Leu Thr Pro Gly Gly Ile Ala Thr Phe Val Arg Gly

225 230 235 240

Ala Ala His Ser Ile Thr Ala Ala Gly Ala Ser Ala Ile Leu Val Met

245 250 255

Gly Phe Pro Val Leu Leu Lys Val Thr Ser Asp Gln Leu Gly Ala Lys

260 265 270

Gly Gly Ala Val Ile Leu Ala Val Thr Leu Thr Arg Ala Pro Leu Leu

275 280 285

Val Pro Leu Ser Ala Met Gln Gly Asn Leu Ile Ala His Phe Val Asp

290 295 300

Arg Arg Thr Gln Arg Leu Arg Ala Leu Ile Ala Pro Ala Leu Val Val

305 310 315 320

Gly Gly Ile Gly Ala Val Gly Met Leu Ala Ala Gly Leu Thr Gly Pro

325 330 335

Trp Leu Leu Arg Val Gly Phe Gly Pro Asp Tyr Gln Thr Gly Gly Ala

340 345 350

Leu Leu Ala Trp Leu Thr Ala Ala Ala Val Ala Ile Ala Met Leu Thr

355 360 365

Leu Thr Gly Ala Ala Ala Val Ala Ala Ala Leu His Arg Ala Tyr Leu

370 375 380

Leu Gly Trp Val Ser Ala Thr Val Ala Ser Thr Leu Leu Leu Leu Leu

385 390 395 400

Pro Met Pro Leu Glu Thr Arg Thr Val Ile Ala Leu Leu Phe Gly Pro

405 410 415

Thr Val Gly Ile Ala Ile His Val Ala Ala Leu Ala Arg Arg Pro Asp

420 425 430

<210> 29

<211> 340

<212> PRT

<213> gmdA氨基酸序列

<400> 29

Met Lys Arg Ala Leu Ile Thr Gly Ile Thr Gly Gln Asp Gly Ser Tyr

1 5 10 15

Leu Ala Glu Leu Leu Leu Ala Lys Gly Tyr Glu Val His Gly Leu Ile

20 25 30

Arg Arg Ala Ser Thr Phe Asn Thr Ser Arg Ile Asp His Leu Tyr Val

35 40 45

Asp Pro His Gln Pro Gly Ala Arg Leu Phe Leu His Tyr Gly Asp Leu

50 55 60

Ile Asp Gly Thr Arg Leu Val Thr Leu Leu Ser Thr Ile Glu Pro Asp

65 70 75 80

Glu Val Tyr Asn Leu Ala Ala Gln Ser His Val Arg Val Ser Phe Asp

85 90 95

Glu Pro Val His Thr Gly Asp Thr Thr Gly Met Gly Ser Met Arg Leu

100 105 110

Leu Glu Ala Val Arg Leu Ser Arg Val His Cys Arg Phe Tyr Gln Ala

115 120 125

Ser Ser Ser Glu Met Phe Gly Ala Ser Pro Pro Pro Gln Asn Glu Leu

130 135 140

Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Gly Ala Ala Lys Val Tyr Ser

145 150 155 160

Tyr Trp Ala Thr Arg Asn Tyr Arg Glu Ala Tyr Gly Leu Phe Ala Val

165 170 175

Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg Gly Glu Thr Phe

180 185 190

Val Thr Arg Lys Ile Thr Arg Ala Val Ala Arg Ile Lys Ala Gly Ile

195 200 205

Gln Ser Glu Val Tyr Met Gly Asn Leu Asp Ala Val Arg Asp Trp Gly

210 215 220

Tyr Ala Pro Glu Tyr Val Glu Gly Met Trp Arg Met Leu Gln Thr Asp

225 230 235 240

Glu Pro Asp Asp Phe Val Leu Ala Thr Gly Arg Gly Phe Thr Val Arg

245 250 255

Glu Phe Ala Arg Ala Ala Phe Glu His Ala Gly Leu Asp Trp Gln Gln

260 265 270

Tyr Val Lys Phe Asp Gln Arg Tyr Leu Arg Pro Thr Glu Val Asp Ser

275 280 285

Leu Ile Gly Asp Ala Thr Lys Ala Ala Glu Leu Leu Gly Trp Arg Ala

290 295 300

Ser Val His Thr Asp Glu Leu Ala Arg Ile Met Val Asp Ala Asp Met

305 310 315 320

Ala Ala Leu Glu Cys Glu Gly Lys Pro Trp Ile Asp Lys Pro Met Ile

325 330 335

Ala Gly Arg Thr

340

<210> 30

<211> 322

<212> PRT

<213> epiA氨基酸序列

<400> 30

Met Asn Ala His Thr Ser Val Gly Pro Leu Asp Arg Ala Ala Arg Val

1 5 10 15

Tyr Ile Ala Gly His Arg Gly Leu Val Gly Ser Ala Leu Leu Arg Thr

20 25 30

Phe Ala Gly Ala Gly Phe Thr Asn Leu Leu Val Arg Ser Arg Ala Glu

35 40 45

Leu Asp Leu Thr Asp Arg Ala Ala Thr Phe Asp Phe Val Leu Glu Ser

50 55 60

Arg Pro Gln Val Val Ile Asp Ala Ala Ala Arg Val Gly Gly Ile Leu

65 70 75 80

Ala Asn Asp Thr Tyr Pro Ala Asp Phe Leu Ser Glu Asn Leu Gln Ile

85 90 95

Gln Val Asn Leu Leu Asp Ala Ala Val Ala Ala Arg Val Pro Arg Leu

100 105 110

Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro Lys Leu Ala Pro Gln Pro

115 120 125

Ile Pro Glu Ser Ala Leu Leu Thr Gly Pro Leu Glu Pro Thr Asn Asp

130 135 140

Ala Tyr Ala Ile Ala Lys Ile Ala Gly Ile Leu Ala Val Gln Ala Val

145 150 155 160

Arg Arg Gln His Gly Leu Pro Trp Ile Ser Ala Met Pro Thr Asn Leu

165 170 175

Tyr Gly Pro Gly Asp Asn Phe Ser Pro Ser Gly Ser His Leu Leu Pro

180 185 190

Ala Leu Ile Arg Arg Tyr Asp Glu Ala Lys Ala Ser Gly Ala Pro Asn

195 200 205

Val Thr Asn Trp Gly Thr Gly Thr Pro Arg Arg Glu Leu Leu His Val

210 215 220

Asp Asp Leu Ala Ser Ala Cys Leu Tyr Leu Leu Glu His Phe Asp Gly

225 230 235 240

Pro Thr His Val Asn Val Gly Thr Gly Ile Asp His Thr Ile Gly Glu

245 250 255

Ile Ala Glu Met Val Ala Ser Ala Val Gly Tyr Ser Gly Glu Thr Arg

260 265 270

Trp Asp Pro Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val

275 280 285

Ser Val Leu Arg Glu Ala Gly Trp Arg Pro Ser Ile Ala Leu Arg Asp

290 295 300

Gly Ile Glu Ala Thr Val Ala Trp Tyr Arg Glu His Ala Gly Thr Val

305 310 315 320

Arg Gln

<210> 31

<211> 243

<212> PRT

<213> Rv1513氨基酸序列

<400> 31

Met Arg Leu Ala Arg Arg Ala Arg Asn Ile Leu Arg Arg Asn Gly Ile

1 5 10 15

Glu Val Ser Arg Tyr Phe Ala Glu Leu Asp Trp Glu Arg Asn Phe Leu

20 25 30

Arg Gln Leu Gln Ser His Arg Val Ser Ala Val Leu Asp Val Gly Ala

35 40 45

Asn Ser Gly Gln Tyr Ala Arg Gly Leu Arg Gly Ala Gly Phe Ala Gly

50 55 60

Arg Ile Val Ser Phe Glu Pro Leu Pro Gly Pro Phe Ala Val Leu Gln

65 70 75 80

Arg Ser Ala Ser Thr Asp Pro Leu Trp Glu Cys Arg Arg Cys Ala Leu

85 90 95

Gly Asp Val Asp Gly Thr Ile Ser Ile Asn Val Ala Gly Asn Glu Gly

100 105 110

Ala Ser Ser Ser Val Leu Pro Met Leu Lys Arg His Gln Asp Ala Phe

115 120 125

Pro Pro Ala Asn Tyr Val Gly Ala Gln Arg Val Pro Ile His Arg Leu

130 135 140

Asp Ser Val Ala Ala Asp Val Leu Arg Pro Asn Asp Ile Ala Phe Leu

145 150 155 160

Lys Ile Asp Val Gln Gly Phe Glu Lys Gln Val Ile Ala Gly Gly Asp

165 170 175

Ser Thr Val His Asp Arg Cys Val Gly Met Gln Leu Glu Leu Ser Phe

180 185 190

Gln Pro Leu Tyr Glu Gly Gly Met Leu Ile Arg Glu Ala Leu Asp Leu

195 200 205

Val Asp Ser Leu Gly Phe Thr Leu Ser Gly Leu Gln Pro Gly Phe Thr

210 215 220

Asp Pro Arg Asn Gly Arg Met Leu Gln Ala Asp Gly Ile Phe Phe Arg

225 230 235 240

Gly Ser Asp

<210> 32

<211> 262

<212> PRT

<213> Rv1514c氨基酸序列

<400> 32

Met Thr Ser Ala Pro Thr Val Ser Val Ile Thr Ile Ser Phe Asn Asp

1 5 10 15

Leu Asp Gly Leu Gln Arg Thr Val Lys Ser Val Arg Ala Gln Arg Tyr

20 25 30

Arg Gly Arg Ile Glu His Ile Val Ile Asp Gly Gly Ser Gly Asp Asp

35 40 45

Val Val Ala Tyr Leu Ser Gly Cys Glu Pro Gly Phe Ala Tyr Trp Gln

50 55 60

Ser Glu Pro Asp Gly Gly Arg Tyr Asp Ala Met Asn Gln Gly Ile Ala

65 70 75 80

His Ala Ser Gly Asp Leu Leu Trp Phe Leu His Ser Ala Asp Arg Phe

85 90 95

Ser Gly Pro Asp Val Val Ala Gln Ala Val Glu Ala Leu Ser Gly Lys

100 105 110

Gly Pro Val Ser Glu Leu Trp Gly Phe Gly Met Asp Arg Leu Val Gly

115 120 125

Leu Asp Arg Val Arg Gly Pro Ile Pro Phe Ser Leu Arg Lys Phe Leu

130 135 140

Ala Gly Lys Gln Val Val Pro His Gln Ala Ser Phe Phe Gly Ser Ser

145 150 155 160

Leu Val Ala Lys Ile Gly Gly Tyr Asp Leu Asp Phe Gly Ile Ala Ala

165 170 175

Asp Gln Glu Phe Ile Leu Arg Ala Ala Leu Val Cys Glu Pro Val Thr

180 185 190

Ile Arg Cys Val Leu Cys Glu Phe Asp Thr Thr Gly Val Gly Ser His

195 200 205

Arg Glu Pro Ser Ala Val Phe Gly Asp Leu Arg Arg Met Gly Asp Leu

210 215 220

His Arg Arg Tyr Pro Phe Gly Gly Arg Arg Ile Ser His Ala Tyr Leu

225 230 235 240

Arg Gly Arg Glu Phe Tyr Ala Tyr Asn Ser Arg Phe Trp Glu Asn Val

245 250 255

Phe Thr Arg Met Ser Lys

260

<210> 33

<211> 298

<212> PRT

<213> Rv1515c氨基酸序列

<400> 33

Met Ser Thr Asn Pro Gly Pro Ala Glu Gly Ala Asn Gln Val Met Ala

1 5 10 15

Gln Glu His Ser Ala Gly Ala Val Gln Phe Thr Ala His Asn Val Arg

20 25 30

Leu Asp Asp Gly Thr Leu Thr Ile Pro Glu Ser Ser Arg Thr Leu Asp

35 40 45

Glu Ser Ser Trp Phe Ile Ser Ala Arg Gly Ile Leu Glu Thr Val Phe

50 55 60

Pro Gly Asp Lys Ser His Leu Arg Leu Ala Asp Val Gly Cys Leu Glu

65 70 75 80

Gly Gly Tyr Ala Val Gly Phe Ala Arg Met Gly Phe Gln Val Leu Gly

85 90 95

Ile Glu Val Arg Glu Leu Asn Met Ala Ala Cys Asn Tyr Ile Lys Ser

100 105 110

Lys Thr Asn Leu Pro Asn Leu Arg Phe Val His Asp Asn Ala Leu Asn

115 120 125

Ile Ala Asn His Gly Leu Phe Asp Thr Val Phe Cys Cys Gly Leu Phe

130 135 140

Tyr His Leu Glu Asn Pro Lys Gln Tyr Leu Glu Thr Leu Ser Ser Val

145 150 155 160

Thr Asn Lys Leu Leu Ile Leu Gln Thr His Phe Ser Ile Ile Asn Arg

165 170 175

Ser Asp Lys Trp Leu Arg Leu Pro Thr Thr Ala Arg Gln Leu Thr Asp

180 185 190

Arg Leu Leu Arg Arg Pro Ala Pro Val Lys Phe Met Leu Ser Ala Pro

195 200 205

Thr Glu His Glu Gly Leu Pro Gly Arg Trp Phe Thr Glu Phe Ser Asp

210 215 220

Asp Arg Ser Phe Gly Gln Arg Asp Thr Ala Lys Trp Ala Ser Trp Asp

225 230 235 240

Asn Arg Arg Ser Phe Trp Ile Gln Arg Glu His Leu Leu Gln Ala Ile

245 250 255

Lys Asp Val Gly Val Asp Leu Val Met Glu Glu Tyr Asp Asn Leu Glu

260 265 270

Pro Ser Ile Ala Glu Ser Leu Leu Gly Gly Ser Tyr Ala Ala Asn Leu

275 280 285

Arg Gly Thr Phe Ile Gly Ile Lys Thr Arg

290 295

<210> 34

<211> 336

<212> PRT

<213> Rv1516c氨基酸序列

<400> 34

Met Ser Pro Gln Leu Cys Pro Lys Val Ser Ile Val Ser Thr Thr His

1 5 10 15

Asn Gln Ala Gly Tyr Ala Arg Gln Ala Phe Asp Ser Phe Leu Asp Gln

20 25 30

Gln Thr Asp Phe Pro Val Glu Ile Ile Val Ala Asp Asp Ala Ser Thr

35 40 45

Asp Ala Thr Pro Ala Ile Ile Arg Glu Tyr Ala Glu Arg Tyr Pro His

50 55 60

Val Phe Arg Pro Ile Phe Arg Thr Glu Asn Leu Gly Leu Asn Gly Asn

65 70 75 80

Leu Thr Gly Ala Leu Ser Ala Ala Arg Gly Glu Tyr Val Ala Leu Cys

85 90 95

Glu Ala Asp Asp Tyr Trp Ile Asp Pro Leu Lys Leu Ser Lys Gln Val

100 105 110

Ala Phe Leu Asp Arg His Pro Lys Thr Thr Val Cys Phe His Pro Val

115 120 125

Arg Val Ile Trp Glu Asp Gly His Ala Lys Asp Ser Lys Phe Pro Pro

130 135 140

Val Arg Val Arg Gly Asn Leu Ser Leu Asp Ala Leu Ile Leu Met Asn

145 150 155 160

Phe Ile Gln Thr Asn Ser Ala Val Tyr Arg Arg Leu Glu Arg Tyr Asp

165 170 175

Asp Ile Pro Ala Asp Val Met Pro Leu Asp Trp Tyr Leu His Val Arg

180 185 190

His Ala Val His Gly Asp Ile Ala Met Leu Pro Asp Thr Met Ala Val

195 200 205

Tyr Arg Arg His Ala Gln Gly Met Trp His Asn Gln Val Val Asp Pro

210 215 220

Pro Lys Phe Trp Leu Thr Gln Gly Pro Gly His Ala Ala Thr Phe Asp

225 230 235 240

Ala Met Leu Asp Leu Phe Pro Gly Asp Pro Ala Arg Glu Glu Leu Ile

245 250 255

Ala Val Met Ala Asp Trp Ile Leu Arg Gln Ile Ala Asn Val Pro Gly

260 265 270

Pro Glu Gly Arg Ala Ala Leu Gln Glu Thr Ile Ala Arg His Pro Arg

275 280 285

Ile Ala Met Leu Ala Leu Gln His Arg Gly Ala Thr Pro Ala Arg Arg

290 295 300

Leu Lys Thr Gln Trp Arg Lys Leu Ala Ala Ala Thr Pro Ser Arg Arg

305 310 315 320

Gly Leu Val Asp Val Trp Pro Ser Arg Leu Arg Arg Gly Cys Arg Ala

325 330 335

Claims

1.一种编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列，其特征在于，

(1)所述核苷酸序列与SEQ ID NO.1具有至少70％，至少80％，至少90％，至少95％，至少98％，至少99％或100％同一性；或，

2.一种重组质粒，其特征在于，所述重组质粒是将编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列插入到大肠杆菌-分枝杆菌穿梭质粒中构成的重组质粒。

3.根据权利要求2所述的重组质粒，其特征在于，编码包含结核分枝杆菌RD4区完整或部分基因的核苷酸序列可来源于结核分枝杆菌的不同菌株，优选权利要求1所述核苷酸序列；所述核苷酸序列可通过PCR扩增或人工合成获得。

4.一种重组卡介苗菌株，其特征在于，所述卡介苗菌株包含权利要求1所述核苷酸序列或权利要求2或3所述重组质粒。

5.一种重组卡介苗疫苗，其特征在于，所述疫苗包含权利要求4所述重组卡介苗菌株或其组合；以及药学上可接受的佐剂或缓冲液体系。

6.一种重组卡介苗菌株的制备方法，其特征在于，包括以下步骤：(1).扩增或人工合成编码完整或部分结核分枝杆菌RD4区基因的核苷酸序列；(2).将编码完整或部分RD4区蛋白的核苷酸序列插入到大肠杆菌-分枝杆菌穿梭质粒的序列中，构建重组质粒；(3).将步骤(2)获得的重组质粒转化入卡介苗菌株中，即得到重组卡介苗菌株；步骤(1)所述核苷酸序列优选权利要求1所述核苷酸序列。

7.权利要求1所述核苷酸序列、权利要求2或3所述重组质粒、和/或权利要求4所述菌株在制备抗致病性分枝杆菌感染的疫苗中的应用。

8.一组多肽，其特征在于，所述多肽由权利要求1所述核苷酸序列编码产生：

(1)所述多肽包含Rv1501(SEQ ID NO.17)、Rv1502(SEQ ID NO.18)、Rv1503(SEQ IDNO.19)、Rv1504c(SEQ ID NO.20)、Rv1505(SEQ ID NO.21)、Rv1506c(SEQ ID NO.22)、Rv1507c(SEQ ID NO.23)、Rv1507A(SEQ ID NO.24)、Rv1508c(SEQ ID NO.25)；或，

(2)所述多肽包含Rv1501(SEQ ID NO.17)、Rv1502(SEQ ID NO.18)、Rv1503(SEQ IDNO.19)、Rv1504c(SEQ ID NO.20)、Rv1505(SEQ ID NO.21)、Rv1506c(SEQ ID NO.22)、Rv1507c(SEQ ID NO.23)、Rv1507A(SEQ ID NO.24)、Rv1508c(SEQ ID NO.25)、Rv1508A(SEQID NO.26)、Rv1509(SEQ ID NO.27)、Rv1510(SEQ ID NO.28)、gmdA(SEQ ID NO.29)、epiA(SEQ ID NO.30)、Rv1513(SEQ ID NO.31)、Rv1514c(SEQ ID NO.32)、Rv1515c(SEQ IDNO.33)、Rv1516c(SEQ ID NO.34)。

9.权利要求1所述核苷酸序列、权利要求2或3所述重组质粒、和/或权利要求8所述多肽在制备抗致病性分枝杆菌感染的基因工程亚单位疫苗中的应用。

10.根据权利要求7或9所述应用，其特征在于，所述致病性分枝杆菌选自结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌、溃疡分枝杆菌、海洋分枝杆菌中的一种或几种的组合。