CN1703513A - 包括表达丙氨酸脱氢酶、丝氨酸脱水酶和/或谷氨酰胺合成酶的重组卡介苗菌株的结核病疫苗 - Google Patents
包括表达丙氨酸脱氢酶、丝氨酸脱水酶和/或谷氨酰胺合成酶的重组卡介苗菌株的结核病疫苗 Download PDFInfo
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Abstract
本发明涉及一种含有可表达一种核酸的活的重组牛型结核菌卡介苗(Mycobacterium bovis－BCG)菌株，该核酸编码至少一种具有丙氨酸脱氢酶活性、谷氨酰胺合成酶活性或丝氨酸脱水酶活性的蛋白质或多肽。
Description
技术领域
本发明涉及结核病(tuberculosis，TB)疫苗。
背景技术
结核病是一种由侵染性病原体结核杆菌(Mycobacterium tuberculosis)引起的致死性传染病。该病每年引起二百万人的死亡。世界卫生组织(WHO)2001年年报估计在1997年有八百万新增结核病人，而在1999年新增的结核病人达到八百四十万。若按这种趋势发展，估计从2000年至2020年将新增约十亿带菌者，其中二亿人将发病，三千五百万人将死于结核病。HIV/AIDS的传播和对多种药物有抗性的结核病的出现使该病变得更加糟糕。卡介苗(Bacille Calmelle-Guerin，BCG)，一种牛型结核菌(Mycobacteriumbovis)的弱毒株，是目前唯一能得到的预防结核病的疫苗。在侵染性动物模型中，证实卡介苗疫苗能诱导对结核杆菌的保护性免疫反应(Baldwins et al.，1998)。人体实验也证明卡介苗疫苗能持续的保护人在儿童期免受结核病，特别是脑膜炎。然而卡介苗疫苗的保护作用存在争议，因为它在保护成年人免受肺结核病的有效性方面变化很大(Fine，1989；Colditz et al.，1994；Sterneet al.，1998)。在二十世纪四十年代和五十年代该疫苗在发达国家，如英国、丹麦和北美在实施应用上证明是很有效的(有效率为70～80％)。而七十年代在印度的一次规模最大，涉及265,000人的一次临床应用中，卡介苗疫苗在保护人们免受肺结核病方面没有检测到它的保护作用。因此研究一种替代BCG的改进疫苗并用于预防结核病是一个很迫切的课题。
对卡介苗保护作用的变异性有几种解释(Anderson，2001)。最突出的假说是暴露于环境中的分枝杆菌使寄主对它的抗性变得敏感，因此人们获得异源免疫性，这使疫苗的潜在好处不明显(Fine 1995；Fine and Vynnycky1998)。另外一个最近的研究显示对那些被环境中分枝杆菌致敏的动物，卡介苗的增殖被抑制，结果卡介苗疫苗只诱导出暂时的免疫反应，而不能提供抗结核病的保护性免疫反应(Brandt et al.，2002)。这一研究也支持长期观察的结果，即对结核病免疫性的诱导需要卡介苗的生产性侵染。卡介苗是一种活疫苗，灭活的卡介苗无保护作用。象结核杆菌一样卡介苗在被侵染寄主的不同组织中能够形成肉芽瘤和脓肿(Hogan et al.，2001)。结核杆菌和牛型结核菌卡介苗能够在肉芽瘤，一种缺乏营养和低氧压的恶劣环境中生存。这种能力可能是由于细菌能改变代谢适应有限的碳、氮和能源资源(Barclay andWheeler，1989)。一个最近的研究显示脂肪酸作为碳源是结核杆菌在小鼠体内持续生存和激活巨噬细胞所必需的(Mckinney et al.，2000)。在具免疫活性的个体中继续接种疫苗，卡介苗可能在寄主体中持续存在一段时间，然后被寄主清除(Dann and North 1995；Lagranderie et al.，1996；Moisan et al.，2001)。
开发一种新的有效的卡介苗疫苗的关键是提供长期的保护作用(Orme，2001；Young，2000)。现有的卡介苗疫苗在孩子中能抗结核病从而起保护作用，但10至15年后它们的效力减弱了，可能是由于卡介苗疫苗引起的保护性免疫反应逐渐消失(Orme，2001)。发展改进型疫苗的新策略包括使用弱化的分枝杆菌、亚单位疫苗和DNA疫苗(Andern，2001)。然而它们中还没有一种证明比卡介苗更有潜力或更有效。牛型结核菌卡介苗的存活和生长是诱导保护性免疫反应所必需的。实验证明用异烟肼对侵染的小鼠进行早期处理抑制细菌的生长，结果阻止了获得性抗性的发展。为了获得更有效的疫苗，在寄主体内能够长期持续存活的卡介苗菌株是必需的。因此需要新的卡介苗重组菌株。
发明内容
本发明提供的疫苗克服了卡介苗菌株利用自然产生的氨基酸作为生长碳源有限能力。另外即使在铵存在时，L-丙氨酸、D-丙氨酸或L-丝氨酸仍然抑制卡介苗菌株的生长。在卡介苗菌株中表达功能性丙氨酸脱氢酶[SEQ IDNO：1；SEQ ID NO：2]可解除丙氨酸对卡介苗的生长抑制作用。同样在卡介苗菌株中表达功能性L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]可解除L-丝氨酸对卡介苗的生长抑制作用。这种抑制作用的机制是通过阻滞谷氨酰胺合成酶而实现的。在卡介苗中谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ IDNO：14]的过表达可解除丙氨酸和L-丝氨酸对卡介苗的生长抑制作用。表达或过表达功能性丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]、功能性L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]和/或谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的重组卡介苗菌株在寄主体内存活和持续存在的时间更长，从而诱导长期的保护性免疫反应。这种持续性重组卡介苗菌株提供更有效的抗结核病和抗其它分枝杆菌侵染的疫苗。
本发明涉及一种重组牛型结核菌卡介苗菌株，这种重组牛型结核菌卡介苗表达一种编码丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]、L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]和/或谷氨酰胺合成酶[SEQ ID NO：7]到[SEQID NO：14]的DNA。我们发现由于缺乏功能性丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]，卡介苗不能利用丙氨酸(L-丙氨酸或D-丙氨酸)作为生长的唯一氮源。我们进一步发现丙氨酸(L-丙氨酸或D-丙氨酸)抑制所有的卡介苗疫苗株的生长。所述的抑制作用可通过在卡介苗中表达功能性丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]而被解除。同样卡介苗菌株不能利用L-丝氨酸作为生长的唯一氮源，并且卡介苗的生长被L-丝氨酸抑制。在卡介苗菌株中表达L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]可解除L-丝氨酸引起的生长抑制作用。
丙氨酸(L-丙氨酸或D-丙氨酸)和L-丝氨酸对卡介苗生长的抑制作用可能是阻滞了谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的活性。在卡介苗菌株中过表达谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]可解除丙氨酸和L-丝氨酸对卡介苗的生长抑制作用。谷氨酰胺合成酶和谷氨酸合成酶提供谷氨酰胺和谷氨酸，它们对于所有氨基酸、蛋白质、嘌呤和嘧啶的生物合成都至关重要。谷氨酰胺合成酶的抑制使细胞生长停止。提供能转化为谷氨酸的氨基酸，如L-谷氨酰胺、L-谷氨酸、L-天冬酰胺和L-天冬氨酸能解除这种抑制。确实，我们的实验数据显示丙氨酸(L-丙氨酸或D-丙氨酸)和L-丝氨酸对卡介苗的生长抑制能通过在培养基中添加L-谷氨酰胺、L-谷氨酸、L-天冬酰胺或L-天冬氨酸而解除。
因为卡介苗是活疫苗，表达或过表达丙氨酸脱氢酶[SEQ ID NO：1；SEQID NO：2]、L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]和/或谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]能使它在人寄主体内存活更长时间，因而能诱导长期记忆免疫。这些重组卡介苗菌株提供了非常有用的疫苗。
本发明涉及一种活的重组牛型结核菌卡介苗菌株，它含有一种能表达的核酸，该核酸编码至少一种具有丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]活性、谷氨酰胺合成酶[SEQ ID NO：7到SEQ ID NO：14]活性或L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]活性的蛋白质或多肽。
本发明还涉及一种活的重组牛型结核菌卡介苗菌株，它含有一种能表达的核酸，该核酸编码至少一种选自丙氨酸脱氢酶[SEQ ID NO：1；SEQ IDNO：2]、谷氨酰胺合成酶[SEQ ID NO：7到SEQ ID NO：14]和L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]所组成的组中的蛋白质或多肽。
本发明进一步涉及一种活的重组牛型结核菌卡介苗菌株，它含有一种能表达的核酸，该核酸含有至少一种全部或部分的核酸分子选自如下组中：[SEQ ID NO：1]、[SEQ ID NO：2]、[SEQ ID NO：5]、[SEQ ID NO：6]、[SEQ IDNO：7]、[SEQ ID NO：8]、[SEQ ID NO：9]、[SEQ ID NO：10]、[SEQ ID NO：11]、[SEQ ID NO：12]、[SEQ ID NO：13]和[SEQ ID NO：14]。
在一个具体的实施方式中，活的重组卡介苗疫苗选自：牛型结核菌-BCG-Russia(Mycobaxterium bovis-BCG-Russia)、牛型结核菌-BCG-Moreau(Mycobaxterium bovis-BCG-Moreau)、牛型结核菌-BCG-Japan(Mycobaxteriumbovis-BCG-Japan)、牛型结核菌-BCG-Sweden(Mycobaxterium bovis-BCG-Sweden)、牛型结核菌-BCG-Birkhaug(Mycobaxterium bovis-BCG-Birkhaug)、牛型结核菌-BCG-Prague(Mycobaxterium bovis-BCG-Prague)、牛型结核菌-BCG-Glaxo(Mycobaxterium bovis-BCG-Glaxo)、牛型结核菌-BCG-Denmark(Mycobaxterium bovis-BCG-Denmark)、牛型结核菌-BCG-Tice(Mycobaxterium bovis-BCG-Tice)、牛型结核菌-BCG-Frappier(Mycobaxterium bovis-BCG-Frappier)、牛型结核菌-BCG-Connaught(Mycobaxterium bovis-BCG-Connaught)、牛型结核菌-BCG-Phipps(Mycobaxterium bovis-BCG-Phipps)和牛型结核菌-BCG-Pasteur(Mycobaxterium bovis-BCG-Pasteur)所组成的组中。
本发明的另一方面提供一种含有一个活的重组牛型结核菌卡介苗菌株的药物组合物，该菌株含有一种能表达的核酸，该核酸编码至少一种具有丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]活性、谷氨酰胺合成酶[SEQ IDNO：7到SEQ ID NO：14]活性和L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]活性的蛋白质或多肽。
本发明还涉及一种活的重组牛型结核菌卡介苗菌株，它含有一种能表达的核酸，该核酸编码至少一种选自丙氨酸脱氢酶[SEQ ID NO：1；SEQ IDNO：2]、谷氨酰胺合成酶[SEQ ID NO：7到SEQ ID NO：14]和L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]所组成的组中的蛋白质或多肽。
本发明的另一方面提供一种含有一个活的重组牛型结核菌卡介苗菌株的药物组合物，该菌株含有一种能表达的核酸，该核酸含有至少一种选自如下组中的核酸分子的全部或部分：[SEQ ID NO：1]、[SEQ ID NO：2]、[SEQ IDNO：5]、[SEQ ID NO：6]、[SEQ ID NO：7]、[SEQ ID NO：8]、[SEQ ID NO：9]，[SEQ ID NO：10]、[SEQ ID NO：11]、[SEQ ID NO：12]、[SEQ ID NO：13]和[SEQID NO：14]。
本发明的另一方面也提供一种疫苗或致免疫的组合物，用于治疗或预防哺乳动物免受分枝杆菌的侵害。它含有一个活的重组牛型结核菌卡介苗菌株，该菌株含有一种能表达的核酸，该核酸编码至少一种具有丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]活性、谷氨酰胺合成酶[SEQ ID NO：7到SEQ IDNO：14]活性或L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]活性的蛋白质或多肽。
另一方面，本发明也提供一种疫苗或致免疫的组合物，用于治疗或预防哺乳动物免受分枝杆菌的侵害。它含有一个活的重组牛型结核菌卡介苗菌株，该菌株含有一种能表达的核酸，该核酸编码至少一种选自丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]、谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ IDNO：14]和L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]所组成的组中的蛋白质或多肽。
本发明的另一方面也提供一种疫苗或致免疫的组合物，用于治疗或预防哺乳动物免受分枝杆菌的侵害。它含有一个活的重组牛型结核菌卡介苗菌株，该菌株含有一种能表达的核酸，该核酸含有至少一种选自如下组中的核酸分子的全部或部分：[SEQ ID NO：1]、[SEQ ID NO：2]、[SEQ ID NO：5]、[SEQ ID NO：6]、[SEQ ID NO：7]、[SEQ ID NO：8]、[SEQ ID NO：9]、[SEQ IDNO：10]、[SEQ ID NO：11]、[SEQ ID NO：12]、[SEQ ID NO：13]和[SEQ IDNO：14]。在一个优选的具体实施方式中，该疫苗或致免疫的组合物，能用于治疗或预防哺乳动物免受分枝杆菌的侵害。在另一个优选的具体实施方式中，本发明的疫苗或产生免疫的组合物，进一步含有药物上可接受的载体。在另一个优选的具体实施方式中，该疫苗或致免疫的组合物，进一步含有佐剂。在另一个具体实施方式中，该疫苗或致免疫的组合物，进一步含有来源于一个或多个其它病原体的致免疫的物质。
本发明另一方面涉及一种用于治疗或预防哺乳动物免受结核杆菌或牛型结核菌侵害的方法。其中包括给哺乳动物供给本发明的疫苗或致免疫的组合物。一个具体实施方式下哺乳动物是牛，另一个具体实施方式下哺乳动物是人。还有一种具体实施方式中，疫苗或致免疫的组合物是与佐剂一起给予的。
本发明进一步的方面是一种用于治疗或预防哺乳动物免受癌症的方法。其中包括给哺乳动物施用本发明的疫苗或致免疫的组合物。一种具体实施方式下癌症为膀胱癌。另一种具体实施方式中，疫苗或致免疫的组合物是与佐剂一起给予的。
本发明也涉及一个含有本发明的活的重组卡介苗菌株的测试试剂盒。
本发明进一步涉及一种抑制牛型结核菌卡介苗生长的培养基成分，它包括丙氨酸作为唯一生长氮源。另一种具体实施方式中，丝氨丝是唯一的生长氮源。另一具体实施方式中，本发明的培养基组合物还包括一种碳源、铁、镁和SO4。在另一种具体实施方式中，碳源选自甘油、右旋糖(dextrose)、柠檬酸盐和葡萄糖(glucose)所组成的组中。
本发明涉及一种抑制牛型结核菌卡介苗生长的方法，其中包括如下的步骤：(a)获得一个含有分枝杆菌的样品；和(b)在选择性培养基上培养该样品。一种具体实施方式中，选择培养基是以丙氨酸为唯一氮源。另一种具体实施方式中，选择培养基是以丝氨酸为唯一氮源。
本发明另一方面涉及一种培养牛型结核菌卡介苗的方法，其中包括如下步骤：(a)获得一个含有分枝杆菌的样品；和(b)在不同的培养基上培养该样品。一种具体实施方式中，该培养基含有组氨酸。
附图说明
本发明的优选实施方式将结合以下附图进行描述。
图1、ald基因的克隆
首先一个4.5kb的含有ald基因[SEQ ID NO：1]的结核杆菌基因组DNASca I片段被连到线性化的Ecl136II pUC19，产生pUC-ALD。然后连接含有ald基因[SEQ ID NO：1]的1.9kb的KpnI片段到线性化的KpnI pMD31，产生分枝杆菌质粒pALD。
图2、sdaA基因的克隆
sdaA基因[SEQ ID NO：5]的克隆由两步完成。首先9.5kb BamHI结核杆菌基因组DNA片段被连到线性化的BamHI pMD31，产生pSDA1。然后用PstI断裂pSDA1，随后通过10.9kb PstI片段的自连产生质粒pSDAA。
图3、在GAS中L-丙氨酸对卡介苗生长的抑制
在7H9/ADC/甘油/吐温-80液体培养基中BCG-Japan、BCG-Frappier、BCG-Pasteur生长到稳定阶段，分别接种到两份5ml培养体积的GAS、无L-丙氨酸的GAS和添加了27mM L-天冬酰胺的GAS中。然后培养到细胞密度为每毫升2×107个细胞。在37℃不断摇动培养16天，然后取2ml细胞培养液离心，将细胞沉淀冻干，称细胞干重。
图4、在含有NH4Cl(5g/L)的Sauton中增加L-丙氨酸的浓度抑制卡介苗的生长
在7H9/ADC/甘油/吐温-80液体培养基中a)BCG-Japan、b)BCG-Frappier和c)BCG-Pasteur生长到稳定阶段。洗涤细胞，然后重新悬浮于Sauton基础培养基(无氮源)中。重新悬浮的每种菌株的细胞接种到两份5ml培养体积的添加了NH4Cl的Sauton培养基和浓度增加的L-丙氨酸的Sauton培养基中。培养物在37℃不断摇动培养30天，然后称细胞干重。
图5、在GAS中D-丙氨酸对卡介苗生长的抑制
在7H9/ADC/甘油/吐温-80液体培养基中BCG-Japan、BCG-Frappier、BCG-Pasteur生长到稳定阶段，分别接种到5ml培养体积的以D-丙氨酸替代L-丙氨酸的GAS、不含L-丙氨酸的GAS和添加27mM L-天冬酰胺的GAS(含D-丙氨酸)。然后培养到细胞密度为每毫升2×107个细胞。培养物在37℃不断摇动培养13天，然后称细胞干重。
图6、在GAS培养基中表达丙氨酸脱氢酶[SEQ ID NO：1]的重组卡介苗菌株的生长
对BCG--Frappier/ald、BCG--Pasteur/ald、BCG--Frappier/pMD31、BCG--Pasteur/pMD31、BCG--Frappier和BCG--Pasteur的生长进行了比较。在7H9/ADC/甘油/吐温-80液体培养基中，不同菌株的细胞培养到稳定阶段，洗涤。然后重新悬浮于Sauton基础培养基(无氮源)中。重新悬浮的细胞接种到两份5ml培养体积的无L-丙氨酸的GAS、含L-丙氨酸的GAS和以D-丙氨酸替代L-丙氨酸的GAS中。培养物在37℃不断摇动培养15天，然后称细胞干重。
图7、在GAS中L-丝氨酸对卡介苗生长的抑制
在7H9/ADC/甘油/吐温-80液体培养基中BCG-Japan、BCG-Frappier和BCG-Pasteur生长到稳定阶段，分别接种到两份5ml培养体积的以L-丝氨酸替代L-丙氨酸的GAS、无L-丙氨酸的GAS和添加27mM L-天冬酰胺的GAS(含L-丝氨酸)中。然后培养到细胞密度为每毫升2×107个细胞。在37℃不断摇动培养15天，然后称细胞干重。
图8、在含有L-丝氨酸的GAS培养基中表达L-丝氨酸脱水酶[SEQ IDNO：5]的重组卡介苗菌株的生长
对BCG-Japan/sdaA、BCG-Frappier/sdaA、BCG-Pasteur/sdaA、BCG-Japan、BCG-Frappier和BCG-Pasteur的生长进行了比较。在7H9/ADC/甘油/吐温-80液体培养基中，不同菌株的细胞培养到稳定阶段，洗涤细胞。然后重新悬浮于Sauton基础培养基(无氮源)中。重新悬浮的细胞接种到两份5ml培养体积的无L-丙氨酸的GAS、以L-丝氨酸替代L-丙氨酸的GAS和添加27mM L-天冬酰胺的GAS(含L-丝氨酸)中。培养物在37℃不断摇动培养15天，然后称细胞干重。
图9、结核杆菌(M.tb)的ald基因[SEQ ID NO：1；SEQ ID NO：2]和牛型结核菌(M.bovis)的ald基因[SEQ ID NO：3；SEQ ID NO：4]的A]核苷酸和B]氨基酸序列图
在牛型结核菌ald基因[SEQ ID NO：3]中，点的缺失引起的移码突变用箭头指示。这一突变对核苷酸密码子和氨基酸的影响的部分被加重。
具体实施方式
卡介苗疫苗株利用氨基酸作为生长氮源的能力是有限的。另外我们发现自然产生的氨基酸L-丙氨酸和L-丝氨酸抑制卡介苗菌株的生长。在卡介苗中功能性L-丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]的表达可解除由丙氨酸引起的生长抑制。在卡介苗中表达功能性L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]可解除由L-丝氨酸引起的生长抑制。另外过表达谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]可解除由丙氨酸和丝氨酸引起的生长抑制。这些新发现是很有意义的，因为表达或过表达丙氨酸脱氢酶[SEQ IDNO：1；SEQ ID NO：2]，L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]和/或谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的重组卡介苗菌株将在作为寄主的人体内存活得更好，能够诱导长期的免疫记忆，提供更有效的预防结核病的疫苗，特别是在成年人中保护他们免受肺结核病侵害。
早就知道灭活的卡介苗菌株的投放只能引起弱的暂时性免疫反应。保护性免疫反应要求卡介苗在被接种疫苗的寄主体内存活和繁殖。这一观点被最近的一个侵染性动物模型的研究所证实。该研究显示预先暴露于活的环境中的分枝杆菌阻滞卡介苗在侵染的小鼠体内增殖。结果卡介苗只诱导暂时的免疫反应，不能提供抗结核病的保护性免疫反应(Brandt et al.，2002)。活的卡介苗不断的分泌多种不同的抗原，这可能对于诱导保护性免疫反应很重要。增殖的卡介苗不断地产生大量抗原使活疫苗优于亚单位疫苗和DNA疫苗，它们只瞬时产生少量的抗原。因此卡介苗在寄主体内增殖和持续存在是卡介苗有效性的重要决定因素。
为了在寄主体内生长和持续存在，卡介苗必需能利用寄主体内能够得到的营养物质。脂肪酸分解代谢的一个主要的酶-异柠檬酸裂合酶，是在侵染的慢性阶段结核杆菌持续存在所要求的。这一要求依赖于寄主的完全免疫反应(McKinney et al.，2000)。在另一研究中，缺乏一种硝酸盐呼吸作用所需的主要的酶-厌氧硝酸盐还原酶的牛型结核菌卡介苗菌株不能在免疫耐受小鼠的肺、肝和肾组织中持续存在(Fritz et al.，2002)。我们发现，卡介苗菌株只能利用少数的几种氨基酸作为生长的氮源，所有的卡介苗菌株的生长都被自然产生的L-丙氨酸和L-丝氨酸所抑制，说明卡介苗在寄主体内生长和持续存在的能力受到限制。在人体内卡介苗生长能够得到的L-丙氨酸的浓度估计为0.33～0.42mM(Barclay and Wheeler，1989)，这个浓度足够抑制BCG-Pasteur或BCG-Frappier的生长，并可显著地降低BCG-Japan的生长(图4)。在寄主细胞外液存在的L-丝氨酸的浓度为大约0.1mM(Barclay andWheeler，1989)，这个浓度可显著地抑制卡介苗的生长。因为卡介苗的增殖是产生保护性免疫反应所必需的，在寄主体内氨基酸的这种抑制可能阻止长效保护性免疫的发展，因此不能保护成年人免受肺结核病。
牛型结核菌卡介苗也用于治疗膀胱癌。大量随机临床实验表明膀胱内注入卡介苗可阻止或延迟肿瘤的复发(回顾Lamm，2000；Lockyer and Gillatt，2001)。为什么卡介苗能发挥这种作用还不清楚。然而抗癌反应需要完整的T-细胞反应。涉及增加的包括TNFα和IL-6 Thl-型细胞因子的表达(回顾Prescott et al.，2000)。最有效的治疗状况包括卡介苗的增殖放大，这说明延长暴露于细菌的时间是需要的。同样，对于保持吞噬卡介苗能力的肿瘤对这种处理最易受影响(de Boer et al 1996)，说明细菌与肿瘤的相互作用是重要的。因此证明增加持久性的卡介苗菌株可能提供增强的抗肿瘤能力。
我们的研究表明卡介苗不能利用丙氨酸(L-丙氨酸或D-丙氨酸)作为唯一氮源的原因是它缺乏功能性丙氨酸脱氢酶[SEQ ID NO：1；SEQ IDNO：2]。一个编码L-丙氨酸脱氢酶(ald)[SEQ ID NO：1]的基因(Rv2708)在结核杆菌基因组中被鉴定。来源于结核杆菌的这种酶的活性在体外用生化方法得到证明。Ald分解L-丙氨酸产生丙酮酸盐和铵，对L-丙氨酸具有高度特异性(Hutter and Singh，1999)。这种酶在结核杆菌的培养上清液中检测到，而在牛型结核菌BCG-Japan和BCG-Copenhagen中未检测到，尽管通过DNASouthern印记显示在这两株菌中均存在该基因(Anderson et al.，1992)。同样我们没有在本报告中12个卡介苗菌株的任何一个中检测到丙氨酸脱氢酶活性(数据没有列出)。在这些卡介苗菌株中丙氨酸脱氢酶功能的缺失可能是在ald基因[SEQ ID NO：3]内出现了突变，这可能起源于牛型结核菌菌株。在发表的牛型结核菌基因组序列[SEQ ID NO：3]中在ald基因内发现了一个移码突变(图9)。因此在卡介苗菌株中不能产生全长的L-丙氨酸脱氢酶[SEQID NO：2；SEQ ID NO：4]，结果导致卡介苗不能分解丙氨酸。同样卡介苗不能利用L-丝氨酸作为唯一氮源可能是由于编码L-丝氨丝脱水酶的基因sdaA[SEQ ID NO：5；SEQ ID NO：6]发生了突变或表达的改变。在卡介苗中表达结核杆菌的sdaA基因[SEQ ID NO：5；SEQ ID NO：6]使卡介苗菌株能以L-丝氨酸为唯一氮源生长，解除L-丝氨酸对卡介苗生长的抑制(图8)。丙氨酸和丝氨酸对卡介苗生长的抑制是由谷氨酰胺合成酶[SEQ ID NO：7]到[SEQID NO：14]的抑制引起的。在卡介苗中过表达谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]可解除L-丝氨酸、L-丙氨酸和D-丙氨酸引起的生长抑制。
BCG-Frappier和BCG-Pasteur较BCG-Japan更易受到丙氨酸的抑制，可能是由于谷氨酰胺合成酶的表达水平或活性的不同。BCG-Japan与BCG-Frappier或BCG-Pasteur存在遗传差异(Behr et al.，1999)。经过13年对牛型结核菌的一个分离株在体外经过230次传代Calmette和Guérin在1921年得到了卡介苗疫苗。从1924年开始卡介苗被传到世界许多实验室。这些实验室用不同的方法和处理在体外对该菌传代，直到1961年冻干得到菌种的方法被建立。这种实践的结果是不同的卡介苗亚株被建立，它们在生化和遗传上存在差异(Oettinger et al.，1999；Behr et al.，1999)。我们的数据显示卡介苗利用氨基酸作为氮源的能力是不同的，例如BCG-Japan能够利用包括L-精氨酸和L-赖氨酸的碱性氨基酸生长，而BCG-Pasteur和BCG-Frappier则不能。这些不同可能导致在不同的临床应用中卡介苗的有效性不同。
总而言之，我们用表达功能性丙氨酸脱氢酶[SEQ ID NO：1；SEQ IDNO：2]、功能性L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]和/或谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的重组卡介苗菌株作为疫苗预防结核病和其它分枝杆菌的侵染。这些重组卡介苗疫苗将诱导抵抗结核病的长期保护性免疫反应。
核酸分子的改变
修饰
在本申请中公开的核酸分子DNA序列可进行多种修饰，这对本领域的技术人员来说是显而易见的。本发明包括在本申请中公开的能够在细菌和哺乳动物细胞中指导表达的序列(或片段)的核苷酸修饰。这些修饰包括核苷酸的替换、插入或缺失，或改变核苷酸的相对位置或顺序。
核酸分子可编码在丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶中变化的保守性氨基酸。本发明包括功能相当的核酸分子，它们编码在丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶中变化的保守性氨基酸和产生在丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶中变化沉默氨基酸。从经验上鉴定保守性氨基酸组的方法已有报道(如，Wu，thomas D.“DiscoveringEmperically Conserved Amino Acid Substitution Groups in Databases of Proteinfamilies”)(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi？cmd＝Retrieve&db＝PubMed&list-uids＝887723&dopt＝Abstract)。
在丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶中核酸分子可编码非保守性氨基酸的替代、插入或缺失。本发明包括功能相当的核酸分子，这些核酸分子导致在[SEQ ID NO：2、6、8、10、12 or 14]中的氨基酸序列内的非保守氨基酸的变化。功能相当的核酸分子包括编码多肽的DNA和RNA，多肽和蛋白质含有非保守氨基酸替代(优选化学性质相近的替代)、插入或缺失。但它们仍保持与[SEQ ID NO：2]所示的丙氨酸脱氢酶、[SEQ ID NO：8、10、12或14]所示的谷氨酰胺合成酶或[SEQ ID NO：6]所示的L-丝氨酸脱水酶相同或相近的丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶活性。
这些DNA或RNA可编码丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶的片段或变异物。
这些片段可用作免疫剂或产生免疫的组合物。
这些片段和变异物的丙氨酸脱氢酶，谷氨酰胺合成酶或L-丝氨酸脱水酶的类似活性已被以下描述的分析所证实。
序列一致性
本发明的这些核酸分子还包括具有与本发明的一个能在细菌或哺乳动物细胞内表达的核酸分子至少约：60％一致性、70％一致性、80％一致性、90％一致性、95％一致性、96％一致性、97％一致性、98％一致性、或最优选的含有99％或99.5％一致性的核酸分子(或其片段)。一致性反映的是线性的两个核苷酸序列的相似性，因此能够得到它们的最佳匹配。一致性是根据现有技术已知的方法计算的。例如一个核苷酸序列(称为序列A)与[SEQ IDNO：1]的一部分有90％的一致性，就是说序列A与[SEQ ID NO：1]的这部分一致而仅在这一部分中每100个核苷酸中可能包括最多10个突变(如被其它核苷酸替代)。
序列一致性(优选每个结构没有编码核酸分子的插入)优选在SEQ IDNO：1到SEQ ID NO：14中提供的序列或它们的互补序列具有至少70％一致性、至少80％一致性、至少90％一致性、至少95％一致性、至少96％一致性、至少97％一致性、至少98％一致性、或最优选的含有至少99％或99.5％的一致性。序列一致性的计算优选生物信息学的GCG程序(威斯康星大学)。其它程序也可用于序列一致性的计算，如Clustal W程序(优选用缺损参数；Thompson，JD et al.，Nucleic Acid Res.22：4673-4680)，BLAST P，BLAST X算法，在基因组研究所的鸟结核分枝杆菌(Mycobacterium avium)BLASTN(http:tigrblast.tigr.org/)，在Wellcome Trust Sanger研究所的牛型结核菌(Mycobacterium bovis)，牛型结核菌-BCG(Pastuer)，海鱼分枝杆菌(M.marinum)，麻风分枝杆菌(M.leprae)，结核杆菌(M.tuberculosis)BLASTN (
http://www.sanger.ac.uk/projects/Microbes/)，在Pasteur(Tuberculist)研究所的结核杆菌BLAST搜索(http://genolist.pasteur.fr/TubercuList/)，在Pasteur(Leproma)研究所的麻风分枝杆菌BLAST搜索(
http://genolist. pasteur.fr/Leproma/)，在明尼苏达大学的微生物基因组计划的约内氏杆菌(M.Paratuberculosis)BLAST(
http://www.cbc.umn.edu/ResearchProjects/Ptb/和http://www.cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mtpbhome.html)，在美国国家生物技术信息中心多种的BLAST搜索(
http://www.ncbi.nlm.nih.gov/ BLAST)和基因组网(GenomeNet)(化学研究所-生物信息中心)多种的BLAST搜索(http://blast.genone.ad.jp)。
因为遗传密码具有简并性，在[SEQ ID NO：1]中的核酸序列不是编码具有脱氢酶活性多肽的唯一序列；在[SEQ ID NO：7、9、11和13]中的核酸序列不是编码具有谷氨酰胺合成酶活性多肽的唯一序列；和在[SEQ ID NO：5]中的核酸序列不是编码具有L-丝氨酸脱水酶活性多肽的唯一序列。本发明包括具有与在[SEQ ID NO：1、5、7、9、11和13]中描述的核酸分子相同的主要的遗传信息的核酸分子。与在本申请中描述的序列比，含有一个或多个氨基酸变化，并如在[SEQ ID NO：2、6、8、10、12和14]中所示的多肽的核酸分子(包括RNA分子)包括在本发明的范围内。
编码丙氨酸脱氢酶、谷氨酰胺合成酶或L-丝氨酸脱水酶的核酸的其它功能相等形式可用传统的DNA-DNA或DNA-RNA杂交技术分离得到。
杂交
本发明包括与本申请中描述的一个核酸分子具有充分一致性序列的DNA，在严谨的杂交条件下进行杂交。
本发明包括与在本申请中描述的核酸分子在严谨条件下杂交具有足够一致性序列的DNA(杂交技术是现有技术中众所周知的)。本发明也包括与在[SEQ D NO：1]到[SEQ D NO：14]中的一个或多个序列或它们的互补序列杂交的核酸分子。这些核酸分子优选在高严谨条件下杂交(见Sambrook et al.Molecular Cloning：A Laboratory Manual，Most Recent Edition，Cold SpringHarbor Laboratory Press，Cold Spring Harbor，N.Y.)。高严谨洗涤在50～65℃低盐条件(大约0.2％SSC)下进行。
疫苗
活的重组疫苗的制备是本领域技术人员所公知的。该疫苗通常被制成液体溶液或悬液状针剂；还可制成适合溶解于或悬浮于注射液体中的固体。药剂还可能是乳状体或包入脂质体的蛋白质。活的免疫产生成分通常与一些药剂学上可接受的与活性成分相容的成型剂混合。适合的赋型剂通常是水、盐水、右旋糖、甘油、乙醇等以及它们的组合。另外，如果需要疫苗中可含有少量辅助物质，如润湿剂或乳化剂，pH缓冲剂和/或增强疫苗有效性的辅剂。有效的辅剂的例子包括但不限于：氢氧化铝、N-乙酰-胞壁酰-L-苏氨酰-D-异谷氨酰胺(苏氨酸-MDP)、N-乙酰-正胞壁酰-L-丙氨酰基-D-异谷氨酰胺(CGP11637，或称为正-MDP)、N-乙酰胞壁酰-L-丙氨酰基-D-异谷氨酰胺酰-L-丙氨酸-2-(1’-2’-二棕榈酰基-锡-丙三氧基-3-羟磷酰氧基)-乙酸胺(N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1’-2’-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine)(CGP 19835A，又称为MTP-PE)和RIBI.RIBI含有从细菌内抽提出的三种成分，在2％鲨烯/吐温-80TM乳浊液中的单磷酰脂A，海藻糖二梅菌酸酯(dimycolate)和细胞壁骨架(MPL+TDM+CWS)。
佐剂的有效性可通过测定抗致免疫的多肽的抗体的量而确定。该致免疫的多肽含有结核杆菌抗原序列，该抗原序列是由注入体内的含有不同佐剂的活的重组牛型结核菌卡介苗产生的。疫苗通常是通过肠胃外给药的，如皮下或肌肉内注射。其它形式的适合于给药的模式包括栓剂和在一些情况下以口服的形式。作为栓剂，传统的粘结剂和载体包括例如聚亚烷基二醇或甘油三脂。这种栓剂可为含有0.5～10％(优选1～2％)的活性成分的混合物。口服剂通常用的成型剂例如：药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。这些组合物为溶液、悬液、片剂、丸剂、胶囊、持续释放剂或粉末等形式，并含有效成分10～95％(优选25～75％)。
疫苗用与剂型相容的方法给药，从而起到预防和/或保护作用。
疫苗可单剂给药，也可多剂给药。多剂给药包括一个1～10次给药的首次过程，和间隔一段时间如1～4个月的第二次给药以保持和/或加强免疫反应，如果需要，几个月后可以再次给予一剂或多剂。剂量的控制应根据个体的需要和具体情况而定。
另外活的重组牛型结核菌卡介苗疫苗也和其它免疫调节剂如免疫球蛋白质一起给药。本发明的主题也是一个多价疫苗配方，是包括上面定义的活的重组牛型结核菌卡介苗疫苗与另一个疫苗，特别是另一个如上所定义的重组活的牛型结核菌-卡介苗疫苗的混合物或进行混合。这些疫苗包括不同的插入序列。
药物组合物
本发明的药物组合物可用于治疗或预防哺乳动物免受结核杆菌或牛型结核菌的侵害。本发明的药物组合物可用于治疗犯有退行性疾病(degenerative disease)和例如癌症的不适或异常身体状态的病人。
药物组合物可通过片剂、气雾剂给药，气管内滴注和静脉注射方法给人或动物用药。
培养基组合物
本发明抑制牛型结核菌卡介苗生长的培养基组合物包括丙氨酸或丝氨酸作为唯一氮源。当丙氨酸为唯一氮源时它的浓度至少为0.03mM，当丝氨酸为唯一氮源时它的浓度至少为0.03mM。
培养基组合物还进一步含有约1.35～约1.65g/L的碳源，优选为至少1.5g/L；约0.045～约0.055g/L，优选至少0.05g/L的铁；约0.45～约0.55g/L，优选至少0.5g/L的镁和约0.045～约0.055g/L，优选至少0.05g/L的SO4。
试剂盒
用于免疫诊断和含有适合标记试剂的试剂盒是由含有本发明的活的重组牛型结核菌卡介苗菌株，在合适的容器中和其它用于分析所需的试剂和材料及一套合适的分析说明书所包装组成的。任何免疫测试方法都可考虑使用，如ELISA、西部印记(Western blot)、夹心(sandwich)分析方法，这些是本领域技术人员所了解的的方法。
材料和方法
菌株和培养条件：研究中采用十二种牛型结核菌卡介苗菌株：BCG-Japan、BCG-Russia、BCG-Moreau、BCG-Sweden、BCG-Birkhaug、BCG-Frappier、BCG-Pasteur、BCG-Glaxo、BCG-Phipps、BCG-Tice、BCG-Denmark和BCG-Prague是从Marcel Behr博士(McGill大学)得到的。菌株的鉴定已被详细地描述过(Behr et al.，1999)。Middlebrook 7H9培养基(Difco)每升含有硫酸铵0.5g、L-谷氨酸0.5g、柠檬酸钠0.1g、维生素B6(pyridoxine)1mg、生物素0.5mg、磷酸二钠2.5g、柠檬酸铵铁40mg、硫酸镁50mg、氯化钙0.5mg、硫酸锌1mg、硫酸铜1mg和甘油2ml；以及在灭菌后加入5g(部分V(fraction V)；牛)白蛋白，2g右旋糖和0.05％吐温80。Sauton培养基每升含有L-天冬酰胺4g、硫酸单钾0.5g、硫酸镁0.5g、柠檬酸铵铁50mg、柠檬酸2g、硫酸锌1mg和甘油60ml；灭菌后加入0.05％吐温80。甘油-丙氨酸-盐(GAS)培养基每升含有氯化铵2g、L-丙氨酸1g、BactoCasitone(Didco)0.3g、磷酸二钾4g、柠檬酸2g、柠檬酸铵铁50mg、六水合氯化镁1.2g、硫酸钾0.6g、10M氢氧化钠1.8ml和10ml甘油。灭菌后加入0.05％吐温80。卡介苗在37℃连续摇动培养3～4周。
ald的克隆：ald[SEQ ID NO：1]的克隆由二步完成(图1)。首先一个含有ald基因的结核杆菌基因组的4.5kb的Sca I DNA片段被连到线性化的Ecl136II pUC19上，产生pUC-ALD。然后连接含有ald基因[SEQ ID NO：1]的1.9kb的KpnI片段到线性化的KpnI pMD31，产生分枝杆菌质粒pLAD(Yuet al.，1998)。pALD质粒被电转入牛型结核菌卡介苗，重组的牛型结核菌卡介苗在添加有富含10％油酸(oleic)/血清/右旋糖/过氧化氢酶(OADC)和25μg/ml卡那霉素的Middlebrook 7H9琼脂培养基(Difco)上选择培养。
sdaA的克隆：sdaA[SEQ ID NO：5]的克隆由二步完成。首先结核杆菌基因组DNA的9.5kb BamHI片段被连到线性化的BamHI pMD31产生pSDA1。然后用PstI断裂pSDA1，随后10.9kb PstI片段自连产生pSDAA质粒。pSDAA质粒被电转入牛型结核菌卡介苗，重组的牛型结核菌卡介苗在添加有富含10％油酸(oleic)/血清/右旋糖/过氧化氢酶(OADC)和25μg/ml卡那霉素的Middlebrook 7H9琼脂培养基(Difco)上选择培养。
实施例1
在甘油-丙氨酸-盐(GAS)培养基中卡介苗的生长：在我们的研究过程中，我们发现虽然比在7H9培养基中长得慢，BCG-Japan在GAS培养基中能够生长。BCG-Frappier和BCG-Pasteur不能在在GAS培养基中能生长，延长培养时间也不能生长(2个月)。其它BCG株在GAS培养基中的生长也被检测。结果显示BCG-Japan、BCG-Russia、BCG-Moreau、BCG-Sweden和BCG-Birkhaug能在GAS培养基中生长，而BCG-Frappier、BCG-Pasteur、BCG-Glaxo、BCG-Phipps、BCG-Tice、BCG-Denmark和BCG-Prague不能在GAS培养基中生长。这是一个有趣的发现，因为12种卡介苗菌株均能在7H9和Sauton broth培养基中生长(表I)。为了弄清为什么有的菌株不能在GAS培养基中生长，对比了GAS、7H9和Sauton培养基的化学成分。添加在7H9和Sauton培养基中有而在GAS培养基中没有的硫酸锌(1mg/l)或丙酮酸钠0.5％)是牛型结核菌大菌落生长所要求的，都不能支持卡介苗菌株在GAS培养基中的生长(数据没有列出)。另外对氮源进行了比较。L-天冬酰胺(4g/l)是Sauton培养基的唯一氮源，而氯化铵(2g/l)和L-丙氨酸(1g/l)是GAS培养基的主要氮源。L-天冬酰胺(4g/l)被加入GAS培养基，BCG-Frappier、BCG-Pasteur、BCG-Glaxo、BCG-Phipps、BCG-Tice、BCG-Denmark和BCG-Prague能快速生长(表I)。添加L-天冬氨酸，L-谷氨酰胺或L-谷氨酸，但不加其它氨基酸于GAS培养基中也能支持这些卡介苗菌株的生长(表I)。这些结果表明特定卡介苗菌株不能在GAS培养基中的生长是由于菌株不能利用所提供的氮源。
实施例2
作为卡介苗生长氮源的氨基酸：上面的结果促使我们检测卡介苗菌株利用不同氨基酸作为唯一氮源的能力。因为GAS培养基含有少量的BactoCasitone(0.3g/l)，这是不同氨基酸和肽的混合物，所以我们选用Sauton培养基，它具有确定的组分，作此研究。原始配方里的L-天冬酰胺分别被每一种氨基酸以同样的浓度(27mM)替代，pH调到7.0。氯化铵以27mM或1mM作为唯一氮源也被测定。三个代表菌株：BCG-Japan、BCG-Pasteur和BCG-Frappier的实验结果总结在表II中。与表I的结果一致当L-天冬酰胺、L-天冬氨酸、L-谷氨酰胺和L-谷氨酸被用作唯一氮源时三种卡介苗菌株均长得很快。BCG-Japan在碱性氨基酸(如L-精氨酸，L-赖氨酸)中能够生长，而BCG-Pasteur和BCG-Frappier则不能。更有趣的是没有一种卡介苗菌株能够利用L-丙氨酸、L-丝氨丝、L-亮氨酸、L-异亮氨酸、L-蛋氨酸或L-甘氨酸作为唯一氮源。而其它分枝杆菌，包括病原结核杆菌、鸟结核分枝杆菌和非病原菌包皮垢分枝杆菌(M.smegmatis)能够在这些氨基酸上生长。这些结果说明卡介苗疫苗菌株只能利用有限的几种氨基酸作为生长氮源；有的卡介苗菌株，如BCG-Pasteur和BCG-Frappier只能在四种氨基酸上生长(表II)。这种限制可能限制了卡介苗在(寄主)体内的生长和持续存在。
实施例3
L-丙氨酸、D-丙氨酸或L-丝氨丝抑制卡介苗的生长：从上面实验中一个惊人的发现是所有卡介苗菌株均能在以低浓度(1mM)或高浓度(27mM)的氯化铵作为唯一氮源上生长(表II)。这与在GAS培养基中得到的结果是相矛盾的。在GAS培养基中37mM的氯化铵不支持BCG-Pasteur和BCG-Frappier的生长(表I)。因为GAS培养基中也含有L-丙氨酸，而L-丙氨酸不能被卡介苗菌株用于生长(表II)。唯一可能的解释是L-丙氨酸实际上抑制卡介苗菌株的生长。为了证明这一点，制成了一种L-丙氨酸被去除的GAS培养基，然后检测卡介苗菌株在这种培养基中的生长。正如预期的那样，不能在原始的含有L-丙氨酸的GAS中生长的BCG-Frappier和BCG-Pasteur则能在没有L-丙氨酸的GAS中迅速生长(图3)。BCG-Japan也能在没有L-丙氨酸GAS培养基中更迅速生长(图3)。同样的结果也出现在本报告所列的其它9个卡介苗菌株中。
为了进一步证明该结果，不断增加浓度的L-丙氨酸被加到含有氯化铵(5g/l)的Sauton培养基中，测定BCG-Japan，BCG-Pasteur和BCG-Frappier的生长(图4)。让人吃惊的是，即使在很低的浓度(0.25mM)下，L-丙氨酸完全抑制BCG-Frappier和BCG-Pasteur的生长。虽然对BCG-Japan的抑制没有那么严重，但当L-丙氨酸增加到0.5mM时对生长的抑制显著，而当浓度增加到8～16mM时生长被完全抑制(图4)。总结一下，这些结果清楚地表明L-丙氨酸抑制卡介苗菌株的生长。我们进一步发现D-丙氨酸也抑制卡介苗菌株的生长。在GAS培养基中D-丙氨酸的存在使BCG-Pasteur和BCG-Frappier停止生长，并显著地降低BCG-Japan的生长(图5)。相似地，L-丝氨丝也显著地抑制BCG-Japan、BCG-Pasteur和BCG-Frappier的生长(图7)。
实施例4
L-丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]在卡介苗中的表达解除L-丙氨酸和D-丙氨酸对卡介苗的生长抑制：丙氨酸是很多分枝杆菌，包括结核杆菌、鸟结核分枝杆菌和包皮垢分枝杆菌的良好氮源。D-丙氨酸的降解始于消旋为L-丙氨酸，然后被L-丙氨酸脱氢酶分解为铵和丙酮酸。有趣的是功能性L-丙氨酸脱氢酶在结核杆菌和包皮垢分枝杆菌中可检测到，而在BCG-Japan或BCG-Copenhagen中没有检测到(Andersen et al.，1992；Hutterand Dick，1998)。我们没有在本研究所列的卡介苗菌株中检测到L-丙氨酸脱氢酶活性(数据没有显示)。卡介苗菌株不能利用L-丙氨酸或D-丙氨酸作为唯一的生长氮源是由于缺乏功能性L-丙氨酸脱氢酶。为了证明这一点，在结核杆菌基因组中编码L-丙氨酸脱氢酶[SEQ ID NO：2]的基因ald[SEQ IDNO：1]被克隆到一个穿梭载体，转入BCG-Pasteur和BCG-Frappier中。得到的重组卡介苗菌株被培养于含有L-丙氨酸或D-丙氨酸的GAS培养基中测定其生长能力。两个重组菌株，BCG-Frappier/ald和BCG-Pasteur/ald均在含有L-丙氨酸或D-丙氨酸的GAS培养基中生长迅速(图6)，而只含有克隆载体的菌株不能生长。这一结果表明功能性L-丙氨酸脱氢酶[SEQ IDNO：1；SEQ ID NO：2]在卡介苗菌株中的表达解除L-丙氨酸和D-丙氨酸对卡介苗生长的抑制。
实施例5
L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]在卡介苗中的表达解除L-丝氨酸对卡介苗的生长抑制：L-丝氨酸被结核杆菌、鸟结合分枝杆菌和包皮垢分枝杆菌用作生长的唯一氮源而牛型结核菌卡介苗不能。卡介苗不能用L-丝氨酸作为生长的唯一氮源可能是由于卡介苗中的编码L-丝氨酸脱水酶的基因sdaA[SEQ ID NO：5]发生突变或改变表达。在卡介苗中表达结核杆菌的sdaA[SEQ ID NO：5；SEQ ID NO：6]使卡介苗菌株能够在以L-丝氨酸作为生长的唯一氮源的培养基中生长，解除L-丝氨酸对卡介苗的生长抑制(图8)。
实施例6
L-丙氨酸、D-丙氨酸和L-丝氨酸对卡介苗生长的抑制可能是由于谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的活性被抑制。谷氨酰胺合成酶在细菌的氮代谢中起中心作用(Reitzer，1996)。先后与谷氨酸合成酶和谷氨酰胺合成酶作用催化了谷酰胺和谷氨酸的合成，几乎能为所有的氨基酸、蛋白质和核苷酸提供氮。在大肠埃希氏杆菌(Escherichia coli)和产气克雷伯氏菌(Klebsiella aerogenes)中谷氨酰胺合成酶受反馈抑制调控，纯化的谷氨酰胺合成酶受L-丙氨酸、L-丝氨酸和甘氨酸抑制(Reitzer，1996)。谷氨酰胺合成酶在结核杆菌和牛型结核菌中被鉴定为细胞外蛋白质(Harth etal.，1994)。这可能是由于没被降解的L-丙氨酸抑制了谷氨酰胺合成酶，结果阻止卡介苗的生长。假如这是正确的话，那么L-丝氨酸不能被卡介苗分解(表I)，而抑制BCG生长的机理可能也是如此。为支持这一假说，在含有氯化铵作为唯一氮源的GAS培养基中加入L-丝氨酸抑制BCG-Frappier、BCG-Pasteur和BCG-Japan(图7)。进一步的，如果谷氨酰胺合成酶是L-丙氨酸和L-丝氨酸抑制的耙标，那么能够转化为谷氨酸的氨基酸将减弱这种作用，正如在产气克雷伯氏菌(K.aerogenes)中证实的那样(Janes and Bender，1998)。确实，加入谷氨酸和能够分解产生谷氨酸的氨基酸(L-谷氨酰胺、L-天冬酰胺和L-天冬氨酸)能够使卡介苗菌株在丙氨酸存在条件下生长(表I)。但是在那些不能分解产生谷氨酸的氨基酸(如L-赖氨酸、L-蛋氨酸、L-亮氨酸)中则不能生长。与BCG-Japan相比BCG-Frappier和BCG-Pasteur更易受丙氨酸和丝氨酸抑制，这可能是由于谷氨酰胺合成酶[SEQ ID NO：7]到[SEQ ID NO：14]的表达水平或活性不同所致，如BCG-Japan比BCG-Frappier和BCG-Pasteur产生更多的谷氨酰胺合成酶或产生的酶的活性更高。
本发明已被详细描述并给出了具体实施方式作为参考；但是本领域的普通技术人员应理解，可在不超出本发明的精神和范围的条件下进行变化。例如，当提及蛋白质的应用时，很清楚，肽和多肽也经常被应用。同样的，当基因在应用中被描述时，很清楚的是，核酸或核酸片段也经常是可被使用的。
所有出版物(包括Genbank中的条目)、专利和专利申请的全部内容在此引入作为参考，以达到每个出版物、专利或专利申请被明确的并分别的指明其全部内容在此引入作为参考的同样效果。
表I
在培养基7H9、Sauton和甘油-丙氨酸-盐(GAS)中结核杆菌、包皮垢分枝杆菌和牛型结核菌卡介苗亚菌株生长的比较。
| 分枝杆菌a | 7H9 | Sauton | GAS | GAS+L-Asnb | GAS+L-Aspb | GAS+L-Glub | GAS+L-Glnb |
| 结核杆菌c包皮垢分枝杆菌BCG-RussiaBCG-MoreauBCG-JapanBCG-SwedenBCG-BirkhaugBCG-PragueBCG-GlaxoBCG-DenmarkBCG-TiceBCG-FrappierBCG-PhippsBCG-Pasteur | ++++++++++++++ | ++++++++++++++ | +++++++------- | ++++++++++++++ | ++++++++++++++ | ++++++++++++++ | ++++++++++++++ |
a每5ml培养物中含有1×107个包皮垢分枝杆菌或牛型结核菌卡介苗亚菌株的细胞。
b在GAS中添加的L-Asn，L-Asp、L-Glu和L-Gln的终浓度为27mM。
c根据研究文献。
表II
牛型结核菌BCG-Japan、BCG-Frappier、BCG-Pasteur、结核杆菌、鸟结核分枝杆菌和包皮垢分枝杆菌的生长比较
| 培养基 | BCG-Japanb | BCG-Frappierb | BCG-Pasteurb | 结核杆菌c | 鸟结核分枝杆菌c | 包皮垢分枝杆菌b |
| Sauton基 | - | - | - | - | - | - |
| 组1 | ||||||
| Sauton+L-AsnSauton+L-AspSauton+L-GluSauton+L-GlnSauton+L-CysSauton+NH4Cl | ++++++++++++++++++ | ++++++++++++++++++ | ++++++++++++++++++ | ++++++++++++++++++ | ++++++++++++++++++ | ++++++++++++++++++ |
| 组2 | ||||||
| Sauton+L-ArgSauton+L-HisSauton+L-LysSauton+L-ProSauton+GABASauton+L-鸟氨酸 | ++++++++++++ | ------ | ------ | ++++++NANANANA | +++++++++-NANA | ++++++++++++++++++ |
| 组3 | ||||||
| Sauton+L-AlaSauton+L-SerSauton+L-LeuSauton+L-IleSauton+L-MetSauton+甘氨酸 | ------ | ------ | ------ | ++++++++++++NA+++ | +++++++++++++++NA | ++++++++++++++++++ |
| 组4 | ||||||
| Sauton+L-TrpSauton+L-PheSauton+L-TyrSauton+L-ValSauton+L-Thr | ----- | ----- | ----- | -+++-NANA | ----- | ----- |
a所有氨基酸，L-鸟氨酸和GABA的终浓度为27mM。NH4Cl浓度在1mM、27mM和96mM进行测试。
b每5ml培养物中含有1×107个包皮垢分枝杆菌或牛型结核菌BCG亚菌株的细胞。
c根据研究文献。
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序列表
<110>成都永安制药有限公司；刘军
<120>Recombinant BCG Strains Expressing Alanine Dehydrogenase，
Serine dehydratase and/or Glutamine Synthetase as TB Vaccines
<130>
<150>US 60/372,450
<151>2002-04-16
<160>14
<170>PatentIn version 3.0
<210>1
<211>1116
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1116)
<223>Sequence is identical to GenBank entries GI：3089350 and MTU92472
<400>1
atg cgc gtc ggt att ccg acc gag acc aaa aac aac gaa ttc cgg gtg 48
Met Arg Val Gly Ile Pro Thr Glu Thr Lys Asn Asn Glu Phe Arg Val
1 5 10 15
gcc atc acc ccg gcc ggc gtc gcg gaa cta acc cgt cgt ggc cat gag 96
Ala Ile Thr Pro Ala Gly Val Ala Glu Leu Thr Arg Arg Gly His Glu
20 25 30
gtg ctc atc cag gca ggt gcc gga gag ggc tcg gct atc acc gac gcg 144
Val Leu Ile Gln Ala Gly Ala Gly Glu Gly Ser Ala Ile Thr Asp Ala
35 40 45
gat ttc aag gcg gca ggc gcg caa ctg gtc ggc acc gcc gac cag gtg 192
Asp Phe Lys Ala Ala Gly Ala Gln Leu Val Gly Thr Ala Asp Gln Val
50 55 60
tgg gcc gac gct gat tta ttg ctc aag gtc aaa gaa ccg ata gcg gcg 240
Trp Ala Asp Ala Asp Leu Leu Leu Lys Val Lys Glu Pro Ile Ala Ala
65 70 75 80
gaa tac ggc cgc ctg cga cac ggg cag atc ttg ttc acg ttc ttg cat 288
Glu Tyr Gly Arg Leu Arg His Gly Gln Ile Leu Phe Thr Phe Leu His
85 90 95
ttg gcc gcg tca cgt gct tgc acc gat gcg ttg ttg gat tcc ggc acc 336
Leu Ala Ala Ser Arg Ala Cys Thr Asp Ala Leu Leu Asp Ser Gly Thr
100 105 110
acg tca att gcc tac gag acc gtc cag acc gcc gac ggc gca cta ccc 384
Thr Ser Ile Ala Tyr Glu Thr Val Gln Thr Ala Asp Gly Ala Leu Pro
115 120 125
ctg ctt gcc ccg atg agc gaa gtc gcc ggt cga ctc gcc gcc cag gtt 432
Leu Leu Ala Pro Met Ser Glu Val Ala Gly Arg Leu Ala Ala Gln Val
130 135 140
ggc gct tac cac ctg atg cga acc caa ggg ggc cgc ggt gtg ctg atg 480
Gly Ala Tyr His Leu Met Arg Thr Gln Gly Gly Arg Gly Val Leu Met
145 150 155 160
ggc ggg gtg ccc ggc gtc gaa ccg gcc gac gtc gtg gtg atc ggc gcc 528
Gly Gly Val Pro Gly Val Glu Pro Ala Asp Val Val Val Ile Gly Ala
165 170 175
ggc acc gcc ggc tac aac gca gcc cgc atc gcc aac ggc atg ggc gcg 576
Gly Thr Ala Gly Tyr Asn Ala Ala Arg Ile Ala Asn Gly Met Gly Ala
180 185 190
acc gtt acg gtt cta gac atc aac atc gac aaa ctt cgg caa ctc gac 624
Thr Val Thr Val Leu Asp Ile Asn Ile Asp Lys Leu Arg Gln Leu Asp
195 200 205
gcc gag ttc tgc ggc cgg atc cac act cgc tac tca tcg gcc tac gag 672
Ala Glu Phe Cys Gly Arg Ile His Thr Arg Tyr Ser Ser Ala Tyr Glu
210 215 220
ctc gag ggt gcc gtc aaa cgt gcc gac ctg gtg att ggg gcc gtc ctg 720
Leu Glu Gly Ala Val Lys Arg Ala Asp Leu Val Ile Gly Ala Val Leu
225 230 235 240
gtg cca ggc gcc aag gca ccc aaa tta gtc tcg aat tca ctt gtc gcg 768
Val Pro Gly Ala Lys Ala Pro Lys Leu Val Ser Asn Ser Leu Val Ala
245 250 255
cat atg aaa cca ggt gcg gta ctg gtg gat ata gcc atc gac cag ggc 816
His Met Lys Pro Gly Ala Val Leu Val Asp Ile Ala Ile Asp Gln Gly
260 265 270
ggc tgt ttc gaa ggc tca cga cog acc acc tac gac cac ccg acg ttc 864
Gly Cys Phe Glu Gly Ser Arg Pro Thr Thr Tyr Asp His Pro Thr Phe
275 280 285
gcc gtg cac gac acg ctg ttt tac tgc gtg gcg aac atg ccc gcc tcg 912
Ala Val His Asp Thr Leu Phe Tyr Cys Val Ala Asn Met Pro Ala Ser
290 295 300
gtg ccg aag acg tcg acc tac gcg ctg acc aac gcg acg atg ccg tat 960
Val Pro Lys Thr Ser Thr Tyr Ala Leu Thr Asn Ala Thr Met Pro Tyr
305 310 315 320
gtg ctc gag ctt gcc gac cat ggc tgg cgg gcg gcg tgc cgg tcg aat 1008
Val Leu Glu Leu Ala Asp His Gly Trp Arg Ala Ala Cys Arg Ser Asn
325 330 335
ccg gca cta gcc aaa ggt ctt tcg acg cac gaa ggg gcg tta ctg tcc 1056
Pro Ala Leu Ala Lys Gly Leu Ser Thr His Glu Gly Ala Leu Leu Ser
340 345 350
gaa cgg gtg gcc acc gac ctg ggg gtg ccg ttc acc gag ccc gcc agc 1104
Glu Arg Val Ala Thr Asp Leu Gly Val Pro Phe Thr Glu Pro Ala Ser
355 360 365
gtg ctg gcc tga 1116
Val Leu Ala
370
<210>2
<211>371
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to SwissProt entry SP：DHA_MYCTU
<400>2
Met Arg Val Gly Ile Pro Thr Glu Thr Lys Asn Asn Glu Phe Arg Val
1 5 10 15
Ala Ile Thr Pro Ala Gly Val Ala Glu Leu Thr Arg Arg Gly His Glu
20 25 30
Val Leu Ile Gln Ala Gly Ala Gly Glu Gly Ser Ala Ile Thr Asp Ala
35 40 45
Asp Phe Lys Ala Ala Gly Ala Gln Leu Val Gly Thr Ala Asp Gln Val
50 55 60
Trp Ala Asp Ala Asp Leu Leu Leu Lys Val Lys Glu Pro Ile Ala Ala
65 70 75 80
Glu Tyr Gly Arg Leu Arg His Gly Gln Ile Leu Phe Thr Phe Leu His
85 90 95
Leu Ala Ala Ser Arg Ala Cys Thr Asp Ala Leu Leu Asp Ser Gly Thr
100 105 110
Thr Ser Ile Ala Tyr Glu Thr Val Gln Thr Ala Asp Gly Ala Leu Pro
115 120 125
Leu Leu Ala Pro Met Ser Glu Val Ala Gly Arg Leu Ala Ala Gln Val
130 135 140
Gly Ala Tyr His Leu Met Arg Thr Gln Gly Gly Arg Gly Val Leu Met
145 150 155 160
Gly Gly Val Pro Gly Val Glu Pro Ala Asp Val Val Val Ile Gly Ala
165 170 175
Gly Thr Ala Gly Tyr Asn Ala Ala Arg Ile Ala Asn Gly Met Gly Ala
180 185 190
Thr Val Thr Val Leu Asp Ile Asn Ile Asp Lys Leu Arg Gln Leu Asp
195 200 205
Ala Glu Phe Cys Gly Arg Ile His Thr Arg Tyr Ser Ser Ala Tyr Glu
210 215 220
Leu Glu Gly Ala Val Lys Arg Ala Asp Leu Val Ile Gly Ala Val Leu
225 230 235 240
Val Pro Gly Ala Lys Ala Pro Lys Leu Val Ser Asn Ser Leu Val Ala
245 250 255
His Met Lys Pro Gly Ala Val Leu Val Asp Ile Ala Ile Asp Gln Gly
260 265 270
Gly Cys Phe Glu Gly Ser Arg Pro Thr Thr Tyr Asp His Pro Thr Phe
275 280 285
Ala Val His Asp Thr Leu Phe Tyr Cys Val Ala Asn Met Pro Ala Ser
290 295 300
Val Pro Lys Thr Ser Thr Tyr Ala Leu Thr Asn Ala Thr Met Pro Tyr
305 310 315 320
Val Leu Glu Leu Ala Asp His Gly Trp Arg Ala Ala Cys Arg Ser Asn
325 330 335
Pro Ala Leu Ala Lys Gly Leu Ser Thr His Glu Gly Ala Leu Leu Ser
340 345 350
Glu Arg Val Ala Thr Asp Leu Gly Val Pro Phe Thr Glu Pro Ala Ser
355 360 365
Val Leu Ala
370
<210>3
<211>399
<212>DNA
<213>牛型结核杆菌(Mycobacterium bovis)
<220>
<221>CDS
<222>(1)..(399)
<400>3
atg cgc gtc ggt att ccg acc gag acc aaa aac aac gaa ttc cgg gtg 48
Met Arg Val Gly Ile Pro Thr Glu Thr Lys Asn Asn Glu Phe Arg Val
1 5 10 15
gcc atc acc ccg gcc ggc gtc gcg gaa cta acc cgt cgt ggc cat gag 96
Ala Ile Thr Pro Ala Gly Val Ala Glu Leu Thr Arg Arg Gly His Glu
20 25 30
gtg ctc atc cag gca ggt gcc gga gag ggc tcg gct atc acc gac gcg 144
Val Leu Ile Gln Ala Gly Ala Gly Glu Gly Ser Ala Ile Thr Asp Ala
35 40 45
gat ttc aag gcg gca ggc gcg caa ctg gtc ggc acc gcc gac cag gtg 192
Asp Phe Lys Ala Ala Gly Ala Gln Leu Val Gly Thr Ala Asp Gln Val
50 55 60
tgg gcc gac gct gat tta ttg ctc aag gtc aaa gaa ccg ata gcg gcg 240
Trp Ala Asp Ala Asp Leu Leu Leu Lys Val Lys Glu Pro Ile Ala Ala
65 70 75 80
gaa tac ggc cgc ctg cga cac ggg cga tct tgt tca cgt tct tgc att 288
Glu Tyr Gly Arg Leu Arg His Gly Arg Ser Cys Ser Arg Ser Cys Ile
85 90 95
tgg ccg cgt cac gtg ctt gca ccg atg cgt tgt tgg att ccg gca cca 336
Trp Pro Arg His Val Leu Ala Pro Met Arg Cys Trp Ile Pro Ala Pro
100 105 110
cgt caa ttg cct acg aga ccg tcc aga ccg ccg acg gcg cac tac ccc 384
Arg Gln Leu Pro Thr Arg Pro Ser Arg Pro Pro Thr Ala His Tyr Pro
115 120 125
tgc ttg ccc cga tga 399
Cys Leu Pro Arg
130
<210>4
<211>132
<212>PRT
<213>牛型结核杆菌(Mycobacterium bovis)
<400>4
Met Arg Val Gly Ile Pro Thr Glu Thr Lys Asn Asn Glu Phe Arg Val
1 5 10 15
Ala Ile Thr Pro Ala Gly Val Ala Glu Leu Thr Arg Arg Gly His Glu
20 25 30
Val Leu Ile Gln Ala Gly Ala Gly Glu Gly Ser Ala Ile Thr Asp Ala
35 40 45
Asp Phe Lys Ala Ala Gly Ala Gln Leu Val Gly Thr Ala Asp Gln Val
50 55 60
Trp Ala Asp Ala Asp Leu Leu Leu Lys Val Lys Glu Pro Ile Ala Ala
65 70 75 80
Glu Tyr Gly Arg Leu Arg His Gly Arg Ser Cys Ser Arg Ser Cys Ile
85 90 95
Trp Pro Arg His Val Leu Ala Pro Met Arg Cys Trp Ile Pro Ala Pro
100 105 110
Arg Gln Leu Pro Thr Arg Pro Ser Arg Pro Pro Thr Ala His Tyr Pro
115 120 125
Cys Leu Pro Arg
130
<210>5
<211>1386
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1386)
<223>Sequence is identical to the complement of nucleotides 13172-14551
of GenBank entry GB：MTV030[AL021428]
Sequence is identical to the complement of nucleotides 13195-14580
of GenBank entry GB：AE006919
<400>5
atg acc atc agc gtc ttc gac ctg ttc acc atc ggc atc ggg ccg tcc 48
Met Thr Ile Ser Val Phe Asp Leu Phe Thr Ile Gly Ile Gly Pro Ser
1 5 10 15
agt tcc cac acc gtg gga ccg atg cgc gcg gca aac cag ttc gta gtt 96
Ser Ser His Thr Val Gly Pro Met Arg Ala Ala Asn Gln Phe Val Val
20 25 30
gcg ctg cgc cgc cgg ggc cac ctg gat gac ctc gag gcg atg cga gtg 144
Ala Leu Arg Arg Arg Gly His Leu Asp Asp Leu Glu Ala Met Arg Val
35 40 45
gat ctg ttc ggc tcg ctc gcg gcc acc gga gcc ggt cat ggc acc atg 192
Asp Leu Phe Gly Ser Leu Ala Ala Thr Gly Ala Gly His Gly Thr Met
50 55 60
tcg gcg ata ttg ctg ggg ctg gaa ggc tgc cag cca gaa acg att acc 240
Ser Ala Ile Leu Leu Gly Leu Glu Gly Cys Gln Pro Glu Thr Ile Thr
65 70 75 80
acc gaa cac aag gaa cgc cgg ctc gcc gag atc gca gcg tcc ggc gtg 288
Thr Glu His Lys Glu Arg Arg Leu Ala Glu Ile Ala Ala Ser Gly Val
85 90 95
acg cga atc ggc ggt gtc att ccg gtc ccg ctg acc gag cgt gat atc 336
Thr Arg Ile Gly Gly Val Ile Pro Val Pro Leu Thr Glu Arg Asp Ile
100 105 110
gac ctg cat ccc gac atc gtt ctg cca acg cat ccc aac gga atg acg 384
Asp Leu His Pro Asp Ile Val Leu Pro Thr His Pro Asn Gly Met Thr
115 120 125
ttc act gcc gcg ggc cca cac ggc cgc gtc ttg gcc acc gag act tat 432
Phe Thr Ala Ala Gly Pro His Gly Arg Val Leu Ala Thr Glu Thr Tyr
130 135 140
ttt tcg gtg ggc gga ggg ttc atc gtc acg gaa cag acc agc ggc aac 480
Phe Ser Val Gly Gly Gly Phe Ile Val Thr Glu Gln Thr Ser Gly Asn
145 150 155 160
agc ggc caa cat cca tgc tca gtt gcc ctt ccc tac gtg tcg gcc caa 528
Ser Gly Gln His Pro Cys Ser Val Ala Leu Pro Tyr Val Ser Ala Gln
165 170 175
gaa ctg ctg gac atc tgt gac cgc ctc gac gtg tca att agc gaa gcg 576
Glu Leu Leu Asp Ile Cys Asp Arg Leu Asp Val Ser Ile Ser Glu Ala
180 185 190
gcg ctg cgc aac gaa aca tgt tgc cgc acc gag aac gag gta cgc gcc 624
Ala Leu Arg Asn Glu Thr Cys Cys Arg Thr Glu Asn Glu Val Arg Ala
195 200 205
gcg ctg ctg cac ctg cgc gac gtc atg gtt gag tgc gaa cag cgg agc 672
Ala Leu Leu His Leu Arg Asp Val Met Val Glu Cys Glu Gln Arg Ser
210 215 220
atc gct cgc gaa ggg ttg ctt cct ggc ggc ctc cgg gtg cgc cgg cga 720
Ile Ala Arg Glu Gly Leu Leu Pro Gly Gly Leu Arg Val Arg Arg Arg
225 230 235 240
gcg aag gtg tgg tat gac cgc ttg aac gcc gaa gac ccc act cgc aag 768
Ala Lys Val Trp Tyr Asp Arg Leu Asn Ala Glu Asp Pro Thr Arg Lys
245 250 255
ccg gaa ttc gct gag gac tgg gtc aac ctg gtc gcg ctg gca gtc aac 816
Pro Glu Phe Ala Glu Asp Trp Val Asn Leu Val Ala Leu Ala Val Asn
260 265 270
gag gag aac gcc tcc ggt ggg cgc gtc gtc acc gcc ccg acc aac ggt 864
Glu Glu Asn Ala Ser Gly Gly Arg Val Val Thr Ala Pro Thr Asn Gly
275 280 285
gcc gcc ggc atc gtg ccg gcg gtc ctg cac tac gca atc cac tac acg 912
Ala Ala Gly Ile Val Pro Ala Val Leu His Tyr Ala Ile His Tyr Thr
290 295 300
tcg gcc ggc gcg ggg gac ccc gac gat gtc acc gtg cga ttc ctg ctc 960
Ser Ala Gly Ala Gly Asp Pro Asp Asp Val Thr Val Arg Phe Leu Leu
305 310 315 320
act gct gga gcc atc gga tcg ttg ttc aag gag cga gca tcg atc tcc 1008
Thr Ala Gly Ala Ile Gly Ser Leu Phe Lys Glu Arg Ala Ser Ile Ser
325 330 335
gga gcc gag gtc ggc tgt cag ggc gag gtc ggc tcc gcg gcc gcc atg 1056
Gly Ala Glu Val Gly Cys Gln Gly Glu Val Gly Ser Ala Ala Ala Met
340 345 350
gcc gcc gcc gga ttg gct gaa atc ctc ggc ggc aca ccg cga caa gtg 1104
Ala Ala Ala Gly Leu Ala Glu Ile Leu Gly Gly Thr Pro Arg Gln Val
355 360 365
gaa aac gcc gcc gag atc gcc atg gaa cac agc ctc ggc ctg acc tgt 1152
Glu Asn Ala Ala Glu Ile Ala Met Glu His Ser Leu Gly Leu Thr Cys
370 375 380
gac ccc atc gcc ggg ctg gtg cag atc ccc tgc atc gaa cgc aac gcg 1200
Asp Pro Ile Ala Gly Leu Val Gln Ile Pro Cys Ile Glu Arg Asn Ala
385 390 395 400
att tcc gcc ggc aag gcc atc aac gcc gca cgg atg gca ttg cgc ggc 1248
Ile Ser Ala Gly Lys Ala Ile Asn Ala Ala Arg Met Ala Leu Arg Gly
405 410 415
gac ggc atc cat cgc gtc acc ctc gac cag gtc atc gac acc atg cgc 1296
Asp Gly Ile His Arg Val Thr Leu Asp Gln Val Ile Asp Thr Met Arg
420 425 430
gcc acc ggc gcg gac atg cac acc aag tac aag gaa acc tcg gcc ggc 1344
Ala Thr Gly Ala Asp Met His Thr Lys Tyr Lys Glu Thr Ser Ala Gly
435 440 445
ggg ctc gcc atc aac gtc gca gtc aac atc gtc gag tgt tga 1386
Gly Leu Ala Ile Asn Val Ala Val Asn Ile Val Glu Cys
450 455 460
<210>6
<211>461
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to SwissProt entry SP：SDHL_MYCTU
Sequence is identical to GenBank entries GP：AE006919_13 and GP：MTV030_11
<400>6
Met Thr Ile Ser Val Phe Asp Leu Phe Thr Ile Gly Ile Gly Pro Ser
1 5 10 15
Ser Ser His Thr Val Gly Pro Met Arg Ala Ala Asn Gln Phe Val Val
20 25 30
Ala Leu Arg Arg Arg Gly His Leu Asp Asp Leu Glu Ala Met Arg Val
35 40 45
Asp Leu Phe Gly Ser Leu Ala Ala Thr Gly Ala Gly His Gly Thr Met
50 55 60
Ser Ala Ile Leu Leu Gly Leu Glu Gly Cys Gln Pro Glu Thr Ile Thr
65 70 75 80
Thr Glu His Lys Glu Arg Arg Leu Ala Glu Ile Ala Ala Ser Gly Val
85 90 95
Thr Arg Ile Gly Gly Val Ile Pro Val Pro Leu Thr Glu Arg Asp Ile
100 105 110
Asp Leu His Pro Asp Ile Val Leu Pro Thr His Pro Asn Gly Met Thr
115 120 125
Phe Thr Ala Ala Gly Pro His Gly Arg Val Leu Ala Thr Glu Thr Tyr
130 135 140
Phe Ser Val Gly Gly Gly Phe Ile Val Thr Glu Gln Thr Ser Gly Asn
145 150 155 160
Ser Gly Gln His Pro Cys Ser Val Ala Leu Pro Tyr Val Ser Ala Gln
165 170 175
Glu Leu Leu Asp Ile Cys Asp Arg Leu Asp Val Ser Ile Ser Glu Ala
180 185 190
Ala Leu Arg Asn Glu Thr Cys Cys Arg Thr Glu Asn Glu Val Arg Ala
195 200 205
Ala Leu Leu His Leu Arg Asp Val Met Val Glu Cys Glu Gln Arg Ser
210 215 220
Ile Ala Arg Glu Gly Leu Leu Pro Gly Gly Leu Arg Val Arg Arg Arg
225 230 235 240
Ala Lys Val Trp Tyr Asp Arg Leu Asn Ala Glu Asp Pro Thr Arg Lys
245 250 255
Pro Glu Phe Ala Glu Asp Trp Val Asn Leu Val Ala Leu Ala Val Asn
260 265 270
Glu Glu Asn Ala Ser Gly Gly Arg Val Val Thr Ala Pro Thr Asn Gly
275 280 285
Ala Ala Gly Ile Val Pro Ala Val Leu His Tyr Ala Ile His Tyr Thr
290 295 300
Ser Ala Gly Ala Gly Asp Pro Asp Asp Val Thr Val Arg Phe Leu Leu
305 310 315 320
Thr Ala Gly Ala Ile Gly Ser Leu Phe Lys Glu Arg Ala Ser Ile Ser
325 330 335
Gly Ala Glu Val Gly Cys Gln Gly Glu Val Gly Ser Ala Ala Ala Met
340 345 350
Ala Ala Ala Gly Leu Ala Glu Ile Leu Gly Gly Thr Pro Arg Gln Val
355 360 365
Glu Asn Ala Ala Glu Ile Ala Met Glu His Ser Leu Gly Leu Thr Cys
370 375 380
Asp Pro Ile Ala Gly Leu Val Gln Ile Pro Cys Ile Glu Arg Asn Ala
385 390 395 400
Ile Ser Ala Gly Lys Ala Ile Asn Ala Ala Arg Met Ala Leu Arg Gly
405 410 415
Asp Gly Ile His Arg Val Thr Leu Asp Gln Val Ile Asp Thr Met Arg
420 425 430
Ala Thr Gly Ala Asp Met His Thr Lys Tyr Lys Glu Thr Ser Ala Gly
435 440 445
Gly Leu Ala Ile Asn Val Ala Val Asn Ile Val Glu Cys
450 455 460
<210>7
<211>1437
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1437)
<223>Sequence is identical to GenBank entry GB：MTU87280[U87280]
Sequence is identical to nucleotides 163-1599 of GenBank entry
GB：MTCY427[Z70692]
Sequence is identical to nucleotides 93-1529 of GenBank entry GB：AE007073
<400>7
gtg acg gaa aag acg ccc gac gac gtc ttc aaa ctt gcc aag gac gag 48
Met Thr Glu Lys Thr Pro Asp Asp Val Phe Lys Leu Ala Lys Asp Glu
1 5 10 15
aag gtc gaa tat gtc gac gtc cgg ttc tgt gac ctg cct ggc atc atg 96
Lys Val Glu Tyr Val Asp Val Arg Phe Cys Asp Leu Pro Gly Ile Met
20 25 30
cag cac ttc acg att ccg gct tcg gcc ttt gac aag agc gtg ttt gac 144
Gln His Phe Thr Ile Pro Ala Ser Ala Phe Asp Lys Ser Val Phe Asp
35 40 45
gac ggc ttg gcc ttt gac ggc tcg tcg att cgc ggg ttc cag tcg atc 192
Asp Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Gln Ser Ile
50 55 60
cac gaa tcc gac atg ttg ctt ctt ccc gat ccc gag acg gcg cgc atc 240
His Glu Ser Asp Met Leu Leu Leu Pro Asp Pro Glu Thr Ala Arg Ile
65 70 75 80
gac ccg ttc cgc gcg gcc aag acg ctg aat atc aac ttc ttt gtg cac 288
Asp Pro Phe Arg Ala Ala Lys Thr Leu Asn Ile Asn Phe Phe Val His
85 90 95
gac ccg ttc acc ctg gag ccg tac tcc cgc gac ccg cgc aac atc gcc 336
Asp Pro Phe Thr Leu Glu Pro Tyr Ser Arg Asp Pro Arg Asn Ile Ala
100 105 110
cgc aag gcc gag aac tac ctg atc agc act ggc atc gcc gac acc gca 384
Arg Lys Ala Glu Asn Tyr Leu Ile Ser Thr Gly Ile Ala Asp Thr Ala
115 120 125
tac ttc ggc gcc gag gcc gag ttc tac att ttc gat tcg gtg agc ttc 432
Tyr Phe Gly Ala Glu Ala Glu Phe Tyr Ile Phe Asp Ser Val Ser Phe
130 135 140
gac tcg cgc gcc aac ggc tcc ttc tac gag gtg gac gcc atc tcg ggg 480
Asp Ser Arg Ala Asn Gly Ser Phe Tyr Glu Val Asp Ala Ile Ser Gly
145 150 155 160
tgg tgg aac acc ggc gcg gcg acc gag gcc gac ggc agt ccc aac cgg 528
Trp Trp Asn Thr Gly Ala Ala Thr Glu Ala Asp Gly Ser Pro Asn Arg
165 170 175
ggc tac aag gtc cgc cac aag ggc ggg tat ttc cca gtg gcc ccc aac 576
Gly Tyr Lys Val Arg His Lys Gly Gly Tyr Phe Pro Val Ala Pro Asn
180 185 190
gac caa tac gtc gac ctg cgc gac aag atg ctg acc aac ctg atc aac 624
Asp Gln Tyr Val Asp Leu Arg Asp Lys Met Leu Thr Asn Leu Ile Asn
195 200 205
tcc ggc ttc atc ctg gag aag ggc cac cac gag gtg ggc agc ggc gga 672
Ser Gly Phe Ile Leu Glu Lys Gly His His Glu Val Gly Ser Gly Gly
210 215 220
cag gcc gag atc aac tac cag ttc aat tcg ctg ctg cac gcc gcc gac 720
Gln Ala Glu Ile Asn Tyr Gln Phe Asn Ser Leu Leu His Ala Ala Asp
225 230 235 240
gac atg cag ttg tac aag tac atc atc aag aac acc gcc tgg cag aac 768
Asp Met Gln Leu Tyr Lys Tyr Ile Ile Lys Asn Thr Ala Trp Gln Asn
245 250 255
ggc aaa acg gtc acg ttc atg ccc aag ccg ctg ttc ggc gac aac ggg 816
Gly Lys Thr Val Thr Phe Met Pro Lys Pro Leu Phe Gly Asp Asn Gly
260 265 270
tcc ggc atg cac tgt cat cag tcg ctg tgg aag gac ggg gcc ccg ctg 864
Ser Gly Met His Cys His Gln Ser Leu Trp Lys Asp Gly Ala Pro Leu
275 280 285
atg tac gac gag acg ggt tat gcc ggt ctg tcg gac acg gcc cgt cat 912
Met Tyr Asp Glu Thr Gly Tyr Ala Gly Leu Ser Asp Thr Ala Arg His
290 295 300
tac atc ggc ggc ctg tta cac cac gcg ccg tcg ctg ctg gcc ttc acc 960
Tyr Ile Gly Gly Leu Leu His His Ala Pro Ser Leu Leu Ala Phe Thr
305 310 315 320
aac ccg acg gtg aac tcc tac aag cgg ctg gtt ccc ggt tac gag gcc 1008
Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu Ala
325 330 335
ccg atc aac ctg gtc tat agc cag cgc aac cgg tcg gca tgc gtg cgc 1056
Pro Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Cys Val Arg
340 345 350
atc ccg atc acc ggc agc aac ccg aag gcc aag cgg ctg gag ttc cga 1104
Ile Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Leu Glu Phe Arg
355 360 365
agc ccc gac tcg tcg ggc aac ccg tat ctg gcg ttc tcg gcc atg ctg 1152
Ser Pro Asp Ser Ser Gly Asn Pro Tyr Leu Ala Phe Ser Ala Met Leu
370 375 380
atg gca ggc ctg gac ggt atc aag aac aag atc gag ccg cag gcg ccc 1200
Met Ala Gly Leu Asp Gly Ile Lys Asn Lys Ile Glu Pro Gln Ala Pro
385 390 395 400
gtc gac aag gat ctc tac gag ctg ccg ccg gaa gag gcc gcg agt atc 1248
Val Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile
405 410 415
ccg cag act ccg acc cag ctg tca gat gtg atc gac cgt ctc gag gcc 1296
Pro Gln Thr Pro Thr Gln Leu Ser Asp Val Ile Asp Arg Leu Glu Ala
420 425 430
gac cac gaa tac ctc acc gaa gga ggg gtg ttc aca aac gac ctg atc 1344
Asp His Glu Tyr Leu Thr Glu Gly Gly Val Phe Thr Asn Asp Leu Ile
435 440 445
gag acg tgg atc agt ttc aag cgc gaa aac gag atc gag ccg gtc aac 1392
Glu Thr Trp Ile Ser Phe Lys Arg Glu Asn Glu Ile Glu Pro Val Asn
450 455 460
atc cgg ccg cat ccc tac gaa ttc gcg ctg tac tac gac gtt taa 1437
Ile Arg Pro His Pro Tyr Glu Phe Ala Leu Tyr Tyr Asp Val
465 470 475
<210>8
<211>478
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to SwissProt entry SP：GLNl_MYCTU
Sequence is identical to PIR entry PIR：H70775
Sequence is identical to PRF entry PRF：2323405A
<400>8
Met Thr Glu Lys Thr Pro Asp Asp Val Phe Lys Leu Ala Lys Asp Glu
1 5 10 15
Lys Val Glu Tyr Val Asp Val Arg Phe Cys Asp Leu Pro Gly Ile Met
20 25 30
Gln His Phe Thr Ile Pro Ala Ser Ala Phe Asp Lys Ser Val Phe Asp
35 40 45
Asp Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Gln Ser Ile
50 55 60
His Glu Ser Asp Met Leu Leu Leu Pro Asp Pro Glu Thr Ala Arg Ile
65 70 75 80
Asp Pro Phe Arg Ala Ala Lys Thr Leu Asn Ile Asn Phe Phe Val His
85 90 95
Asp Pro Phe Thr Leu Glu Pro Tyr Ser Arg Asp Pro Arg Asn Ile Ala
100 105 110
Arg Lys Ala Glu Asn Tyr Leu Ile Ser Thr Gly Ile Ala Asp Thr Ala
115 120 125
Tyr Phe Gly Ala Glu Ala Glu Phe Tyr Ile Phe Asp Ser Val Ser Phe
130 135 140
Asp Ser Arg Ala Asn Gly Ser Phe Tyr Glu Val Asp Ala Ile Ser Gly
145 150 155 160
Trp Trp Asn Thr Gly Ala Ala Thr Glu Ala Asp Gly Ser Pro Asn Arg
165 170 175
Gly Tyr Lys Val Arg His Lys Gly Gly Tyr Phe Pro Val Ala Pro Asn
180 185 190
Asp Gln Tyr Val Asp Leu Arg Asp Lys Met Leu Thr Asn Leu Ile Asn
195 200 205
Ser Gly Phe Ile Leu Glu Lys Gly His His Glu Val Gly Ser Gly Gly
210 215 220
Gln Ala Glu Ile Asn Tyr Gln Phe Asn Ser Leu Leu His Ala Ala Asp
225 230 235 240
Asp Met Gln Leu Tyr Lys Tyr Ile Ile Lys Asn Thr Ala Trp Gln Asn
245 250 255
Gly Lys Thr Val Thr Phe Met Pro Lys Pro Lau Phe Gly Asp Asn Gly
260 265 270
Ser Gly Met His Cys His Gln Ser Leu Trp Lys Asp Gly Ala Pro Leu
275 280 285
Met Tyr Asp Glu Thr Gly Tyr Ala Gly Leu Ser Asp Thr Ala Arg His
290 295 300
Tyr Ile Gly Gly Leu Leu His His Ala Pro Ser Leu Leu Ala Phe Thr
305 310 315 320
Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu Ala
325 330 335
Pro Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Cys Val Arg
340 345 350
Ile Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Leu Glu Phe Arg
355 360 365
Ser Pro Asp Ser Ser Gly Asn Pro Tyr Leu Ala Phe Ser Ala Met Leu
370 375 380
Met Ala Gly Leu Asp Gly Ile Lys Asn Lys Ile Glu Pro Gln Ala Pro
385 390 395 400
Val Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile
405 410 415
Pro Gln Thr Pro Thr Gln Leu Ser Asp Val Ile Asp Arg Leu Glu Ala
420 425 430
Asp His Glu Tyr Leu Thr Glu Gly Gly Val Phe Thr Asn Asp Leu Ile
435 440 445
Glu Thr Trp Ile Ser Phe Lys Arg Glu Asn Glu Ile Glu Pro Val Asn
450 455 460
Ile Arg Pro His Pro Tyr Glu Phe Ala Leu Tyr Tyr Asp Val
465 470 475
<210>9
<211>1341
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1341)
<223>Sequence is identical to complement of nucleotides 4950-6290
of GenBank entry GB：MTCY427[Z70692]
Sequence is identical to complement of nucleotides 4880-6220
of GenBank entry GB：AE007073
<400>9
atg gac cga cag aag gaa ttc gtt ctt cgt acc ctg gaa gaa cgc gac 48
Met Asp Arg Gln Lys Glu Phe Val Leu Arg Thr Leu Glu Glu Arg Asp
1 5 10 15
atc cgc ttc gtc cgg ctg tgg ttc aca gac gtg ctc ggt ttc ctc aag 96
Ile Arg Phe Val Arg Leu Trp Phe Thr Asp Val Leu Gly Phe Leu Lys
20 25 30
tcg gtc gcc atc gcc cca gcc gaa ctc gag ggc gcc ttc gag gaa ggc 144
Ser Val Ala Ile Ala Pro Ala Glu Leu Glu Gly Ala Phe Glu Glu Gly
35 40 45
atc ggc ttc gac gga tcc tcg atc gag ggc ttt gcg cgg gtc tcg gaa 192
Ile Gly Phe Asp Gly Ser Ser Ile Glu Gly Phe Ala Arg Val Ser Glu
50 55 60
tcc gat acg gtg gcg cac ccg gac ccg tcg acc ttc cag gtg ctg ccc 240
Ser Asp Thr Val Ala His Pro Asp Pro Ser Thr Phe Gln Val Leu Pro
65 70 75 80
tgg gcc acc agt tcc ggc cac cac cac tca gcg cgg atg ttt tgc gac 288
Trp Ala Thr Ser Ser Gly His His His Ser Ala Arg Met Phe Cys Asp
85 90 95
atc acc atg ccg gac ggc tcg ccg tcg tgg gcg gac ccg cgg cac gtg 336
Ile Thr Met Pro Asp Gly Ser Pro Ser Trp Ala Asp Pro Arg His Val
100 105 110
ttg cgg cgg cag ctg acg aag gcc ggc gaa ctc ggc ttc tcc tgc tac 384
Leu Arg Arg Gln Leu Thr Lys Ala Gly Glu Leu Gly Phe Ser Cys Tyr
115 120 125
gtg cat ccc gaa atc gag ttc ttc ctg ctc aag ccc gga ccc gag gac 432
Val His Pro Glu Ile Glu Phe Phe Leu Leu Lys Pro Gly Pro Glu Asp
130 135 140
ggg tcg gtg ccc gtc ccg gtc gac aac gcc ggc tat ttc gac caa gcg 480
Gly Ser Val Pro Val Pro Val Asp Asn Ala Gly Tyr Phe Asp Gln Ala
145 150 155 160
gtg cac gac tcc gcc ttg aac ttt cgc cgc cac gcg atc gat gcc ctg 528
Val His Asp Ser Ala Leu Asn Phe Arg Arg His Ala Ile Asp Ala Leu
165 170 175
gaa ttc atg ggc atc tcg gtg gag ttc agc cat cac gaa ggc gca ccc 576
Glu Phe Met Gly Ile Ser Val Glu Phe Ser His His Glu Gly Ala Pro
180 185 190
ggc cag cag gag atc gac ctg cgg ttt gcc gac gct ctg tcg atg gct 624
Gly Gln Gln Glu Ile Asp Leu Arg Phe Ala Asp Ala Leu Ser Met Ala
195 200 205
gac aac gtg atg acc ttc cgc tac gtc atc aaa gaa gtc gcg ctg gaa 672
Asp Asn Val Met Thr Phe Arg Tyr Val Ile Lys Glu Val Ala Leu Glu
210 215 220
gag ggc gcc cgg gcg tcg ttc atg ccc aag cca ttc ggc cag cac ccg 720
Glu Gly Ala Arg Ala Ser Phe Met Pro Lys Pro Phe Gly Gln His Pro
225 230 235 240
ggc tcg gcg atg cac acc cac atg agc ctg ttc gag ggt gat gtc aac 768
Gly Ser Ala Met His Thr His Met Ser Leu Phe Glu Gly Asp Val Asn
245 250 255
gcg ttc cac agc gct gat gat ccg ctg cag ctg tcg gaa gtg ggt aaa 816
Ala Phe His Ser Ala Asp Asp Pro Leu Gln Leu Ser Glu Val Gly Lys
260 265 270
tcg ttc atc gcc ggg atc ctg gag cac gct tgc gag atc agc gcg gtc 864
Ser Phe Ile Ala Gly Ile Leu Glu His Ala Cys Glu Ile Ser Ala Val
275 280 285
aca aat cag tgg gtc aac tct tac aag cgg ctg gtg cag ggc ggc gaa 912
Thr Asn Gln Trp Val Asn Ser Tyr Lys Arg Leu Val Gln Gly Gly Glu
290 295 300
gcg ccc acg gcc gcg tcg tgg ggg gcc gcc aac cga tcc gcc cta gtg 960
Ala Pro Thr Ala Ala Ser Trp Gly Ala Ala Asn Arg Ser Ala Leu Val
305 310 315 320
cgg gtg ccg atg tac acg ccg cac aag acc tcg tcg cgg cgg gtc gaa 1008
Arg Val Pro Met Tyr Thr Pro His Lys Thr Ser Ser Arg Arg Val Glu
325 330 335
gta cgc agc cct gat tcg gcg tgc aat ccc tat ctg aca ttc gcc gtg 1056
Val Arg Ser Pro Asp Ser Ala Cys Asn Pro Tyr Leu Thr Phe Ala Val
340 345 350
ctg ctg gcc gcg gga ttg cgg ggt gta gag aag ggt tac gtg ctg ggc 1104
Leu Leu Ala Ala Gly Leu Arg Gly Val Glu Lys Gly Tyr Val Leu Gly
355 360 365
ccg cag gcc gag gac aac gta tgg gac ctc aca ccc gag gaa cgc cga 1152
Pro Gln Ala Glu Asp Asn Val Trp Asp Leu Thr Pro Glu Glu Arg Arg
370 375 380
gcg atg ggg tac cga gaa ttg ccg tcc agt ttg gat agt gcg ctg cgc 1200
Ala Met Gly Tyr Arg Glu Leu Pro Ser Ser Leu Asp Ser Ala Leu Arg
385 390 395 400
gcc atg gag gcc tcc gaa ctc gtc gcg gag gcc ttg ggg gag cac gtt 1248
Ala Met Glu Ala Ser Glu Leu Val Ala Glu Ala Leu Gly Glu His Val
405 410 415
ttt gac ttt ttc ttg cgc aac aag cgc acg gag tgg gcg aac tac cgc 1296
Phe Asp Phe Phe Leu Arg Asn Lys Arg Thr Glu Trp Ala Asn Tyr Arg
420 425 430
agc cac gtc acg cca tac gag ctg cgc acc tac ctg tcg ctg tag 1341
Ser His Val Thr Pro Tyr Glu Leu Arg Thr Tyr Leu Ser Leu
435 440 445
<210>10
<211>446
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to SwissProt entry SP：GLN2_MYCTU
Sequence is identical to PIR entry PIR：B70776
<400>10
Met Asp Arg Gln Lys Glu Phe Val Leu Arg Thr Leu Glu Glu Arg Asp
1 5 10 15
Ile Arg Phe Val Arg Leu Trp Phe Thr Asp Val Leu Gly Phe Leu Lys
20 25 30
Ser Val Ala Ile Ala Pro Ala Glu Leu Glu Gly Ala Phe Glu Glu Gly
35 40 45
Ile Gly Phe Asp Gly Ser Ser Ile Glu Gly Phe Ala Arg Val Ser Glu
50 55 60
Ser Asp Thr Val Ala His Pro Asp Pro Ser Thr Phe Gln Val Leu Pro
65 70 75 80
Trp Ala Thr Ser Ser Gly His His His Ser Ala Arg Met Phe Cys Asp
85 90 95
Ile Thr Met Pro Asp Gly Ser Pro Ser Trp Ala Asp Pro Arg His Val
100 105 110
Leu Arg Arg Gln Leu Thr Lys Ala Gly Glu Leu Gly Phe Ser Cys Tyr
115 120 125
Val His Pro Glu Ile Glu Phe Phe Leu Leu Lys Pro Gly Pro Glu Asp
130 135 140
Gly Ser Val Pro Val Pro Val Asp Asn Ala Gly Tyr Phe Asp Gln Ala
145 150 155 160
Val His Asp Ser Ala Leu Asn Phe Arg Arg His Ala Ile Asp Ala Leu
165 170 175
Glu Phe Met Gly Ile Ser Val Glu Phe Ser His His Glu Gly Ala Pro
180 185 190
Gly Gln Gln Glu Ile Asp Leu Arg Phe Ala Asp Ala Leu Ser Met Ala
195 200 205
Asp Asn Val Met Thr Phe Arg Tyr Val Ile Lys Glu Val Ala Leu Glu
210 215 220
Glu Gly Ala Arg Ala Ser Phe Met Pro Lys Pro Phe Gly Gln His Pro
225 230 235 240
Gly Ser Ala Met His Thr His Met Ser Leu Phe Glu Gly Asp Val Asn
245 250 255
Ala Phe His Ser Ala Asp Asp Pro Leu Gln Leu Ser Glu Val Gly Lys
260 265 270
Ser Phe Ile Ala Gly Ile Leu Glu His Ala Cys Glu Ile Ser Ala Val
275 280 285
Thr Asn Gln Trp Val Asn Ser Tyr Lys Arg Leu Val Gln Gly Gly Glu
290 295 300
Ala Pro Thr Ala Ala Ser Trp Gly Ala Ala Asn Arg Ser Ala Leu Val
305 310 315 320
Arg Val Pro Met Tyr Thr Pro His Lys Thr Ser Ser Arg Arg Val Glu
325 330 335
Val Arg Ser Pro Asp Ser Ala Cys Asn Pro Tyr Leu Thr Phe Ala Val
340 345 350
Leu Leu Ala Ala Gly Leu Arg Gly Val Glu Lys Gly Tyr Val Leu Gly
355 360 365
Pro Gln Ala Glu Asp Asn Val Trp Asp Leu Thr Pro Glu Glu Arg Arg
370 375 380
Ala Met Gly Tyr Arg Glu Leu Pro Ser Ser Leu Asp Ser Ala Leu Arg
385 390 395 400
Ala Met Glu Ala Ser Glu Leu Val Ala Glu Ala Leu Gly Glu His Val
405 410 415
Phe Asp Phe Phe Leu Arg Asn Lys Arg Thr Glu Trp Ala Asn Tyr Arg
420 425 430
Ser His Val Thr Pro Tyr Glu Leu Arg Thr Tyr Leu Ser Leu
435 440 445
<210>11
<211>1353
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1353)
<223>Sequence is identical to nucleotides 4871-6223
of GenBank entry GB：MTCY180[Z97193]
Sequence is identical to nucleotides 7308-8660
of GenBank entry GB：AE007049
<400>11
atg aca gcc aca ccg ctt gec gcg gcc gcg atc gcc caa ttg gag gca 48
Met Thr Ala Thr Pro Leu Ala Ala Ala Ala Ile Ala Gln Leu Glu Ala
1 5 10 15
gag ggc gtc gac acc gtc atc ggc acc gte gtg aac ccc gcc gga ctc 96
Glu Gly Val Asp Thr Val Ile Gly Thr Val Val Asn Pro Ala Gly Leu
20 25 30
acc cag gcc aag acc gtg ccg ata cgc cgg acc aac aca ttc gcc aat 144
Thr Gln Ala Lys Thr Val Pro Ile Arg Arg Thr Asn Thr Phe Ala Asn
35 40 45
cct ggc ctc ggc gcc agt ccg gtg tgg cat acc ttc tgt atc gac caa 192
Pro Gly Leu Gly Ala Ser Pro Val Trp His Thr Phe Cys Ile Asp Gln
50 55 60
tgc agt att gca ttc acc gca gac atc agt gtg gtc ggc gat caa cgt 240
Cys Ser Ile Ala Phe Thr Ala Asp Ile Ser Val Val Gly Asp Gln Arg
65 70 75 80
ctc cgc atc gat ctg tcc gcc ttg cgc atc atc ggc gac ggg ttg gcg 288
Leu Arg Ile Asp Leu Ser Ala Leu Arg Ile Ile Gly Asp Gly Leu Ala
85 90 95
tgg gcg ccc gcc ggg ttc ttc gag cag gac ggc aca ccg gtc ccc gcc 336
Trp Ala Pro Ala Gly Phe Phe Glu Gln Asp Gly Thr Pro Val Pro Ala
100 105 110
tgc agc cga gga aca ctg agc cgg atc gag gcc gcg ctt gct gat gcc 384
Cys Ser Arg Gly Thr Leu Ser Arg Ile Glu Ala Ala Leu Ala Asp Ala
115 120 125
ggc atc gac gcg gta atc ggc cac gaa gtc gaa ttc ctc ttg gtc gac 432
Gly Ile Asp Ala Val Ile Gly His Glu Val Glu Phe Leu Leu Val Asp
130 135 140
gcg gac ggc cag cgg ctg cct tcg acg ctg tgg gcg cag tac ggt gtc 480
Ala Asp Gly Gln Arg Leu Pro Ser Thr Leu Trp Ala Gln Tyr Gly Val
145 150 155 160
gcc ggg gtg ctc gag cac gag gcg ttc gtc cgc gat gtc aac gcc gcg 528
Ala Gly Val Leu Glu His Glu Ala Phe Val Arg Asp Val Asn Ala Ala
165 170 175
gca acg gca gca ggc ate gct atc gag cag ttc cat ccc gaa tac ggt 576
Ala Thr Ala Ala Gly Ile Ala Ile Glu Gln Phe His Pro Glu Tyr Gly
180 185 190
gcc aac cea ttc gag atc tcg tta gcg ccg cag ccg ccg gtc gcg gcc 624
Ala Asn Gln Phe Glu Ile Ser Leu Ala Pro Gln Pro Pro Val Ala Ala
195 200 205
gcc gat cag ctg gtg ctg acc cgc ctc atc atc ggc cgt acc gcc cgc 672
Ala Asp Gln Leu Val Leu Thr Arg Leu Ile Ile Gly Arg Thr Ala Arg
210 215 220
cgg cac ggg tta cgc gtg agc cta tcg cca gcg ccc ttc gcc gga agt 720
Arg His Gly Leu Arg Val Ser Leu Ser Pro Ala Pro Phe Ala Gly Ser
225 230 235 240
atc gga tcc ggt gcc cac caa cac ttc tcg ctg act atg tcg gaa ggg 768
Ile Gly Ser Gly Ala His Gln His Phe Ser Leu Thr Met Ser Glu Gly
245 250 255
atg ctg ttc tcc ggt ggg act gga gca gct ggc atg acc tcg gcc ggg 816
Met Leu Phe Ser Gly Gly Thr Gly Ala Ala Gly Met Thr Ser Ala Gly
260 265 270
gag gcc gcg gtg gca gga gtg ctt cgc gga cta ccg gac gcc caa ggc 864
Glu Ala Ala Val Ala Gly Val Leu Arg Gly Leu Pro Asp Ala Gln Gly
275 280 285
atc ctg tgc gga tcg atc gtg tcc ggt ctg cga atg cga ccc ggt aac 912
Ile Leu Cys Gly Ser Ile Val Ser Gly Leu Arg Met Arg Pro Gly Asn
290 295 300
tgg gcc gga atc tat gca tgc tgg ggt acc gaa aac cgg gaa gcg gcg 960
Trp Ala Gly Ile Tyr Ala Cys Trp Gly Thr Glu Asn Arg Glu Ala Ala
305 310 315 320
gtg cga ttc gtc aag ggc ggg gct ggc agc gcg tac ggc ggg aac gtg 1008
Val Arg Phe Val Lys Gly Gly Ala Gly Ser Ala Tyr Gly Gly Asn Val
325 330 335
gag gtg aag gtc gtc gac ccg tcg gcc aac ccg tat ctc gcg tcg gcg 1056
Glu Val Lys Val Val Asp Pro Ser Ala Asn Pro Tyr Leu Ala Ser Ala
340 345 350
gcg atc ctc gga ctg gca ctc gac ggc atg aag acc aag gcg gtg ttg 1104
Ala Ile Leu Gly Leu Ala Leu Asp Gly Met Lys Thr Lys Ala Val Leu
355 360 365
ccg tcg gaa aeg acc gta gac ccg aca cag ctg tct gac gtg gat cgt 1152
Pro Ser Glu Thr Thr Val Asp Pro Thr Gln Leu Ser Asp Val Asp Arg
370 375 380
gac cgt gcc ggc att ctg cga ctt gct gcc gat cag gcg gat gca att 1200
Asp Arg Ala Gly Ile Leu Arg Leu Ala Ala Asp Gln Ala Asp Ala Ile
385 390 395 400
gct gta ctg gat agt tcg aaa ctg ctt cgg tgc atc ctt ggc gat ccc 1248
Ala Val Leu Asp Ser Ser Lys Leu Leu Arg Cys Ile Leu Gly Asp Pro
405 410 415
gtg gta gat gcc gtg gtc gcg gta cgc cag tta gag cat gag cgc tac 1296
Val Val Asp Ala Val Val Ala Val Arg Gln Leu Glu His Glu Arg Tyr
420 425 430
ggt gac ctc gat cct gcg cag ctg gcc gac aag ttc cgg atg gct tgg 1344
Gly Asp Leu Asp Pro Ala Gln Leu Ala Asp Lys Phe Arg Met Ala Trp
435 440 445
agt gtg taa 1353
Ser Val
450
<210>12
<211>450
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to PIR entry PIR：C70515
<400>12
Met Thr Ala Thr Pro Leu Ala Ala Ala Ala Ile Ala Gln Leu Glu Ala
1 5 10 15
Glu Gly Val Asp Thr Val Ile Gly Thr Val Val Asn Pro Ala Gly Leu
20 25 30
Thr Gln Ala Lys Thr Val Pro Ile Arg Arg Thr Asn Thr Phe Ala Asn
35 40 45
Pro Gly Leu Gly Ala Ser Pro Val Trp His Thr Phe Cys Ile Asp Gln
50 55 60
Cys Ser Ile Ala Phe Thr Ala Asp Ile Ser Val Val Gly Asp Gln Arg
65 70 75 80
Leu Arg Ile Asp Leu Ser Ala Leu Arg Ile Ile Gly Asp Gly Leu Ala
85 90 95
Trp Ala Pro Ala Gly Phe Phe Glu Gln Asp Gly Thr Pro Val Pro Ala
100 105 110
Cys Ser Arg Gly Thr Leu Ser Arg Ile Glu Ala Ala Leu Ala Asp Ala
115 120 125
Gly Ile Asp Ala Val Ile Gly His Glu Val Glu Phe Leu Leu Val Asp
130 135 140
Ala Asp Gly Gln Arg Leu Pro Ser Thr Leu Trp Ala Gln Tyr Gly Val
145 150 155 160
Ala Gly Val Leu Glu His Glu Ala Phe Val Arg Asp Val Asn Ala Ala
165 170 175
Ala Thr Ala Ala Gly Ile Ala Ile Glu Gln Phe His Pro Glu Tyr Gly
180 185 190
Ala Asn Gln Phe Glu Ile Ser Leu Ala Pro Gln Pro Pro Val Ala Ala
195 200 205
Ala Asp Gln Leu Val Leu Thr Arg Leu Ile Ile Gly Arg Thr Ala Arg
210 215 220
Arg His Gly Leu Arg Val Ser Leu Ser Pro Ala Pro Phe Ala Gly Ser
225 230 235 240
Ile Gly Ser Gly Ala His Gln His Phe Ser Leu Thr Met Ser Glu Gly
245 250 255
Met Leu Phe Ser Gly Gly Thr Gly Ala Ala Gly Met Thr Ser Ala Gly
260 265 270
Glu Ala Ala Val Ala Gly Val Leu Arg Gly Leu Pro Asp Ala Gln Gly
275 280 285
Ile Leu Cys Gly Ser Ile Val Ser Gly Leu Arg Met Arg Pro Gly Asn
290 295 300
Trp Ala Gly Ile Tyr Ala Cys Trp Gly Thr Glu Asn Arg Glu Ala Ala
305 310 315 320
Val Arg Phe Val Lys Gly Gly Ala Gly Ser Ala Tyr Gly Gly Asn Val
325 330 335
Glu Val Lys Val Val Asp Pro Ser Ala Asn Pro Tyr Leu Ala Ser Ala
340 345 350
Ala Ile Leu Gly Leu Ala Leu Asp Gly Met Lys Thr Lys Ala Val Leu
355 360 365
Pro Ser Glu Thr Thr Val Asp Pro Thr Gln Leu Ser Asp Val Asp Arg
370 375 380
Asp Arg Ala Gly Ile Leu Arg Leu Ala Ala Asp Gln Ala Asp Ala Ile
385 390 395 400
Ala Val Leu Asp Ser Ser Lys Leu Leu Arg Cys Ile Leu Gly Asp Pro
405 410 415
Val Val Asp Ala Val Val Ala Val Arg Gln Leu Glu His Glu Arg Tyr
420 425 430
Gly Asp Leu Asp Pro Ala Gln Leu Ala Asp Lys Phe Arg Met Ala Trp
435 440 445
Ser Val
450
<210>13
<211>1374
<212>DNA
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>CDS
<222>(1)..(1374)
<223>Sequence is identical to complement of nucleotides 3104-4477
of GenBank entry GB：MTV003[AL008883]
Sequence is identical to complement of nucleotides 3138-4511
of GenBank entry GB：AE007117
<400>13
gtg acc ggc ccc ggt tcg ccg ccg ttg gcg tgg acc gag ttg gag cga 48
Met Thr Gly Pro Gly Ser Pro Pro Leu Ala Trp Thr Glu Leu Glu Arg
1 5 10 15
ctg gtc gcg gcc ggt gac gtc gac acc gtc atc gtc gcg ttc acc gac 96
Leu Val Ala Ala Gly Asp Val Asp Thr Val Ile Val Ala Phe Thr Asp
20 25 30
atg cag ggc cgg ctg gcc ggc aaa cgg ata tcg ggc cgg cat ttc gtc 144
Met Gln Gly Arg Leu Ala Gly Lys Arg Ile Ser Gly Arg His Phe Val
35 40 45
gac gac ata gcc acc cgc ggc gtc gag tgc tgc agt tat ctg ctg gcc 192
Asp Asp Ile Ala Thr Arg Gly Val Glu Cys Cys Ser Tyr Leu Leu Ala
50 55 60
gtg gac gtc gac ctg aac acg gtg ccc ggc tat gcg atg gcc agt tgg 240
Val Asp Val Asp Leu Asn Thr Val Pro Gly Tyr Ala Met Ala Ser Trp
65 70 75 80
gac acc ggc tac ggc gat atg gtg atg acg ccg gac ttg tcc act ctg 288
Asp Thr Gly Tyr Gly Asp Met Val Met Thr Pro Asp Leu Ser Thr Leu
85 90 95
cgg ctg att cct tgg cta ccg gga acg gcg ctg gtg atc gcc gac ctg 336
Arg Leu Ile Pro Trp Leu Pro Gly Thr Ala Leu Val Ile Ala Asp Leu
100 105 110
gtc tgg gcc gac ggc agc gag gtc gcc gtc tcg ccg cgc agc att ctg 384
Val Trp Ala Asp Gly Ser Glu Val Ala Val Ser Pro Arg Ser Ile Leu
115 120 125
cgc cgt cag ctc gat cgg ctc aag gcg cgc gga ctg gtc gcc gat gtg 432
Arg Arg Gln Leu Asp Arg Leu Lys Ala Arg Gly Leu Val Ala Asp Val
130 135 140
gcc acc gag ctg gag ttc atc gtg ttc gac cag ccg tat cgc cag gca 480
Ala Thr Glu Leu Glu Phe Ile Val Phe Asp Gln Pro Tyr Arg Gln Ala
145 150 155 160
tgg gcc agc ggg tat cgc ggg ctg acc ccg gcc agc gac tac aac atc 528
Trp Ala Ser Gly Tyr Arg Gly Leu Thr Pro Ala Ser Asp Tyr Asn Ile
165 170 175
gac tac gcg ata ttg gca tcc tcg cgg atg gag ccg ttg ctg cgc gac 576
Asp Tyr Ala Ile Leu Ala Ser Ser Arg Met Glu Pro Leu Leu Arg Asp
180 185 190
atc cgg ttg ggt atg gcc ggt gcg ggt ctg cga ttc gag gcg gtc aaa 624
Ile Arg Leu Gly Met Ala Gly Ala Gly Leu Arg Phe Glu Ala Val Lys
195 200 205
ggc gaa tgc aac atg ggc cag cag gag atc ggg ttt cgt tac gac gag 672
Gly Glu Cys Asn Met Gly Gln Gln Glu Ile Gly Phe Arg Tyr Asp Glu
210 215 220
gcg ctg gtc acc tgc gac aac cat gcg atc tac aag aac ggc gcc aag 720
Ala Leu Val Thr Cys Asp Asn His Ala Ile Tyr Lys Asn Gly Ala Lys
225 230 235 240
gaa atc gcc gac cag cac ggc aag agc cta acg ttc atg gcg aaa tac 768
Glu Ile Ala Asp Gln His Gly Lys Ser Leu Thr Phe Met Ala Lys Tyr
245 250 255
gat gaa cgc gaa ggt aat agc tgt cac atc cat gtc tcg ctg cgt ggc 816
Asp Glu Arg Glu Gly Asn Ser Cys His Ile His Val Ser Leu Arg Gly
260 265 270
acg gat ggc tcc gcg gtg ttt gcc gac agt aac ggg ccg cac ggc atg 864
Thr Asp Gly Ser Ala Val Phe Ala Asp Ser Asn Gly Pro His Gly Met
275 280 285
tcg tcg atg ttc cgc agc ttc gtc gcc ggc cag ttg gcc acg ttg cgc 912
Ser Ser Met Phe Arg Ser Phe Val Ala Gly Gln Leu Ala Thr Leu Arg
290 295 300
gaa ttc acg ctg tgc tat gcg ccg acc att aac tcc tac aag cga ttt 960
Glu Phe Thr Leu Cys Tyr Ala Pro Thr Ile Asn Ser Tyr Lys Arg Phe
305 310 315 320
gcc gat agc agt ttc gcg ccg acg gcg ctg gct tgg ggg ctg gac aat 1008
Ala Asp Ser Ser Phe Ala Pro Thr Ala Leu Ala Trp Gly Leu Asp Asn
325 330 335
cgc acc tgc gcc ctg cgg gtg gtt ggc cac ggg caa aac atc cgg gtc 1056
Arg Thr Cys Ala Leu Arg Val Val Gly His Gly Gln Asn Ile Arg Val
340 345 350
gaa tgc cgg gtt ccc ggc ggt gat gtc aac cag tac ctg gcg gtg gcg 1104
Glu Cys Arg Val Pro Gly Gly Asp Val Asn Gln Tyr Leu Ala Val Ala
355 360 365
gct ctc att gct gga ggg ttg tac ggt atc gag cgg ggc ctt cag ctg 1152
Ala Leu Ile Ala Gly Gly Leu Tyr Gly Ile Glu Arg Gly Leu Gln Leu
370 375 380
ccc gag ccc tgt gtc ggc aac gcc tac caa ggc gcc gat gtc gaa cgg 1200
Pro Glu Pro Cys Val Gly Asn Ala Tyr Gln Gly Ala Asp Val Glu Arg
385 390 395 400
ctg ccg gtt acg ctg gcc gac gcc gcg gtg ctg ttc gag gat tct gcg 1248
Leu Pro Val Thr Leu Ala Asp Ala Ala Val Leu Phe Glu Asp Ser Ala
405 410 415
ctg gtg cgc gag gcg ttc ggc gag gat gtt gtc gcg cac tac ctg aac 1296
Leu Val Arg Glu Ala Phe Gly Glu Asp Val Val Ala His Tyr Leu Asn
420 425 430
aac gcg cgt gtg gag ctg gcg gcg ttc aac gcg gcg gtc acc gat tgg 1344
Asn Ala Arg Val Glu Leu Ala Ala Phe Asn Ala Ala Val Thr Asp Trp
435 440 445
gag agg ata cgt gga ttt gag cgc ctc tag 1374
Glu Arg Ile Arg Gly Phe Glu Arg Leu
450 455
<210>14
<211>457
<212>PRT
<213>结核杆菌(Mycobacterium tuberculosis)
<220>
<221>
<222>
<223>Sequence is identical to PIR entry PIR：E70885
<400>14
Met Thr Gly Pro Gly Ser Pro Pro Leu Ala Trp Thr Glu Leu Glu Arg
1 5 10 15
Leu Val Ala Ala Gly Asp Val Asp Thr Val Ile Val Ala Phe Thr Asp
20 25 30
Met Gln Gly Arg Leu Ala Gly Lys Arg Ile Ser Gly Arg His Phe Val
35 40 45
Asp Asp Ile Ala Thr Arg Gly Val Glu Cys Cys Ser Tyr Leu Leu Ala
50 55 60
Val Asp Val Asp Leu Asn Thr Val Pro Gly Tyr Ala Met Ala Ser Trp
65 70 75 80
Asp Thr Gly Tyr Gly Asp Met Val Met Thr Pro Asp Leu Ser Thr Leu
85 90 95
Arg Leu Ile Pro Trp Leu Pro Gly Thr Ala Leu Val Ile Ala Asp Leu
100 105 110
Val Trp Ala Asp Gly Ser Glu Val Ala Val Ser Pro Arg Ser Ile Leu
115 120 125
Arg Arg Gln Leu Asp Arg Leu Lys Ala Arg Gly Leu Val Ala Asp Val
130 135 140
Ala Thr Glu Leu Glu Phe Ile Val Phe Asp Gln Pro Tyr Arg Gln Ala
145 150 155 160
Trp Ala Ser Gly Tyr Arg Gly Leu Thr Pro Ala Ser Asp Tyr Asn Ile
165 170 175
Asp Tyr Ala Ile Leu Ala Ser Ser Arg Met Glu Pro Leu Leu Arg Asp
180 185 190
Ile Arg Leu Gly Met Ala Gly Ala Gly Leu Arg Phe Glu Ala Val Lys
195 200 205
Gly Glu Cys Asn Met Gly Gln Gln Glu Ile Gly Phe Arg Tyr Asp Glu
210 215 220
Ala Leu Val Thr Cys Asp Asn His Ala Ile Tyr Lys Asn Gly Ala Lys
225 230 235 240
Glu Ile Ala Asp Gln His Gly Lys Ser Leu Thr Phe Met Ala Lys Tyr
245 250 255
Asp Glu Arg Glu Gly Asn Ser Cys His Ile His Val Ser Leu Arg Gly
260 265 270
Thr Asp Gly Ser Ala Val Phe Ala Asp Ser Asn Gly Pro His Gly Met
275 280 285
Ser Ser Met Phe Arg Ser Phe Val Ala Gly Gln Leu Ala Thr Leu Arg
290 295 300
Glu Phe Thr Leu Cys Tyr Ala Pro Thr Ile Asn Ser Tyr Lys Arg Phe
305 310 315 320
Ala Asp Ser Ser Phe Ala Pro Thr Ala Leu Ala Trp Gly Leu Asp Asn
325 330 335
Arg Thr Cys Ala Leu Arg Val Val Gly His Gly Gln Asn Ile Arg Val
340 345 350
Glu Cys Arg Val Pro Gly Gly Asp Val Asn Gln Tyr Leu Ala Val Ala
355 360 365
Ala Leu Ile Ala Gly Gly Leu Tyr Gly Ile Glu Arg Gly Leu Gln Leu
370 375 380
Pro Glu Pro Cys Val Gly Asn Ala Tyr Gln Gly Ala Asp Val Glu Arg
385 390 395 400
Leu Pro Val Thr Leu Ala Asp Ala Ala Val Leu Phe Glu Asp Ser Ala
405 410 415
Leu Val Arg Glu Ala Phe Gly Glu Asp Val Val Ala His Tyr Leu Asn
420 425 430
Asn Ala Arg Val Glu Leu Ala Ala Phe Asn Ala Ala Val Thr Asp Trp
435 440 445
Glu Arg Ile Arg Gly Phe Glu Arg Leu
450 455
Claims (29)
1、一种可表达一种核酸的活的重组牛型结核菌卡介苗菌株，该核酸编码至少一种具有丙氨酸脱氢酶活性、谷氨酰胺合成酶活性或L-丝氨酸脱水酶活性的蛋白质或多肽。
2、一种可表达一种核酸的活的重组牛型结核菌卡介苗菌株，该核酸编码至少一种蛋白质或多肽，选自丙氨酸脱氢酶[SEQ ID NO：1；SEQ ID NO：2]、谷氨酰胺合成酶[SEQ ID NO：7到SEQ ID NO：14]和L-丝氨酸脱水酶[SEQ ID NO：5；SEQ ID NO：6]所组成的组中。
3、一种可表达一种核酸的活的重组牛型结核菌卡介苗菌株，该核酸包括至少一种全部或部分核酸分子，选自[SEQ ID NO：1]、[SEQ ID NO：5]、[SEQ IDNO：7]、[SEQ ID NO：9]、[SEQ ID NO：11]和[SEQ ID NO：13]所组成的组中。
4、一种可表达一种核酸的活的重组牛型结核菌卡介苗菌株，该核酸包括一个序列，该序列与至少一种选自[SEQ ID NO：1]、[SEQ ID NO：5]、[SEQ ID NO：7]、[SEQ ID NO：9]、[SEQ ID NO：11]和[SEQ ID NO：13]所组成的组中的核酸具有至少60％的序列一致性。
5、如权利要求3或4所述的活的重组牛型结核菌卡介苗菌株，其中核酸分子已被修饰。
6、如权利要求1、2、3、4或5所述的活的重组牛型结核菌卡介苗菌株，其中该牛型结核菌卡介苗菌株选自以下的组中：牛型结核菌-卡介苗-Russia、牛型结核菌-卡介苗-Moreau、牛型结核菌-卡介苗-Japan、牛型结核菌-卡介苗-Sweden、牛型结核菌-卡介苗-Birkhaug、牛型结核菌-卡介苗-Prague、牛型结核菌-卡介苗-Glaxo、牛型结核菌-卡介苗-Denmark、牛型结核菌-卡介苗-Tice、牛型结核菌-卡介苗-Frappier、牛型结核菌-卡介苗-Connaught、牛型结核菌-卡介苗-Phipps和牛型结核菌-卡介苗-Pasteur。
7、一种包括权利要求1、2、3、4、5或6中的活的重组牛型结核菌卡介苗菌株的药物组合物。
8、一种包括权利要求1、2、3、4、5或6中的活的重组牛型结核菌卡介苗菌株的用于治疗或预防哺乳动物免受分枝杆菌侵害的疫苗或致免疫的组合物。
9、如权利要求8所述的疫苗或致免疫的组合物，其中分枝杆菌是结核杆菌。
10、如权利要求8或9所述的疫苗或致免疫的组合物，进一步包括一种药物上可接受的载体。
11、如权利要求8、9或10所述的疫苗或致免疫的组合物，进一步包括一种佐剂。
12、如权利要求8、9、10或11所述的疫苗或致免疫的组合物，进一步包括来源于一种或多种其它病原体的致免疫的物质。
13、一种治疗或预防哺乳动物免受结核杆菌或牛型结核杆菌侵害的方法，包括给哺乳动物施用如权利要求1、2、3、4、5或6中所述的活的重组牛型结核菌卡介苗菌株。
14、如权利要求13所述的方法，其中哺乳动物是牛。
15、如权利要求13所述的方法，其中哺乳动物是人。
16、如权利要求13所述的方法，其中疫苗或致免疫的组合物是在有佐剂存在下用药的。
17、一种治疗或预防哺乳动物免受癌症侵害的方法，包括给哺乳动物施用如权利要求1、2、3、4、5或6中所述活的重组牛型结核菌卡介苗菌株。
18、如权利要求17所述的方法，其中疫苗或致免疫的组合物是在有佐剂存在下用药的。
19、如权利要求17或18所述的方法，其中癌症是膀胱癌。
20、一种包括权利要求1、2、3、4、5或6中的活的重组牛型结核菌卡介苗菌株的测试试剂盒。
21、一种含有丙氨酸作为唯一生长氮源的抑制牛型结核菌菌株生长的培养基组合物。
22、一种含有丝氨酸作为唯一生长氮源的抑制牛型结核菌菌株生长的培养基组合物。
23、如权利要求21或22所述的培养基组合物，进一步包括：
(a)一种碳源；
(b)铁离子；
(c)镁离子；和
(d)SO4。
24、如权利要求23所述的培养基组合物，其中碳源选自甘油、右旋糖、柠檬酸和葡萄糖所组成的组中。
25、一种抑制牛型结核菌卡介苗生长的方法，包括：
(a)获得一个分枝杆菌的样品；和
(b)在选择性培养基中培养该样品。
26、如权利要求25所述的方法，其中选择性培养基包括以丙氨酸作为唯一生长氮源。
27、如权利要求25所述的方法，其中选择性培养基包括以丝氨酸作为唯一生长氮源。
28、一种培养牛型结核菌卡介苗的方法，包括：
(a)获得一个分枝杆菌样品；和
(b)在不同的培养基中培养该样品。
29、如权利要求28所述的方法，其中不同的培养基中含有组氨酸。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US37245002P | 2002-04-16 | 2002-04-16 | |
| US60/372,450 | 2002-04-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1703513A true CN1703513A (zh) | 2005-11-30 |
| CN100572540C CN100572540C (zh) | 2009-12-23 |
Family
ID=29250855
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB038022761A Expired - Lifetime CN100572540C (zh) | 2002-04-16 | 2003-04-16 | 包括表达丙氨酸脱氢酶、丝氨酸脱水酶和/或谷氨酰胺合成酶的重组卡介苗菌株的结核病疫苗 |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US7611720B2 (zh) |
| JP (1) | JP4233458B2 (zh) |
| CN (1) | CN100572540C (zh) |
| AU (1) | AU2003218838A1 (zh) |
| CA (1) | CA2481108C (zh) |
| GB (1) | GB2403477B (zh) |
| RU (1) | RU2339692C2 (zh) |
| WO (1) | WO2003089462A2 (zh) |
| ZA (1) | ZA200408344B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101040047B (zh) * | 2004-10-13 | 2012-11-07 | 三井化学株式会社 | 编码新型的具有d-丝氨酸合成活性的酶的dna、该酶的制备方法、及利用该酶的d-丝氨酸制备方法 |
| CN114748615A (zh) * | 2022-04-29 | 2022-07-15 | 成都安永鼎业生物技术有限公司 | 一种治疗用重组卡介苗的冻干制剂及其制备方法和用途 |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006000045A1 (en) * | 2004-06-25 | 2006-01-05 | Proteome Systems Intellectual Property Pty Ltd | Novel methods of diagnosis and treatment of m. tuberculosis infection and reagents therefor i |
| US8647641B2 (en) * | 2008-10-20 | 2014-02-11 | University Of Zurich | Mycobacterium tuberculosis vaccine |
| US20130295648A1 (en) * | 2011-01-11 | 2013-11-07 | Bakulesh Mafatlal Khamar | Pharmaceutical composition for treating cancer |
| EP2696891B1 (en) | 2011-04-11 | 2019-06-26 | Alarum Development Ltd | New vaccines for prevention and treatment of tuberculosis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6097998A (en) * | 1997-01-29 | 1998-08-18 | Flohe, Leopold | L-alanine dehydrogenase of mycobacterium marinum |
| US6291190B1 (en) * | 1998-08-25 | 2001-09-18 | The Board Of Trustees Of The Leland Stanford Junior University | Molecular differences between species of the M. tuberculosis complex |
| US6471967B1 (en) * | 2000-04-17 | 2002-10-29 | The Regents Of The University Of California | Recombinant intracellular pathogen vaccines and methods for use |
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2003
- 2003-04-16 AU AU2003218838A patent/AU2003218838A1/en not_active Abandoned
- 2003-04-16 GB GB0425165A patent/GB2403477B/en not_active Expired - Lifetime
- 2003-04-16 CN CNB038022761A patent/CN100572540C/zh not_active Expired - Lifetime
- 2003-04-16 WO PCT/CA2003/000566 patent/WO2003089462A2/en active Application Filing
- 2003-04-16 RU RU2004133751/13A patent/RU2339692C2/ru not_active IP Right Cessation
- 2003-04-16 CA CA2481108A patent/CA2481108C/en not_active Expired - Fee Related
- 2003-04-16 US US10/511,718 patent/US7611720B2/en not_active Expired - Fee Related
- 2003-04-16 JP JP2003586182A patent/JP4233458B2/ja not_active Expired - Fee Related
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2004
- 2004-10-14 ZA ZA2004/08344A patent/ZA200408344B/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101040047B (zh) * | 2004-10-13 | 2012-11-07 | 三井化学株式会社 | 编码新型的具有d-丝氨酸合成活性的酶的dna、该酶的制备方法、及利用该酶的d-丝氨酸制备方法 |
| CN114748615A (zh) * | 2022-04-29 | 2022-07-15 | 成都安永鼎业生物技术有限公司 | 一种治疗用重组卡介苗的冻干制剂及其制备方法和用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| US7611720B2 (en) | 2009-11-03 |
| CA2481108C (en) | 2013-05-28 |
| CA2481108A1 (en) | 2003-10-30 |
| AU2003218838A8 (en) | 2003-11-03 |
| AU2003218838A1 (en) | 2003-11-03 |
| WO2003089462A3 (en) | 2004-05-21 |
| GB2403477B (en) | 2006-08-23 |
| JP2006508633A (ja) | 2006-03-16 |
| JP4233458B2 (ja) | 2009-03-04 |
| RU2339692C2 (ru) | 2008-11-27 |
| RU2004133751A (ru) | 2005-08-10 |
| CN100572540C (zh) | 2009-12-23 |
| ZA200408344B (en) | 2005-11-30 |
| US20070264286A1 (en) | 2007-11-15 |
| WO2003089462A2 (en) | 2003-10-30 |
| GB2403477A (en) | 2005-01-05 |
| GB0425165D0 (en) | 2004-12-15 |
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