专利名称：蝎子电压门控钠通道基因、编码蛋白及其克隆方法
技术领域：
本发明涉及一种电压门控钠通道基因、编码蛋白及其克隆方法。特别是一种东亚钳蝎的电压门控钠通道基因、编码蛋白及其克隆方法。
背景技术：
电压门控钠通道(VGSC)在神经元以及其它可兴奋性细胞动作电位形成和传导过程中扮演着极为重要的角色。当细胞膜去极化时，VGSC被电压依赖性的激活，使胞外钠离子选择性通透进入胞内，形成动作电位的去极化相；VGSC在开放后几个毫秒时段里就会失活，失活的VGSC需要回到静息状态后才能再开放(Catteral，1992)。VGSC的细微突变或异常表达会导致其相应的功能改变，甚至诱发诸如神经肌肉周期性麻痹症，心脏长QT间隔综合征(LQTs)、癫痫以及一些遗传性疾病(Aschroft，2000)。此外，VGSC还被认为参与了神经病理性痛的发生和维持(Lai et al.，2003)。与哺乳动物含多种钠通道基因不同，昆虫钠通道的基因相对较少，只发现一到两个(para基因和DSC基因)，但仅para基因编码的钠通道在体外得以表达和功能鉴定，而DSC基因可能不再编码功能性的钠通道(Goldin，2002)。目前已克隆和测序的昆虫para直系同源钠通道基因(para-orthologous sodium channel gene)有果蝇(Drosophila melanogaster)中的para基因、家蝇(Musca domestica)中的Vsscl基因(也称Msc基因)、烟草蚜虫(Heliothis virescens)中的hscp基因、德国小蠊Blattella germanica中的paraCSMA(Loughney et al.，1989；Ingles et al.，1996；Miyazaki et al.，1996；Park et al.，1999)。昆虫钠通道存在于包括感觉神经元在内的昆虫神经系统，一般不表达在肌肉组织。细胞水平上的研究表明，昆虫钠通道在神经元的胞体与轴突上均有表达(Wicher et al.，2001)。
钠通道呈现出的亚型多样性，为理解钠通道在生理/病理下的功能特性及其动态平衡调控带来了复杂性，同时，也正在引发通道药理与毒理基因组学研究的挑战性。例如，在神经系统中，各亚型的钠通道通常共表达于同一个细胞上(例如DRG神经元)，为了阐明各亚型参与的生理活动，有必要将各通道介导的电流成分分离。但由于钠通道各亚型介导的宏观电流性质接近，单用不同刺激方式无法获得单一电流。体外表达虽可以获得单一亚型的信息，但表达体系以及辅助亚基的存在与否都会对通道的性质产生重大影响，而且失去了通道在体内时的复杂环境。又如，目前临床上癫痫、神经病理性痛等的治疗药物通常以钠通道为靶器，但由于其选择性不高，可能产生严重的副作用。再如，昆虫钠通道是DDT和拟除虫菊酯类杀虫剂的主要作用靶点，但长期使用和滥用，一方面严重的污染了环境和危害公共健康；另一方面导致了昆虫抗药性的出现，其敏感性的变化是由于昆虫的para钠通道基因序列在杀虫剂和神经毒素结合位点处发生了突变(Williamson et al.，1996；Soderlund & Knipple，2003)。因此，开拓对不同通道亚型具有特异选择性作用的配体/调制剂并解析新型通道亚型的基因编码特征，不仅有利于各亚型生理功能的阐明与药理价值的评估，同时也为钠通道关联的临床疾病治疗药物以及高效生物杀虫剂研制提供理论的可行性和新分子先导物。
发明内容
本发明的目的之一在于提供一种蝎子电压门控钠通道基因。
本发明的目的之二在于提供该电压门控钠通道基因的编码蛋白。
本发明的目的之三在于提供该电压门控钠通道基因的克隆方法。
为达到上述目的，本发明采用如下技术方案一种蝎子电压门控钠通道基因，其特征在于，它具有SEQ NO 1所示的碱基序列。
上述的蝎子电压门控钠通道基因的编码蛋白，其特征在于，它具有SEQ NO 2所示的氨基酸序列。
上述的蝎子电压门控钠通道基因的克隆方法，其特征在于该方法的具体步骤为a.从东亚钳蝎的腹神经纤维中提取总RNA；b.用Superscript II反转录酶和Oligo-(dT)引物合成单链cDNA；c.借鉴昆虫Para钠通道的保守氨基酸残基设计退火引物，再采用兼并PCR以及cDNA末端的快速扩增技术，得到全长cDNA。
本发明利用逆转录聚合酶链式反应(RT-PCR)，兼并PCR(Nested-PCR)以及cDNA末端的快速扩增(RACE)技术，得到了东亚钳蝎的全长cDNA。用于电压门控钠通道的表达及药理调制机制的研究，并为离子通道病的防治和进一步开发为医药品奠定基础。
图1为蝎子钠离子通道克隆实验策略。
图2为蝎子钠离子通道(BmNav1)与蜘蛛(OhNav1)，果蝇(Drosopjlia para)(DmNav1)，鼠脑II型(rNav1.2)电压门控钠离子通道基因序列比对图。跨膜片段用框表注出来了，S4电压感受器中正电荷氨基酸残基和结构域III-IV连接片段(DomainIII-IV linker)中在钠离子通道的快失活中的三个关键氨基酸残基都用阴影标注出来了。
具体实施例方式
实施例一电压门控钠通道基因的克隆1.蝎神经总RNA的制备剥离蝎腹部的神经纤维(50～100只)，迅速置于液氮中保存。用50ml离心管抽提总RNA将神经浸泡在冰浴的5ml异硫氰酸胍溶液(含0.2M的醋酸钠<pH＝4>)中，加入5ml冰上预冷的苯酚(pH＝4)，匀浆，冰上放置5min后离心(10000rpm/4℃/10min)，将上清转移至新离心管中，加入5ml冰上预冷的氯仿/异戊醇(V/V＝24/1)，剧烈混匀后再次离心(10000rpm/4℃/10 min)，取上清液转移至新管，缓缓加入等体积的异戊醇，边加边混匀.放置-70℃保存。
2钠通道cDNA的合成大约10ug蝎神经总RNA样品用Superscript II反转录酶和Oligo-(dT)引物合成单链cDNA，得到的一系列重叠片段被确定基本覆盖整个编码区，参见图1；再借鉴昆虫Para钠通道的保守氨基酸残基设计退火引物，进行扩增蝎钠通道的cDNA，PCR在PE-2400(Perkin-Elmer，USA)上进行，再经5’RACE(Rapid Amplificationof the cDNA Ends)和3’RACE获得全长cDNA。所设计的退火引物见表1表1 Primer for Scorpion Sodium Channel
实施例二上述PCR产物用琼脂糖凝胶分析，经割胶回收后克隆到pGEM-T easy载体(Promega，USA)，连接有插入的DNA载体转化至大肠杆菌，最后被纯化并用ABIPRISM 377 DNA测序仪(Perkin-Elmer，USA)测序。
测序结果如下引物AUAP/A1275所得片段正向测序结果aaataaaacctacggtcaaagatggcggacttggtctgagtggttttttctgggttttcgtcgacaatattctcattcgagagtcccggattatgagccagcggatactttaactctgccaaaagaacggagcatggtccagggccccgggccccgtaggtggtagagagcgttcgcctgtctcgtcttcgccatgtcttcggacgagcagtccgaggaggaagttctgcccgcattccgtcccttcaccagggagtcattggcggccatcgaagccaggatcgccgaggctgaatcaagaaaaaaggaatcgcttcagaaaaaaggggaaggcgacgatgaacactacggcacttaccagccggaagtggccgaagccgaccctcaactggaggcgggtctcccattacccaagtatttggaacgtgaatttcctcccgaactattggctacacctatcgaaggtttggacaaattttacgaaaacaaaaggacattcattgtgataagtaaaggtaaggatatcttccggtttagtgcaacaaatgccatgtggat反向测序结果tgaatcttgcataaaagctacagcaagaggatttatacttgagcaattcacatatcttcgagatccgtggaattggctggattttgtagttatttcttta
gcttacattacaatgggagttgaattaggaaatttaagtgctttgaggacttttcgtgtacttagagcattaaaaactgttgctgttattccaggtttaaagactattgttggagctgtcatagaatctgtaaagaatttaaaagatgtgattattttgacagtgttttctctatctctatttgccttacttggtctccagatgtatatgggtgttctaatgcaaaaatgtattgtagaagctcctccaaatactacttatgaagagaagaaagtacattataggaatcaaagtaactggtatctagattctgctggtgaatattttatttgtggaaatgcatcaggagctagacaatgtcctgaaggatatatttgtttgtccaattatggtcccaatccaaatcatggttttacaaattttgacacattttattgggcatttcttg引物S7/A2435所得片段正向测序结果cttggtctccagatgtatatgggtgttctaatgcaaaaatgtattgtagaagctcctccaaatactacttatgaagagaagaaagtacattataggaagtcccaatccaaatcatggttttacaaattttgacacattttattgggcatttcttgcatcatttagacttatgactcaagatttttgggaggtctttaccaaatggtgctaagaacagcaggtccttggcatatatgttttttcatcgtaatcatctttcttggttcattttatttattaaatctcattttggccattgttgctatgtcatatgatgaattacaaaaaagagctgaaaaagaagaagaagatgcacaaaaagaagaagaaaactataatgcagctgtagaacgtaaaagattgttagaagaagaaaaagcaggagtgggtgtagtgaagagttcttc反向测序结果tgagatttattgatatcttctgcgtatgggactgttgtaactgctggttatatatacaaaaaatagttaacatcattgtctttgatccatttatggagttattcattacactggcaataattgtcaacacgctcttcatgtctatagaacatgccgacatggataaaagtcttcaggaattcttaaaaaatggcaactactttttcacagcaacatttgccatagaagcagtaatgaagttggtagcactgagtccaaaatattactttaaagaaggatggaacatatttgattttttaattgtagtactttctcttgttgaactgtgtggagaaaatgtttctttacctggattatcagttttgagatcatttcgtttgcttcgtgtatttaaacttgcaaaatcttggccaacactcaatttattaatatccattatgggaaaaactgtgggtgccttgggaaacctaacttttgtgttgggtatcatcattttcatatttgctgttatgggtatg引物S5/A4248所得片段正向测序结果atcatcattttcatatttgctgttatgggtatgcagttgtttggtgaaaaatatatagaaaataaacatgtgtttcctgataatgctgttccacgctggaattttgttgattttatgcattcatttatgattgtgtttcgtgttttgtgtggagaatggatagaatctatgtggggtcgtatgcttgttgcagaatggccatgtgttcctttttttttatcaactgttataattggaaacctagtggtgctgaatttgtttcctgccttgcttttgtcctcctttggtgccgcaaatctttccatggcttcaccagaaagtgctgatactaagaaactgcaagaagcatttgaacgtataggaagagctatcaattgggttaaaatctgcttcttacgaatcttaaaaaatttaagaccaaaaactagaaatcaaattggagatcaaacaacagatattagagaagatctggaagatgctacaataggacgagagatgttagttgatggtcaggttaaaatgaaagagaaaaaaagccctaaagaaattacag反向测序结果aaaagtacttcacaaatgcttggtgttggcttgattttgttatagttctggtttctctgtttaatctggcagtaagcttaatgggaggtagtgacatatctgcctttaaaactatgcgaaccctgagagctctccgcccacttagggcaatgtcacgtctggagggaatgagagttgtagtaaatgctctcgttcaagct
atccctgccatctttaatgttttacttgtgtgcctcatcttttggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttcaatagctctactgtgcctaacaaatctgtttgttgcttaaacaattttacttgggaaaatccaaaaataaattctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcaatagatactaagcaaaatgaagatgaacaac引物S4/A4448所得片段正向测序结果tggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttcaatagctctactgtgcctaacaaatctgtttgttgcttaaacaattttacttgggaaaatccaaaaataaattctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcaatagatactaagcaaaatgaagatgaacaacctgaatatgaagtcaatcgatatatgtatctatactttgtacttttcatcattataggatcttttttcacgttaaatctcttcattggagtaataatagataatttcaatgaacagaaaaaaaaggcaggaggttctttagaaatgtttatgactgaagatcagaagaaatattataatgctatgaaaaaaatgggagagaagaagccagctaaagctattcctcggccaaggttcaaattacaagcagtagtatttgatcttattactaatacaaaatttgatatggcaatcgtgttgtttattgg反向测序结果ctatccctgccatctttaatgttttacttgtgtgcctcatcttttggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttcaatagctctactgtgcctaacaaatctgtttgttgcttaaacaattttacttgggaaaatccaaaaataaattctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcaatagatactaagcaaaatgaagatgaacaacctgaatatgaagtcaatcgatatatgtatctatactttgtacttttcatcattataggatcttttttcacgttaaatctcttcattggagtaataatagataatttcaatgaacagaaaaaaaaggcaggaggttctttagaaatgtttatgactgaagatcagaagaaatattataatgctatgaaaaaaatgggagag引物S3/A5149所得片段正向测序结果atgtttatgactgaagatcagaagaaatattataatgctatgaaaaaaatgggagagaagaagccagctaaagctattcctcggccaaggttcaaattacaagcagtagtatttgatcttattactaatacaaaatttgatatggcaatcgtgttgtttattggacttaatatgctagtgatggcaatggaacattatcatcagacaccaacatttgatgatattctagaaagattaaatatattctttattgccattttcactacagaatgtgtgttaaaaatgtttgcccttaggtggtattattttaaagaaccatggaatgtatttgattgtgtggttgtaaccttgtccattttaggcattgcaatcaaagatttaatagcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtcaaaggggccaaagggattcgaaccttactttttgctcttgccatgtccttgccagcactttttaatatttgccttctgctctttttagttatgtttatctacgccatatttggaatgtct反向测序结果tattgccattttcactacagaatgtgtgttaaaaatgtttgcccttaggtggtattattttaaagaaccatggaatgtatttgattgtgtggttgtaaccttgtccattttaggcattgcaatcaaagatttaatagcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtcaaaggggccaaagggattcgaaccttactttttgctcttgccatgtccttgccagcactttttaatatttgccttctgctctttttagttatgtttatctacgccatatttggaatgtcttttttcatgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaa
atgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatgaaggcattgaagg引物S2/AS1所得片段正向测序结果tgccagcactttttaatatttgccttctgctctttttagttatgtttatctacgccatatttggaatgtcttttttcatgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatgaaggcattgaaggagattgtggtaaaccaggtgtagcagtggcatatttaatttcttacttaattgtaagttttttaattataattaacatgtatattgctgtcattcttgaaaactacagtcaagctacagaagatgtacaggaaggtttaacagctgatgattatgatatgtattatgaaatatggcaaaaatacgatcctgaaggtacacaatatatcaagtatgaagatttgagtgacttcctagatgctctagaagaaccattgcagattgagaaacctaataagtacaaaatcattgacatggatattac反向测序结果attgtgtggttgtaaccttgtccattttaggcattgcaatcaaagatttaatagcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtcaaaggggccaaagggattcgaaccttactttttgctcttgccatgtccttgccagcactttttaatatttgccttctgctctttttagttatgtttatctacgccatatttggaatgtcttttttcatgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatgaaggcattgaaggagattgtggtaaaccaggtgtagcagtggcatatttaatttcttacttaattgtaagttttttaattataattaacatgtatattgctgtcattcttgaaa引物S5158/NUP所得片段正向测序结果taaaccaggtgtagcagtggcatatttaatttcttacttaattgtaagttttttaattataattaacatgtatattgctgtcattcttgaaaactacagtcaagctacagaagatgtacaggaaggtttaacagctgatgattatgatatgtattatgaaatatggcaaaaatacgatcctgaaggtacacaatatatcaagtatgaagatttgagtgacttcctagatgctctagaagaaccattgcagattgagaaacctaataagtacaaaatcattgacatggatattacaatatgtaaaggtgatcttatgtattgtgtagatattcttgatgcactgaccaaagatttctttgcgaggaaaggtactgctatagaagaatctgcagaacttggagaagttgctcctaggaaagacgttgaaggctatgagccaatcagcagtacgcgcttccgtcaacaggaagaatataatgctcgtgttattcagagagcatggcgacattataaagggtgtagtgctagtg反向测序结果aaattcttgcctgttatacatagcaggagctagtatagaattattacctaactccaatcctagtaacttagttgccattgaaataggcactctagcattcatgaaagttttattagcttcggaaacaatgcaaccacaggatgagacagtgaaagggaatgtgatatattcctttatcatcctgtctgttatagtgaaaaattaaaaaaaaatgaaaaacttatgtttcaagagccagaactcttcctcttactacaaagcatggctttgagaatgacaagtttataatggaagaatctgctatgctgaagattgcttcaagaccttgactcgccatttccatcttaccacccagtttcaatcattttctcaattatgccttttcagcttgtacttactttgtaaaatgtttcacagtttgtaccatactgcctgtaaatacatcattgaataaatttttgataatttatcacaacgaaccgatgctcaagataaaaaaaaaaaaaaaaa
aaaaaaaaaaa蝎子电压门控钠离子通道结构特征分析及功能说明1.蝎子电压门控钠离子通道结构特征分析如附图2所示，蝎子电压门控钠离子通道(BmNav1)蛋白质内部有四个高度相似的同源结构域(D1-D4)，且每一结构域含有六个α螺旋跨膜片段(S1-S6)，具有电压门控钠通道的所有关键性的结构1)BmNav1的S4片段具有钠通道电压感受器的共性，即在每隔2个疏水残基有一个带正电的Arg或Lys残基，且碱性氨基酸残基数目和位置基本对应；2)BmNav1与其它节肢动物电压门控钠通道蛋白在结构域III和IV之间的短链胞内环处(钠通道失活化门控域)高度保守，它们在对应于哺乳动物钠通道rNav1.2的三个关键残基I1488-F1489-M1490处也存在三个疏水性残基(MFM)；3)BmNav1蛋白分子中形成钠通道选择性过滤器的胞内环(DEKA)和胞外环(EEDD)以及与TTX结合的关键残基也都是保守的。通过氨基酸序列比对，我们可以看出，我们所克隆得到的基因序列正是蝎子的电压门控钠离子通道的基因序列.
2.蝎子电压门控钠离子通道功能说明电压门控钠通道(VGSC)在神经元以及其它可兴奋性细胞动作电位形成和传导过程中扮演着极为重要的角色。当细胞膜去极化时，VGSC被电压依赖性的激活，使胞外钠离子选择性通透进入胞内，形成动作电位的去极化相；VGSC在开放后几个毫秒时段里就会失活，失活的VGSC需要回到静息状态后才能再开放。VGSC的细微突变或异常表达会导致其相应的功能改变，甚至诱发诸如神经肌肉周期性麻痹症，心脏长QT间隔综合征(LQTs)、癫痫以及一些遗传性疾病。此外，VGSC还被认为参与了神经病理性痛的发生和维持。
蝎子钠离子通道对于蝎子自身细胞动作电位的产生，传导，神经的兴奋性传递具有重要作用.同时，蝎毒素被发现可以作用在不同物种或者亚型的电压门控钠通道上，但药理实验表明蝎子对蝎毒素表现出不敏感性，新克隆的蝎钠通道无疑为研究蝎毒素对钠通道选择性的分子机制，研究钠离子通道结构与功能之间的关系提供了难得的分子模板。此外，钠离子通道是多种药物和杀虫剂但作用靶点，蝎子钠离子通道的克隆为新型药物和杀虫剂的筛选，测试，研发提供了新的研究体系。
序列表<110>上海大学
<120>蝎子电压门控钠通道基因、编码蛋白及其克隆方法<160>2<210>1<211>8065<212>DNA<213>东亚钳蝎(Buthus Martensii Karsch)<220>
<221>5’UTP<222>(1)...(193)<220>
<221>CDS<222>(194)...(5809)<220>
<221>3’UTP<222>(5810)...(8065)<220>
<221>PolyA_signal<222>(7994)...(7999)<400>1aaataaaacc tacggtcaaa gatggcggac ttggtctgag tggttttttc tgggttttcg 60tcgacaatat tctcattcga gagtcccgga ttatgagcca gcggatactt taactctgcc 120aaaagaacgg agcatggtcc agggccccgg gccccgtagg tggtagagag cgttcgcctg 180tctcgtcttc gccatgtctt cggacgagca gtccgaggag gaagttctgc ccgcattccg 240tcccttcacc agggagtcat tggcggccat cgaagccagg atcgccgagg ctgaatcaag 300aaaaaaggaa tcgcttcaga aaaaagggga aggcgacgat gaacactacg gcacttacca 360gccggaagtg gccgaagccg accctcaact ggaggcgggt ctcccattac ccaagtattt 420ggaacgtgaa tttcctcccg aactattggc tacacctatc gaaggtttgg acaaatttta 480cgaaaacaaa aggacattca ttgtgataag taaaggtaag gatatcttcc ggtttagtgc 540aacaaatgcc atgtggatat tagatccttt caatcctatc cgtcgtgcag ccatcagcat 600cctcgttcat cctgccttta gcttcttggt tattgtcact atccttgtaa actgtgttct 660
tatgacgcaa acaaatgtaa cgcacgaaaa ggaattagag aggcttttta cggcgatata 720tacaattgaa tcttgcataa aagctacagc aagaggattt atacttgagc aattcacata 780tcttcgagat ccgtggaatt ggctggattt tgtagttatt tctttagctt acattacaat 840gggagttgaa ttaggaaatt taagtgcttt gaggactttt cgtgtactta gagcattaaa 900aactgttgct gttattccag gtttaaagac tattgttgga gctgtcatag aatctgtaaa 960gaatttaaaa gatgtgatta ttttgacagt gttttctcta tctctatttg ccttacttgg 1020tctccagatg tatatgggtg ttctaatgca aaaatgtatt gtagaagctc ctccaaatac 1080tacttatgaa gagaagaaag tacattatag gaatcaaagt aactggtatc tagattctgc 1140tggtgaatat tttatttgtg gaaatgcatc aggagctaga caatgtcctg aaggatatat 1200ttgtttgtcc aattatggtc ccaatccaaa tcatggtttt acaaattttg acacatttta 1260ttgggcattt cttgcatcat ttagacttat gactcaagat ttttgggaag gtctttacca 1320aatggtgcta agaacagcag gtccttggca tatatgtttt ttcatcgtaa tcatctttct 1380tggttcattt tatttattaa atctcatttt ggccattgtt gctatgtcat atgatgaatt 1440acaaaaaaga gctgaaaaag aagaagaaga tgcacaaaaa gaagaagaaa actataatgc 1500agctgtagaa cgtaaaagat tgttagaaga agaaaaagca ggagtgggtg tagtgaagag 1560ttcttcagaa tcttcttctg gatcttacga attatttgtg ggacaagata aaactgtaga 1620agaaaaagat gcaagtatta agagtgtaga tggagatagc attggtgaac caaaatctca 1680aataaatggc aaagtaagga aggattcttt ggatgatgtg gaatatagca taaaacaaaa 1740accatatgat agtcctttca tagatcctca aagacaaaca gaattagata tgaaagatgt 1800gatggttttg aatgacatta tagatcaagc tgcaggacgt caaagtagag aaagtggcag 1860agagagggaa ccagaagaga aggagaagtt caaagaaaaa tgcttcaaaa attgcttgag 1920atttattgat atcttctgcg tatgggactg ttgtaactgc tggttatata tacaaaaaat 1980agttaacatc attgtctttg atccatttat ggagttattc attacactgg caataattgt 2040caacacgctc ttcatgtcta tagaacatgc cgacatggat aaaagtcttc aggaattctt 2100aaaaaatggc aactactttt tcacagcaac atttgccata gaagcagtaa tgaagttggt 2160agcactgagt ccaaaatatt actttaaaga aggatggaac atatttgatt ttttaattgt 2220agtactttct cttgttgaac tgtgtggaga aaatgtttct ttacctggat tatcagtttt 2280gagatcattt cgtttgcttc gtgtatttaa acttgcaaaa tcttggccaa cactcaattt 2340attaatatcc attatgggaa aaactgtggg tgccttggga aacctaactt ttgtgttggg 2400tatcatcatt ttcatatttg ctgttatggg tatgcagttg tttggtgaaa aatatataga 2460
aaataaacat gtgtttcctg ataatgctgt tccacgctgg aattttgttg attttatgca 2520ttcatttatg attgtgtttc gtgttttgtg tggagaatgg atagaatcta tgtggggtcg 2580tatgcttgtt gcagaatggc catgtgttcc ttttttttta tcaactgtta taattggaaa 2640cctagtggtg ctgaatttgt ttcctgcctt gcttttgtcc tcctttggtg ccgcaaatct 2700ttccatggct tcaccagaaa gtgctgatac taagaaactg caagaagcat ttgaacgtat 2760aggaagagct atcaattggg ttaaaatctg cttcttacga atcttaaaaa atttaagacc 2820aaaaactaga aatcaaattg gagatcaaac aacagatatt agagaagatc tggaagatgc 2880tacaatagga cgagagatgt tagttgatgg tcaggttaaa atgaaagaga aaaaaagccc 2940taaagaaatt acagatcttg aagttgtagt aggagatgga ttagaaatat caattcaagg 3000aaatggacaa gcaataccaa taaaaatggc taataatact aaaacacttg taaattcttc 3060acatatgtct gctaaagtag aattgaaaga aaaggaaaaa gaaaaattca aagaaaaaga 3120taataaagaa aaggatgaag gatttggaaa taaagtatat cctcaaaaag atgacgatga 3180tgttagtaac aaatctatta ataatgtcaa ggatttaaaa gataatattg agaaggggat 3240tcctaataac aaaccgtcca aagaggaaag ccaaatttca ctgccagaag gatcccaaga 3300agaaaaaaag gatgctagta aagatgatgt tattaatgaa ggaggatttt taaaagatga 3360ggatatactt gaagaagatg cagaaattga gggagataac gcagatgatg aatcatcaaa 3420aattataatt cctgaatatc catctgattg ttttccagac aagtgttatc aacattttcc 3480attttgttta gggaatgaag aatcaaaatt ttggagacac tatactacta tacgtggaaa 3540aagttacaca ttggtggaaa ataaatattt tgaaacattg attattttta tgatattagt 3600cagcagtttg gcattagcat tagaagatgt taatttgaaa aaaagaagat ggttaatgga 3660tattttacaa tatatggaca agatatttac aattttattt ttttttgaaa tgttaatcaa 3720atggctagct tttggtttta aaaagtactt cacaaatgct tggtgttggc ttgattttgt 3780tatagttctg gtttctctgt ttaatctggc agtaagctta atgggaggta gtgacatatc 3840tgcctttaaa actatgcgaa ccctgagagc tctccgccca cttagggcaa tgtcacgtct 3900ggagggaatg agagttgtag taaatgctct cgttcaagct atccctgcca tctttaatgt 3960tttacttgtg tgcctcatct tttggcttat tttttctatt atgggtgttc agttatttgc 4020tgggaaattt cgttattgtg cagataaaga tgggaatcgt ttcaatagct ctactgtgcc 4080taacaaatct gtttgttgct taaacaattt tacttgggaa aatccaaaaa taaattctga 4140taatgtactc aatgcatatt tagctctttt tcaagttgcc acattcaaag gatggattga 4200tataatgagt aatgcaatag atactaagca aaatgaagat gaacaacctg aatatgaagt 4260
caatcgatat atgtatctat actttgtact tttcatcatt ataggatctt ttttcacgtt 4320aaatctcttc attggagtaa taatagataa tttcaatgaa cagaaaaaaa aggcaggagg 4380ttctttagaa atgtttatga ctgaagatca gaagaaatat tataatgcta tgaaaaaaat 4440gggagagaag aagccagcta aagctattcc tcggccaagg ttcaaattac aagcagtagt 4500atttgatctt attactaata caaaatttga tatggcaatc gtgttgttta ttggacttaa 4560tatgctagtg atggcaatgg aacattatca tcagacacca acatttgatg atattctaga 4620aagattaaat atattcttta ttgccatttt cactacagaa tgtgtgttaa aaatgtttgc 4680ccttaggtgg tattatttta aagaaccatg gaatgtattt gattgtgtgg ttgtaacctt 4740gtccatttta ggcattgcaa tcaaagattt aatagcaagt tattttgtgt caccaactct 4800tctgcgtgtg gttcgtgttg taaaagttgg tcgtgtcctc aggttggtca aaggggccaa 4860agggattcga accttacttt ttgctcttgc catgtccttg ccagcacttt ttaatatttg 4920ccttctgctc tttttagtta tgtttatcta cgccatattt ggaatgtctt ttttcatgac 4980tgtaaaacat cgtgcgggca tcgatgataa ttttaatttt gaaacatttg gtcagtcaat 5040gattctttta tttcaaatgt caacatcagc aggttgggat ggtgtgttat taggtatcat 5100ggatgacagt gactgtgtac aaggatctga agatgaaggc attgaaggag attgtggtaa 5160accaggtgta gcagtggcat atttaatttc ttacttaatt gtaagttttt taattataat 5220taacatgtat attgctgtca ttcttgaaaa ctacagtcaa gctacagaag atgtacagga 5280aggtttaaca gctgatgatt atgatatgta ttatgaaata tggcaaaaat acgatcctga 5340aggtacacaa tatatcaagt atgaagattt gagtgacttc ctagatgctc tagaagaacc 5400attgcagatt gagaaaccta ataagtacaa aatcattgac atggatatta caatatgtaa 5460aggtgatctt atgtattgtg tagatattct tgatgcactg accaaagatt tctttgcgag 5520gaaaggtact gctatagaag aatctgcaga acttggagaa gttgctccta ggaaagacgt 5580tgaaggctat gagccaatca gcagtacgcg cttccgtcaa caggaagaat ataatgctcg 5640tgttattcag agagcatggc gacattataa agggtgtagt gctagtgaac ctcgtcagca 5700acaaactgca attgttattg aaagtgatgg ccatgtaact agaaatggtc acaaagttgt 5760gattcattct aggtcaccaa gtgttacaag ccgttccaca gatgtgtgat atttataaaa 5820tagaaaaggg gctttctaca tcaacattat ctaaatagaa attggttgat gattgattgc 5880tgacaatatc tgcatgaagt gtatattttt atattcttta tgcacgagtt gagtaccagt 5940ctgcttgcaa cacctgtact tggttgacat aaaccaatgt tttccatgtt atttttctaa 6000tgactgatta gttgtaatta tggtctattt acactacttg taaatatgca aactaaaagg 6060
gataatgatc ccattcttgt tgctacaata ggataagagt ggaagcatct aagttttaat 6120ttgttttata tatattttga tttatagaca ttttacggat tttagaatgt attttttatt 6180attacatgta atgttattac ataacaaagt gcttttattt acaaattcta gaatttcaaa 6240ggaaaaatgt cagaaattac tagagagact gttaaatckg ttattattat tctgaaaatt 6300tgttttttac ttgtgtatat atataatata tatatatatg gaagcagaat ttatactgct 6360tctataaaca tgttaaattt attcaaaggg aagagatgtg aattatttaa gtttttttta 6420actaatatta atttatttat tttttagcac tttggacatt aaccaatgcc aaatgaaatg 6480aacgttttaa ctagaaaagt tcagtgccca tatttaaaca gcatataaga agtaaaatgt 6540gattccaaga gaagtatgat ctgtatcagt aaagctacat aggtgggttg aagatggtta 6600ttgctacatt agctgttaac acacaaacta aatttgaaag attctgctta tgaacagggt 6660ttcttataag aagaaatata acaatgtagg ggaatgttcc cccctatca gtgataatgt 6720atctatagga atcttcctca gtttttctgc tttctaagct gatggcatta aaaaattttg 6780cacttatgaa tagattcatg ttatcagcat caagctgtga aattgggtgg caaacaactt 6840cagctgtgtt ataagcgtgt tgttataaac ggagtataca gcttcgagga tacgggaggg 6900tgggaaaaat caacgcttaa ttgtcctaat aacctctgca ctgtacctct aagtgtataa 6960cactctacaa agctcagttt tcctgccgct tttaaatgag cagaaaaaga gtgtgtgata 7020agggtaatta atatacaaag ttattggctg agttcagaag ttggactttt ggccaccaga 7080ctttaaaagc agagattatc aaaactctgc aaaatcttaa gagattttat ttatcatgtg 7140cctttctgcg ttgttactgt tgtcacaaga aagcataatc attgcatcat aaatcagctt 7200cccacgcaac aatacttgct ttcttatata ctttccaaca tttttcaagc caaatagata 7260agactgtggt ttcttttcaa agtgcacaac attatttcat aaaaaaaatg aagcagatct 7320caccattggc ttgatgtcta tactgctagt atatataaaa gctcaagact gcaaataatt 7380tataatatcc tataccacac tatgcattgg attttatttg aacacaacta aggtggtggt 7440tgtataatta attctaaatg tcaagaatat aaaagttttg ttagtttctt ctcttctggg 7500aaaaatcatc actccatgta aaaaaatcag aaaaaatcta gcataaattc ttgcctgtta 7560tacatagcag gagctagtat agaattatta cctaactcca atcctagtaa cttagttgcc 7620attgaaatag gcactctagc attcatgaaa gttttattag cttcggaaac aatgcaacca 7680caggatgaga cagtgaaagg gaatgtgata tattccttta tcatcctgtc tgttatagtg 7740aaaaattaaa aaaaaatgaa aaacttatgt ttcaagagcc agaactcttc ctcttactac 7800aaagcatggc tttgagaatg acaagtttat aatggaagaa tctgctatgc tgaagattgc 7860
ttcaagacct tgactcgcca tttccatctt accacccagt ttcaatcatt ttctcaatta 7920tgccttttca gcttgtactt actttgtaaa atgtttcaca gtttgtacca tactgcctgt 7980aaatacatca ttgaataaat ttttgataat ttatcacaac gaaccgatgc tcaagataaa 8040aaaaaaaaaa aaaaaaaaaa aaaaa 8065<210>2<211>1871<212>PRT<213>东亚钳蝎(Buthus Martensii Karsch)<400>2Met Ser Ser Asp Glu Gln Ser Glu Glu Glu Val Leu Pro Ala Phe Arg Pro Phe Thr Arg1 5 10 15 20Glu Ser Leu Ala Ala Ile Glu Ala Arg Ile Ala Glu Ala Glu Ser Arg Lys Lys Glu Ser25 30 35 40Leu Gln Lys Lys Gly Glu Gly Asp Asp Glu His Tyr Gly Thr Tyr Gln Pro Glu Val Ala45 50 55 60Glu Ala Asp Pro Gln Leu Glu Ala Gly Leu Pro Leu Pro Lys Tyr Leu Glu Arg Glu Phe65 70 75 80Pro Pro Glu Leu Leu Ala Thr Pro Ile Glu Gly Leu Asp Lys Phe Tyr Glu Asn Lys Arg85 90 95 100Thr Phe Ile Val Ile Ser Lys Gly Lys Asp Ile Phe Arg Phe Ser Ala Thr Asn Ala Met105 110 115120Trp Ile Leu Asp Pro Phe Asn Pro Ile Arg Arg Ala Ala Ile Ser Ile Leu Val His Pro125 130 135140Ala Phe Ser Phe Leu Val Ile Val Thr Ile Leu Val Asn Cys Val Leu Met Thr Gln Thr145 150 155160Asn Val Thr His Glu Lys Glu Leu Glu Arg Leu Phe Thr Ala Ile Tyr Thr Ile Glu Ser165 170 175180Cys Ile Lys Ala Thr Ala Arg Gly Phe Ile Leu Glu Gln Phe Thr Tyr Leu Arg Asp Pro185 190 195200Trp Asn Trp Leu Asp Phe Val Val Ile Ser Leu Ala Tyr Ile Thr Met Gly Val Glu Leu205 210 215220Gly Asn Leu Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr Val Ala Val225 230 235240Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Val Ile Glu Ser Val Lys Asn Leu Lys Asp245 250 255260Val Ile Ile Leu Thr Val Phe Ser Leu Ser Leu Phe Ala Leu Leu Gly Leu Gln Met Tyr265 270 275280Met Gly Val Leu Met Gln Lys Cys Ile Val Glu Ala Pro Pro Asn Thr Thr Tyr Glu Glu285 290 295 300
Lys Lys Val His Tyr Arg Asn Gln Ser Asn Trp Tyr Leu Asp Ser Ala Gly Glu Tyr Phe305 310 315 320Ile Cys Gly Asn Ala Ser Gly Ala Arg Gln Cys Pro Glu Gly Tyr Ile Cys Leu Ser Asn325 330 335 340Tyr Gly Pro Asn Pro Asn His Gly Phe Thr Asn Phe Asp Thr Phe Tyr Trp Ala Phe Leu345 350 355 360Ala Ser Phe Arg Leu Met Thr Gln Asp Phe Trp Glu Gly Leu Tyr Gln Met Val Leu Arg365 370 375 380Thr Ala Gly Pro Trp His Ile Arg Phe Phe Ile Val Ile Ile Phe Leu Gly Ser Phe Tyr385 390 395 400Leu Leu Asn Leu Ile Leu Ala Ile Val Ala Met Ser Tyr Asp Glu Leu Gln Lys Arg Ala405 410 415 420Glu Lys Glu Glu Glu Asp Ala Gln Lys Glu Glu Glu Asn Tyr Asn Ala Ala Val Glu Arg425 430 435 440Lys Arg Leu Leu Glu Glu Glu Lys Ala Gly Val Gly Val Val Lys Ser Ser Ser Glu Ser445 450 455 460Ser Ser Gly Ser Tyr Glu Leu Phe Val Gly Gln Asp Lys Thr Val Glu Glu Lys Asp Ala465 470 475 480Ser Ile Lys Ser Val Asp Gly Asp Ser Ile Gly Glu Pro Lys Ser Gln Ile Asn Gly Lys485 490 495 500Val Arg Lys Asp Ser Leu Asp Asp Val Glu Tyr Ser Ile Lys Gln Lys Pro Tyr Asp Ser505 510 515 520Pro Phe Ile Asp Pro Gln Arg Gln Thr Glu Leu Asp Met Lys Asp Val Met Val Leu Asn525 530 535 540Asp Ile Ile Asp Gln Ala Ala Gly Arg Gln Ser Arg Glu Ser Gly Arg Glu Arg Glu Pro545 550 555 560Glu Glu Lys Glu Lys Phe Lys Glu Lys Cys Phe Lys Asn Cys Leu Arg Phe Ile Asp Ile565 570 575 580Phe Cys Val Trp Asp Cys Cys Asn Cys Trp Leu Tyr Ile Gln Lys Ile Val Asn Ile Ile585 590 595 600Val Phe Asp Pro Phe Met Glu Leu Phe Ile Thr Leu Ala Ile Ile Val Asn Thr Leu Phe605 610 615 620Met Ser Ile Glu His Ala Asp Met Asp Lys Ser Leu Gln Glu Phe Leu Lys Asn Gly Asn625 630 635 640Tyr Phe Phe Thr Ala Thr Phe Ala Ile Glu Ala Val Met Lys Leu Val Ala Leu Ser Pro645 650 655 660Lys Tyr Tyr Phe Lys Glu Gly Trp Asn Ile Phe Asp Phe Leu Ile Val Val Leu Ser Leu665 670 675 680Val Glu Leu Cys Gly Glu Asn Val Ser Leu Pro Gly Leu Ser Val Leu Arg Ser Phe Arg685 690 695 700Leu Leu Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Leu Leu Ile Ser Ile705 710 715 720Met Gly Lys Thr Val Gly Ala Leu Gly Asn Leu Thr Phe Val Leu Gly Ile Ile Ile Phe725 730 735 740Ile Phe Ala Val Met Gly Met Gln Leu Phe Gly Glu Lys Tyr Ile Glu Asn Lys His Val
745 750 755 760Phe Pro Asp Asn Ala Val Pro Arg Trp Asn Phe Val Asp Phe Met His Ser Phe Met Ile765 770 775 780Val Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Ser Met Trp Gly Arg Met Leu Val Ala785 790 795 800Glu Trp Pro Cys Val Pro Phe Phe Leu Ser Thr Val Ile Ile Gly Asn Leu Val Val Leu805 810 815 820Asn Leu Phe Pro Ala Leu Leu Leu Ser Ser Phe Gly Ala Ala Asn Leu Ser Met Ala Ser825 830 835 840Pro Glu Ser Ala Asp Thr Lys Lys Leu Gln Glu Ala Phe Glu Arg Ile Gly Arg Ala Ile845 850 855 860Asn Trp Val Lys Ile Cys Phe Leu Arg Ile Leu Lys Asn Leu Arg Pro Lys Thr Arg Asn865 870 875 880Gln Ile Gly Asp Gln Thr Thr Asp Ile Arg Glu Asp Leu Glu Asp Ala Thr Ile Gly Arg885 890 895 900Glu Met Leu Val Asp Gly Gln Val Lys Met Lys Glu Lys Lys Ser Pro Lys Glu Ile Thr905 910 915 920Asp Leu Glu Val Val Val Gly Asp Gly Leu Glu Ile Ser Ile Gln Gly Asn Gly Gln Ala925 930 935 940Ile Pro Ile Lys Met Ala Asn Asn Thr Lys Thr Leu Val Asn Ser Ser His Met Ser Ala945 950 955 960Lys Val Glu Leu Lys Glu Lys Glu Lys Glu Lys Phe Lys Glu Lys Asp Asn Lys Glu Lys965 970 975 980Asp Glu Gly Phe Gly Asn Lys Val Tyr Pro Gln Lys Asp Asp Asp Asp Val Ser Asn Lys985 990 995 1000Ser Ile Asn Asn Val Lys Asp Leu Lys Asp Asn Ile Glu Lys Gly Ile Pro Asn Asn Lys10051010 10151020Pro Ser Lys Glu Glu Ser Gln Ile Ser Leu Pro Glu Gly Ser Gln Glu Glu Lys Lys Asp10251030 10351040Ala Ser Lys Asp Asp Val Ile Asn Glu Gly Gly Phe Leu Lys Asp Glu Asp Ile Leu Glu10451050 10551060Glu Asp Ala Glu Ile Glu Gly Asp Asn Ala Asp Asp Glu Ser Ser Lys Ile Ile Ile Pro10651070 10751080Glu Tyr Pro Ser Asp Cys Phe Pro Asp Lys Cys Tyr Gln His Phe Pro Phe Cys Leu Gly10851090 10851100Asn Glu Glu Ser Lys Phe Trp Arg His Tyr Thr Thr Ile Arg Gly Lys Ser Tyr Thr Leu10951110 11051120Val Glu Asn Lys Tyr Phe Glu Thr Leu Ile Ile Phe Met Ile Leu Val Ser Ser Leu Ala10251130 10351140Leu Ala Leu Glu Asp Val Asn Leu Lys Lys Arg Arg Trp Leu Met Asp Ile Leu Gln Tyr10451150 10551160Met Asp Lys Ile Phe Thr Ile Leu Phe Phe Phe Glu Met Leu Ile Lys Trp Leu Ala Phe10651170 10751180Gly Phe Lys Lys Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Val Ile Val Leu Val10851190 10951200
Ser Leu Phe Asn Leu Ala Val Ser Leu Met Gly Gly Ser Asp Ile Ser Ala Phe Lys Thr12051210 12151220Met Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Met Ser Arg Leu Glu Gly Met Arg12251230 12351240Val Val Val Asn Ala Leu Val Gln Ala Ile Pro Ala Ile Phe Asn Val Leu Leu Val Cys12451250 12551260Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Gln Leu Phe Ala Gly Lys Phe Arg12651270 12751280Tyr Cys Ala Asp Lys Asp Gly Asn Arg Phe Asn Ser Ser Thr Val Pro Asn Lys Ser Val12851290 12951300Cys Cys Leu Asn Asn Phe Thr Trp Glu Asn Pro Lys Ile Asn Ser Asp Asn Val Leu Asn13051310 13151320Ala Tyr Leu Ala Leu Phe Gln Val Ala Thr Phe Lys Gly Trp Ile Asp Ile Met Ser Asn13251330 13351340Ala Ile Asp Thr Lys Gln Asn Glu Asp Glu Gln Pro Glu Tyr Glu Val Asn Arg Tyr Met13451350 13551360Tyr Leu Tyr Phe Val Leu Phe Ile Ile Ile Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile13651370 13751380Gly Val Ile Ile Asp Asn Phe Asn Glu Gln Lys Lys Lys Ala Gly Gly Ser Leu Glu Met13851390 13951400Phe Met Thr Glu Asp Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Met Gly Glu Lys Lys14051410 14151420Pro Ala Lys Ala Ile Pro Arg Pro Arg Phe Lys Leu Gln Ala Val Val Phe Asp Leu Ile14251430 14351440Thr Asn Thr Lys Phe Asp Met Ala Ile Val Leu Phe Ile Gly Leu Asn Met Leu Val Met14451450 14551460Ala Met Glu His Tyr His Gln Thr Pro Thr Phe Asp Asp Ile Leu Glu Arg Leu Asn Ile14651470 14751480Phe Phe Ile Ala Ile Phe Thr Thr Glu Cys Val Leu Lys Met Phe Ala Leu Arg Trp Tyr14851490 14951500Tyr Phe Lys Glu Pro Trp Asn Val Phe Asp Cys Val Val Ala Thr Leu Ser Ile Leu Gly15051510 15151520Ile Ala Ile Lys Asp Leu Leu Ala Ser Tyr Phe Val Ser Pro Thr Leu Leu Arg Val Val15251530 15351540Arg Val Val Lys Val Gly Arg Val Leu Arg Leu Val Lys Gly Ala Lys Gly Ile Arg Thr15451550 15551560Leu Leu Phe Ala Leu Ala Met Ser Leu Pro Ala Leu Phe Asn Ile Cys Leu Leu Leu Phe15651570 15751580Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser Phe Phe Met Thr Val Lys His Arg15851590 15951600Ala Gly Ile Asp Asp Asn Phe Asn Phe Glu Thr Phe Gly Gln Ser Met Ile Leu Leu Phe16051610 16151620Gln Met Ser Thr Ser Ala Gly Trp Asp Gly Val Leu Leu Gly Ile Met Asp Asp Ser Asp16251630 16351640Cys Val Gln Gly Ser Glu Asp Glu Gly Ile Glu Gly Asp Cys Gly Lys Pro Gly Val Ala
16451650 16551660Val Ala Tyr Leu Ile Ser Tyr Leu Ile Val Ser Phe Leu Ile Ile Ile Asn Met Tyr Ile16651670 16751680Ala Val Ile Leu Glu Asn Tyr Ser Gln Ala Thr Glu Asp Val Gln Glu Gly Leu Thr Ala16751690 16851700Asp Asp Tyr Asp Met Tyr Tyr Glu Ile Trp Gln Lys Tyr Asp Pro Glu Gly Thr Gln Tyr16951710 17151720Ile Lys Tyr Glu Asp Leu Ser Asp Phe Leu Asp Ala Leu Glu Glu Pro Leu Gln Ile Glu17251730 17351740Lys Pro Asn Lys Tyr Lys Ile Ile Asp Met Asp Ile Thr Ile Cys Lys Gly Asp Leu Met17451750 17551760Tyr Cys Val Asp Ile Leu Asp Ala Leu Thr Lys Asp Phe Phe Ala Arg Lys Gly Thr Ala17651770 17751780Ile Glu Glu Ser Ala Glu Leu Gly Glu Val Ala Pro Arg Lys Asp Val Glu Gly Tyr Glu17851790 17951800Pro Ile Ser Ser Thr Arg Phe Arg Gln Gln Glu Glu Tyr Asn Ala Arg Val Ile Gln Arg18051810 18151820Ala Trp Arg His Tyr Lys Gly Cys Ser Ala Ser Glu Pro Arg Gln Gln Gln Thr Ala Ile18251830 18351840Val Ile Glu Ser Asp Gly His Val Thr Arg Asn Gly His Lys Val Val Ile His Ser Arg18451850 18551860Ser Pro Ser Val Thr Ser Arg Ser Thr Asp Val18651870
权利要求
1.一种蝎子电压门控钠通道基因，其特征在于，它具有SEQ NO 1所示的碱基序列。
2.一种根据权利要求1所述的蝎子电压门控钠通道基因的编码蛋白，其特征在于，它具有SEQ NO 2所示的氨基酸序列。
3.一种根据权利要求1所述的蝎子电压门控钠通道基因的克隆方法，其特征在于该方法的具体步骤为a.从东亚钳蝎的腹神经纤维中提取总RNA；b.用Superscript II反转录酶和Oligo-(dT)引物合成单链cDNA；c.借鉴昆虫Para钠通道的保守氨基酸残基设计退火引物，再采用兼并PCR以及cDNA末端的快速扩增技术，得到全长cDNA。
全文摘要
本发明涉及一种东亚钳蝎的电压门控钠通道基因、编码蛋白及其克隆方法。本发明的一种电压门控钠通道基因，具有SEQ NO 1所示的碱基序列。该电压门控钠通道基因的编码蛋白，具有SEQ NO 2所示的氨基酸序列。该电压门控钠通道基因的克隆方法的具体步骤为首先从东亚钳蝎的腹神经纤维中提取总RNA；然后用Superscript II反转录酶和Oligo－(dT)引物合成单链cDNA；最后借鉴昆虫Para钠通道的保守氨基酸残基设计退火引物，再采用兼并PCR以及cDNA末端的快速扩增技术，得到全长cDNA。本发明利用逆转录聚合酶链式反应(RT－PCR)，兼并PCR(Nested－PCR)以及cDNA末端的快速扩增(RACE)技术，得到了东亚钳蝎的全长cDNA。用于电压门控钠通道的表达及药理调制机制的研究，并为离子通道病的防治和进一步开发为医药品奠定基础。
文档编号C07K14/435GK1952139SQ20061011638
公开日2007年4月25日 申请日期2006年9月21日 优先权日2006年9月21日
发明者吉永华, 左小潘, 贺明, 刘志睿 申请人:上海大学