电压门控钠通道基因、编码蛋白及其克隆方法 【技术领域】
本发明涉及一种新型电压门控钠通道基因、编码蛋白及其克隆方法。特指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。
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。
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，目前已被克隆和测序的昆虫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Vssc1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。
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。例如，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。
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新型电压门控钠通道基因。
本发明的目的之二在于提供一种新型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新型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：
一种电压门控钠通道基因，其特征在于该基因具有下列核苷酸序列之一：
(1).具有SEQ NO 1所示的DNA序列；
(2).与序列表中序列1限定的核酸序列具有95％以上的同源性，且编码相同功能蛋白质的DNA序列。
一种根据上述的电压门控钠通道基因的编码蛋白，其特征在于该编码蛋白具有序列表中序列2的氨基酸残基序列或者是将序列18-28的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列14-26的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
一种根据上述的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；
a.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；所述的退火引物为下表：
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。并根据序列分析结果，对rNav1.5DIV/S3-S4胞外环相应氨基酸进行点突变，运用电生理手段检验突变体对BmKI的敏感性，借此探明钠通道与特异性调制剂BmKI结合的关键性氨基酸，为蝎钠通道对自身分泌毒素-BmKI不敏感的现象提供合理地解释。证明BmNav1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开发通道型药物提供新型通道分子模板。
本发明的电压门控钠通道基因可以作为研究特异性相关调制剂药理效应及其功能机制的模板。另通过突变实验以及电生理记录检验了突变体对特异性钠通道调制剂BmK I的敏感性，证明该新型钠通道基因可作为膜离子通道调制剂作用机制的研究模板。
【附图说明】
图1为新型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。
图3为蝎子钠离子通道(BmNav1)与蜘蛛(OhNav1)和四种哺乳动物钠通道的DIV/S3-S4胞外环氨基酸序列，位点3毒素结合关键氨基酸用虚线框和实线框标注出来了。
图4为300nM BmK I施加前后rNav1.5及其各突变体I5ms/Ipeak的相对变化值。
图5为AFTII对rNav1.5和K1616S的作用
图6为AaHII对rNav1.5和K1616S的作用
具体实施方案
实施例一：电压门控钠通道基因的克隆，参见图1：
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℃/10min)，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：新型钠通道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 Amplification of the cDNA Ends)和3’RACE获得全长cDNA。所设计的退火引物见表1：
表1Primer for Scorpion Sodium Channel
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ABI PRISM 377DNA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：
tgaatcttgcataaaagctacagcaagaggatttatacttgagcaattcacatatcttcgagatccgtggaattggctggattttgtagttatttctttagcttacattacaatgggagttgaattaggaaatttaagtgctttgaggacttttcgtgtacttagagcattaaaaactgttgctgttattccaggtttaaagactattgttggagctgtcatagaatctgtaaagaatttaaaagatgtgattattttgacagtgttttctctatctctatttgccttacttggtctccagatgtatatgggtgttctaatgcaaaaatgtattgtagaagctcctccaaatactacttatgaagagaagaaagtacattataggaatcaaagtaactggtatctagattctgctggtgaatattttatttgtggaaatgcatcaggagctagacaatgtcctgaaggatatatttgtttgtccaattatggtcccaatccaaatcatggttttacaaattttgacacattttattgggcatttcttg
引物S7/A2435所得片段：
正向测序结果：
cttggtctccagatgtatatgggtgttctaatgcaaaaatgtattgtagaagctcctccaaatactacttatgaagagaagaaagtacattataggaatcaaagtaactggtatctagattctgctggtgaatattttatttgtggaaatgcatcaggagctagacaatgtcctgaaggatatatttgtttgtccaattatggtcccaatccaaatcatggttttacaaattttgacacattttattgggcatttcttgcatcatttagacttatgactcaagatttttgggaaggtctttaccaaatggtgctaagaacagcaggtccttggcatatatgttttttcatcgtaatcatctttcttggttcattttatttattaaatctcattttggccattgttgctatgtcatatgatgaattacaaaaaagagctgaaaaagaagaagaagatgcacaaaaagaagaagaaaactataatgcagctgtagaacgtaaaagattgttagaagaagaaaaagcaggagtgggtgtagtgaagagttcttc
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：
aaaagtacttcacaaatgcttggtgttggcttgattttgttatagttctggtttctctgtttaatctggcagtaagcttaatgggaggtagtgacatatctgcctttaaaactatgcgaaccctgagagctctccgcccacttagggcaatgtcacgtctggagggaatgagagttgtagtaaatgctctcgttcaagctatccctgccatctttaatgttttacttgtgtgcctcatcttttggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttcaatagctctactgtgcctaacaaatctgtttgttgcttaaacaattttacttgggaaaatccaaaaataaattctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcaatagatactaagcaaaatgaagatgaacaac
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：
tattgccattttcactacagaatgtgtgttaaaaatgtttgcccttaggtggtattattttaaagaaccatggaatgtatttgattgtgtggttgtaaccttgtccattttaggcattgcaatcaaagatttaatagcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtcaaaggggccaaagggattcgaaccttactttttgctcttgccatgtccttgccagcactttttaatatttgccttctgctctttttagttatgtttatctacgccatatttggaatgtcttttttcatgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatgaaggcattgaagg
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：
Aaattcttgcctgttatacatagcaggagctagtatagaattattacctaactccaatcctagtaacttagttgccattgaaataggcactctagcattcatgaaagttttattagcttcggaaacaatgcaaccacaggatgagacagtgaaagggaatgtgatatattcctttatcatcctgtctgttatagtgaaaaattaaaaaaaaatgaaaaacttatgtttcaagagccagaactcttcctcttactacaaagcatggctttgagaatgacaagtttataatggaagaatctgctatgctgaagattgcttcaagaccttgactcgccatttccatcttaccacccagtttcaatcattttctcaattatgccttttcagcttgtacttactttgtaaaatgtttcacagtttgtaccatactgcctgtaaatacatcattgaataaatttttgataatttatcacaacgaaccgatgctcaagataaaaaaaaaaaaaaaaaaaaaaaaaaaa。
实施例二：新型电压门控钠离子通道结构特征分析及功能说明
1.新型电压门控钠离子通道结构特征
如附图2所示，新型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.新型电压门控钠离子通道功能特征
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。
3.新型电压门控钠离子通道对自分泌调制剂不敏感机制
电压门控钠通道DIV/S3-S4胞外环靠近N-端的一个酸性氨基酸残基(Asp/Glu)对位点3毒素的结合至关重要。经序列分析：所有蝎位点三毒素敏感的钠通道，包括昆虫VGSCs、Nav1.4、Nav1.5、Nav1.6和hNav1.7等，在相应位置均为Asp；而对蝎位点三毒素不敏感的钠通道，如Nav1.2和Nav1.3在相应的位置上被Glu占据(附图3)。
由此提示，DIV/S3-S4胞外环的带负电荷残基Asp决定了VGSCs对位点三毒素的敏感性。另有研究提示：蝎神经纤维的钠离子通道对自分泌毒素-BmK I不敏感，即BmK I缺失对自身钠通道的调制能力。令人费解的是，与已知所有位点三毒素敏感的钠通道一样，蝎钠通道DIV/S3-S4胞外环相应位置上却同为Asp。因而推测：决定蝎钠通道对BmK I不敏感的氨基酸残基可能并不是哺乳动物VGSCs中常见的Glu/Asp。
以rNav1.5模版构建突变体，通过电生理记录检验各个突变体对特异性调制剂BmK I敏感性变化。结果表明：1)VGSCs DIV/S3-S4胞外连接环的Asp1612对通道与毒素的敏感性非常重要，说明DIV/S3-S4是BmK I毒素结合的重要区域，第一次用分子生物学的方法确证了BmKI是一个位点三毒素；2)除VGSCs DIV/S3-S4连接环关键性氨基酸Asp1612外，此区域正电荷Lys1616对特异性调制剂BmK I的敏感性同样至关重要；3)对BmK I敏感性起决定作用的Lys1616，对通道与同为位点三毒素的AFT II以及AaH II并不重要。决定特异性调制剂BmK I与钠通道结合的氨基酸除了已知的Asp外，Lys1616的作用同样至关重要，进而，蝎钠通道Lys氨基酸的改变可能是蝎钠通道对自身毒素BmK I不敏感的分子基础；而该位点的正电荷氨基酸对其他位点三毒素并不重要，表明位点三毒素在钠通道上的受体并不完全一致，而仅是部分重叠。
参见图4、图5、图6，图4说明野生型rNav1.5通道对BmKI敏感性最强，I5ms/Ipeak的相对变化值最大。突变体D1612E大大削弱了通道对BmKI的敏感性，说明DIV/S3-S4胞外连接环的Asp1612对通道与毒素的敏感性非常重要，证明BmKI是位点三毒素。rNav1.5的1611和1615位的氨基酸突变(S1611K，S1611A，Q1615A，Q1515N)能不同程度地降低I5ms/Ipeak的相对变化值，提示两个位点可能对通道与BmKI的敏感性起微调作用，但不起决定性作用。rNav1.5的1616位点的任何一种突变(K1616S，K1616A，K1616L)均使通道BmKI的敏感性显著性下降，说明该位点Lys1616对BmKI的敏感性同样至关重要。图5说明AFT II能极显著地延缓rNav1.5(A，B)和K1616S(C，D)的失活化过程。图6说明AaH II能极显著地延缓rNav1.5(A，B)和K1616S(C，D)的失活化过程。
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>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>1
aaataaaacc tacggtcaaa gatggcggac ttggtctgag tggttttttc tgggttttcg 60
tcgacaatat tctcattcga gagtcccgga ttatgagcca gcggatactt taactctgcc 120
aaaagaacgg agcatggtcc agggccccgg gccccgtagg tggtagagag cgttcgcctg 180
tctcgtcttc gccatgtctt cggacgagca gtccgaggag gaagttctgc ccgcattccg 240
tcccttcacc agggagtcat tggcggccat cgaagccagg atcgccgagg ctgaatcaag 300
aaaaaaggaa tcgcttcaga aaaaagggga aggcgacgat gaacactacg gcacttacca 360
gccggaagtg gccgaagccg accctcaact ggaggcgggt ctcccattac ccaagtattt 420
ggaacgtgaa tttcctcccg aactattggc tacacctatc gaaggtttgg acaaatttta 480
cgaaaacaaa aggacattca ttgtgataag taaaggtaag gatatcttcc ggtttagtgc 540
aacaaatgcc atgtggatat tagatccttt caatcctatc cgtcgtgcag ccatcagcat 600
cctcgttcat cctgccttta gcttcttggt tattgtcact atccttgtaa actgtgttct 660
tatgacgcaa acaaatgtaa cgcacgaaaa ggaattagag aggcttttta cggcgatata 720
tacaattgaa tcttgcataa aagctacagc aagaggattt atacttgagc aattcacata 780
tcttcgagat ccgtggaatt ggctggattt tgtagttatt tctttagctt acattacaat 840
gggagttgaa ttaggaaatt taagtgcttt gaggactttt cgtgtactta gagcattaaa 900
aactgttgct gttattccag gtttaaagac tattgttgga gctgtcatag aatctgtaaa 960
gaatttaaaa gatgtgatta ttttgacagt gttttctcta tctctatttg ccttacttgg 1020
tctccagatg tatatgggtg ttctaatgca aaaatgtatt gtagaagctc ctccaaatac 1080
tacttatgaa gagaagaaag tacattatag gaatcaaagt aactggtatc tagattctgc 1140
tggtgaatat tttatttgtg gaaatgcatc aggagctaga caatgtcctg aaggatatat 1200
ttgtttgtcc aattatggtc ccaatccaaa tcatggtttt acaaattttg acacatttta 1260
ttgggcattt cttgcatcat ttagacttat gactcaagat ttttgggaag gtctttacca 1320
aatggtgcta agaacagcag gtccttggca tatatgtttt ttcatcgtaa tcatctttct 1380
tggttcattt tatttattaa atctcatttt ggccattgtt gctatgtcat atgatgaatt 1440
acaaaaaaga gctgaaaaag aagaagaaga tgcacaaaaa gaagaagaaa actataatgc 1500
agctgtagaa cgtaaaagat tgttagaaga agaaaaagca ggagtgggtg tagtgaagag 1560
ttcttcagaa tcttcttctg gatcttacga attatttgtg ggacaagata aaactgtaga 1620
agaaaaagat gcaagtatta agagtgtaga tggagatagc attggtgaac caaaatctca 1680
aataaatggc aaagtaagga aggattcttt ggatgatgtg gaatatagca taaaacaaaa 1740
accatatgat agtcctttca tagatcctca aagacaaaca gaattagata tgaaagatgt 1800
gatggttttg aatgacatta tagatcaagc tgcaggacgt caaagtagag aaagtggcag 1860
agagagggaa ccagaagaga aggagaagtt caaagaaaaa tgcttcaaaa attgcttgag 1920
atttattgat atcttctgcg tatgggactg ttgtaactgc tggttatata tacaaaaaat 1980
agttaacatc attgtctttg atccatttat ggagttattc attacactgg caataattgt 2040
caacacgctc ttcatgtcta tagaacatgc cgacatggat aaaagtcttc aggaattctt 2100
aaaaaatggc aactactttt tcacagcaac atttgccata gaagcagtaa tgaagttggt 2160
agcactgagt ccaaaatatt actttaaaga aggatggaac atatttgatt ttttaattgt 2220
agtactttct cttgttgaac tgtgtggaga aaatgtttct ttacctggat tatcagtttt 2280
gagatcattt cgtttgcttc gtgtatttaa acttgcaaaa tcttggccaa cactcaattt 2340
attaatatcc attatgggaa aaactgtggg tgccttggga aacctaactt ttgtgttggg 2400
tatcatcatt ttcatatttg ctgttatggg tatgcagttg tttggtgaaa aatatataga 2460
aaataaacat gtgtttcctg ataatgctgt tccacgctgg aattttgttg attttatgca 2520
ttcatttatg attgtgtttc gtgttttgtg tggagaatgg atagaatcta tgtggggtcg 2580
tatgcttgtt gcagaatggc catgtgttcc ttttttttta tcaactgtta taattggaaa 2640
cctagtggtg ctgaatttgt ttcctgcctt gcttttgtcc tcctttggtg ccgcaaatct 2700
ttccatggct tcaccagaaa gtgctgatac taagaaactg caagaagcat ttgaacgtat 2760
aggaagagct atcaattggg ttaaaatctg cttcttacga atcttaaaaa atttaagacc 2820
aaaaactaga aatcaaattg gagatcaaac aacagatatt agagaagatc tggaagatgc 2880
tacaatagga cgagagatgt tagttgatgg tcaggttaaa atgaaagaga aaaaaagccc 2940
taaagaaatt acagatcttg aagttgtagt aggagatgga ttagaaatat caattcaagg 3000
aaatggacaa gcaataccaa taaaaatggc taataatact aaaacacttg taaattcttc 3060
acatatgtct gctaaagtag aattgaaaga aaaggaaaaa gaaaaattca aagaaaaaga 3120
taataaagaa aaggatgaag gatttggaaa taaagtatat cctcaaaaag atgacgatga 3180
tgttagtaac aaatctatta ataatgtcaa ggatttaaaa gataatattg agaaggggat 3240
tcctaataac aaaccgtcca aagaggaaag ccaaatttca ctgccagaag gatcccaaga 3300
agaaaaaaag gatgctagta aagatgatgt tattaatgaa ggaggatttt taaaagatga 3360
ggatatactt gaagaagatg cagaaattga gggagataac gcagatgatg aatcatcaaa 3420
aattataatt cctgaatatc catctgattg ttttccagac aagtgttatc aacattttcc 3480
attttgttta gggaatgaag aatcaaaatt ttggagacac tatactacta tacgtggaaa 3540
aagttacaca ttggtggaaa ataaatattt tgaaacattg attattttta tgatattagt 3600
cagcagtttg gcattagcat tagaagatgt taatttgaaa aaaagaagat ggttaatgga 3660
tattttacaa tatatggaca agatatttac aattttattt ttttttgaaa tgttaatcaa 3720
atggctagct tttggtttta aaaagtactt cacaaatgct tggtgttggc ttgattttgt 3780
tatagttctg gtttctctgt ttaatctggc agtaagctta atgggaggta gtgacatatc 3840
tgcctttaaa actatgcgaa ccctgagagc tctccgccca cttagggcaa tgtcacgtct 3900
ggagggaatg agagttgtag taaatgctct cgttcaagct atccctgcca tctttaatgt 3960
tttacttgtg tgcctcatct tttggcttat tttttctatt atgggtgttc agttatttgc 4020
tgggaaattt cgttattgtg cagataaaga tgggaatcgt ttcaatagct ctactgtgcc 4080
taacaaatct gtttgttgct taaacaattt tacttgggaa aatccaaaaa taaattctga 4140
taatgtactc aatgcatatt tagctctttt tcaagttgcc acattcaaag gatggattga 4200
tataatgagt aatgcaatag atactaagca aaatgaagat gaacaacctg aatatgaagt 4260
caatcgatat atgtatctat actttgtact tttcatcatt ataggatctt ttttcacgtt 4320
aaatctcttc attggagtaa taatagataa tttcaatgaa cagaaaaaaa aggcaggagg 4380
ttctttagaa atgtttatga ctgaagatca gaagaaatat tataatgcta tgaaaaaaat 4440
gggagagaag aagccagcta aagctattcc tcggccaagg ttcaaattac aagcagtagt 4500
atttgatctt attactaata caaaatttga tatggcaatc gtgttgttta ttggacttaa 4560
tatgctagtg atggcaatgg aacattatca tcagacacca acatttgatg atattctaga 4620
aagattaaat atattcttta ttgccatttt cactacagaa tgtgtgttaa aaatgtttgc 4680
ccttaggtgg tattatttta aagaaccatg gaatgtattt gattgtgtgg ttgtaacctt 4740
gtccatttta ggcattgcaa tcaaagattt aatagcaagt tattttgtgt caccaactct 4800
tctgcgtgtg gttcgtgttg taaaagttgg tcgtgtcctc aggttggtca aaggggccaa 4860
agggattcga accttacttt ttgctcttgc catgtccttg ccagcacttt ttaatatttg 4920
ccttctgctc tttttagtta tgtttatcta cgccatattt ggaatgtctt ttttcatgac 4980
tgtaaaacat cgtgcgggca tcgatgataa ttttaatttt gaaacatttg gtcagtcaat 5040
gattctttta tttcaaatgt caacatcagc aggttgggat ggtgtgttat taggtatcat 5100
ggatgacagt gactgtgtac aaggatctga agatgaaggc attgaaggag attgtggtaa 5160
accaggtgta gcagtggcat atttaatttc ttacttaatt gtaagttttt taattataat 5220
taacatgtat attgctgtca ttcttgaaaa ctacagtcaa gctacagaag atgtacagga 5280
aggtttaaca gctgatgatt atgatatgta ttatgaaata tggcaaaaat acgatcctga 5340
aggtacacaa tatatcaagt atgaagattt gagtgacttc ctagatgctc tagaagaacc 5400
attgcagatt gagaaaccta ataagtacaa aatcattgac atggatatta caatatgtaa 5460
aggtgatctt atgtattgtg tagatattct tgatgcactg accaaagatt tctttgcgag 5520
gaaaggtact gctatagaag aatctgcaga acttggagaa gttgctccta ggaaagacgt 5580
tgaaggctat gagccaatca gcagtacgcg cttccgtcaa caggaagaat ataatgctcg 5640
tgttattcag agagcatggc gacattataa agggtgtagt gctagtgaac ctcgtcagca 5700
acaaactgca attgttattg aaagtgatgg ccatgtaact agaaatggtc acaaagttgt 5760
gattcattct aggtcaccaa gtgttacaag ccgttccaca gatgtgtgat atttataaaa 5820
tagaaaaggg gctttctaca tcaacattat ctaaatagaa attggttgat gattgattgc 5880
tgacaatatc tgcatgaagt gtatattttt atattcttta tgcacgagtt gagtaccagt 5940
ctgcttgcaa cacctgtact tggttgacat aaaccaatgt tttccatgtt atttttctaa 6000
tgactgatta gttgtaatta tggtctattt acactacttg taaatatgca aactaaaagg 6060
gataatgatc ccattcttgt tgctacaata ggataagagt ggaagcatct aagttttaat 6120
ttgttttata tatattttga tttatagaca ttttacggat tttagaatgt attttttatt 6180
attacatgta atgttattac ataacaaagt gcttttattt acaaattcta gaatttcaaa 6240
ggaaaaatgt cagaaattac tagagagact gttaaatckg ttattattat tctgaaaatt 6300
tgttttttac ttgtgtatat atataatata tatatatatg gaagcagaat ttatactgct 6360
tctataaaca tgttaaattt attcaaaggg aagagatgtg aattatttaa gtttttttta 6420
actaatatta atttatttat tttttagcac tttggacatt aaccaatgcc aaatgaaatg 6480
aacgttttaa ctagaaaagt tcagtgccca tatttaaaca gcatataaga agtaaaatgt 6540
gattccaaga gaagtatgat ctgtatcagt aaagctacat aggtgggttg aagatggtta 6600
ttgctacatt agctgttaac acacaaacta aatttgaaag attctgctta tgaacagggt 6660
ttcttataag aagaaatata acaatgtagg ggaatgttcc ccccttatca gtgataatgt 6720
atctatagga atcttcctca gtttttctgc tttctaagct gatggcatta aaaaattttg 6780
cacttatgaa tagattcatg ttatcagcat caagctgtga aattgggtgg caaacaactt 6840
cagctgtgtt ataagcgtgt tgttataaac ggagtataca gcttcgagga tacgggaggg 6900
tgggaaaaat caacgcttaa ttgtcctaat aacctctgca ctgtacctct aagtgtataa 6960
cactctacaa agctcagttt tcctgccgct tttaaatgag cagaaaaaga gtgtgtgata 7020
agggtaatta atatacaaag ttattggctg agttcagaag ttggactttt ggccaccaga 7080
ctttaaaagc agagattatc aaaactctgc aaaatcttaa gagattttat ttatcatgtg 7140
cctttctgcg ttgttactgt tgtcacaaga aagcataatc attgcatcat aaatcagctt 7200
cccacgcaac aatacttgct ttcttatata ctttccaaca tttttcaagc caaatagata 7260
agactgtggt ttcttttcaa agtgcacaac attatttcat aaaaaaaatg aagcagatct 7320
caccattggc ttgatgtcta tactgctagt atatataaaa gctcaagact gcaaataatt 7380
tataatatcc tataccacac tatgcattgg attttatttg aacacaacta aggtggtggt 7440
tgtataatta attctaaatg tcaagaatat aaaagttttg ttagtttctt ctcttctggg 7500
aaaaatcatc actccatgta aaaaaatcag aaaaaatcta gcataaattc ttgcctgtta 7560
tacatagcag gagctagtat agaattatta cctaactcca atcctagtaa cttagttgcc 7620
attgaaatag gcactctagc attcatgaaa gttttattag cttcggaaac aatgcaacca 7680
caggatgaga cagtgaaagg gaatgtgata tattccttta tcatcctgtc tgttatagtg 7740
aaaaattaaa aaaaaatgaa aaacttatgt ttcaagagcc agaactcttc ctcttactac 7800
aaagcatggc tttgagaatg acaagtttat aatggaagaa tctgctatgc tgaagattgc 7860
ttcaagacct tgactcgcca tttccatctt accacccagt ttcaatcatt ttctcaatta 7920
tgccttttca gcttgtactt actttgtaaa atgtttcaca gtttgtacca tactgcctgt 7980
aaatacatca ttgaataaat ttttgataat ttatcacaac gaaccgatgc tcaagataaa 8040
aaaaaaaaaa aaaaaaaaaa aaaaa 8065
<210>2
<211>1871
<212>PRT
<213>东亚钳蝎(Buthus Martensii Karsch)
<400>2
Met Ser Ser Asp Glu Gln Ser Glu Glu Glu
1 5 10
Val Leu Pro Ala Phe Arg Pro Phe Thr Arg
15 20
Glu Ser Leu Ala Ala Ile Glu Ala Arg Ile
25 30
Ala Glu Ala Glu Ser Arg Lys Lys Glu Ser
35 40
Leu Gln Lys Lys Gly Glu Gly Asp Asp Glu
45 50
His Tyr Gly Thr Tyr Gln Pro Glu Val Ala
55 60
Glu Ala Asp Pro Gln Leu Glu Ala Gly Leu
65 70
Pro Leu Pro Lys Tyr Leu Glu Arg Glu Phe
75 80
Pro Pro Glu Leu Leu Ala Thr Pro Ile Glu
85 90
Gly Leu Asp Lys Phe Tyr Glu Asn Lys Arg
95 100
Thr Phe Ile Val Ile Ser Lys Gly Lys Asp
105 110
Ile Phe Arg Phe Ser Ala Thr Asn Ala Met
115 120
Trp Ile Leu Asp Pro Phe Asn Pro Ile Arg
125 130
Arg Ala Ala Ile Ser Ile Leu Val His Pro
135 140
Ala Phe Ser Phe Leu Val Ile Val Thr Ile
145 150
Leu Val Asn Cys Val Leu Met Thr Gln Thr
155 160
Asn Val Thr His Glu Lys Glu Leu Glu Arg
165 170
Leu Phe Thr Ala Ile Tyr Thr Ile Glu Ser
175 180
Cys Ile Lys Ala Thr Ala Arg Gly Phe Ile
185 190
Leu Glu Gln Phe Thr Tyr Leu Arg Asp Pro
195 200
Trp Asn Trp Leu Asp Phe Val Val Ile Ser
205 210
Leu Ala Tyr Ile Thr Met Gly Val Glu Leu
215 220
Gly Asn Leu Ser Ala Leu Arg Thr Phe Arg
225 230
Val Leu Arg Ala Leu Lys Thr Val Ala Val
235 240
Ile Pro Gly Leu Lys Thr Ile Val Gly Ala
245 250
Val Ile Glu Ser Val Lys Asn Leu Lys Asp
255 260
Val Ile Ile Leu Thr Val Phe Ser Leu Ser
265 270
Leu Phe Ala Leu Leu Gly Leu Gln Met Tyr
275 280
Met Gly Val Leu Met Gln Lys Cys Ile Val
285 290
Glu Ala Pro Pro Asn Thr Thr Tyr Glu Glu
295 300
Lys Lys Val His Tyr Arg Asn Gln Ser Asn
305 310
Trp Tyr Leu Asp Ser Ala Gly Glu Tyr Phe
315 320
Ile Cys Gly Asn Ala Ser Gly Ala Arg Gln
325 330
Cys Pro Glu Gly Tyr Ile Cys Leu Ser Asn
335 340
Tyr Gly Pro Asn Pro Asn His Gly Phe Thr
345 350
Asn Phe Asp Thr Phe Tyr Trp Ala Phe Leu
355 360
Ala Ser Phe Arg Leu Met Thr Gln Asp Phe
365 370
Trp Glu Gly Leu Tyr Gln Met Val Leu Arg
375 380
Thr Ala Gly Pro Trp His Ile Arg Phe Phe
385 390
Ile Val Ile Ile Phe Leu Gly Ser Phe Tyr
395 400
Leu Leu Asn Leu Ile Leu Ala Ile Val Ala
405 410
Met Ser Tyr Asp Glu Leu Gln Lys Arg Ala
415 420
Glu Lys Glu Glu Glu Asp Ala Gln Lys Glu
425 430
Glu Glu Asn Tyr Asn Ala Ala Val Glu Arg
435 440
Lys Arg Leu Leu Glu Glu Glu Lys Ala Gly
445 450
Val Gly Val Val Lys Ser Ser Ser Glu Ser
455 460
Ser Ser Gly Ser Tyr Glu Leu Phe Val Gly
465 470
Gln Asp Lys Thr Val Glu Glu Lys Asp Ala
475 480
Ser Ile Lys Ser Val Asp Gly Asp Ser Ile
485 490
Gly Glu Pro Lys Ser Gln Ile Asn Gly Lys
495 500
Val Arg Lys Asp Ser Leu Asp Asp Val Glu
505 510
Tyr Ser Ile Lys Gln Lys Pro Tyr Asp Ser
515 520
Pro Phe Ile Asp Pro Gln Arg Gln Thr Glu
525 530
Leu Asp Met Lys Asp Val Met Val Leu Asn
535 540
Asp Ile Ile Asp Gln Ala Ala Gly Arg Gln
545 550
Ser Arg Glu Ser Gly Arg Glu Arg Glu Pro
555 560
Glu Glu Lys Glu Lys Phe Lys Glu Lys Cys
565 570
Phe Lys Asn Cys Leu Arg Phe Ile Asp Ile
575 580
Phe Cys Val Trp Asp Cys Cys Asn Cys Trp
585 590
Leu Tyr Ile Gln Lys Ile Val Asn Ile Ile
595 600
Val Phe Asp Pro Phe Met Glu Leu Phe Ile
605 610
Thr Leu Ala Ile Ile Val Asn Thr Leu Phe
615 620
Met Ser Ile Glu His Ala Asp Met Asp Lys
625 630
Ser Leu Gln Glu Phe Leu Lys Asn Gly Asn
635 640
Tyr Phe Phe Thr Ala Thr Phe Ala Ile Glu
645 650
Ala Val Met Lys Leu Val Ala Leu Ser Pro
655 660
Lys Tyr Tyr Phe Lys Glu Gly Trp Asn Ile
665 670
Phe Asp Phe Leu Ile Val Val Leu Ser Leu
675 680
Val Glu Leu Cys Gly Glu Asn Val Ser Leu
685 690
Pro Gly Leu Ser Val Leu Arg Ser Phe Arg
695 700
Leu Leu Arg Val Phe Lys Leu Ala Lys Ser
705 710
Trp Pro Thr Leu Asn Leu Leu Ile Ser Ile
715 720
Met Gly Lys Thr Val Gly Ala Leu Gly Asn
725 730
Leu Thr Phe Val Leu Gly Ile Ile Ile Phe
735 740
Ile Phe Ala Val Met Gly Met Gln Leu Phe
745 750
Gly Glu Lys Tyr Ile Glu Asn Lys His Val
755 760
Phe Pro Asp Asn Ala Val Pro Arg Trp Asn
765 770
Phe Val Asp Phe Met His Ser Phe Met Ile
775 780
Val Phe Arg Val Leu Cys Gly Glu Trp Ile
785 790
Glu Ser Met Trp Gly Arg Met Leu Val Ala
795 800
Glu Trp Pro Cys Val Pro Phe Phe Leu Ser
805 810
Thr Val Ile Ile Gly Asn Leu Val Val Leu
815 820
Asn Leu Phe Pro Ala Leu Leu Leu Ser Ser
825 830
Phe Gly Ala Ala Asn Leu Ser Met Ala Ser
835 840
Pro Glu Ser Ala Asp Thr Lys Lys Leu Gln
845 850
Glu Ala Phe Glu Arg Ile Gly Arg Ala Ile
855 860
Asn Trp Val Lys Ile Cys Phe Leu Arg Ile
865 870
Leu Lys Asn Leu Arg Pro Lys Thr Arg Asn
875 880
Gln Ile Gly Asp Gln Thr Thr Asp Ile Arg
885 890
Glu Asp Leu Glu Asp Ala Thr Ile Gly Arg
895 900
Glu Met Leu Val Asp Gly Gln Val Lys Met
905 910
Lys Glu Lys Lys Ser Pro Lys Glu Ile Thr
915 920
Asp Leu Glu Val Val Val Gly Asp Gly Leu
925 930
Glu Ile Ser Ile Gln Gly Asn Gly Gln Ala
935 940
Ile Pro Ile Lys Met Ala Asn Asn Thr Lys
945 950
Thr Leu Val Asn Ser Ser His Met Ser Ala
955 960
Lys Val Glu Leu Lys Glu Lys Glu Lys Glu
965 970
Lys Phe Lys Glu Lys Asp Asn Lys Glu Lys
975 980
Asp Glu Gly Phe Gly Asn Lys Val Tyr Pro
985 990
Gln Lys Asp Asp Asp Asp Val Ser Asn Lys
995 1000
Ser Ile Asn Asn Val Lys Asp Leu Lys Asp
1005 1010
Asn Ile Glu Lys Gly Ile Pro Asn Asn Lys
1015 1020
Pro Ser Lys Glu Glu Ser Gln Ile Ser Leu
1025 1030
Pro Glu Gly Ser Gln Glu Glu Lys Lys Asp
1035 1040
Ala Ser Lys Asp Asp Val Ile Asn Glu Gly
1045 1050
Gly Phe Leu Lys Asp Glu Asp Ile Leu Glu
1055 1060
Glu Asp Ala Glu Ile Glu Gly Asp Asn Ala
1065 1070
Asp Asp Glu Ser Ser Lys Ile Ile Ile Pro
1075 1080
Glu Tyr Pro Ser Asp Cys Phe Pro Asp Lys
1085 1090
Cys Tyr Gln His Phe Pro Phe Cys Leu Gly
1085 1100
Asn Glu Glu Ser Lys Phe Trp Arg His Tyr
1095 1110
Thr Thr Ile Arg Gly Lys Ser Tyr Thr Leu
1105 1120
Val Glu Asn Lys Tyr Phe Glu Thr Leu Ile
1025 1130
Ile Phe Met Ile Leu Val Ser Ser Leu Ala
1035 1140
Leu Ala Leu Glu Asp Val Asn Leu Lys Lys
1045 1150
Arg Arg Trp Leu Met Asp Ile Leu Gln Tyr
1055 1160
Met Asp Lys Ile Phe Thr Ile Leu Phe Phe
1065 1170
Phe Glu Met Leu Ile Lys Trp Leu Ala Phe
1075 1180
Gly Phe Lys Lys Tyr Phe Thr Asn Ala Trp
1085 1190
Cys Trp Leu Asp Phe Val Ile Val Leu Val
1095 1200
Ser Leu Phe Asn Leu Ala Val Ser Leu Met
1205 1210
Gly Gly Ser Asp Ile Ser Ala Phe Lys Thr
1215 1220
Met Arg Thr Leu Arg Ala Leu Arg Pro Leu
1225 1230
Arg Ala Met Ser Arg Leu Glu Gly Met Arg
1235 1240
Val Val Val Asn Ala Leu Val Gln Ala Ile
1245 1250
Pro Ala Ile Phe Asn Val Leu Leu Val Cys
1255 1260
Leu Ile Phe Trp Leu Ile Phe Ser Ile Met
1265 1270
Gly Val Gln Leu Phe Ala Gly Lys Phe Arg
1275 1280
Tyr Cys Ala Asp Lys Asp Gly Asn Arg Phe
1285 1290
Asn Ser Ser Thr Val Pro Asn Lys Ser Val
1295 1300
Cys Cys Leu Asn Asn Phe Thr Trp Glu Asn
1305 1310
Pro Lys Ile Asn Ser Asp Asn Val Leu Asn
1315 1320
Ala Tyr Leu Ala Leu Phe Gln Val Ala Thr
1325 1330
Phe Lys Gly Trp Ile Asp Ile Met Ser Asn
1335 1340
Ala Ile Asp Thr Lys Gln Asn Glu Asp Glu
1345 1350
Gln Pro Glu Tyr Glu Val Asn Arg Tyr Met
1355 1360
Tyr Leu Tyr Phe Val Leu Phe Ile Ile Ile
1365 1370
Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile
1375 1380
Gly Val Ile Ile Asp Asn Phe Asn Glu Gln
1385 1390
Lys Lys Lys Ala Gly Gly Ser Leu Glu Met
1395 1400
Phe Met Thr Glu Asp Gln Lys Lys Tyr Tyr
1405 1410
Asn Ala Met Lys Lys Met Gly Glu Lys Lys
1415 1420
Pro Ala Lys Ala Ile Pro Arg Pro Arg Phe
1425 1430
Lys Leu Gln Ala Val Val Phe Asp Leu Ile
1435 1440
Thr Asn Thr Lys Phe Asp Met Ala Ile Val
1445 1450
Leu Phe Ile Gly Leu Asn Met Leu Val Met
1455 1460
Ala Met Glu His Tyr His Gln Thr Pro Thr
1465 1470
Phe Asp Asp Ile Leu Glu Arg Leu Asn Ile
1475 1480
Phe Phe Ile Ala Ile Phe Thr Thr Glu Cys
1485 1490
Val Leu Lys Met Phe Ala Leu Arg Trp Tyr
1495 1500
Tyr Phe Lys Glu Pro Trp Asn Val Phe Asp
1505 1510
Cys Val Val Ala Thr Leu Ser Ile Leu Gly
1515 1520
Ile Ala Ile Lys Asp Leu Leu Ala Ser Tyr
1525 1530
Phe Val Ser Pro Thr Leu Leu Arg Val Val
1535 1540
Arg Val Val Lys Val Gly Arg Val Leu Arg
1545 1550
Leu Val Lys Gly Ala Lys Gly Ile Arg Thr
1555 1560
Leu Leu Phe Ala Leu Ala Met Ser Leu Pro
1565 1570
Ala Leu Phe Asn Ile Cys Leu Leu Leu Phe
1575 1580
Leu Val Met Phe Ile Tyr Ala Ile Phe Gly
1585 1590
Met Ser Phe Phe Met Thr Val Lys His Arg
1595 1600
Ala Gly Ile Asp Asp Asn Phe Asn Phe Glu
1605 1610
Thr Phe Gly Gln Ser Me tIle Leu Leu Phe
1615 1620
Gln Met Ser Thr Ser Ala Gly Trp Asp Gly
1625 1630
Val Leu Leu Gly Ile Met Asp Asp Ser Asp
1635 1640
Cys Val Gln Gly Ser Glu Asp Glu Gly Ile
1645 1650
Glu Gly Asp Cys Gly Lys Pro Gly Val Ala
1655 1660
Val Ala Tyr Leu Ile Ser Tyr Leu Ile Val
1665 1670
Ser Phe Leu Ile Ile Ile Asn Met Tyr Ile
1675 1680
Ala Val Ile Leu Glu Asn Tyr Ser Gln Ala
1675 1690
Thr Glu Asp Val Gln Glu Gly Leu Thr Ala
1685 1700
Asp Asp Tyr Asp Met Tyr Tyr Glu Ile Trp
1695 1710
Gln Lys Tyr Asp Pro Glu Gly Thr Gln Tyr
1715 1720
Ile Lys Tyr Glu Asp Leu Ser Asp Phe Leu
1725 1730
Asp Ala Leu Glu Glu Pro Leu Gln Ile Glu
1735 1740
Lys Pro Asn Lys Tyr Lys Ile Ile Asp Met
1745 1750
Asp Ile Thr Ile Cys Lys Gly Asp Leu Met
1755 1760
Tyr Cys Val Asp Ile Leu Asp Ala Leu Thr
1765 1770
Lys Asp Phe Phe Ala Arg Lys Gly Thr Ala
1775 1780
Ile Glu Glu Ser Ala Glu Leu Gly Glu Val
1785 1790
Ala Pro Arg Lys Asp Val Glu Gly Tyr Glu
1795 1800
Pro Ile Ser Ser Thr Arg Phe Arg Gln Gln
1805 1810
Glu Glu Tyr Asn Ala Arg Val Ile Gln Arg
1815 1820
Ala Trp Arg His Tyr Lys Gly Cys Ser Ala
1825 1830
Ser Glu Pro Arg Gln Gln Gln Thr Ala Ile
1835 1840
Val Ile Glu Ser Asp Gly His Val Thr Arg
1845 1850
Asn Gly His Lys Val Val Ile His Ser Arg
1855 1860
Ser Pro Ser Val Thr Ser Arg Ser Thr Asp
1865 1870
Val