專利名稱：：電壓門控鈉通道基因、編碼蛋白及其克隆方法
技術(shù)領(lǐng)域：
：本發(fā)明涉及一種新型電壓門控鈉通道基因、編碼蛋白及其克隆方法。特指東亞鉗蝎的電壓門控鈉通道基因、編碼蛋白及其克隆方法。
背景技術(shù)：
：電壓門控鈉通道(VGSC)在神經(jīng)元以及其它可興奮性細(xì)胞動作電位形成和傳導(dǎo)過程中扮演著極為重要的角色。VGSC的細(xì)微突變或異常表達(dá)會導(dǎo)致其相應(yīng)的功能改變，甚至誘發(fā)諸如神經(jīng)肌肉周期性麻痹癥，心臟長QT間隔綜合征(LQTs)、癲癇以及一些遺傳性疾病(Aschroft，2000)。此外，VGSC還被認(rèn)為參與了神經(jīng)病理性痛的發(fā)生和維持。與哺乳動物含多種鈉通道基因不同，昆蟲鈉通道的基因相對較少，目前已被克隆和測序的昆蟲；ara直系同源鈉通道基因(/ara"orthologoussodiumchannelgene)有果蟲雖(Zro5^p力j7a歷eZs/7c^35^e力中的基因、家蟲場(Myscaobwest/ca)中的Ksvsrl基因(也稱他c基因)、煙草頓蟲(Heliothisvirescens)中的/卿基因、德國小蠊腸"el/ager翻ica中的/^ra，。鈉通道呈現(xiàn)出的亞型多樣性，為理解鈉通道在生理/病理下的功能特性及其動態(tài)平衡調(diào)控帶來了復(fù)雜性。例如，目前臨床上癲癇、神經(jīng)病理性痛等的治療藥物通常以鈉通道為靶器，但由于其選擇性不高，可能產(chǎn)生嚴(yán)重的副作用。再如，昆蟲鈉通道是DDT和擬除蟲菊酯類殺蟲劑的主要作用靶點，但長期使用和濫用，一方面嚴(yán)重的污染了環(huán)境和危害公共健康；另一方面導(dǎo)致了昆蟲抗藥性的出現(xiàn)，其敏感性的變化是由于昆蟲的para鈉通道基因序列在殺蟲劑和神經(jīng)毒素結(jié)合位點處發(fā)生了突變。因此，開拓對不同通道亞型具有特異選擇性作用的配體/調(diào)制劑并解析新型通道亞型的基因編碼特征，不僅有利于各亞型生理功能的闡明與藥理價值的評估，同時也為鈉通道關(guān)聯(lián)的臨床疾病治療藥物以及高效生物殺蟲劑研制提供理論的可行性和新分子先導(dǎo)物。
發(fā)明內(nèi)容本發(fā)明的目的之一在于提供一種新型電壓門控鈉通道基因。本發(fā)明的目的之二在于提供一種新型電壓門控鈉通道基因的編碼蛋白。本發(fā)明的目的之三在于提供新型電壓門控鈉通道基因的克隆方法。為達(dá)到上述目的，本發(fā)明采用如下技術(shù)方案3一種電壓門控鈉通道基因，其特征在于該基因具有下列核苷酸序列之一-(1).具有SEQNO1所示的DNA序列；(2).與序列表中序列1限定的核酸序列具有95%以上的同源性，且編碼相同功能蛋白質(zhì)的DNA序列。一種根據(jù)上述的電壓門控鈉通道基因的編碼蛋白，其特征在于該編碼蛋白具有序列表中序列2的氨基酸殘基序列或者是將序列18-28的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代、缺失或添加且具有與序列14-26的氨基酸殘基序列相同活性的由序列2衍生的蛋白質(zhì)。一種根據(jù)上述的電壓門控鈉通道基因的克隆方法，其特征在于該方法的具體步驟為a.從東亞鉗蝎的腹神經(jīng)纖維中提取總RNA;b.用SuperscriptII反轉(zhuǎn)錄酶和Oligo-(dT)引物合成單鏈c腿;a.借鑒昆蟲Para鈉通道的保守氨基酸殘基設(shè)計退火引物，再采用兼并PCR以及cDNA末端的快速擴(kuò)增技術(shù)，得到全長cDNA;所述的退火引物為下表NameNucleotidesequence<table>tableseeoriginaldocumentpage4</column></row><table>本發(fā)明利用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)，兼并PCR(Nested-PCR)以及cDNA末端的快速擴(kuò)增(RACE)技術(shù)，得到了東亞鉗蝎的全長cDNA。并根據(jù)序列分析結(jié)果，對rNavl.5DIV/S3-S4胞外環(huán)相應(yīng)氨基酸進(jìn)行點突變，運用電生理手段檢驗突變體對BmKI的敏感性，借此探明鈉通道與特異性調(diào)制劑BmKI結(jié)合的關(guān)鍵性氨基酸，為蝎鈉通道對自身分泌毒素一BmKI不敏感的現(xiàn)象提供合理的解釋。證明BmNavl用于電壓門控鈉通道的表達(dá)及藥理調(diào)制機制的研究，并為離子通道病的防治和開發(fā)通道型藥物提供新型通道分子模板。本發(fā)明的電壓門控鈉通道基因可以作為研究特異性相關(guān)調(diào)制劑藥理效應(yīng)及其功能機制的模板。另通過突變實驗以及電生理記錄檢驗了突變體對特異性鈉通道調(diào)制劑3mKI的敏感性，證明該新型鈉通道基因可作為膜離子通道調(diào)制劑作用機制的研究模板。圖1為新型鈉離子通道克隆實驗策略。圖2為蝎子鈉離子通道(BmNavl)與蜘蛛(OhNavl)，果蠅(Drosopjliapara)(DmNavl)，鼠腦II型(rNavl.2)電壓門控鈉離子通道基因序列比對圖.跨膜片段用框表注出來了，S4電壓感受器中正電荷氨基酸殘基和結(jié)構(gòu)域III-IV連接片段(DomainIII-IVlinker)中在鈉離子通道的快失活中的三個關(guān)鍵氨基酸殘基都用陰影標(biāo)注出來了。圖3為蝎子鈉離子通道(BmNavl)與蜘蛛(OhNavl)和四種哺乳動物鈉通道的DIV/S3-S4胞外環(huán)氨基酸序列，位點3毒素結(jié)合關(guān)鍵氨基酸用虛線框和實線框標(biāo)注出來了。圖4為300nMBmKI施加前后rNavl.5及其各突變體1^/Ip^的相對變化值。圖5為AFTII對rNavl.5和K1616S的作用圖6為AaHII對rNavl.5和K1616S的作用具體實施方案實施例一電壓門控鈉通道基因的克隆，參見圖1:1.蝎神經(jīng)總RNA的制備剝離蝎腹部的神經(jīng)纖維(50100只)，迅速置于液氮中保存。用50ml離心管抽提總RNA:將神經(jīng)浸泡在冰浴的5ml異硫氰酸胍溶液(含0.2M的醋酸鈉〈pH=4〉)中，加入5ml冰上預(yù)冷的苯酚(pH4),勻漿，冰上放置5min后離心(10000rpm/4°C/10tnin),將上清轉(zhuǎn)移至新離心管中，加入5ml冰上預(yù)冷的氯仿/異戊醇(V/V=24/l)，劇烈混勻后再次離心(10000rpm/4°C/10min),取上清液轉(zhuǎn)移至新管，緩緩加入等體積的異戊醇，邊加邊混勻.放置-7(TC保存。.2:新型鈉通道cDNA的合成大約10ug蝎神經(jīng)總RNA樣品用SuperscriptII反轉(zhuǎn)錄酶和Oligo-(dT)引物合成單鏈cDNA，得到的一系列重疊片段被確定基本覆蓋整個編碼區(qū)，參見圖1;再借鑒昆蟲Para鈉通道的保守氨基酸殘基設(shè)計退火引物，進(jìn)行擴(kuò)增蝎鈉通道的cDNA，PCR在PE-2400(Perkin-Elmer,USA)上進(jìn)行，再經(jīng)5，RACE(RapidAmplificationofthecDNAEnds)和3，RACE獲得全長cDNA。所設(shè)計的退火引物見表l:表1PrimerforScorpionSodiumChannelNameNucleotidesequenceDegenerateprimersS15'-AT3CC5SACIS2TGTCJTTCIGS3TTCACWGAYCARAA-3'S4CTHATYTTYicrcS5V—ATTATCATCTTCATCTTT<JCCGT-3'S6G故AYTTGGAYTWS75,-ATSGGSCT3CAGMTTTAYATGWAS1TCMAGYACIGCIATRTACATRTT"3'AS25，-TCTCRTAGTACATRTCRTA效—3'SpecificprlmsrsAS1495'-TCCTTCAATGCCTTCATCTTC-3'A44485'-TCTCTCCCATTTTTTTCATAGCWA4248TGTTCATCTTCATTTTGC—3'A2451TCACCAAACAACTGC-3'A2435TACCCATAACAGCAAA-3'S49175'—TTTGCCTTCTGCTCTTTT一3'5'RACEand3'RACEA1967ACCAGCAGTTACAACAGTA1351CCAAGSTGT-3'A1275A<5AAATsecCAATAAAWAUAPCACGCSACTAGTAC-3'AAP5"郞CCACTC<5ACT站TAOS鵬GIIGGG'二一一'CA(TAXCAACGCAGAGTAC《則30MoW-3'S51385'-XfeAAWT效AGCA(TATAT"-3'HUP5,-AAGCAeT郎TATCAAUPMLong:5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCACA<5AGT"3'_Shot:t::5'-CTAATACGACTCACTATAGGGC-3'_上述PCR產(chǎn)物用瓊脂糖凝膠分析，經(jīng)割膠回收后克隆到pGEM-Teasy載體(Promega,USA)，連接有插入的DNA載體轉(zhuǎn)化至大腸桿菌，最后被純化并用ABIPRISM377DNA測序儀(Perkin-Elmer，USA)測序。測序結(jié)果如下引物AUAP/A1275所得片段正向測序結(jié)果aaataaaacctacggtcaaagatggcggacttggtctgagtggttttttctgggttttcgtcgacaatattctcattcgagagtcccggattatgagccagcggatactttaactctgccaaaagaacggagcatggtccagggccccgggccccgtaggtggtagagagcgttcgcctgtctcgtcttcgccatgtcttcggacgagcagtccgaggaggaagttctgcccgcattccgtcccttcaccagggagtcattggcggccatcgaagccaggatcgccgaggctgaatcaagaaaaaaggaatcg6cttcagaaaaaaggggaaggcgacgatgaacactacggcacttaccagccggaagtggccgaagccgaccctcaactggaggcgggtctcccattacccaagtatttggaacgtgaatttcctcccgaactattggctacacctatcgaaggtttggacaaattttacgaaaacaaaaggacattcattgtgataagtaaaggtaaggatatcttccggtttagtgcaacaaatgccatgtggat反向測序結(jié)果tga^tcttgc3taBaagcctggattttgtagttatttctttagcttacattacaatgggagttgaattaggaaatttaagtgctttgaggacttttcgaatttaaaagatgtgattattttgacagtgttttctctatctctatttgccttacttggtctccagatgtatatgggtgttctaatgcaaaaatgtattgtagaagctcctccaaatactacttatgaagagaagaaagtacattataggaatcaaagtaactggtatctagattctgctggtgaatattttatttgtggaaatgcatcaggagctagacaatgtcctgaaggata引物S7/A2435所得片段正向測序結(jié)果-cttggtctccsgatgtatatgggtgttctaatgcaaBaatgtattgtELgaagctcctccaaatactacttatgaagagaageiaagtacattataggaatcaaagtaactggtatctagattctgctggtgaatattttatttgtggaaatgcatcgacacattttattgggcatttcttgcatcatttagacttatgactcaagatttttgggaaggtctttaccaaatggtgctaagaacagcaggtccttggcatatatgttttttcatcgtaatcatctttcttggttcattttatttattaaatctcattttggccattgttgctatgtcatatgatgaattaca^犯犯gagctgaaaBagaagaagaagatgcacaaa犯g犯gaagaaaactataatgcagctgtagaacgtaaaagattgttagaagaagaaaaagcaggagtgggtgtagtgaagagttcttc反向測序結(jié)果tgagatttattgatatcttctgcgtatgggactgttgtaactgctggttatatat3C犯aaa^t8gttaacatcattgtctttgatccattteLtggagttattcattacactggcaataattgtcaacacgctcttcatgt,ctatagaacatgccgeicELtggeLt^aaglxtlxaggaattctl^Eiaa&a/tggcaactactttttcacagcaacstttgccataga^gcagtaatgaagttggtagcactgagtccaaaatattactttaaagaaggatggaacatatttgattttttaattgtagtactttctcttgttgaactgtgtggagaaaatgtttctttacctggattatcagttttgagatcatttcgtttgcttcgtgtatttcttttgtgttgggtatcatcattttcatatttgctgttatgggtatg引物S5/A4248所得片段正向測序結(jié)果atcatcattttcatatttgctgttatgggtatgcagttgtttggtgaaaaatatatagaaaataaacatgtgtttcctgataatgctgttccacgctggaattttgttgattttatgcatteattta/tgattgtgtttcgtgttttgtgtggagaatggatagaatctatgtggggtcgtatgcttgttgcagaMggccatgtgttcctttttttttatcaactgttataattggaaacctagtggtgctgaatttgtttcctgccttgcttttgtcctcctttggtgccgcaaatctttccatggcttcaccagaaagtgctgatactaaga已actgcasgaagcatttgaacgtataggaagagctatcaattgggttaaa已tctgcttctta^cga^a^tcttaEL3a3attt33ga^cc3aa^33ct3ga3atc33attgg3gatC333C33C3g3ta^ttag3gaELgalctggaagatgctacsataggacgagsgatgttagttgatggtcaggttaaaatgaaagagaaaaaaagccctaaagaasttac已g反向測序結(jié)果aaaagtacttcacaaatgcttggtgttggcttgattttgttatagttctggtttctctgtttkatctggcagtaagcgtctggagggaatgagagttgtagtaaatgctctcgttcaagctatccctgccatctttaatgttttacttgtgtgcctcatcttttggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttc犯tagctctactgtgcctaacaaatctgtttgttgctt3aacaattttacttgggaa犯tccaaa犯taaattctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaagga/tggattgatataatgagtaatgcaatag3t3ct犯gcaaaatg3agatg^c犯c引物S4/A4448所得片段正向測序結(jié)果-tggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatctgataatgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcatcattatagga^tcttttttcacgttaBatctcttcattggagtaataatagat犯tttc犯tgaacagaaaaaaaaggcccagctaaagct3ttcctcggccaaggttcaaattacaagcagt3gtatttgatcttattactastacaaaatttgatatggcaatcgtgttgtttattgg8200910053876.1反向測序結(jié)果ctatccctgccatctttaatgttttacttgtgtgcctcatcttttggcttattttttctattatgggtgttcagttatttgctgggaaatttcgttattgtgcagataaagatgggaatcgtttc已atagctctax;tgtgcctaacaa6Ltctgtttgttgctt已aacaattttacttggga已aatccaasaa1:a8attctgELta^atgtactcaatgcatatttagctctttttcaagttgccacattcaaaggatggattgatataatgagtaatgcaatagatactaagcaa已atgaagatgaacaacctgaattggagtaataatagat犯tttc犯tgaacag3aaaaaaaggcaggaggttctttag3aatgtttatg3ctgEiag3tc引物S3/A5149所得片段正向測序結(jié)果-3tgttt3tg8ctgaagELtc鄧aBg^atatteit犯tgctatga犯犯aBtgggagag3agaagccagctaaELgct3gtttattggactt^tatgctagtgatggcaatggaacattatcatcagacaccaacatttg已tgatattctagaaagaaaccatggaatgtatttgattgtgtggttgtaBCcttgtccattttaggcattgcaatcaaagatttaat已gcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtcaaaggggccaaatgtttatctacgccatatttggaatgtct反向測序結(jié)果tattgccattttcactacagaatgtgtgttaaaaatgtttgcccttaggtggtattattttaaagaaccatggaatgtatttgeLttgtgtggttgtaaccttgtccattttaggcattgcaatcaaagatttaatagcaagttattttgtgtcaccaactcttctgcgtgtggttcgtgttgtaa犯gttggtcgtgtcctcaggttggtca^aggggccaaagggattcgaacgccatatttggaatgtcttttttcatgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatgaaggcattgaagg引物S2/AS1所得片段正向測序結(jié)果catgactgtaaaacatcgtgcgggcatcgatgataattttaattttgaaacatttggtcagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttattaggtatcatggatgacagtgactgtgtacaaggatctgaagatga已ggcattgaaggagattgtggtaaaccaggtgtagcagtggcatattt已atttcttacttaattgtaagttttttaattataattaacatgtatattgctgtcattcttgaaaactacagtc犯gctacagaagatgtacaggaaggtttaacagctgatgattatgatgLtgt已ttatgaaatatggcaaaaatacgatcctgaaggtacacaatatatcaagtatg6L已gatttac反向測序結(jié)果tcttctgcgtgtggttcgtgttgtaaaagttggtcgtgtcctcaggttggtca^aggggccaaagggattcgaaccttagtcaatgattcttttatttcaaatgtcaacatcagcaggttgggatggtgtgttatt已ggtatcatggatgacagtgactgtgtacaaggatctgaagatga^ggcattgaaggagattgtggta犯ccaggtgtagcagtggcatatttaBtttctt引物S5158/而P所得片段正向測序結(jié)果-gctgtcattcttgaaaactacagtcaagctacagaagatgtacaggaaggtttaacagctgatgattatgatatgtattatgaaatatggcaaaaatacgatcctgaaggtacacaatatatcaagtatgaagatttgagtgacttcctagatgctctagaagaaccattgcagattgagaaacctaataagtacaaaatcattg3catggatattacaatatgtaaaggtgaitcttatgtattgtgtagatattcttgatgcactgaccaaagatttctttgcgaggaaaggtactgctatagaagaatctgcagaacttggagaagttgctcctaggaaagacgttgaaggctatgagccaatcagc3gtacgcgcttccgtcaacaggaagaatataatgctcgtgttattcagagagcatggcgacattataaagggtgtagtgctagtg反向測序結(jié)果AaattcttgcctgttatacatagcaggagctagtatagaattattacctaactccaatcctagtaacttagttgccattgaaataggcactctagcattcatgaaagttttattagcttcggaaacaatgcaaccacaggatragacagtgaaagggaatgtgatatsttcctttatcatcctgtctgttatagtgaBaaattaaaaaaaaatgaaaaacttatgtttcaagagccagaactcttcctcttactacaaagcatggctttgagaatgacaagtttataatggaageLatctgctatgctgaeLgattgcttcaagaccttgactcgccatttccatcttaccacccagtttcaatcattttctcaattatgccttttcagcttgtacttactttgtaaaBtgtttcacagtttgtaccatactgcctgtaaatacatcattgaataaa/tttttgataatttatcacaacgaaccgatgctcaagataaaaaaaaaaaaaaaaaaaaaaaaaaaa。實施例二新型電壓門控鈉離子通道結(jié)構(gòu)特征分析及功能說明1.新型電壓門控鈉離子通道結(jié)構(gòu)特征如附圖2所示，新型電壓門控鈉離子通道(BmNavl)蛋白質(zhì)內(nèi)部有四個高度相似的同源結(jié)構(gòu)域(Dl-D4)，且每一結(jié)構(gòu)域含有六個oc螺旋跨膜片段(Sl-S6)，具有電壓門控鈉通道的所有關(guān)鍵性的結(jié)構(gòu)1)BmNavl的S4片段具有鈉通道電壓感受器的共性，即在每隔2個疏水殘基有一個帶正電的Arg或Lys殘基，且堿性氨基酸殘基數(shù)目和位置基本對應(yīng)；.2)BmNavl與其它節(jié)肢動物電壓門控鈉通道蛋白在結(jié)構(gòu)域III和IV之間的短鏈胞內(nèi)環(huán)處(鈉通道失活化門控域)高度保守，它們在對應(yīng)于哺乳動物鈉通道rNavl.2的三個關(guān)鍵殘基I1488-F1489-M自處也存在三個疏水性殘基(MFM);3)BmNavl蛋白分子中形成鈉通道選擇性過濾器的胞內(nèi)環(huán)(DEKA)和胞外環(huán)(EEDD)以及與TTX結(jié)合的關(guān)鍵殘基也都是保守的。通過氨基酸序列比對，我們可以看出，我們所克隆得到的基因序列正是蝎子的電壓門控鈉離子通道的基因序列。2.新型電壓門控鈉離子通道功能特征電壓門控鈉通道(VGSC)在神經(jīng)元以及其它可興奮性細(xì)胞動作電位形成和傳導(dǎo)過程中扮演著極為重要的角色。當(dāng)細(xì)胞膜去極化時，VGSC被電壓依賴性的激活，使胞外鈉離子選擇性通透進(jìn)入胞內(nèi)，形成動作電位的去極化相；VGSC在開放后幾個毫秒時段里就會失活，失活的VGSC需要回到靜息狀態(tài)后才能再開放。VGSC的細(xì)微突變或異常表達(dá)會導(dǎo)致其相應(yīng)的功能改變，甚至誘發(fā)諸如神經(jīng)肌肉周期性麻痹癥，心臟長QT間隔綜合征(LQTs)、.癲癇以及一些遺傳性疾病。此外，VGSC還被認(rèn)為參與了神經(jīng)病理性痛的發(fā)生和維持。3.新型電壓門控鈉離子通道對自分泌調(diào)制劑不敏感機制電壓門控鈉通道DIV/S3-S4胞外環(huán)靠近N-端的一個酸性氨基酸殘基(Asp/Glu)對位點3毒素的結(jié)合至關(guān)重要。經(jīng)序列分析:所有蝎位點三毒素敏感的鈉通道，包括昆蟲VGSCs、Nav1.4、Navl.5、Navl,6和hNavl.7等，在相應(yīng)位置均為Asp;而對蝎位點三毒素不敏感的鈉通道，如Navl.2和Navl.3在相應(yīng)的位置上被Glu占據(jù)(附圖3)。由此提示，DIV/S3-S4胞外環(huán)的帶負(fù)電荷殘基Asp決定了VGSCs對位點三毒素的敏感性。另有研究提示蝎神經(jīng)纖維的鈉離子通道對自分泌毒素一BmKI不敏感，即BmKI缺失對自身鈉通道的調(diào)制能力。令人費解的是，與已知所有位點三毒素敏感的鈉通道一樣，蝎鈉通道DIV/S3-S4胞外環(huán)相應(yīng)位置上卻同為Asp。因而推測決定蝎鈉通道對BmKI不敏感的氨基酸殘基可能并不是哺乳動物VGSCs中常見的Glu/Asp。以rNavl.5模版構(gòu)建突變體，通過電生理記錄檢驗各個突變體對特異性調(diào)制劑BmKI敏感性變化。結(jié)果表明1)VGSCsDIV/S3-S4胞外連接環(huán)的Asp皿對通道與毒素的敏感性非常重要，說明DIV/S3-S4是BraKI毒素結(jié)合的重要區(qū)域，第一次用分子生物學(xué)的方法確證了BmKI是一個位點三毒素；2)除VGSCsDIV/S3-S4連接環(huán)關(guān)鍵性氨基酸Asp皿外，此區(qū)域正電荷Lys，對特異性調(diào)制劑BmKI的敏感性同樣至關(guān)重要；3)對BmKI敏感性起決定作用的Lys1616，對通道與同為位點三毒素的AFTII以及AaHII并不重要。決定特異性調(diào)制劑BmKI與鈉通道結(jié)合的氨基酸除了已知的Asp外，Lys,的作用同樣至關(guān)重要，進(jìn)而，蝎鈉通道Lys氨基酸的改變可能是蝎鈉通道對自身毒素BmKI不敏感的分子基礎(chǔ)；而該位點的正電荷氨基酸對其他位點三毒素并不重要，表明位點三毒素在鈉通道上的受體并不完全一致，而僅是部分重疊。參見圖4、圖5、圖6，圖4說明野生型rNavl.5通道對BmKI敏感性最強，變化值最大。突變體D1612E大大削弱了通道對BmKI的敏感性，說明DIV/S3-S4胞外連接環(huán)的Asp腿對通道與毒素的敏感性非常重要，證明BmKI是位點三毒素。rNav1.5,的1611和1615位的氨基酸突變(S1611K，S1611A，Q1615A，Q1515N)能不同程度地降低I5ms/IPMk的相對變化值，提示兩個位點可能對通道與BmKI的敏感性起微調(diào)作用，但不起決定性作用。rNavl.5的1616位點的任何一種突變(K1616S，K1616A，K1616L)均使通道BmKI的敏感性顯著性下降，說明該位點Lys,對BmKI的敏感性同樣至關(guān)重要。圖5說明AFTII能極顯著地延緩rNavl.5(A，B)和K1616S(C，D)的失活化過程。圖6說明AaHII能極顯著地延緩rNavl.5(A，B)和K1616S(C，D)的失活化過程。蝎子鈉離子通道對于蝎子自身細(xì)胞動作電位的產(chǎn)生，傳導(dǎo)，神經(jīng)的興奮性傳遞具有重要作用。同時，蝎毒素被發(fā)現(xiàn)可以作用在不同物種或者亞型的電壓門控鈉通道上，但藥理實驗表明蝎子對蝎毒素表現(xiàn)出不敏感性，新克隆的鈉通道無疑為研究蝎毒素對辨通道選擇性的分子機制，研究鈉離子通道結(jié)構(gòu)與功能之間的關(guān)系提供了難得的分子模板。此外，鈉離子通道是多種藥物和殺蟲劑但作用靶點，蝎子鈉離子通道的克隆為新型藥物和殺蟲劑的篩選，測試，研發(fā)提供了新的研究體系。序列表<110>上海大學(xué)<120〉電壓門控鈉通道基因、編碼蛋白及其克隆方法〈160〉2(193)(5809)(8065)<210>1〈211>8065〈212〉DNA〈213〉東亞鉗蝎(Buthus〈220〉〈221>5'野<222>(1)...〈220〉〈221>CDS<222>(194).<220〉〈221〉3'UTP〈222〉(5810)〈220>〈221〉PolyA—signal<222>(7994「.(7999)〈400〉1aaataaaacctcgacaatataa犯gaacggtctcgtcttctcccttcaccgccggaagtggg咖gtgaaaacaaatgcccctcgttcattatgacgcaatcttcgagatgggagttg3aaactgttgcttctccagatgtggtgaatatttgtttgtccMartensiiKarsch)tacggtxa8Lagatggcggacttggtctgagtggttttttctgggttttcg60tctcattcgagagtcccggattatgagccagcggatsctttaactctgcc120agcatggtccagggccccgggccccgt鄉(xiāng)tggt卿gagcgttcgcctg180gccatgtcttcggacgagcagtccgaggaggaagttctgcccgcattccg240郷gagtcattggcggccatcg鄉(xiāng)ccagg3tcgccgaggCtg33tC^g300tcgcttcagaa^aaaggggaaggcg3cg3tgaacactacggcacttacca360gccgaagccgaccctcaactgg鄉(xiāng)cgggtctcccattacccaagtattt420tttcctcccg犯ctattggctacacctatcgaaggtttgg■aggacattc3ttgtgataaggatatcttccggtttagtgc540atgtggatattagatcctttcaatcctatccgtcgtgcagccatcagcat600cctgcctttagcttcttggttattgtcactatccttgtaaactgtgttct660cgca^cg^aaggaaU卿gsggctttttacggcgatstei720a3gct3cagcatacttgagcaattcacata780ccgtggaattggctggattttgtagttatttctttagcttacattacaat840tt￡iggaaMttaagtgctttgaggacttttcgtgtacttagagcattaaa900gttattccaggtttaaagactattgttggagctgtcatag33tctgt3aa960ttttgacagtgttttctctatctctatttgccttacttgg1020t由tgggtgttctaatgca犯aatgt3ttgtagaagctcctccaaatac1080g卿卿鄉(xiāng)tacattatagg肌tcsaagtaactggtatctagattctgc1140tttatttgtggM3tgC3tC鄉(xiāng)agct卿caatgtcctgsaggatatat1200aattatggtcccaatccaaatcatggttttscaaattttgacacatttta126013ttgggcatttaatggtgctatggttcatttagctgtagaattcttc3gaaaataaatggcaccatatgatgatggttttgag卿ggga3atttattgatagttaacatccaacacgctcagcactgagtagtactttctgagatcatttattaatatcctatcatcattaaataaacatttcatttatgtatgcttgttcctagtggtgttccatggctagga卿gcttacaataggstaaag3已attacatatgtcttgttagtaactcctaataacag犯aB3aagggatatacttaattataattattttgtttaaagttacacacagcagtttgtattttacaa已tggctsgcttst￡igttctgtgcctttaaacttgcatcat3g犯C3gC3gt已tttattaagCtga^3￡Lgcgtaaaagattcttcttctggc犯gt3ttaagtcctttcaccagaagagaatcttctgcgattgtctttgttcatgtctaaactacttttccaaaatattcttgttgaaccgtttgcttcattatgggaattcatatttggtgtttcctgattgtgtttcgcagaatggcctg犯tttgttcacc已g3犯atcaattgggcgag卿tgtacagatcttggcaataccaagct犯agtagaaggatgaagaaatctattaaaaccgtccagatgctagtaga卿卿tgcctgaatatcgggaatg卿ttggtgga^gcattagcatt已tatggac已tttggttttagtttctctgt3cteitgcgaattagacttatgtccttggcaatctcatttttgttsgaag3gatcttacga卿gtgt聊aggattcttttagatcctcatagatc犯gc3gg卿agttta/tggg已ctgatccatttattagaacatgctcacagcaac3ctttaaag￡itgtgtgg卿gtgtatttaaaaactgtgggctgttatgggat犯tgctgtgtgttttgtgcatgtgttccttcctgccttgtgctgatacttaaaatctgg3gatca^ctagttgatggaagttgtagtaattgaaagagatttgg￡L￡L8LataatgtcaaaagatgatgtCEigaa3ttg3catctgattgaatcaaaattataaatat11agatatttacttaatctggcccctgagagcgactcaagattatatgttttggccattgtttgcac^a犯ag犯aaagcaattatttgtgtgg卿tagcggatg已tgtgtgcaggacgtttgtaactgcggagtt已ttccgacatggatatttgccataaggatgg咖aaatgtttct￡LCttgC3￡L3atgccttgggatatgcagttgtccscgctggtgg卿atggtttttttttagcttttgtcctaagaaactgcttcttacga犯cagatatttC3ggtt3犯3gg卿tgg3taataatactggatttaaaaccaaatttcat3ttaa^tgaaggg卿taacttttccagacttggagacactg犯3CELttgtaatttgaaaaattttatttcacaaatgct3gt犯gCtt3tctccgcccattttggg鄉(xiāng)ttcatcgta已gctatgtcatgaagaagaaaggagtgggtgattggtgaaccaaagtagagtgcttcaaaatggtt3tatsattscactgggaagcagtaaatatttgattttacctggattcttggccaatttggtgaaa犯ttttgttgtcaactgttatcctttggtgcaag^gc￡itatc11aaaaaag8g3a^tcttagaaatatgaaaaattcag3t犯t3ttgctgccag犯gggaggattttgc卿tgatgaagtgttatcattatttttattttttgaaatggtgttggcatggg鄉(xiāng)t3ctt3gggca3gtctttaccatcatctttctactataatgc￡L￡L￡LCtgt￡Lg￡Lcaaaatctcataaaacaaaatgaaagatgta犯gtggcagattgcttgagtacaaaaaatcaataattgtaggaattctttgaagttggtttttaattgttatcagttttcactcaatttttgtgUgggattttstgcatgtggggtcgtaattgga^accgcaeiatctttgaacgtatatttaagacctgga卿tgc833aa3gccccaattcaaggtaaattcttcaagaaaaagaatgacgatga卿aggggstgatcccaagataaaagatgaaatcatcaaaaacattttcctacgtgga朋tgatattagtggtt肌tggatgttaatcaattgattttgtgtgacatatctgtcacgtct13201380144015001560162016801740180018601920198020402100216022202280234024002460252025802640270027602820288029403000306031203180324033003360342034803540360036603720378038403900ggagggaatgtttacttgtgtggg333ttttaacaaatcttaatgtactcC￡La_tCg￡Lt3taaatctcttcttctttagaaggg聊gaagatttgatctttatgctagtgcctt鄉(xiāng)tgggtccattttatctgcgtgtgagggattcgaccttctgctcgattcttttaggatg織gtaccaggtgt已taacatgtataggtttaacaaggtacacaa￡LttgC￡Lg￡Lttaggtgstcttgaaaggtacttgaaggctattgttatttcagacaaactgcagattcattcttg3C33tatCctgcttgcaatgactgatteig￡Ltaatga_tcttgttttataattacatgtagg犯犯3tgttgttttttactctataaacaactaatattaaacgttttaa卿gttgtagtgcctcatctcgttattgtggtttgUgctaatgc犯tagatgtatctatattggagtaaatgtttatga33gCC3gCt3attactaataatggc犯tggatattctttatattattttaggcattgcaagttcgtgttgaccttacttttttttagttacgtgcgggcatttcaaatgtgactgtgtacgc已gtggcatattgctgtcagctgatgattg￡Lg￡L￡L￡LCCt￡Latgtattgtggctatagaaggagccaatca卿gcatggcattgttattgaggtcaccaagctttctacatgcstgaagtcacctgtactgttgt已attaccattcttgttatattttgaatgttattaccaga犯ttacttgtg"t￡Lt￡Lttgttaaatttatttatttatctag犯犯gtt犯atgctcttttggcttatC3g3taaagatagctcttttactttgtactCtg^g3tC3aagctattccaacattatcattgccatttttca^agattttsaaagttggttgctcttgctgtttatctatcgatgat犯caacatcagca3ggatctgaattt犯tttcttcttg犯aaatgatatgta3tgMg3tttataagtacaatagatattctaatctgcagagcagtacgcgg3C3ttat犯a犯gtg8tgggtgttscaagtcaacattatgtatattttttggttgacattggtctattttgctacaata^tttat3g3c33ta3C3a^gttag3gagactatataa"tata3ttc3a3gggtttttagcactcagtgcccacgttcaagcttttttctatttgggaatcgttacttgggaatcaagttgcctttcatcatttttcei8tgaatcggcc卿gtatggcaatctcagacacc3cactacagaag犯tgtattttcgtgtcctccatgtccttgcgccatatttttttaatttt鄉(xiāng)ttgggat卿tg鄉(xiāng)gctt3Ctt犯ttctacagtcaattatgaaatagagtgacttcaatcattg已ctgatgcactgcttccgtcaa鄉(xiāng)gtgtagtcceLtgtaactccgttccacactaaatag犯犯acc犯tgtacactacttgggata卿gtttttacggatgcttttatttgttaaatckg鄉(xiāng)卿tgtgtttggacatttatttaaacaatccctgccaatgggtgttcttcaatagctaatccaaaaa3c3ttcaa^gga￡LC￡L￡LCCtgataggatcttt3taMgct3ttcaaattacgtgttgtttaeicatttgatgtgtgtgttaagattgtgtggtattttgtgt鄉(xiāng)ttggtcaccagcactttggaatgtcttga^aca^tttgggtgtgttat￡LUg￡L￡Lgg￡Lggtaagtttttgctacagaagtggca^a^tctagatgctcacc3犯g3ttgttgctcctacsgga^gaatgctagtgaacBgaaatggtcgatgtgtgatattggttgattgcacgagtttttccatgtttaaatatgcaggaagcatcttttag犯tgtacaaattctattattattataattatttaaaacc犯tgccgcatataagatctttaatgt已gttatttgcctactgtgccgatggattga犯tettg犯gtttttcacgttaggcaggaggttggacttaa3t3ttCt8g3aaatgtttgcttgt犯ccttcaccaactctttaatatttgttttcatgacgtcagtc已attaggtatcatattgtggtaaacgatcctgatctttgcgaggg犯卿cgtat犯tgctcgctcgtcagcaacaaagttgtat11ataaaagattgattgcgagtaccsgtatttttctaaaactaaeiaggaagttttaatattttttattgastttcaaatctgaaaattttatactgctgttttttttaaaatgaaatgagtaaaatgt39604020408041404200426043204380444045004560462046804740480048604920498050405100516052205280534054005460552055805640570057605820588059406000606061206180624063006360642064806540ttgctacattatctataggacscttatga已cagctgtgtttgggaaaaatcsctctacaactttaaaagccctttctgcgcccacgc33c卿ctgtggtcaccattggctataatatcctgtat3￡Ltt3aaaaatcatct已catagcagattg犯atagcaggatg卿3犯gC3tggCttcaagaxxttgccttttcaaaatacatcaaaaaaaaaaagaagtatgstagctgttaacatcttcctcatagattcatgata^gcgtgtc犯cgctt犯agctcagtttat3taca^3gttgttELCtgtaatacttgctttcttttcaattgatgtctat3taccac3c3ttct3aatgactccatgtagagctagtatgcactctagccsgtg犯aggtttg已gaatgtgactcgccagcttgtacttttg犯taaBtctgtatcagtacacaa&ctaaca3tgt8gggtttttctgcttatcagcattgtt3taa3cttgtcctaattcctgccgctttattggctga^actctgcttcttatata3gtgc3ca^ctactgctagttatgcattggtc犯gaatat犯犯犯tcagagaattattagaatgtgata犯acttatgtac犯gtttattttccatcttsctttgtaaattttgataataaaaa8065aaagctacata^Ltttg^sgggaatgttcctttctaagctcaagctgtgaggagtatacaaacctctgcatttaaatgag已gttcagaagaaaatc11aaaagcstaatcctttccaacaattatttcatatatataaaaattttatttg犯aagttttgaaaaaatctacct犯ctccagttttattagtattcctttattcaagagcc犯tggaagaaaccacccagtatgtttcacattatcacaacaggtgggttgattctgcttaccccttatcagatggcattaaattgggtgggcttcgaggactgtacctctttggacttttgagattttatattgcatcsttttttcaagcgctcaagactaacacaactattagtttcttgcataaattc3tCCt3gt^cttcggaaactcatcctgtcagaactcttctctgctatgcttca^tcattgtttgtaccag犯ccgatgcaBgatggtt3tgaacagggtgtgataatgt犯aaattttgcaaacaac11tacgggaggggtgtgtg3t3ggccaccagattatcatgtgaaatcagcttaagcagatctaggtggtggtctcttctgggttgcctgttacttagttgcca_3tgcaBcc3tgttatagtgctcttactactgaagattgcttctcaattatactgcctgttcaaga_ta_aa6600666067206780684069006960702070807140720072607320738074407500756076207680774078007860792079808040〈210〉2〈211〉1871〈212>PRT<213〉東亞鉗蝎(Buthus〈400〉2SerMetSer1ValLeuGluSerAlaGluLeuGinHisTyrProLeuAlaLysGlyAspGluGin5AlaPheArg15AlaAlalie25GluSerArg35LysGlyGlu45ThrTyrGin55MartensiiSerGluProPheGluAlaLysLysGlyAspProGluKarsch)GluThrArgGluAspValGlu10Arg20lie30Ser40Glu50Ala60GluAlaProLeuProProGlyLeuThrPheliePheTrplieArgAlaAlaPheLeuValAsnVeilLeuPheCyslieLeuGluTrpAsnLeuAlaGlyAsnValLeulieProVallieVallieLeuPheAspProProLysGluLeuAspLyslieValArgPheLeuAspAlalieSerPheAsnCysThrHisThrAlaLysAlaGinPheTrpLeuTyrlieLeuSerArgAlaGlyLeuGluSerlieLeuAlaLeuGinLeu65TyrLeu75LeuAla85PheTyr95lieSer105SerAla115ProPhe125Serlie135LeuVal145ValLeu155GluLys165lieTyr175ThrAla185ThrTyr195AspPhe205ThrMet215AlaLeu225!LeuLys235LysThr245ValLys255ThrVal265LeuGly275GluAlaGluArgThrProGluAsnLysGlyThrAsnAsnProLeuVallieValMetThrGluLeuThrlieArgGlyLeuArgValValGlyValArgThrThrVallieValAsnLeuPheSerLeuGinGlyLeu70GluPhe80lieGlu90LysArg100LysAsp110AlaMet120lieArg130HisPro140Thrlie150GinThr160GluArg170GluSer180Phelie190AspPro200lieSer210GluLeu220PheArg230AlaVal240GlyAla250LysAsp260LeuSer270MetTyr28017MetGlyValLeuMetGinLysCyslieVal285290GluAlaProProAsnThrThrTyrGluGlu295300LysLysValHisTyrArgAsnGinSerAsn305310TrpTyrLeuAspSerAlaGlyGluTyrPhe315320lieCysGlyAsnAlaSerGlyAlaArgGin325330CysProGluGlyTyrlieCysLeuSerAsn335340TyrGlyProAsnProAsnHisGlyPheThr345350AsnPheAspThrPheTyrTrpAlaPheLeu355360AlaSerPheArgLeuMetThrGinAspPhe365370TrpGluGlyLeuTyrGinMetValLeuArg375380ThrAlaGlyProTrpHislieArgPhePhe385390lieVallieliePheLeuGlySerPheTyr395400LeuLeuAsnLeulieLeuAlalieValAla405410MetSerTyrAspGluLeuGinLysArgAla415420GluLysGluGluGluAspAlaGinLysGlu425430GluGluAsnTyrAsnAlaAlaValGluArg435440LysArgLeuLeuGluGluGluLysAlaGly445450ValGlyValValLysSerSerSerGluSer455460SerSerGlySerTyrGluLeuPheValGly465470GinAspLysThrValGluGluLysAspAla475480SerlieLysSerValAspGlyAspSerlie485490GlyGluProLysSerGinlieAsnGlyLys495500說明書第16/23頁ValArgTyrSerProPheLeuAspAsplieSerArgGluGluPheLysPheCysLeuTyrValPheThrLeuMetSerSerLeuTyrPheAlaValLysTyrPheAspValGluProGlyLeuLeuTrpProLysAsplieLyslieAspMetLyslieAspGluSerLysGluAsnCysValTrplieGinAspProAlalielieGluGinGluPheThrMetLysTyrPhePheLeuLeuCysLeuSerArgValThrLeuSerLeu505GinLys515ProGin525AspVal535GinAla545GlyArg555LysPhe565LeuArg575AspCys585Lyslie595PheMet605lieVal615HisAla625PheLeu635AlaThr645LeuVal655LysGlu665lieVal675GlyGlu685ValLeu695PheLys705AsnLeu715AspAspProTyrArgGinMetValAlaGlyGluArgLysGluPhelieCysAsnValAsnGluLeuAsnThrAspMetLysAsnPheAlaAlaLeuGlyTrpValLeuAsnValArgSerLeuAlaLeulieValGlu510AspSer520ThrGlu530LeuAsn540ArgGin550GluPro560LysCys570Asplie580CysTrp590lielie600Phelie610LeuPhe620AspLys630GlyAsn640lieGlu650SerPro660Asnlie670SerLeu680Ser!>eu690PheArg700LysSer710Serlie72019MetGlyLeuThrliePheGlyGluPheProPheValValPheGluSerGluTrpThrValAsnLeuPheGlyProGluGluAlaAsnTrpLeuLysGinlieGluAspGluMetLysGluAspLeuGlulieLysThrPheValAlaVall>ysTyrAspAsnAspPheArgValMetTrpProCyslieliePheProAlaAleiSerAlaPheGluValLysAsnLeuGlyAspLeuGluLeuValLysLysGluValSerlieValGly725LeuGly735MetGly745lieGlu755AlaVal765MetHis775LeuCys785GlyArg795ValPro805GlyAsn815AlaLeu825AsnLeu835AspThr845Arglie855lieCys865ArgPro875GinThr885AspAla895AspGly905SerPro915ValVal925GinGly935AlaLeulielieMetGinAsnLysProArgSerPheGlyGluMetLeuPhePheLeuValLeuLeuSerMetLysLysGlyArgPheLeuLysThrThrAspThrlieGinValLysGluGlyAspAsnGlyGlyAsn730liePhe740LeuPhe750HisVal760TrpAsn770Metlie780Trplie790ValAla800LeuSer810ValLeu820SerSer830AlaSer840LeuGin850Alalie860Arglie870ArgAsn880lieArg890GlyArg■LysMet910lieThr920GlyLeu930GinAla94020lieProlieLysMetAlaAsnAsnThrLys945950ThrLeuValAsnSerSerHisMetSerAla955%0LysValGluLeuLysGluLysGluLysGlu965970LysPheLysGluLysAspAsnLysGluLys975980AspGluGlyPheGlyAsnLysValTyrPro985990GinLysAspAspAspAspValSerAsnLys9951000SerlieAsnAsnValLysAspLeuLysAsp10051010AsnlieGluLysGlylieProAsnAsnLys10151020ProSerLysGluGluSerGinlieSerLeu10251030ProGluGlySerGinGluGluLysLysAsp10351040AlaSerLysAspAspVallieAsnGluGly10451050GlyPheLeuLysAspGluAsplieLeuGlu10551060GluAspAlaGlulieGluGlyAspAsnAla10651070AspAspGluSerSerLyslielieliePro10751080GluTyrProSerAspCysPheProAspLys10851090CysTyrGinHisPheProPheCysLeuGly10851100AsnGluGluSerLysPheTrpArgHisTyr10951110ThrThrlieArgGlyLysSerTyrThrLeu11051120ValGluAsnLysTyrPheGluThrLeulie10251130liePheMetlieLeuValSerSerLeuAla10351140LeuAlaLeuGluAspValAsnLeuLysLys10451150ArgArgTrpLeuMetAsplieLeuGinTyr10551160MetAspLysliePheThrlieLeuPhePhe10651170PheGluMetLeulieLysTrpLeuAlaPhe10751180GlyPheLysLysTyrPheThrAsnAlaTrp10851190CysTrpLeuAspPheVallieValLeuVal10951200SerLeuPheAsnLeuAlaValSerLeuMet12051210GlyGlySerAsplieSerAlaPheLysThr12151220MetArgThrLeuArgAlaLeuArgProLeu12251230ArgAlaMetSerArgLeuGluGlyMetArg12351240ValValValAsnAlaLeuValGinAlalie12451250ProAlaliePheAsnValLeuLeuValCys12551260LeuliePheTrpLeuliePheSerlieMet12651270GlyValGinLeuPheAlaGlyLysPheArg12751280TyrCysAlaAspLysAspGlyAsnArgPhe12851290AsnSerSerThrValProAsnLysSerVal12951300CysCysLeuAsnAsnPheThrTrpGluAsn13051310ProLyslieAsnSerAspAsnValLeuAsn13151320AlaTyrLeuAlaLeuPheGinValAlaThr13251330PheLysGlyTrplieAsplieMetSerAsn13351340AlalieAspThrLysGinAsnGluAspGlu13451350GinProGluTyrGluValAsnArgTyrMet13551360TyrLeuTyrPheValLeuPhelielielie13651370GlySerPhePheThrLeuAsnLeuPhelie13751380GlyValLysLysPheMetAsnAlaProAlaLysLeuThrAsnLeuPheAlaMetPheAspPhePheValLeuTyrPheCysVallieAlaPheValArgValLeuValLeuLeuAIelLeuLeuValMetSerlieLysThrMetlysGinThrlieGluAsplieLysLysVallieSerValLysPhePheMetPhelieAlaGluLysAlaAlaLysGlyHislieAlaMetGluAlaLysProLysGlyAlaAsnPhePheAspAsn1385GlyGly1395AspGin1405LysMet1415liePro1425ValVal1435PheAsp1445LeuAsn1455TyrHis1465LeuGlu1475liePhe1485PheAla1495ProTrp1505ThrLeu1515AspLeu1525ThrLeu1535ValGly1545AlaLys1555LeuAla1565lieCys1575lieTyr1585MetThr1595PheAsnGluSerLeuGluLysLysTyrGlyGluLysArgProArgPheAspLeuMetAlalieMetLeuValGinThrProArgLeuAsnThrThrGluLeuArgTrpAsnValPheSerlieLeuGin1390Met1400Tyr1410Cys1420Phe1430lie1440Val1450Met1460Thr1470lie1480Cys1490Tyr1500Asp1510Gly1520LeuAlaSerTyr1530LeuArgValVal1540ArgValLeuArg1550GlylieArgThr1560MetSerLeuPro1570LeuLeuLeuPhe1580AlaliePheGly1590ValLysHisArg1600AlaGlylieAspAspAsnPheAsnPheGlu16051610ThrPheGlyGinSerMetlieLeuLeuPhe16151620GinMetSerThrSerAlaGlyTrpAspGly16251630ValLeuLeuGlylieMetAspAspSerAsp16351640CysValGinGlySerGluAspGluGlylie16451650GluGlyAspCysGlyLysProGlyValAla16551660ValAlaTyrLeulieSerTyrLeulieVal16651670SerPheLeulielielieAsnMetTyrlie16751680AlaVallieLeuGluAsnTyrSerGinAla16751690ThrGluAspValGinGluGlyLeuThrAla16851700AspAspTyrAspMetTyrTyrGlulieTrp16951710GinLysTyrAspProGluGlyThrGinTyr17151720lieLysTyrGluAspLeuSerAspPheLeu17251730AspAlaLeuGluGluProLeuGinlieGlu17351740LysProAsnLysTyrLyslielieAspMet17451750AsplieThrlieCysLysGlyAspLeuMet17551760TyrCysValAsplieLeuAspAlaLeuThr17651770LysAspPhePheAlaArgLysGlyThrAla17751780lieGluGluSerAlaGluLeuGlyGluVal17851790AlaProArgLysAspValGluGlyTyrGlu17951800ProlieSerSerThrArgPheArgGinGin18051810GluGluTyrAsnAlaArgVallieGinArg1815182024AlaTrpArgSerGluProVallieGluAsnGlyHisSerProSerValHisTyrLysGlyCysSerAla18251830ArgGinGinGinThrAlalie18351840SerAspGlyHisValThrArg18451850LysValVallieHisSerArg18551860ValThrSerArgSerThrAsp18651870權(quán)利要求1.一種電壓門控鈉通道基因，其特征在于該基因具有下列核苷酸序列之一(1).具有SEQNO1所示的DNA序列；(2).與序列表中序列1限定的核酸序列具有95％以上的同源性，且編碼相同功能蛋白質(zhì)的DNA序列。2.—種根據(jù)權(quán)利要求1所述的電壓門控鈉通道基因的編碼蛋白，其特征在于該編碼蛋白具有序列表中序列2的氨基酸殘基序列或者是將序列18-28的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代、缺失或添加且具有與序列14-26的氨基酸殘基序列相同活性的由序列2衍生的蛋白質(zhì)。3.—種根據(jù)權(quán)利要求1所述的電壓門控鈉通道基因的克隆方法，其特征在于該方法的具體步驟為a.從東亞鉗蝎的腹神經(jīng)纖維中提取總RNA;b.用SuperscriptII反轉(zhuǎn)錄酶和Oligo-(dT)引物合成單鏈cDNA;c.借鑒昆蟲Para鈉通道的保守氨基酸殘基設(shè)計退火引物，再采用兼并PCR以及cDNA末端的快速擴(kuò)增技術(shù)，得到全長cDNA;所述的退火引物為下表所示N3m6Muclsotidessqu6柳Degener彼primers<table>tableseeoriginaldocumentpage2</column></row><table>全文摘要本發(fā)明涉及一種東亞鉗蝎的電壓門控鈉通道基因、編碼蛋白及其克隆方法。本發(fā)明的一種電壓門控鈉通道基因具有SEQNO1所示的堿基序列，其編碼蛋白具有SEQNO2所示的氨基酸序列。該電壓門控鈉通道基因的克隆方法的具體步驟為首先從東亞鉗蝎的腹神經(jīng)纖維中提取總RNA；然后用SuperscriptII反轉(zhuǎn)錄酶和Oligo-(dT)引物合成單鏈cDNA；最后借鑒昆蟲Para鈉通道的保守氨基酸殘基設(shè)計退火引物，再采用兼并PCR以及cDNA末端的快速擴(kuò)增技術(shù)，得到全長cDNA。本發(fā)明利用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)，兼并PCR(Nested-PCR)以及cDNA末端的快速擴(kuò)增(RACE)技術(shù)，得到了東亞鉗蝎的全長cDNA。另通過突變實驗以及電生理記錄檢驗了突變體對特異性鈉通道調(diào)制劑BmKI的敏感性，證明該新型鈉通道基因可作為膜離子通道調(diào)制劑作用機制的研究模板。文檔編號C12N15/12GK101654675SQ20091005387公開日2010年2月24日申請日期2009年6月26日優(yōu)先權(quán)日2009年6月26日發(fā)明者吉永華,周智磊,峰姜,左小潘,楊宏天申請人:上海大學(xué)