CN115141787B - 一种潜艇微生物传感器及其制备方法和应用 - Google Patents

一种潜艇微生物传感器及其制备方法和应用 Download PDF

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CN115141787B
CN115141787B CN202210639825.2A CN202210639825A CN115141787B CN 115141787 B CN115141787 B CN 115141787B CN 202210639825 A CN202210639825 A CN 202210639825A CN 115141787 B CN115141787 B CN 115141787B
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杨建明
李美洁
吕书喆
汤若昊
王兆宝
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Abstract

本发明公开了一种潜艇微生物传感器及其制备方法和应用。所述潜艇微生物传感器包括含有感应金属镉Cd(II)的感应蛋白Cad的感应元件以及电信号元件蛋白mtrCAB，并敲除乳酸脱氢酶1dh基因，增加了细胞内可释放电子，过表达孔蛋白基因OprF增强细菌粘附电极和增加膜磁导率。本发明所构建的潜艇微生物传感器能够感应低浓度的金属镉Cd(II)离子，而产生强电信号，进而实现了对低浓度金属镉离子的实时检测。与其他检测方法相比，所述潜艇微生物传感器检测金属镉离子操作更简便、信号收集更容易，能够用于海洋中对潜艇外壳氧化还原反应释放的金属镉离子的检测，因此具有广泛的应用前景。

Description

一种潜艇微生物传感器及其制备方法和应用
技术领域
本发明属于基因工程和分子生物学技术领域，具体涉及一种潜艇微生物传感器及其制备方法和应用。
背景技术
海上作战行动中，潜艇因其先进的性能、优越的隐蔽性、持久的续航能力，被称为“深海神器”。每个国家都在潜心研究潜艇技术，以便有效扩展的海上军力。除此之外，如何高效快速的监测敌方的作战潜艇，从而对敌方潜艇实施驱逐和捕获，对我国海洋国防安全具有重要战略意义。当前传统的潜艇探测方法主要包括：声呐探测、红外探测、电磁探测等方法，其中声呐探测是目前各国所使用的最主要方法。然而，在使用声呐探测进行潜艇探测时亦易暴露自身位置。
潜艇在海洋中长期潜行的情况下，一些部位很容易受到海水的腐蚀，继而释放出一定量的金属离子。生物感应技术是利用基因工程手段对菌株进行改造，使得该微生物在感应特定化合物条件下可以发生被检测的变化，从而达到对特定化合物进行检测的目的。通过对潜艇释放的金属离子进行感应，构建微生物传感器，可以达到对潜艇进行实时检测的目的。感应元件包括负责基因转录的启动子、核糖体结合位点、终止子、转录调控蛋白等等，可特异性地感应金属离子，引起下游报告元件产生信号。一般常用的报告元件包括绿色荧光蛋白(GFP)、黄色荧光蛋白(YFP)和红色荧光蛋白(RFP)，分别产生绿色荧光、黄色荧光和红色荧光等可以被检测的感应信号。但是这种信号输出方式并不能应对海洋中复杂的环境和众多的干扰因素。
因此，需要对报告元件和宿主进行优化，获得一种方便于海水中进行实时检测的潜艇微生物感应器。
发明内容
本发明提供了一种潜艇微生物传感器及其制备方法和应用。所述潜艇生物传感器中含有感应金属离子Cd(II)的感应元件以及电信号元件蛋白mtrCAB，敲除了宿主大肠杆菌的乳酸脱氢酶ldh基因，过表达孔蛋白基因OprF，从而增加了细胞内电流信号强度。本发明提供的潜艇微生物传感器，可以实现对海洋中的金属镉离子的实时荧光检测，从而进一步对潜艇进行实时监测。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种潜艇生物感应器，所述潜艇生物感应器中包含电信号元件蛋白基因、感应元件、孔蛋白基因，并同时敲除了大肠杆菌乳酸脱氢酶基因。
进一步的，所述电信号元件蛋白基因为mtrCAB基因，其核苷酸序列如SEQ ID NO.1所示；所述感应元件为cad基因，其核苷酸序列如SEQ ID NO.3所示。
进一步的，所述孔蛋白基因为oprF基因，其核苷酸序列如SEQ ID NO.6所示；所述大肠杆菌乳酸脱氢酶基因为ldh基因，其核苷酸序列如SEQ ID NO.4所示。
进一步的，所述潜艇生物感应器具有感应金属镉离子Cd(II)的作用。
本发明还提供了所述的潜艇生物感应器的制备方法，包括以下步骤：
(1)将电信号元件蛋白mtrCAB基因利用PCR扩增、纯化回收，其与质粒pACYCDuet-1同时使用EcoR I和Hind III进行双酶切，将mtrCAB片段与质粒酶切片段进行无缝连接，连接产物转化大肠杆菌感受态细胞，于含抗生素的LB固体平板上筛选阳性克隆，得到重组质粒p-mtrCAB；
(2)扩增感应元件Cad基因，纯化回收后，其与重组质粒p-mtrCAB利用EcoR I进行单酶切，将质粒酶切片段与Cad进行无缝连接，连接产物转化大肠杆菌感受态细胞，于含抗生素的LB固体平板上筛选阳性克隆，得到重组质粒p-Cad-mtrCAB；
(3)扩增大肠杆菌基因组中ldh基因的上游片段ldh-up和下游片段ldh-down，扩增孔蛋白基因oprF，将三个片段进行overlap PCR获得ldh-up-oprF-ldh-down片段，通过无缝克隆连接至***质粒pRE112的Hind III位点，得到pRE112-Δldh-oprF质粒；
(4)将pRE112-Δldh-oprF转化供体菌E.coli χ7213，宿主E.coli BL21(DE3)作为受体菌；将供体菌和受体菌进行活化，ddH2O洗涤，1％DAP重悬菌体，涂布于无抗性平板，进行接合；ddH2O洗涤重悬，涂布于氯霉素平板上，菌落PCR验证；将阳性克隆于无抗性LB中进行培养，涂布于10％蔗糖平板上进行筛选，将阳性克隆于蔗糖平板和氯霉素平板上进行筛选，最终获得重组大肠杆菌E.coliBL21(DE3)Δldh::oprF；
(5)将重组质粒p-Cad-mtrCAB转化E.coli BL21(DE3)Δldh::oprF感受态细胞，于含抗生素的LB固体平板上筛选到的阳性克隆，得到工程菌株ECCad，即为潜艇微生物传感器。
本发明还提供了所述的潜艇生物感应器在用于金属Cd(II)离子的实时检测中的应用。
进一步的，所述潜艇生物感应器能够检测到的Cd(II)离子的浓度不低于0.1μmol/L。
本发明还提供了所述的潜艇生物感应器用于制备实时检测海洋中潜艇的探测装置中的应用。
进一步的，所述探测装置的搭建方法为：在阳极室和阴极室中间放置密封圈，将二者夹紧进行组装，阳极材料由导线分别固定在阳极室和阴极室中；阳极室内接种活化的潜艇微生物传感器，并施加电压促进阳极材料上生物膜的形成；阴极室内装载电解液。
进一步的，所述探测装置通过实时检测海洋中潜艇释放的Cd(II)离子，并转化为变化的电信号输出，以此达到实时检测海洋中潜艇中的目的。
进一步的，所述探测装置能够感应到的Cd(II)离子的浓度不低于0.1μmol/L。
本发明与现有技术相比，具有以下优点和有益效果：
本发明将金属离子感应元件与胞外电子传递路径mtrCAD进行连接，并且利用了孔蛋白提高膜通透性，提高细菌粘附电极和增加膜磁导率，并且敲除了乳酸合成途径，增加了细胞内可释放电子，放大了电流信号，从而产生易于被检测到的电流信号。本发明构建的潜艇微生物传感器可以检测微量的金属离子，并产生瞬时的电信号输出，进而实现对海洋中潜艇所释放金属离子的检测，且该方法操作方便、简单、安全性高、灵敏度高。
附图说明
图1为构建的载体p-Cad-mtrCAB的质粒图谱。
图2为构建的工程菌株ECCad诱导产电检测结果
具体实施方式
下面结合具体实施例对本发明做进一步说明，但本发明不受实施例的限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过购买获得的常规产品。
实施例1：载体p-Cad-mtrCAB的构建
1、p-mtrCAB表达载体的构建
以S.oneidensis基因组为模版，引物mtrCAB-F和引物mtrCAB-R，进行聚合酶链式反应(PCR)，扩增mtrCAB片段，其PCR扩增体系如下所示：
PCR程序为：95℃3min；30×(95℃15s，55℃15s，72℃4min)；72℃5min：16℃∞。
引物序列如下所示：
mtrCAB-F：5’-CAAGGAGAAAAAAATGATGAACGCACAAAAATC-3’(SEQ ID NO.8)；
mtrCAB-R：5’-GCCGCAAGCTTTTAGAGTTTGTAACTCATGCTC-3’(SEQ ID NO.9)。
利用限制性内切酶1EcoR I(TaKaRa，货号1611)和限制性内切酶2 Hind III(TaKaRa，货号1615)双酶切pACYCDuet-1质粒，其酶切体系为：
酶切体系置于37℃孵育1h，进行胶回收纯化。
利用无缝克隆将mtrCAB片段克隆pACYCDuet-1质粒上，其体系如下所示：
连接体系置于50℃孵育30min。连接产物转化E.coli DH5α感受态，涂布到含有34mg/L氯霉素的LB固体平板上，PCR筛选阳性克隆，从阳性克隆中提取重组质粒p-mtrCAB，再通过限制性酶切和测序进行鉴定。mtrCAB的核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO.2所示。
2、p-Cad-mtrCAB表达载体的构建
合成感应元件cad，序列如SEQ ID NO.3所示，利用引物Cad-F和引物Cad-R进行聚合酶链式反应(PCR)，扩增Cad片段，其PCR扩增体系如下所示：
PCR程序为：95℃3min；30循环×(95℃15s，58℃15s，72℃4min)；72℃5min；16℃∞。
引物序列如下所示：
Cad-F：5’-CCGAATTCGTGTATTTTTTAATAAATTATTTTAC-3’(SEQ ID NO.10)；
Cad-R：5’-CATGAATTCTCAGACATTGACCTTCACTTCT-3’(SEQ ID NO.11)。
PCR产物利用胶回收纯化试剂盒(Vazyme，货号DC301-01)进行胶回收纯化。
利用限制性内切酶1 EcoR I(TaKaRa，货号1611)酶切p-mtrCAB质粒，其酶切体系为：
酶切体系置于37℃孵育1h，进行胶回收纯化。
利用无缝克隆将Cad片段克隆p-mtrCAB质粒上，其体系如下所示：
连接体系置于50℃孵育30min。连接产物转化E.coli DH5α感受态，涂布到含有34mg/L氯霉素的LB固体平板上，PCR筛选阳性克隆，从阳性克隆中提取重组质粒p-Cad-mtrCAB(图1)，再通过限制性酶切和测序进行鉴定。
实施例2：宿主E.coli BL21(DE3)Δldh::oprF的构建
(1)以大肠杆菌基因组为模板，扩增ldh基因的上游片段ldh-up和下游片段ldh-down，扩增孔蛋白基因oprF，将三个片段进行overlap PCR获得ldh-up-oprF-ldh-down片段，通过无缝克隆连接至***质粒pRE112的Sac I位点，获得pRE112-Δldh-oprF质粒；其中，ldh基因的核苷酸序列如SEQ ID NO.4所示，其氨基酸序列如SEQ ID NO.5所示；孔蛋白基因oprF的核苷酸序列如SEQ ID NO.6所示，其氨基酸序列如SEQ ID NO.7所示。
引物序列如下所示：
ldh-up-F：5’-AGCCAAGTAATTTCCACTCCTTGTGGTGGC-3’(SEQ ID NO.12)：
ldh-up-R：5’-GCATGCGATATCGAGCTCACTGGTCGCGCAGAACATCTTTC-3’(SEQ IDNO.13)；
ldh-down-F：5’-ATGAATTCCCGGGAGAGCTCCGTTATGATGGCGTCGCTAG-3’(SEQ IDNO.14)；
ldh-down-R：5’-TCAGTTTCATAAATTACGGATGGCAGAGTA-3’(SEQ ID NO.15)；
oprF-F：5’-TCCGTAATTTATGAAACTGAAGAACACCTTAG-3’(SEQ ID NO.16)：
oprF-R：5’-CAAGGAGTGGAAATTACTTGGCTTCAGCTTCTACT-3’(SEQ ID NO.17)。
(2)将构建的pRE112-Δldh-oprF转化供体菌E.coli χ7213，宿主E.coli BL21(DE3)作为受体菌；将供体菌和受体菌进行活化，将上述活化的菌按1％的接种量转接于10ml LB液体培养基中，37℃，180rpm，大约2.5h，至OD600值到0.5。
(3)各取菌液1.5ml于离心管中，10000rpm，离心1min，弃上清，在短暂离心一次，彻底去掉上清残余LB，用1mL ddH2O悬浮沉淀，10000rpm，离心1min，弃上清，再用1mL ddH2O悬浮沉淀，10000rpm，离心1min，弃上清，以除去抗生素。
(4)用500μL的ddH2O重新悬浮菌体沉淀后，各取100μL混合均匀，10000rpm，离心1min，弃上清，用50μL的1％DAP重新悬浮菌体沉淀，涂布于无抗性的LB平板上，于37℃培养12h。ddH2O洗涤重悬，将重悬菌液稀释10-1、10-2、10-3、10-4倍，各倍数取30μL涂布于氯霉素平板上，菌落PCR验证；将阳性克隆于无抗性LB中进行培养，涂布于10％蔗糖平板上进行筛选，将阳性克隆于蔗糖平板和氯霉素平板上进行筛选，最终获得重组大肠杆菌E.coli BL21(DE3)Δldh::oprF。
实施例3：潜艇微生物感应器的构建
将p-Cad-mtrCAB重组质粒转化E..coli BL21(DE3)Δldh::oprF感受态细胞，涂布到含34mg/L氯霉素的LB固体平板上，通过PCR筛选获得阳性克隆，由此获得含有载体p-Cad-mtrCAB的工程菌株ECCad。
实施例4：潜艇生物感应器检测金属镉离子的应用
(1)检测装置的搭建
将活化的ECCad按1％接种量接种至含有LB培养基的阳极室内，外加0.3V的电压促进碳纤维毡阳极上生物膜的形成。在阳极室和阴极室中间放置密封圈，再将二者夹紧进行组装，并确保溶液不向外泄露。碳毡由导线连接分别位于阳极室和阴极室中，其中在阳极室中的位置与进水口相平行，在阴极室中垂直于底部，阳极室装载含有LB培养基，阴极室装载由K3[Fe(CN)6]、K2HPO4和KH2PO4所配置的电解液。电化学工作站用于输出电信号的检测。
(2)以电信号作为输出的检测
将构建的装置中通入金属离子Cd(II)浓度含量为5μmol/L的缓冲溶液，利用电化学工作站检测电信号输出变化，每1小时检测一次，一共检测12小时。
结果如图2所示，在5μmol/L金属离子Cd(II)存在的情况下与无金属离子存在时有显著性的差别，7小时是检测电压达到了最高0.252V，后期金属离子全部排除后电流密度又降低到平稳值。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 青岛农业大学
<120> 一种潜艇微生物传感器及其制备方法和应用
<141> 2022-06-08
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5192
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgatgaacg cacaaaaatc aaaaatcgca ctgctgctcg cagcaagtgc cgtcacaatg 60
gccttaaccg gctgtggtgg aagcgatggt aataacggca atgatggtag tgatggtggt 120
gagccagcag gtagcatcca gacgttaaac ctagatatca ctaaagtaag ctatgaaaat 180
ggtgcaccta tggtcactgt tttcgccact aacgaagccg acatgccagt gattggtctc 240
gcaaatttag aaatcaaaaa agcactgcaa ttaataccgg aaggggcgac aggcccaggt 300
aatagcgcta actggcaagg cttaggctca tcaaagagct atgtcgataa taaaaacggt 360
agctatacct ttaaattcga cgccttcgat agtaataagg tctttaatgc tcaattaacg 420
caacgcttta acgttgtttc tgctgcgggt aaattagcag acggaacgac cgttcccgtt 480
gccgaaatgg ttgaagattt cgacggccaa ggtaatgcgc cgcaatatac aaaaaatatc 540
gttagccacg aagtatgtgc ttcttgccac gtagaaggtg aaaagattta tcaccaagct 600
actgaagtcg aaacttgtat ttcttgccac actcaagagt ttgcggatgg tcgcggcaaa 660
ccccatgtcg cctttagtca cttaattcac aatgtgcata atgccaacaa agcttggggc 720
aaagacaata aaatccctac agttgcacaa aatattgtcc aagataattg ccaagtttgt 780
cacgttgaat ccgacatgct caccgaggca aaaaactggt cacgtattcc aacaatggaa 840
gtctgttcta gctgtcacgt agacatcgat tttgctgcgg gtaaaggcca ctctcaacaa 900
ctcgataact ccaactgtat cgcctgccat aacagcgact ggactgctga gttacacaca 960
gccaaaacca ccgcaactaa gaacttgatt aatcaatacg gtatcgagac tacctcgaca 1020
attaataccg aaactaaagc agccacaatt agtgttcaag ttgtagatgc gaacggtact 1080
gctgttgatc tcaagaccat cctgcctaaa gtgcaacgct tagagatcat caccaacgtt 1140
ggtcctaata atgcaacctt aggttatagt ggcaaagatt caatatttgc aatcaaaaat 1200
ggagctcttg atccaaaagc tactatcaat gatgctggca aactggttta taccactact 1260
aaagacctca aacttggcca aaacggcgca gacagcgaca cagcatttag ctttgtaggt 1320
tggtcaatgt gttctagcga aggtaagttt gtagactgtg cagaccctgc atttgatggt 1380
gttgatgtaa ctaagtatac cggcatgaaa gcggatttag cctttgctac tttgtcaggt 1440
aaagcaccaa gtactcgcca cgttgattct gttaacatga cagcctgtgc caattgccac 1500
actgctgagt tcgaaattca caaaggcaaa caacatgcag gctttgtgat gacagagcaa 1560
ctatcacaca cccaagatgc taacggtaaa gcgattgtag gccttgacgc atgtgtgact 1620
tgtcatactc ctgatggcac ctatagcttt gccaaccgtg gtgcgctaga gctaaaacta 1680
cacaaaaaac acgttgaaga tgcctacggc ctcattggtg gcaattgtgc ctcttgtcac 1740
tcagacttca accttgagtc tttcaagaag aaaggcgcat tgaatactgc cgctgcagca 1800
gataaaacag gtctatattc tacgccgatc actgcaactt gtactacctg tcacacagtt 1860
ggcagccagt acatggtcca tacgaaagaa accctggagt ctttcggtgc agttgttgat 1920
ggcacaaaag atgatgctac cagtgcggca cagtcagaaa cctgtttcta ctgccatacc 1980
ccaacagttg cagatcacac taaagtgaaa atgtaatttg cccaagcagg gggagctcgc 2040
tccccctttc ttgaattttg ttgggacaaa ttgggaagcc tattatgaag aactgcctaa 2100
aaatgaaaaa cctactgccg gcacttacca tcacaatggc aatgtctgca gttatggcat 2160
tagtcgtcac accaaacgct tatgcgtcga agtgggatga gaaaatgacg ccagagcaag 2220
tcgaagccac cttagataag aagtttgccg aaggcaacta ctcccctaaa ggcgccgatt 2280
cttgcttgat gtgccataag aaatccgaaa aagtcatgga ccttttcaaa ggtgtccacg 2340
gtgcgattga ctcctctaag agtccaatgg ctggcctgca atgtgaggca tgccacggcc 2400
cactgggtca gcacaacaaa ggcggcaacg agccgatgat cacttttggt aagcaatcaa 2460
ccttaagtgc cgacaagcaa aacagcgtat gtatgagctg tcaccaagac gataagcgta 2520
tgtcttggaa tggcggtcac catgacaatg ccgatgttgc ttgtgcttct tgtcaccaag 2580
tacacgtcgc aaaagatcct gtgttatcta aaaacacgga aatggaagtc tgtactagct 2640
gccatacaaa gcaaaaagcg gatatgaata aacgctcaag tcacccactc aaatgggcac 2700
aaatgacctg tagcgactgt cacaatcccc atgggagcat gacagattcc gatcttaaca 2760
agcctagcgt gaatgatacc tgttattcct gtcacgccga aaaacgcggc ccaaaacttt 2820
gggagcatgc acccgtcact gagaattgtg tcacttgcca caatcctcac ggtagtgtga 2880
atgacggtat gctgaaaacc cgtgcgccac agctatgtca gcaatgtcac gccagcgatg 2940
gccacgccag caacgcctac ttaggtaaca ctggattagg ttcaaatgtc ggtgacaatg 3000
cctttactgg tggaagaagc tgcttaaatt gccatagtca ggttcatggt tctaaccatc 3060
catctggcaa gctattacag cgctaaggag acgagaaaat gaaatttaaa ctcaatttga 3120
tcactctagc gttattagcc aacacaggct tggccgtcgc tgctgatggt tatggtctag 3180
cgaatgccaa tactgaaaaa gtgaaattat ccgcatggag ctgtaaaggc tgcgtcgttg 3240
aaacgggcac atcaggcact gtgggtgtcg gtgtcggtta taacagcgaa gaggatattc 3300
gctctgccaa tgcctttggt acatccaatg aagtggcggg taaatttgat gccgatttaa 3360
actttaaagg tgaaaagggt tatcgtgcca gtgttgatgc ttatcaactc ggtatggatg 3420
gcggtcgctt agatgtcaat gcgggcaaac aaggccagta caacgtcaat gtgaactatc 3480
gccaaattgc tacctacgac agcaatagcg ccctatcgcc ctacgcgggt attggtggca 3540
ataacctcac gttaccggat aactggataa cagcaggttc aagcaaccaa atgccactct 3600
tgatggacag cctcaatgcc ctcgaactct cacttaaacg tgagcgcacg gggttgggat 3660
ttgaatatca aggtgaatcc ctgtggagca cctatgttaa ctacatgcgt gaagagaaaa 3720
ccggcttaaa acaagcctct ggtagcttct tcaaccaatc gatgatgtta gcagagccgg 3780
tggattacac cactgacacc attgaagcgg gtgtcaaact caagggtgat cgttggttta 3840
ccgcactcag ttacaatggg tcaatattca aaaacgaata caaccaattg gactttgaaa 3900
atgcttttaa ccccaccttt ggtgctcaaa cccaaggtac gatggcactc gatccggata 3960
accagtcaca caccgtgtcg ctgatgggac agtacaacga tggcagcaac gcactgtcgg 4020
gtcgtattct gaccggacaa atgagccaag atcaggcgtt agtgacggat aactaccgtt 4080
atgctaatca gctcaatacc gatgccgtcg atgccaaagt cgatctactg ggtatgaacc 4140
tgaaagtcgt tagcaaagtg agcaatgatc ttcgcttaac aggtagttac gattattacg 4200
accgtgacaa taatacccaa gtagaagaat ggactcagat cagcatcaac aatgtcaacg 4260
gtaaggtggc ttataacacc ccttacgata atcgtacgca acgctttaaa gttgccgcag 4320
attatcgcat tacccgcgat atcaaactcg atggtggtta tgacttcaaa cgtgaccaac 4380
gtgattatca agaccgtgaa accacggatg aaaataccgt ttgggcccgt ttacgtgtaa 4440
acagcttcga tacttgggac atgtgggtaa aaggcagtta cggtaaccgt gacggctcac 4500
aataccaagc gtctgaatgg acctcttctg aaaccaacag cctgttacgt aagtacaatc 4560
tggctgaccg tgacagaact caagtcgaag cacggatcac ccattcgcca ttagaaagcc 4620
tgactatcga tgttggtgcc cgttacgcgt tagatgatta taccgatact gtgattggat 4680
taactgagtc aaaagacacc agttatgatg ccaacatcag ttatatgatc accgctgact 4740
tactggcaac cgccttctac aattaccaaa ccattgagtc tgaacaggcg ggtagcagca 4800
attacagcac cccaacgtgg acaggcttta tagaagatca ggtagatgtg gtcggtgcag 4860
gtatcagcta caacaatctg ctggagaaca agttacgcct aggactggac tacacctatt 4920
ccaactccga cagtaacact caagtcagac aaggtatcac tggcgactat ggtgattatt 4980
ttgccaaagt gcataacatt aacttatacg ctcaatatca agccaccgag aaactcgcgc 5040
tgcgcttcga ttacaaaatt gagaactata aggacaatga cgccgcaaat gatatcgccg 5100
ttgatggcat ttggaacgtc gtaggttttg gtagtaacag ccatgactac accgcacaaa 5160
tgctgatgct gagcatgagt tacaaactct aa 5192
<210> 2
<211> 1701
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Lys Asn Cys Leu Lys Met Lys Asn Leu Leu Pro Ala Leu Thr Ile
1 5 10 15
Thr Met Ala Met Ser Ala Val Met Ala Leu Val Val Thr Pro Asn Ala
20 25 30
Tyr Ala Ser Lys Trp Asp Glu Lys Met Thr Pro Glu Gln Val Glu Ala
35 40 45
Thr Leu Asp Lys Lys Phe Ala Glu Gly Asn Tyr Ser Pro Lys Gly Ala
50 55 60
Asp Ser Cys Leu Met Cys His Lys Lys Ser Glu Lys Val Met Asp Leu
65 70 75 80
Phe Lys Gly Val His Gly Ala Ile Asp Ser Ser Lys Ser Pro Met Ala
85 90 95
Gly Leu Gln Cys Glu Ala Cys His Gly Pro Leu Gly Gln His Asn Lys
100 105 110
Gly Gly Asn Glu Pro Met Ile Thr Phe Gly Lys Gln Ser Thr Leu Ser
115 120 125
Ala Asp Lys Gln Asn Ser Val Cys Met Ser Cys His Gln Asp Asp Lys
130 135 140
Arg Met Ser Trp Asn Gly Gly His His Asp Asn Ala Asp Val Ala Cys
145 150 155 160
Ala Ser Cys His Gln Val His Val Ala Lys Asp Pro Val Leu Ser Lys
165 170 175
Asn Thr Glu Met Glu Val Cys Thr Ser Cys His Thr Lys Gln Lys Ala
180 185 190
Asp Met Asn Lys Arg Ser Ser His Pro Leu Lys Trp Ala Gln Met Thr
195 200 205
Cys Ser Asp Cys His Asn Pro His Gly Ser Met Thr Asp Ser Asp Leu
210 215 220
Asn Lys Pro Ser Val Asn Asp Thr Cys Tyr Ser Cys His Ala Glu Lys
225 230 235 240
Arg Gly Pro Lys Leu Trp Glu His Ala Pro Val Thr Glu Asn Cys Val
245 250 255
Thr Cys His Asn Pro His Gly Ser Val Asn Asp Gly Met Leu Lys Thr
260 265 270
Arg Ala Pro Gln Leu Cys Gln Gln Cys His Ala Ser Asp Gly His Ala
275 280 285
Ser Asn Ala Tyr Leu Gly Asn Thr Gly Leu Gly Ser Asn Val Gly Asp
290 295 300
Asn Ala Phe Thr Gly Gly Arg Ser Cys Leu Asn Cys His Ser Gln Val
305 310 315 320
His Gly Ser Asn His Pro Ser Gly Lys Leu Leu Gln Arg Met Met Asn
325 330 335
Ala Gln Lys Ser Lys Ile Ala Leu Leu Leu Ala Ala Ser Ala Val Thr
340 345 350
Met Ala Leu Thr Gly Cys Gly Gly Ser Asp Gly Asn Asn Gly Asn Asp
355 360 365
Gly Ser Asp Gly Gly Glu Pro Ala Gly Ser Ile Gln Thr Leu Asn Leu
370 375 380
Asp Ile Thr Lys Val Ser Tyr Glu Asn Gly Ala Pro Met Val Thr Val
385 390 395 400
Phe Ala Thr Asn Glu Ala Asp Met Pro Val Ile Gly Leu Ala Asn Leu
405 410 415
Glu Ile Lys Lys Ala Leu Gln Leu Ile Pro Glu Gly Ala Thr Gly Pro
420 425 430
Gly Asn Ser Ala Asn Trp Gln Gly Leu Gly Ser Ser Lys Ser Tyr Val
435 440 445
Asp Asn Lys Asn Gly Ser Tyr Thr Phe Lys Phe Asp Ala Phe Asp Ser
450 455 460
Asn Lys Val Phe Asn Ala Gln Leu Thr Gln Arg Phe Asn Val Val Ser
465 470 475 480
Ala Ala Gly Lys Leu Ala Asp Gly Thr Thr Val Pro Val Ala Glu Met
485 490 495
Val Glu Asp Phe Asp Gly Gln Gly Asn Ala Pro Gln Tyr Thr Lys Asn
500 505 510
Ile Val Ser His Glu Val Cys Ala Ser Cys His Val Glu Gly Glu Lys
515 520 525
Ile Tyr His Gln Ala Thr Glu Val Glu Thr Cys Ile Ser Cys His Thr
530 535 540
Gln Glu Phe Ala Asp Gly Arg Gly Lys Pro His Val Ala Phe Ser His
545 550 555 560
Leu Ile His Asn Val His Asn Ala Asn Lys Ala Trp Gly Lys Asp Asn
565 570 575
Lys Ile Pro Thr Val Ala Gln Asn Ile Val Gln Asp Asn Cys Gln Val
580 585 590
Cys His Val Glu Ser Asp Met Leu Thr Glu Ala Lys Asn Trp Ser Arg
595 600 605
Ile Pro Thr Met Glu Val Cys Ser Ser Cys His Val Asp Ile Asp Phe
610 615 620
Ala Ala Gly Lys Gly His Ser Gln Gln Leu Asp Asn Ser Asn Cys Ile
625 630 635 640
Ala Cys His Asn Ser Asp Trp Thr Ala Glu Leu His Thr Ala Lys Thr
645 650 655
Thr Ala Thr Lys Asn Leu Ile Asn Gln Tyr Gly Ile Glu Thr Thr Ser
660 665 670
Thr Ile Asn Thr Glu Thr Lys Ala Ala Thr Ile Ser Val Gln Val Val
675 680 685
Asp Ala Asn Gly Thr Ala Val Asp Leu Lys Thr Ile Leu Pro Lys Val
690 695 700
Gln Arg Leu Glu Ile Ile Thr Asn Val Gly Pro Asn Asn Ala Thr Leu
705 710 715 720
Gly Tyr Ser Gly Lys Asp Ser Ile Phe Ala Ile Lys Asn Gly Ala Leu
725 730 735
Asp Pro Lys Ala Thr Ile Asn Asp Ala Gly Lys Leu Val Tyr Thr Thr
740 745 750
Thr Lys Asp Leu Lys Leu Gly Gln Asn Gly Ala Asp Ser Asp Thr Ala
755 760 765
Phe Ser Phe Val Gly Trp Ser Met Cys Ser Ser Glu Gly Lys Phe Val
770 775 780
Asp Cys Ala Asp Pro Ala Phe Asp Gly Val Asp Val Thr Lys Tyr Thr
785 790 795 800
Gly Met Lys Ala Asp Leu Ala Phe Ala Thr Leu Ser Gly Lys Ala Pro
805 810 815
Ser Thr Arg His Val Asp Ser Val Asn Met Thr Ala Cys Ala Asn Cys
820 825 830
His Thr Ala Glu Phe Glu Ile His Lys Gly Lys Gln His Ala Gly Phe
835 840 845
Val Met Thr Glu Gln Leu Ser His Thr Gln Asp Ala Asn Gly Lys Ala
850 855 860
Ile Val Gly Leu Asp Ala Cys Val Thr Cys His Thr Pro Asp Gly Thr
865 870 875 880
Tyr Ser Phe Ala Asn Arg Gly Ala Leu Glu Leu Lys Leu His Lys Lys
885 890 895
His Val Glu Asp Ala Tyr Gly Leu Ile Gly Gly Asn Cys Ala Ser Cys
900 905 910
His Ser Asp Phe Asn Leu Glu Ser Phe Lys Lys Lys Gly Ala Leu Asn
915 920 925
Thr Ala Ala Ala Ala Asp Lys Thr Gly Leu Tyr Ser Thr Pro Ile Thr
930 935 940
Ala Thr Cys Thr Thr Cys His Thr Val Gly Ser Gln Tyr Met Val His
945 950 955 960
Thr Lys Glu Thr Leu Glu Ser Phe Gly Ala Val Val Asp Gly Thr Lys
965 970 975
Asp Asp Ala Thr Ser Ala Ala Gln Ser Glu Thr Cys Phe Tyr Cys His
980 985 990
Thr Pro Thr Val Ala Asp His Thr Lys Val Lys Met Met Lys Phe Lys
995 1000 1005
Leu Asn Leu Ile Thr Leu Ala Leu Leu Ala Asn Thr Gly Leu Ala Val
1010 1015 1020
Ala Ala Asp Gly Tyr Gly Leu Ala Asn Ala Asn Thr Glu Lys Val Lys
1025 1030 1035 1040
Leu Ser Ala Trp Ser Cys Lys Gly Cys Val Val Glu Thr Gly Thr Ser
1045 1050 1055
Gly Thr Val Gly Val Gly Val Gly Tyr Asn Ser Glu Glu Asp Ile Arg
1060 1065 1070
Ser Ala Asn Ala Phe Gly Thr Ser Asn Glu Val Ala Gly Lys Phe Asp
1075 1080 1085
Ala Asp Leu Asn Phe Lys Gly Glu Lys Gly Tyr Arg Ala Ser Val Asp
1090 1095 1100
Ala Tyr Gln Leu Gly Met Asp Gly Gly Arg Leu Asp Val Asn Ala Gly
1105 1110 1115 1120
Lys Gln Gly Gln Tyr Asn Val Asn Val Asn Tyr Arg Gln Ile Ala Thr
1125 1130 1135
Tyr Asp Ser Asn Ser Ala Leu Ser Pro Tyr Ala Gly Ile Gly Gly Asn
1140 1145 1150
Asn Leu Thr Leu Pro Asp Asn Trp Ile Thr Ala Gly Ser Ser Asn Gln
1155 1160 1165
Met Pro Leu Leu Met Asp Ser Leu Asn Ala Leu Glu Leu Ser Leu Lys
1170 1175 1180
Arg Glu Arg Thr Gly Leu Gly Phe Glu Tyr Gln Gly Glu Ser Leu Trp
1185 1190 1195 1200
Ser Thr Tyr Val Asn Tyr Met Arg Glu Glu Lys Thr Gly Leu Lys Gln
1205 1210 1215
Ala Ser Gly Ser Phe Phe Asn Gln Ser Met Met Leu Ala Glu Pro Val
1220 1225 1230
Asp Tyr Thr Thr Asp Thr Ile Glu Ala Gly Val Lys Leu Lys Gly Asp
1235 1240 1245
Arg Trp Phe Thr Ala Leu Ser Tyr Asn Gly Ser Ile Phe Lys Asn Glu
1250 1255 1260
Tyr Asn Gln Leu Asp Phe Glu Asn Ala Phe Asn Pro Thr Phe Gly Ala
1265 1270 1275 1280
Gln Thr Gln Gly Thr Met Ala Leu Asp Pro Asp Asn Gln Ser His Thr
1285 1290 1295
Val Ser Leu Met Gly Gln Tyr Asn Asp Gly Ser Asn Ala Leu Ser Gly
1300 1305 1310
Arg Ile Leu Thr Gly Gln Met Ser Gln Asp Gln Ala Leu Val Thr Asp
1315 1320 1325
Asn Tyr Arg Tyr Ala Asn Gln Leu Asn Thr Asp Ala Val Asp Ala Lys
1330 1335 1340
Val Asp Leu Leu Gly Met Asn Leu Lys Val Val Ser Lys Val Ser Asn
1345 1350 1355 1360
Asp Leu Arg Leu Thr Gly Ser Tyr Asp Tyr Tyr Asp Arg Asp Asn Asn
1365 1370 1375
Thr Gln Val Glu Glu Trp Thr Gln Ile Ser Ile Asn Asn Val Asn Gly
1380 1385 1390
Lys Val Ala Tyr Asn Thr Pro Tyr Asp Asn Arg Thr Gln Arg Phe Lys
1395 1400 1405
Val Ala Ala Asp Tyr Arg Ile Thr Arg Asp Ile Lys Leu Asp Gly Gly
1410 1415 1420
Tyr Asp Phe Lys Arg Asp Gln Arg Asp Tyr Gln Asp Arg Glu Thr Thr
1425 1430 1435 1440
Asp Glu Asn Thr Val Trp Ala Arg Leu Arg Val Asn Ser Phe Asp Thr
1445 1450 1455
Trp Asp Met Trp Val Lys Gly Ser Tyr Gly Asn Arg Asp Gly Ser Gln
1460 1465 1470
Tyr Gln Ala Ser Glu Trp Thr Ser Ser Glu Thr Asn Ser Leu Leu Arg
1475 1480 1485
Lys Tyr Asn Leu Ala Asp Arg Asp Arg Thr Gln Val Glu Ala Arg Ile
1490 1495 1500
Thr His Ser Pro Leu Glu Ser Leu Thr Ile Asp Val Gly Ala Arg Tyr
1505 1510 1515 1520
Ala Leu Asp Asp Tyr Thr Asp Thr Val Ile Gly Leu Thr Glu Ser Lys
1525 1530 1535
Asp Thr Ser Tyr Asp Ala Asn Ile Ser Tyr Met Ile Thr Ala Asp Leu
1540 1545 1550
Leu Ala Thr Ala Phe Tyr Asn Tyr Gln Thr Ile Glu Ser Glu Gln Ala
1555 1560 1565
Gly Ser Ser Asn Tyr Ser Thr Pro Thr Trp Thr Gly Phe Ile Glu Asp
1570 1575 1580
Gln Val Asp Val Val Gly Ala Gly Ile Ser Tyr Asn Asn Leu Leu Glu
1585 1590 1595 1600
Asn Lys Leu Arg Leu Gly Leu Asp Tyr Thr Tyr Ser Asn Ser Asp Ser
1605 1610 1615
Asn Thr Gln Val Arg Gln Gly Ile Thr Gly Asp Tyr Gly Asp Tyr Phe
1620 1625 1630
Ala Lys Val His Asn Ile Asn Leu Tyr Ala Gln Tyr Gln Ala Thr Glu
1635 1640 1645
Lys Leu Ala Leu Arg Phe Asp Tyr Lys Ile Glu Asn Tyr Lys Asp Asn
1650 1655 1660
Asp Ala Ala Asn Asp Ile Ala Val Asp Gly Ile Trp Asn Val Val Gly
1665 1670 1675 1680
Phe Gly Ser Asn Ser His Asp Tyr Thr Ala Gln Met Leu Met Leu Ser
1685 1690 1695
Met Ser Tyr Lys Leu
1700
<210> 3
<211> 572
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtgtattttt taataaatta ttttacttat tgaaatgtat tattttctaa tgtcataccc 60
tggtcaaaac cgttcgtttt tgagactaga attttatgcc ctacttactt cttttatttt 120
cattcaaata tttgcttgca tgatgagtcg aaaatggtta taatacactc aaataaatat 180
ttgaatgaag atgggatgat aatatgaaaa agaaagatac ttgtgaaatt ttttgttatg 240
acgaagaaaa ggttaatcga atacaagggg atttacaaac agttgatatt tctggtgtta 300
gccaaatttt aaaggctatt gccgatgaaa atagagcaaa aattacttac gctctgtgtc 360
aggatgaaga gttgtgtgtt tgtgatatag caaatatctt aggtgttacg atagcaaatg 420
catctcatca tttacgtacg ctttataagc aaggggtggt caactttaga aaagaaggaa 480
aactagcttt atattcttta ggtgatgaac atatcaggca gataatgatg atcgccctag 540
cacataagaa agaagtgaag gtcaatgtct ga 572
<210> 4
<211> 1716
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgtcttcca tgacaacaac tgataataaa gcctttttga atgaacttgc tcgtctggtg 60
ggttcttcac acctgctcac cgatcccgca aaaacggccc gctatcgcaa gggcttccgt 120
tctggtcagg gcgacgcgct ggctgtcgtt ttccctggct cactactaga attgtggcgg 180
gtgctgaaag cctgcgtcac cgccgacaaa attattctga tgcaggccgc caatacaggc 240
ctgaccgaag gatcgacgcc aaacggtaac gattatgatc gcgatgtcgt tatcatcagc 300
accctgcgtc tcgacaagct gcacgttctt ggcaagggcg aacaggtgct ggcctatccg 360
ggcaccacgc tctattcgct ggaaaaagcc ctcaaaccgc tgggacgcga accgcactca 420
gtgattggat catcgtgtat aggcgcatcg gtcatcggcg gtatttgtaa caactccggc 480
ggctcgctgg tgcaacgtgg cccggcgtat accgaaatgt cgttattcgc gcgtataaat 540
gaagacggca aactgacgct ggtgaaccat ctggggattg atctgggcga aacgccggag 600
cagatcctta gcaagctgga tgatgatcgc atcaaagatg acgatgtgcg tcacgatggt 660
cgtcacgccc acgattatga ctatgtccac cgcgttcgtg atattgaagc cgacacgccc 720
gcacgttata acgccgatcc tgatcggtta tttgaatctt ctggttgcgc cgggaagctg 780
gcggtctttg cagtacgtct tgataccttc gaagcggaaa aaaatcagca ggtgttttat 840
atcggcacca accagccgga agtgctgacc gaaatccgcc gtcatattct ggctaacttc 900
gaaaatctgc cggttgccgg ggaatatatg caccgggata tctacgatat tgcggaaaaa 960
tacggcaaag acaccttcct gatgattgat aagttaggca ccgacaagat gccgttcttc 1020
tttaatctca agggacgcac cgatgcgatg ctggagaaag tgaaattctt ccgtccgcat 1080
tttactgacc gtgcgatgca aaaattcggt cacctgttcc ccagccattt accgccgcgc 1140
atgaaaaact ggcgcgataa atacgagcat catctgctgt taaaaatggc gggcgatggc 1200
gtgggcgaag ccaaatcgtg gctggtggat tatttcaaac aggccgaagg cgatttcttt 1260
gtctgtacgc cggaggaagg cagcaaagcg tttttacacc gtttcgccgc tgcgggcgca 1320
gcaattcgtt atcaggcggt gcattccgat gaagtcgaag acattctggc gttggatatc 1380
gctctgcggc gtaacgacac cgagtggtat gagcatttac cgccggagat cgacagccag 1440
ctggtgcaca agctctatta cggccatttt atgtgctatg tcttccatca ggattacata 1500
gtgaaaaaag gcgtggatgt gcatgcgtta aaagaacaga tgctggaact gctacagcag 1560
cgcggcgcgc agtaccctgc cgagcataac gtcggtcatt tgtataaagc accggagacg 1620
ttgcagaagt tctatcgcga gaacgatccg accaacagca tgaatccggg gatcggtaaa 1680
accagtaaac ggaaaaactg gcaggaagtg gagtaa 1716
<210> 5
<211> 571
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser Ser Met Thr Thr Thr Asp Asn Lys Ala Phe Leu Asn Glu Leu
1 5 10 15
Ala Arg Leu Val Gly Ser Ser His Leu Leu Thr Asp Pro Ala Lys Thr
20 25 30
Ala Arg Tyr Arg Lys Gly Phe Arg Ser Gly Gln Gly Asp Ala Leu Ala
35 40 45
Val Val Phe Pro Gly Ser Leu Leu Glu Leu Trp Arg Val Leu Lys Ala
50 55 60
Cys Val Thr Ala Asp Lys Ile Ile Leu Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Asn Gly Asn Asp Tyr Asp Arg Asp Val
85 90 95
Val Ile Ile Ser Thr Leu Arg Leu Asp Lys Leu His Val Leu Gly Lys
100 105 110
Gly Glu Gln Val Leu Ala Tyr Pro Gly Thr Thr Leu Tyr Ser Leu Glu
115 120 125
Lys Ala Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Ile Gly Gly Ile Cys Asn Asn Ser Gly
145 150 155 160
Gly Ser Leu Val Gln Arg Gly Pro Ala Tyr Thr Glu Met Ser Leu Phe
165 170 175
Ala Arg Ile Asn Glu Asp Gly Lys Leu Thr Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Glu Thr Pro Glu Gln Ile Leu Ser Lys Leu Asp Asp
195 200 205
Asp Arg Ile Lys Asp Asp Asp Val Arg His Asp Gly Arg His Ala His
210 215 220
Asp Tyr Asp Tyr Val His Arg Val Arg Asp Ile Glu Ala Asp Thr Pro
225 230 235 240
Ala Arg Tyr Asn Ala Asp Pro Asp Arg Leu Phe Glu Ser Ser Gly Cys
245 250 255
Ala Gly Lys Leu Ala Val Phe Ala Val Arg Leu Asp Thr Phe Glu Ala
260 265 270
Glu Lys Asn Gln Gln Val Phe Tyr Ile Gly Thr Asn Gln Pro Glu Val
275 280 285
Leu Thr Glu Ile Arg Arg His Ile Leu Ala Asn Phe Glu Asn Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Asp Ile Tyr Asp Ile Ala Glu Lys
305 310 315 320
Tyr Gly Lys Asp Thr Phe Leu Met Ile Asp Lys Leu Gly Thr Asp Lys
325 330 335
Met Pro Phe Phe Phe Asn Leu Lys Gly Arg Thr Asp Ala Met Leu Glu
340 345 350
Lys Val Lys Phe Phe Arg Pro His Phe Thr Asp Arg Ala Met Gln Lys
355 360 365
Phe Gly His Leu Phe Pro Ser His Leu Pro Pro Arg Met Lys Asn Trp
370 375 380
Arg Asp Lys Tyr Glu His His Leu Leu Leu Lys Met Ala Gly Asp Gly
385 390 395 400
Val Gly Glu Ala Lys Ser Trp Leu Val Asp Tyr Phe Lys Gln Ala Glu
405 410 415
Gly Asp Phe Phe Val Cys Thr Pro Glu Glu Gly Ser Lys Ala Phe Leu
420 425 430
His Arg Phe Ala Ala Ala Gly Ala Ala Ile Arg Tyr Gln Ala Val His
435 440 445
Ser Asp Glu Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Thr Glu Trp Tyr Glu His Leu Pro Pro Glu Ile Asp Ser Gln
465 470 475 480
Leu Val His Lys Leu Tyr Tyr Gly His Phe Met Cys Tyr Val Phe His
485 490 495
Gln Asp Tyr Ile Val Lys Lys Gly Val Asp Val His Ala Leu Lys Glu
500 505 510
Gln Met Leu Glu Leu Leu Gln Gln Arg Gly Ala Gln Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Lys Ala Pro Glu Thr Leu Gln Lys Phe
530 535 540
Tyr Arg Glu Asn Asp Pro Thr Asn Ser Met Asn Pro Gly Ile Gly Lys
545 550 555 560
Thr Ser Lys Arg Lys Asn Trp Gln Glu Val Glu
565 570
<210> 6
<211> 1053
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaaactga agaacacctt aggcgttgtc atcggctcgc tggttgccgc ttcggcaatg 60
aacgcctttg cccagggcca gaactcggta gagatcgaag ccttcggcaa gcgctacttc 120
accgacagcg ttcgcaacat gaagaacgcg gacctgtacg gcggctcgat cggttacttc 180
ctgaccgacg acgtcgagct ggcgctgtcc tacggtgagt accatgacgt tcgtggcacc 240
tacgaaaccg gcaacaagaa ggtccacggc aacctgacct ccctggacgc catctaccac 300
ttcggtaccc cgggcgtagg tctgcgtccg tacgtgtcgg ctggtctggc tcaccagaac 360
atcaccaaca tcaacagcga cagccaaggc cgtcagcaga tgaccatggc caacatcggc 420
gctggtctga agtactactt caccgagaac ttcttcgcca aggccagcct cgacggccag 480
tacggtctgg agaagcgtga caacggtcac cagggcgagt ggatggctgg cctgggcgtc 540
ggcttcaact tcggtggttc gaaagccgct ccggctccgg aaccggttgc cgacgtttgc 600
tccgactccg acaacgacgg cgtttgcgac aacgtcgaca agtgcccgga taccccggcc 660
aacgtcaccg ttgacgccaa cggctgcccg gctgtcgccg aagtcgtacg cgtacagctg 720
gacgtgaagt tcgacttcga caagtccaag gtcaaagaga acagctacgc tgacatcaag 780
aacctggctg acttcatgaa gcagtacccg tccacttcca ccaccgttga aggtcacacc 840
gactccgtcg gcaccgacgc ttacaaccag aagctgtccg agcgtcgtgc caacgccgtt 900
cgtgacgtac tggtcaacga gtacggtgta gaaggtggtc gcgtgaacgc tgttggttac 960
ggcgagtccc gcccggttgc cgacaacgcc accgctgaag gccgcgctat caaccgtcgc 1020
gttgaagccg aagtagaagc tgaagccaag taa 1053
<210> 7
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Lys Leu Lys Asn Thr Leu Gly Val Val Ile Gly Ser Leu Val Ala
1 5 10 15
Ala Ser Ala Met Asn Ala Phe Ala Gln Gly Gln Asn Ser Val Glu Ile
20 25 30
Glu Ala Phe Gly Lys Arg Tyr Phe Thr Asp Ser Val Arg Asn Met Lys
35 40 45
Asn Ala Asp Leu Tyr Gly Gly Ser Ile Gly Tyr Phe Leu Thr Asp Asp
50 55 60
Val Glu Leu Ala Leu Ser Tyr Gly Glu Tyr His Asp Val Arg Gly Thr
65 70 75 80
Tyr Glu Thr Gly Asn Lys Lys Val His Gly Asn Leu Thr Ser Leu Asp
85 90 95
Ala Ile Tyr His Phe Gly Thr Pro Gly Val Gly Leu Arg Pro Tyr Val
100 105 110
Ser Ala Gly Leu Ala His Gln Asn Ile Thr Asn Ile Asn Ser Asp Ser
115 120 125
Gln Gly Arg Gln Gln Met Thr Met Ala Asn Ile Gly Ala Gly Leu Lys
130 135 140
Tyr Tyr Phe Thr Glu Asn Phe Phe Ala Lys Ala Ser Leu Asp Gly Gln
145 150 155 160
Tyr Gly Leu Glu Lys Arg Asp Asn Gly His Gln Gly Glu Trp Met Ala
165 170 175
Gly Leu Gly Val Gly Phe Asn Phe Gly Gly Ser Lys Ala Ala Pro Ala
180 185 190
Pro Glu Pro Val Ala Asp Val Cys Ser Asp Ser Asp Asn Asp Gly Val
195 200 205
Cys Asp Asn Val Asp Lys Cys Pro Asp Thr Pro Ala Asn Val Thr Val
210 215 220
Asp Ala Asn Gly Cys Pro Ala Val Ala Glu Val Val Arg Val Gln Leu
225 230 235 240
Asp Val Lys Phe Asp Phe Asp Lys Ser Lys Val Lys Glu Asn Ser Tyr
245 250 255
Ala Asp Ile Lys Asn Leu Ala Asp Phe Met Lys Gln Tyr Pro Ser Thr
260 265 270
Ser Thr Thr Val Glu Gly His Thr Asp Ser Val Gly Thr Asp Ala Tyr
275 280 285
Asn Gln Lys Leu Ser Glu Arg Arg Ala Asn Ala Val Arg Asp Val Leu
290 295 300
Val Asn Glu Tyr Gly Val Glu Gly Gly Arg Val Asn Ala Val Gly Tyr
305 310 315 320
Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala
325 330 335
Ile Asn Arg Arg Val Glu Ala Glu Val Glu Ala Glu Ala Lys
340 345 350
<210> 8
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caaggagaaa aaaatgatga acgcacaaaa atc 33
<210> 9
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gccgcaagct tttagagttt gtaactcatg ctc 33
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccgaattcgt gtatttttta ataaattatt ttac 34
<210> 11
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
catgaattct cagacattga ccttcacttc t 31
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agccaagtaa tttccactcc ttgtggtggc 30
<210> 13
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gcatgcgata tcgagctcac tggtcgcgca gaacatcttt c 41
<210> 14
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgaattccc gggagagctc cgttatgatg gcgtcgctag 40
<210> 15
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tcagtttcat aaattacgga tggcagagta 30
<210> 16
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tccgtaattt atgaaactga agaacacctt ag 32
<210> 17
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caaggagtgg aaattacttg gcttcagctt ctact 35

Claims (7)

1.一种潜艇生物感应器，其特征在于，所述潜艇生物感应器中包含电信号元件蛋白基因、感应元件、孔蛋白基因，并同时敲除了大肠杆菌乳酸脱氢酶基因；所述电信号元件蛋白基因为mtrCAB基因，其核苷酸序列如SEQ ID NO.1所示；所述感应元件为cad基因，其核苷酸序列如SEQ ID NO.3所示；所述孔蛋白基因为oprF基因，其核苷酸序列如SEQ ID NO.6所示；所述大肠杆菌乳酸脱氢酶基因为ldh基因，其核苷酸序列如SEQ ID NO.4所示；所述潜艇生物感应器具有感应金属镉离子Cd(II)的作用。
2.权利要求1所述的潜艇生物感应器的制备方法，其特征在于，所述制备方法包括以下步骤：
（1）扩增电信号元件蛋白基因mtrCAB基因，回收与双酶切质粒片段连接，得到重组质粒p-mtrCAB；扩增感应元件cad基因，回收与单酶切重组质粒p-mtrCAB连接，得到重组质粒p-Cad-mtrCAB；
（2）扩增大肠杆菌ldh基因的上下游片段和孔蛋白基因oprF基因，共连接的片段克隆至***质粒上，得到的重组质粒转化菌株，得到重组菌株；
（3）将步骤（1）的重组质粒p-Cad-mtrCAB转化步骤（2）的重组菌株，筛选阳性克隆，最终得到的工程菌株为潜艇微生物传感器。
3.权利要求1所述的潜艇生物感应器在用于金属Cd(Ⅱ)离子的实时检测中的应用。
4.权利要求1所述的潜艇生物感应器用于制备实时检测海洋中潜艇的探测装置中的应用。
5.根据权利要求4所述的应用，其特征在于，所述探测装置的搭建方法为：在阳极室和阴极室中间放置密封圈，将二者夹紧进行组装，阳极材料由导线分别固定在阳极室和阴极室中；阳极室内接种活化的潜艇微生物传感器，并施加电压促进阳极材料上生物膜的形成；阴极室内装载电解液。
6.根据权利要求5所述的应用，其特征在于，所述探测装置通过实时检测海洋中潜艇释放的Cd(Ⅱ)离子，并转化为变化的电信号输出，以此达到实时检测海洋中潜艇中的目的。
7.根据权利要求6所述的应用，其特征在于，所述探测装置能够感应到的Cd(Ⅱ)离子的浓度不低于0.1 μmol/L。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110684789A (zh) * 2019-10-24 2020-01-14 南京林业大学 融合基因、重组载体及其制备方法、镉离子全细胞生物传感器及其制备方法与应用
WO2020021740A1 (ja) * 2018-07-26 2020-01-30 国立研究開発法人物質・材料研究機構 細胞外電子移動作用に関与する酵素及びその利用
CN112301049A (zh) * 2020-11-02 2021-02-02 中国科学技术大学 高产血红素的重组质粒和基因工程菌株及其构建方法、高产血红素的方法
CN112608922A (zh) * 2020-12-30 2021-04-06 中国科学技术大学 调控异化金属还原菌电子流的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020021740A1 (ja) * 2018-07-26 2020-01-30 国立研究開発法人物質・材料研究機構 細胞外電子移動作用に関与する酵素及びその利用
CN110684789A (zh) * 2019-10-24 2020-01-14 南京林业大学 融合基因、重组载体及其制备方法、镉离子全细胞生物传感器及其制备方法与应用
CN112301049A (zh) * 2020-11-02 2021-02-02 中国科学技术大学 高产血红素的重组质粒和基因工程菌株及其构建方法、高产血红素的方法
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