CN106868032A - 一种可异位展示两种多肽的噬菌体双展示载体 - Google Patents

一种可异位展示两种多肽的噬菌体双展示载体 Download PDF

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CN106868032A
CN106868032A CN201710162188.3A CN201710162188A CN106868032A CN 106868032 A CN106868032 A CN 106868032A CN 201710162188 A CN201710162188 A CN 201710162188A CN 106868032 A CN106868032 A CN 106868032A
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毛传斌
李龑
杨明英
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Zhejiang University ZJU
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Abstract

本发明公开了一种可异位展示两种多肽的噬菌体双展示载体，本发明通过基因工程技术成功构建了丝状噬菌体双展示载体fd388。该展示载体含有fd噬菌体的全基因组，且其gene3和gene8的起始密码子一端含有酶切位点，能够直接插入外源多肽基因以完成噬菌体展示。同时fd388展示载体含有四环素抗性基因Tn10，从而达到阳性克隆筛选的目的。fd388可以展示两种不同功能的多肽以应用于多种生物学，如肿瘤靶向治疗、重大疾病分子标志物超灵敏检测、干细胞定向分化和纳米颗粒合成和自组装等的应用。

Description

一种可异位展示两种多肽的噬菌体双展示载体
技术领域
本发明属于分子生物学领域，尤其涉及一种可异位展示两种多肽的噬菌体双展示载体。
背景技术
1985年密苏里大学George Smith博士开创了著名的噬菌体展示技术，经过近三十年的研究该技术也被不断地完善。噬菌体展示技术是指通过基因工程技术将外源多肽或蛋白的DNA序列插入到噬菌体外壳蛋白结构基因的适当位置，重组的噬菌体基因组进入宿主菌以后利用宿主菌的表达体系，使外源多肽或蛋白和噬菌体外壳蛋白以融合蛋白的方式表达，并在噬菌体自组装过程将外源多肽或蛋白展示到噬菌体外壳上。被展示在噬菌体外壳上的的外源多肽和蛋白因为空间位阻较小，所以能保持相对独立的空间结构和生物活性。基于以上优势噬菌体基因工程化被广泛应用于靶向筛选、确定抗原表位，研制多肽和蛋白药物，探索蛋白受体和配体作用，蛋白质-DNA相互作用和递呈抗原制备抗体等领域。
丝状噬菌体是一种由外壳蛋白围绕基因组形成的900nm、直径为6.5nm的生物纳米纤维。它像其他纳米颗粒一样具有较大的比表面积，所以能携带大量的分子药物；同时，它分子量较大具有很强的免疫原性，比较容易机体产生免疫反应，很容易被机体清除。因为噬菌体是特异性针对细菌的病毒颗粒，所以对人体而言是一种安全的纳米材料。并且噬菌体还可以通过噬菌体展示技术和生物淘选获得与目标靶向物(如：纳米颗粒、蛋白质、细胞和器官)特异性识别的结合肽。故丝状噬菌体是一种具有巨大潜能的靶向阻断药物载体。但目前很多研究只利用了它的靶向性，而没有在一个噬菌体上同时展示上靶向性功能肽和治疗性功能肽。
发明内容
本发明的目的在于针对现有技术的不足，提供一种可异位展示两种多肽的噬菌体双展示载体。
本发明的目的是通过以下技术方案来实现的：一种可异位展示两种多肽的噬菌体双展示载体，它通过以下方法制备得到：
(1)基于fd丝状噬菌体fUSE55基因组1022位置的BsrGI单酶切位点和2244的BamHI单酶切位点，利用BsrGI/BamHI双酶切获得含有重组gene3的长为1223bp的基因片段，所述fUSE55的序列如SEQ ID NO.1所示；
(2)基于fd丝状噬菌体f88-4基因组1022位置的BsrG I单酶切位点和2221的BamHI单酶切位点。利用BsrGI/BamHI双酶切敲除掉f88-4的野生gene3，获得剩下长为8034bp的基因片段，所述f88-4的序列如SEQ ID NO.2所示；
(3)构建双展示载体fd388：将步骤1得到的长为1223bp的基因片段和步骤2得到的长为8034bp的基因片段按1:3的比例，通过T4DNA连接酶16℃过夜连接；
(4)将连接产物转化进入JM109感受态细胞，并涂抹在四环素抗性平板上37℃过夜获取阳性单克隆，从而得到长为9257bp的可异位展示两种多肽的噬菌体双展示载体fd388，fd388的序列如SEQ ID NO.3所示。
本发明的有益效果是，本发明利用基因工程技术将fd丝状噬菌体单展示载体fUSE55基因组的重组gene3取代另一个fd丝状噬菌体单展示载体f88-4的野生gene3，得到了双展示噬菌体载体fd388。该展示载体含有fd噬菌体的全基因组，且其gene3和gene8的起始密码子端含有单酶切位点(SfiI，PstI和HindIII)，能够直接插入外源多肽基因以完成噬菌体展示。且fd388载体因为携带了f88-4载体的基因Tn10，所以继承了f88-4的四环素抗性，能为后续阳性克隆筛选带来很大的方便。经过不同长度多肽的成功展示验证，可以展望fd388双展示载体可以展示制备出多种双功能噬菌体，并应用于肿瘤靶向治疗、重大疾病分子标志物超灵敏检测、干细胞定向分化和纳米颗粒合成和自组装等领域。
附图说明
图1为fd388双展示载体构建琼脂糖凝胶电泳图；
图2为实例1中利用fd388双展示载体构建的能同时特异性识别乳腺癌和angiogenin的双功能性fd388-AR-WR噬菌体的表征图；其中，(a)为TEM图，(b)AFM图，(c)为SDS-PAGE电泳银染图。
具体实施方式
下面通过实施例对本发明作进一步的详细说明，以下实施例是对本发明的解释而本发明并不局限于以下实施例。
实施例1：
1、获得重组gene3：按照New England Biolab公司的BsrGI/BamHI双酶切体系加入fUSE55载体(序列如SEQ ID NO.1所示)37℃双酶切3h；利用Qiagen公司的质粒回收试剂盒回收小片段以获得包含有重组gene3的长为1222bp的DNA片段1。其中，所述fUSE55是通过overlap PCR实验将含有2个SfiI酶切位点(SEQ ID NO.4：GCCGACGTGGCCCGGCCTCTGGGGCC)的DNA片段插入野生fd噬菌体的gene3的5’端获得。
2、获得噬菌体基因组其他部分：按照New England Biolab公司的BsrGI/BamHI双酶切体系加入f88-4载体(序列如SEQ ID NO.2所示)37℃双酶切3h；利用Qiagen公司的质粒回收试剂盒回收大片段以获得包含有噬菌体基因组中除去DNA片段1剩下的长为8034bp的DNA片段2。其中f88-4载体是通过overlap PCR反应将PstI和HindIII酶切位点插入野生gene8的5’端获得工程化gene8，并将工程化的gene8插入野生的fd噬菌体获得。
3、构建双展示载体fd388：将DNA片段1和DNA片段2按1:3的比例，通过T4DNA连接酶16℃过夜连接。
4、将连接产物转化进入JM109感受态细胞，并涂抹在四环素抗性平板上37℃过夜获取阳性单克隆，得到可异位展示两种多肽的噬菌体双展示载体。然后挑取单克隆进行DNA测序，得到可异位展示两种多肽的噬菌体双展示载体的DNA序列(序列如SEQ ID NO.3所示)。图1为fd388双展示载体构建电泳图，第一泳道为marker；第二泳道为fUSE55经过BsrGI/BamHI双酶切后回收的1222bp小片段；第三泳道为f88-4经过BsrGI/BamHI双酶切后回收的8034bp大片段；第四泳道为连接成功的fd388双展示载体质粒。
p3展示：将测序成功的fd388载体克隆得到大量质粒，并用New England Biolab公司的SfiI限制性内切酶37℃过夜酶切；利用Qiagen公司的质粒回收试剂盒回收大片段，将其与公司合成的带有SfiI粘性末端的编码乳腺癌靶向多肽的DNA序列通过T4DNA连接酶进行连接；连接产物转化入JM109感受态细胞，挑选克隆进行DNA测序。
p8展示：将p3成功展示的质粒克隆，并用New England Biolab公司的PstI和HindIII限制性内切酶37℃过夜双酶切；利用Qiagen公司的质粒回收试剂盒回收大片段，将其与公司合成的带有PstI和HindIII粘性末端的编码Angiogenin识别肽的DNA序列通过T4DNA连接酶进行连接；连接产物转化入JM109感受态细胞，挑选克隆进行DNA测序。
噬菌体表征：通过TEM、AFM和琼脂糖凝胶电泳银染，结果如图2所示。
实施例2：
fd388丝状噬菌体双展示载体构建和实施例1一致。验证fd388是否能成功双展示多肽时，我们在p3展示了黑色素瘤靶向多肽；在P8展示了肿瘤治疗免疫靶点蛋白PD-1L特异识别肽。
SEQUENCE LISTING
<110> 浙江大学
<120> 一种可异位展示两种多肽的噬菌体双展示载体
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 9206
<212> DNA
<213> 人工设计
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aattccttcc ataattcaga aatataaccc aaatagtcag gattatattg atgaattgcc 4560
atcatctgat attcaggaat atgatgataa ttccgctcct tctggtggtt tctttgttcc 4620
gcaaaatgat aatgttactc aaacatttaa aattaataac gttcgcgcaa aggatttaat 4680
aagggttgta gaattgtttg ttaaatctaa tacatctaaa tcctcaaatg tattatctgt 4740
tgatggttct aacttattag tagttagcgc ccctaaagat attttagata accttccgca 4800
atttctttct actgttgatt tgccaactga ccagatattg attgaaggat taattttcga 4860
ggttcagcaa ggtgatgctt tagatttttc ctttgctgct ggctctcagc gcggcactgt 4920
tgctggtggt gttaatactg accgtctaac ctctgtttta tcttctgcgg gtggttcgtt 4980
cggtattttt aacggcgatg ttttagggct atcagttcgc gcattaaaga ctaatagcca 5040
ttcaaaaata ttgtctgtgc ctcgtattct tacgctttca ggtcagaagg gttctatttc 5100
tgttggccag aatgtccctt ttattactgg tcgtgtaact ggtgaatctg ccaatgtaaa 5160
taatccattt cagacggttg agcgtcaaaa tgttggtatt tctatgagtg tttttcccgt 5220
tgcaatggct ggcggtaata ttgttttaga tataaccagt aaggccgata gtttgagttc 5280
ttctactcag gcaagtgatg ttattactaa tcaaagaagt attgcgacaa cggttaattt 5340
gcgtgatggt cagactcttt tgctcggtgg cctcactgat tacaaaaaca cttctcaaga 5400
ttctggtgtg ccgttcctgt ctaaaatccc tttaatcggc ctcctgttta gctcccgttc 5460
tgattctaac gaggaaagca cgttgtacgt gctcgtcaaa gcaaccatag tacgcgccct 5520
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 5580
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttctccg 5640
gctttccccg tcaagctcta aatcggggga tctcgggaaa agcgttggtg accaaaggtg 5700
ccttttatca tcactttaaa aataaaaaac aattactcag tgcctgttat aagcagcaat 5760
taattatgat tgatgcctac atcacaacaa aaactgattt aacaaatggt tggtctgcct 5820
tagaaagtat atttgaacat tatcttgatt atattattga taataataaa aaccttatcc 5880
ctatccaaga agtgatgcct atcattggtt ggaatgaact tgaaaaaatt agccttgaat 5940
acattactgg taaggtaaac gccattgtca gcaaattgat ccaagagaac caacttaaag 6000
cttatgatga tgatgtgctt aaaaacttac tcaatggctg gtttatgcat atcgcaatac 6060
atgcgaaaaa cctaaaagag cttgccgata aaaaaggcca atttattgct atttaccgcg 6120
gctttttatt gagcttgaaa gataaataaa atagataggt tttatttgaa gctaaatctt 6180
ctttatcgta aaaaatgccc tcttgggtta tcaagagggt cattatattt cgcggaataa 6240
catcatttgg tgacgaaata actaagcact tgtctcctgt ttactcccct gagcttgagg 6300
ggttaacatg aaggtcatcg atagcaggat aataatacag taaaacgcta aaccaataat 6360
ccaaatccag ccatcccaaa ttggtagtga atgattataa ataacagcaa acagtaatgg 6420
gccaataaca ccggttgcat tggtaaggct caccaataat ccctgtaaag caccttgctg 6480
atgactcttt gtttggatag acatcactcc ctgtaatgca ggtaaagcga tcccaccacc 6540
agccaataaa attaaaacag ggaaaactaa ccaaccttca gatataaacg ctaaaaaggc 6600
aaatgcacta ctatctgcaa taaatccgag cagtactgcc gttttttcgc cccatttagt 6660
ggctattctt cctgccacaa aggcttggaa tactgagtgt aaaagaccaa gacccgctaa 6720
tgaaaagcca accatcatgc tattccatcc aaaacgattt tcggtaaata gcacccacac 6780
cgttgcggga atttggccta tcaattgcgc tgaaaaataa ataatcaaca aaatgggcat 6840
cgttttaaat aaagtgatgt ataccgaatt cgattgcgtc tcaaccccta cttcggtatc 6900
tgtattatca cgtgtatttt tggtttcacg gaaccaaaac ataaccacaa ggaaagtgac 6960
aatatttagc aacgcagcga taaaaaaggg actatgcggt gaaatctctc ctgcaaaacc 7020
accaataata ggccccgcta ttaaaccaag cccaaaactt gcccctaacc aaccgaacca 7080
cttcacgcgt tgagaagctg aggtggtatc ggcaatgacc gatgccgcga cagccccagt 7140
agctcctgtg atccctgaaa gcaaacggcc taaatacagc atccaaagcg cacttgaaaa 7200
agccagcaat aagtaatcca gcgatgcgcc tattaatgac aacaacagca ctgggcgccg 7260
accaaatcgg tcagacattt ttccaagcca aggagcaaag ataacctgca ttaacgcata 7320
aagtgcaagc aatacgccaa agtggttagc gatatcttcc gaagcaataa attcacgtaa 7380
taacgttggc aagactggca tgataaggcc aatccccatg gcatcgagta acgtaattac 7440
caatgcgatc tttgtcgaac tattcatttc acttttctct atcactgata gggagtggta 7500
aaataactct atcaatgata gagtgtcaac aaaaattagg aattaatgat gtctagatta 7560
gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt 7620
ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat 7680
gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat 7740
actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa 7800
agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg 7860
cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt 7920
ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc 7980
gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat 8040
agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca 8100
gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa 8160
agtgggtctt aaaagcagca taaccttttt ccgtgatggt aacttcacgg taaccaagat 8220
gtcgagttaa ccacccttta gattcataaa gcgaaaataa tgcggctcca acgtacccac 8280
ctaaatggaa acggcgttca ctccaatcta aacacgcaca acagatttta cgtgaatgtt 8340
tggaaggaac gtcaattccc atttcatgaa aatattgaat accacttaat gtgatcattg 8400
aaccattttc agtgatccat tgctgttgac aaagggaatc atagatccct ttagggttcc 8460
gatttagtgc tttacggcac ctcgacctcc aaaaacttga tttgggtgat ggttcacgta 8520
gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 8580
atagtggact cttgttccaa actggaacaa cactcacaac taactcggcc tattcttttg 8640
atttataagg atttttgtca ttttctgctt actggttaaa aaataagctg atttaacaaa 8700
tatttaacgc gaaatttaac aaaacattaa cgtttacaat ttaaatattt gcttatacaa 8760
tcatcctgtt tttggggctt ttctgattat caatcggggt acatatgatt gacatgctag 8820
ttttacgatt accgttcatc gattctcttg tttgctccag actttcaggt aatgacctga 8880
tagcctttgt agacctctca aaaatagcta ccctctccgg catgaattta tcagctagaa 8940
cggttgaata tcatattgac ggtgatttga ctgtctccgg cctttctcac ccgtttgaat 9000
ctttgcctac tcattactcc ggcattgcat ttaaaatata tgagggttct aaaaattttt 9060
atccctgcgt tgaaattaag gcttcaccag caaaagtatt acagggtcat aatgtttttg 9120
gtacaaccga tttagcttta tgctctgagg ctttattgct taattttgct aactctctgc 9180
cttgcttgta cgatttattg gatgtt 9206
<210> 2
<211> 9234
<212> DNA
<213> 人工设计
<400> 2
aacgctacta ccattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120
cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180
gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300
ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttgaaacgcg atatttgaag 360
tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420
cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540
aaacatttta caattacccc ctctggcaaa acttcctttg caaaagcctc tcgctatttt 600
ggtttctatc gtcgtctggt taatgagggt tatgatagtg ttgctcttac catgcctcgt 660
aattcctttt ggcgttatgt atctgcatta gttgagtgtg gtattcctaa atctcaattg 720
atgaatcttt ccacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780
tcctcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840
aaatgattaa agttgaaatt aaaccgtctc aagcgcaatt tactacccgt tctggtgttt 900
ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960
aatatccggt gcttgtcaag attactctcg acgaaggtca gccagcgtat gcgcctggtc 1020
tgtacaccgt gcatctgtcc tcgttcaaag ttggtcagtt cggttctctt atgattgacc 1080
gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140
caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200
caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta 1260
gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320
caaagcctcc gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380
cgatcccgca aaagcggcct ttgactccct gcaagcctca gcgaccgaat atatcggtta 1440
tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500
attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560
tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620
tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aacctcatac agaaaattca 1680
tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt 1740
ctgtggaatg ctacaggcgt tgtggtttgt actggtgacg aaactcagtg ttacggtaca 1800
tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1860
tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1920
attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1980
aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2040
cagaataata ggttccgaaa taggcagggt gcattaactg tttatacggg cactgttact 2100
caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2160
tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2220
gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2280
gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggcgg ctctgagggt 2340
ggcggttctg agggtggcgg ctctgagggt ggcggttccg gtggcggctc cggttccggt 2400
gattttgatt atgaaaaaat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2460
gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2520
gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2580
ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2640
ttaatgaata atttccgtca atatttacct tctttgcctc agtcggttga atgtcgccct 2700
tatgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2760
ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttcgacg 2820
tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2880
tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttactttcc 2940
ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3000
ggcttaactc aattcttgtg ggttatctct ctgatattag cgcacaatta ccctctgatt 3060
ttgttcaggg cgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3120
tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3180
attgggataa ataaatatgg ctgtttattt tgtaactggc aaattaggct ctggaaagac 3240
gctcgttagc gttggtaaga ttcaggataa aattgtagct gggtgcaaaa tagcaactaa 3300
tcttgattta aggcttcaaa acctcccgca agtcgggagg ttcgctaaaa cgcctcgcgt 3360
tcttagaata ccggataagc cttctatttc tgatttgctt gctattggtc gtggtaatga 3420
ttcctacgac gaaaataaaa acggtttgct tgttcttgat gaatgcggta cttggtttaa 3480
tacccgttca tggaatgaca aggaaagaca gccgattatt gattggtttc ttcatgctcg 3540
taaattggga tgggatatta tttttcttgt tcaggattta tctattgttg ataaacaggc 3600
gcgttctgca ttagctgaac acgttgttta ttgtcgccgt ctggacagaa ttactttacc 3660
ctttgtcggc actttatatt ctcttgttac tggctcaaaa atgcctctgc ctaaattaca 3720
tgttggtgtt gttaaatatg gtgattctca attaagccct actgttgagc gttggcttta 3780
tactggtaag aatttatata acgcatatga cactaaacag gctttttcca gtaattatga 3840
ttcaggtgtt tattcatatt taacccctta tttatcacac ggtcggtatt tcaaaccatt 3900
aaatttaggt cagaagatga aattaactaa aatatatttg aaaaagtttt ctcgcgttct 3960
ttgtcttgcg ataggatttg catcagcatt tacatatagt tatataaccc aacctaagcc 4020
ggaggttaaa aaggtagtct ctcagaccta tgattttgat aaattcacta ttgactcttc 4080
tcagcgtctt aatctaagct atcgctatgt tttcaaggat tctaagggaa aattaattaa 4140
tagcgacgat ttacagaagc aaggttattc catcacatat attgatttat gtactgtttc 4200
aattaaaaaa ggtaattcaa atgaaattgt taaatgtaat taattttgtt ttcttgatgt 4260
ttgtttcatc atcttctttt gctcaagtaa ttgaaatgaa taattcgcct ctgcgcgatt 4320
tcgtgacttg gtattcaaag caaacaggtg aatctgttat tgtctcacct gatgttaaag 4380
gtacagtgac tgtatattcc tctgacgtta agcctgaaaa tttacgcaat ttctttatct 4440
ctgttttacg tgctaataat tttgatatgg ttggctcaat tccttccata attcagaaat 4500
ataacccaaa tagtcaggat tatattgatg aattgccatc atctgatatt caggaatatg 4560
atgataattc cgctccttct ggtggtttct ttgttccgca aaatgataat gttactcaaa 4620
catttaaaat taataacgtt cgcgcaaagg atttaataag ggttgtagaa ttgtttgtta 4680
aatctaatac atctaaatcc tcaaatgtat tatctgttga tggttctaac ttattagtag 4740
ttagcgcccc taaagatatt ttagataacc ttccgcaatt tctttctact gttgatttgc 4800
caactgacca gatattgatt gaaggattaa ttttcgaggt tcagcaaggt gatgctttag 4860
atttttcctt tgctgctggc tctcagcgcg gcactgttgc tggtggtgtt aatactgacc 4920
gtctaacctc tgttttatct tctgcgggtg gttcgttcgg tatttttaac ggcgatgttt 4980
tagggctatc agttcgcgca ttaaagacta atagccattc aaaaatattg tctgtgcctc 5040
gtattcttac gctttcaggt cagaagggtt ctatttctgt tggccagaat gtccctttta 5100
ttactggtcg tgtaactggt gaatctgcca atgtaaataa tccatttcag acggttgagc 5160
gtcaaaatgt tggtatttct atgagtgttt ttcccgttgc aatggctggc ggtaatattg 5220
ttttagatat aaccagtaag gccgatagtt tgagttcttc tactcaggca agtgatgtta 5280
ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggtcag actcttttgc 5340
tcggtggcct cactgattac aaaaacactt ctcaagattc tggtgtgccg ttcctgtcta 5400
aaatcccttt aatcggcctc ctgtttagct cccgttctga ttctaacgag gaaagcacgt 5460
tgtacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 5520
ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 5580
ttcgctttct tcccttcctt tctcgccacg ttctccggct ttccccgtca agctctaaat 5640
cgggggagct cgagcttact ccccatcccc ctgttgacaa ttaatcatcg gctcgtataa 5700
tgtgtggaat tgtgagcgga taacaatttc ttaatggaaa cttcctcatg aaaaagtctt 5760
tagttcttaa agcatctgtt gctgttgcga ctcttgttcc tatgctaagc tttgccaacg 5820
tccctgcaga aggtgatgac ccggctaaag ctgcttttga ctctcttcag gcttctgcta 5880
ctgaatacat cggctacgct tgggctatgg tggttgttat cgttggtgct actattggca 5940
tcaaactttt caaaaaattc acttctaaag cgtcttaatg aactcagata cccagcccgc 6000
ctaatgagcg ggcttttttt taagctagct tatgatgatg atgtgcttaa aaacttactc 6060
aatggctggt ttatgcatat cgcaatacat gcgaaaaacc taaaagagct tgccgataaa 6120
aaaggccaat ttattgctat ttaccgcggc tttttattga gcttgaaaga taaataaaat 6180
agataggttt tatttgaagc taaatcttct ttatcgtaaa aaatgccctc ttgggttatc 6240
aagagggtca ttatatttcg cggaataaca tcatttggtg acgaaataac taagcacttg 6300
tctcctgttt actcccctga gcttgagggg ttaacatgaa ggtcatcgat agcaggataa 6360
taatacagta aaacgctaaa ccaataatcc aaatccagcc atcccaaatt ggtagtgaat 6420
gattataaat aacagcaaac agtaatgggc caataacacc ggttgcattg gtaaggctca 6480
ccaataatcc ctgtaaagca ccttgctgat gactctttgt ttggatagac atcactccct 6540
gtaatgcagg taaagcgatc ccaccaccag ccaataaaat taaaacaggg aaaactaacc 6600
aaccttcaga tataaacgct aaaaaggcaa atgcactact atctgcaata aatccgagca 6660
gtactgccgt tttttcgccc catttagtgg ctattcttcc tgccacaaag gcttggaata 6720
ctgagtgtaa aagaccaaga cccgctaatg aaaagccaac catcatgcta ttccatccaa 6780
aacgattttc ggtaaatagc acccacaccg ttgcgggaat ttggcctatc aattgcgctg 6840
aaaaataaat aatcaacaaa atgggcatcg ttttaaataa agtgatgtat accgaattcg 6900
attgcgtctc aacccctact tcggtatctg tattatcacg tgtatttttg gtttcacgga 6960
accaaaacat aaccacaagg aaagtgacaa tatttagcaa cgcagcgata aaaaagggac 7020
tatgcggtga aatctctcct gcaaaaccac caataatagg ccccgctatt aaaccaagcc 7080
caaaacttgc ccctaaccaa ccgaaccact tcacgcgttg agaagctgag gtggtatcgg 7140
caatgaccga tgccgcgaca gccccagtag ctcctgtgat ccctgaaagc aaacggccta 7200
aatacagcat ccaaagcgca cttgaaaaag ccagcaataa gtaatccagc gatgcgccta 7260
ttaatgacaa caacagcact gggcgccgac caaatcggtc agacattttt ccaagccaag 7320
gagcaaagat aacctgcatt aacgcataaa gtgcaagcaa tacgccaaag tggttagcga 7380
tatcttccga agcaataaat tcacgtaata acgttggcaa gactggcatg ataaggccaa 7440
tccccatggc atcgagtaac gtaattacca atgcgatctt tgtcgaacta ttcatttcac 7500
ttttctctat cactgatagg gagtggtaaa ataactctat caatgataga gtgtcaacaa 7560
aaattaggaa ttaatgatgt ctagattaga taaaagtaaa gtgattaaca gcgcattaga 7620
gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc agaagctagg 7680
tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc tcgacgcctt 7740
agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag gggaaagctg 7800
gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa gtcatcgcga 7860
tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa 7920
tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat atgcactcag 7980
cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc aagtcgctaa 8040
agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac aagctatcga 8100
attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat tgatcatatg 8160
cggattagaa aaacaactta aatgtgaaag tgggtcttaa aagcagcata acctttttcc 8220
gtgatggtaa cttcacggta accaagatgt cgagttaacc accctttaga ttcataaagc 8280
gaaaataatg cggctccaac gtacccacct aaatggaaac ggcgttcact ccaatctaaa 8340
cacgcacaac agattttacg tgaatgtttg gaaggaacgt caattcccat ttcatgaaaa 8400
tattgaatac cacttaatgt gatcattgaa ccattttcag tgatccattg ctgttgacaa 8460
agggaatcat agatcccttt agggttccga tttagtgctt tacggcacct cgacctccaa 8520
aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 8580
cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 8640
ctcacaacta actcggccta ttcttttgat ttataaggat ttttgtcatt ttctgcttac 8700
tggttaaaaa ataagctgat ttaacaaata tttaacgcga aatttaacaa aacattaacg 8760
tttacaattt aaatatttgc ttatacaatc atcctgtttt tggggctttt ctgattatca 8820
atcggggtac atatgattga catgctagtt ttacgattac cgttcatcga ttctcttgtt 8880
tgctccagac tttcaggtaa tgacctgata gcctttgtag acctctcaaa aatagctacc 8940
ctctccggca tgaatttatc agctagaacg gttgaatatc atattgacgg tgatttgact 9000
gtctccggcc tttctcaccc gtttgaatct ttgcctactc attactccgg cattgcattt 9060
aaaatatatg agggttctaa aaatttttat ccctgcgttg aaattaaggc ttcaccagca 9120
aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct 9180
ttattgctta attttgctaa ctctctgcct tgcttgtacg atttattgga tgtt 9234
<210> 3
<211> 9257
<212> DNA
<213> 人工设计
<400> 3
aacgctacta ccattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120
cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180
gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300
ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttgaaacgcg atatttgaag 360
tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420
cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540
aaacatttta caattacccc ctctggcaaa acttcctttg caaaagcctc tcgctatttt 600
ggtttctatc gtcgtctggt taatgagggt tatgatagtg ttgctcttac catgcctcgt 660
aattcctttt ggcgttatgt atctgcatta gttgagtgtg gtattcctaa atctcaattg 720
atgaatcttt ccacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780
tcctcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840
aaatgattaa agttgaaatt aaaccgtctc aagcgcaatt tactacccgt tctggtgttt 900
ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960
aatatccggt gcttgtcaag attactctcg acgaaggtca gccagcgtat gcgcctggtc 1020
tgtacaccgt gcatctgtcc tcgttcaaag ttggtcagtt cggttctctt atgattgacc 1080
gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140
caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200
caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta 1260
gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320
caaagcctcc gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380
cgatcccgca aaagcggcct ttgactccct gcaagcctca gcgaccgaat atatcggtta 1440
tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500
attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560
tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620
tattctcact cggccgacgt ggcccggcct ctggggccga aactgttgaa agttgtttag 1680
caaaacctca tacagaaaat tcatttacta acgtctggaa agacgacaaa actttagatc 1740
gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtggtt tgtactggtg 1800
acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct gaaaatgagg 1860
gtggtggctc tgagggtggc ggttctgagg gtggcggttc tgagggtggc ggtactaaac 1920
ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct ctcgacggca 1980
cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt gaggagtctc 2040
agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag ggtgcattaa 2100
ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat taccagtaca 2160
ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc agagactgcg 2220
ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc caatcgtctg 2280
acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct ggtggcggct 2340
ctgagggtgg cggctctgag ggtggcggtt ctgagggtgg cggctctgag ggtggcggtt 2400
ccggtggcgg ctccggttcc ggtgattttg attatgaaaa aatggcaaac gctaataagg 2460
gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg 2520
attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac gtttccggcc 2580
ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa atggctcaag 2640
tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta ccttctttgc 2700
ctcagtcggt tgaatgtcgc ccttatgtct ttggcgctgg taaaccatat gaattttcta 2760
ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta tatgttgcca 2820
cctttatgta tgtattttcg acgtttgcta acatactgcg taataaggag tcttaatcat 2880
gccagttctt ttgggtattc cgttattatt gcgtttcctc ggtttccttc tggtaacttt 2940
gttcggctat ctgcttactt tccttaaaaa gggcttcggt aagatagcta ttgctatttc 3000
attgtttctt gctcttatta ttgggcttaa ctcaattctt gtgggttatc tctctgatat 3060
tagcgcacaa ttaccctctg attttgttca gggcgttcag ttaattctcc cgtctaatgc 3120
gcttccctgt ttttatgtta ttctctctgt aaaggctgct attttcattt ttgacgttaa 3180
acaaaaaatc gtttcttatt tggattggga taaataaata tggctgttta ttttgtaact 3240
ggcaaattag gctctggaaa gacgctcgtt agcgttggta agattcagga taaaattgta 3300
gctgggtgca aaatagcaac taatcttgat ttaaggcttc aaaacctccc gcaagtcggg 3360
aggttcgcta aaacgcctcg cgttcttaga ataccggata agccttctat ttctgatttg 3420
cttgctattg gtcgtggtaa tgattcctac gacgaaaata aaaacggttt gcttgttctt 3480
gatgaatgcg gtacttggtt taatacccgt tcatggaatg acaaggaaag acagccgatt 3540
attgattggt ttcttcatgc tcgtaaattg ggatgggata ttatttttct tgttcaggat 3600
ttatctattg ttgataaaca ggcgcgttct gcattagctg aacacgttgt ttattgtcgc 3660
cgtctggaca gaattacttt accctttgtc ggcactttat attctcttgt tactggctca 3720
aaaatgcctc tgcctaaatt acatgttggt gttgttaaat atggtgattc tcaattaagc 3780
cctactgttg agcgttggct ttatactggt aagaatttat ataacgcata tgacactaaa 3840
caggcttttt ccagtaatta tgattcaggt gtttattcat atttaacccc ttatttatca 3900
cacggtcggt atttcaaacc attaaattta ggtcagaaga tgaaattaac taaaatatat 3960
ttgaaaaagt tttctcgcgt tctttgtctt gcgataggat ttgcatcagc atttacatat 4020
agttatataa cccaacctaa gccggaggtt aaaaaggtag tctctcagac ctatgatttt 4080
gataaattca ctattgactc ttctcagcgt cttaatctaa gctatcgcta tgttttcaag 4140
gattctaagg gaaaattaat taatagcgac gatttacaga agcaaggtta ttccatcaca 4200
tatattgatt tatgtactgt ttcaattaaa aaaggtaatt caaatgaaat tgttaaatgt 4260
aattaatttt gttttcttga tgtttgtttc atcatcttct tttgctcaag taattgaaat 4320
gaataattcg cctctgcgcg atttcgtgac ttggtattca aagcaaacag gtgaatctgt 4380
tattgtctca cctgatgtta aaggtacagt gactgtatat tcctctgacg ttaagcctga 4440
aaatttacgc aatttcttta tctctgtttt acgtgctaat aattttgata tggttggctc 4500
aattccttcc ataattcaga aatataaccc aaatagtcag gattatattg atgaattgcc 4560
atcatctgat attcaggaat atgatgataa ttccgctcct tctggtggtt tctttgttcc 4620
gcaaaatgat aatgttactc aaacatttaa aattaataac gttcgcgcaa aggatttaat 4680
aagggttgta gaattgtttg ttaaatctaa tacatctaaa tcctcaaatg tattatctgt 4740
tgatggttct aacttattag tagttagcgc ccctaaagat attttagata accttccgca 4800
atttctttct actgttgatt tgccaactga ccagatattg attgaaggat taattttcga 4860
ggttcagcaa ggtgatgctt tagatttttc ctttgctgct ggctctcagc gcggcactgt 4920
tgctggtggt gttaatactg accgtctaac ctctgtttta tcttctgcgg gtggttcgtt 4980
cggtattttt aacggcgatg ttttagggct atcagttcgc gcattaaaga ctaatagcca 5040
ttcaaaaata ttgtctgtgc ctcgtattct tacgctttca ggtcagaagg gttctatttc 5100
tgttggccag aatgtccctt ttattactgg tcgtgtaact ggtgaatctg ccaatgtaaa 5160
taatccattt cagacggttg agcgtcaaaa tgttggtatt tctatgagtg tttttcccgt 5220
tgcaatggct ggcggtaata ttgttttaga tataaccagt aaggccgata gtttgagttc 5280
ttctactcag gcaagtgatg ttattactaa tcaaagaagt attgcgacaa cggttaattt 5340
gcgtgatggt cagactcttt tgctcggtgg cctcactgat tacaaaaaca cttctcaaga 5400
ttctggtgtg ccgttcctgt ctaaaatccc tttaatcggc ctcctgttta gctcccgttc 5460
tgattctaac gaggaaagca cgttgtacgt gctcgtcaaa gcaaccatag tacgcgccct 5520
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 5580
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttctccg 5640
gctttccccg tcaagctcta aatcggggga gctcgagctt actccccatc cccctgttga 5700
caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcttaatgg 5760
aaacttcctc atgaaaaagt ctttagttct taaagcatct gttgctgttg cgactcttgt 5820
tcctatgcta agctttgcca acgtccctgc agaaggtgat gacccggcta aagctgcttt 5880
tgactctctt caggcttctg ctactgaata catcggctac gcttgggcta tggtggttgt 5940
tatcgttggt gctactattg gcatcaaact tttcaaaaaa ttcacttcta aagcgtctta 6000
atgaactcag atacccagcc cgcctaatga gcgggctttt ttttaagcta gcttatgatg 6060
atgatgtgct taaaaactta ctcaatggct ggtttatgca tatcgcaata catgcgaaaa 6120
acctaaaaga gcttgccgat aaaaaaggcc aatttattgc tatttaccgc ggctttttat 6180
tgagcttgaa agataaataa aatagatagg ttttatttga agctaaatct tctttatcgt 6240
aaaaaatgcc ctcttgggtt atcaagaggg tcattatatt tcgcggaata acatcatttg 6300
gtgacgaaat aactaagcac ttgtctcctg tttactcccc tgagcttgag gggttaacat 6360
gaaggtcatc gatagcagga taataataca gtaaaacgct aaaccaataa tccaaatcca 6420
gccatcccaa attggtagtg aatgattata aataacagca aacagtaatg ggccaataac 6480
accggttgca ttggtaaggc tcaccaataa tccctgtaaa gcaccttgct gatgactctt 6540
tgtttggata gacatcactc cctgtaatgc aggtaaagcg atcccaccac cagccaataa 6600
aattaaaaca gggaaaacta accaaccttc agatataaac gctaaaaagg caaatgcact 6660
actatctgca ataaatccga gcagtactgc cgttttttcg ccccatttag tggctattct 6720
tcctgccaca aaggcttgga atactgagtg taaaagacca agacccgcta atgaaaagcc 6780
aaccatcatg ctattccatc caaaacgatt ttcggtaaat agcacccaca ccgttgcggg 6840
aatttggcct atcaattgcg ctgaaaaata aataatcaac aaaatgggca tcgttttaaa 6900
taaagtgatg tataccgaat tcgattgcgt ctcaacccct acttcggtat ctgtattatc 6960
acgtgtattt ttggtttcac ggaaccaaaa cataaccaca aggaaagtga caatatttag 7020
caacgcagcg ataaaaaagg gactatgcgg tgaaatctct cctgcaaaac caccaataat 7080
aggccccgct attaaaccaa gcccaaaact tgcccctaac caaccgaacc acttcacgcg 7140
ttgagaagct gaggtggtat cggcaatgac cgatgccgcg acagccccag tagctcctgt 7200
gatccctgaa agcaaacggc ctaaatacag catccaaagc gcacttgaaa aagccagcaa 7260
taagtaatcc agcgatgcgc ctattaatga caacaacagc actgggcgcc gaccaaatcg 7320
gtcagacatt tttccaagcc aaggagcaaa gataacctgc attaacgcat aaagtgcaag 7380
caatacgcca aagtggttag cgatatcttc cgaagcaata aattcacgta ataacgttgg 7440
caagactggc atgataaggc caatccccat ggcatcgagt aacgtaatta ccaatgcgat 7500
ctttgtcgaa ctattcattt cacttttctc tatcactgat agggagtggt aaaataactc 7560
tatcaatgat agagtgtcaa caaaaattag gaattaatga tgtctagatt agataaaagt 7620
aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc 7680
cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat 7740
aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt 7800
tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga 7860
tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa 7920
aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta 7980
gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa 8040
gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg 8100
ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta 8160
ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct 8220
taaaagcagc ataacctttt tccgtgatgg taacttcacg gtaaccaaga tgtcgagtta 8280
accacccttt agattcataa agcgaaaata atgcggctcc aacgtaccca cctaaatgga 8340
aacggcgttc actccaatct aaacacgcac aacagatttt acgtgaatgt ttggaaggaa 8400
cgtcaattcc catttcatga aaatattgaa taccacttaa tgtgatcatt gaaccatttt 8460
cagtgatcca ttgctgttga caaagggaat catagatccc tttagggttc cgatttagtg 8520
ctttacggca cctcgacctc caaaaacttg atttgggtga tggttcacgt agtgggccat 8580
cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 8640
tcttgttcca aactggaaca acactcacaa ctaactcggc ctattctttt gatttataag 8700
gatttttgtc attttctgct tactggttaa aaaataagct gatttaacaa atatttaacg 8760
cgaaatttaa caaaacatta acgtttacaa tttaaatatt tgcttataca atcatcctgt 8820
ttttggggct tttctgatta tcaatcgggg tacatatgat tgacatgcta gttttacgat 8880
taccgttcat cgattctctt gtttgctcca gactttcagg taatgacctg atagcctttg 8940
tagacctctc aaaaatagct accctctccg gcatgaattt atcagctaga acggttgaat 9000
atcatattga cggtgatttg actgtctccg gcctttctca cccgtttgaa tctttgccta 9060
ctcattactc cggcattgca tttaaaatat atgagggttc taaaaatttt tatccctgcg 9120
ttgaaattaa ggcttcacca gcaaaagtat tacagggtca taatgttttt ggtacaaccg 9180
atttagcttt atgctctgag gctttattgc ttaattttgc taactctctg ccttgcttgt 9240
acgatttatt ggatgtt 9257
<210> 4
<211> 26
<212> DNA
<213> 人工设计
<400> 4
gccgacgtgg cccggcctct ggggcc 26

Claims (1)

1.一种可异位展示两种多肽的噬菌体双展示载体，其特征在于，它通过以下方法制备得到：
(1)基于fd丝状噬菌体fUSE55基因组1022位置的BsrGI单酶切位点和2244的BamHI单酶切位点，利用BsrGI/BamHI双酶切获得含有重组gene3的长为1223bp的基因片段，所述fUSE55的序列如SEQ ID NO.1所示。
(2)基于fd丝状噬菌体f88-4基因组1022位置的BsrG I单酶切位点和2221的BamHI单酶切位点。利用BsrGI/BamHI双酶切敲除掉f88-4的野生gene3，获得剩下长为8034bp的基因片段，所述f88-4的序列如SEQ ID NO.2所示。
(3)构建双展示载体fd388：将步骤1得到的长为1223bp的基因片段和步骤2得到的长为8034bp的基因片段按1:3的比例，通过T4DNA连接酶16℃过夜连接。
(4)将连接产物转化进入JM109感受态细胞，并涂抹在四环素抗性平板上37℃过夜获取阳性单克隆，从而可以得到长为9257bp的可异位展示两种多肽的噬菌体双展示载体fd388，fd388的序列如SEQ ID NO.3所示。
CN201710162188.3A 2017-03-17 2017-03-17 一种可异位展示两种多肽的噬菌体双展示载体 Pending CN106868032A (zh)

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Application publication date: 20170620