CN114107394A - 一种慢病毒转移载体、表达PylRS及tRNACUA的细胞系及制备方法与应用 - Google Patents
一种慢病毒转移载体、表达PylRS及tRNACUA的细胞系及制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种慢病毒转移载体、表达PylRS及tRNACUA的细胞系及制备方法与应用。首先构建编码吡咯赖氨酸氨酰tRNA合成酶及tRNACUA的慢病毒转移载体(SEQ ID NO：2)，然后通过细胞转染拯救携载了上述目的元件的慢病毒，并将慢病毒以高MOI感染HEK293T细胞，传代5次后挑单克隆做PCR鉴定获得稳定细胞系。为了验证细胞系对目的蛋白掺入非天然氨基酸的效果，制备了绿色荧光蛋白报告质粒pcDNA3.1‑EGFP‑Y39TAG(SEQ ID NO：3)，通过该报告质粒转染验证了上述稳定细胞系的功能。
Description
技术领域
本发明属于生物技术领域，涉及一种慢病毒转移载体、表达PylRS及 tRNACUA的细胞系及制备方法及应用。
背景技术
目前的生物学研究，尤其是合成生物学研究中，研究人员不再仅仅局限于对已有的生物系统进行修改，而是逐步进行人工设计和改造，合成大量自然界不存在或未发现的生物元件、生物装置及生物系统。但人工添加的元件及装置常常与天然存在的生物系统不兼容或者受到天然系统的干扰，甚至会导致所在生物系统崩溃，为了避免人工构建的体系与天然系统的相互干扰，研究人员建立了一个与自然界的生物系统及其组成交叉很少或没有交叉的体系—正交系统。
非天然氨基酸系统是指可在目的蛋白特定位点掺入除20种天然氨基酸之外的非天然氨基酸的技术方法。非天然氨基酸插入系统一般由以下部分组成：空白密码子、正交氨酰tRNA合成酶(aaRS)、tRNA及目标非天然氨基酸。空白密码子是指生物体内不编码氨基酸的终止密码子或编码氨基酸但是很少出现的罕用密码子，最常用的是琥珀终止密码子UAG，其通常作为终止密码子，未被用于编码天然氨基酸。aaRS/tRNA对应该能够识别目标非天然氨基酸，并将其插入到合适的位点。aaRS/tRNA的正交性要求aaRS不能识别宿主内源的tRNAs或者氨基酸，只能氨酰化其配体tRNA，而tRNA和目标非天然氨基酸不能被内源 aaRSs氨酰化。另外，目标空白密码子只能被其对应的外源tRNA识别，而不能被其他内源tRNA识别。
目前最常用的非天然氨基酸插入系统是使用UAG编码吡咯赖氨酸(Pyl)及其类似物，例如NAEK(Nε-2-azidoethyloxycarbonyl-L-lysine,Nε-2-叠氮乙氧羰基 -L-赖氨酸)。通常在细胞水平做蛋白表达过程中，若使非天然氨基酸掺入到目的蛋白的特定位点，需满足以下条件：
1.目的蛋白中要掺入非天然氨基酸的DNA位点上含有TAG密码子；
2.提供质粒补偿非天然氨基酸对应的正交氨酰tRNA合成酶(aaRS)和 tRNA；
3.培养基中添加非天然氨基酸。
发明内容
本发明旨在构建慢病毒转移载体、包装慢病毒、通过慢病毒感染建立表达吡咯赖氨酸(Pyl)及其类似物对应的正交氨酰tRNA合成酶(aaRS)和tRNA 的细胞系、并通过绿色荧光蛋白报告质粒在验证上述的细胞系对目的蛋白掺入非天然氨基酸的效果的应用。
本发明的一个目的是提供一种慢病毒转移载体，用于编码吡咯赖氨酸氨酰 tRNA合成酶及tRNACUA，所述的慢病毒转移载体由骨架载体和插入其克隆位点PacI和KpnI之间的如SEQ ID.No.1所示的核苷酸序列，如SEQ ID.No.1所示的核苷酸序列包含hUbC启动子-PylRS-WPRE片段及2个拷贝的U6启动子- tRNACUA片段，所述慢病毒转移载体的核苷酸序列如SEQ ID.No.2所示，骨架载体为FUGW载体。
所述FUGW载体的addgene编号为14883。
本发明的另一个目的是提供一种慢病毒，携载所述慢病毒转移载体，由慢病毒包装质粒及权利要求1所述的慢病毒转移载体转染细胞后分离后获得。
优选地，所述慢病毒包装质粒由：addgene编号为12251的pMDLg/pRRE、 addgene编号为12253的pRSV-Rev、addgene编号为12259的pMD2.G组成。
本发明的第三个目的是提供表达PylRS及tRNACUA的细胞系，所述慢病毒转染所述细胞后得到表达PylRS及tRNACUA的细胞系。
优选地，所述的细胞是真核细胞。
优选地，所述的真核细胞是哺乳动物细胞。
优选地，所述的哺乳动物细胞是293T细胞。
优选地，MOI＝10的所述慢病毒的病毒液感染HEK293T细胞，所述慢病毒的病毒液以感染复数MOI＝10感染HEK293T细胞，传代5次后挑单克隆做PCR 鉴定获得所述的表达PylRS及tRNACUA的细胞系。
本发明的第四个目的是提供一种绿色荧光蛋白报告质粒在验证上述的细胞系对目的蛋白掺入非天然氨基酸的效果的应用，所述绿色荧光蛋白报告质粒为 addgene编号为129020的pcDNA3.1-EGFP在EGFP的39位氨基酸位点引入 TAG密码子得到的质粒，即pcDNA3.1-EGFP-Y39TAG，其核苷酸序列如SEQ ID NO：3所示。
本发明的第五个目的是提供一种制备上述的细胞系的方法，包括以下步骤：
(1)人工合成核苷酸序列如SEQ ID NO.1所示的基因片段，利用试剂盒One StepCloning Kit重组克隆将所述基因片段连入经PacI、KpnI线性化的addgene编号为14883的FUGW载体获得pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒转移载体，并测序；
(2)将测序验证正确的慢病毒核心质粒pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)与addgene编号为12251的pMDLg/pRRE、addgene编号为12253的 pRSV-Rev、addgene编号为12259的pMD2.G共转染293T细胞，并在48h和72h 收集上清，用滤膜过滤，得到FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒，将收集到的FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒测定感染滴度，并以MOI＝10的病毒液感染HEK293T细胞，并培养传代，传代5次后进行保种，挑单克隆做PCR鉴定，鉴定结果为阳性的为获得的所述表达PylRS及tRNACUA的细胞系。
本发明的第六个目的是提供上述的细胞系在将非天然氨基酸掺入到目的蛋白的特定位点的应用，所述非天然氨基酸为NAEK，NAEK的叠氮基团能与含炔基的功能分子通过“点击化学”反应形成共价键，从而借助功能分子对蛋白进行功能修饰，使一些功能分子缀合在蛋白表面，从而改变蛋白的部分特性。
本发明的主要创新点如下：
1.构建了编码吡咯赖氨酸氨酰tRNA合成酶及tRNACUA的慢病毒转移载体；
2.建立了编码吡咯赖氨酸氨酰tRNA合成酶及tRNACUA的细胞系；
3.构建了非天然氨基酸系统绿色荧光蛋白报告质粒pcDNA3.1(+)-EGFP- Y39TAG，可方便地检测非天然氨基酸系统的有效性。
附图说明
图1为pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒质粒图谱；
图2为pcDNA3.1(+)-EGFP-Y39TAG质粒图谱；
图3为pcDNA3.1(+)-EGFP-Y39TAG转染验证HEK293T-PylRS-tRNACUA功能结果图。
具体实施方式
以下结合实施例对本发明作进一步说明，但不用来限制本发明的范围。实施例中采用的仪器、试剂等，如无特别说明，均为现有技术中已有的常规仪器、试剂，可通过商业途径获得。
首先对本发明所设计的术语解释如下：
“UPRT基因片段”包含hUbC启动子-PylRS-WPRE片段及2个拷贝的U6启动子-tRNACUA片段，是从hUbC、PylRS、WPRE、tRNACUA中抽取字母UPRT形成的组合。
“FUGW载体”慢病毒载体滴度高，可容纳较大片段的外源性目的基因，宿主细胞范围较广，在宿主中表达时间长、毒性低、不易诱发宿主免疫反应，不仅可以高效感染分裂期细胞，而且可以感染非分裂期细胞，还可以高效感染质粒难转染的原代细胞和胚胎干细胞，并可在体内长期稳定地表达。
“试剂盒One Step Cloning Kit”是一款简单、快速、高效的DNA定向克隆产品，该试剂盒可以将PCR产物定向克隆至任意载体的任意位点，可高效克隆50 bp-10kb片段。将载体在克隆位点进行线性化，并在插入片段PCR引物5’端引入线性化克隆载体末端序列，使得插入片段PCR产物5’和3’最末端分别带有和线性化克隆载体两末端对应的完全一致的序列(15bp-20bp)。将这种两端带有载体末端序列的PCR产物和线性化克隆载体按一定比例混合，在重组酶Exnase的催化下，仅需反应20min即可进行转化，完成定向克隆。克隆载体酶切产物或 PCR产物和插入片段PCR产物可以不进行DNA纯化直接用于重组克隆，极大的简化了实验步骤。试剂盒中包含有重组反应所需缓冲液和重组酶Exnase。
“pMDLg/pRRE、pRSV-Rev、pMD2.G”第三代慢病毒包装质粒。
“pcDNA3.1(+)EGFP”将此载体转染至HEK293T细胞、Hela细胞、CHO 细胞等多株细胞，24小时后观察荧光，可见多且亮的绿色荧光。
实施例
1.pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒质粒的构建
由生工(Sangon)生物工程(上海)股份有限公司合成UPRT基因片段，其核苷酸序列如SEQ ID NO.1所示，利用试剂盒One Step Cloning Kit(Vazyme公司)重组克隆将UPRT连入经PacI、KpnI线性化的FUGW载体(addgene编号#14883)获得pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒转移载体，重组克隆所需引物为UPRT-F:GGTTAATTAAGAGGGCCTATTTCCCATGATTCCT TCATA、UPRT-R:AAGGTACCTGAGGTTCCGCGCACATTTC)。
获得的pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒转移载体经基因测序验证无误。
2.HEK293T-PylRS-tRNACUA稳定细胞系的建立
HEK293T细胞在10cm培养皿中培养至70～80％融合时，将细胞培养基更换为无血清培养基。在无菌离心管中加入2.5ml无血清Opti-MEM，按比例加入测序验证正确的慢病毒核心质粒pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)与骨架质粒(pMDLg/pRRE(addgene编号#12251)、pRSV-Rev(addgene编号#12253)、 pMD2.G(addgene编号#12259))；取另一支离心管，加入2.5ml无血清DMEM，再加入300μl Lipofectamine 2000转染试剂(Invitrogen，12566014)混匀；室温放置5分钟后将两管混合，室温放置20～25分钟；将转染混合物逐滴加入15cm 培养皿中混匀，在培养箱中温育4-6h，吸弃转染液，加入18ml含10％FBS的 DMEM培养液，放回培养箱中培养，并在48h和72h收集上清，用0.22μm滤膜过滤，得到FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒。
将HEK293T细胞按3×104细胞/孔的浓度接种96孔板，混匀后在37℃5％ CO2培养箱培养24h，将收集到的FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒用含有10％FBS的DMEM培养液10倍稀释3-5个梯度，再加入终浓度为 5μg/ml的Polybrene；吸弃培养液，每孔加入100μl不同稀释倍数的病毒液，同时设立空白对照组，继续培养24h；吸弃病毒稀释液，每孔加入100μl含FBS的 DME吗培养液，继续培养72h；提取基因组DNA进行实时荧光定量PCR检测，结合稀释倍数计算得到的FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒的滴度；
测定感染滴度后以感染复数MOI＝10感染HEK293T细胞，置于35℃，3％ CO2细胞培养箱中继续培养24h小时后换新鲜的培养基，72h后观察细胞状态并进行传代，传代5次后进行保种。挑10个单克隆做PCR鉴定(Pyl-F： ggtccaaaatctacatcgagatggcc，Pyl-R：gtaaactcttccaggtgctctttgcc)，鉴定结果均为阳性，认为已获得稳定细胞系。将得到的稳定细胞系命名为HEK293T-PylRS- tRNACUA。由于慢病毒核心未携载抗性基因和荧光标签，无法通过荧光分选或药物筛选来分出整合了目的基因的细胞，为保证细胞系感染效果，使用高MOI 感染后通过PCR鉴定获得稳定细胞系。
3.pcDNA3.1(+)-EGFP-Y39TAG非天然氨基酸系统荧光报告质粒的构建
使用Fast Mutagenesis System定点突变试剂盒(全式金，FM111-01)对 pcDNA3.1(+)EGFP(addgene编号#129020)质粒做定点突变，以在EGFP的39 位氨基酸位点引入TAG密码子。
用于点突变的引物序列如下：
MuTAG-F:GGGCGATGCCACCTAgGGCAAGCTGAC；
MuTAG-R:CTAGGTGGCATCG CCCTCGCCCTCG；
质粒经测序验证无误。
4.pcDNA3.1(+)-EGFP-Y39TAG转染验证HEK293T-PylRS-tRNACUA功能
将pcDNA3.1(+)-EGFP-Y39TAG转染HEK293T-PylRS-tRNACUA，培养基中添加非天然氨基酸NAEK(购自上海蓝鸟)，终浓度0.1mM，对照组不添加非天然氨基酸，实验组可见绿色荧光蛋白表达，对照组无荧光表达，证明构建的细胞系HEK293T-PylRS-tRNACUA可正常表达吡咯赖氨酸对应的aaRS及tRNA。如图3所示。
质粒pcDNA3.1(+)-EGFP-Y39TAG中，将绿色荧光蛋白基因表达框中第39 位氨基酸突变为琥珀终止密码子TAG，质粒转染细胞后，若无对应的tRNACUA 识别TAG密码子/无对应的氨酰tRNA合成酶/无对应的非天然氨基酸，则 EGFP蛋白翻译将终止在39位氨基酸，不会产生荧光，若提供非天然氨基酸功能元件及对应非天然氨基酸，则EGFP阅读框可完整表达，可见绿色荧光。
上述细胞系能将非天然氨基酸NAEK掺入到目的蛋白的特定位点，而NAEK 的叠氮基团可与炔基发生点击化学反应，可借助这一反应对蛋白进行修饰，使一些功能分子缀合在蛋白表面，从而改变蛋白的部分特性。
序列表
<110> 中国科学院精密测量科学与技术创新研究院
<120> 一种慢病毒转移载体、表达PylRS及tRNACUA的细胞系及制备方法与应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3943
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 1
ttaattaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 60
ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt 120
gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 180
actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 240
ggaaaggacg aaacaccgga aacctgatca tgtagatcga atggactcta aatccgttca 300
gccgggttag attcccgggg tttccgccat ttttcgggga aatgtgcgcg gaaccgcgcc 360
gggttttggc gcctcccgcg ggcgcccccc tcctcacggc gagcgctgcc acgtcagacg 420
aagggcgcag gagcgttcct gatccttccg cccggacgct caggacagcg gcccgctgct 480
cataagactc ggccttagaa ccccagtatc agcagaagga cattttagga cgggacttgg 540
gtgactctag ggcactggtt ttctttccag agagcggaac aggcgaggaa aagtagtccc 600
ttctcggcga ttctgcggag ggatctccgt ggggcggtga acgccgatga ttatataagg 660
acgcgccggg tgtggcacag ctagttccgt cgcagccggg atttgggtcg cggttcttgt 720
ttgtggatcg ctgtgatcgt cacttggtga gttgcgggct gctgggctgg ccggggcttt 780
cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga gagaccgcca agggctgtag 840
tctgggtccg cgagcaaggt tgccctgaac tgggggttgg ggggagcgca caaaatggcg 900
gctgttcccg agtcttgaat ggaagacgct tgtaaggcgg gctgtgaggt cgttgaaaca 960
aggtgggggg catggtgggc ggcaagaacc caaggtcttg aggccttcgc taatgcggga 1020
aagctcttat tcgggtgaga tgggctgggg caccatctgg ggaccctgac gtgaagtttg 1080
tcactgactg gagaactcgg gtttgtcgtc tggttgcggg ggcggcagtt atgcggtgcc 1140
gttgggcagt gcacccgtac ctttgggagc gcgcgcctcg tcgtgtcgtg acgtcacccg 1200
ttctgttggc ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc 1260
tccgtcgcag gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg 1320
acctctggtg aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct 1380
atcttcttaa gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt 1440
tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc 1500
agtgttagac tagtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagacg 1560
aagcttgggc tgcaggtcga ctctagagga tccaccatgg acaagaagcc cctgaacacc 1620
ctgatcagcg ccacaggact gtggatgtcc agaaccggca ccatccacaa gatcaagcac 1680
cacgaggtgt cccggtccaa aatctacatc gagatggcct gcggcgatca cctggtcgtc 1740
aacaacagca gaagcagccg gacagccaga gccctgcggc accacaagta cagaaagacc 1800
tgcaagcggt gcagagtgtc cgacgaggac ctgaacaagt tcctgaccaa ggccaacgag 1860
gaccagacca gcgtgaaagt gaaggtggtg tccgccccca cccggaccaa gaaagccatg 1920
cccaagagcg tggccagagc ccccaagccc ctggaaaaca ccgaagccgc tcaggcccag 1980
cccagcggca gcaagttcag ccccgccatc cccgtgtcta cccaggaaag cgtcagcgtc 2040
cccgccagcg tgtccaccag catctctagc atctcaaccg gcgccacagc ttctgccctg 2100
gtcaagggca acaccaaccc catcaccagc atgtctgccc ctgtgcaggc ctctgcccca 2160
gccctgacca agtcccagac cgaccggctg gaagtgctcc tgaaccccaa ggacgagatc 2220
agcctgaaca gcggcaagcc cttccgggag ctggaaagcg agctgctgag ccggcggaag 2280
aaggacctcc agcaaatcta cgccgaggaa cgggagaact acctgggcaa gctggaaaga 2340
gagatcaccc ggttcttcgt ggaccggggc ttcctggaaa tcaagagccc catcctgatc 2400
cccctggagt acatcgagcg gatgggcatc gacaacgaca ccgagctgag caagcagatt 2460
ttccgggtgg acaagaactt ctgcctgcgg cccatgctgg cccccaacct ggccaactac 2520
ctgcggaaac tggatcgcgc tctgcccgac cccatcaaga ttttcgagat cggcccctgc 2580
taccggaaag agagcgacgg caaagagcac ctggaagagt ttacaatgct gaacttttgc 2640
cagatgggca gcggctgcac cagagagaac ctggaatcca tcatcaccga ctttctgaac 2700
cacctgggga tcgacttcaa gatcgtgggc gacagctgca tggtgttcgg cgacaccctg 2760
gacgtgatgc acggcgacct ggaactgtct agcgccgtcg tgggacccat ccctctggac 2820
cgggagtggg gcatcgataa gccctggatc ggagccggct tcggcctgga acggctgctg 2880
aaagtcaagc acgactttaa gaacatcaag cgggctgcca gaagcgagag ctactacaac 2940
ggcatcagca ccaacctgtg acgatatcaa gcttatcgat aatcaacctc tggattacaa 3000
aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 3060
cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 3120
cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 3180
tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 3240
ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 3300
cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 3360
ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 3420
tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 3480
ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 3540
tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag ggcctatttc 3600
ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta 3660
atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc 3720
ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 3780
cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggaa 3840
acctgatcat gtagatcgaa tggactctaa atccgttcag ccgggttaga ttcccggggt 3900
ttccgccatt tttcggggaa atgtgcgcgg aacctcaggt acc 3943
<210> 2
<211> 11117
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 2
gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600
tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780
gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400
gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460
tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520
agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580
agatccagtt tggttaatta agagggccta tttcccatga ttccttcata tttgcatata 2640
cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta 2700
gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta 2760
tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct 2820
ttatatatct tgtggaaagg acgaaacacc ggaaacctga tcatgtagat cgaatggact 2880
ctaaatccgt tcagccgggt tagattcccg gggtttccgc catttttcgg ggaaatgtgc 2940
gcggaaccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcctcac ggcgagcgct 3000
gccacgtcag acgaagggcg caggagcgtt cctgatcctt ccgcccggac gctcaggaca 3060
gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta 3120
ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag 3180
gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga 3240
tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg 3300
tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagttgcgg gctgctgggc 3360
tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg 3420
ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc 3480
gcacaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtaagg cgggctgtga 3540
ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt 3600
cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct 3660
gacgtgaagt ttgtcactga ctggagaact cgggtttgtc gtctggttgc gggggcggca 3720
gttatgcggt gccgttgggc agtgcacccg tacctttggg agcgcgcgcc tcgtcgtgtc 3780
gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 3840
cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 3900
cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 3960
gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 4020
ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 4080
atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 4140
tttttgttag acgaagcttg ggctgcaggt cgactctaga ggatccacca tggacaagaa 4200
gcccctgaac accctgatca gcgccacagg actgtggatg tccagaaccg gcaccatcca 4260
caagatcaag caccacgagg tgtcccggtc caaaatctac atcgagatgg cctgcggcga 4320
tcacctggtc gtcaacaaca gcagaagcag ccggacagcc agagccctgc ggcaccacaa 4380
gtacagaaag acctgcaagc ggtgcagagt gtccgacgag gacctgaaca agttcctgac 4440
caaggccaac gaggaccaga ccagcgtgaa agtgaaggtg gtgtccgccc ccacccggac 4500
caagaaagcc atgcccaaga gcgtggccag agcccccaag cccctggaaa acaccgaagc 4560
cgctcaggcc cagcccagcg gcagcaagtt cagccccgcc atccccgtgt ctacccagga 4620
aagcgtcagc gtccccgcca gcgtgtccac cagcatctct agcatctcaa ccggcgccac 4680
agcttctgcc ctggtcaagg gcaacaccaa ccccatcacc agcatgtctg cccctgtgca 4740
ggcctctgcc ccagccctga ccaagtccca gaccgaccgg ctggaagtgc tcctgaaccc 4800
caaggacgag atcagcctga acagcggcaa gcccttccgg gagctggaaa gcgagctgct 4860
gagccggcgg aagaaggacc tccagcaaat ctacgccgag gaacgggaga actacctggg 4920
caagctggaa agagagatca cccggttctt cgtggaccgg ggcttcctgg aaatcaagag 4980
ccccatcctg atccccctgg agtacatcga gcggatgggc atcgacaacg acaccgagct 5040
gagcaagcag attttccggg tggacaagaa cttctgcctg cggcccatgc tggcccccaa 5100
cctggccaac tacctgcgga aactggatcg cgctctgccc gaccccatca agattttcga 5160
gatcggcccc tgctaccgga aagagagcga cggcaaagag cacctggaag agtttacaat 5220
gctgaacttt tgccagatgg gcagcggctg caccagagag aacctggaat ccatcatcac 5280
cgactttctg aaccacctgg ggatcgactt caagatcgtg ggcgacagct gcatggtgtt 5340
cggcgacacc ctggacgtga tgcacggcga cctggaactg tctagcgccg tcgtgggacc 5400
catccctctg gaccgggagt ggggcatcga taagccctgg atcggagccg gcttcggcct 5460
ggaacggctg ctgaaagtca agcacgactt taagaacatc aagcgggctg ccagaagcga 5520
gagctactac aacggcatca gcaccaacct gtgacgatat caagcttatc gataatcaac 5580
ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 5640
cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 5700
tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 5760
ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 5820
gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 5880
cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 5940
ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 6000
ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 6060
cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 6120
gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc 6180
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 6240
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 6300
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 6360
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 6420
cgaaacaccg gaaacctgat catgtagatc gaatggactc taaatccgtt cagccgggtt 6480
agattcccgg ggtttccgcc atttttcggg gaaatgtgcg cggaacctca ggtaccttta 6540
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 6600
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 6660
acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca 6720
ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc 6780
aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg 6840
gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga 6900
gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 6960
tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 7020
gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 7080
tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc 7140
ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 7200
tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 7260
gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 7320
caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 7380
ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 7440
ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 7500
tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 7560
gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 7620
gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 7680
ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 7740
ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 7800
tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 7860
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 7920
tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 7980
catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 8040
ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 8100
gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 8160
ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac 8220
gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga 8280
actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg 8340
gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact 8400
tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg 8460
tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt 8520
ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg 8580
gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact 8640
tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct 8700
atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 8760
gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 8820
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 8880
gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 8940
gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 9000
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 9060
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 9120
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 9180
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 9240
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 9300
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 9360
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 9420
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 9480
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 9540
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9600
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9660
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9720
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9780
ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 9840
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9900
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9960
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 10020
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 10080
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 10140
gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 10200
tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 10260
cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 10320
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 10380
gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 10440
caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 10500
gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 10560
ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10620
tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 10680
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 10740
tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10800
cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10860
ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10920
aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 10980
tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 11040
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 11100
gaaaagtgcc acctgac 11117
<210> 3
<211> 6142
<212> DNA
<213> Artificial Sequence(人工序列)
<400> 3
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 960
ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 1020
ctagggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 1080
caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 1140
gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 1200
cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 1260
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 1320
gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 1380
gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 1440
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 1500
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 1560
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 1620
gtaaaagctt ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat 1680
ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg 1740
actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 1800
ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 1860
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 1920
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 1980
agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 2040
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2100
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2160
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 2220
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2280
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 2340
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2400
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 2460
agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 2520
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2580
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2640
ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2700
atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 2760
ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 2820
atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 2880
ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 2940
gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 3000
ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg 3060
ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 3120
ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 3180
agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 3240
gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 3300
gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 3360
tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 3420
cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 3480
ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 3540
agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 3600
cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 3660
cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 3720
cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 3780
gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 3840
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 3900
ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 3960
ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4020
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4080
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4140
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4200
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4260
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4320
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4380
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4440
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4500
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4560
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 4620
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4680
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4740
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4800
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 4860
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4920
tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 4980
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5040
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5100
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5160
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5220
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5280
accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5340
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5400
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5460
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5520
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5580
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 5640
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5700
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5760
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5820
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 5880
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 5940
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6000
actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6060
cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6120
ccgaaaagtg ccacctgacg tc 6142
Claims (12)
1.一种慢病毒转移载体，用于编码吡咯赖氨酸氨酰tRNA合成酶及tRNACUA，所述的慢病毒转移载体由骨架载体和插入其克隆位点PacI和KpnI之间的如SEQ ID.No.1所示的核苷酸序列，如SEQ ID.No.1所示的核苷酸序列包含hUbC启动子-PylRS-WPRE片段及2个拷贝的U6启动子-tRNACUA片段，所述慢病毒转移载体的核苷酸序列如SEQ ID.No.2所示，骨架载体为FUGW载体。
2.如权利要求1所述的慢病毒转移载体，其特征在于，所述FUGW载体的addgene编号为14883。
3.一种慢病毒，其特征在于，携载所述慢病毒转移载体，由慢病毒包装质粒及权利要求1所述的慢病毒转移载体转染细胞后分离后获得。
4.如权利要求3所述的慢病毒，其特征在于，所述慢病毒包装质粒由：addgene编号为12251的pMDLg/pRRE、addgene编号为12253的pRSV-Rev、addgene编号为12259的pMD2.G组成。
5.一种表达PylRS及tRNACUA的细胞系，其特征在于，所述慢病毒转染所述细胞后得到表达PylRS及tRNACUA的细胞系。
6.如权利要求3～5所述的表达PylRS及tRNACUA的细胞系，其特征在于，所述细胞是真核细胞。
7.如权利要求6所述的表达PylRS及tRNACUA的细胞系，其特征在于，所述的真核细胞是哺乳动物细胞。
8.如权利要求7所述的表达PylRS及tRNACUA的细胞系，其特征在于，所述的哺乳动物细胞是HEK293T细胞。
9.如权利要求8所述的表达PylRS及tRNACUA的细胞系，其特征在于，所述慢病毒的病毒液以感染复数MOI＝10感染HEK293T细胞，传代5次后挑单克隆做PCR鉴定获得所述的表达PylRS及tRNACUA的细胞系。
10.一种绿色荧光蛋白报告质粒在验证权利要求4～9任一项所述的细胞系对目的蛋白掺入非天然氨基酸的效果的应用，其特征在于，所述绿色荧光蛋白报告质粒为addgene编号为129020的pcDNA3.1-EGFP在EGFP的39位氨基酸位点引入TAG密码子得到的质粒，其核苷酸序列如SEQ ID NO：3所示。
11.一种制备权利要求4～9任一项所述的细胞系的方法，其特征在于，包括以下步骤：
(1)人工合成核苷酸序列如SEQ ID NO.1所示的基因片段，利用试剂盒One StepCloning Kit重组克隆将所述基因片段连入经PacI、KpnI线性化的addgene编号为14883的FUGW载体获得pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒转移载体，并测序；
(2)将测序验证正确的慢病毒核心质粒pL-FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)与addgene编号为12251的pMDLg/pRRE、addgene编号为12253的pRSV-Rev、addgene编号为12259的pMD2.G共转染293T细胞，并在48h和72h收集上清，用滤膜过滤，得到FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒，将收集到的FUGW-hUBC-PylRS-WPRE-2(U6-PyltRNA)慢病毒测定感染滴度，并以MOI＝10的病毒液感染HEK293T细胞，并培养传代，传代5次后进行保种，挑单克隆做PCR鉴定，鉴定结果为阳性的为获得的所述表达PylRS及tRNACUA的细胞系。
12.如权利要求4～9任一项所述的细胞系在将非天然氨基酸掺入到目的蛋白的特定位点的应用，其特征在于，所述非天然氨基酸为NAEK，NAEK的叠氮基团能与含炔基的功能分子通过“点击化学”反应形成共价键，从而借助功能分子对蛋白进行功能修饰。
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