CN116926123A - 自复制mRNA增强型TIL细胞及其制备方法与应用 - Google Patents
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Abstract
本发明公开一种自复制mRNA增强型TIL细胞及其制备方法与应用。本发明提供的自复制mRNA增强型TIL细胞主要通过构建TIL细胞的高效培养和富集体系获得TIL细胞，然后将含有目的基因的自复制mRNA直接转染至TIL细胞中制得，用于肿瘤细胞的过继免疫治疗时，既能发挥传统TIL细胞的治疗效果，又可以在较低剂量下实现抗原的高表达，达到产生较强免疫反应的能力，显著提高TIL细胞的抗肿瘤活性，使TIL免疫治疗方法能够适用于免疫原性较差的肿瘤如乳腺癌、结肠癌、卵巢癌、前列腺癌和胰腺癌等的治疗。
Description
技术领域
本发明属于生物技术领域，具体涉及一种自复制mRNA增强型TIL细胞的制备方法及其应用。
背景技术
肿瘤浸润淋巴细胞(Tumor Infiltrating Lymphocytes，TIL)，是肿瘤间质中的异质性淋巴细胞，包括T细胞及NK细胞等。大多数情况下以T细胞为主，是机体淋巴细胞侵入到肿瘤组织中，并对肿瘤起识别、抵抗和攻击作用的细胞群体。
肿瘤的TIL免疫治疗在一些恶性肿瘤如宫颈癌、黑色素瘤等中取得了显著成效，因其具有靶向性高、特异性强、毒副作用小、疗效显著等优势，目前已经成为一种新型有效的免疫治疗方法。但是相比在黑色素瘤和宫颈癌的成功，TIL用于治疗其他癌症的治疗效果仍有巨大的差距。这可能是因为乳腺癌、结肠癌、卵巢癌、前列腺癌和胰腺癌等等许多癌症的免疫原性较差，传统TIL因其自身缺陷而无法惠及大量免疫原性较差的患者，在这类患者自身肿瘤组织中收获的TIL，回输后只有一小部分对肿瘤有反应，因此在利用TIL治疗肿瘤时，给这类患者提供他们自己的异质性TIL似乎是徒劳的。
因此，需要对TIL进行改造，以提高其用于对肿瘤进行过继性细胞免疫治疗时的治疗效果。
发明内容
本发明的主要目的在于提供一种自复制mRNA增强型TIL细胞的制备方法，以解决上述技术问题中的至少一个。
根据本发明的一个方面，提供了一种自复制mRNA增强型TIL细胞的制备方法，包括如下步骤：
将肿瘤样品处理成组织块，然后加入含5000-10000IU/mL IL-2的完全培养基中培养，得到TIL细胞；
根据α病毒家族的基因组设计自复制mRNA序列，合成并利用该自复制mRNA序列构建自复制载体，然后将目的基因插入自复制载体中，得到重组质粒；
利用重组质粒通过体外转录合成含有目的基因的自复制mRNA；
将含有目的基因的自复制mRNA通过电穿孔法转染至TIL细胞中，即得自复制mRNA增强型TIL细胞。
其中，目的基因可以选自PD-1、CBLB(或称为CBL-B)、CISH (Cytokine-inducibleSRC homology 2(SH2)domain protein，细胞因子诱导的含SH2结构域蛋白)、CCR2、CCL5(或称为RANTES)、CXCL1、CXCL8 中的至少一种。
本发明的研究思路为：
先是构建TIL细胞的高效培养和富集体系，TIL细胞在经过高效培养和富集体系富集和培养后可以快速扩增，细胞在第10天就可以完全进入扩增状态，继续培养即可获得足够的可以用于改造和治疗的TIL细胞。通过流式细胞术对培养得到的TIL细胞的表型进行验证的结果表明，经本发明方法分离和培养得到的TIL细胞，其中接近90％细胞为CD3阳性T细胞，由此说明，本发明提供的方法还可以保证培养细胞的分型准确。
然后将含有目的基因的自复制mRNA导入TIL细胞内，制得增强型TIL 细胞。
本发明提供的增强型TIL细胞用于肿瘤细胞的过继免疫治疗时，既能发挥传统TIL细胞的治疗效果，又可以在较低剂量下实现抗原的高表达，达到产生较强免疫反应的能力，显著提高TIL细胞的抗肿瘤活性的效果，使TIL免疫治疗方法能够适用于免疫原性较差的肿瘤如乳腺癌、结肠癌、卵巢癌、前列腺癌和胰腺癌等的治疗。
根据α病毒家族的基因组设计得到的自复制mRNA序列包含可编码α病毒自复制组件的基因，但缺乏制造具有传染性的α病毒颗粒的结构蛋白的基因，使用安全。
在一些实施方式中，α病毒可以选自辛德毕斯病毒(Sin-dbis virus)、屈曲病毒(Chikungunya virus)、东方马脑脊髓炎病毒(Eastern equine encephali- tis virus)、西方马脑脊髓炎病毒(Western equineencephalitis virus)、马雅鲁病毒(Mayarovirus)、生里基森林病毒(Semliki forest virus)、委内瑞拉马脑脊髓炎病毒(Venezuelan equineencephalitis virus，VEEV)中的至少一种。
在一些实施方式中，由根据委内瑞拉马脑脊髓炎病毒(venezuelan equineencephalomyelitis virus，VEEV)基因组设计并合成得到的自复制mRNA构建的自复制载体的核苷酸序列如SEQ ID NO:3所示。
在一些实施方式中，完全培养基可以为含有8-10％v/v FBS的基础培养基，基础培养基可以选自RPMI 1640、DMEM中的至少一种。
在一些实施方式中，将含有目的基因的自复制mRNA转染至TIL细胞的方法可以为任意一种将基因导入细胞中的方法，如电穿孔法、脂质体转染法等。
在一些实施方式中，目的基因可以为PD-1。根据人偏好密码子表进行密码子优化后得到的目的基因PD-1片段的核苷酸序列可以如SEQ ID NO:1 和SEQ ID NO:2所示。
在一些实施方式中，以PD-1为目的基因制得的重组质粒的核苷酸序列可以如SEQID NO:4或SEQ ID NO:5所示。
在一些实施方式中，利用重组质粒通过体外转录合成含有目的基因的自复制mRNA包括如下步骤：利用体外转录试剂盒将重组质粒通过体外转录合成含有目的基因的自复制mRNA。
在一些实施方式中，体外转录试剂盒可以选自MEGAscript T7 TranscriptionKit试剂盒。
通过本发明制备方法制得的自复制mRNA增强型TIL细胞可用于制备治疗肿瘤的药物用于治疗肿瘤，包括但不限于乳腺癌、结肠癌、卵巢癌、前列腺癌、胰腺癌等免疫原性较差的肿瘤的过继性细胞免疫治疗。
体外实验证明，相比于传统TIL，本发明提供的增强型TIL与U251(人胶质瘤细胞)在效靶比2:1和8：1时都能展现出更强的细胞杀伤能力，同时细胞因子的分泌量也远高于传统TIL。
此外，本发明提供的自复制mRNA增强型TIL细胞还可以与手术、放化疗等传统肿瘤治疗手段联合，进一步提高肿瘤的治疗效果。
附图说明
图1为TIL细胞培养第3天(D3)、第6天(D6)和第10天(D10) 的生长情况图；
图2为直接mRNA电转后第1天、第3天、第5天和第7天的TIL细胞的荧光图；
图3为转染后mRNA转染效率随时间变化的变化图；
图4为质粒系统转染后第2天(Day2)和第5天(Day5)的TIL细胞的荧光图。
具体实施方式
下面结合实施方式对本发明作进一步详细的说明。实施例仅用于解释而不以任何方式限制本发明。如无特殊说明，实施例中所用原料和试剂为可以通过市售获得的常规产品；实施例中未注明具体条件的实验方法，通常按照本领域常规条件或按照制造厂商建议的条件。
实施例1TIL细胞的富集和培养
包括如下步骤：
收集新切除的肿瘤标本，立即在无菌条件下进行处理。具体处理步骤包括：去除肿瘤标本周围的正常组织和坏死区域，从标本的不同区域取下大小为1-2mm3的小组织块，24孔板的每一孔放置一块。每孔加2mL完全培养基(含10％FBS的Gibco RPMI 1640培养基)和6000IU/mL的IL-2，然后将24孔板置于37℃、5％CO2培养箱中培养。培养起始后的第5-6天为所有孔进行半量换液。之后，根据TI L生长情况，每隔1-2天进行一次半量换液。一旦孔内TIL长满，就将各个长满孔内的TIL收集起来。TIL 细胞培养第3天、第6天和第10天的生长情况见图1。
TIL细胞在经过上述培养后可以快速扩增，细胞在第10天就完全进入扩增状态，后续可达到回输剂量。
通过流式细胞验证培养的TIL细胞表型，结果表明，培养得到的TIL 细胞中，接近90％细胞为CD3阳性T细胞，培养细胞的分型准确。
实施例2自复制mRNA增强型TIL细胞的制备
包括如下步骤：
1、合成目的基因PD-1片段
查询目的蛋白氨基酸序列，根据人偏好密码子表进行密码子优化得到优化后的序列，交由基因合成公司合成序列。目的基因PD-1片段的核苷酸序列如SEQ ID NO:1或SEQ IDNO:2所示。
2、构建SM-1自复制载体
根据α病毒家族的基因组设计并合成得到自复制mRNA序列，然后利用自复制mRNA序列构建SM-1自复制载体。
构建得到的SM-1自复制载体的核苷酸序列如SEQ ID NO:3所示。
3、制备重组质粒SMPD-1
将目的基因PD1片段插入SM-1自复制载体中，制得重组质粒SMPD-1。
构建得到的重组质粒SMPD-1的核苷酸序列如SEQ ID NO:4和SEQ ID NO:5所示。
上述步骤2和3中SM-1自复制载体和重组质粒SMPD-1的构建和制备均由基因合成公司完成。
4、体外转录合成含有目的基因的自复制mRNA
基于MEGAscriptT7 Transcription Kit试剂盒提供的方法，对重组质粒SMPD-1进行体外转录，制得含有目的基因的自复制mRNA。
5、将含有目的基因的自复制mRNA通过电穿孔法直接转染至TIL细胞中，即得自复制mRNA增强型TIL细胞。
试验例1mRNA转染验证
以EGFP mRNA作为试验样本转染TIL细胞，验证采用mRNA直接转染TIL细胞的转染效果。
试验方法：
(1)预先将培养基加入12孔板内，每孔2mL，随后将12孔板置于细胞培养箱内在37℃、5％CO2的条件下预热1小时；
(2)配制电转液；
(3)取实施例1获得的TIL细胞至EP管内，每个EP管内加入5×106个细胞，1200rpm离心5min，弃上清，随后用500μL生理盐水重悬细胞，重复离心步骤洗涤细胞沉淀；
(4)向步骤(2)中配置好的电转液加入EGFP mRNA 10μg，随后室温静置10min以内；
(5)用步骤(4)中配置好的含mRNA的电转液重悬EP管内的TIL，每管100μL，小心吸取细胞重悬液转入电转杯内，将电转杯放入电转槽内，启动电转程序，开始电转；
(6)电转完成后小心取出电转杯，吸取细胞悬液转移至EP管中，每管加入预热的培养基，随后转入步骤(1)中的、内含有预热培养基的12孔板中，37℃、5％CO2培养箱培养；
(7)对电转后第1天、第3天、第5天和第7天的TIL细胞进行荧光拍照，结果如图2所示；并取样进行流式细胞仪检测电转阳性率，结果如图3所示。
结果表明，转染mRNA后阳性率极高并且随时间降低。
试验例2
作为试验例1的对比实验，本试验采用EGFP质粒系统转染TIL细胞。
方法参考试验例1，不同的是，本试验步骤(4)中，是加入EGFP质粒5μg。
对电转后第2天和第5天的TIL细胞进行荧光拍照，结果如图4所示。
图中结果表明，质粒系统转染TIL细胞转染效率较低，维持在50-60％之间，但转染后细胞损伤很大难以恢复，后续对TIL细胞大量扩增带来很大不确定因素。
综合试验例1和试验例2的结果可知，相比质粒系统转染的低效和高细胞毒性，直接用mRNA转染TIL细胞体现出高效和低细胞毒性，mRNA 在D5天转染效率大大下降，D9天完全检测不到，可以有效避免细胞毒性和潜在的转基因影响。
采用mRNA转染TIL细胞，其细胞毒性极低有利于TIL细胞后续大量扩增，转染效率极高，安全性极佳。
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
序列表
<110> 上海兰艾生物医疗科技有限公司
<120> 自复制mRNA增强型TIL细胞及其制备方法与应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7478
<212> DNA
<213> 人工序列
<400> 1
acaggcgcgc tttagcgcga tacgatgtgt cacacagtgc gcgtagacat tagacagcat 60
gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg gctggcggcc 120
aggatggttc ttaggtaggt ggggtcggcg gtcaggtgtc ccagagccag gggtctggag 180
ggaccttcca ccctcagtcc ctggcaggtc ggggggtgct gaggcgggcc tggccctggc 240
agcccagggg tcccggagcg aggggtcgcg cgctactctc tgagatctcg cgctatagat 300
cagacgatag acgatacaga tacgatcgta gctagctgca tagcatagtg gagggacctt 360
tcactctcag tccctggcag gtcggggggt gctgtggcag gcccagcctt ggcccccatg 420
ggttcctctc tgggccccaa ctcctcccct ggcccaagtc ccctctttgc tcctgggcag 480
ctcagccaga atgacgggtc aggctggcca gaaccccaaa ccttggccgt ggggagtctg 540
cgtggcggct ctgccttgcc caggcatcct tggtcctcac tcgagttttc ctaaggcttt 600
acgacgtcaa agtttagatg agctggtgat atttttctca ttatatccaa agtgtacctg 660
ttcgagtgag gacagttctt ctgtctccag gatccctcct gggtggggat tgtgcccgcc 720
tgggtctctt agggcccgcg cgccggatat atagcgcgcc cgctcctgga ccctctgtgc 780
ccggctggcc ctctgtgccc agccagcctg cacctgggga tgccaaggcc tggggagggt 840
ggtttcaccc aggccaagcc taagacagtc cctctgggcc ctgctgggta ccggggtgtg 900
acaccactgg gaggacaaga tgaggggcac ccctggggcc gccctgacac cccctcgagg 960
ctcctgcccc gggggtcctg gtgccccttc actgtggcag gcgactgggg gttccccacc 1020
tcggcccctc cagggccgtg cacctcttaa ggttggcccg tgggtcactc aggactcaga 1080
actggagatt ctgggcaaaa ggcaaagagc aaagggccct ccccgaccca ggcgggaaag 1140
ataagagcaa ggtgtccgca gcctgacact cgtgcctcag gtgcccgcgc ttgtgccgga 1200
caagactctc acaggtggca tgcctcggtt tccccactgg taacagcaca gggcactcgt 1260
cctgtgggtc ctgacccaga tgtggccacc ccggccgcag tggtcttcat tccaggatgc 1320
ctcttttccc tcctgatcta ttcactgcgt tcgccattcg gtcattcccg gggccaccac 1380
tcccacctca ggtgtgtgct tcccttgtgt tttatgagat atccccaacc cggctgctta 1440
ttggccccgt ccgagggcag gagcataaat aagagcctcc cagtcagcgc tgccactgct 1500
gagctctggg ccttcgacag gacttggccc cttactggcg tcctgaggat taaatcgggt 1560
catctgggac tataggcacg tgccaccctg ccaggctaat ttcttccatt ttttttagag 1620
acagggtctc gctatgttcc ccaggctggg ctcaaatggt cctcccacct cagcctcccc 1680
aagtactggg attacaggca taagccactg catctggcct ccatgacaca tatttttaaa 1740
gtctgatttt taaagtcaaa cttttgaagt cagattttaa acggactatt ttgaaaaata 1800
tacaaaaacg tttaaaaaca atgaatatcc ctcacctaga atcaataact aagaatattg 1860
acacatttgc tttggggact gggcggctgg agctgccatg acaaagctcc gccgaccgga 1920
ctgagggctg gacgtgcagg atggagctcc gcagggtcgg ctcccctgtg ctctgagggg 1980
ctctgctcag cctctcccgg ctgtggctta aaaacagagc ctgtcagggt cggctccctg 2040
tgctccgaga gggctctgct cagcttctcc tggctggggg gttttgtggc caccctctgt 2100
gttcctgggt tcagaagcat cccccaggct ctgccttcat ctctgcacgg gtgactctgt 2160
acaggaagcc aggcctgctg gtcaatggcc acccagccct gtgccctcat cttacctagt 2220
cccagctgcc gtcaccatgg ggttaggact tccacatagg aatctgtggg gacacggttc 2280
ggcccacagc ccttcccacc tccacacaca cacacggcag ccttcttctg tgcgcttctg 2340
gaaagcaatg tccccaccgc cacctcacag gcctgttcca cagccccggg atgggctctg 2400
tctccctcct gaccctgcat agggcacagc cctctctcat caacccacga tcctacgtgg 2460
atccgagagt cccatagaaa caccccaaat ctaacttgat ccaggaccag ccagtggtca 2520
cttctgaata ttcaccttcc tagtagacac taccagccaa gggaggccag gaagccttcc 2580
tggaggaggt ggcctgagga ctggggtgag gcaggccctg cgtgggggtc gccacccagc 2640
acccccacac tgggtgggag ccagtctctg agactggctg ggggaggtgg gagagggggc 2700
tgcttgaact gcagacaccg aggtctagcc cccaccccac ccagccagtt ggtggaggca 2760
ggggaggccg aggggcccag ctggacctgc tccccggggt ggattccaaa ataggggggt 2820
tggggggggc ggaacaggag cccagggtcc tggcttgagg cccagtggct gagggctggt 2880
gcaagccaga caggaaaagg gttgagcctg tcagcgccag cacagatcaa gtcaggagca 2940
ggtccctcca ccaatgtgtg caaataaata gcagctaagt ttccagttac aagaacaatg 3000
cacagatggt cccagggaca ttgcggtgtg gacacacagc ggccattgtc ctgtcgccag 3060
cacctcgccc tacagctggg gggtccctta gcacttccta gccatgcagg gtccctgctc 3120
acagtacccg tgatgacttc tgttcctcac ctgcctgtct gtcccgacag ctgcatggca 3180
gccctggcct gggagatgga gaccccgagg ggctgcctgc ggtggtgggg ttatatatat 3240
atatcggcgc tcgctctatt aattactctc ggccgttctc gagagtcgcg ccacacacat 3300
ctctcgagag ctctacacgt gtgtaccatc tcggggcccc ctagagggca gggcatttcc 3360
cctgctctgt gccgagtcca cccagcccca gcctaggccc agtaagggct gcagcccacc 3420
ctgtcccagg ctgcctccca ggagccctct tggccctgat gccagaagcc catcttcctc 3480
cattcaggca ggtctctgag tgccctggcc tggctgcctg ctggccctga gagtcacact 3540
accccacagc cctccttggt caaaatccac tctggagtgg ctggaagatt ccccgggccc 3600
acgccgcaca cgcctatgca gggagcttcc cctggccggc cggcagacaa gggcggtctc 3660
agagaggggg ctcacctcag cagccccttg tgtagctggc cctcgcccct gccacctctg 3720
ggaacaccac caggaagctg ggggacaggc acgcaggtga aggaggcgag cgcttgtcag 3780
ccgggaggcc atgggcacag agggaacagg gacaccctgg gtggcctcaa ggtcacttca 3840
aacccctcac tcgtcccctg ggagggtgcc cagtgaggtt ggcactacca cccacctctg 3900
gtgtccagcc agcacgccgt gggccagcct ggctgcaggg acacgagggc agcagccccc 3960
tcctcctctg agctggttgc tccttgagtc atcaccaccg cctgccacgg aggccgcctg 4020
tcccaggaag cagagggacc gcagctgtgg caaccagggc ctggtctctg tgtcacctcg 4080
ctggggggcc gtgcccaggc ctgagacgga actgagtgct ggcggaagag ccgcgacccc 4140
cacgttgcca tgcgggtatc ccaagccctg accctggcag gcatatgttt caggaggtcc 4200
ttgtcttggg agcccagggt cgggggcccc gtgtctgtcc acatccgagt caatggccca 4260
tctcgtctct gaagcatctt tgctgtgagc tctagtcccc actgtcttgc tggaaaatgt 4320
ggaggcccca ctgcccactg cccagggcag caatgcccat accacgtggt cccagctccg 4380
agcttgtcct gaaaaggggg caaagactgg accctgagcc tgccaagggg ccacactcct 4440
cccagggctg gggtctccat gggcagcccc ccacccaccc agaccagtta cactcccctg 4500
tgccagagca gtgcagacag gaccaggcca ggatgcccaa gggtcagggg ctggggatgg 4560
gtagccccca aacagccctt tctgggggaa ctggcctcaa cggggaaggg ggtgaaggct 4620
cttagtagga aatcagggag acccaagtca gagccaggtg ctgtgcagaa gctgcagcct 4680
cacgtagaag gaagaggctc tgcagtggag gccagtgccc atccccgggt ggcagaggcc 4740
ccagcagaga cttctcaatg acattccagc tggggtggcc cttccagagc ccttgctgcc 4800
cgagggatgt gagcaggtgg ccggggaggc tttgtggggc cacccagccc cttcctcacc 4860
tctctccatc tctcagactc cccagacacc tgctcgtggt gaccgaaggg gacaacgcca 4920
ccttcacctg cagcttctcc aacacatcgg agagcttcgt gctaaactgg taccgcatga 4980
gccccagcaa ccagacggac aagctggccg ccttccccga ggaccgcagc cagcccggcc 5040
aggactgccg cttccgtgtc acacaactgc ccaacgggcg tgacttccac atgagcgtgg 5100
tggggccatc tccctggccc ccaaggcgca gatcaaagag agcctgcggg cagagctcag 5160
ggtgacaggt gcggcctcgg aggccccggg gcaggggtga gctgagccgg tcctggggtg 5220
ggtgtcccct cctgcacagg atcaggagct ccagggtcgt agggcaggga ccccccagct 5280
ccagtccagg gctctgtcct gcacctgggg aatggtgacc ggcatctctg tcctctagct 5340
ctggaagcac cccagcccct ctagtctgcc ctcacccctg accctgaccc tccaccctga 5400
ccccgtccta acccctgacc tttgtgccct tcccacccca gcccctcacc caggccagcc 5460
ggccagttcc aaaccctggt ggttggtgtc gtgggcggcc tgctgggcag cctggtgctg 5520
ctagtctggg tcctggccgt catctgctcc cgggccgcac gaggtaacgt catcccagcc 5580
cctcggcctg ccctgcccta accctgctgg cggccctcac tcccgcctcc ccttcctcca 5640
cccttccctc accccacccc acctcccccc atctccccgc caggctaagt ccctgatgaa 5700
ggcccctgga ctaagacccc ccacctagga gcacggctca gggtcggcct ggtgacccct 5760
aggagccagg cgcaccggcc agcccctggt gagtctcact cttttcctgc atgatccact 5820
gtgccttcct tcctgggtgg gcagaggtgg aaggacaggc tgggaccaca cggcctgcag 5880
gactcacatt ctattatagc caggacccca cctccccagc ccccaggcag caacctcaat 5940
ccctaaagcc atgatctggg gccccagccc acctgcggtc tccgggggtg cccggcccat 6000
gtgtgtgcct gcctgcggtc tccaggggtg cctggcccac gcgtgtgccc gcctgcggtc 6060
tctgggggtg cccggcccac atatgtgcct gcctgcggtc tccaggtgtg cccggcccat 6120
gcgtgtgccc acctgcgagg gcgtggggtg ggcttggtca tttcttatct tacattggag 6180
acaggagagc ttgaaaagtc acagggggac attgagccag agaggagggg tggtgtcccc 6240
agatcacaca gagggcagtg gtgggacagc tcagggtaag cagctcatag tggggggccc 6300
aggttcggtg ccggtactgc agccaggctg tggagccgcg ggcctccttc ctgcggtggg 6360
ccgtggggct gactccctct ccctttctcc tcaaagaagg aggacccctc agccgtgggc 6420
gaccggggct ccagcctgca cctgcaccag gcacagcccc accacaggac tcatgtctca 6480
atgcccacag tgagcccagg cagcaggtgt caccgtcccc tacagggagg gccagatgca 6540
gtcactgctt caggtcctgc cagcacagag ctgcctgcgt ccagctccct gaatctctgc 6600
tgctgctgct gctgctgctg ctgctgcctg cggcccgggg ctgatgcctg aacttggggg 6660
ctggttggag atggccttgg agcagccaag gtgcccctgg cagtggcatc ccgaaacgcc 6720
ctggacgcag ggcccaagac tgggcacagg atcgcgtatc gggggggttt tatatatagg 6780
gcgcgctggt gtccccccgc gcgcaaattc tctctgggtg tgtcccaaaa gggagagaga 6840
cttctctgcg cgcgttatat atatacgcgc ctcgctcgct cgctctagct gtacgctagc 6900
gtgcatgcat atagggaagg tcagaagagc tcctggctgt ggtgggcagg gcaggaaacc 6960
cctccacctt tacacatgcc caggcagcac ctcaggccct ttgtggggca gggaagctga 7020
ggcagtaagc gggcaggcag agctggaggc ctttcaggcc cagccagcac tctggcctcc 7080
tgccgccgca ttccacccca gcccctcaca ccactcggga gagggacatc ctacggtccc 7140
aaggtcagga gggcagggct ggggttgact caggcccctc ccagctgtgg ccacctgggt 7200
gggagctctc cttggaaccc attcctgaaa ttatttaaag gggttggccg ggctcccacc 7260
agggcctggg tgggaaggta caggcgttcc cccggggcct agtacccccg ccgtggccta 7320
tccactcctc acatccacac actgcacccc cactcctggg gcagggccac cagcatccag 7380
gcggccagca ggcacctgag tggctgggac aagggatccc ccttccctgt ggttctatta 7440
tattataatt ataattaaat atgagagcat gctaagga 7478
<210> 2
<211> 7114
<212> DNA
<213> 人工序列
<400> 2
acaggcgcgc tttagcgcga tacgatgtgt cacacagtgc gcgtagacat tagacagcat 60
gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg gctggcggcc 120
aggatggtcc cagagccagg ggtctggcct ttaatatacg acgcgcgctc ttctcgagag 180
agagtctctc gagaagagtc tcctgagagc ttcctgatag tgcgcgtaga taacagatac 240
agatacgata gcatagagag ggaccttcca ccctcagtcc ctggcaggtc ggggggtgct 300
gaggcgggcc tggccctggc agcccagggg tcccggagcg aggggtcgcg cgctactctc 360
tgagatctcg cgctatagat cagacgatag acgatacaga tacgatcgta gctagctgca 420
tagcatagtg gagggacctt tcactctcag tccctggcag gtcggggggc ccaagtcccc 480
tctttgctcc tgggcagctc agccagaatg acgggtcagg ctggccagaa ccccaaacct 540
tggccgtggg gagtctgcgt ggcggctctg ccttgcccag gcatccttgg tcttctatac 600
gacgcgcgct cttctcgaga gagagtctct cgagaagagt ctcctgagag cttcctgata 660
gtgcgcgtag ataacagata cagatacgat agcatagagc tcactcgagt tttcctaagg 720
ctttacgacg tcaaagttta gatgagctgg tgatattttt ctcattatat ccaaagtgta 780
cctgttcgag tgaggacagt tctttagggc ccgcgcgccg gatatatagc gcgcccgctc 840
ctggaccctc tgtgcccggc tggccctctg tgcccagcca gcctgcacct ggggatgcca 900
aggcctgggg agggtggttt cacccaggcc aagcctaaga cagtccctct gggccctgct 960
gggtaccggg gtgtgacacc actgggagga caagatgagg ggcacccctg gggccgccct 1020
gacaccccct cgaggctcct gccccggggg tcctggtgcc ccttcactgt ggcaggcgac 1080
tgggggttcc ccacctcggc ccctccaggg ccgtgcacct cttaaggttg gcccgcccga 1140
cccaggcggg aaagataaga gcaaggtgtc cgcagcctga cactcgtgcc tcaggtgccc 1200
gcgcttgtgc cggacaagac tctcacaggt ggcatgcctc ggtttcccca ctggtaacag 1260
cacagggcac tcgtcctgtg ggtcctgacc cagatgtggc caccccggcc gcagtggtct 1320
tcattccagg atgcctcttt tccctcctga tctattcact gcgttcgcca ttcggtcatt 1380
cccggggcca ccactcccac ctcaggtgtg tgcttccctt gtgttttatg agatatcccc 1440
aacccggctg cttattggcc ccgtcccctt cgacaggact tggcccctta ctggcgtcct 1500
gaggattaaa tcgggtcatc tgggactata ggcacgtgcc accctgccag gctaatttct 1560
tccatttttt ttagagacag ggtctcgcta tgttccccag gctgggctca aatggtcctc 1620
ccacctcagc ctccccaagt actgggatta caggcataag ccactgcatc tggcctccat 1680
gacaccagat tttaaacgga ctattttgaa aaatatacaa aaacgtttaa aaacaatgaa 1740
tatccctcac ctagaatcaa taactaagaa tattgacaca tttgctttgg ggactgggcg 1800
gctggagctg ccatgacaaa gctccgccga ccggactgag ggctggacgt gcaggatgga 1860
gctccgcagg gtcggctccc ctgtgctctg aggggctctg ctcagcctct cccggctgtg 1920
gcttaaaaac agagcctgtc agggtcggct ccctgtgctc cgagagggct ctgctcagct 1980
tctcctggct ggggggtttt gtggccaccc tctgtgttct ctctatatat tatacgacgc 2040
gcgctcttct cgagagagag tctctcgaga agagtctcct gagagcttcc tgatagtgcg 2100
cgtagataac agatacagat acgatagcat agagtcctgg gttcagaagc atcccccagg 2160
ctctgccttc atctctgcac gggtgactct gtacaggaag ccaggcctgc tggtcaatgg 2220
ccacccagcc ctgtgcagga cttccacata ggaatctgtg gggacacggt tcggcccaca 2280
gcccttccca cctccacaca cacacacggc agccttcttc tgtgcgcttc tggaaagcaa 2340
tgtcctgtct ccctcctgac cctgcatagg gcacagccct ctctcatcaa cccacgatcc 2400
tacgtggatc cgagagtccc atagaaacac cccaaatcta acttgatcca ggaccagcca 2460
gtggtcactt ctgaatattc accttcctag tagacactac cagccaaggg aggccaggaa 2520
gccttcctgg aggaggtggc ctgaggactg gggtgaggca ggccctgcgt gggggtcgcc 2580
acccagcacc cccacactgg gtgggagcca gtctctgaga ctggctgggg gaggtgggag 2640
agggggctgc ttgaactgca gacaccgagg tctagccccc acccaggggc ccagctggac 2700
ctgctccccg gggtggattc caaaataggg gggttggggg gggcggaaca ggagcccagg 2760
gtcctggctt gaggcccagt ggctgagggc tggtacagat caagtcagga gcaggtccct 2820
ccaccaatgt gtgcaaataa atagcagcta agtttccagt tacaagaaca atgcacagat 2880
ggtcccaggg acattgcggt gtggacacac ttatatatta tacgacgcgc gctcttctcg 2940
agagagagtc tctcgagaag agtctcctga gagcttcctg atagtgcgcg tagataacag 3000
atacagatac gatagcatag agagcggcca ttgtcctgtc gccagcacct cgccctacag 3060
ctggggggtc ccttagcact tcctagccat gcagggtccc tgctcacagt acccgtgatg 3120
gcagccctgg cctgggagat ggagaccccg aggggctgcc tgcggtggtg gggttatata 3180
tatatatcgg cgctcgctct attaattact ctcggccgtt ctcgagagtc gcgccacaca 3240
catctctcga gagctctaca cgtgtgtacc atctcggggc cccctagagg gcagcaccct 3300
gtcccaggct gcctcccagg agccctcttg gccctgatgc cacagccctg agagtcacac 3360
taccccacag ccctccttgg tcaaaatcca ctctggagtg gctggaagat tccccgggcc 3420
cacgccgcac acgcctatgc agggagcttc ccctggccgg ccggcagaca agggcggtct 3480
cagagagggg gctcacctca gcagcccctt gtgtagctgg caccaggaag ctgggggaca 3540
ggcacgcagg tgaaggaggc gagcgcttgt cagccgggag gccatgggca cagagggaac 3600
agggacaccc tgggtggcct caaggtcact tcaaacccct cactcgtccc ctgggagggt 3660
gcccagtgag agggacacga gggcagcagc cccctcctcc tctgagctgg ttgctccttg 3720
agtcatcacc accgcctgcc acggaggccg cctgtcccag gaagcagagg gaccgcagct 3780
gtggcaacca gggcctggtc tctgtgtcac ctcgctgggg ggccgtgccc aggcctgaga 3840
cggaactgag tgctggcgga agagccgcga cccccacgtt gccatgcggg tatcccaagc 3900
cctgaccctg gcaggcatat gtttcaggag gtccttgtct tgggagccca gggtcggggg 3960
ccccgtgtct gtccacatcc gagtcaatgg cccatctcgt ctactcctcc cagggctggg 4020
gtctccatgg gcagcccccc acccacccag accagttaca ctcccctgtg ccagagcagt 4080
gcagacagga ccaggccagg atgcccaagg gtcaggggct ggggatgggt agcccccaaa 4140
cagccctttc tgggggaact ggcctcaacg gggaaggggg tgaaggctct tagtaggaaa 4200
tcagggagac ccaagtcaga gccaggtgct gtgcagaagc tgcagcctca cgtagaagga 4260
agaggctctg cagtggaggc cagtgcccat ccccgggtgg cagaggcccc agcagagact 4320
tctcaatgac attccagctg gggtggccct tccagagccc ttgctgcccg agggatgtga 4380
gcaggtggcc ggggaggctt tgtggggcca cccagcccct tcctcacctc tctccatctc 4440
tcagactccc cagacacctg ctcgtggtga ccgaagggga caacgccacc ttcacctgca 4500
gcttctccaa cacatcggag agcttcgtgc taaactggta ccgcatgagc cccagcaacc 4560
agacggacaa gctggccgcc ttccccgagg accgcagcca gcccggccag gactgccgct 4620
tccgtgtcac acaactgccc aacgggcgtg acttccacat gagcggagag cctgcgggca 4680
gagctcaggg tgacaggtgc ggcctcggag gccccggggc aggggtgagc tgagccggtc 4740
ctggggtggg tgtcccctcc tgcacaggat caggagctcc ctatacgacg cgcgctcttc 4800
tcgagagaga gtctctcgag aagagtctcc tgagagcttc ctgatagtgc gcgtagataa 4860
cagatacaga tacgatagca tagaggctct gtcctgcacc tggggaatgg tgaccggcat 4920
ctctgtcctc tagctctgga agcaccccag cccctctagt ctgccctcac ccctgaccct 4980
gaccctccac cctgaccccg tcctaacccc tgacctttgt gcccttccca ccccagcccc 5040
tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg cggcctgctg 5100
ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccgggc cgcacgaggt 5160
aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc ctcactcccg 5220
caccccacct ccccccatct ccccgccagg ctaagtccct gatgaaggcc cctggactaa 5280
gaccccccac ctaggagcac ggctcagggt cggcctggtg acccctagga gccaggcgca 5340
ccggccagcc cctggtgagt ctcactcttt tcctgcatga tccactgtgc cttccttcct 5400
gggtgggcag aggtggaagg acaggctggg accacacggc ctgcaggact cacattctat 5460
tatagccagg accccacctc cccagccccc aggcagcaac ctcaatccct aaagccatga 5520
tctggggccc cagcccacct gcggtcgcgg tctccagggg tgcctggccc acgcgtgtgc 5580
ccgcctgcgg tctctggggg tgcccggccc acatatgtgc ctgcctgcgg tctccaggtg 5640
tgcccggccc attatacgac gcgcgctctt ctcgagagag agtctctcga gaagagtctc 5700
ctgagagctt cctgatagtg cgcgtagata acagatacag atacgatagc atagaggcgt 5760
gtgcccacct gcgagggcgt ggggtgggct tggtcatttc ttatcttaca ttggagacag 5820
gagagcttga aaagtcacag ggggacattg agccagagag gaggggtggt gtccccagat 5880
cacacagagg gcagtggtgg gacagctcag ggtaagcagc tcatagtggg gggcccaggt 5940
tcggtgccgg tactgcagcc aggctgtgga gccgcgggcc tccttcctgc ggtgggccgt 6000
ggggctgact ccctctccct ttctcctcaa agaaggagga cccctcagcc gtgggcgacc 6060
ggggctccag cctgcacctg caccaggcac agccccacca caggactcat gtctcaatgc 6120
ccacagtgag cccaggcagc aggtgtcacc gtcccctaca gggagggcca gatgcagtca 6180
ctgcttcagg tcctgccagc acagagctgc ctgcgtccag ctccctgaat ctctgctgct 6240
gctgctgctg ctgctgctgc tgcctgcggc ccggggctga tgcctgaact tgggggctgg 6300
ttggagatgg ccttggagca gccaaggtgc ccctggcagt ggcatcccga aacgccctgg 6360
acgcagggcc caagactggg cacaggatcg cgtatcgggg gggttttata tatagggcgc 6420
gctggtgtcc ccccgcgcgc aaattctctc tgggtgtgtc ccaaaaggga gagagacttc 6480
tctgcgcgcg ttatatatat acgcgcctcg ctcgctcgct ctagctgtac gctagcgtgc 6540
atgcatatag ggaaggtcag aagagctcct ggctgtggtg ggcagggcag gaaacccctc 6600
cacctttaca catgcccagg cagcacctca ggccctttgt ggggcaggga agctgaggca 6660
gtaagcgggc aggcagagct ggaggccttt caggcccagc cagcactctg gcctcctgcc 6720
gccgcattcc accccagccc ctcacaccac tcgggagagg gacatcctac ggtcccaagg 6780
tcaggagggc agggctgggg ttgactcagg cccctcccag ctgtggccac ctgggtggga 6840
gctctccttg gaacccattc ctgaaattat ttaaaggggt tggccgggct cccaccaggg 6900
cctgggtggg aaggtacagg cgttcccccg gggcctagta cccccgccgt ggcctatcca 6960
ctcctcacat ccacacactg cacccccact cctggggcag ggccaccagc atccaggcgg 7020
ccagcaggca cctgagtggc tgggacaagg gatccccctt ccctgtggtt ctattatatt 7080
ataattataa ttaaatatga gagcatgcta agga 7114
<210> 3
<211> 11023
<212> DNA
<213> 人工序列
<400> 3
atgggcggcg catgagagaa gcccagacca atgagctttg cagcggagct tcccgcagtt 60
tgaggtagaa gccaagcagg tcactgataa tgaccatgct aatgccagag cgttttcgca 120
tctggcttca aaactgatcg aaacggaggt ggacccatcc gacacgatcc ttgacattgg 180
aagtgcgccc gcccgcagaa tgtattctaa gcacaagtat cattgtatct gtccgatgag 240
atgtgcggaa gatcctgaag aaaaactgta aggaaataac tgataaggaa ttggacaaga 300
aaatgaagga gctcgccgcc gtcatgagcg accctgacct ggaaactgag actatgtgcc 360
tccacgacga cgagtcgtgt cgctacgaag ggcaagtcgc tgtttaccag gatgtatacg 420
cggttgacgg accgacaagt ctctatcacc aagccaataa gggagttaga gtcgagaact 480
tggctggacc aactgggccg acgaaaccgt gttaacggct cgtaacatag gcctatgcag 540
ctctgacgtt atggagcggt cacgtagagg gatgtccatt cttagaaaga agtatttgaa 600
accatccaac aatgttctat tctctgttgg ctcgaccatc taccacgaga agagggactt 660
actgaggagc tggcacctgc cgtctgtatt tcacttacgt ggcaagcaaa attacacatg 720
tcggtgtgag actatagtta gttgcgacgg gtacgtcgtt aaaagaatag ctatcagtcc 780
aggcctgtat gggaagcctt caggcgacag acacattgaa cggggagagg gtctcttttc 840
ccgtgtgcac gtatgtgcca gctacattgt gtgaccaaat gactggcata ctggcaacag 900
atgtcagtgc ggacgacgcg caaaaagtga acagcgattt ccactcattc gtgctgccca 960
ggataggcag taacacattg gagatcgggc tgagaacaag aatcaggaaa atgttagagg 1020
agcacaagga gccgtcacct ctcattaccg ccgaggacgt acaagaagct aagtgcgcag 1080
ccgatgaggc taaggaggtg cgtgaagccg aggagttgcg cgcagctcta ccacctttgg 1140
cagctgatgt tgaggaaaat tatcttgcat ccaccctctc gctgaacaag tcatagtgat 1200
aacacactct ggccgaaaag ggcgttatgc cgtggaacca taccatggta aagtagtggt 1260
gccagaggga catgcaatac ccgtccagga ctttcaagct ctgagtgaaa gtgccaccat 1320
tgtgtacaac gaacgtgagt tcgtaaacag gtacctgcac catattgcca cacatggagg 1380
agcgctgaac actgatgaag aatattacaa aactgtcaag cccagcgagc acgacggcga 1440
atacctgtac gacatcgaca ggaaacagtg cgtcaagaaa gaactagtca ctgggctagg 1500
gctcacaggc gagctggtgg atcctccctt ccatgaattc gcctacgaga gtctgagaac 1560
acgaccagcc gctgtggact cagtgctctt gaatggatgc aaacaccccg tagagaccct 1620
gtatattgac gaagcttttg cttgtcatgc aggtactctc agagcgctca tagccattat 1680
aagacctaaa aaggcagtgc tctgcgggga tcccaaacaa ccacgagatt tgcacacaag 1740
tcttccacaa aagcatctct cgccgttgca ctaacagttg caaatagatt acaaaggcaa 1800
cgaaataatg acggcagctg cctctcaagg gctgacccgt aaaggtgtgt atgccgttcg 1860
gtacaaggtg aatgaaaatc ctctgtacgc acccacctca gaacatgtga acgtcctact 1920
gacccagacc ggaccctacc gacgtcttcc agaataaggc aaacgtgtgt tgggccaagg 1980
ctttagtgcc ggtgctgaag accgctggca tagacatgac cactgaacaa tggaacactg 2040
tggattattt tgaaacggac aaagctcact cagcagagat agtattgaac caactatgcg 2100
tgaggttctt tggactcgat ctggactccg gtctattttc tgcacccact gttccgttat 2160
ccattaggaa taatcactgg gataactccc cgtcgcctaa catgtacggg ctgaataaag 2220
aagtggtccg tcagctctct cgcaggtacc cacaactgcc tcgggcagtt gccactggaa 2280
gagtctatga catgaacact ggtacactgc gcaattatga tccgcgcata aacctagtac 2340
ctgtaaacag aagactgcct catgctttag tcctccacca taatgaacac ccacagagtg 2400
acttttcttc attcgtcagc aaattgaagg gcagaactgt cctggtggtc ggggaaaagt 2460
tgtccgtccc aggcaaaatg gttgactggt tgtcagaccg gcctgaggct accttcagag 2520
ctcggctgga tttaggcatc ccaggtgatg tgcccaaata tgacataata tttgttaatg 2580
tgaggacccc atataaatac catcactatc agcagtgtga agaccatgcc attaagctta 2640
gcatgttgac caagaaagct tgtctgcatc tgaatcccgg cggaacctgt gtcagcatag 2700
gttatggtta cgctgacagg gccagcgaaa gcatcattgg tgctatagcg cggctgttca 2760
agttttcccg ggtatgcaaa ccgaaatcct cacttgaaga gacgggtacg cacaatcctt 2820
acaagctttc atcaaccttg accaacattt atacaggttc cagactccac gaagccggat 2880
gtgcaccctc atatcatgtg gtgcgagggg atattgccac ggccaccgaa ggagtgatta 2940
taaatgctgc taacagcaaa ggacaacctg gcggaggggt gtgcggagcg ctgtataaga 3000
aattcccgga aagcttcgat ttacagccga tcgaagtagg aaaagcgcga ctggtcaaag 3060
gtgcagctaa acatatcatt catgccgtag gaccaaactt caacaaagtt tcggaggttg 3120
aaggtgacaa aataacaatt acaagtcagt agcgattcca ctgttgtcca ccggcatctt 3180
ttccgggaac aaagatcgac taacccaatc attgaaccat ttgctgacag ctttagacac 3240
cactgatgca gatgtagcca tatactgcag ggacaagaaa tgggaaatga ctctcaagga 3300
agcagtggct aggagagaag cagtggagga gatatgcata tccgacgact cttcagtgac 3360
agaacctgat gcagagctgg tgagggtgca tccgaagagt tctttggctg gaaggaaggg 3420
ctacagcaca agcgatggca aaactttctc atatttggaa gggaccaagt ttcaccaggc 3480
ggccaaggat atagcagaaa ttaatgccat gtggcccgtt gcaacggagg ccaatgagca 3540
ggtatgcatg tgagtcggaa gcctccacac cacctagcac gctgccttgc ttgtgcatcc 3600
atgccatgac tccagaaaga gtacagcgcc taaaagcctc acgtccagaa caaattactg 3660
tgtgctcatc ctttccattg ccgaagtata gaatcactgg tgtgcagaag atccaatgct 3720
cccagcctat attgttctca ccgaaagtgc ctgcgtatat tcatccaagg aagtatctcg 3780
tggaaacacc accggtagac gagactccgg agccatcggc agagaaccaa tccacagagg 3840
ggacacctga acaaccacca cttataaccg aggatgagac caggactggt ccattcctca 3900
tgcatccgac tttgatgtgg acagtttatc catacttgac accctggagg gagctagcgt 3960
gaccagcggg gcaacgtcag ccgagactaa ctcttacttc gcaaagagta tggagtttct 4020
ggcgcgaccg gtgcctgcgc ctcgaacagt attcaggaac cctccacatc ccgctccgcg 4080
cacaagaaca ccgtcacttg cacccagcag ggcctgctcg aatagggtga ttacaagaga 4140
ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg catacatctt 4200
ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa cggtgctatc 4260
cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc tcgaccaaga 4320
aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta acagaagcag 4380
ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta ttctgcaagg 4440
cctagggcat tatttgaagg cagaaggaaa aggcgccgcg tctctctctc atatatagga 4500
agagtctcgc tcgctctatt atatcttgga gtgctaccga accctgcatc ctgttccttt 4560
gtattcatct agtgtgaacc gtgccttttc aagccccaag gtcgcagtgg aagcctgtaa 4620
cgccatgttg aaagagaact ttccgactgt ggcttcttac tgtattattc cagagtacga 4680
tgcctatttg gacatggttg acggagcttc atgctgctta gacactgcca gtttttgccc 4740
tgcaaagctg cgcagctttc caaagaaaca ctcctatttg gaacccacaa tacgatcggc 4800
agtgccttca gcgatccaga acacgctcca gaacgtcctg gcagctgcca caaaaagaaa 4860
ttgcaatgtc acgcaaatga gagaattgcc cgtattggat tcggcggcct ttaatgtgga 4920
atgcttcaag aaatatgcgt gtaataatga atattgggaa acgtttaaag aaaaccccat 4980
caggcttact gaagaaaacg tggtaaatta cattaccaaa ttaaaaggac caaaagctgc 5040
tgcttgccga tccgctagca acagcgtatc tgtgcggaat ccaccgagag ctggttagga 5100
gattaaatgc ggtcctgctt ccgaacattc atacactgtt tgatatgtcg gctgaagact 5160
ttgacgctat tatagccgag cacttccagc ctggggattg tgttctggaa actgacatcg 5220
cgtcgtttga taaaagtgag gacgacgcca tggctctgac cgcgttaatg attctggaag 5280
acttaggtgt ggacgcagag ctgttgacgc tgattgaggc ggctttcggc gaaatttcat 5340
caatacattt gcccactaaa actaaattta aattcggagc catgatgaaa tctggaatgt 5400
tcctcacact gtttgtgaac acagtcatta acattgtaat cgcaagcaga gtgttgagag 5460
aacggctaac cggatcacca tgtgcagcat tgcatatata tgatagatac agatagatac 5520
attggagatg acaatatcgt gaaaggagtc aaatcggaca aattaatggc agacaggtgc 5580
gccacctggt tgaatatgga agtcaagatt atagatgctg tggtgggcga gaaagcgtgt 5640
gactccgtga ccggcacagc gtgccgtgtg gcagaccccc taaaaaggct gtttaagctt 5700
ggcaaacctc tggcagcaga cgatggtatt ctttcagagc tgatcatagt tatggccatg 5760
actactctag ctagcagtgt taaatcattc agctacctga gaggggcccc tataactctc 5820
tacggctaac ctgaatggac tacgacatag tctagtccgc caagatgttc gtgttcctcg 5880
tgctgctgcc cctggtgtct agccagtgcg tgaacctgac caccagaacc cagctgcctc 5940
cagcctacac caacagcttc accaggggag tgtactaccc cgacaaggtg ttccggagca 6000
gcgtgctgca tagcacccag gacctgttcc tgcccttctt cagcaacgtg acttggttcc 6060
acgccatcca cgtcagcgga aagcgctttg acaacccagt gctgcccttc aacgacggag 6120
tgtacttcgc cagcaccgag aagtccaaca tcatccgggg ttggatcttc ggcaccacac 6180
gtcgtgatca aggtctgcga gttccagttt tgcaacgacc ccttcctggg cgtgtactac 6240
cacaagaaca acaagtcttg gatggagagc gagttccggg tgtacagcag cgccaacaat 6300
tgcaccttcg agtacgtgtc ccagcccttc ctgatggacc tggaaggcaa gcagggcaac 6360
ttcaagaacc tgcgggagtt cgtgttcaag agcgacagag atatattata agagagagac 6420
acgaacatcg acggctactt cttcagcgct ctggaacctc tggtggatct gccaatcggc 6480
atcaacatca cccggttcca gaccctgctg gctctgcaca gaagctacct gaccccaggc 6540
gattcttcta gcggctggac agcaggagcc gccgcttatt acgtgggcta tctgcagccc 6600
cggaccttcc tgctgaagta caacgagaac ggcaccatca ccgacgcagt ggattgcgct 6660
gcgaggtgtt caacgccacc agattcgcca gcgtctacgc ctggaaccgg aagcggatca 6720
gcaattgcgt ggccgactac agcgtgctgt acaacagcgc cagcttcagc accttcaagt 6780
gctacggcgt gtcccctacc aagctgaacg atctgtgctt caccaacgtg tacgccgaca 6840
gcttcgtgat caggggcgac gaagtgcgac agatcgcccc aggacagaca ggaaagatcg 6900
ccgattacaa ctacaagctg cccgacgact tcaccggttg cgtgatcgct tggaacgtcg 6960
gcggcaacta caactacctg taccggctgt tccggaagtc caacctgaag cccttcgagc 7020
gggacatcag cacagagatc taccaggccg gcagcacacc ttgtaacggc gtggagggct 7080
tcaactgcta cttccctctg cagagctacg gcttccagcc tacaaacggc gtgggatacc 7140
agccttacag ggtggtggtg ctgagcttcg aactgctgca cgctcgtgaa gaacaagtgc 7200
gtgaatttca acttcaacgg cctgaccgga accggcgtgc tgaccgagag caacaagaag 7260
ttcctgccct tccagcagtt cggcagagac atcgcagaca ccacagacgc cgtgagagat 7320
cctcagaccc tggagatcct ggacatcacc ccttgcagct ttggcggagt gtccgtgatc 7380
accccaggaa ccaacaccag caatcaggtg gccgtgctgt accaggacgt gaattgcacc 7440
gaggtgccag tggccattca cgcagatcag ctgaccccta cctggagagt gtacagcacc 7500
ggaagcaacg tgttccagac cagggcaggt tgcctgatcg gagccgagca cgtgaacaac 7560
agctacgagt gcgatatccc tatcggcgcc ggcatttgcg cctcttacca gacccagacc 7620
aacagcccta ggagagccag aagcgtggcc tctcagagca tcatcgccta caccatgagc 7680
ctgggagccg agaacagcgt ggcctacagc aacaacagca tcgccatccc caccaacttc 7740
accatcagcg tgaccaccga gattctgccc gtgtctatga ccaagaccag cgtcgactgc 7800
accatgtaca tctgcggcga cagcaccgag tgctctaacc tgctgctgca gtacggctcc 7860
ttttgcaccc agctgaacag ggctctgaca ggcatcgcag tggagcagga caagaacacc 7920
caggaggtct tcgcccaggt caagcagatt gccgccagag acctgatttg cgcccagaag 7980
ttcaacggcc tgacagtgct gcctcctctg ctgaccgacg agatgatcgc ccagtataca 8040
agcgctctgc tggcaggaac catcaccaga tggcctaccg cttcaacgga atcggcgtga 8100
cccagaacgt gctctacgag aaccagaagc tgatcgccaa ccagttcaac agcgccatcg 8160
gcaagatcca ggacagcgcg tattatacac taagaataga tatgcctgag cagcacagct 8220
agcgctctgg gcaagctgca ggatgtggtg aaccagaacg cccaggctct gaacaccctg 8280
gtgaagcagc tgagcagcaa cttcggagcc atcagcagcg tgctgaacga catcctgagc 8340
aggctggaca aggtggaagc cgaagtgcag atcgacaggc tgatcaccgg cagactgcag 8400
tctctgcaga cctacgtgac ccagcagctg atcagagccg ccgagatcag agctagcgct 8460
aatctggccg ccaccaagat gagcgagtgc gtgctgggac agagcaagag agtggacttc 8520
tgcggcaagg gctaccacct gatgagcttt cctcagagcg ctcctcacgg agtggtgttt 8580
ctgcacgtga cctacgtgcc agcccaggag aagaacttca ccacagcccc agccatttgc 8640
cacgacgcgt gacccagcgg aacttctacg agccccagat catcaccacc gacaacacct 8700
tcgtgagcgg caattgcgac gtggtcatcg gcatcgtgaa caacaccgtg tacgaccccc 8760
gcgctatata gacacagtag atgatagtgc agccagagct ggactccttc aaggaggagc 8820
tggacaagta cttcaagaac cacaccagcc cagacgtgga cctgggagat atcagagagc 8880
ctgatcgacc tgcaggagct gggcaagtac gagcagtaca tcaagtggcc ttggtacatt 8940
tggctgggct tcatcgccgg cctgatcgca atcgtcatgg tgaccatcat gctctgttgc 9000
atgaccagtt gctgcagctg cctgaagggt tgctgcagtt gcgggagttg ctgcaagttc 9060
gacgaggacg acagcgagcc agtgctgaaa ggcgtgaagc tgcactacac gcgacagcag 9120
acgatatata tacatgataa ggcgcgccca cccagcggcc gcatacagca gcaattggca 9180
agctgcttac atagaactcg cggcgattgg catgccgcct taaaattttt attttatttt 9240
tcttttcttt tccgaatcgg attttgtttt taatatttca aaaaaaaaaa aaaaaaaaaa 9300
aaaaaaaaaa aaaaaaaaaa gaagagctag ggataacagg gtaattgagc aaaaggccag 9360
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9420
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9480
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9540
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 9600
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 9660
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 9720
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 9780
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 9840
agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 9900
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 9960
cagattacgc gcagaaaaaa aggatctcaa gaaggcggtg gtttttttgt ttgcaagcag 10020
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10080
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10140
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10200
gagtaaactt ggtctgacag ttcgcggcgc tctctctctc ttatatagag acacgagaaa 10260
aactatcgag catcaaatgt aatttcccct cgtcaaaaat aaggttatca agtgagaaat 10320
caccatgagt gacgactgaa tggtgagaat ggcaaaagtt tatgcatttc tttccagact 10380
tgttcaacag gccagccatt agtgtcatca aaatcactcg catcaaccaa accgttattc 10440
attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa 10500
acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct 10560
gaatcaggat attcttctaa tacctggaat gctgttttcc cagggatcgc agtggtgagt 10620
aaccatgcat catcaggagt aggataaaat gcttgatggt cggaagaggc ataaattccg 10680
tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat 10740
gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg 10800
attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat 10860
ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcacattt cccgcgcggc 10920
gcggatatta taattattat catgacatta acctataaaa ataggcgtat cacgaggccc 10980
tttcgtctag ggataacagg gtaattaata cgactcacta tag 11023
<210> 4
<211> 16789
<212> DNA
<213> 人工序列
<400> 4
atgggcggcg catgagagaa gcccagacca atgagctttg cagcggagct tcccgcagtt 60
tgaggtagaa gccaagcagg tcactgataa tgaccatgct aatgccagag cgttttcgca 120
tctggcttca aaactgatcg aaacggaggt ggacccatcc gacacgatcc ttgacattgg 180
aagtgcgccc gcccgcagaa tgtattctaa gcacaagtat cattgtatct gtccgatgag 240
atgtgcggaa gatcctgaag aaaaactgta aggaaataac tgataaggaa ttggacaaga 300
aaatgaagga gctcgccgcc gtcatgagcg accctgacct ggaaactgag actatgtgcc 360
tccacgacga cgagtcgtgt cgctacgaag ggcaagtcgc tgtttaccag gatgtatacg 420
cggttgacgg accgacaagt ctctatcacc aagccaataa gggagttaga gtcgagaact 480
tggctggacc aactgggccg acgaaaccgt gttaacggct cgtaacatag gcctatgcag 540
ctctgacgtt atggagcggt cacgtagagg gatgtccatt cttagaaaga agtatttgaa 600
accatccaac aatgttctat tctctgttgg ctcgaccatc taccacgaga agagggactt 660
actgaggagc tggcacctgc cgtctgtatt tcacttacgt ggcaagcaaa attacacatg 720
tcggtgtgag actatagtta gttgcgacgg gtacgtcgtt aaaagaatag ctatcagtcc 780
aggcctgtat gggaagcctt caggcgacag acacattgaa cggggagagg gtctcttttc 840
ccgtgtgcac gtatgtgcca gctacattgt gtgaccaaat gactggcata ctggcaacag 900
atgtcagtgc ggacgacgcg caaaaagtga acagcgattt ccactcattc gtgctgccca 960
ggataggcag taacacattg gagatcgggc tgagaacaag aatcaggaaa atgttagagg 1020
agcacaagga gccgtcacct ctcattaccg ccgaggacgt acaagaagct aagtgcgcag 1080
ccgatgaggc taaggaggtg cgtgaagccg aggagttgcg cgcagctcta ccacctttgg 1140
cagctgatgt tgaggaaaat tatcttgcat ccaccctctc gctgaacaag tcatagtgat 1200
aacacactct ggccgaaaag ggcgttatgc cgtggaacca taccatggta aagtagtggt 1260
gccagaggga catgcaatac ccgtccagga ctttcaagct ctgagtgaaa gtgccaccat 1320
tgtgtacaac gaacgtgagt tcgtaaacag gtacctgcac catattgcca cacatggagg 1380
agcgctgaac actgatgaag aatattacaa aactgtcaag cccagcgagc acgacggcga 1440
atacctgtac gacatcgaca ggaaacagtg cgtcaagaaa gaactagtca ctgggctagg 1500
gctcacaggc gagctggtgg atcctccctt ccatgaattc gcctacgaga gtctgagaac 1560
acgaccagcc gctgtggact cagtgctctt gaatggatgc aaacaccccg tagagaccct 1620
gtatattgac gaagcttttg cttgtcatgc aggtactctc agagcgctca tagccattat 1680
aagacctaaa aaggcagtgc tctgcgggga tcccaaacaa ccacgagatt tgcacacaag 1740
tcttccacaa aagcatctct cgccgttgca ctaacagttg caaatagatt acaaaggcaa 1800
cgaaataatg acggcagctg cctctcaagg gctgacccgt aaaggtgtgt atgccgttcg 1860
gtacaaggtg aatgaaaatc ctctgtacgc acccacctca gaacatgtga acgtcctact 1920
gacccagacc ggaccctacc gacgtcttcc agaataaggc aaacgtgtgt tgggccaagg 1980
ctttagtgcc ggtgctgaag accgctggca tagacatgac cactgaacaa tggaacactg 2040
tggattattt tgaaacggac aaagctcact cagcagagat agtattgaac caactatgcg 2100
tgaggttctt tggactcgat ctggactccg gtctattttc tgcacccact gttccgttat 2160
ccattaggaa taatcactgg gataactccc cgtcgcctaa catgtacggg ctgaataaag 2220
aagtggtccg tcagctctct cgcaggtacc cacaactgcc tcgggcagtt gccactggaa 2280
gagtctatga catgaacact ggtacactgc gcaattatga tccgcgcata aacctagtac 2340
ctgtaaacag aagactgcct catgctttag tcctccacca taatgaacac ccacagagtg 2400
acttttcttc attcgtcagc aaattgaagg gcagaactgt cctggtggtc ggggaaaagt 2460
tgtccgtccc aggcaaaatg gttgactggt tgtcagaccg gcctgaggct accttcagag 2520
ctcggctgga tttaggcatc ccaggtgatg tgcccaaata tgacataata tttgttaatg 2580
tgaggacccc atataaatac catcactatc agcagtgtga agaccatgcc attaagctta 2640
gcatgttgac caagaaagct tgtctgcatc tgaatcccgg cggaacctgt gtcagcatag 2700
gttatggtta cgctgacagg gccagcgaaa gcatcattgg tgctatagcg cggctgttca 2760
agttttcccg ggtatgcaaa ccgaaatcct cacttgaaga gacgggtacg cacaatcctt 2820
acaagctttc atcaaccttg accaacattt atacaggttc cagactccac gaagccggat 2880
gtgcaccctc atatcatgtg gtgcgagggg atattgccac ggccaccgaa ggagtgatta 2940
taaatgctgc taacagcaaa ggacaacctg gcggaggggt gtgcggagcg ctgtataaga 3000
aattcccgga aagcttcgat ttacagccga tcgaagtagg aaaagcgcga ctggtcaaag 3060
gtgcagctaa acatatcatt catgccgtag gaccaaactt caacaaagtt tcggaggttg 3120
aaggtgacaa aataacaatt acaagtcagt agcgattcca ctgttgtcca ccggcatctt 3180
ttccgggaac aaagatcgac taacccaatc attgaaccat ttgctgacag ctttagacac 3240
cactgatgca gatgtagcca tatactgcag ggacaagaaa tgggaaatga ctctcaagga 3300
agcagtggct aggagagaag cagtggagga gatatgcata tccgacgact cttcagtgac 3360
agaacctgat gcagagctgg tgagggtgca tccgaagagt tctttggctg gaaggaaggg 3420
ctacagcaca agcgatggca aaactttctc atatttggaa gggaccaagt ttcaccaggc 3480
ggccaaggat atagcagaaa ttaatgccat gtggcccgtt gcaacggagg ccaatgagca 3540
ggtatgcatg tgagtcggaa gcctccacac cacctagcac gctgccttgc ttgtgcatcc 3600
atgccatgac tccagaaaga gtacagcgcc taaaagcctc acgtccagaa caaattactg 3660
tgtgctcatc ctttccattg ccgaagtata gaatcactgg tgtgcagaag atccaatgct 3720
cccagcctat attgttctca ccgaaagtgc ctgcgtatat tcatccaagg aagtatctcg 3780
tggaaacacc accggtagac gagactccgg agccatcggc agagaaccaa tccacagagg 3840
ggacacctga acaaccacca cttataaccg aggatgagac caggactggt ccattcctca 3900
tgcatccgac tttgatgtgg acagtttatc catacttgac accctggagg gagctagcgt 3960
gaccagcggg gcaacgtcag ccgagactaa ctcttacttc gcaaagagta tggagtttct 4020
ggcgcgaccg gtgcctgcgc ctcgaacagt attcaggaac cctccacatc ccgctccgcg 4080
cacaagaaca ccgtcacttg cacccagcag ggcctgctcg aatagggtga ttacaagaga 4140
ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg catacatctt 4200
ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa cggtgctatc 4260
cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc tcgaccaaga 4320
aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta acagaagcag 4380
ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta ttctgcaagg 4440
cctagggcat tatttgaagg cagaaggaaa aggcgccgcg tctctctctc atatatagga 4500
agagtctcgc tcgctctatt atatcttgga gtgctaccga accctgcatc ctgttccttt 4560
gtattcatct agtgtgaacc gtgccttttc aagccccaag gtcgcagtgg aagcctgtaa 4620
cgccatgttg aaagagaact ttccgactgt ggcttcttac tgtattattc cagagtacga 4680
tgcctatttg gacatggttg acggagcttc atgctgctta gacactgcca gtttttgccc 4740
tgcaaagctg cgcagctttc caaagaaaca ctcctatttg gaacccacaa tacgatcggc 4800
agtgccttca gcgatccaga acacgctcca gaacgtcctg gcagctgcca caaaaagaaa 4860
ttgcaatgtc acgcaaatga gagaattgcc cgtattggat tcggcggcct ttaatgtgga 4920
atgcttcaag aaatatgcgt gtaataatga atattgggaa acgtttaaag aaaaccccat 4980
caggcttact gaagaaaacg tggtaaatta cattaccaaa ttaaaaggac caaaagctgc 5040
tgcttgccga tccgctagca acagcgtatc tgtgcggaat ccaccgagag ctggttagga 5100
gattaaatgc ggtcctgctt ccgaacattc atacactgtt tgatatgtcg gctgaagact 5160
ttgacgctat tatagccgag cacttccagc ctggggattg tgttctggaa actgacatcg 5220
cgtcgtttga taaaagtgag gacgacgcca tggctctgac cgcgttaatg attctggaag 5280
acttaggtgt ggacgcagag ctgttgacgc tgattgaggc ggctttcggc gaaatttcat 5340
caatacattt gcccactaaa actaaattta aattcggagc catgatgaaa tctggaatgt 5400
tcctcacact gtttgtgaac acagtcatta acattgtaat cgcaagcaga gtgttgagag 5460
aacggctaac cggatcacca tgtgcagcat tgcatatata tgatagatac agatagatac 5520
attggagatg acaatatcgt gaaaggagtc aaatcggaca aattaatggc agacaggtgc 5580
gccacctggt tgaatatgga agtcaagatt atagatgctg tggtgggcga gaaagcgtgt 5640
gactccgtga ccggcacagc gtgccgtgtg gcagaccccc taaaaaggct gtttaagctt 5700
ggcaaacctc tggcagcaga cgatggtatt ctttcagagc tgatcatagt tatggccatg 5760
actactctag ctagcagtgt taaatcattc agctacctga gaggggcccc tataactctc 5820
tacggctaac ctgaatggac tacgacatag tctagtccgc caagatgttc gtgttcctcg 5880
tgctgctgcc cctggtgtct agccagtgcg tgaacctgac caccagaacc cagctgcctc 5940
cagcctacac caacagcttc accaggggag tgtactaccc cgacaaggtg ttccggagca 6000
gcgtgctgca tagcacccag gacctgttcc tgcccttctt cagcaacgtg acttggttcc 6060
acgccatcca cgtcagcgga aagcgctttg acaacccagt gctgcccttc aacgacggag 6120
tgtacttcgc cagcaccgag aagtccaaca tcatccgggg ttggatcttc ggcaccacac 6180
gtcgtgatca aggtctgcga gttccagttt tgcaacgacc ccttcctggg cgtgtactac 6240
cacaagaaca acaagtcttg gatggagagc gagttccggg tgtacagcag cgccaacaat 6300
tgcaccttcg agtacgtgtc ccagcccttc ctgatggacc tggaaggcaa gcagggcaac 6360
ttcaagaacc tgcgggagtt cgtgttcaag agcgacagag atatattata agagagagac 6420
acgaacatcg acggctactt cttcagcgct ctggaacctc tggtggatct gccaatcggc 6480
atcaacatca cccggttcca gaccctgctg gctctgcaca gaagctacct gaccccaggc 6540
gattcttcta gacaggcgcg gcgcgtagac attagacagc atgcagatcc cacaggcgcc 6600
ctggccagtc gtctgggcgg tgctacaact gggctggcgg ccaggatggt tcttaggtag 6660
gtggggtcgg cggtcaggtg tcccagagcc aggggtctgg agggaccctc agtccctggc 6720
aggtcggggg gtgctgaggc gggcctggcc ctggcagccc aggggtcccg gagcgagggg 6780
tcgcgcgcta ctctctgaga tctcgcgcta tagatcagac gatagacgat acagatacga 6840
tcgtagctag ctgcatagca tagtggaggg acctttcact ctcagtccct ggcaggtcgg 6900
ggggtgctgt ggcaggccca gccttggccc ccatgggttc ctctctgggc cccaactcct 6960
cccctggccc aagtcccctc tttgctcctg ggcagctcag ccagaatgac gggtcaggct 7020
ggccagaacc ccaaaccttg gccgtgggga gtctgcgtgg cggctctgcc ttgcccaggc 7080
atccttggtc ctcactcgag ttttcctaag gctttacgac gtcaaagttt agatgagctg 7140
gtgatatttt tctcattata tccaaggtgg tttcacccag gccaagccta agacagtccc 7200
tctgggccct gctgggtacc ggggtgtgac accactggga ggacaagatg aggggcaccc 7260
ctggggccgc cctgacaccc cctcgaggct cctgccccgg gggtcctggt gccccttcac 7320
tgtggcaggc gactgggggt tccccacctc ggcccctcca gggccgtgca cctcttaagg 7380
ttggcccgtg ggtcactcag gactcagaac tggagattct gggcaaaagg caaagagcaa 7440
agggccctcc ccgacccagg cgggaaagat aagagcaagg tgtccgcagc ctgacactcg 7500
tgcctcaggt gcccgcgctt gtgccggaca agactctcac aggtggcatg cctcggtttc 7560
cccactggta acagcacagg gcactcgtcc gacccagatg tggccacccc ggccgcagtg 7620
gtcttcattc caggatgcct cttttccctc ctgatctatt cactgcgttc gccattcggt 7680
cattcccggg gccaccactc ccacctcagg tgtgtgcttc ccttgtgttt tatgagatag 7740
aggagcataa ataagagcct cccagtcagc gctgccactg ctgagctctg ggccttcgac 7800
aggacttggc cccttactgg cgtcctgagg attaaatcgg gtcatctggg actataggca 7860
cgtgccaccc tgccaggcta atttcttcca ttttttttag agacagggtc tcgctatgtt 7920
ccccaggctg ggctcaaatg gtcctcccac ctcagcctcc ccaagtactg ggattacagg 7980
cataagccac tgcatctggc ctccatgaca catattttta aagtctgatt tttaaagtca 8040
aacttttgaa gtcagatttt aaacggacta ttttgaaaaa tatacaaaaa cgtttaaaaa 8100
caatgaatat ccctcaccta gaatcaataa ctaagaatat tgacacattt gctttgggga 8160
ctgggcggct ggagctgcca tgacaaagct ccgccgaccg gactgagggc tggacgtgca 8220
ggatggagct ccgcagggtc ggctcccctg tgctctgagg ggctctgctc agcctctccc 8280
ggctgtggct taaaaacaga gcctgtcagg gtcggctccc tgtgctccga gagggctctg 8340
ctcagcttct cctggctggg gggttttgtg gccaccctct gtgttcctgg gttcagaagc 8400
atcccccagg ctctgccttc atctctgcac gggtgactct gtacaggaag ccaggcctgc 8460
tggtcaatgg ccacccagcc ctgtgccctc atcttaccta gtcccagctg ccgtcaccat 8520
ggggttagga cttccacata ggaatctgtg gggacacggt tcggcccaca gcccttccca 8580
cctccacaca cacacacggc agccttcttc tgtgcgcttc tggaaagcaa tgtccccacc 8640
gccacctcac aggcctgttc cacagccccg ggatgggctc tgtctccctc ctgaccctgc 8700
atagggcaca gccctctctc atcaacccac gatcctacgt ggatccgaga gtcccataga 8760
aacaccccaa atctaacttg atccaggacc agccagtggt cacttctgaa tattcacctt 8820
cctagtagac actaccagcc aagggaggcc aggaagcctt cctggaggag gtggcctgag 8880
gactggggtg aggcaggccc tgcgtggggg tcgccaccca gcacccccac actgggtggg 8940
agccagtctc tgagactggc tgggggaggt gggagagggg gctgcttgaa ctgcagacac 9000
cgaggtctag cccccacccc acccagccag ttggtggagg caggggaggc cgaggggccc 9060
agctggacct gctccccggg gtggattcca aaataggggg gttggggggg gcggaacagg 9120
agcccagggt cctggcttga ggcccagtgg ctgagggctg gtgcaagcca gacaggaaaa 9180
gggttgagcc tgtcagcgcc agcacagatc aagtcaggcc atgcagggtc cctgctcccg 9240
tgatgacttc tgttcctcac ctgcctgtct gtcccgacag ctgcatggca gccctggcct 9300
gggagatgga gaccccgagg ggctgcctgc ggtggtgggg ttatatatat atatcggcgc 9360
tcgctctatt aattactctc ggccgttctc gagagtcgcg ccacacacat ctctcgagag 9420
ctctacacgt gtgtaccatc tcggggcccc ctagagggca gggcatttcc cctgctctgt 9480
gccgagtcca cccagcccca gcctaggccc agtaagggct gcagcccacc ctgtcccagg 9540
ctgcctccca ggagccctct tggccctgat gccagaagcc catcttcctc cattcaggca 9600
ggtctctgag tgccctggcc tggctgcctg ctggccctga gagtcacact accccacagc 9660
cctccttggt caaaatccac tctggagtgg ctggaagatt ccccgggccc acgccgcaca 9720
cgcctatgca gggagcttcc cctggccggc cggcagacaa gggcggtctc agagaggggg 9780
ctcacctcag cagccccttg tgtagctggc cctcgcccct gccacctctg ggaacaccac 9840
caggaagctg ggggacaggc acgcaggtga aggaggcgag cgcttgtcag ccgggaggcc 9900
atgggcacag agggaacagg gacaccctgg gtggcctcaa ggtcacttca aacccctcac 9960
tcgtcccctg ggagggtgcc cagtgaggtt ggcactacca cccacctctg gtgtccagcc 10020
agcacgccgt gggccagcct ggctgcaggg acacgagggc agcagccccc tcctcctctg 10080
agctggttgc tccttgagtc atcaccaccg cctgccacgg aggccgcctg tcccaggaag 10140
cagagggacc gcagctgtgg caaccagggc ctggtctctg tgtcacctcg ctggggggcc 10200
gtgcccaggc ctgagacgga actgagtgct ggcggaagag ccgcgacccc cacgttgcca 10260
tgcgggtatc ccaagccctg accctggcag gcatatgttt caggaggtcc ttgtcttggg 10320
agcccagggt cgggggcccc gtgtctgtcc acatccgagt caatggccca tctcgtctct 10380
gaagcatctt tgctgtgagc tctagtcccc actgtcttgc tggaaaatgt ggaggcccca 10440
ctgcccactg cccagggcag caatgcccat accacgtggt cccagctccg agcttgtcct 10500
gaaaaggggg caaagactgg accctgagcc tgccaagggg ccacactcct cccagggctg 10560
gggtctccat gggcgctctt agtaggaaat cagggagacc caagtcagag ccaggtgctg 10620
tgcagaagct gcagcctcac gtagaaggaa gaggctctgc agtggaggcc agtgcccatc 10680
cccgggtggc agaggcccca gcagagactt ctcaatgaca ttccagctgg ggtggccctt 10740
ccagagccct tgctgcccga gggatgtgag caggtggctt tgtggggcca cccagcccct 10800
tcctcacctc tctccatctc tcagactccc cagacacctg ctcgtggtga ccgaagggga 10860
caacgccacc ttcacctgca gcttctccaa cacatcggag agcttcgtgc taaactggta 10920
ccgcatgagc cccagcaacc agacggacaa gctggccgcc ttccccgagg accgcagcca 10980
gcccggccag gactgccgct tccgtgtcac acaactgccc aacgggcgtg acttccacat 11040
gagcgtggtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 11100
gagctcaggg tgacaggtgc ggcctcggag gccccggggc aggggtgagc cctcacccct 11160
gaccctgacc ctccaccctg accccgtcct aacccctgac ctttgtgccc ttcccacccc 11220
agcccctcac ccaggccagc cggccagttc caaaccctgg tggttggtgt cgtgggcggc 11280
ctgctgggca gcctggtgct gctagtctgg gtcctggccg tcatctgctc ccgggccgca 11340
cgaggtaacg tcatcccagc ccctcggcct gccctgccct aaccctgctg gcggccctct 11400
caatccctaa agccatgatc tggggcccca gcccacctgc ggtctccggg ggtgcccggc 11460
ccatgtgtgt gcctgcctgc ggtctccagg ggtgcctggc ccacgcgtgt gcccgcctgc 11520
ggtctctggg ggtgcccggc ccacatatgt gcctgcctgc ggtctccggc ccatgcgtgt 11580
gcccacctgc gagggcgtcc caccacagga ctcatgtctc aatgcccaca gtgagcccag 11640
gcagcaggtg tcaccgtccc ctacagggag ggccagatgc agtcactgct tcaggtcctg 11700
ccagcacaga gctgcctgcg tccagctccc tgaatctctg ctgctgctgc tgctgctgct 11760
gctgctgcct gcggcccggg gctgatgcct gaacttgggg gctggttgga gatggccttg 11820
gagcagccaa ggtgcccctg gcagtggcat cccgaaacgc cctggacgca gggcccaaga 11880
ctgggcacag gatcgcgtat cgggggggtt ttatatatag ggcgcgctgg tgtccccccg 11940
cgcgcaaatt ctctctgggt gtgtcccaaa agggagagag acttctctgc gcgcgttata 12000
tatatacgcg cctcgctcgc tcgctctagc tgtacgctag cgtgctctcc ttggaaccca 12060
ttcctgaaat tatttaaagg ggttggccgg gctcccacca gggcctgggt gggaaggtac 12120
aggcgttccc ccggggccta gtacccccgc cgtggcctat ccactcctca catccacaca 12180
ctgcaccccc actcctgggg cagggccacc agcatccagg cggccagcag gcacctgagt 12240
ggctgggaca agggatcccc cttccctgtg gttctattat attataatta taattaaata 12300
tgagagcatg ctaaggacgg ctggacagca ggagccgccg cttattacgt gggctatctg 12360
cagccccgga ccttcctgct gaagtacaac gagaacggca ccatcaccga cgcagtggat 12420
tgcgctgcga ggtgttcaac gccaccagat tcgccagcgt ctacgcctgg aaccggaagc 12480
ggatcagcaa ttgcgtggcc gactacagcg tgctgtacaa cagcgccagc ttcagcacct 12540
tcaagtgcta cggcgtgtcc cctaccaagc tgaacgatct gtgcttcacc aacgtgtacg 12600
ccgacagctt cgtgatcagg ggcgacgaag tgcgacagat cgccccagga cagacaggaa 12660
agatcgccga ttacaactac aagctgcccg acgacttcac cggttgcgtg atcgcttgga 12720
acgtcggcgg caactacaac tacctgtacc ggctgttccg gaagtccaac ctgaagccct 12780
tcgagcggga catcagcaca gagatctacc aggccggcag cacaccttgt aacggcgtgg 12840
agggcttcaa ctgctacttc cctctgcaga gctacggctt ccagcctaca aacggcgtgg 12900
gataccagcc ttacagggtg gtggtgctga gcttcgaact gctgcacgct cgtgaagaac 12960
aagtgcgtga atttcaactt caacggcctg accggaaccg gcgtgctgac cgagagcaac 13020
aagaagttcc tgcccttcca gcagttcggc agagacatcg cagacaccac agacgccgtg 13080
agagatcctc agaccctgga gatcctggac atcacccctt gcagctttgg cggagtgtcc 13140
gtgatcaccc caggaaccaa caccagcaat caggtggccg tgctgtacca ggacgtgaat 13200
tgcaccgagg tgccagtggc cattcacgca gatcagctga cccctacctg gagagtgtac 13260
agcaccggaa gcaacgtgtt ccagaccagg gcaggttgcc tgatcggagc cgagcacgtg 13320
aacaacagct acgagtgcga tatccctatc ggcgccggca tttgcgcctc ttaccagacc 13380
cagaccaaca gccctaggag agccagaagc gtggcctctc agagcatcat cgcctacacc 13440
atgagcctgg gagccgagaa cagcgtggcc tacagcaaca acagcatcgc catccccacc 13500
aacttcacca tcagcgtgac caccgagatt ctgcccgtgt ctatgaccaa gaccagcgtc 13560
gactgcacca tgtacatctg cggcgacagc accgagtgct ctaacctgct gctgcagtac 13620
ggctcctttt gcacccagct gaacagggct ctgacaggca tcgcagtgga gcaggacaag 13680
aacacccagg aggtcttcgc ccaggtcaag cagattgccg ccagagacct gatttgcgcc 13740
cagaagttca acggcctgac agtgctgcct cctctgctga ccgacgagat gatcgcccag 13800
tatacaagcg ctctgctggc aggaaccatc accagatggc ctaccgcttc aacggaatcg 13860
gcgtgaccca gaacgtgctc tacgagaacc agaagctgat cgccaaccag ttcaacagcg 13920
ccatcggcaa gatccaggac agcgcgtatt atacactaag aatagatatg cctgagcagc 13980
acagctagcg ctctgggcaa gctgcaggat gtggtgaacc agaacgccca ggctctgaac 14040
accctggtga agcagctgag cagcaacttc ggagccatca gcagcgtgct gaacgacatc 14100
ctgagcaggc tggacaaggt ggaagccgaa gtgcagatcg acaggctgat caccggcaga 14160
ctgcagtctc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagct 14220
agcgctaatc tggccgccac caagatgagc gagtgcgtgc tgggacagag caagagagtg 14280
gacttctgcg gcaagggcta ccacctgatg agctttcctc agagcgctcc tcacggagtg 14340
gtgtttctgc acgtgaccta cgtgccagcc caggagaaga acttcaccac agccccagcc 14400
atttgccacg acgcgtgacc cagcggaact tctacgagcc ccagatcatc accaccgaca 14460
acaccttcgt gagcggcaat tgcgacgtgg tcatcggcat cgtgaacaac accgtgtacg 14520
acccccgcgc tatatagaca cagtagatga tagtgcagcc agagctggac tccttcaagg 14580
aggagctgga caagtacttc aagaaccaca ccagcccaga cgtggacctg ggagatatca 14640
gagagcctga tcgacctgca ggagctgggc aagtacgagc agtacatcaa gtggccttgg 14700
tacatttggc tgggcttcat cgccggcctg atcgcaatcg tcatggtgac catcatgctc 14760
tgttgcatga ccagttgctg cagctgcctg aagggttgct gcagttgcgg gagttgctgc 14820
aagttcgacg aggacgacag cgagccagtg ctgaaaggcg tgaagctgca ctacacgcga 14880
cagcagacga tatatataca tgataaggcg cgcccaccca gcggccgcat acagcagcaa 14940
ttggcaagct gcttacatag aactcgcggc gattggcatg ccgccttaaa atttttattt 15000
tatttttctt ttcttttccg aatcggattt tgtttttaat atttcaaaaa aaaaaaaaaa 15060
aaaaaaaaaa aaaaaaaaaa aaaaaagaag agctagggat aacagggtaa ttgagcaaaa 15120
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 15180
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 15240
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 15300
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 15360
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 15420
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 15480
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 15540
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 15600
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 15660
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 15720
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag gcggtggttt ttttgtttgc 15780
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 15840
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 15900
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 15960
atatatgagt aaacttggtc tgacagttcg cggcgctctc tctctcttat atagagacac 16020
gagaaaaact atcgagcatc aaatgtaatt tcccctcgtc aaaaataagg ttatcaagtg 16080
agaaatcacc atgagtgacg actgaatggt gagaatggca aaagtttatg catttctttc 16140
cagacttgtt caacaggcca gccattagtg tcatcaaaat cactcgcatc aaccaaaccg 16200
ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa 16260
ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt 16320
tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccagg gatcgcagtg 16380
gtgagtaacc atgcatcatc aggagtagga taaaatgctt gatggtcgga agaggcataa 16440
attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt 16500
tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg 16560
cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt 16620
tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc acatttcccg 16680
cgcggcgcgg atattataat tattatcatg acattaacct ataaaaatag gcgtatcacg 16740
aggccctttc gtctagggat aacagggtaa ttaatacgac tcactatag 16789
<210> 5
<211> 18017
<212> DNA
<213> 人工序列
<400> 5
atgggcggcg catgagagaa gcccagacca atgagctttg cagcggagct tcccgcagtt 60
tgaggtagaa gccaagcagg tcactgataa tgaccatgct aatgccagag cgttttcgca 120
tctggcttca aaactgatcg aaacggaggt ggacccatcc gacacgatcc ttgacattgg 180
aagtgcgccc gcccgcagaa tgtattctaa gcacaagtat cattgtatct gtccgatgag 240
atgtgcggaa gatcctgaag aaaaactgta aggaaataac tgataaggaa ttggacaaga 300
aaatgaagga gctcgccgcc gtcatgagcg accctgacct ggaaactgag actatgtgcc 360
tccacgacga cgagtcgtgt cgctacgaag ggcaagtcgc tgtttaccag gatgtatacg 420
cggttgacgg accgacaagt ctctatcacc aagccaataa gggagttaga gtcgagaact 480
tggctggacc aactgggccg acgaaaccgt gttaacggct cgtaacatag gcctatgcag 540
ctctgacgtt atggagcggt cacgtagagg gatgtccatt cttagaaaga agtatttgaa 600
accatccaac aatgttctat tctctgttgg ctcgaccatc taccacgaga agagggactt 660
actgaggagc tggcacctgc cgtctgtatt tcacttacgt ggcaagcaaa attacacatg 720
tcggtgtgag actatagtta gttgcgacgg gtacgtcgtt aaaagaatag ctatcagtcc 780
aggcctgtat gggaagcctt caggcgacag acacattgaa cggggagagg gtctcttttc 840
ccgtgtgcac gtatgtgcca gctacattgt gtgaccaaat gactggcata ctggcaacag 900
atgtcagtgc ggacgacgcg caaaaagtga acagcgattt ccactcattc gtgctgccca 960
ggataggcag taacacattg gagatcgggc tgagaacaag aatcaggaaa atgttagagg 1020
agcacaagga gccgtcacct ctcattaccg ccgaggacgt acaagaagct aagtgcgcag 1080
ccgatgaggc taaggaggtg cgtgaagccg aggagttgcg cgcagctcta ccacctttgg 1140
cagctgatgt tgaggaaaat tatcttgcat ccaccctctc gctgaacaag tcatagtgat 1200
aacacactct ggccgaaaag ggcgttatgc cgtggaacca taccatggta aagtagtggt 1260
gccagaggga catgcaatac ccgtccagga ctttcaagct ctgagtgaaa gtgccaccat 1320
tgtgtacaac gaacgtgagt tcgtaaacag gtacctgcac catattgcca cacatggagg 1380
agcgctgaac actgatgaag aatattacaa aactgtcaag cccagcgagc acgacggcga 1440
atacctgtac gacatcgaca ggaaacagtg cgtcaagaaa gaactagtca ctgggctagg 1500
gctcacaggc gagctggtgg atcctccctt ccatgaattc gcctacgaga gtctgagaac 1560
acgaccagcc gctgtggact cagtgctctt gaatggatgc aaacaccccg tagagaccct 1620
gtatattgac gaagcttttg cttgtcatgc aggtactctc agagcgctca tagccattat 1680
aagacctaaa aaggcagtgc tctgcgggga tcccaaacaa ccacgagatt tgcacacaag 1740
tcttccacaa aagcatctct cgccgttgca ctaacagttg caaatagatt acaaaggcaa 1800
cgaaataatg acggcagctg cctctcaagg gctgacccgt aaaggtgtgt atgccgttcg 1860
gtacaaggtg aatgaaaatc ctctgtacgc acccacctca gaacatgtga acgtcctact 1920
gacccagacc ggaccctacc gacgtcttcc agaataaggc aaacgtgtgt tgggccaagg 1980
ctttagtgcc ggtgctgaag accgctggca tagacatgac cactgaacaa tggaacactg 2040
tggattattt tgaaacggac aaagctcact cagcagagat agtattgaac caactatgcg 2100
tgaggttctt tggactcgat ctggactccg gtctattttc tgcacccact gttccgttat 2160
ccattaggaa taatcactgg gataactccc cgtcgcctaa catgtacggg ctgaataaag 2220
aagtggtccg tcagctctct cgcaggtacc cacaactgcc tcgggcagtt gccactggaa 2280
gagtctatga catgaacact ggtacactgc gcaattatga tccgcgcata aacctagtac 2340
ctgtaaacag aagactgcct catgctttag tcctccacca taatgaacac ccacagagtg 2400
acttttcttc attcgtcagc aaattgaagg gcagaactgt cctggtggtc ggggaaaagt 2460
tgtccgtccc aggcaaaatg gttgactggt tgtcagaccg gcctgaggct accttcagag 2520
ctcggctgga tttaggcatc ccaggtgatg tgcccaaata tgacataata tttgttaatg 2580
tgaggacccc atataaatac catcactatc agcagtgtga agaccatgcc attaagctta 2640
gcatgttgac caagaaagct tgtctgcatc tgaatcccgg cggaacctgt gtcagcatag 2700
gttatggtta cgctgacagg gccagcgaaa gcatcattgg tgctatagcg cggctgttca 2760
agttttcccg ggtatgcaaa ccgaaatcct cacttgaaga gacgggtacg cacaatcctt 2820
acaagctttc atcaaccttg accaacattt atacaggttc cagactccac gaagccggat 2880
gtgcaccctc atatcatgtg gtgcgagggg atattgccac ggccaccgaa ggagtgatta 2940
taaatgctgc taacagcaaa ggacaacctg gcggaggggt gtgcggagcg ctgtataaga 3000
aattcccgga aagcttcgat ttacagccga tcgaagtagg aaaagcgcga ctggtcaaag 3060
gtgcagctaa acatatcatt catgccgtag gaccaaactt caacaaagtt tcggaggttg 3120
aaggtgacaa aataacaatt acaagtcagt agcgattcca ctgttgtcca ccggcatctt 3180
ttccgggaac aaagatcgac taacccaatc attgaaccat ttgctgacag ctttagacac 3240
cactgatgca gatgtagcca tatactgcag ggacaagaaa tgggaaatga ctctcaagga 3300
agcagtggct aggagagaag cagtggagga gatatgcata tccgacgact cttcagtgac 3360
agaacctgat gcagagctgg tgagggtgca tccgaagagt tctttggctg gaaggaaggg 3420
ctacagcaca agcgatggca aaactttctc atatttggaa gggaccaagt ttcaccaggc 3480
ggccaaggat atagcagaaa ttaatgccat gtggcccgtt gcaacggagg ccaatgagca 3540
ggtatgcatg tgagtcggaa gcctccacac cacctagcac gctgccttgc ttgtgcatcc 3600
atgccatgac tccagaaaga gtacagcgcc taaaagcctc acgtccagaa caaattactg 3660
tgtgctcatc ctttccattg ccgaagtata gaatcactgg tgtgcagaag atccaatgct 3720
cccagcctat attgttctca ccgaaagtgc ctgcgtatat tcatccaagg aagtatctcg 3780
tggaaacacc accggtagac gagactccgg agccatcggc agagaaccaa tccacagagg 3840
ggacacctga acaaccacca cttataaccg aggatgagac caggactggt ccattcctca 3900
tgcatccgac tttgatgtgg acagtttatc catacttgac accctggagg gagctagcgt 3960
gaccagcggg gcaacgtcag ccgagactaa ctcttacttc gcaaagagta tggagtttct 4020
ggcgcgaccg gtgcctgcgc ctcgaacagt attcaggaac cctccacatc ccgctccgcg 4080
cacaagaaca ccgtcacttg cacccagcag ggcctgctcg aatagggtga ttacaagaga 4140
ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg catacatctt 4200
ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa cggtgctatc 4260
cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc tcgaccaaga 4320
aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta acagaagcag 4380
ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta ttctgcaagg 4440
cctagggcat tatttgaagg cagaaggaaa aggcgccgcg tctctctctc atatatagga 4500
agagtctcgc tcgctctatt atatcttgga gtgctaccga accctgcatc ctgttccttt 4560
gtattcatct agtgtgaacc gtgccttttc aagccccaag gtcgcagtgg aagcctgtaa 4620
cgccatgttg aaagagaact ttccgactgt ggcttcttac tgtattattc cagagtacga 4680
tgcctatttg gacatggttg acggagcttc atgctgctta gacactgcca gtttttgccc 4740
tgcaaagctg cgcagctttc caaagaaaca ctcctatttg gaacccacaa tacgatcggc 4800
agtgccttca gcgatccaga acacgctcca gaacgtcctg gcagctgcca caaaaagaaa 4860
ttgcaatgtc acgcaaatga gagaattgcc cgtattggat tcggcggcct ttaatgtgga 4920
atgcttcaag aaatatgcgt gtaataatga atattgggaa acgtttaaag aaaaccccat 4980
caggcttact gaagaaaacg tggtaaatta cattaccaaa ttaaaaggac caaaagctgc 5040
tgcttgccga tccgctagca acagcgtatc tgtgcggaat ccaccgagag ctggttagga 5100
gattaaatgc ggtcctgctt ccgaacattc atacactgtt tgatatgtcg gctgaagact 5160
ttgacgctat tatagccgag cacttccagc ctggggattg tgttctggaa actgacatcg 5220
cgtcgtttga taaaagtgag gacgacgcca tggctctgac cgcgttaatg attctggaag 5280
acttaggtgt ggacgcagag ctgttgacgc tgattgaggc ggctttcggc gaaatttcat 5340
caatacattt gcccactaaa actaaattta aattcggagc catgatgaaa tctggaatgt 5400
tcctcacact gtttgtgaac acagtcatta acattgtaat cgcaagcaga gtgttgagag 5460
aacggctaac cggatcacca tgtgcagcat tgcatatata tgatagatac agatagatac 5520
attggagatg acaatatcgt gaaaggagtc aaatcggaca aattaatggc agacaggtgc 5580
gccacctggt tgaatatgga agtcaagatt atagatgctg tggtgggcga gaaagcgtgt 5640
gactccgtga ccggcacagc gtgccgtgtg gcagaccccc taaaaaggct gtttaagctt 5700
ggcaaacctc tggcagcaga cgatggtatt ctttcagagc tgatcatagt tatggccatg 5760
actactctag ctagcagtgt taaatcattc agctacctga gaggggcccc tataactctc 5820
tacggctaac ctgaatggac tacgacatag tctagtccgc caagatgttc gtgttcctcg 5880
tgctgctgcc cctggtgtct agccagtgcg tgaacctgac caccagaacc cagctgcctc 5940
cagcctacac caacagcttc accaggggag tgtactaccc cgacaaggtg ttccggagca 6000
gcgtgctgca tagcacccag gacctgttcc tgcccttctt cagcaacgtg acttggttcc 6060
acgccatcca cgtcagcgga aagcgctttg acaacccagt gctgcccttc aacgacggag 6120
tgtacttcgc cagcaccgag aagtccaaca tcatccgggg ttggatcttc ggcaccacac 6180
gtcgtgatca aggtctgcga gttccagttt tgcaacgacc ccttcctggg cgtgtactac 6240
cacaagaaca acaagtcttg gatggagagc gagttccggg tgtacagcag cgccaacaat 6300
tgcaccttcg agtacgtgtc ccagcccttc ctgatggacc tggaaggcaa gcagggcaac 6360
ttcaagaacc tgcgggagtt cgtgttcaag agcgacagag atatattata agagagagac 6420
acgaacatcg acggctactt cttcagcgct ctggaacctc tggtggatct gccaatcggc 6480
atcaacatca cccggttcca gaccctgctg gctctgcaca gaagctacct gaccccaggc 6540
gattcttcta gacaggcgcg ctttagcgcg atacgatgtg tcacacagtg cgcgtagaca 6600
ttagacagca tgcagatccc acaggcgccc tggccagtcg tctgggcggt gctacaactg 6660
ggctggcggc caggatggtc ccagagccag gggtctggcc tttaatatac gacgcgcgct 6720
cttctcgaga gagagtctct cgagaagagt ctcctgagag cttcctgata gtgcgcgtag 6780
ataacagata cagatacgat agcatagaga gggaccttcc accctcagtc cctggcaggt 6840
cggggggtgc tgaggcgggc ctggccctgg cagcccaggg gtcccggagc gaggggtcgc 6900
gcgctactct ctgagatctc gcgctataga tcagacgata gacgatacag atacgatcgt 6960
agctagctgc atagcatagt ggagggacct ttcactctca gtccctggca ggtcgggggg 7020
cccaagtccc ctctttgctc ctgggcagct cagccagaat gacgggtcag gctggccaga 7080
accccaaacc ttggccgtgg ggagtctgcg tggcggctct gccttgccca ggcatccttg 7140
gtcttctata cgacgcgcgc tcttctcgag agagagtctc tcgagaagag tctcctgaga 7200
gcttcctgat agtgcgcgta gataacagat acagatacga tagcatagag ctcactcgag 7260
ttttcctaag gctttacgac gtcaaagttt agatgagctg gtgatatttt tctcattata 7320
tccaaagtgt acctgttcga gtgaggacag ttctttaggg cccgcgcgcc ggatatatag 7380
cgcgcccgct cctggaccct ctgtgcccgg ctggccctct gtgcccagcc agcctgcacc 7440
tggggatgcc aaggcctggg gagggtggtt tcacccaggc caagcctaag acagtccctc 7500
tgggccctgc tgggtaccgg ggtgtgacac cactgggagg acaagatgag gggcacccct 7560
ggggccgccc tgacaccccc tcgaggctcc tgccccgggg gtcctggtgc cccttcactg 7620
tggcaggcga ctgggggttc cccacctcgg cccctccagg gccgtgcacc tcttaaggtt 7680
ggcccgcccg acccaggcgg gaaagataag agcaaggtgt ccgcagcctg acactcgtgc 7740
ctcaggtgcc cgcgcttgtg ccggacaaga ctctcacagg tggcatgcct cggtttcccc 7800
actggtaaca gcacagggca ctcgtcctgt gggtcctgac ccagatgtgg ccaccccggc 7860
cgcagtggtc ttcattccag gatgcctctt ttccctcctg atctattcac tgcgttcgcc 7920
attcggtcat tcccggggcc accactccca cctcaggtgt gtgcttccct tgtgttttat 7980
gagatatccc caacccggct gcttattggc cccgtcccct tcgacaggac ttggcccctt 8040
actggcgtcc tgaggattaa atcgggtcat ctgggactat aggcacgtgc caccctgcca 8100
ggctaatttc ttccattttt tttagagaca gggtctcgct atgttcccca ggctgggctc 8160
aaatggtcct cccacctcag cctccccaag tactgggatt acaggcataa gccactgcat 8220
ctggcctcca tgacaccaga ttttaaacgg actattttga aaaatataca aaaacgttta 8280
aaaacaatga atatccctca cctagaatca ataactaaga atattgacac atttgctttg 8340
gggactgggc ggctggagct gccatgacaa agctccgccg accggactga gggctggacg 8400
tgcaggatgg agctccgcag ggtcggctcc cctgtgctct gaggggctct gctcagcctc 8460
tcccggctgt ggcttaaaaa cagagcctgt cagggtcggc tccctgtgct ccgagagggc 8520
tctgctcagc ttctcctggc tggggggttt tgtggccacc ctctgtgttc tctctatata 8580
ttatacgacg cgcgctcttc tcgagagaga gtctctcgag aagagtctcc tgagagcttc 8640
ctgatagtgc gcgtagatac gggtgactct gtacaggaag ccaggcctgc tggtcaatgg 8700
ccacccagcc ctgtgcagga cttccacata ggaatctgtg gggacacggt tcggcccaca 8760
gcccttccca cctccacaca cacacacggc agccttcttc tgtgcgcttc tggaaagcaa 8820
tgtcctgtct ccctcctgac cctgcatagg gcacagccct ctctcatcaa cccacgatcc 8880
tacgtggatc cgagagtccc atagaaacac cccaaatcta acttgatcca ggaccagcca 8940
gtggtcactt ctgaatattc accttcctag tagacactac cagccaaggg aggccaggaa 9000
gccttcctgg aggaggtggc ctgaggactg gggtgaggca ggccctgcgt gggggtcgcc 9060
acccagcacc cccacactgg gtgggagcca gtctctgaga ctggctgggg gaggtgggag 9120
agggggctgc ttgaactgca gacaccgagg tctagccccc acccaggggc ccagctggac 9180
ctgctccccg gggtggattc caaaataggg gggttggggg gggcggaaca ggagcccagg 9240
gtcctggctt gaggcccagt ggctgagggc tggtacagat caagtcagga gcaggtccct 9300
ccaccaatgt gtgcaaataa atagcagcta agtttccagt tacaagaaca atgcacagat 9360
ggtcccaggg acattgcggt gtggacacac ttatatatta tacgacgcgc gctcttctcg 9420
agagagagtc tctcgagaag agtctcctga gagcttcctg atagtgcgcg tagataacag 9480
atacagatac gatagcatag agagcggcca ttgtcctgtc gccagcacct cgccctacag 9540
ctggggggtc ccttagcact tcctagccat gcagggtccc tgctcacagt acccgtgatg 9600
gcagccctgg cctgggagat ggagaccccg aggggctgcc tgcggtggtg gggttatata 9660
tatatatcgg cgctcgctct attaattact ctcggccgtt ctcgagagtc gcgccacaca 9720
catctctcga gagctctaca cgtgtgtacc atctcggggc cccctagagg gcagcaccct 9780
gtcccaggct gcctcccagg agccctcttg gccctgatgc cacagccctg agagtcacac 9840
taccccacag ccctccttgg tcaaaatcca ctctggagtg gctggaagat tccccgggcc 9900
cacgccgcac acgcctatgc agggagcttc ccctggccgg ccggcagaca agggcggtct 9960
cagagagggg gctcacctca gcagcccctt gtgtagctgg caccaggaag ctgggggaca 10020
ggcacgcagg tgaaggaggc gagcgcttgt cagccgggag gccatgggca cagagggaac 10080
agggacaccc tgggtggcct caaggtcact tcaaacccct cactcgtccc ctgggagggt 10140
gcccagtgag agggacacga gggcagcagc cccctcctcc tctgagctgg ttgctccttg 10200
agtcatcacc accgcctgcc acggaggccg cctgtcccag gaagcagagg gaccgcagct 10260
gtggcaacca gggcctggtc tctgtgtcac ctcgctgggg ggccgtgccc aggcctgaga 10320
cggaactgag tgctggcgga agagccgcga cccccacgtt gccatgcggg tatcccaagc 10380
cctgaccctg gcaggcatat gtttcaggag gtccttgtct tgggagccca gggtcggggg 10440
ccccgtgtct gtccacatcc gagtcaatgg cccatctcgt ctactcctcc cagggctggg 10500
gtctccatgg gcagcccccc acccacccag accagttaca ctcccctgtg ccagagcagt 10560
gcagacagga ccaggccagg atgcccaagg gtcaggggct ggggatgggt agcccccaaa 10620
cagccctttc tgggggaact ggcctcaacg gggaaggggg tgaaggctct tagtaggaaa 10680
tcagggagac ccaagtcaga gccaggtgct gtgcagaagc tgcagcctca cgtagaagga 10740
agaggctctg cagtggaggc cagtgcccat ccccgggtgg cagaggcccc agcagagact 10800
tctcaatgac attccagctg gggtggccct tccagagccc ttgctgcccg agggatgtga 10860
gcaggtggcc ggggaggctt tgtggggcca cccagcccct tcctcacctc tctccatctc 10920
tcagactccc cagacacctg ctcgtggtga ccgaagggga caacgccacc ttcacctgca 10980
gcttctccaa cacatcggag agcttcgtgc taaactggta ccgcatgagc cccagcaacc 11040
agacggacaa gctggccgcc ttccccgagg accgcagcca gcccggccag gactgccgct 11100
tccgtgtcac acaactgccc aacgggcgtg acttccacat gagcggagag cctgcgggca 11160
gagctcaggg tgacaggtgc ggcctcggag gccccggggc aggggtgagc tgagccggtc 11220
ctggggtggg tgtcccctcc tgcacaggat caggagctcc ctatacgacg cgcgctcttc 11280
tcgagagaga gtctctcgag aagagtctcc tgagagcttc ctgatagtgc gcgtagataa 11340
cagatacaga tacgatagca tagaggctct gtcctgcacc tggggaatgg tgaccggcat 11400
ctctgtcctc tagctctgga agcaccccag cccctctagt ctgccctcac ccctgaccct 11460
gaccctccac cctgaccccg tcctaacccc tgacctttgt gcccttccca ccccagcccc 11520
tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg cggcctgctg 11580
ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccgggc cgcacgaggt 11640
aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc ctcactcccg 11700
caccccacct ccccccatct ccccgccagg ctaagtccct gatgaaggcc cctggactaa 11760
gaccccccac ctaggagcac ggctcagggt cggcctggtg acccctagga gccaggcgca 11820
ccggccagcc cttttcctgc atgatccact gtgccttcct tcctgggtgg gcagaggtgg 11880
aaggacaggc tgggaccaca cggcctgcag gactcacatt ctattatagc caggacccca 11940
cctccccagc ccccaggcag caacctcaat ccctaaagcc atgatctggg gccccagccc 12000
acctgcggtc gcggtctcca ggggtgcctg gcccacgcgt gtgcccgcct gcggtctctg 12060
ggggtgcccg gcccacatat gtgcctgcct gcggtctcca ggtgtgcccg gcccattata 12120
cgacgcgcgc tcttctcgag agagagtctc tcgagaagag tctcctgaga gcttcctgat 12180
agtgcgcgta gataacagat acagatacga tagcatagag gcgtgtgccc acctgcgagg 12240
gcgtggggtg ggcttggtca tttcttatct tacattggag acaggagagc ttgaaaagtc 12300
acagggggac attgagccag agaggagggg tggtgtcccc agatcacaca gagggcagtg 12360
gtgggacagc tcagggtaag cagctcatag tggggggccc aggttcggtg ccggtactgc 12420
agccaggctg tggagccgcg ggcctccttc ctgcggtggg ccgtggggct gactccctct 12480
ccctttctcc tcaaagaagg aggacccctc agccgtgggc gaccggggct ccagcctgca 12540
cctgcaccag gcacagcccc accacaggac tcatgtctca atgcccacag tgagcccagg 12600
cagcaggtgt caccgtcccc tacagggagg gccagatgca gtcactgctt caggtcctgc 12660
cagcacagag ctgcctgcgt ccagctccct gaatctctgc tgctgctgct gctgctgctg 12720
ctgctgcctg cggcccgggg ctgatgcctg aacttggggg ctggttggag atggccttgg 12780
agcagccaag gtgcccctgg cagtggcatc ccgaaacgcc ctggacgcag ggcccaagac 12840
tgggcacagg atcgcgtatc gggggggttt tatatatagg gcgcgctggt gtccccccgc 12900
gcgcaaattc tctctgggtg tgtcccaaaa gggagagaga cttctctgcg cgcgttatat 12960
atatacgcgc ctcgctcgct cgctctagct gtacgctagc gttataggga aggtcagaag 13020
agctcctggc tgtggtgggc agggcaggaa acccctccac ctttacacat gcccaggcag 13080
cacctcaggc cctttgtggg gcagggaagc tgaggcagta agcgggcagg cagagctgga 13140
ggcctttcag gcccagccag cactctggcc tcctgccgcc gcattccacc ccagcccctc 13200
acaccactcg ggagagggac atcctacggt cccaaggtca ggagggcagg gctggggttg 13260
actcaggccc ctcccagctg tggccacctg ggtgggagct ctccttggaa cccattcctg 13320
aaattattta aaggggttgg ccgggctccc accagggcct gggtgggaag gtacaggcgt 13380
tcccccgggg cctagtaccc ccgccgtggc ctatccactc ctcacatcca cacactgcac 13440
ccccactcct ggggcagggc caccagcatc caggcggcca gcaggcacct gagtggctgg 13500
gacaagggat cccccttccc tgtggttcta ttatattata attataatta aatatgagag 13560
catgctaagg acggctggac agcaggagcc gccgcttatt acgtgggcta tctgcagccc 13620
cggaccttcc tgctgaagta caacgagaac ggcaccatca ccgacgcagt ggattgcgct 13680
gcgaggtgtt caacgccacc agattcgcca gcgtctacgc ctggaaccgg aagcggatca 13740
gcaattgcgt ggccgactac agcgtgctgt acaacagcgc cagcttcagc accttcaagt 13800
gctacggcgt gtcccctacc aagctgaacg atctgtgctt caccaacgtg tacgccgaca 13860
gcttcgtgat caggggcgac gaagtgcgac agatcgcccc aggacagaca ggaaagatcg 13920
ccgattacaa ctacaagctg cccgacgact tcaccggttg cgtgatcgct tggaacgtcg 13980
gcggcaacta caactacctg taccggctgt tccggaagtc caacctgaag cccttcgagc 14040
gggacatcag cacagagatc taccaggccg gcagcacacc ttgtaacggc gtggagggct 14100
tcaactgcta cttccctctg cagagctacg gcttccagcc tacaaacggc gtgggatacc 14160
agccttacag ggtggtggtg ctgagcttcg aactgctgca cgctcgtgaa gaacaagtgc 14220
gtgaatttca acttcaacgg cctgaccgga accggcgtgc tgaccgagag caacaagaag 14280
ttcctgccct tccagcagtt cggcagagac atcgcagaca ccacagacgc cgtgagagat 14340
cctcagaccc tggagatcct ggacatcacc ccttgcagct ttggcggagt gtccgtgatc 14400
accccaggaa ccaacaccag caatcaggtg gccgtgctgt accaggacgt gaattgcacc 14460
gaggtgccag tggccattca cgcagatcag ctgaccccta cctggagagt gtacagcacc 14520
ggaagcaacg tgttccagac cagggcaggt tgcctgatcg gagccgagca cgtgaacaac 14580
agctacgagt gcgatatccc tatcggcgcc ggcatttgcg cctcttacca gacccagacc 14640
aacagcccta ggagagccag aagcgtggcc tctcagagca tcatcgccta caccatgagc 14700
ctgggagccg agaacagcgt ggcctacagc aacaacagca tcgccatccc caccaacttc 14760
accatcagcg tgaccaccga gattctgccc gtgtctatga ccaagaccag cgtcgactgc 14820
accatgtaca tctgcggcga cagcaccgag tgctctaacc tgctgctgca gtacggctcc 14880
ttttgcaccc agctgaacag ggctctgaca ggcatcgcag tggagcagga caagaacacc 14940
caggaggtct tcgcccaggt caagcagatt gccgccagag acctgatttg cgcccagaag 15000
ttcaacggcc tgacagtgct gcctcctctg ctgaccgacg agatgatcgc ccagtataca 15060
agcgctctgc tggcaggaac catcaccaga tggcctaccg cttcaacgga atcggcgtga 15120
cccagaacgt gctctacgag aaccagaagc tgatcgccaa ccagttcaac agcgccatcg 15180
gcaagatcca ggacagcgcg tattatacac taagaataga tatgcctgag cagcacagct 15240
agcgctctgg gcaagctgca ggatgtggtg aaccagaacg cccaggctct gaacaccctg 15300
gtgaagcagc tgagcagcaa cttcggagcc atcagcagcg tgctgaacga catcctgagc 15360
aggctggaca aggtggaagc cgaagtgcag atcgacaggc tgatcaccgg cagactgcag 15420
tctctgcaga cctacgtgac ccagcagctg atcagagccg ccgagatcag agctagcgct 15480
aatctggccg ccaccaagat gagcgagtgc gtgctgggac agagcaagag agtggacttc 15540
tgcggcaagg gctaccacct gatgagcttt cctcagagcg ctcctcacgg agtggtgttt 15600
ctgcacgtga cctacgtgcc agcccaggag aagaacttca ccacagcccc agccatttgc 15660
cacgacgcgt gacccagcgg aacttctacg agccccagat catcaccacc gacaacacct 15720
tcgtgagcgg caattgcgac gtggtcatcg gcatcgtgaa caacaccgtg tacgaccccc 15780
gcgctatata gacacagtag atgatagtgc agccagagct ggactccttc aaggaggagc 15840
tggacaagta cttcaagaac cacaccagcc cagacgtgga cctgggagat atcagagagc 15900
ctgatcgacc tgcaggagct gggcaagtac gagcagtaca tcaagtggcc ttggtacatt 15960
tggctgggct tcatcgccgg cctgatcgca atcgtcatgg tgaccatcat gctctgttgc 16020
atgaccagtt gctgcagctg cctgaagggt tgctgcagtt gcgggagttg ctgcaagttc 16080
gacgaggacg acagcgagcc agtgctgaaa ggcgtgaagc tgcactacac gcgacagcag 16140
acgatatata tacatgataa ggcgcgccca cccagcggcc gcatacagca gcaattggca 16200
agctgcttac atagaactcg cggcgattgg catgccgcct taaaattttt attttatttt 16260
tcttttcttt tccgaatcgg attttgtttt taatatttca aaaaaaaaaa aaaagaagag 16320
ctagggataa cagggtaatt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 16380
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 16440
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 16500
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 16560
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 16620
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 16680
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 16740
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 16800
cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 16860
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 16920
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 16980
tcaagaaggc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 17040
tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 17100
ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 17160
aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttcgcg 17220
gcgctctctc tctcttatat agagacacga gaaaaactat cgagcatcaa atgtaatttc 17280
ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatggtga 17340
gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattagtgtc 17400
atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg 17460
aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag 17520
gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg 17580
gaatgctgtt ttcccaggga tcgcagtggt gagtaaccat gcatcatcag gagtaggata 17640
aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 17700
tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 17760
ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 17820
ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt 17880
tcccgttgaa tatggctcac atttcccgcg cggcgcggat attataatta ttatcatgac 17940
attaacctat aaaaataggc gtatcacgag gccctttcgt ctagggataa cagggtaatt 18000
aatacgactc actatag 18017
Claims (9)
1.自复制mRNA增强型TIL细胞的制备方法，其特征在于，包括如下步骤：
将肿瘤样品处理成组织块，然后加入含5000-10000IU/mL IL-2的完全培养基中培养，得到TIL细胞；
根据α病毒家族的基因组设计自复制mRNA序列，并利用所述自复制mRNA序列构建自复制载体，然后将目的基因插入自复制载体中，得到重组质粒；
利用重组质粒通过体外转录合成含有目的基因的自复制mRNA；
将含有目的基因的自复制mRNA转染至TIL细胞中，即得自复制mRNA增强型TIL细胞；
其中，所述目的基因选自PD-1、CBLB、CISH、CCR2、CCL5、CXCL1、CXCL8中的至少一种。
2.根据权利要求1所述的制备方法，其特征在于，所述自复制载体的核苷酸序列如SEQID NO:3所示。
3.根据权利要求1或2所述的制备方法，其特征在于，所述完全培养基为含有8-10％v/vFBS的基础培养基，所述基础培养基选自RPMI 1640、DMEM中的至少一种。
4.根据权利要求1所述的制备方法，其特征在于，所述将含有目的基因的自复制mRNA转染至TIL细胞的方法选自电穿孔法、脂质体转染法中的一种。
5.根据权利要求4所述的制备方法，其特征在于，所述目的基因为PD-1。
6.根据权利要求5所述的制备方法，其特征在于，所述重组质粒的核苷酸序列如SEQ IDNO:4或SEQ ID NO:5所示。
7.根据权利要求1-6任一项所述的自复制mRNA增强型TIL细胞的制备方法制得的自复制mRNA增强型TIL细胞。
8.根据权利要求7所述的自复制mRNA增强型TIL细胞在制备治疗肿瘤的药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述肿瘤选自乳腺癌、结肠癌、卵巢癌、前列腺癌、胰腺癌中的至少一种。
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