CN111424054B - 改进的大型载体在人类细胞中传递方法的发现及应用 - Google Patents
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Abstract
本发明涉及基因组工程方面技术领域，尤其是改进的大型载体（6‑15 kb）在人类细胞中传递方法，包括向大型载体(6‑15 kb)中添加小型(1‑3 kb)载体、通过电穿孔等量共转染小(1‑3 kb)载体和大载体(6‑15 kb)。本技术方案结构设计合理，可以克服现有方法的局限性，显著提高细胞转染效率和细胞存活率，利用小载体改善细胞转染的方法将会在临床生物医学和工业生物技术中得到广泛应用，具有很好的推广应用前景。
Description
技术领域
本发明涉及基因编辑技术领域，尤其涉及一种新的细胞转染方法研发及其应用，尤其是改进了大型载体在人类细胞转染方法。
背景技术
CRISPR是原核生物基因组内的一段重复序列，是原核生物抵抗病毒入侵而进化出来的免疫武器， 来抵御病毒将自身基因整合到原核生物基因组内，从而产生了CRISPR-Cas9系统，通过该系统细菌可以精准敲除或者抑制病毒在原核生物中复制和繁殖。
CRISPR-Cas9技术与哺乳动物的二次免疫应答类似，细菌在抵抗病毒或外源质粒入侵时，会产生相应的“记忆”，来抵抗该种外源遗传物质的再次入侵，而这种获得性免疫正是由细菌的CRISPR-Cas9系统实现的在细菌的基因组上，存在着串联间隔排列的“重复序列”，这些重复序列相对保守，我们称之为CRISPR序列（Clustered RegularlyInterspersed Short Palindromic Repeats—成簇的规律间隔的短回文重复序列）。其中包括了可变的间隔序列和相对保守的重复序列，病毒或外源质粒上，存在“原间隔序列”，“间隔序列”正是与它们互相对应。“原间隔序列”的选取并不是随机的，这些原间隔序列的两端向外延伸的几个碱基往往都很保守，我们称为PAM（Protospacer adjacent motifs-原间隔序列临近基序）。当病毒或外源质粒DNA首次入侵到细菌体内时，细菌会对外源DNA潜在的PAM序列进行扫描识别，将临近PAM的序列作为候选的“原间隔序列”，将其整合到细菌基因组上CRISPR序列中的两个“重复序列”之间。这就是“间隔序列”产生的过程。当外源质粒或病毒再次入侵宿主菌时，会诱导CRISPR序列的表达。同时，在CRISPR序列附近还有一组保守的蛋白编码基因，称为Cas基因。CRISPR序列的转录产物CRISPR-RNA和Cas基因的表达产物等一起合作，通过对PAM序列的识别，以及“间隔序列”与外源DNA的碱基互补配对，来找到外源DNA上的靶序列，并对其切割，降解外源DNA。这也就实现了对病毒或外源质粒再次入侵的。
正是基于细菌的这种后天免疫防御机制，CRISPR-Cas9技术应运而生，从而被科学家们利用。引导Cas9核酸酶实现对多种细胞基因组的特定位点进行修饰。在CRISPR-Cas9技术中，我们把即将被编辑的细胞基因组DNA看作病毒或外源DNA。基因编辑的实现只需要两个工具——向导RNA（guide RNA， gRNA）和Cas9蛋白。
其中，向导RNA的设计并不是随机的，待编辑的区域附近需要存在相对保守的PAM序列（即三碱基序列NGG，其中N可以是任意碱基而这种特征的序列在每128bp的随机DNA序列中就重复出现一次。研究结果表明，Cas9还可以剪切线性和超螺旋的质粒，其剪切效率堪比限制性内切酶。），而且向导RNA要与PAM上游的序列碱基互补配对。以基因敲除为例，在待敲除基因的上下游各设计一条向导RNA（向导RNA1，向导RNA2），将其与含有Cas9蛋白编码基因的质粒一同转入细胞中，向导RNA通过碱基互补配对可以靶向PAM附近的目标序列，Cas9蛋白会使该基因上下游的DNA双链断裂。
对于DNA双链的断裂这一生物事件，生物体自身存在着DNA损伤修复的应答机制，会将断裂上下游两端的序列连接起来，从而实现了细胞中目标基因的敲除。
CRISPR-Cas9以其简单的设计、目标位点的特异性和高通量应用的可扩展性，彻底改变了生物系统的基因工程技术，颠覆了以往的基因编辑技术，它可以在体内和体外删除、增强或抑制基因表达。
CRISPR-Cas9系统的组成部分(包括导向RNAs )通常编码在大型染色体外表达载体(12- 19kb)上，这些载体通过转染的方法被传递到细胞中。由于体积大，这些载体很难成功的进入到细胞核中，并导致细胞死亡。
标准细胞转染方法的发展集中在改进技术方面，如物理介导方法包括电穿孔法发、基因枪和显微注射等，化学方法包括磷酸钙共沉淀法，脂体转染法等，生物介导包括原生质体转染法和病毒介导转染法等。其中病毒介导的转染效率最高，细胞毒性低等特点，但当用于研究或临床应用时，会引起巨大的生物安全问题和伦理问题。我们的方法能够显著提高细胞转染效率和细胞存活率，克服现有方法的局限性，并在临床生物医学和工业生物技术中具有很好的市场应用前景。
发明内容
为了能更好的介绍本发明的创新理念和技术手段，方便他人的理解和应用，下面将对本发明进行详细的说明。
CRISPR-Cas基因组工程是一项功能强大、用途广泛、适应性强的技术，但主要依赖于将大型载体(12- 19kb)导入人类细胞，导致转染效率和细胞存活率低下。在这里，我们提出了一种改进的无毒和非病毒的传递方法，可将大型载体的转染效率提高40倍，细胞存活率提高6倍。该技术的核心是在转染混合物中加入外源性的特定大小的小质粒。
优选的，我们通过电穿孔的转染方法将一个大型载体(6-15 kb)转染到人的细胞中。
优选的，我们向大型载体(6-15 kb)的混合液中添加了适量的小型载体( 1-3kb)，发现转染效率和细胞存活率显著提高(图1a-b)。
优选的，我们通过电转的方式将大载体(6-15 kb)和等量的小载体（1-4kb）共转染到Huh7细胞(人肝癌细胞系)，共转染效率从14.2%(图1c)提高到45.1%(图1d)，细胞死亡率从60.3%(图1c)降低至18.5%(图1d)，可以看出效果十分显著。
优选的，共转染的细胞不仅仅是Huh7细胞系还包括了HepG2（肝），PC3（前列腺），MCF7（乳腺），HEK293（肾），A549（肺），神经元（SH-SY5Y）、白血病（HL-60）以及外周血单个核细胞和纯化的CD8+T细胞。
优选的，共转染方法在其它难以转染和原代细胞类型中更为常用。
为了能更好的介绍本发明的创新理念和技术手段，方便他人的理解和应用，下面将对本发明进行详细的说明。
CRISPR-Cas基因组工程是一项功能强大、用途广泛、适应性强的技术，但主要依赖于将大型载体(12- 19kb)导入人类细胞，导致转染效率和细胞存活率低下。在这里，我们提出了一种改进的无毒和非病毒的传递方法，可将大型载体的转染效率提高40倍，细胞存活率提高6倍。该技术的核心是在转染混合物中加入外源性的特定大小的小质粒。
优选的，我们通过电穿孔的转染方法将一个大型载体(6-15 kb)转染到人的细胞中。
优选的，我们向大型载体(6-15 kb)的混合液中添加了适量的小型载体( 1-3kb)，发现转染效率和细胞存活率显著提高(图1a-b)。
优选的，我们通过电转的方式将大载体(6-15 kb)和等量的小载体（1-4kb）共转染到Huh7细胞(人肝癌细胞系)，共转染效率从14.2%(图1c)提高到45.1%(图1d)，细胞死亡率从60.3%(图1c)降低至18.5%(图1d)，可以看出效果十分显著。
优选的，共转染的细胞不仅仅是Huh7细胞系还包括了HepG2（肝），PC3（前列腺），MCF7（乳腺），HEK293（肾），A549（肺），神经元（SH-SY5Y）、白血病（HL-60）以及外周血单个核细胞和纯化的CD8+T细胞。
优选的，共转染方法在其它难以转染和原代细胞类型中更为常用。
附图说明
图1:CRISPR-cas9大载体与小载体共转染可以提高转染效率,a-b，细胞转染设置示意图。电穿孔介导的CRISPR-GFP大载体(6-15kb)转染，没有小(1-3kb)载体。c-d，流式细胞仪显示GFP+和死细胞标记物7AAD染色的Huh7细胞的门控。分别用GFP+细胞数和死细胞标记物7AAD染色细胞数来评估转染效率和细胞存活率。e，折线图显示了在A549、Huh7和MCF7细胞(n = 3，均值+/- SEM)中，大小不同的载体(1.8-6.5 kb)与大载体共转染后，转染效率和细胞存活率增长的百分比。f-h，折线图演示了(f)转染效率，(g)细胞存活率和(h)整体增加的百分比，通过在A549, Huh7, MCF7细胞中共转染大GFP载体(6.5 -15 kb)，不添加(灰色)和(绿色)小载体(1-3 kb)(n = 6 - 8,均值+ / - SEM,statistics: paired 2-tailedt-test, *p < 0.05, **p<0.01, ***p<0.001)。
图2:小型载体于CRISR大型载体共转染可提高多种类型细胞的转染效率,a，采用大型CRISPR-GFP (15kb)和小(3kb)载体结合电穿孔人类癌细胞系和新分离的初级免疫细胞。b-c，柱状图显示了(b)转染效率和(c)细胞存活率在共转染CRISPR-GFP 大载体(6-15kb)中没有或有小载体(1-3kb)的受试贴壁细胞和非贴壁细胞后，百分比的增长(n=1-6,均值+/-SEM)。加入小载体后(1-3kb)，通过降低转染效率或细胞存活率来对细胞类型进行排序。d-e，柱状图显示了添加经FC-富集排列的小载体(1-3kb)后，(d)GFP+和(e)活细胞数量的变化(FC)。f，图显示了斯皮尔曼等级相关系数(p)和假定值(p)的转染效率(x轴)和细胞活力(y轴)(百分比)没有(灰色)或有(绿色)共转染小载体(1-3kb) (n = 26)。g，模型解释了大的CRISPR载体(紫色)与小的载体(蓝色)的转染效率的增加。膜和核孔被小载体包覆，从而促进高效的核内传递和后面的大载体的分子活性。
图3:pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP8大载体图谱。
图4:pUC19小载体图谱。
图5:pBlueScript小载体图谱。
具体实施方式
此具体实施方式仅为介绍本发明的构思，为解释性和说明性，不能限制本发明的使用范围。
1.细胞培养和细胞系：HepG2、Huh7、PC3、SH-SY5Y、HEK293、MCF7、HL-60、A549癌细胞系均来自美国菌种保藏中心(ATCC)。所有的细胞系在使用支原体检测或分枝杆菌探针进行周期性检测时都没有支原体。
2.细胞在37°C和5% CO2气氛下的T-75烧瓶中培养。
3.使用补充了1/100青霉素/链霉素(P/S, Sigma)和10%胎牛血清(Hyclone)的培养基。
4.Huh7、HepG2、A549、HEK293、MCF7在Dulbecco改良的Eagle培养基中培养(DMEM,Sigma)，HL-60在RPMI培养基1640中培养(Sigma)，PC3和SH-SY-5Y在DMEM:F-12(1:1)培养基中培养(Gibco)。
5.细胞以约70-90%的融合速度分裂，吸取培养基。
6.用磷酸缓冲盐水(PBS, Sigma)轻轻冲洗细胞。
7.用3 mL胰蛋白酶DETA溶液(Sigma)分离3到5分钟。
8.在细胞传代之前，胰蛋白酶在至少10倍富余的培养基中倍灭活。所用载体包括（文中仅提供三种载体的核酸序列及其图谱）：
pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP8 (15.0 kb),
FC3-GFP(6.2kb),
pX458-prickle-GFP(9.2kb),
pCAGGs-jmj1dc-IRES-GFP(13.3kb),
pUC19-no-LacZ(1757bp),
pUC19(2686bp),
pBlueScript(2961bp),
Cterm-Halo(3473bp),
Halo-STMN1(4448bp),
PKM-Halo(5057bp),
Halo-CCT3(5633bp),
Halo-CTCF (6209bp),
N-D9-C-Term-Halo (6531bp)。
9.电穿孔：细胞电穿孔采用NEON氖电穿孔系统(Invitrogen)。收集生长至70-90%融合度的细胞。
10.在室温下500g离心5分钟。
11.细胞用PBS重悬，计数。
12.室温下500 g离心5分钟。
13.取适量质粒DNA(见下表)转入无菌1.5 mL微离心管。
14.从细胞微球中吸取PBS后，在复苏缓冲液R中复苏至1.0×107 个细胞/mL。
15.将细胞轻轻混合，得到单细胞悬浮液。
16.加入含有质粒DNA的试管中。这些细胞与质粒温和混合，没有产生任何气泡。为了避免不必要的细胞死亡，电穿孔细胞被直接镀入没有任何抗生素的预加热的酚红培养基中。
17.24小时后用GFP流式细胞仪检测转染效率，电穿孔设置如下。
| cells | #of cells | μg GFP vector | μg small vector | Voltage | ms | pulses | Further optimized from manufacturer’s settings |
| Huh7 | 106 | 7.5 | 7.5 | 1000 | 40 | 2 | Yes |
| HepG2 | 106 | 2.5 | 7.5 | 1200 | 30 | 2 | Yes |
| A549 | 106 | 5 | 5 | 1230 | 30 | 2 | No |
| HEK293 | 106 | 5 | 5 | 1100 | 20 | 2 | No |
| MCF7 | 106 | 5 | 5 | 1100 | 30 | 2 | No |
| HL60 | 106 | 5 | 5 | 1350 | 35 | 1 | No |
| PC3 | 106 | 5 | 5 | 1450 | 10 | 3 | No |
| SH-SY5Y | 106 | 5 | 5 | 1200 | 20 | 3 | No |
| PBMCs | 106 | 5 | 5 | 2150 | 20 | 1 | No |
| CD8 T cells | 106 | 5 | 5 | 2100 | 20 | 1 | No |
18. 脂质体转染：脂质体转染使用脂质体3000按照制造商的说明书进行，所有细胞均置于24孔板中(110,000/well HepG2 and MCF7, 70,000/ well Huh7, and 50,000/well A549). Mix 1: 25 μL Opti-MEM (Gibco) + 1.5 μL Lipofectamine 3000. Mix 2:25 μL Opti-MEM + 250 ng pBluescript + 250 ng GFP-vector + 1 μL P3000 reagent。
19.转染效率和细胞活力测定：收集所有可能死亡的细胞，包括上清液和贴壁细胞(通过胰酶消化收集)，用PBS 和1% 牛血清白蛋白（Sigma）冲洗细胞。在100μL缓冲液中用5μL 7-AAD活性染色液（eBioscience）染色15分钟。细胞是直接获得的，不需要清洗FACSNavios上的染色缓冲液(Beckman Coulter)。在一些实验中，使用Zoe荧光细胞成像仪(Bio-Rad)拍摄显微图像。
20.初级免疫细胞分离：根据机构指南，在获得志愿者知情同意后获得Buffycoats血沉棕黄层。如前所述，使用Ficoll-Paque (GE Healthcare)分离PBMCs 。按照制造商的说明使用磁性联合细胞分选(Miltenyi)分离CD8T细胞。电穿孔前，PBMCs和CD8 T细胞在37℃和5% CO2气氛中静置过夜，并补充1/100青霉素/链霉素和10%胎牛血清。
序列表
<110> 拜澳泰克（沈阳）生物医学集团有限公司
<120> 改进的大型载体在人类细胞中传递方法的发现及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2961
<212> DNA
<213> 未知(人工序列)
<400> 1
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540
gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600
taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660
gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg 720
atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt 780
gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 840
atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 900
cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 960
gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 1020
gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 1080
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1140
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1200
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1260
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1320
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1380
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1440
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1500
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1560
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1620
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 1680
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1740
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1800
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 1860
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 1920
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 1980
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 2040
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 2100
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 2160
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 2220
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 2280
ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2340
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 2400
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 2460
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 2520
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 2580
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 2640
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 2700
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 2760
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 2820
gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 2880
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 2940
catttccccg aaaagtgcca c 2961
<210> 2
<211> 14982
<212> DNA
<213> 未知(人工序列)
<400> 2
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540
tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 600
tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 660
cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 720
gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 780
ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 840
ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 900
aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 960
gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1020
ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1080
gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1140
agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1200
atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1260
acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1320
atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1380
ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1440
ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1500
atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1560
ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1620
cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1680
atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1740
agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1800
attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1860
aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1920
ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1980
ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2040
ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2100
gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2160
cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2220
gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2280
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2340
cagagatcca gtttggttaa ttaataactt cgtatagcat acattatacg aagttattaa 2400
aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa 2460
cttgctattt ctagctctaa aactgagacg tccgtctccg gtgtttcgtc ctttccacaa 2520
gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat 2580
tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt 2640
attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct ctctaacagc 2700
cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt aattaattaa 2760
cccgtgtcgg ctccagatct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc 2820
cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc 2880
cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta 2940
tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc 3000
agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc 3060
gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc 3120
gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt 3180
gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg 3240
gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga 3300
actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac 3360
gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga 3420
acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg 3480
gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg 3540
tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga 3600
gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg 3660
gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc 3720
tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga 3780
ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt 3840
tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg 3900
aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta 3960
aattctggcc gtttttggct tttttgttag acgaagcttg ggctgcaggt cgactctaga 4020
gccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag 4080
gatgacgatg acaagatggc ccccaagaag aagaggaagg tgggccgcgg aatggacaag 4140
aagtactcca ttgggctcgc catcggcaca aacagcgtcg gctgggccgt cattacggac 4200
gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 4260
aagaagaacc tcattggcgc cctcctgttc gactccgggg aaaccgccga agccacgcgg 4320
ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 4380
gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 4440
tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 4500
gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 4560
gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 4620
tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 4680
ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 4740
tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 4800
aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 4860
ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 4920
cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 4980
gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 5040
gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 5100
cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 5160
cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 5220
gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 5280
gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 5340
actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc 5400
aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 5460
ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 5520
tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 5580
aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 5640
aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 5700
ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 5760
cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 5820
ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 5880
gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 5940
aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 6000
acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 6060
gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 6120
agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 6180
aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 6240
tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac 6300
atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 6360
gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 6420
gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 6480
attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 6540
acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 6600
gtggatcagg aactggacat caatcggctc tccgactacg acgtggatgc catcgtgccc 6660
cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 6720
agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 6780
cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 6840
gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag 6900
acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 6960
gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 7020
tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 7080
gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 7140
cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 7200
aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 7260
aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 7320
gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 7380
cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 7440
ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 7500
aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 7560
ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 7620
ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 7680
gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 7740
tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 7800
aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 7860
aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 7920
cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 7980
gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 8040
gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 8100
ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 8160
gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 8220
cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtggc tagcgatgct 8280
aagtcactga ctgcctggtc ccggacactg gtgaccttca aggatgtgtt tgtggacttc 8340
accagggagg agtggaagct gctggacact gctcagcaga tcctgtacag aaatgtgatg 8400
ctggagaact ataagaacct ggtttccttg ggttatcagc ttactaagcc agatgtgatc 8460
ctccggttgg agaagggaga agagccctgg ctggtggaga gagaaattca ccaagagacc 8520
catcctgatt cagagactgc atttgaaatc aaatcatcag ttccgaaaaa gaaacgcaaa 8580
gttgctagcg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 8640
cctggtaccg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 8700
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 8760
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 8820
cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 8880
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 8940
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 9000
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 9060
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 9120
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 9180
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 9240
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 9300
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 9360
aagaccggtt gataatagat aacttcgtat agcatacatt atacgaagtt atgaattcga 9420
tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 9480
tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 9540
tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 9600
tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 9660
tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 9720
cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 9780
gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc 9840
ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 9900
cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 9960
gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 10020
cccgcatcga taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa 10080
tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg 10140
ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct 10200
tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca 10260
agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta 10320
cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc 10380
agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc 10440
tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag 10500
ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg 10560
gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 10620
tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 10680
taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt 10740
aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 10800
cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 10860
aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 10920
aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 10980
ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct 11040
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 11100
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 11160
gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 11220
ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 11280
gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 11340
tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 11400
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 11460
aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag 11520
aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc 11580
cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc 11640
cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 11700
ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca 11760
gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg 11820
tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc 11880
ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt 11940
ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 12000
ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 12060
ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 12120
cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 12180
gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 12240
cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta 12300
cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 12360
gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 12420
aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 12480
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 12540
tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 12600
tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 12660
aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 12720
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 12780
gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 12840
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 12900
tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 12960
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 13020
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 13080
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 13140
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 13200
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 13260
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 13320
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 13380
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 13440
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 13500
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 13560
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 13620
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 13680
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 13740
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 13800
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 13860
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 13920
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 13980
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 14040
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 14100
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 14160
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 14220
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 14280
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 14340
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 14400
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 14460
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 14520
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 14580
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 14640
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 14700
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga 14760
gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 14820
gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 14880
aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 14940
gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt 14982
<210> 3
<211> 2961
<212> DNA
<213> 未知(人工序列)
<400> 3
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540
gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600
taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660
gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg 720
atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt 780
gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 840
atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 900
cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 960
gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 1020
gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 1080
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1140
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1200
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1260
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1320
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1380
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1440
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1500
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1560
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1620
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 1680
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1740
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1800
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 1860
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 1920
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 1980
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 2040
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 2100
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 2160
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 2220
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 2280
ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2340
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 2400
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 2460
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 2520
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 2580
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 2640
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 2700
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 2760
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 2820
gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 2880
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 2940
catttccccg aaaagtgcca c 2961
Claims (3)
1.改进的大型载体6-15 kb在人类细胞中传递方法，其特征在于，通过电穿孔等量共转染1-3 kb小载体和6-15 kb大载体。
2.根据权利要求1所述的方法，其特征在于，1-3kb小载体作用在于提高转染效率和细胞存活率。
3.权利要求1和2任一所述的方法，其特征在于，在贴壁和非贴壁癌细胞系中转染，所述细胞系为肝癌细胞Huh7和HepG2，前列腺癌细胞PC3，乳腺癌细胞MCF7，肾癌细胞HEK293，肺癌细胞A549，神经母细胞瘤细胞SH-SY5Y、白血病细胞HL-60以及外周血单个核细胞和纯化的CD8+T细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010315646.4A CN111424054B (zh) | 2020-04-21 | 2020-04-21 | 改进的大型载体在人类细胞中传递方法的发现及应用 |
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| CN103865942A (zh) * | 2012-12-18 | 2014-06-18 | 常州碳宇纳米科技有限公司 | 一种可提高基因转染效率的纳米粒子和基于该粒子的基因转染试剂的制备 |
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| GB8904009D0 (en) * | 1989-02-22 | 1989-04-05 | Celltech Ltd | Vector |
| CN103865942A (zh) * | 2012-12-18 | 2014-06-18 | 常州碳宇纳米科技有限公司 | 一种可提高基因转染效率的纳米粒子和基于该粒子的基因转染试剂的制备 |
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