CN110452893B - 一种高保真CRISPR/AsCpf1突变体的构建及其应用 - Google Patents

Abstract

本发明公开了一种高保真CRISPR/AsCpf1突变体的构建及其应用。所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和/或第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸，其氨基酸序列如SEQ ID NO：1‑3所示。所述的AsCpf1突变体的编码基因，其核苷酸序列如SEQ ID NO：4所示。所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用。一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，所述AsCpf1蛋白为上述所述的AsCpf1突变体。CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中的应用。本发明构建的新型AsCpf1突变体既保留野生型AsCpf1的基因编辑效率，同时比野生型AsCpf1具有较高的特异性。

Description

一种高保真CRISPR/AsCpf1突变体的构建及其应用

技术领域

本发明涉及生物技术领域，具体涉及一种高保真CRISPR/AsCpf1突变体的构建及其应用。

背景技术

CRISPR/Cpf1是与CRISPR/Cas9系统相似的DNA编辑技术，它与CRISPR/Cas9一样，属于二类CRISPR系统核酸内切酶。但是，和Cas9相比，Cpf1更小，结构也更简单，而且Cpf1还具有Cas9没有的一些特性，如Cpf1切割形成的是粘性末端，PAM区为TTTN，且自身具有自加工CrRNA能力等特点，使得Cpf1不但可以帮助弥补CRISPR/Cas9系统的一些缺陷，而且可能在某些方面的应用上，比CRISPR/Cas9更有优势。目前CRISPR系统应用广泛，特别是它们逐渐被应用于疾病的基因治疗及改善退行性病变等方面。虽然CRISPR系统可以对各种细胞、组织、器官等进行高效基因编辑，但它们也会在一些与靶位点序列相似的位置产生非靶向的切割，这就是所谓的“脱靶效应”。这些脱靶效应会引起一些不可预测的突变，这就使得，如果要将这些核酸酶应用于临床，则可能存在潜在风险。基于此，开发高保真的CRISPR系统就显得非常关键。目前对于高保真Cas9已经开发了好几个版本，例如eCas9，Cas9-HF，HypaCas9，evoCas9等等。而对于Cpf1来说，其目前开发的高保真版本则很少。所以，本发明专利基于点突变技术，通过一系列突变改造，找到了一个高效，且可以降低脱靶效应的新型Cpf1突变体。

发明内容

本发明的第一个目的是提供一种新的，具有降低脱靶效应的AsCpf1突变体(AsCpf1-KA突变体)，即高保真的CRISPR/AsCpf1突变体。

为了达到上述目的，本发明所采用的技术方案为，一种AsCpf1突变体，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；

或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸；

或所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。

进一步地，所述AsCpf1突变体为对AsCpf1蛋白氨基酸序列的第951位精氨酸和第955位精氨酸进行突变，替换成不与靶位点DNA之间形成氢键的氨基酸。

进一步地，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：1所示；

或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：2所示；

或所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，以及第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。

进一步地，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K(951R->K)，以及第955位的精氨酸R变为丙氨酸A(955R->A)，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。

2016年Feng Zhang小组解析了AsCpf1蛋白(属于Cpf1家族中常用一个菌属)的三维结构，申请人通过研究分析，发现AsCpf1的氨基酸951的R和955的R可与基因组的靶位点DNA形成非特异性的氢键结合，这可能导致AsCpf1在进行基因编辑时，会在一些与靶位点序列相似的位置形成非特异性结合，从而产生非靶向切割。因此申请人对这几个位置进行突变，破坏蛋白与靶位点之间形成的非特异性的氢键结合，使得AsCpf1在基因编辑时对gRNA与靶位点之间核苷酸的互补配对要求加强，从而降低脱靶效应。

本发明的第二个目的是提供一种上述AsCpf1突变体的编码基因。

为了达到上述目的，本发明所采用的技术方案为，上述所述的AsCpf1突变体的编码基因，其核苷酸序列如SEQ ID NO：4所示。

本发明的第三个目的是提供一种上述所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用。本发明的第四个目的是提供一种CRISPR/AsCpf1基因编辑系统。

为了达到上述目的，本发明所采用的技术方案为，一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，所述AsCpf1蛋白为上述所述的AsCpf1突变体。

进一步地，所述CRISPR/AsCpf1基因编辑系统还含有用于启动编码AsCpf1突变体基因的sgRNA表达的U6启动子、AsCpf1基因编辑的必须元件CrRNA、用于启动编码AsCpf1突变体基因表达的真核启动子CAG、剪切钛序列P2A，以及监测质粒表达的报告基因mcherry。具体效用如下：

1、U6启动子，作用为启动AsCpf1突变体基因的sgRNA的表达；

2、AsCpf1的scaffold，即CrRNA，AsCpf1基因编辑的必须元件之一，同时也是导向目标靶序列的元件；

3、真核启动子CAG，用来启动AsCpf1突变体基因的表达；

4、剪切钛序列P2A，蛋白连接柔性序列；

5、红色荧光蛋白mCherry，荧光报告基因。

作为特异性验证，本发明实施例选取了DNMT1(DNA甲基转移酶1)的site3和另一个Match-site6。方法上采取了目标gRNA错配碱基和目标gRNA已知脱靶位点两种方法。而技术方面采用了常用的基因型分析方法PAGE和T7E1，通过转染野生型AsCpf1质粒(pu6-CAG-AsCpf1-mCherry野生型AsCpf1质粒)与AsCpf1突变体质粒(pu6-CAG-AsCpf1-KA-mCherry突变体AsCpf1-KA质粒)，在HEK-293T(人肾上皮细胞系)与MCF7(人乳腺癌细胞系)中进行验证。结果证明，不管是在错配碱基或是在已知的脱靶位点上，且在不同细胞株293T或MCF7中，突变的AsCpf1突变体(AsCpf1-KA)的特异性都要比野生型AsCpf1好。

本发明的第五个目的是提供一种上述CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中的应用。

与现有技术相比，本发明的优势在于：

本发明提供的AsCpf1-KA突变体(即AsCpf1突变体)的基因编辑脱靶效率要远低于野生型的AsCpf1。不管是在gRNA与靶位点不完全匹配的DNMT1-Site3上，其切割(脱靶的切割)效率要远低于野生型；还是在完全匹配的Match-site6位点上，PAGE和T7E1结果显示，以及在不同的细胞系(293T或MCF7细胞系)中，都可以明显观察到AsCpf1-KA突变体保真性更好，它能降低Match-site6的2个已知脱靶位点到检测限以下的水平。以上结果表明，AsCpf1-KA突变体不但具有较好的在靶切割活性，而且其特异性比野生型AsCpf1更好，是一种高保真的CRISPR核酸酶。

附图说明

图1为pU6-CAG-AsCpf1-mCherry野生型AsCpf1质粒图谱。

图2为pU6-CAG-AsCpf1-KA-mCherry突变体AsCpf1-KA质粒图谱。

图3为野生型AsCpf1(AsCpf1-WT)和AsCpf1-KA突变体(即AsCpf1突变体)在DNMT1-site3位点的在靶gRNA与错配gRNA的PAGE胶图。

图4为野生型AsCpf1和AsCpf1-KA突变体在Match-site6位点的在靶gRNA与2个脱靶位点的PAGE胶图，其中Blank为空白对照，WT为野生型AsCpf1，KA为AsCpf1-KA突变体。

图5为野生型AsCpf1和AsCpf1-KA突变体在Match-site6位点的在靶gRNA与2个脱靶位点的T7E1胶图，其中Blank为空白对照，WT为野生型AsCpf1，KA为AsCpf1-KA突变体。(左：细胞系HEK293T，右：细胞系MCF7，黑色箭头表示脱靶切割情况)

具体实施方式

下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。除非特别说明，下面实施例中所用的技术均为本领域内的技术人员已知的常规技术，所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。

实施例1重组表达质粒构建

pU6-CAG-AsCpf1-mCherry序列如SEQ ID NO：5所示。

以野生型pU6-CAG-AsCpf1-mCherry(图1)为载体(其核苷酸序列如SEQ ID NO：5所示)，利用Gibson Assembly原理技术，设计相对应突变引物，进行PCR，最后连接得到pU6-CAG-AsCpf1-KA-mCherry(图2)。具体如下：

先将pU6-CAG-AsCpf1-mCherry载体进行PmI I和BamHI酶切得到骨架载体。以pU6-CAG-AsCpf1-mCherry为模板，PCR分别扩增得到含突变碱基的片段，PCR引物序列如下：

AsCpf1-Pml I-F：5'-ACCAGCGACAAGTTCTTTTTCCACGTGCCTATCA-3'；(SEQ ID NO：6)

AsCpf1-KA-R：5'-CACAGACCAGGCCTGAGCGGCCGCCACCTTCTCCTTCTCCCTGT TG-3'；(SEQ ID NO：7)

AsCpf1-KA-F：5'-GAAGGAGAAGGTGGCGGCCGCTCAGGCCTGGTCTGTGGTGGGC-3'；(SEQ IDNO：8)

AsCpf1-BamH I-R：5'-AAGCGTAATCTGGAACATCGTATGGGTAGGATCC-3'；(SEQ ID NO：9)

以质粒pU6-CAG-AsCpf1-mCherry为模板，NEB Q5酶进行PCR(50μl体系如下：5×reaction buffer：10μl；5×enchance GC buffer：10μl；dNTP Mix 2.5μm each：4μl；F+R：2+2μl；Template：2ng DNA；水：up to 50μl。反应条件：98℃15s，35cycle(98℃10s，58℃30s，72℃1kb/s)，72℃10min，4℃hold)，跑胶，将得到的单一产物，应用PCR纯化试剂盒进行纯化。

其中AsCpf1-Pml I-F与AsCpf1-KA-R进行PCR，其产物命名为PCR-片段1；

AsCpf1-KA-F与AsCpf1-Bam H I-R进行PCR，其产物命名为PCR-片段2。

利用Gibson Assembly试剂盒将PCR片段1，PCR片段2以及使用限制性内切酶PmI I和BamHI对pU6-CAG-AsCpf1-mCherry载体进行双酶切后得到的骨架载体行3片段GibsonAssembly反应即可得到目标质粒pU6-CAG-AsCpf1-KA-mCherry(图2)，该质粒只是对pU6-CAG-AsCpf1-mCherry载体中的AsCpf1蛋白的氨基酸序列的第951位的精氨酸R变为赖氨酸K(951R->K)，以及第955位的精氨酸R变为丙氨酸A(955R->A)，突变后的AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。

实施例2特异性验证

为了验证得到的突变体AsCpf1-KA(即AsCpf1突变体)保真性比野生型AsCpf1好，设计以下实验进行特异性验证：

(1)文献报道(BP Kleinstiver,et al.Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells,Nartue.2016)，野生型AsCpf1对一些即使不完全匹配的gRNA也能进行切割，特别是1，2，8，9，19，20，21，22，23等位置。也就是说野生型AsCpf1能容忍一些gRNA的碱基错配，即特异性一般。所以，我们参考文献设计了针对DNMT1-site3这个位点的gRNA进行了验证，其中包括完全匹配的在靶位点(ON)，和错配1(mm1)，错配8(mm8)，错配9(mm9)，错配19(mm19)，错配20(mm20)等位置的gRNA，具体序列如下：

AsCpf1-gRNA-DNMT1-3-ON：CTGATGGTCCATGTCTGTTACTC；(SEQ ID NO：10)

AsCpf1-gRNA-DNMT1-3-mm1：GTGATGGTCCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：11)

AsCpf1-gRNA-DNMT1-3-mm8：CTGATGGACCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：12)

AsCpf1-gRNA-DNMT1-3-mm9：CTGATGGTGCATGTCTGTTACTC(下划线为错配位置)；(SEQ ID NO：13)

AsCpf1-gRNA-DNMT1-3-mm19：CTGATGGTCCATGTCTGTAACTC(下划线为错配位置)；(SEQ ID NO：14)

AsCpf1-gRNA-DNMT1-3-mm20：CTGATGGTCCATGTCTGTTTCTC(下划线为错配位置)；(SEQ ID NO：15)

分别以pU6-pCAG-AsCpf1-mCherry和pU6-pCAG-AsCpf1-KA-mCherry为载体，用BaeI酶切得到9452bp的载体，合成相对应的gRNA-oligo-F和R，并将其退火得到gRNA的DNA序列，利用T4ligase试剂盒将载体与完全匹配或带错配的gRNA相连，即可得到质粒：

pU6-CAG-AsCpf1-mCherry-ON(即pU6-CAG-AsCpf1-mCherry与AsCpf1-gRNA-DNMT1-3-ON连接，以下以此类推)，pU6-CAG-AsCpf1-mCherry-mm1，pU6-CAG-AsCpf1-mCherry-mm8，pU6-CAG-AsCpf1-mCherry-mm9，pU6-CAG-AsCpf1-mCherry-mm19，pU6-CAG-AsCpf1-mCherry-mm20；pU6-CAG-AsCpf1-KA-mCherry-ON，pU6-CAG-AsCpf1-KA-mCherry-mm1，pU6-CAG-AsCpf1-KA-mCherry-mm8，pU6-CAG-AsCpf1-KA-mCherry-mm9，pU6-CAG-AsCpf1-KA-mCherry-mm19，pU6-CAG-AsCpf1-KA-mCherry-mm20，

将上述构建好的12个表达质粒用转染试剂PEI转进HEK293T，48小时后，消化细胞，用SDS裂解法提取基因组DNA，以基因组DNA为模板进行PCR扩增，引物如下：

DNMT1-3-PAGE-F：5'-CAAGTGCTTAGAGCAGGCGT-3'；(SEQ ID NO：16)

DNMT1-3-PAGE-R：5'-GTGACGGGAGGGCAGAACTA-3'；(SEQ ID NO：17)

PCR反应体系如下：

gDNA：50ng；F+R引物：0.5μl；2×PCR mix：5μl；水：up to 10μl。

反应条件：95℃5min；40cycle(95℃30s，58℃30s，72℃20s)，72℃10min，95℃5min，然后自然降温至室温。

1)取PCR产物2ul加样，恒流12m Ah，跑PAGE胶，当溴酚蓝条带移动到距凝胶前沿约1cm时，停止电泳；

2)到达时间点后，将PAGE胶从玻璃板轻轻剥出，放入含GelRed的溶液中浸泡3min，然后于紫外下拍照并观察；

3)PAGE胶进行基因型鉴定，观察编辑效率和特异性情况，结果见图3。结果显示，野生型AsCpf1用错配的gRNA(1，8，9，19，20位)也能检测出明显的切割效果(出现明显的杂带，特别是20位错配其基本与不错配的gRNA显示出一样编辑效率28％)；而改造的AsCpf1-KA用错配的gRNA(1，8，9，19，20位)检测出的切割效率明显比野生型低，基本都保持在10％以下，但同时也保持了几乎与野生型(28％)一样的效率，达24％编辑效率。

(2)另外，有文献报道野生型AsCpf1在Match-site6上存在着明显的脱靶位点，于是申请人挑出了其中2个进行验证。在靶的gRNA和2个脱靶gRNA位置及序列如下：

Chr3：Match-site6-ON：GGGTGATCAGACCCAACAGCAGG；(SEQ ID NO：18)

Chr2：Match-site6-OT1：GGGTGATCAGACCCAACACCAGG(下划线为脱靶位置)；(SEQID NO：19)

Chr8：Match-site6-OT2：GGGTGATCAGACCCAACACCAGG(下划线为脱靶位置)；(SEQID NO：20)

同样，分别以pU6-pCAG-AsCpf1-mCherry和pU6-pCAG-AsCpf1-KA-mCherry为载体，用BaeI酶切得到9452bp的载体，合成在靶的gRNA-oligo-F和R，退火得到site6-ON-gRNA的DNA序列，利用T4ligase试剂盒将载体与gRNA的DNA序列相连，即可得到质粒：

pU6-CAG-AsCpf1-mCherry-site6-ON

pU6-CAG-AsCpf1-KA-mCherry-site6-ON

将上述构建好的2个表达质粒用转染试剂PEI转进HEK293T或MCF7，48小时后，消化细胞，用SDS裂解法提取基因组DNA，以基因组DNA为模板进行PCR扩增，然后应用T7E1和PAGE胶进行编辑效率及特异性分析，其中所用引物如下：

Site6-ON-F：5'-CCACATCCTCACCACCTGTT-3'；(SEQ ID NO：21)

Site6-ON-R：5'-CCCACAGCCATCCAGCTC-3'；(SEQ ID NO：22)

Site6-OT1-PAGE-F：5'-ACACTACGATGGTCCCTGGTGC-3'；(SEQ ID NO：23)

Site6-OT1-PAGE-R：5'-TGGATGCTGGATGGCGTCACAT-3'；(SEQ ID NO：24)

Site6-OT1-T7E1-F：5'-AGCCAATATTATTACATTGCCGTT-3'；(SEQ ID NO：25)

Site6-OT1-T7E1-R：5'-TGGCGTCACATTAGTGCCAT-3'；(SEQ ID NO：26)

Site6-OT2-PAGE-F：5'-GACTTGGCTAGCTTGGGGAC-3'；(SEQ ID NO：27)

Site6-OT2-PAGE-R：5'-GCTGTGAGAAACCCCATGTT-3'；(SEQ ID NO：28)

Site6-OT2-T7E1-F：5'-GACAGTTCAGACCCTTGGGG-3'；(SEQ ID NO：29)

Site6-OT2-T7E1-R：5'-TGCTGTGAGAAACCCCATGTT-3'；(SEQ ID NO：30)

T7E1鉴定方法具体如下：

1)提取细胞基因组DNA；

2)取50ng gDNA为模板行PCR，体系如下：gDNA：50ng；F+R引物：2μl；2×PCR mix：15μl；水：up to 30μl；

反应条件：95℃5min；38cycle(95℃30s，58℃30s，72℃20s)，72℃10min，4℃Hold；

3)纯化PCR产物，之后取300ng DNA行退火步骤：

纯化DNA：300ng；NEB buffer 2：2ul；水：up to 20μl；

反应条件：95℃5min，接着自然降温至室温，然后加入0.3ul T7E1内切酶37℃反应4h，之后跑胶于紫外下拍照观察。

PAGE胶鉴定方法具体如下：

1)提取细胞基因组DNA；

2)取50ng gDNA为模板行PCR，体系如下：gDNA：50ng；PAGE-F+R引物：0.5μl；2×PCRmix：5μl；水：up to 10μl；

反应条件：95℃5min；40cycle(95℃30s，58℃30s，72℃20s)，72℃10min，95℃5min，然后自然降温至室温；

3)取PCR产物2ul加样，恒流12m Ah，跑PAGE胶，当溴酚蓝条带移动到距凝胶前沿约1cm时，停止电泳；

4)到达时间点后，将PAGE胶从玻璃板轻轻剥出，放入含GelRed的溶液中浸泡3min，然后于紫外下拍照并观察。

PAGE胶和T7E1显示编辑效率和特异性情况见图4和图5。结果，与预期一致，突变体AsCpf1-KA的基因编辑脱靶效率要远低于野生型的AsCpf1。不管是在gRNA与靶位点不完全匹配的DNMT1-site3上，其切割(脱靶的切割)效率要远低于野生型；还是在完全匹配的Match-site6位点上，PAGE和T7E1结果显示，以及在不同的细胞系(293T或MCF7细胞系)中，都可以明显观察到突变体AsCpf1-KA保真性更好，它能降低Match-site6的2个已知脱靶点到检测限以下的水平，黑色箭头所示。所有这些结果都说明了突变体AsCpf1-KA不但具有较好的在靶切割活性，而且其特异性比野生型AsCpf1更好，是一种高保真的CRISPR核酸酶。

以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

SEQUENCE LISTING

<110> 南方医科大学

<120> 一种高保真CRISPR/AsCpf1突变体的构建及其应用

<130> CP11901466C

<160> 30

<170> PatentIn version 3.3

<210> 1

<211> 1352

<212> PRT

<213> 人工序列

<400> 1

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Lys Val Ala Ala Arg Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys

1295 1300 1305

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1310 1315 1320

Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp

1325 1330 1335

Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1340 1345 1350

<210> 2

<211> 1352

<212> PRT

<213> 人工序列

<400> 2

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Ala Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys

1295 1300 1305

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1310 1315 1320

Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp

1325 1330 1335

Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1340 1345 1350

<210> 3

<211> 1352

<212> PRT

<213> 人工序列

<400> 3

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Lys Val Ala Ala Ala Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys

1295 1300 1305

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1310 1315 1320

Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp

1325 1330 1335

Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1340 1345 1350

<210> 4

<211> 4059

<212> DNA

<213> 人工序列

<400> 4

atgacacagt tcgagggctt taccaacctg tatcaggtga gcaagacact gcggtttgag 60

ctgatcccac agggcaagac cctgaagcac atccaggagc agggcttcat cgaggaggac 120

aaggcccgca atgatcacta caaggagctg aagcccatca tcgatcggat ctacaagacc 180

tatgccgacc agtgcctgca gctggtgcag ctggattggg agaacctgag cgccgccatc 240

gactcctata gaaaggagaa aaccgaggag acaaggaacg ccctgatcga ggagcaggcc 300

acatatcgca atgccatcca cgactacttc atcggccgga cagacaacct gaccgatgcc 360

atcaataaga gacacgccga gatctacaag ggcctgttca aggccgagct gtttaatggc 420

aaggtgctga agcagctggg caccgtgacc acaaccgagc acgagaacgc cctgctgcgg 480

agcttcgaca agtttacaac ctacttctcc ggcttttatg agaacaggaa gaacgtgttc 540

agcgccgagg atatcagcac agccatccca caccgcatcg tgcaggacaa cttccccaag 600

tttaaggaga attgtcacat cttcacacgc ctgatcaccg ccgtgcccag cctgcgggag 660

cactttgaga acgtgaagaa ggccatcggc atcttcgtga gcacctccat cgaggaggtg 720

ttttccttcc ctttttataa ccagctgctg acacagaccc agatcgacct gtataaccag 780

ctgctgggag gaatctctcg ggaggcaggc accgagaaga tcaagggcct gaacgaggtg 840

ctgaatctgg ccatccagaa gaatgatgag acagcccaca tcatcgcctc cctgccacac 900

agattcatcc ccctgtttaa gcagatcctg tccgatagga acaccctgtc tttcatcctg 960

gaggagttta agagcgacga ggaagtgatc cagtccttct gcaagtacaa gacactgctg 1020

agaaacgaga acgtgctgga gacagccgag gccctgttta acgagctgaa cagcatcgac 1080

ctgacacaca tcttcatcag ccacaagaag ctggagacaa tcagcagcgc cctgtgcgac 1140

cactgggata cactgaggaa tgccctgtat gagcggagaa tctccgagct gacaggcaag 1200

atcaccaagt ctgccaagga gaaggtgcag cgcagcctga agcacgagga tatcaacctg 1260

caggagatca tctctgccgc aggcaaggag ctgagcgagg ccttcaagca gaaaaccagc 1320

gagatcctgt cccacgcaca cgccgccctg gatcagccac tgcctacaac cctgaagaag 1380

caggaggaga aggagatcct gaagtctcag ctggacagcc tgctgggcct gtaccacctg 1440

ctggactggt ttgccgtgga tgagtccaac gaggtggacc ccgagttctc tgcccggctg 1500

accggcatca agctggagat ggagccttct ctgagcttct acaacaaggc cagaaattat 1560

gccaccaaga agccctactc cgtggagaag ttcaagctga actttcagat gcctacactg 1620

gcctctggct gggacgtgaa taaggagaag aacaatggcg ccatcctgtt tgtgaagaac 1680

ggcctgtact atctgggcat catgccaaag cagaagggca ggtataaggc cctgagcttc 1740

gagcccacag agaaaaccag cgagggcttt gataagatgt actatgacta cttccctgat 1800

gccgccaaga tgatcccaaa gtgcagcacc cagctgaagg ccgtgacagc ccactttcag 1860

acccacacaa cccccatcct gctgtccaac aatttcatcg agcctctgga gatcacaaag 1920

gagatctacg acctgaacaa tcctgagaag gagccaaaga agtttcagac agcctacgcc 1980

aagaaaaccg gcgaccagaa gggctacaga gaggccctgt gcaagtggat cgacttcaca 2040

agggattttc tgtccaagta taccaagaca acctctatcg atctgtctag cctgcggcca 2100

tcctctcagt ataaggacct gggcgagtac tatgccgagc tgaatcccct gctgtaccac 2160

atcagcttcc agagaatcgc cgagaaggag atcatggatg ccgtggagac aggcaagctg 2220

tacctgttcc agatctataa caaggacttt gccaagggcc accacggcaa gcctaatctg 2280

cacacactgt attggaccgg cctgttttct ccagagaacc tggccaagac aagcatcaag 2340

ctgaatggcc aggccgagct gttctaccgc cctaagtcca ggatgaagag gatggcacac 2400

cggctgggag agaagatgct gaacaagaag ctgaaggatc agaaaacccc aatccccgac 2460

accctgtacc aggagctgta cgactatgtg aatcacagac tgtcccacga cctgtctgat 2520

gaggccaggg ccctgctgcc caacgtgatc accaaggagg tgtctcacga gatcatcaag 2580

gataggcgct ttaccagcga caagttcttt ttccacgtgc ctatcacact gaactatcag 2640

gccgccaatt ccccatctaa gttcaaccag agggtgaatg cctacctgaa ggagcacccc 2700

gagacaccta tcatcggcat cgatcggggc gagagaaacc tgatctatat cacagtgatc 2760

gactccaccg gcaagatcct ggagcagcgg agcctgaaca ccatccagca gtttgattac 2820

cagaagaagc tggacaacag ggagaaggag aaggtggcgg ccgctcaggc ctggtctgtg 2880

gtgggcacaa tcaaggatct gaagcagggc tatctgagcc aggtcatcca cgagatcgtg 2940

gacctgatga tccactacca ggccgtggtg gtgctggaga acctgaattt cggctttaag 3000

agcaagagga ccggcatcgc cgagaaggcc gtgtaccagc agttcgagaa gatgctgatc 3060

gataagctga attgcctggt gctgaaggac tatccagcag agaaagtggg aggcgtgctg 3120

aacccatacc agctgacaga ccagttcacc tcctttgcca agatgggcac ccagtctggc 3180

ttcctgtttt acgtgcctgc cccatataca tctaagatcg atcccctgac cggcttcgtg 3240

gaccccttcg tgtggaaaac catcaagaat cacgagagcc gcaagcactt cctggagggc 3300

ttcgactttc tgcactacga cgtgaaaacc ggcgacttca tcctgcactt taagatgaac 3360

agaaatctgt ccttccagag gggcctgccc ggctttatgc ctgcatggga tatcgtgttc 3420

gagaagaacg agacacagtt tgacgccaag ggcacccctt tcatcgccgg caagagaatc 3480

gtgccagtga tcgagaatca cagattcacc ggcagatacc gggacctgta tcctgccaac 3540

gagctgatcg ccctgctgga ggagaagggc atcgtgttca gggatggctc caacatcctg 3600

ccaaagctgc tggagaatga cgattctcac gccatcgaca ccatggtggc cctgatccgc 3660

agcgtgctgc agatgcggaa ctccaatgcc gccacaggcg aggactatat caacagcccc 3720

gtgcgcgatc tgaatggcgt gtgcttcgac tcccggtttc agaacccaga gtggcccatg 3780

gacgccgatg ccaatggcgc ctaccacatc gccctgaagg gccagctgct gctgaatcac 3840

ctgaaggaga gcaaggatct gaagctgcag aacggcatct ccaatcagga ctggctggcc 3900

tacatccagg agctgcgcaa caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 3960

aagaaaaagg gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct 4020

gattatgcat acccatacga tgtccccgac tatgcctga 4059

<210> 5

<211> 9485

<212> DNA

<213> 人工序列

<400> 5

aattgaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac 60

gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag 120

tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 180

gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt 240

tatatatctt gtggaaagga cgaaacaccg taatttctac tcttgtagat atcaccgcct 300

acgtcagtac ctacaagctt tttttacgcg ttgacattga ttattgacta gttattaata 360

gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 420

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 480

gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggacta 540

tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 600

tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 660

ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgggtcgagg 720

tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 780

atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg 840

ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca 900

gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 960

ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc 1020

ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag 1080

gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 1140

ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc 1200

gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg 1260

cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 1320

gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg 1380

gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc 1440

ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt 1500

cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc 1560

ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg 1620

ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc 1680

ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc 1740

cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg 1800

ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc 1860

tccatctcca gcctcggggc tgtccgcagg gggacggctg ccttcggggg ggacggggca 1920

gggcggggtt cggcttctgg cgtgtgaccg gcggctctag tgcctctgct aaccatgttc 1980

atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc 2040

attttggcaa agaattcatt taggtgacac tatagataat acgactcact atagggatga 2100

cacagttcga gggctttacc aacctgtatc aggtgagcaa gacactgcgg tttgagctga 2160

tcccacaggg caagaccctg aagcacatcc aggagcaggg cttcatcgag gaggacaagg 2220

cccgcaatga tcactacaag gagctgaagc ccatcatcga tcggatctac aagacctatg 2280

ccgaccagtg cctgcagctg gtgcagctgg attgggagaa cctgagcgcc gccatcgact 2340

cctatagaaa ggagaaaacc gaggagacaa ggaacgccct gatcgaggag caggccacat 2400

atcgcaatgc catccacgac tacttcatcg gccggacaga caacctgacc gatgccatca 2460

ataagagaca cgccgagatc tacaagggcc tgttcaaggc cgagctgttt aatggcaagg 2520

tgctgaagca gctgggcacc gtgaccacaa ccgagcacga gaacgccctg ctgcggagct 2580

tcgacaagtt tacaacctac ttctccggct tttatgagaa caggaagaac gtgttcagcg 2640

ccgaggatat cagcacagcc atcccacacc gcatcgtgca ggacaacttc cccaagttta 2700

aggagaattg tcacatcttc acacgcctga tcaccgccgt gcccagcctg cgggagcact 2760

ttgagaacgt gaagaaggcc atcggcatct tcgtgagcac ctccatcgag gaggtgtttt 2820

ccttcccttt ttataaccag ctgctgacac agacccagat cgacctgtat aaccagctgc 2880

tgggaggaat ctctcgggag gcaggcaccg agaagatcaa gggcctgaac gaggtgctga 2940

atctggccat ccagaagaat gatgagacag cccacatcat cgcctccctg ccacacagat 3000

tcatccccct gtttaagcag atcctgtccg ataggaacac cctgtctttc atcctggagg 3060

agtttaagag cgacgaggaa gtgatccagt ccttctgcaa gtacaagaca ctgctgagaa 3120

acgagaacgt gctggagaca gccgaggccc tgtttaacga gctgaacagc atcgacctga 3180

cacacatctt catcagccac aagaagctgg agacaatcag cagcgccctg tgcgaccact 3240

gggatacact gaggaatgcc ctgtatgagc ggagaatctc cgagctgaca ggcaagatca 3300

ccaagtctgc caaggagaag gtgcagcgca gcctgaagca cgaggatatc aacctgcagg 3360

agatcatctc tgccgcaggc aaggagctga gcgaggcctt caagcagaaa accagcgaga 3420

tcctgtccca cgcacacgcc gccctggatc agccactgcc tacaaccctg aagaagcagg 3480

aggagaagga gatcctgaag tctcagctgg acagcctgct gggcctgtac cacctgctgg 3540

actggtttgc cgtggatgag tccaacgagg tggaccccga gttctctgcc cggctgaccg 3600

gcatcaagct ggagatggag ccttctctga gcttctacaa caaggccaga aattatgcca 3660

ccaagaagcc ctactccgtg gagaagttca agctgaactt tcagatgcct acactggcct 3720

ctggctggga cgtgaataag gagaagaaca atggcgccat cctgtttgtg aagaacggcc 3780

tgtactatct gggcatcatg ccaaagcaga agggcaggta taaggccctg agcttcgagc 3840

ccacagagaa aaccagcgag ggctttgata agatgtacta tgactacttc cctgatgccg 3900

ccaagatgat cccaaagtgc agcacccagc tgaaggccgt gacagcccac tttcagaccc 3960

acacaacccc catcctgctg tccaacaatt tcatcgagcc tctggagatc acaaaggaga 4020

tctacgacct gaacaatcct gagaaggagc caaagaagtt tcagacagcc tacgccaaga 4080

aaaccggcga ccagaagggc tacagagagg ccctgtgcaa gtggatcgac ttcacaaggg 4140

attttctgtc caagtatacc aagacaacct ctatcgatct gtctagcctg cggccatcct 4200

ctcagtataa ggacctgggc gagtactatg ccgagctgaa tcccctgctg taccacatca 4260

gcttccagag aatcgccgag aaggagatca tggatgccgt ggagacaggc aagctgtacc 4320

tgttccagat ctataacaag gactttgcca agggccacca cggcaagcct aatctgcaca 4380

cactgtattg gaccggcctg ttttctccag agaacctggc caagacaagc atcaagctga 4440

atggccaggc cgagctgttc taccgcccta agtccaggat gaagaggatg gcacaccggc 4500

tgggagagaa gatgctgaac aagaagctga aggatcagaa aaccccaatc cccgacaccc 4560

tgtaccagga gctgtacgac tatgtgaatc acagactgtc ccacgacctg tctgatgagg 4620

ccagggccct gctgcccaac gtgatcacca aggaggtgtc tcacgagatc atcaaggata 4680

ggcgctttac cagcgacaag ttctttttcc acgtgcctat cacactgaac tatcaggccg 4740

ccaattcccc atctaagttc aaccagaggg tgaatgccta cctgaaggag caccccgaga 4800

cacctatcat cggcatcgat cggggcgaga gaaacctgat ctatatcaca gtgatcgact 4860

ccaccggcaa gatcctggag cagcggagcc tgaacaccat ccagcagttt gattaccaga 4920

agaagctgga caacagggag aaggagaggg tggcagcaag gcaggcctgg tctgtggtgg 4980

gcacaatcaa ggatctgaag cagggctatc tgagccaggt catccacgag atcgtggacc 5040

tgatgatcca ctaccaggcc gtggtggtgc tggagaacct gaatttcggc tttaagagca 5100

agaggaccgg catcgccgag aaggccgtgt accagcagtt cgagaagatg ctgatcgata 5160

agctgaattg cctggtgctg aaggactatc cagcagagaa agtgggaggc gtgctgaacc 5220

cataccagct gacagaccag ttcacctcct ttgccaagat gggcacccag tctggcttcc 5280

tgttttacgt gcctgcccca tatacatcta agatcgatcc cctgaccggc ttcgtggacc 5340

ccttcgtgtg gaaaaccatc aagaatcacg agagccgcaa gcacttcctg gagggcttcg 5400

actttctgca ctacgacgtg aaaaccggcg acttcatcct gcactttaag atgaacagaa 5460

atctgtcctt ccagaggggc ctgcccggct ttatgcctgc atgggatatc gtgttcgaga 5520

agaacgagac acagtttgac gccaagggca cccctttcat cgccggcaag agaatcgtgc 5580

cagtgatcga gaatcacaga ttcaccggca gataccggga cctgtatcct gccaacgagc 5640

tgatcgccct gctggaggag aagggcatcg tgttcaggga tggctccaac atcctgccaa 5700

agctgctgga gaatgacgat tctcacgcca tcgacaccat ggtggccctg atccgcagcg 5760

tgctgcagat gcggaactcc aatgccgcca caggcgagga ctatatcaac agccccgtgc 5820

gcgatctgaa tggcgtgtgc ttcgactccc ggtttcagaa cccagagtgg cccatggacg 5880

ccgatgccaa tggcgcctac cacatcgccc tgaagggcca gctgctgctg aatcacctga 5940

aggagagcaa ggatctgaag ctgcagaacg gcatctccaa tcaggactgg ctggcctaca 6000

tccaggagct gcgcaacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga 6060

aaaagggatc ctacccatac gatgttccag attacgctta tccctacgac gtgcctgatt 6120

atgcataccc atacgatgtc cccgactatg ccctcgagag caccggtggc agcggagcta 6180

ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctgccggta 6240

tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg 6300

tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc 6360

gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct 6420

tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc 6480

ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg 6540

tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg 6600

gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa 6660

tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg 6720

ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg 6780

aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca 6840

acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac 6900

gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa ctgcagcgcg 6960

gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 7020

acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 7080

gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 7140

gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 7200

caattccaca caacatacga gccggaagca taaagtgtaa agcctagggt gcctaatgag 7260

tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 7320

cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 7380

gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 7440

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 7500

agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 7560

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 7620

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 7680

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 7740

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 7800

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 7860

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 7920

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 7980

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 8040

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 8100

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 8160

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 8220

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 8280

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 8340

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 8400

tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 8460

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 8520

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 8580

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 8640

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 8700

gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 8760

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 8820

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 8880

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 8940

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 9000

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 9060

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 9120

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 9180

tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 9240

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 9300

gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc ctagggtcga 9360

ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg 9420

tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga 9480

ccgac 9485

<210> 6

<211> 34

<212> DNA

<213> 人工序列

<400> 6

accagcgaca agttcttttt ccacgtgcct atca 34

<210> 7

<211> 46

<212> DNA

<213> 人工序列

<400> 7

cacagaccag gcctgagcgg ccgccacctt ctccttctcc ctgttg 46

<210> 8

<211> 43

<212> DNA

<213> 人工序列

<400> 8

gaaggagaag gtggcggccg ctcaggcctg gtctgtggtg ggc 43

<210> 9

<211> 34

<212> DNA

<213> 人工序列

<400> 9

aagcgtaatc tggaacatcg tatgggtagg atcc 34

<210> 10

<211> 23

<212> DNA

<213> 人工序列

<400> 10

ctgatggtcc atgtctgtta ctc 23

<210> 11

<211> 23

<212> DNA

<213> 人工序列

<400> 11

gtgatggtcc atgtctgtta ctc 23

<210> 12

<211> 23

<212> DNA

<213> 人工序列

<400> 12

ctgatggacc atgtctgtta ctc 23

<210> 13

<211> 23

<212> DNA

<213> 人工序列

<400> 13

ctgatggtgc atgtctgtta ctc 23

<210> 14

<211> 23

<212> DNA

<213> 人工序列

<400> 14

ctgatggtcc atgtctgtaa ctc 23

<210> 15

<211> 23

<212> DNA

<213> 人工序列

<400> 15

ctgatggtcc atgtctgttt ctc 23

<210> 16

<211> 20

<212> DNA

<213> 人工序列

<400> 16

caagtgctta gagcaggcgt 20

<210> 17

<211> 20

<212> DNA

<213> 人工序列

<400> 17

gtgacgggag ggcagaacta 20

<210> 18

<211> 23

<212> DNA

<213> 人工序列

<400> 18

gggtgatcag acccaacagc agg 23

<210> 19

<211> 23

<212> DNA

<213> 人工序列

<400> 19

gggtgatcag acccaacacc agg 23

<210> 20

<211> 23

<212> DNA

<213> 人工序列

<400> 20

gggtgatcag acccaacacc agg 23

<210> 21

<211> 20

<212> DNA

<213> 人工序列

<400> 21

ccacatcctc accacctgtt 20

<210> 22

<211> 18

<212> DNA

<213> 人工序列

<400> 22

cccacagcca tccagctc 18

<210> 23

<211> 22

<212> DNA

<213> 人工序列

<400> 23

acactacgat ggtccctggt gc 22

<210> 24

<211> 22

<212> DNA

<213> 人工序列

<400> 24

tggatgctgg atggcgtcac at 22

<210> 25

<211> 24

<212> DNA

<213> 人工序列

<400> 25

agccaatatt attacattgc cgtt 24

<210> 26

<211> 20

<212> DNA

<213> 人工序列

<400> 26

tggcgtcaca ttagtgccat 20

<210> 27

<211> 20

<212> DNA

<213> 人工序列

<400> 27

gacttggcta gcttggggac 20

<210> 28

<211> 20

<212> DNA

<213> 人工序列

<400> 28

gctgtgagaa accccatgtt 20

<210> 29

<211> 20

<212> DNA

<213> 人工序列

<400> 29

gacagttcag acccttgggg 20

<210> 30

<211> 21

<212> DNA

<213> 人工序列

<400> 30

tgctgtgaga aaccccatgt t 21

Claims (7)

1.一种AsCpf1突变体，其特征在于，所述AsCpf1突变体为AsCpf1蛋白氨基酸序列的第951位的精氨酸R变为赖氨酸K，以及第955位的精氨酸R变为丙氨酸A，所述AsCpf1突变体的氨基酸序列如SEQ ID NO：3所示。

2.一种权利要求1所述的AsCpf1突变体的编码基因。

3.根据权利要求2所述的编码基因，其特征在于，其核苷酸序列如SEQ ID NO：4所示。

4.权利要求2或3所述的编码基因在构建CRISPR/AsCpf1基因编辑系统中的应用。

5.一种CRISPR/AsCpf1基因编辑系统，含有编码AsCpf1蛋白的基因，其特征在于，所述AsCpf1蛋白为权利要求1所述的AsCpf1突变体。

6.根据权利要求5所述的CRISPR/AsCpf1基因编辑系统，其特征在于，所述CRISPR/AsCpf1基因编辑系统还含有用于启动编码AsCpf1突变体基因的sgRNA表达的U6启动子、AsCpf1基因编辑的必须元件CrRNA、用于启动编码AsCpf1突变体基因表达的真核启动子CAG和剪切钛序列P2A。

7.权利要求5或6所述的CRISPR/AsCpf1基因编辑系统在基因编辑中降低脱靶效应中的应用。

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