CN108588123A - CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用 - Google Patents
CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用 Download PDFInfo
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Abstract
本发明公开了CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用，所述基因敲除猪为敲除了GGTA1基因、CMAH基因和β4GalNT2基因的猪，所述CRISPR/Cas9载体组合包括GGTA1‑CRISPR/Cas9载体、CMAH‑CRISPR/Cas9载体和β4GalNT2‑CRISPR/Cas9载体。通过设计特异性靶向的SgRNA序列，三个基因敲除效率分别为56％、63％和41％，敲除与免疫排斥反应有关的三个基因，得到三基因敲除猪，其红细胞与人血清中免疫球蛋白结合显著降低，对克服超急性免疫排斥反应有显著作用，有效解决了临床缺血的问题，为临床输血提供宝贵的材料来源。
Description
技术领域
本发明属于基因工程技术领域，具体涉及CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用。
背景技术
现代医学不断进步，输血在临床上被大量使用，但同样面临越来越多的问题，包括艾滋病、乙肝、丙肝等高发的传染病导致严重的输血风险。血液需求不断增多而献血量相对减少，血源资源日渐紧张。从19世纪至今，异种输血研究致力于用动物红细胞(RBCs)研发出人RBCs输血的替代品。在事故或战况下急性失血时，异种输血后能维持2-3天的生理功能，延长治疗的时间窗；病人对人RBCs敏感需要持续输血情况下，异种输血可以延长寿命和RBCs的功能。目前已有大量异种输血研究，用α-半乳糖苷酶和甲氧基聚乙二醇等方法修饰Gal抗原，或用基因敲除Gal和non-Gal抗原等方法修饰动物RBCs，为异种输血提供可能。猪红细胞(pRBCs)已广泛用于异种输血红细胞的研发。pRBCs异种输血有一定的优势，pRBCs和人RBCs具有大量的相似性。尽管pRBCs寿命比人的RBCs短，但是他们的直径和数量是相似的。猪的血型系统A-O(H)跟人的ABO血型系统是相关的。猪血红蛋白与人类血红蛋白具有85％的序列相同和相似的三维结构。同时，饲养在无特定病原体和生物安全条件下，pRBCs不携带人类的病原微生物，pRBCs不表达MHC抗原，即猪白细胞抗原(SLA)，因此降低了免疫原性。pRBCs没有细胞核，不可能携带猪内源性逆转录病毒。
但是pRBCs用于临床输血还有许多免疫学问题。将野生型pRBCs输注到灵长类动物体中会产生抗体-抗原结合，补体激活，以及输血细胞的直接裂解。因为野生型pRBCs表达了人类具有天然溶血抗体的Gal和non-Gal抗原。Gal是由α1,3-galactosyltransferase(GGTA1)产生的一种末端低聚糖，类似于A、B和O糖类，是灵长类天然抗体的主要目标抗原，当猪器官或细胞移植到非人灵长类时，产生一致的超急性排斥反应。目前还发现了一些引起急性排斥反应的抗non-Gal天然抗体，尽管与抗Gal抗体相比，抗non-Gal抗体相关的细胞毒性显著降低。其中一些抗原在pRBCs上表达，如N-glycolylneuraminic acid(NeuGc)和b1,4N-acetylgalactosaminyltransferase(B4GalNT2)。B4GalNT2是产生Sda血型抗原的一种糖基转移酶。NeuGc是一种糖偶联唾液酸，存在于人以外的所有哺乳动物体内，几乎100％的人类身上都可以检测到抗NeuGc抗体。因此，如果pRBCs能成功地为人类输血，那么α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)抗原就需要从pRBCs膜上消失。
发明内容
发明目的：为了解决现有异种红细胞临床输血中存在的免疫排斥反应，本发明提供了一种CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用。
技术方案：本发明所述CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用，所述基因敲除猪为敲除了GGTA1基因、CMAH基因和β4GalNT2基因的猪，所述CRISPR/Cas9载体组合包括GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体；所述GGTA1-CRISPR/Cas9载体含有SEQ ID No:1所示的特异性靶向GGTA1基因的SgRNA核苷酸序列，所述CMAH-CRISPR/Cas9载体含有SEQ ID No:2所示的特异性靶向CMAH基因的SgRNA核苷酸序列，所述β4GalNT2-CRISPR/Cas9载体含有SEQ ID No:3所示的特异性靶向β4GalNT2基因的SgRNA核苷酸序列。
优选地，所述血液制品为红细胞。
其中，所述GGTA1-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:4所示；所述CMAH-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:5所示；所述β4GalNT2-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:6所示。
其中，所述CRISPR/Cas9载体组合按如下方法构建得到：
(1)用BbsI酶酶切pX330质粒，酶切后的质粒使用琼脂糖凝胶分离，然后用胶回收试剂盒纯化回收酶切产物；
(2)在SgRNA核苷酸序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后退火得到双链片段；
(3)将步骤(1)得到的酶切产物和步骤(2)得到的双链片段使用连接酶进行连接；
(4)用质粒安全核酸外切酶处理步骤(3)得到的体系，去除错误连接的质粒；
(5)将步骤(4)得到的重组质粒转化到感受态细胞中进行培养；
(6)从步骤(5)培养的感受态细胞中提取重组质粒进行测序，确定载体构建成功；
当所述CRISPR/Cas9载体为GGTA1-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:1所示；当所述CRISPR/Cas9载体为CMAH-CRISPR/Cas9时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:2所示；当所述CRISPR/Cas9载体为β4GalNT2-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:3所示。
CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用，包括以下步骤：
(1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
(2)对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞；
(3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的猪卵母细胞中培养至囊胚阶段；
(4)将步骤(3)得到的囊胚移植到代孕猪中进行饲养，生产；
(5)静脉抽取步骤(4)生产的断奶后小猪的血液，置于抗凝管中保存，分离出红细胞。
所述红细胞的分离步骤如下：取抗凝管中保存的血液加入离心管中，用PBS溶液稀释，然后加入Ficoll-paque分离液形成分离体系，离心得到四层溶液，从上到下依次为血浆层、单核细胞层、Ficoll-paque层和红细胞层，弃去上三层，并用PBS溶液清洗红细胞，即得红细胞溶液。优选地，所述分离体系中血液、PBS溶液和Ficoll-paque分离液的体积比为2:2:3。
优选地，所述离心条件为：19℃，400g离心40min。
有益效果：(1)通过设计特异性靶向GGTA1基因、CMAH基因和靶向β4GalNT2基因的SgRNA序列，三基因敲除效率分别为56％、63％和41％；(2)通过对猪的基因进行改造，敲除与免疫排斥反应有关的三个基因(GGTA1/CMAH/β4GalNT2)，敲除方式为移码突变，可彻底敲除上述三个基因，得到三基因敲除猪，并取得其红细胞；(3)三基因敲除猪的红细胞与人血清中免疫球蛋白结合显著降低，对克服超急性免疫排斥反应有显著作用，有效解决了临床缺血的问题，为临床输血提供宝贵的材料来源。
附图说明
图1为GGTA1、CMAH和β4GalNT2三基因的CRISPR/Cas9靶点示意图；
图2为GGTA1-CRISPR/Cas9载体示意图；
图3为CMAH-CRISPR/Cas9载体示意图；
图4为β4GalNT2-CRISPR/Cas9载体示意图；
图5为体细胞核移植后出生的三基因敲除小猪出生时和断奶后的照片；
图6为用特异性结合α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)的抗体检测敲除猪PBMC中抗原的表达情况；
图7为GGTA1/CMAH/β4GalNT2三基因敲除猪(TKO)、野生型猪(WT)，GGTA1敲除猪(GGTA1-KO)和人分别分离PBMC，经人的血清孵育PBMC 2小时后，用抗IgG和IgM的抗体结合，流式细胞术检测与人的免疫球蛋白结合情况；
图8为GGTA1/CMAH/β4GalNT2三基因敲除猪(TKO)红细胞表面αGal,βGal和Neu5GC三种抗原流式结果；
图9为GGTA1/CMAH/β4GalNT2三基因敲除猪(TKO)红细胞与人的AB型血清中IgM和IgG结合结果；
图10健康人群(A,B,AB和O型)血清与TKO猪pRBCs红细胞凝集试验结果。
具体实施方式
实施例1构建CRISPR/Cas9载体
首先根据GGTA1/CMAH/β4GalNT2基因的DNA序列，合成靶向GGTA1，CMAH和β4GalNT2基因的sgRNA(single guide RNA)，以pX330为骨架质粒，分别构建GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体。
一、GGTA1-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪GGTA1基因序列，选取GGTA1基因的3号外显子exon3作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计SgRNA序列为GAAAATAATGAATGTCAA，见图1，核苷酸序列如SEQ ID No:1所示。
GGTA1-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在GGTA1基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列：在SgRNA核苷酸序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列：
5’–CACCGAAAATAATGAATGTCAA–3’
3’–CTTTTATTACTTACAGTTCAAA–5’。
SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、步骤三合成的正向和反向寡核苷酸序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃
4、按照以下体系启动连接反应：室温反应10min
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，公司测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为GGTA1-PX330，全核苷酸序列如SEQ ID No:4所示。
二、CMAH-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪CMAH基因序列，选取CMAH基因的6号外显子exon6作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计SgRNA向导序列为GAGTAAGGTACGTGATCTGT，见图1，核苷酸序列SEQ ID No:2所示。
CMAH-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在CMAH基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列：在SgRNA核苷酸序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列：
5’–CACCGGAGTAAGGTACGTGATCTGT–3’
3’–CCTCATTCCATGCACTAGACACAAA–5’
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、步骤三合成的正向和反向寡核苷酸序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，公司进行测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为CMAH-PX330，全核苷酸序列如SEQ ID No:5所示。
三、β4GalNT2-CRISPR/Cas9载体按如下方法制备得到：
首先根据Genbank中公布的猪β4GalNT2基因序列，选取β4GalNT2基因的8号外显子exon8作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计向导序列GGTAGTACTCACGAACACTC见图1，核苷酸序列SEQ ID No:3所示。
β4GalNT2-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在β4GalNT2基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列：在SgRNA核苷酸序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列：
5’–CACC GGTAGTACTCACGAACACTC–3’
3’–CCATCATGAGTGCTTGTGAGCAAA–5’
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、步骤三合成的正向和反向寡核苷酸序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，从公司测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为β4GalNT2-PX330，全核苷酸序列如SEQ ID No:6所示。
这种能广泛存在哺乳动物中分别表达GGTA1/CMAH/β4GalNT2基因的转基因片段(见图2、3和4)包含U6启动子，CMV联合鸡β-肌动蛋白(CMV-chicken-β-actin enhancer)基因的增强子，且带有哺乳动物细胞中筛选用的抗性基因—新霉素(Neomycin)基因和原核细胞中筛选用的抗性基因—氨苄青霉素(ampicillin)基因。这种能广泛性表达的β-骨骼肌肌动蛋白(CMV-chicken-β-actin promoter)基因的U6启动子可保证下游基因广泛性表达。
实施例2利用体细胞克隆的方法构建GGTA1/CMAH/β4GalNT2三基因敲除猪
将构建好的GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体通过与tdTomato质粒共转染猪胎儿成纤维细胞。通过G418筛选获得单细胞克隆，测序鉴定获得GGTA1/CMAH/β4GalNT2三基因敲除的猪胎儿成纤维细胞，通过体细胞核移植(SCNT)制备GGTA1/CMAH/β4GalNT2三基因敲除的长白猪。提取刚出生的小猪基因组，利用PCR引物进行扩增，连接T载体进行基因型鉴定。
步骤一、猪原代成纤维细胞复苏
1、从液氮中取出冻存的原代猪成纤维细胞，在37℃水浴中解冻；
2、将解冻的细胞转入无菌的15mL离心管中，然后加入3mL细胞培养基，1500rpm离心5min；
其中，细胞完全培养基的配方为：16％胎牛血清(Gibco)+84％DMEM培养基(Gibco)，16％和84％为体积百分比。
3、弃去上清，加入2mL完全培养基重悬细胞沉淀，然后将重悬的细胞铺入6cm细胞培养皿中，补加2mL完全培养基，置于37℃，5％CO2(体积百分比)的恒温培养箱中进行培养；
4、将细胞培养至长满皿底90％左右时使用0.05％(5g/100mL)的胰蛋白酶将细胞消化下来，然后加入完全培养基终止消化，将细胞悬液转入15mL离心管中，1500rpm离心5min，弃去上清，使用2mL完全培养基重悬细胞，对细胞计数，将细胞总量调整至1.5×106以备下一步核转染实验。
步骤二、使用构建好的GGTA1-PX330，CMAH-PX330，β4GalNT2-PX330和tdTomato质粒(Clontech,PT4069-5)共转染猪原代成纤维细胞
使用哺乳动物成纤维细胞核转染试剂盒(Lonza)与Lonza NucleofactorTM2b核转仪进行核转染实验
1、配制核转染反应液，体系如下：
核转染基本溶液 82μL
补充成分 8μL
2、将构建好的三个质粒与Tdtomato质粒分别按照质量比5:1的比例加入本步骤1获得的100μL核转反应液中混匀，过程中注意切勿产生气泡；
3、将步骤一制备得到的细胞悬液使用DPBS杜氏磷酸缓冲液(Gibco)洗两遍，37℃消化2min，用含体积百分比为10％胎牛血清的DMEM完全培养基终止消化后，1500rpm离心5min，弃去上清，使用本步骤2中含有质粒的核转反应液重悬细胞，重悬过程中要避免气泡的产生；
4、将该核转体系小心加入到试剂盒带有的电转杯中，注意防止气泡。先用含有100μLPBS的电转杯放置于Lonza核转仪的杯槽内，选择U023核转程序调试程序后，将含有细胞的电转杯电击转染后立即在超净台内将电转杯中液体轻柔吸出，转入到1mL含体积百分比为16％胎牛血清的DMEM完全培养基中，轻轻混匀；
5、准备含8mL完全培养基的培养皿(10cm)若干，吸取核转后的细胞悬液加入含有完全培养基的培养皿中，混匀，在显微镜下观察细胞数量，计数，使得培养皿在显微镜下一个视野内约有50～60个细胞，其余皿均按照此细胞悬液最终用量加入，混匀后放置于37℃，5％CO2的恒温培养箱中进行培养。
步骤三、三基因敲除细胞系的筛选
1、将步骤二所得细胞培养24h后将细胞培养基更换为含有1mg/mL G418的完全培养基，放置于37℃，5％CO2的恒温培养箱中进行培养，每2～3天更换一次细胞培养基，期间根据细胞生长状况逐渐降低G418的药物浓度，G418终浓度为0.3mg/mL，培养10～14天左右培养皿中会陆续长出G418抗性的单克隆细胞系；
2、使用克隆环挑取细胞系，将挑取的单克隆细胞系接种于铺有0.3mg/mLG418完全培养基的24孔板中，放置于37℃，5％CO2的恒温培养箱中进行培养，每2～3天换一次细胞培养基；
3、待24孔板的孔中细胞长满孔底，使用胰蛋白酶消化并收集细胞，其中4/5细胞接种到含有0.3mg/mL G418完全培养基的12孔板或6孔板中(根据细胞量)，剩余的1/5的细胞留在24孔板中继续培养；
4、待12孔板或6孔板细胞铺满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，使用细胞冻存液(90％胎牛血清+10％DMSO，体积比)将细胞冻存；
步骤四、三基因敲除细胞系的基因鉴定
1、待24孔板中细胞长满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，然后在细胞中加入25ml NP-40裂解液裂解细胞提取细胞基因组DNA，裂解程序为：55℃60min——95℃5min——4℃，反应结束后基因组DNA于-20℃保存；
2、针对GGTA1/CMAH/β4GalNT2基因靶点信息设计相应的PCR引物，PCR引物序列分别为：
GGTA1
正向引物为：5’-CCTTAGTATCCTTCCCAACCCAGAC-3’
反向引物为：5’-GCTTTCTTTACGGTGTCAGTGAATCC-3’
PCR目的产物长度为428bp；
CMAH
正向引物为：5’-CTTGGAGGTGATTTGAGTTGGG-3’
反向引物为：5’-CATTTTCTTCGGAGTTGAGGGC-3’
PCR目的产物长度为485bp；
β4GalNT2
正向引物为：5’-CCCAAGGATCCTGCTGCC-3’
反向引物为：5’-CGCCGTGTAAAGAAACCTCC-3’；
PCR目的产物长度为399bp；
3、使用PCR反应扩增GGTA1/CMAH/β4GalNT2靶点基因，PCR反应体系如下：
反应条件如下
CMAH靶点基因的扩增同上述步骤；β4GalNT2靶点基因的扩增同上述步骤。
4、将PCR反应产物进行琼脂糖凝胶电泳(1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)，电泳结束后在紫外线下切下目的条带，然后使用胶回收试剂盒(QIAGEN)回收目的条带，并使用NanoDrop 200测定回收的PCR产物的浓度；
5、将回收的PCR产物使用TAKARA pMDTM18-T Vector Cloning Kit链接T载体，T载体反应体系如下：
pMD18-T vector 1μL
胶回收PCR产物 81.7ng*
ddH2O 补齐体系至10uL
*注：TAKARA pMDTM18-T Vector Cloning Kit说明书上对Insert DNA(本次为胶回收PCR产物)用量的要求0.1～0.3pM，本次选取0.2pM，用量计算方法为：Insert DNA的使用量(ng)＝nmol数×660×Insert DNA 的bp数。
T载体链接的反应条件为16℃反应30min；
6、将本步骤5所得的T载体链接产物使用感受态细胞(TIANGEN)进行转化，转化后将感受态细胞涂布于Amp抗性的LB琼脂固体培养基上，37℃恒温培养箱培养过夜；
从培养过夜的培养基上挑取10～15个单克隆菌落送测序公司进行测序，然后将测序结果与靶点GGTA1/CMAH/β4GalNT2信息进行比对，从而判断该细胞系是否为GGTA1/CMAH/β4GalNT2基因敲除细胞系；
本次挑取的单克隆细胞系共27个，其中三个基因同时敲除的双等位基因敲除细胞系1个，编号为50#，该克隆基因型情况见表1：
表1 GGTA1/CMAH/β4GalNT2基因敲除的长白猪成纤维细胞的基因鉴定
敲除GGTA1、CMAH、β4GalNT2基因的敲除效率分别为56％、63％和41％。
GGTA1/CMAH/β4GalNT2三基因敲除和GGTA1/CMAH两基因敲除相比，与人的IgM，IgG的结合明显降低，因此三基因敲除是必须的。
步骤五、体细胞核移植
1、从屠宰场购买六月龄以上的母猪卵巢，人工抽取卵泡中未成熟的卵母细胞，在显微镜下挑取质量较好的卵母细胞并置于38.5℃，5％CO2恒温培养箱中培养42～44h至卵母细胞成熟；
2、利用显微操作系统将本步骤(1)中成熟的卵母细胞去核，然后复苏步骤四获得的GGTA1/CMAH/β4GalNT2敲除单克隆细胞系，将GGTA1/CMAH/β4GalNT2敲除细胞作为核供体注入去核卵母细胞中，每个去核卵母细胞注射一个GGTA1/CMAH/β4GalNT2敲除细胞；
3、将注射好的细胞利用电融合技术将核移植后得重构胚胎激活，将胚胎置于38.5℃培养箱培养5天发育成桑椹胚；
4、将发育情况良好的胚胎移植到代孕母猪的子宫中，小心护理代孕母猪，移植一个月后使用B超检测受体猪的怀孕情况，期间及时监控直至代孕母猪分娩。
步骤六、三基因基因敲除巴马小型猪的基因型分析
1、GGTA1/CMAH/β4GalNT2基因敲除小猪出生后剪取小猪耳部组织，然后使用血液/细胞/组织基因组DNA提取试剂盒(TIANGEN)提取小猪基因组DNA；
2、使用本步骤1中所得小猪基因组DNA进行PCR反应，PCR反应条件同步骤四3，然后将PCR反应产物送测序公司进行测序，将测序结果与GGTA1/CMAH/β4GalNT2基因靶点序列进行比对。
本次共出生8只长白公猪编号为1-8，出生的8头公猪与细胞基因型结果一致。
实施例3 GGTA1/CMAH/β4GalNT2三基因敲除猪的表型分析
1、敲除野生型猪体内的GGTA1、CMAH和β4GalNT2基因，可以有效地降低异种移植过程中的超急性免疫排斥反应
小猪断奶以后，抽血、分离外周血单个核细胞(PBMC)，通过流式细胞仪测定小猪的基因敲除情况，以及与人血清中免疫球蛋白(IgM，IgG)的结合情况，发现实施例2制得的三基因敲除小猪的α-1,3-半乳糖基转移酶(GGTA1)，CMP-N-乙酰神经氨酸羟化酶(CMAH)和β-1,4-N-乙酰氨基半乳糖转移酶2(β4GalNT2)三种抗原成功被敲除，如图6所示，其中PBSControl为空白对照，Isotype Control为chicken IgY，WT为野生型猪，GGTA1-KO为GGTA1基因敲除猪，CMAH-KO为CMAH基因敲除猪，β4GalNT2-KO为β4GalNT2基因敲除猪，结果显示，GGTA1/CMAH/β4GalNT2敲除猪不表达这三种抗原，代表三基因成功敲除。
通过以下方法分离PBMC：取100μL抗凝血，加3倍体积的红细胞裂解液(BD，用去离子水稀释10倍)，室温裂解5min～10min。离心后，弃上清，用预冷洗涤液0.1％FBS(溶剂为PBS，0.1％即0.1g FBS/100mL PBS)(增强细胞沉降)，漂洗，离心，即获得PBMC沉淀。
2、外周血单个核细胞(PBMC)经流式细胞仪检测三基因敲除小猪和对照野生型小猪的PBMC与人血清中免疫球蛋白结合水平，结果说明与野生型猪相比，三基因敲除猪的PBMC与人的免疫球蛋白结合水平明显降低，接近于人的水平
商业化的人血清56℃水浴锅灭活30min后，孵育已获得的PBMC，冰上孵育2h，5000rpm转离心5min，用PBS洗三次，体积比为10％山羊血清4℃封闭30min后，PBS洗三次。孵育人特异性的免疫球蛋白抗体后，PBS洗掉抗体，重悬，上机检测平均荧光强度。结果发现相较于野生型猪的PBMC来说，三基因敲除猪的PBMC与人的免疫球蛋白结合水平大大降低，与正常情况下人的水平差异不大，如图6所示，GGTA1/CMAH/β4GalNT2三基因敲除猪对克服超急性免疫排斥反应有显著作用。
3、TKO猪pRBC相关表型试验
(1)红细胞分离：几个工作人员合作固定小猪，用无菌注射器从前腔静脉丛抽取5mL血液，置于抗凝管中，4℃保存一周。取2mL抗凝血，加到15mL离心管中，再加2mL PBS溶液稀释混匀。缓慢将稀释好的血液加入装有3mL Ficoll-paque分离液(GE公司)的15mL离心管中，分上下两层，上层为血液，下层为Ficoll-paque分离液。19℃，400g离心40min后取出，此时液相分为四层，从上到下依次为血浆层、单核细胞层、Ficoll-paque层和红细胞层。弃去上层液，留下红细胞，并加7mL PBS溶液重悬混匀，19℃，400g离心10min后取出，弃去上层液。加5mL PBS溶液重悬混匀，19℃，400g离心10min后取出，弃去上层液，加2mL PBS重悬即可使用。
(2)IB4和DBA凝集素孵育：取1×105红细胞于1.5mL EP管中，3000rpm离心5min，弃去上清液。用PBS稀释好的IB4凝集素或DBA凝集素稀释液(稀释比例1：1000)200μL重悬细胞沉淀，4℃避光孵育1h，将未孵育凝集素的样品作为空白对照。用PBS溶液洗两次，离心的沉淀用200μL PBS溶液重悬，使用BD FACSCalibur流式细胞仪检测，使用FlowJo 10.0软件进行分析，结果见图8，抗原流式结果显示为阴性，和人的O型红细胞无差别。
(3)Neu5Gc抗体孵育：取1×105红细胞于1.5mL EP管中，3000rpm离心5min，弃去上清液。用稀释好的0.5％封闭液(不含哺乳动物血清)200μL重悬细胞，4℃避光孵育30min。用PBS溶液洗两次后，再用PBS溶液稀释好的鸡抗Neu5Gc抗体(Purified anti-Neu5GcAntibody(biolegend,146903))稀释液(稀释比例1：1000)200μL重悬细胞沉淀并4℃孵育1h，将未孵育抗体的样品作为空白对照。用PBS溶液洗两次后，再用PBS溶液稀释好的山羊抗鸡IgY抗体(invitrogen，A11039)稀释液(稀释比例1：1000)200μL重悬细胞沉淀并避光4℃孵育1h，3000rpm离心5min，弃去上清。用PBS溶液洗两次后，离心的沉淀用200μL PBS溶液重悬。使用BD FACSCalibur流式细胞仪检测，使用FlowJo 10.0软件进行分析，结果见图8，抗原流式结果显示为阴性，和人的O型红细胞无差别。
(4)人IgG/IgM结合实验：预先将人AB血清在56℃孵育30min灭活。取1×105红细胞于1.5mL EP管中，3000rpm离心5min，弃去上清液。用PBS溶液稀释好的15％(v/v)人AB血清稀释液200μL重悬细胞沉淀并4℃孵育1h，将未孵育人AB血清的样品作为空白对照。用PBS溶液洗两次后，再用10％(v/v)即用型正常山羊血清200μL重悬细胞，4℃孵育30min。用PBS溶液洗两次后，再用PBS溶液稀释好的山羊抗人IgG或IgM抗体(anti-human IgM(invitrogen,A18842)；anti-human IgG(invitrogen,A18830)稀释液(稀释比例1:1000)200μL重悬细胞沉淀并避光4℃孵育1h。3000rpm离心min，弃去上清。用PBS溶液洗两次后，离心的沉淀用200μL PBS重悬。使用BD FACSCalibur流式细胞仪检测，使用FlowJo 10.0软件进行分析，结果见图9，人和TKO猪红细胞的结合能力远低于WT猪红细胞对人IgG和IgM的结合能力。
(5)用间接抗人球蛋白法检测红细胞凝集：25μL 2％(v/v)pRBCs(野生型猪和TKO猪)悬液与50μL正常人群(A，B，AB和O型)血清分别加入玻璃试管中，37℃孵育30min，1500g30s离心弃上清，生理盐水洗3遍，加抗人IgG抗体(上海血液生物医药有限责任公司)25μL，1000g 15s常温离心，轻轻摇晃后观察凝集情况。结果见图10，其中1代表液体澄清，红细胞成逗号样滑落；1+代表液体浑浊，细胞分散；2+代表液体浑浊，细胞凝集成小块；3+代表液体澄清，凝集块可散开；4+代表液体澄清，凝集块不可散开。
序列表
<110> 南京医科大学
<120> CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaaaataatg aatgtcaa 18
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagtaaggta cgtgatctgt 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggtagtactc acgaacactc 20
<210> 4
<211> 8505
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 60
tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 120
aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 180
aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 240
taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 300
taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 360
agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 420
tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 480
cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 540
agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 600
gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 660
aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 720
tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga 780
gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt 840
agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat 900
catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa 960
ggacgaaaca ccgaaaataa tgaatgtcaa gttttagagc tagaaatagc aagttaaaat 1020
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 1080
gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 1140
gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 1200
cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 1260
gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 1320
tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 1380
agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 1440
ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 1500
cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 1560
gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 1620
ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 1680
ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga 1740
cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 1800
ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1860
tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 1920
tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 1980
ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 2040
agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 2100
agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 2160
agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 2220
agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 2280
tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 2340
agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 2400
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 2460
acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 2520
acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 2580
tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 2640
tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 2700
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 2760
atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2820
tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 2880
tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 2940
accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 3000
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 3060
gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 3120
ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 3180
acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 3240
acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 3300
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 3360
ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 3420
tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 3480
ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 3540
agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 3600
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 3660
tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 3720
agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3780
tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3840
aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 3900
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 3960
cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 4020
acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 4080
ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 4140
agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 4200
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 4260
ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 4320
tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 4380
ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 4440
tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 4500
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 4560
tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 4620
agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 4680
ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 4740
ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4800
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4860
cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 4920
acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 4980
ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 5040
cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 5100
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 5160
agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 5220
actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 5280
agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 5340
ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 5400
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 5460
aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 5520
tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 5580
tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 5640
ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 5700
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 5760
acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5820
agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 5880
actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 5940
acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 6000
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 6060
tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 6120
acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 6180
agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 6240
agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 6300
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 6360
ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 6420
gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 6480
ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 6540
gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 6600
agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 6660
gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 6720
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 6780
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 6840
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 6900
ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 6960
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 7020
actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 7080
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 7140
aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 7200
tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 7260
ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 7320
ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 7380
taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 7440
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 7500
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 7560
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 7620
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 7680
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 7740
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 7800
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 7860
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 7920
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 7980
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 8040
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 8100
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 8160
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 8220
gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 8280
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 8340
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 8400
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 8460
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctc 8505
<210> 5
<211> 8508
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 60
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 120
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 180
aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 240
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 300
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 360
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 420
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 480
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 540
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 600
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 660
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 720
atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 780
agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 840
tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 900
tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa 960
aggacgaaac accgagtaag gtacgtgatc tgtgttttag agctagaaat agcaagttaa 1020
aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt 1080
agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 1140
gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct 1200
gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt 1260
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 1320
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg 1380
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 1440
ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 1500
ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 1560
gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 1620
ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 1680
cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg 1740
cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 1800
tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1860
aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1920
aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg 1980
actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg 2040
ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca 2100
agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg 2160
acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca 2220
tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc 2280
ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc 2340
aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag 2400
agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg 2460
tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg 2520
tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca 2580
agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca 2640
agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg 2700
ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg 2760
aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg 2820
ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca 2880
aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg 2940
gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga 3000
gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca 3060
agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc 3120
tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca 3180
ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga 3240
tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc 3300
ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc 3360
tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga 3420
tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg 3480
cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg 3540
acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc 3600
ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg 3660
agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg 3720
agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc 3780
agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg 3840
tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg 3900
acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc 3960
tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt 4020
tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga 4080
gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc 4140
tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga 4200
cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc 4260
acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg 4320
tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa 4380
tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc 4440
ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa 4500
acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt 4560
acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc 4620
ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga 4680
accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact 4740
ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg 4800
ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg 4860
aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt 4920
acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg 4980
tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc 5040
acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta 5100
agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg 5160
ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca 5220
tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga 5280
tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg 5340
tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag 5400
gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa 5460
agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg 5520
tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc 5580
tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg 5640
aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc 5700
tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg 5760
gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg 5820
agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca 5880
agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg 5940
ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca 6000
gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg 6060
ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc 6120
tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt 6180
ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga 6240
aaaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6300
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6360
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6420
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc 6480
tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6540
ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6600
ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6660
tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 6720
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6780
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6840
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6900
cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 6960
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7020
caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 7080
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7140
aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7200
gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7260
ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7320
aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7380
ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7440
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7500
atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7560
caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7620
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7680
tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 7740
tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 7800
gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 7860
tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 7920
gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 7980
aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8040
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8100
atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8160
caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8220
ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8280
attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8340
agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8400
aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8460
catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctc 8508
<210> 6
<211> 8508
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 60
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 120
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 180
aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 240
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 300
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 360
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 420
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 480
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 540
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 600
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 660
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 720
atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 780
agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 840
tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 900
tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa 960
aggacgaaac accggtagta ctcacgaaca ctcgttttag agctagaaat agcaagttaa 1020
aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt 1080
agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 1140
gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct 1200
gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt 1260
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 1320
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg 1380
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 1440
ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 1500
ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 1560
gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 1620
ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 1680
cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg 1740
cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 1800
tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1860
aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1920
aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg 1980
actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg 2040
ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca 2100
agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg 2160
acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca 2220
tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc 2280
ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc 2340
aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag 2400
agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg 2460
tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg 2520
tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca 2580
agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca 2640
agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg 2700
ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg 2760
aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg 2820
ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca 2880
aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg 2940
gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga 3000
gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca 3060
agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc 3120
tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca 3180
ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga 3240
tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc 3300
ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc 3360
tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga 3420
tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg 3480
cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg 3540
acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc 3600
ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg 3660
agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg 3720
agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc 3780
agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg 3840
tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg 3900
acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc 3960
tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt 4020
tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga 4080
gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc 4140
tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga 4200
cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc 4260
acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg 4320
tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa 4380
tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc 4440
ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa 4500
acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt 4560
acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc 4620
ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga 4680
accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact 4740
ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg 4800
ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg 4860
aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt 4920
acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg 4980
tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc 5040
acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta 5100
agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg 5160
ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca 5220
tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga 5280
tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg 5340
tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag 5400
gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa 5460
agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg 5520
tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc 5580
tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg 5640
aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc 5700
tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg 5760
gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg 5820
agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca 5880
agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg 5940
ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca 6000
gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg 6060
ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc 6120
tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt 6180
ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga 6240
aaaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6300
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6360
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6420
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc 6480
tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6540
ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6600
ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6660
tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 6720
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6780
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6840
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6900
cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 6960
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7020
caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 7080
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7140
aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7200
gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7260
ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7320
aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7380
ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7440
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7500
atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7560
caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7620
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7680
tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 7740
tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 7800
gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 7860
tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 7920
gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 7980
aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8040
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8100
atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8160
caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8220
ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8280
attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8340
agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8400
aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8460
catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctc 8508
Claims (8)
1.CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用，所述基因敲除猪为敲除了GGTA1基因、CMAH基因和β4GalNT2基因的猪，所述CRISPR/Cas9载体组合包括GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体；所述GGTA1-CRISPR/Cas9载体含有SEQ ID No:1所示的特异性靶向GGT A1基因的SgRNA核苷酸序列，所述CMAH-CRISPR/Cas9载体含有SEQ ID No:2所示的特异性靶向CMAH基因的SgRNA核苷酸序列，所述β4GalNT2-CRISPR/Cas9载体含有SEQ ID No:3所示的特异性靶向β4GalNT2基因的SgRNA核苷酸序列。
2.根据权利要求1所述的应用，其特征在于，所述血液制品为红细胞。
3.根据权利要求1所述的应用，其特征在于，所述GGTA1-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:4所示；所述CMAH-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:5所示；所述β4GalNT2-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:6所示。
4.根据权利要求1所述的应用，其特征在于，所述CRISPR/Cas9载体组合按如下方法构建得到：
(1)用BbsI酶酶切pX330质粒，酶切后的质粒使用琼脂糖凝胶分离，然后用胶回收试剂盒纯化回收酶切产物；
(2)在SgRNA核苷酸序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后退火得到双链片段；
(3)将步骤(1)得到的酶切产物和步骤(2)得到的双链片段使用连接酶进行连接；
(4)用质粒安全核酸外切酶处理步骤(3)得到的体系，去除错误连接的质粒；
(5)将步骤(4)得到的重组质粒转化到感受态细胞中进行培养；
(6)从步骤(5)培养的感受态细胞中提取重组质粒进行测序，确定载体构建成功；
当所述CRISPR/Cas9载体为GGTA1-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:1所示；当所述CRISPR/Cas9载体为CMAH-CRISPR/Cas9时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:2所示；当所述CRISPR/Cas9载体为β4GalNT2-CRISPR/Cas9载体时，步骤(2)中所述SgRNA核苷酸序列如SEQ ID No:3所示。
5.根据权利要求1所述的应用，其特征在于，包括以下步骤：
(1)将CRISPR/Cas9载体组合转化至猪的胎儿成纤维细胞中；
(2)对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行PCR扩增基因测序，获得敲除GGTA1基因、CMAH基因和β4GalNT2基因的成纤维细胞；
(3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的猪卵母细胞中培养至囊胚阶段；
(4)将步骤(3)得到的囊胚移植到代孕猪中进行饲养，生产；
(5)静脉抽取步骤(4)生产的断奶后小猪的血液，置于抗凝管中保存，分离出红细胞。
6.根据权利要求5所述的应用，其特征在于，所述红细胞的分离步骤如下：取抗凝管中保存的血液加入离心管中，用PBS溶液稀释，然后加入Ficoll-paque分离液形成分离体系，离心得到四层溶液，从上到下依次为血浆层、单核细胞层、Ficoll-paque层和红细胞层，弃去上三层，并用PBS溶液清洗红细胞，即得红细胞溶液。
7.根据权利要求6所述的应用，其特征在于，所述分离体系中血液、PBS溶液和Ficoll-paque分离液的体积比为2:2:3。
8.根据权利要求6所述的应用，其特征在于，所述离心条件为：19℃，400g离心40min。
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| CN201810426018.6A CN108588123A (zh) | 2018-05-07 | 2018-05-07 | CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用 |
| JP2020563606A JP7285578B2 (ja) | 2018-05-07 | 2019-05-07 | 遺伝子ノックアウトブタ由来の血液製剤およびその使用 |
| US17/053,198 US20210254004A1 (en) | 2018-05-07 | 2019-05-07 | Blood product derived from gene knockout pig and use thereof |
| EP19799696.0A EP3791887A4 (en) | 2018-05-07 | 2019-05-07 | BLOOD PRODUCT DERIVED FROM A GENE KNOCKOUT PIG AND ITS USE |
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- 2018-05-07 CN CN201810426018.6A patent/CN108588123A/zh active Pending
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2019
- 2019-05-07 EP EP19799696.0A patent/EP3791887A4/en active Pending
- 2019-05-07 US US17/053,198 patent/US20210254004A1/en active Pending
- 2019-05-07 CN CN201980030378.7A patent/CN112105369A/zh active Pending
- 2019-05-07 WO PCT/CN2019/085773 patent/WO2019214591A1/zh unknown
- 2019-05-07 JP JP2020563606A patent/JP7285578B2/ja active Active
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2019214591A1 (zh) | 2019-11-14 |
| JP7285578B2 (ja) | 2023-06-02 |
| US20210254004A1 (en) | 2021-08-19 |
| EP3791887A9 (en) | 2022-08-31 |
| CN112105369A (zh) | 2020-12-18 |
| EP3791887A1 (en) | 2021-03-17 |
| JP2021523175A (ja) | 2021-09-02 |
| EP3791887A4 (en) | 2022-01-26 |
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