CN115247163A - 用于构建gp130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统及其应用 - Google Patents
用于构建gp130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统及其应用 Download PDFInfo
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Abstract
本发明公开了用于构建GP130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统及其应用。该基因编辑系统,包含按照本发明所述方法制备的高效Cas9蛋白、经筛选的针对GP130基因的高效靶点gRNA以及含有GP130突变位点的单链Donor DNA,并对该系统各成分的最佳用量配比进行了优化,最终获得靶标位点点突变的单细胞克隆的比率为22.5%,远高于常规的点突变效率(<5%)。
Description
技术领域
本发明属于基因编辑技术领域,具体涉及CRISPR/Cas9系统及ssODN同源重组技术在构建GP130基因突变的胃癌模型猪核移植供体细胞中的应用。
背景技术
胃癌(gastric cancer)是最常见的消化道恶性肿瘤。根据世界卫生组织国际癌症研究机构(IARC)发布的2020年全球最新癌症负担数据显示,胃癌占2020年内全球新增癌症病例的6.7%,位居第五,在国内则占比10.5%,位列第三,给我国人民造成了严重的健康威胁及经济负担,成为了当今最紧迫的健康问题之一。随着现代医疗的不断发展,尽管在癌症诊断、治疗和寿命方面有了进步,但对死亡率的改善程度一直不大。缺乏对疾病自然史的了解是造成这种局限性的主要原因,目前在分子水平上还不清楚胃癌中具体哪些变化可能导致肿瘤的化生、侵袭和转移。
白细胞介素6(interleukin6,IL-6)是细胞因子网络中的重要成员,其家族包括IL-6、IL-11、IL-27、IL-31、抑瘤素M(OSM)、白血病抑制因子(LIF)、睫状神经营养因子(CNTF)、心肌营养素1(CT-1)和心肌营养素样细胞因子1(CLCF1),这些细胞因子在免疫、造血和神经系统中表现出多效的生物活性,同时在人体代谢、自身免疫细胞分化、疾病治疗等方面都有重要作用。GP130是IL-6家族细胞因子功能受体复合物的常见信号转导成分,几乎表达于体内各种组织细胞上,其主要的作用之一就是利用胞外区结合IL-6-IL-6R复合物,向细胞内传导信号,从而参与上述重要的生理过程。已有研究结果显示,在小鼠中GP130基因Y757F突变(对应猪GP130 Y779)可引起与人类胃癌疾病表型相似的症状,经比对该位置在不同物种间高度保守。这表明GP130突变会导致胃癌的发生,然而其致病机制尚不清楚。
研究由GP130突变导致胃癌或其他疾病发生发展的细胞和分子机制及研发相应的药物均需要在动物模型的基础上进行,目前常用的动物模型为小鼠模型,然而小鼠不论从体型、器官大小、生理、病理等方面都与人相差巨大,不能真实地模拟人类正常的生理、病理状态。而猪作为大动物,是人类长期以来主要的肉食供应动物,其体型大小和生理功能与人类近似,易于大规模繁殖饲养,而且在伦理道德及动物保护等方面要求较低,是理想的人类疾病模型动物。
基因编辑是近年来不断取得重大发展的一种生物技术,其包括从基于同源重组的基因编辑到基于核酸酶的ZFN、TALEN、CRISPR/Cas9等编辑技术,其中CRISPR/Cas9技术是当前最先进的基因编辑技术。目前,基因编辑技术被越来越多地应用到动物模型的制作上。
同源重组(HDR)是通过序列同源性交换DNA序列信息:即修复模板中包含所需插入片段,修复模板的两端则是与插入位点附近具有序列同源性的重组臂。过去通常使用双链DNA(dsDNA)作为修复模板,但最近的研究揭示了单链寡核苷酸脱氧核苷酸(ssODN)作为HDR供体模板的优越性。首先,ssODN作为供体模板比dsDNA模板的插入位点特异性高,dsDNA模板容易产生随机插入。其次,ssODN对同源重组臂的长度要求比dsDNA模板更短,单侧30~60个碱基的重组臂设计可以获得高效且稳定的HDR,相比类似的dsDNA模板,其提供的插入效率更高。第三,dsDNA容易被NHEJ修复途径合并,从而导致同源臂的复制或者dsDNA模板的部分整合,而ssODN就不易产生这种现象。另外,dsDNAs对培养的细胞是有害的,线型或者质粒dsDNAs的转染效率较低,并使细胞产生不良反应,而ssODN模板在这些方面就更有优势。
因此,本发明采用CRISPR/Cas9技术联合ssODN同源重组技术进行了GP130基因的点突变基因编辑,模拟胃癌的自然发病遗传特征,并获得了GP130基因精确点突变的单细胞克隆,为后期通过体细胞核移植动物克隆技术培育胃癌疾病模型猪奠定了基础。该模型猪将为研究胃癌的发病机制及药物研发提供有力的实验工具。
发明内容
本发明的目的是提供一种编码含Cas9蛋白的特异融合蛋白的特异融合基因。
本发明的又一目的是提供用于构建GP130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统。
本发明的又一目的是提供该基因编辑系统的应用。
含Cas9蛋白的特异融合蛋白;所述的特异融合蛋白自N端至C端依次包括如下元件:用于目的蛋白分泌表达的信号肽,增加目的蛋白可溶性表达的分子伴侣融合蛋白,用于蛋白纯化的标签蛋白,用于去除融合标签,从融合蛋白中获取天然形式Cas9蛋白的内切蛋白酶识别位点,引导Cas9蛋白进入细胞核的核定位信号1,Cas9蛋白(spCas9),引导Cas9蛋白进入细胞核的核定位信号2。
所述特异融合蛋白中,用于目的蛋白分泌表达的信号肽选自大肠杆菌碱性磷酸酶(phoA)信号肽、金黄色葡萄球菌蛋白A信号肽、大肠杆菌外膜蛋白(ompa)信号肽或任何其他原核基因的信号肽,优选为碱性磷酸酶(phoA)信号肽。
所述特异融合蛋白中,增加目的蛋白可溶性表达的分子伴侣融合蛋白可为任何帮助形成二硫键的蛋白,优选为硫氧还原蛋白Trx,进一步优选为TrxA。
所述特异融合蛋白中,用于去除融合标签,从融合蛋白中获取天然形式Cas9蛋白的内切蛋白酶识别位点选自肠激酶(Enterokinase)、因子Xa(Factor Xa),凝血酶(Thrombin)、TEV蛋白酶(TEV protease)、HRV 3C蛋白酶(HRV 3C protease)、WELQut蛋白酶或任何其他内切蛋白酶的识别位点,进一步优选为肠激酶识别位点。
所述特异融合蛋白中,便于目的蛋白纯化的蛋白标签选自His标签、GST标签、Flag标签、HA标签、c-Myc标签或其他任何蛋白标签,进一步优选为His蛋白标签。
所述特异融合蛋白中,引导Cas9蛋白进入细胞核的核定位信号可为任何真核细胞核定位信号,进一步优选为SV40核定位信号和/或nucleoplasmin核定位信号。
所述特异融合蛋白中,cas蛋白选自Casl-lO、Cpfl或其他类型Cas蛋白,优选为Cas9,进一步优选为spCas9。
所述特异融合蛋白自N端至C端依次包括如下元件:碱性磷酸酶(phoA)信号肽(phoA:SP),硫氧还原蛋白A(TrxA),His标签蛋白,肠激酶酶切位点(EK),核定位信号(SV40NLS),Cas9蛋白(spCas9),核定位信号(nucleoplasmin NLS)。
一种编码所述特异融合蛋白的特异融合基因。
作为本发明的一种优选,所述的特异融合基因序列如SEQ ID NO.1中第5209-9849位核苷酸所示,或者如SEQ ID NO.2所示。
一种原核Cas9高效表达载体pKG-GE4,含有本发明所述的特异融合基因。
作为本发明的一种优选,所述的原核Cas9高效表达载体pKG-GE4的主要元件有:T7启动子(T7 Promoter),Lac操纵子(Lac Operator),核糖体结合位点(RBS)、所述特异融合基因、T7终止子序列元件。
质粒pKG-GE4中,由T7启动子启动所述特异融合基因的表达。
质粒pKG-GE4中,由Lac操纵子控制所述特异融合基因的诱导表达。
质粒pKG-GE4中,所述特异融合基因下游具有T7终止子序列元件。
作为本发明的进一步优选,所述的原核Cas9高效表达载体pKG-GE4的主要元件有:
T7启动子(T7 Promoter),Lac操纵子(Lac Operator),核糖体结合位点(RBS),碱性磷酸酶(phoA)信号肽(phoA:SP),硫氧还原蛋白A(TrxA),His标签蛋白,肠激酶酶切位点(EK),核定位信号(SV40 NLS),Cas9蛋白(spCas9),核定位信号(nucleoplasmin NLS),T7终止子(T7 terminator),载体骨架(包括Amp抗性元件、ori复制起始子及LacI组成型表达元件等)。
作为本发明的更进一步优选,本发明所述的原核Cas9高效表达载体pKG-GE4序列如SEQ ID NO.1所示。
在本发明所述的原核Cas9高效表达载体pKG-GE4中,特异融合基因具体如SEQ IDNO.1中第5209-9849位核苷酸所示,其中碱性磷酸酶信号肽的编码序列如SEQ ID NO.1中第5209-5271位核苷酸所示,TrxA蛋白的编码序列如SEQ ID NO.1中第5272-5598位核苷酸所示,His-Tag的编码序列如SEQ ID NO:1中第5620-5637位核苷酸所示,肠激酶酶切位点的编码序列如SEQ ID NO.1中第5638-5652位核苷酸所示,核定位信号(SV40 NLS)的编码序列如SEQ ID NO.1中第5656-5670位核苷酸所示,spCas9蛋白的编码序列如SEQ ID NO.1中第5701-9801位核苷酸所示,核定位信号(nucleoplasmin NLS)的编码序列如SEQ ID NO.1中第9802-9849位核苷酸所示。T7启动子如SEQ ID NO.1中第5121-5139位核苷酸所示。Lac操纵子如SEQ ID NO.1中第5140-5164位核苷酸所示。RBS如SEQ ID NO.1中第5178-5201位核苷酸所示,T7终止子如SEQ ID NO.1第9902-9949位核苷酸所示。
经过上述各项优化设计及改造,pKG-GE4载体所表达得到的Cas9蛋白活性比商品Cas9蛋白有了极显著的提高。
一种制备Cas9蛋白的方法,包含以下步骤:
(1)将鉴定正确的pKG-GE4质粒转化入大肠杆菌表达菌株BL21(DE3),培养菌体,加入IPTG,在25℃的温度下诱导所述的基因工程菌表达可溶性目的蛋白,并收集菌体沉淀;
(2)粗提融合蛋白,然后采用Ni-NTA琼脂糖柱进行融合蛋白的纯化;
(3)用带his标签的重组牛肠激酶酶切融合蛋白,并用Ni-NTA树脂纯化得到酶切后去除重组牛肠激酶和TrxA-His的NLS-spCas9-NLS目的蛋白。
一种用于构建GP130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统,包含按照本发明所述方法制备的Cas9蛋白,针对GP130基因的gRNA以及含有GP130突变位点的单链Donor DNA。
作为本发明的一种优选,所述的针对GP130基因的gRNA的靶点选自SEQ ID NO.15所示的GP130-E16-gRNA4和SEQ ID NO.16所示的GP130-E16-gRNA7。
作为本发明的进一步优选,所述的针对GP130基因的gRNA分别由如SEQ ID NO.25所示的GP130-T7-gRNA4转录模板和如SEQ ID NO.26所示的GP130-T7-gRNA7转录模板经体外gRNA转录得到。
作为本发明的进一步优选,所述的含有GP130突变位点的单链Donor DNA序列如SEQ ID NO.27所示。
作为本发明的进一步优选,GP130-E16-gRNA4:GP130-E16-gRNA7:Cas9蛋白:单链Donor DNA的质量比为1:1:4:2。
本发明所述的基因编辑系统在构建GP130基因突变的猪重组细胞中的应用。
一种重组细胞,由本发明所述的基因编辑系统共转染猪原代成纤维细胞经验证后所得。
本发明所述的基因编辑系统、本发明所述的重组细胞在构建GP130基因突变的胃癌模型猪中的应用。将所述重组细胞作为核移植供体细胞进行体细胞克隆,所得到的克隆猪即为胃癌模型猪。
本发明还保护用所述重组细胞所制备的模型猪的猪组织、猪器官和/或猪细胞。
本发明还保护所述重组细胞、所述猪组织、所述猪器官、所述猪细胞或者用所述重组细胞制备的胃癌模型猪在筛选胃癌治疗药物、胃癌治疗药物的药效评价、胃癌基因治疗和/或细胞治疗的疗效评价或胃癌的发病机制研究中的应用。
与现有技术相比,本发明至少具有如下有益效果:
(1)本发明研究对象(猪)比其他动物(大小鼠、灵长类)具有更好的应用性。
大小鼠等啮齿类动物不论从体型、器官大小、生理、病理等方面都与人相差巨大,无法真实地模拟人类正常的生理、病理状态。研究表明,95%以上在大小鼠中验证有效的药物在人类临床试验中是无效的。就大动物而言,灵长类是与人亲缘关系最近的动物,但其体型小、性成熟晚(6-7岁开始交配),且为单胎动物,群体扩繁速度极慢,饲养成本很高。另外,灵长类动物克隆效率低、难度大、成本高。
而猪作为模型动物就没有上述缺点,猪是除灵长类外与人亲缘关系最近的动物,其体型、体重、器官大小等与人相近,在解剖学、生理学、免疫学、营养代谢、疾病发病机制等方面与人类极为相似。同时,猪的性成熟早(4-6个月),繁殖力高,一胎多仔,在2-3年内即可形成一个较大群体。另外,猪的克隆技术非常成熟,克隆及饲养成本也较灵长类低得多。因此猪是非常适合作为人类疾病模型的动物。
(2)本发明所构建的pET32a-T7lac-phoA:SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter(简称pKG-GE4)载体,使用了能够高效表达目的蛋白的强启动子T7lac来进行目的蛋白的表达,用细菌周质蛋白碱性磷酸酶(phoA)的信号肽来引导目的蛋白分泌表达至细菌周质腔中,从而与细菌胞内蛋白分离,且分泌到细菌周质腔中的目的蛋白为可溶性表达。同时还采用硫氧还原蛋白TrxA与Cas9蛋白融合表达,TrxA能帮助所共表达的目的蛋白形成二硫键,提高蛋白的稳定性、折叠的正确性,增加目的蛋白的溶解性及活性。为了方便目的蛋白的纯化,设计了His标签,可以通过一步法Ni柱亲和层析纯化目的蛋白,极大地简化了目的蛋白的纯化流程。同时在His标签后设计了一个肠激酶酶切位点,便于切除所融合的TrxA-His多肽片段,得到天然形式的Cas9蛋白。利用带His标签的肠激酶酶切融合蛋白后,可通过一次亲和层析除去TrxA-His多肽片段及带His标签的肠激酶,得到天然形式的Cas9蛋白,避免了多次纯化透析对目的蛋白的伤害和损耗。同时,本发明也在Cas9的N端及C端分别设计了一个NLS位点,使Cas9能更有效地进入细胞核进行基因编辑。另外,本发明选择了E.coliBL21(DE3)菌株为目的蛋白表达菌株,该菌株可高效表达克隆于含有噬菌体T7启动子的表达载体(如pET-32a)的外源基因。同时,对于Cas9蛋白的密码子,本发明进行了密码子优化,使之完全适应表达菌株的密码子偏好,从而提高目的蛋白的表达水平。另外,本发明在细菌生长至一定数量后,再用IPTG在低温下诱导目的蛋白的表达,可避免目的蛋白过早表达对宿主菌生长的影响,低温下诱导表达也显著提高所表达的目的蛋白的可溶性。经过上述各项优化设计及实验实施,所得到的Cas9蛋白活性比商品Cas9蛋白有了极显著的提高。
(3)采用本发明构建并表达的Cas9高效蛋白联合体外转录的gRNA进行基因编辑,并对Cas9和gRNA的最佳用量配比进行了优化,配合合成的ssODN作为Donor DNA,最终获得靶标位点点突变的单细胞克隆的比率为22.5%,高于常规的点突变效率(<5%)。
(4)利用本发明所得到的靶基因点突变单细胞克隆株进行体细胞核移植动物克隆可直接得到含靶标基因点突变的克隆猪,并且该突变可稳定遗传。
在小鼠模型制作中采用的受精卵显微注射基因编辑材料后再进行胚胎移植的方法,因其直接获得点突变后代的概率非常低(低于1%),需要进行后代的杂交选育,这不太适用于妊娠期较长的大动物(如猪)模型制作。因此,本发明采用技术难度大、挑战性高的原代细胞体外编辑并筛选阳性编辑单细胞克隆的方法,后期再通过体细胞核移植动物克隆技术直接获得相应疾病模型猪,可大大缩短模型猪制作周期并节省人力、物力、财力。
本发明为通过基因编辑手段获得类似人胃癌疾病发展进程的GP130点突变模型猪奠定了坚实的基础,将有助于研究并揭示GP130突变导致胃癌的发病机制,并可用于进行药物筛选、药效检测、基因及细胞治疗等研究,能够为进一步的临床应用提供有效的实验数据,进而为预防和治疗人类胃癌提供有力的实验手段。本发明对可用于人类胃癌治疗药物的研发及临床前测试具有重大应用价值。
附图说明
图1为质粒pET-32a的结构示意图。
图2为质粒pKG-GE4的结构示意图。
图3为实施例3中步骤3.3.3的电泳图。
图4为实施例3中步骤3.4.3的电泳图。
图5为实施例4中步骤4.2.3的电泳图。
图6为实施例4中步骤4.2.4的电泳图。
图7为实施例4中步骤4.6.4的电泳图。
图8为实施例5中步骤5.1.3的电泳图。
图9为实施例5中步骤5.6.3的电泳图。
图10为实施例5中步骤5.6.4判定为野生型的示例性测序峰图。
图11为实施例5中步骤5.6.4判定为杂合突变型的示例性测序峰图。
图12为实施例5中步骤5.6.4判定为双等位基因不同变异的纯合突变型示例性测序峰图。
图13为实施例5中步骤5.6.4判定为双等位基因相同变异的纯合突变型示例性测序峰图。
图14为实施例5中步骤5.6.4判定为靶标位点点突变的杂合突变型示例性测序峰图。
图15为实施例5中步骤5.6.4判定为靶标位点点突变的纯合突变型示例性测序峰图。
具体实施方式
实施例1 原核Cas9高效表达载体(简称pKG-GE4)的构建
质粒pET32a-T7lac-phoA:SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter(简称pKG-GE4,质粒图谱如图2)是以质粒pET-32a(结构示意图见图1)作为骨架进行改造而来,主要改造如下:①保留了TrxA蛋白的编码区域,可以帮助所表达的目的蛋白形成二硫键,增加目的蛋白溶解性及活性,但是在此序列之前加入碱性磷酸酶(phoA)的信号肽(SP)序列,该SP可以引导所表达的目的蛋白分泌至细菌的膜周质腔中,并可被原核周质信号肽酶酶切;②在TrxA蛋白编码序列之后增加His-Tag标签基团,可用于所表达的目的蛋白的富集;③在His-Tag标签下游增加肠激酶(EK)酶切位点DDDDK(Asp-Asp-Asp-Asp-Lys),纯化出的蛋白将在肠激酶作用下去除His-Tag标签和上游所融合的TrxA蛋白。④插入密码子优化后的适宜大肠杆菌BL21(DE3)菌株表达的Cas9蛋白的编码序列,同时在该基因的上游和下游均增加核定位信号编码序列(NLS),增加后期纯化出的Cas9蛋白的核定位能力。
pKG-GE4载体构建方法如下:
(1)骨架载体的制备
质粒pET-32a用XbaI、XhoI酶切,回收载体片段(约5329bp左右)。
(2)全基因合成插入序列
全基因合成如SEQ ID NO.2所示序列,依次包含前述的碱性磷酸酶(phoA)信号肽(phoA:SP)序列、TrxA蛋白编码序列、His-Tag标签基团序列、EK酶切位点序列、核定位信号(SV40 NLS)序列、Cas9蛋白(spCas9)编码序列、核定位信号(nucleoplasmin NLS)序列,在全基因合成的N端和C端分别包含与骨架载体序列同源的25个碱基对。
(3)全基因合成片段与骨架载体连接
将步骤(1)回收的骨架载体和步骤(2)全基因合成的序列重组得到质粒pKG-GE4,核苷酸序列见SEQ ID NO.1。SEQ ID NO.1中,第5121-5139位核苷酸组成T7启动子,第5140-5164位核苷酸编码Lac操纵子(lac operator),第5178-5201位核苷酸编码核糖体结合位点(RBS),第5209-5271位核苷酸编码phoA(碱性磷酸酶)信号肽(signal peptide,SP),第5272-5598位核苷酸编码TrxA蛋白,第5620-5637位核苷酸编码His-Tag标签,第5638-5652位核苷酸编码肠激酶酶切位点,第5656-5670位核苷酸编码SV40核定位信号(NLS),第5701-9801位核苷酸编码spCas9蛋白(其密码子已优化为适宜在大肠杆菌BL21(DE3)菌株中进行表达),第9802-9849位核苷酸编码nucleoplasmin核定位信号(NLS),第9902-9949位核苷酸编码T7终止子。
实施例2 pKG-GE4融合蛋白TrxA-His-EK-NLS-spCas9-NLS的诱导表达、纯化、酶切及pKG-GE4-Cas9蛋白的纯化
2.1 pKG-GE4融合蛋白TrxA-His-EK-NLS-spCas9-NLS的诱导表达
将鉴定正确的pKG-GE4质粒转化入大肠杆菌表达菌株BL21(DE3)(武汉灵淼生物公司)中,涂氨苄抗性(AmpR)平板,培养过夜后,挑选单菌落,接种到含100μg/ml氨苄青霉素的LB液体培养基中,在37℃、200转/min条件下培养过夜,然后将过夜培养的菌液接种到500mLLB培养基中,接种比例为1:200,30℃、230转/min条件下培养至OD600达到1.0左右,加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG)诱导BL21(DE3)菌株表达目的蛋白,然后25℃培养12小时进行目的蛋白的低温诱导可溶性表达。4℃,10000g离心15分钟收集菌体,用PBS洗涤菌体并离心收集菌体沉淀。
2.2 pKG-GE4融合蛋白TrxA-His-EK-NLS-spCas9-NLS的纯化
2.2.1 融合蛋白的粗提
粗提缓冲液为20mM Tris-HCl pH 8.0,0.5M NaCl,5mM Imidazole,1mM PMSF。粗提方法:按每克湿菌加入10ml上述缓冲液,悬浮细菌,均质机破碎,1000par循环三次。然后4℃,15000g离心细菌悬液30min,收集上清,经0.22μm滤膜过滤用于下一步的亲和层析蛋白纯化。
2.2.2 融合蛋白的纯化
采用Ni-NTA琼脂糖柱(金斯瑞,L00250/L00250-C,填料为10ml)进行融合蛋白的纯化。首先用5个柱体积平衡液(20mM Tris-HCl pH 8.0,0.5M NaCl,5mM Imidazole)以1ml/min的流速对Ni柱进行平衡,然后将上述过滤后的菌液上清上样到平衡后的Ni柱,再用5个柱体积平衡液洗涤Ni柱(流速为1ml/min),然后用5个柱体积缓冲液(20mM Tris-HCl pH8.0,0.5M NaCl,50mM Imidazole)洗去杂蛋白(流速为1ml/min),最后用10个柱体积的洗脱液(20mM Tris-HCl pH 8.0,0.5M NaCl,500mM Imidazole)洗脱目的蛋白(流速为0.5-1ml/min)。
2.3 pKG-GE4融合蛋白(TrxA-His-EK-NLS-spCas9-NLS)的酶切与pKG-GE4-Cas9蛋白的纯化
(1)取15ml步骤2.2.2收集的过柱后溶液(共约90-100ml),使用Amicon超滤管(Sigma,UFC9100)将其浓缩至200μl,然后用25mM Tris-HCl(pH8.0,为下一步的重组牛肠激酶酶切反应的最佳缓冲体系)稀释至1ml,使其NaCl和Imidazole的浓度均降低至100mM,利于后续的重组牛肠激酶酶切反应。所有过柱后溶液,共使用6个超滤管进行蛋白浓缩,总计得到1.2ml蛋白浓缩液,并最终稀释至6ml。
(2)将商品来源的带His标签的重组牛肠激酶(生工生物,C620031,重组牛肠激酶轻链,带His标签)加入到步骤(1)得到的溶液中,25℃酶切时间16小时。每50μg蛋白量配比加入2个单位的肠激酶。
(3)将完成步骤(2)的溶液(共计6ml),按每毫升溶液与80μl Ni-NTA树脂(金斯瑞,L00250/L00250-C)比例混匀,在室温下旋转混匀15min,然后7000g离心3min,上清与树脂分离,收集上清液,即为酶切后去除TrxA-His的NLS-spCas9-NLS目的蛋白。酶切后的TrxA-His多肽片段及带His标签的肠激酶EK均结合在Ni-NTA树脂上,从而将上清中的Cas9蛋白分离纯化。
(4)把步骤(3)得到的上清液,使用Amicon超滤管(Sigma,UFC9100)将其浓缩至200μl,然后加入事先配制的酶贮存液(含10mM Tris,300mM NaCl,0.1mM EDTA,1mM DTT,50%甘油,pH7.4)中,调整蛋白终浓度为5mg/ml,即为NLS-spCas9-NLS蛋白溶液(命名为pKG-GE4-Cas9蛋白),并于-80℃保存备用。
实施例3 pKG-GE4-Cas9与gRNA最佳用量配比优化及与商品Cas9蛋白的切割效率比较
3.1 TTN基因靶点gRNA设计及转录
3.1.1 使用Benchling对TTN基因进行gRNA靶点设计,经预筛确定选用以下两条gRNA靶点序列:
TTN-gRNA1:AGAGCACAGTCAGCCTGGCG(SEQ ID NO.3)
TTN-gRNA2:CTTCCAGAATTGGATCTCCG(SEQ ID NO.4)
3.1.2 设计并合成gRNA分子不同区段序列(由基因合成公司合成)
T7-gRNA1:GGCTTGTCGGACTCTTCGCTATTACGCCAGCTGGCGAAGGGGGAT
T7-gRNA2:TGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTCGCCAGC
T7-gRNA3:ACGCCAGGGTTTTCCCAGTCACGACGTTAGGAAATTAATACGACTCACTATAGG
TTN-g1T7-gRNA4:TTCTAGCTCTAAAACCGCCAGGCTGACTGTGCTCTCCTATAGTGAGTCGTATTAATTTC
TTN-g1T7-gRNA5:CCTGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT
TTN-g2T7-gRNA4:TTCTAGCTCTAAAACCGGAGATCCAATTCTGGAAGCCTATAGTGAGTCGTATTAATTTC
TTN-g2T7-gRNA5:ATCTCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT
T7-gRNA6:AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTAT
3.1.3 设计用于鉴定包含TTN gRNA靶点片段的引物
TTN-F55:TACGGAATTGGGGAGCCAGCGGA(SEQ ID NO.5)
TTN-R560:CAAAGTTAACTCTCTGTGTCT(SEQ ID NO.6)
3.1.4 转录模板的扩增
TTN-T7-gRNA1转录模板序列如SEQ ID NO.7所示,是使用T7-gRNA1、T7-gRNA2、T7-gRNA3、TTN-g1T7-gRNA4、TTN-g1T7-gRNA5、T7-gRNA6共计6条合成引物采用重叠延伸PCR扩增技术制作的,该序列中含有T7启动子,可启动相关序列的转录。扩增后将目的条带切胶后按照Fast Pure Gel DNA Extraction Mini Kit(Vazyme,DC301)说明书进行操作,回收产物作为转录模板。
TTN-T7-gRNA2转录模板序列如SEQ ID NO.8所示,是使用T7-gRNA1、T7-gRNA2、T7-gRNA3、TTN-g2T7-gRNA4、TTN-g2T7-gRNA5、T7-gRNA6共计6条合成引物采用重叠延伸PCR扩增技术制作的,该序列中含有T7启动子,可启动相关序列的转录。扩增后将目的条带切胶后按照Fast Pure Gel DNA Extraction Mini Kit(Vazyme,DC301)说明书进行操作,回收产物作为转录模板。
3.1.5 gRNA的转录
以步骤3.1.4制备的转录模板用Transcript Aid T7 High Yield TranscriptionKit(Fermentas,K0441)进行体外gRNA的转录,然后用MEGA clearTM Transcription Clean-Up Kit(Thermo,AM1908)进行所转录的gRNA的回收纯化,操作步骤按说明书进行,所获产物即为可用于细胞电转的gRNA。
3.2 猪原代成纤维细胞制备
3.2.1 取刚出生从江香猪耳组织0.5g,去除毛发及骨组织,75﹪酒精浸泡30-40s;
3.2.2 用含5%P/S(Gibco Penicillin-Streptomycin)的PBS洗涤5次,不含P/S的PBS洗一次;
其中5%P/S的PBS配方为:5%P/S(Gibco Penicillin-Streptomycin)+95%PBS,5%、95%为体积百分比。
3.2.3 用剪刀将组织剪碎,加入5mL 0.1%的胶原酶(Sigma)溶液,37℃摇床消化1h;
3.2.4 500g离心5min,去上清,将沉淀用1mL完全培养基重悬,铺入含10mL完全培养基并已用0.2%明胶(VWR)封盘的10cm细胞培养皿中。
其中,细胞完全培养基的配方为:15%胎牛血清(Gibco)+83%DMEM培养基(Gibco)+1%P/S(Gibco Penicillin-Streptomycin)+1%HEPES(Solarbio),15%、83%、1%、1%为体积百分比。
3.2.5 置于37℃,5%CO2(体积百分比)、5%O2(体积百分比)的恒温培养箱中进行培养;
3.2.6 将细胞培养至长满皿底60%左右时使用0.25%(Gibco)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,400g离心4min,弃去上清,得到细胞沉淀,以备下一步的细胞转染实验。
3.3 gRNA与pKG-GE4-Cas9用量配比优化
3.3.1 共转染分组情况
第一组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和pKG-GE4-Cas9蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:0.5μg TTN-T7-gRNA1:0.5μg TTN-T7-gRNA2:4μg pKG-GE4-Cas9。
第二组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和pKG-GE4-Cas9蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:0.75μg TTN-T7-gRNA1:0.75μg TTN-T7-gRNA2:4μg pKG-GE4-Cas9。
第三组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和pKG-GE4-Cas9蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2:4μgpKG-GE4-Cas9。
第四组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和pKG-GE4-Cas9蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1.25μg TTN-T7-gRNA1:1.25μg TTN-T7-gRNA2:4μg pKG-GE4-Cas9。
第五组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2。
3.3.2 共转染操作方法
使用哺乳动物细胞转染试剂盒(Neon kit)与Neon TM transfection system电转仪进行转染实验。
1)按照上述分组配制电转DNA,混匀过程中注意切勿产生气泡;
2)将3.2.6制备得到的细胞沉淀使用1ml PBS缓冲液(Solarbio)清洗,并转移到1.5ml离心管中,600g离心6min,弃去上清,使用11μL电转基本溶液Opti-MEM重悬细胞,重悬过程中要避免气泡的产生;
3)吸取10μL细胞悬液,加入至步骤1)中的电转DNA液中混匀,混匀过程中注意切勿产生气泡;
4)将试剂盒带有的电转杯放置于Neon TM transfection system电转仪杯槽内,加入3mL Buffer E;
5)用电转枪吸取10μL步骤3)得到的混合液,插入电击杯内,选择电转程序(1450V10ms3pulse),电击转染后立即将电转枪中混合液转入到6孔板中,每孔含3mL的完全培养液(15%胎牛血清(Gibco)+83%DMEM培养基(Gibco)+1%P/S(Gibco Penicillin-Streptomycin)+1%HEPES(Solarbio));
6)混匀后放置于37℃,5%CO2、5%O2的恒温培养箱中进行培养;
7)电转后12-18h换液,36-48h用0.25%(Gibco)的胰蛋白酶消化并收集细胞于1.5mL离心管中。
3.3.3 基因编辑效率分析
提取3.3.2中收集的细胞基因组DNA,采用TTN-F55和TTN-R560组成的引物对进行PCR扩增,然后进行1%琼脂糖凝胶电泳(见图3)。505bp条带为野生型条带(WT),254bp左右(条带505bp理论缺失251bp)为缺失突变条带(MT)。
基因缺失突变效率=(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)×100%。据此计算,第一组基因缺失突变效率为19.9%,第二组基因缺失突变效率为39.9%,第三组基因缺失突变效率为79.9%,第四组基因缺失突变效率为44.3%。
结果表明,当两个gRNA与pKG-GE4-Cas9蛋白的质量比为1:1:4,实际用量为1μg:1μg:4μg时基因编辑效率最高,确定两个gRNA与pKG-GE4-Cas9蛋白的最适用量为1μg:1μg:4μg。
3.4 pKG-GE4-Cas9蛋白和商品Cas9蛋白的基因编辑效率对比
3.4.1 共转染分组情况
Cas9-A组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和商品Cas9-A蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2:4μgCas9-A。
pKG-GE4组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和pKG-GE4-Cas9蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2:4μg pKG-GE4-Cas9。
Cas9-B组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2和商品Cas9-B蛋白共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2:4μgCas9-B。
Control组:将转录的TTN-T7-gRNA1、TTN-T7-gRNA2共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μg TTN-T7-gRNA1:1μg TTN-T7-gRNA2。
3.4.2 共转染操作方法
同本实施例中步骤3.3.2。
3.4.3 基因编辑效率分析
提取3.4.2中收集的细胞基因组DNA,采用TTN-F55和TTN-R560组成的引物对进行PCR扩增,然后进行1%琼脂糖凝胶电泳(见图4)。505bp条带为野生型条带(WT),254bp左右(条带505bp理论缺失251bp)为缺失突变条带(MT)。
基因缺失突变效率=(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)×100%。据此计算,采用商品Cas9-A蛋白的基因缺失突变效率为28.5%,采用pKG-GE4-Cas9蛋白的基因缺失突变效率为85.6%,采用商品Cas9-B蛋白的基因缺失突变效率为16.6%。
结果表明,与采用商品的Cas9蛋白相比,采用本发明制备的pKG-GE4-Cas9蛋白使得基因编辑效率显著提高。
实施例4 GP130基因高效gRNA靶点的筛选
4.1 基因组DNA的提取
使用Vazyme公司FastPure Cell/Tissue DNA Isolation Mini Kit(VazymeCat.DC102-01)分别进行18只猪(雄性A、B、C、D、E、F、G、H雌性1、2、3、4、5、6、7、8、9、10)耳组织的基因组DNA的柱式提取,使用NanoDrop进行定量,-20℃保存备用。
4.2 GP130基因预设点突变位点及邻近基因组序列保守性分析
4.2.1 猪GP130基因信息
白细胞介素6细胞因子家族信号转换器基因(IL6ST,又称GP130);位于16号染色体;GeneID为100037294,Sus scrofa。猪GP130基因编码蛋白氨基酸序列如SEQ ID NO.9所示。猪基因组DNA中,拟突变Y777F位置由第16外显子编码(猪GP130基因编码蛋白的第16外显子序列如SEQ ID NO.10所示)。
4.2.2 GP130基因预设点突变位点外显子及邻近基因组序列PCR扩增引物设计
根据查到的猪GP130基因组序列
(https://www.ncbi.nlm.nih.gov/nuccore/NC_010458.4?report=genbank& from=35101304&to=35151832&strand=true),设计引物扩增前述18只猪基因组样品GP130基因外显子16的位点。
使用Oligo7进行引物设计,设计结果如下:
GP130-E16-F128:TTTCACTGATGTAAGTGTTGTGG(SEQ ID NO.11)
GP130-E16-R545:TGGACTGGTTTCGTGTTGACT(SEQ ID NO.12)
GP130-E16-F131:CACTGATGTAAGTGTTGTGGAAA(SEQ ID NO.13)
GP130-E16-R432:AATCTAACAAGGGCTGGGTGG(SEQ ID NO.14)
4.2.3 GP130基因组PCR扩增引物筛选
使用猪(雌性1#)耳朵组织提取的基因组为模板,使用设计的两条上游引物和两条下游引物组合,Max酶(Vazyme公司货号:P505)进行PCR,产物进行1%琼脂糖凝胶电泳以筛选好的扩增引物,结果如图5,组1:GP130-E16-F128/GP130-E16-R432;组2:GP130-E16-F128/GP130-E16-R545;组3:GP130-E16-F131/GP130-E16-R432;组4:GP130-E16-F131/GP130-E16-R545;优选GP130-E16-F131/GP130-E16-R545引物对进行目的片段扩增。
4.2.4 18只猪GP130基因片段PCR扩增
分别以18只猪的基因组DNA为模板(雄性A、B、C、D、E、F、G、H雌性1、2、3、4、5、6、7、8、9、10),采用引物GP130-E16-F131/GP130-E16-R545及Max酶进行GP130基因组片段的扩增,产物进行1%琼脂糖凝胶电泳,结果如图6。
4.2.5 GP130基因序列保守性分析
将上述PCR扩增产物使用扩增引物进行测序(通用生物公司测序),将测序结果与公共数据库中的GP130基因序列进行比对分析,选择18只猪中共有的保守区进行gRNA靶点的设计。
4.3 gRNA靶点设计及表达载体构建
4.3.1 使用Benchling进行靶点gRNA设计
设计靶点已避开可能的突变位点,使用Benchling(https://benchling.com/)进行靶点gRNA设计。
GP130基因敲除gRNA靶点设计如下:
GP130-E16-gRNA1:TGTGTACCACAGTGGAATAC
GP130-E16-gRNA2:CACTGTCCAGTATTCCACTG
GP130-E16-gRNA3:GTAGCCACTGTGTACCACAG
GP130-E16-gRNA4:TATTCCACTGTGGTACACAG(SEQ ID NO.15)
GP130-E16-gRNA5:GACACAGTAGTGGTATTGGA
GP130-E16-gRNA6:GGACACAGTAGTGGTATTGG
GP130-E16-gRNA7:TCATCACTGCTAGAAATGCT(SEQ ID NO.16)
GP130-E16-gRNA8:CTTCGTGCATGTCATCTTCT
合成的GP130基因共6个靶点的插入序列互补DNA Oligo如下:
GP130-E16-gRNA1-S:caccgTGTGTACCACAGTGGAATAC
GP130-E16-gRNA1-A:aaacGTATTCCACTGTGGTACACAc
GP130-E16-gRNA2-S:caccgCACTGTCCAGTATTCCACTG
GP130-E16-gRNA2-A:aaacCAGTGGAATACTGGACAGTGc
GP130-E16-gRNA3-S:caccGTAGCCACTGTGTACCACAG
GP130-E16-gRNA3-A:aaacCTGTGGTACACAGTGGCTAC
GP130-E16-gRNA4-S:caccgTATTCCACTGTGGTACACAG(SEQ ID NO.17)
GP130-E16-gRNA4-A:aaacCTGTGTACCACAGTGGAATAc(SEQ ID NO.18)
GP130-E16-gRNA5-S:caccGACACAGTAGTGGTATTGGA
GP130-E16-gRNA5-A:aaacTCCAATACCACTACTGTGTC
GP130-E16-gRNA6-S:caccGGACACAGTAGTGGTATTGG
GP130-E16-gRNA6-A:aaacCCAATACCACTACTGTGTCC
GP130-E16-gRNA7-S:caccgTCATCACTGCTAGAAATGCT(SEQ ID NO.19)
GP130-E16-gRNA7-A:aaacAGCATTTCTAGCAGTGATGAc(SEQ ID NO.20)
GP130-E16-gRNA8-S:caccgCTTCGTGCATGTCATCTTCT
GP130-E16-gRNA8-A:aaacAGAAGATGACATGCACGAAGc
GP130-E16-gRNA1-S、GP130-E16-gRNA1-A、GP130-E16-gRNA2-S、GP130-E16-gRNA2-A、GP130-E16-gRNA3-S、GP130-E16-gRNA3-A、GP130-E16-gRNA4-S、GP130-E16-gRNA4-A、GP130-E16-gRNA5-S、GP130-E16-gRNA5-A、GP130-E16-gRNA6-S、GP130-E16-gRNA6-A、GP130-E16-gRNA7-S、GP130-E16-gRNA7-A、GP130-E16-gRNA8-S、GP130-E16-gRNA8-A均为单链DNA分子。
4.3.2 gRNA载体构建
1)将合成的GP130-E16-gRNA1-S和GP130-E16-gRNA1-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA(构建方法详见CN112442515A具体实施方式1.2构建MSTN和FNDC5基因gRNA靶点载体来检测所改造的cas9载体的效率)连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA1)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA1序列所对应的gRNA。
2)将合成的GP130-E16-gRNA2-S和GP130-E16-gRNA2-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA2)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA2序列所对应的的gRNA。
3)将合成的GP130-E16-gRNA3-S和GP130-E16-gRNA3-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA3)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA3序列所对应的gRNA。
4)将合成的GP130-E16-gRNA4-S和GP130-E16-gRNA4-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA4)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA4序列所对应的gRNA。
5)将合成的GP130-E16-gRNA5-S和GP130-E16-gRNA5-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA5)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA5序列所对应的gRNA。
6)将合成的GP130-E16-gRNA6-S和GP130-E16-gRNA6-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA6)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA6序列所对应的gRNA。
7)将合成的GP130-E16-gRNA7-S和GP130-E16-gRNA7-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA7)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA7序列所对应的gRNA。
8)将合成的GP130-E16-gRNA8-S和GP130-E16-gRNA8-A混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架pKG-U6gRNA连接,得到质粒pKG-U6gRNA(GP130-E16-gRNA8)。该质粒将会在所转染的细胞中转录出与GP130-E16-gRNA8序列所对应的gRNA。
4.3.3 gRNA载体鉴定
从LB平板上挑取单克隆置入加有相应抗生素的LB培养液内,37℃恒温摇床内培养12-16h后小提质粒送通用公司测序,经序列比对确认pKG-U6gRNA(GP130-E16-gRNA1)、pKG-U6gRNA(GP130-E16-gRNA2)、pKG-U6gRNA(GP130-E16-gRNA3)、pKG-U6gRNA(GP130-E16-gRNA4)、pKG-U6gRNA(GP130-E16-gRNA5)、pKG-U6gRNA(GP130-E16-gRNA6)、pKG-U6gRNA(GP130-E16-gRNA7)和pKG-U6gRNA(GP130-E16-gRNA8)载体均构建成功。
4.4 猪原代成纤维细胞制备
同实施例3中3.2。
4.5 使用构建好的gRNA质粒、Cas9质粒(pKG-GE3)共转染猪原代成纤维细胞。
4.5.1 共转染分组情况
第一组:将质粒pKG-U6gRNA(GP130-E16-gRNA1)、质粒pKG-GE3(构建方法见CN112442515A中具体实施方式1.1Cas9高效表达载体的构建)共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA1):1.08μg质粒pKG-GE3。
第二组:将质粒pKG-U6gRNA(GP130-E16-gRNA2)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA2):1.08μg质粒pKG-GE3。
第三组:将质粒pKG-U6gRNA(GP130-E16-gRNA3)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA3):1.08μg质粒pKG-GE3。
第四组:将质粒pKG-U6gRNA(GP130-E16-gRNA4)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA4):1.08μg质粒pKG-GE3。
第五组:将质粒pKG-U6gRNA(GP130-E16-gRNA5)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA5):1.08μg质粒pKG-GE3。
第六组:将质粒pKG-U6gRNA(GP130-E16-gRNA6)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA6):1.08μg质粒pKG-GE3。
第七组:将质粒pKG-U6gRNA(GP130-E16-gRNA7)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA7):1.08μg质粒pKG-GE3。
第八组:将质粒pKG-U6gRNA(GP130-E16-gRNA8)、质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(GP130-E16-gRNA8):1.08μg质粒pKG-GE3。
第九组:猪原代成纤维细胞,同等电转参数不加质粒进行电转操作。
4.5.2 共转染操作方法
同实施例3中3.3.2。
4.6 GP130基因不同靶点的编辑效率分析
4.6.1 分别向步骤4.5.2中收集在1.5mL离心管中的5组细胞加入10μL KAPA2G裂解液裂解细胞,得到释放出基因组DNA的细胞裂解液
KAPA2G裂解液配制体系如下:
10×extract Buffer 1μL
Enzyme 0.2μL
ddH2O 8.8μL
75℃15min—95℃5min—4℃,反应结束后细胞裂解液于-20℃保存;
4.6.2 采用前述针对GP130基因E4的引物对GP130-E16-F131/GP130-E16-R545,并以上述细胞裂解液为DNA模板,进行PCR扩增GP130基因靶点区域,检测细胞靶基因突变情况,目的PCR产物长度为414bp;
4.6.3 使用常规PCR反应扩增GP130靶点基因;
4.6.4 GP130基因不同靶点的编辑效率分析
将PCR反应产物进行1%琼脂糖凝胶电泳,如图7,将目的产物切胶回收后送测序公司进行测序,然后将测序结果利用网页版Synthego ICE工具分析测序峰图得出GP130-E16-gRNA1、GP130-E16-gRNA2、GP130-E16-gRNA3、GP130-E16-gRNA4、GP130-E16-gRNA5、GP130-E16-gRNA6、GP130-E16-gRNA7、GP130-E16-gRNA8不同靶点的编辑效率依次为20%、24%、43%、48%、14%、10%、52%、4%。结果表明,GP130-E16-gRNA4和GP130-E16-gRNA7编辑效率较高。
实施例5 制备GP130基因点突变的从江香猪单细胞克隆
5.1 GP130基因高效gRNA靶点模板制备及转录
5.1.1 选用实施例4中筛到的两个高效gRNA靶点
GP130-E16-gRNA4:TATTCCACTGTGGTACACAG(SEQ ID NO.15)
GP130-E16-gRNA7:TCATCACTGCTAGAAATGCT(SEQ ID NO.16)
5.1.2 设计并合成靶点gRNA转录模板的不同区段序列(由基因合成公司合成)
T7-gRNA1、T7-gRNA2、T7-gRNA3、T7-gRNA6序列同实施例3中步骤3.1.2;
GP130-g4T7-gRNA4:
TTCTAGCTCTAAAACCTGTGTACCACAGTGGAATACCTATAGTGAGTCGTATTAATTTC(SEQ IDNO.21)
GP130-g4T7-gRNA5:
TACACAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT(SEQ IDNO.22)
GP130-g7T7-gRNA4:
TTCTAGCTCTAAAACAGCATTTCTAGCAGTGATGACCTATAGTGAGTCGTATTAATTTC(SEQ IDNO.23)
GP130-g7T7-gRNA5:
AAATGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT(SEQ IDNO.24)
5.1.3 转录模板的扩增
GP130-T7-gRNA4转录模板序列如SEQ ID NO.25所示,是使用T7-gRNA1、T7-gRNA2、T7-gRNA3、GP130-g4T7-gRNA4、GP130-g4T7-gRNA5、T7-gRNA6共计6条合成引物采用重叠延伸PCR扩增技术制作的,该序列中含有T7启动子,可启动相关序列的转录。扩增结果如图8所示,将目的条带切胶后按照Fast Pure Gel DNA Extraction Mini Kit(Vazyme,DC301)说明书进行操作,回收产物作为转录模板。
GP130-T7-gRNA7转录模板序列如SEQ ID NO.26所示,是使用T7-gRNA1、T7-gRNA2、T7-gRNA3、GP130-g7T7-gRNA4、GP130-g7T7-gRNA5、T7-gRNA6共计6条合成引物采用重叠延伸PCR扩增技术制作的,该序列中含有T7启动子,可启动相关序列的转录。扩增结果如图8所示,将目的条带切胶后按照Fast Pure Gel DNA Extraction Mini Kit(Vazyme,DC301)说明书进行操作,回收产物作为转录模板。
5.1.4 高效gRNA的转录
以步骤5.1.3制备的转录模板用Transcript Aid T7 High Yield TranscriptionKit(Fermentas,K0441)进行体外gRNA的转录,然后用MEGA clearTM Transcription Clean-Up Kit(Thermo,AM1908)进行所转录的gRNA的回收纯化,操作步骤按说明书进行,所获产物即为可用于细胞电转的gRNA。
5.2 合成含有GP130突变位点的单链Donor DNA
合成对应人GP130 Y689F氨基酸突变的单链DNA作为Donor DNA,该单链DNA除靶位点突变外还含有GP130-E16-gRNA4和GP130-E16-gRNA靶点PAM或PAM邻近的3’端序列同义突变,命名为GP130-mutant-ss183,序列如SEQ ID NO.27所示。
5.3 制备猪原代成纤维细胞
同实施例3中3.2。
5.4 转染猪原代成纤维细胞
使用转录的GP130-T7-gRNA4和GP130-T7-gRNA7、pKG-GE4-Cas9蛋白、GP130-mutant-ss183Donor DNA共转染猪原代成纤维细胞。配比:约10万个猪原代成纤维细胞:1μgGP130-T7-gRNA4:1μg GP130-E16-gRNA7:4μg pKG-GE4-Cas9蛋白:2μg GP130-mutant-ss183。共转染操作方法同实施例3中3.3.2。
5.5 筛选GP130-mutant-ss183 Donor DNA发生同源重组(HDR)的单细胞克隆株
5.5.1 将步骤5.4所得电转48h的群体细胞,使用胰蛋白酶进行消化,完全培养基中和,500g离心5min,去上清,将沉淀用200μL完全培养基重悬,并适当稀释,用口吸管挑取单细胞转移到每孔含100μL完全培养基的96孔板中,每孔放置一个细胞。
5.5.2 37℃、5%CO2、5%O2的恒温培养箱中进行培养,每2~3天换一次细胞培养基,期间用显微镜观察每孔细胞生长情况,排除无细胞及非单细胞克隆的孔;
5.5.3 待96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2/3细胞接种到含有完全培养基的6孔板中,剩余的1/3细胞收集在1.5mL离心管中备后续基因型测定;
5.5.4 待6孔板长至80%汇合度时使用0.25%(Gibco)的胰蛋白酶消化并收集细胞,使用细胞冻存液(90%完全培养基+10%DMSO,体积比)将细胞冻存。
5.6 单细胞克隆的基因型鉴定
5.6.1 在步骤5.5.3收集在1.5mL离心管中得到的细胞中加入10μL KAPA2G裂解液裂解细胞,得到释放出基因组DNA的细胞裂解液。
KAPA2G裂解液配制体系如下:
10×extract Buffer 1μL
Enzyme 0.2μL
ddH2O 8.8μL
75℃ 15min—95℃ 5min—4℃,反应结束后细胞裂解液于-20℃保存;
5.6.2 采用前述针对GP130基因E6的引物对GP130-E16-F131/GP130-E16-R545,并以上述细胞裂解液为DNA模板,进行PCR扩增GP130基因靶点区域,检测单细胞克隆的靶基因突变情况,目的PCR产物长度为414bp;
5.6.3 将PCR产物进行电泳,电泳结果如图9,泳道编号与单细胞克隆编号一致。回收PCR扩增产物并测序。
5.6.4 将测序结果与GP130靶标位点突变序列信息进行比对,从而判断该单细胞克隆株是否为靶标位点成功突变株。
编号为2、6、22、36、37的单细胞克隆的基因型为野生型。编号为1、3、8、9、12、16、17、23、25、26、28、30、32、33、35、38的单细胞克隆的基因型为杂合突变型。编号为4、11、18、21、24、34、39的单细胞克隆的基因型为双等位基因不同变异的纯合突变型。编号为5、7、10、13、14、15、19、20、27、29、31、40的单细胞克隆的基因型为双等位基因相同变异的纯合突变型。其中,9、17、23、32、35的单细胞克隆为靶标位点点突变的杂合突变型,13、27、29、40的单细胞克隆为靶标位点点突变的纯合突变型。得到GP130基因编辑单细胞克隆的比率为87.5%,得到靶标位点点突变的单细胞克隆的比率为22.5%。
示例性的测序比对结果如图10至图15,其中图10是克隆号为GP130-ss183-2的正向测序和反向测序同时与靶标位点野生型序列的比对结果,判定为野生型;图11是克隆号为GP130-ss183-3的正向测序和反向测序同时与靶标位点野生型序列的比对结果,判定为杂合突变型;图12是克隆号为GP130-ss183-4的正向测序和反向测序同时与靶标位点野生型序列的比对结果,为双等位基因不同变异的纯合突变型;图13是克隆号为GP130-ss183-7的正向测序和反向测序同时与靶标位点野生型序列的比对结果,为双等位基因相同变异的纯合突变型;图14是克隆号为GP130-ss183-23的正向测序与靶标位点野生型序列的比对结果,为靶标位点点突变的杂合突变型;图15是克隆号为GP130-ss183-13的正向测序与靶标位点野生型序列的比对结果,为靶标位点点突变的纯合突变型。
通过具体序列的分析,GP130各单细胞克隆基因型如表1:
表1 GP130基因点突变单细胞克隆的基因型测定结果
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 南京启真基因工程有限公司
<120> 用于构建GP130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统及其应用
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9974
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160
ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatgt gaaacaaagc 5220
actattgcac tggcactctt accgttactg tttacccctg tgacaaaagc catgagcgat 5280
aaaattattc acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg 5340
atcctcgtcg atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg 5400
gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa 5460
aaccctggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 5520
aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 5580
ctcgacgcta acctggccgg ttctggttct ggccatatgc accatcatca tcatcatgac 5640
gatgacgata agatgcccaa aaagaaacga aaggtgggta tccacggagt cccagcagcc 5700
gacaaaaaat atagcatcgg cctggacatc ggtaccaaca gcgttggctg ggcagtgatc 5760
actgatgaat acaaagttcc atccaaaaaa tttaaagtac tgggcaacac cgaccgtcac 5820
tctatcaaaa aaaacctgat tggtgctctg ctgtttgaca gcggcgaaac tgctgaggct 5880
acccgtctga aacgtacggc tcgccgtcgc tacactcgtc gtaaaaaccg catctgttat 5940
ctgcaggaaa ttttctctaa cgaaatggca aaagttgatg atagcttctt tcatcgtctg 6000
gaagagagct tcctggtgga agaagataaa aaacacgaac gtcacccgat tttcggtaac 6060
attgtggatg aggttgccta ccacgagaaa tatccgacca tctaccatct gcgtaaaaaa 6120
ctggttgata gcactgacaa agcggatctg cgtctgatct acctggctct ggcacacatg 6180
atcaaattcc gtggtcactt cctgatcgaa ggtgatctga accctgataa ctccgacgtg 6240
gacaaactgt tcattcagct ggttcagacc tataaccagc tgttcgaaga aaacccgatc 6300
aacgcgtccg gtgtagacgc taaggcaatt ctgtctgcgc gtctgtctaa gtctcgtcgt 6360
ctggaaaacc tgattgcgca actgccaggt gaaaagaaaa acggcctgtt cggcaatctg 6420
atcgccctgt ccctgggtct gactccgaac tttaaatcca actttgacct ggcggaagat 6480
gccaagctgc agctgagcaa agatacctat gacgatgacc tggataacct gctggcacag 6540
atcggtgatc agtatgccga tctgttcctg gccgcgaaaa acctgtctga tgcgattctg 6600
ctgtctgata tcctgcgcgt taacactgaa attactaaag cgccgctgag cgcatccatg 6660
attaaacgtt acgatgaaca ccaccaggat ctgaccctgc tgaaagcgct ggtgcgtcag 6720
cagctgccgg aaaaatacaa ggagatcttc ttcgaccaga gcaaaaacgg ttacgcgggc 6780
tacattgatg gtggtgcatc tcaggaggaa ttctacaaat tcattaaacc gatcctggaa 6840
aaaatggatg gtactgaaga gctgctggtt aaactgaatc gtgaagatct gctgcgcaaa 6900
cagcgtacct tcgataacgg ttccatcccg catcagattc atctgggcga actgcacgct 6960
atcctgcgcc gtcaggaaga cttttatccg ttcctgaaag acaaccgtga gaaaattgaa 7020
aaaatcctga ccttccgtat tccgtactat gtaggtccgc tggcgcgtgg taactcccgt 7080
ttcgcttgga tgacccgcaa aagcgaagaa accatcaccc cgtggaattt cgaagaagtc 7140
gttgacaaag gcgcgtccgc gcagtctttc atcgaacgca tgacgaactt cgacaaaaac 7200
ctgccgaacg agaaagtgct gccgaaacac tctctgctgt acgagtactt cactgtgtac 7260
aacgaactga ccaaagtgaa atacgtcacc gaaggtatgc gtaaaccggc attcctgtcc 7320
ggtgagcaaa aaaaagcaat cgtggatctg ctgttcaaaa ccaaccgtaa agtaaccgtg 7380
aaacagctga aggaagacta tttcaagaaa atcgaatgtt ttgattctgt tgaaatctcc 7440
ggcgtggaag atcgcttcaa tgcgtccctg ggtacgtatc acgacctgct gaaaattatc 7500
aaagacaaag attttctgga caacgaggaa aacgaagaca tcctggagga tattgtactg 7560
accctgaccc tgttcgaaga ccgtgagatg atcgaagaac gcctgaaaac ctacgcccac 7620
ctgttcgatg acaaggtaat gaagcagctg aaacgtcgtc gttataccgg ctggggtcgt 7680
ctgtcccgta aactgatcaa tggcatccgt gataaacagt ctggcaaaac catcctggac 7740
ttcctgaaat ccgacggttt cgcgaatcgt aacttcatgc aactgattca tgacgattct 7800
ctgactttca aagaagacat ccagaaagca caggtttccg gccagggtga ctctctgcac 7860
gagcacattg ccaatctggc tggttctccg gctattaaaa agggtattct gcagactgtg 7920
aaagtagttg atgagctggt caaagtaatg ggccgtcaca agccggaaaa cattgtgatc 7980
gaaatggcac gtgaaaacca gacgacccag aaaggtcaga aaaactctcg tgaacgcatg 8040
aaacgtatcg aagaaggcat caaagaactg ggctctcaga tcctgaagga acaccctgta 8100
gaaaataccc agctgcagaa cgaaaagctg tatctgtatt acctgcagaa cggccgcgat 8160
atgtatgtgg accaggaact ggatatcaac cgcctgtccg attacgatgt agatcacatc 8220
gtgccgcaaa gcttcctgaa agacgacagc attgacaaca aagtactgac ccgttctgat 8280
aagaaccgtg gcaaatccga taacgtcccg tctgaagaag ttgttaaaaa aatgaaaaac 8340
tattggcgtc agctgctgaa cgcgaaactg atcacccagc gtaagttcga caatctgact 8400
aaagctgagc gcggtggtct gtccgaactg gataaagcgg gttttatcaa acgccagctg 8460
gttgaaaccc gtcagatcac gaagcacgtt gcgcagattc tggactctcg tatgaacacc 8520
aaatacgacg aaaacgacaa actgatccgc gaggttaagg ttatcaccct gaaaagcaaa 8580
ctggtatccg attttcgtaa agactttcag ttctacaaag tgcgcgaaat taacaactat 8640
caccacgctc acgatgcata tctgaatgca gttgttggca cggcgctgat caaaaagtat 8700
ccgaaactgg aatctgaatt cgtatacggc gattacaaag tgtatgacgt tcgtaagatg 8760
atcgcaaaat ccgagcagga aattggtaag gcgacggcga aatacttctt ttattccaat 8820
attatgaact ttttcaaaac cgaaatcacc ctggcgaatg gtgaaattcg taaacgcccg 8880
ctgatcgaaa ccaacggtga aactggtgaa atcgtttggg acaaaggccg cgacttcgcg 8940
accgtgcgta aagttctgtc tatgccgcaa gtgaacatcg tcaagaagac cgaagtacaa 9000
accggcggtt ttagcaaaga gagcattctg ccaaaacgta actccgacaa actgatcgcg 9060
cgcaagaaag actgggatcc gaaaaaatac ggtggtttcg attctccaac cgttgcttat 9120
tccgttctgg tggtagccaa agttgagaaa ggtaaaagca aaaaactgaa atccgtaaag 9180
gaactgctgg gtattactat catggagcgt agctccttcg aaaaaaaccc gatcgatttt 9240
ctggaagcga aaggctataa agaagtcaaa aaggacctga tcatcaaact gccaaaatac 9300
agcctgttcg agctggaaaa cggccgtaaa cgtatgctgg catctgcggg cgaactgcag 9360
aaaggcaacg agctggctct gccgtccaaa tacgtgaact ttctgtacct ggcctctcac 9420
tacgaaaaac tgaaaggttc cccggaagac aacgaacaga aacagctgtt cgtagagcag 9480
cacaaacact acctggacga gatcatcgaa cagatttctg aattttctaa acgtgtgatt 9540
ctggctgatg cgaatctgga taaagttctg tctgcctata acaagcatcg tgacaaaccg 9600
atccgcgaac aggctgagaa catcatccac ctgttcactc tgactaacct gggcgcgcca 9660
gcggctttca agtactttga taccaccatt gaccgcaagc gttacacctc cactaaagaa 9720
gtgctggacg cgactctgat ccaccagtcc atcaccggtc tgtacgagac ccgtatcgat 9780
ctgagccagc tgggcggtga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 9840
aagaaaaagt gacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9900
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9960
aactatatcc ggat 9974
<210> 2
<211> 4694
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttaactttaa gaaggagata tacatgtgaa acaaagcact attgcactgg cactcttacc 60
gttactgttt acccctgtga caaaagccat gagcgataaa attattcacc tgactgacga 120
cagttttgac acggatgtac tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga 180
gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat gaaatcgctg acgaatatca 240
gggcaaactg accgttgcaa aactgaacat cgatcaaaac cctggcactg cgccgaaata 300
tggcatccgt ggtatcccga ctctgctgct gttcaaaaac ggtgaagtgg cggcaaccaa 360
agtgggtgca ctgtctaaag gtcagttgaa agagttcctc gacgctaacc tggccggttc 420
tggttctggc catatgcacc atcatcatca tcatgacgat gacgataaga tgcccaaaaa 480
gaaacgaaag gtgggtatcc acggagtccc agcagccgac aaaaaatata gcatcggcct 540
ggacatcggt accaacagcg ttggctgggc agtgatcact gatgaataca aagttccatc 600
caaaaaattt aaagtactgg gcaacaccga ccgtcactct atcaaaaaaa acctgattgg 660
tgctctgctg tttgacagcg gcgaaactgc tgaggctacc cgtctgaaac gtacggctcg 720
ccgtcgctac actcgtcgta aaaaccgcat ctgttatctg caggaaattt tctctaacga 780
aatggcaaaa gttgatgata gcttctttca tcgtctggaa gagagcttcc tggtggaaga 840
agataaaaaa cacgaacgtc acccgatttt cggtaacatt gtggatgagg ttgcctacca 900
cgagaaatat ccgaccatct accatctgcg taaaaaactg gttgatagca ctgacaaagc 960
ggatctgcgt ctgatctacc tggctctggc acacatgatc aaattccgtg gtcacttcct 1020
gatcgaaggt gatctgaacc ctgataactc cgacgtggac aaactgttca ttcagctggt 1080
tcagacctat aaccagctgt tcgaagaaaa cccgatcaac gcgtccggtg tagacgctaa 1140
ggcaattctg tctgcgcgtc tgtctaagtc tcgtcgtctg gaaaacctga ttgcgcaact 1200
gccaggtgaa aagaaaaacg gcctgttcgg caatctgatc gccctgtccc tgggtctgac 1260
tccgaacttt aaatccaact ttgacctggc ggaagatgcc aagctgcagc tgagcaaaga 1320
tacctatgac gatgacctgg ataacctgct ggcacagatc ggtgatcagt atgccgatct 1380
gttcctggcc gcgaaaaacc tgtctgatgc gattctgctg tctgatatcc tgcgcgttaa 1440
cactgaaatt actaaagcgc cgctgagcgc atccatgatt aaacgttacg atgaacacca 1500
ccaggatctg accctgctga aagcgctggt gcgtcagcag ctgccggaaa aatacaagga 1560
gatcttcttc gaccagagca aaaacggtta cgcgggctac attgatggtg gtgcatctca 1620
ggaggaattc tacaaattca ttaaaccgat cctggaaaaa atggatggta ctgaagagct 1680
gctggttaaa ctgaatcgtg aagatctgct gcgcaaacag cgtaccttcg ataacggttc 1740
catcccgcat cagattcatc tgggcgaact gcacgctatc ctgcgccgtc aggaagactt 1800
ttatccgttc ctgaaagaca accgtgagaa aattgaaaaa atcctgacct tccgtattcc 1860
gtactatgta ggtccgctgg cgcgtggtaa ctcccgtttc gcttggatga cccgcaaaag 1920
cgaagaaacc atcaccccgt ggaatttcga agaagtcgtt gacaaaggcg cgtccgcgca 1980
gtctttcatc gaacgcatga cgaacttcga caaaaacctg ccgaacgaga aagtgctgcc 2040
gaaacactct ctgctgtacg agtacttcac tgtgtacaac gaactgacca aagtgaaata 2100
cgtcaccgaa ggtatgcgta aaccggcatt cctgtccggt gagcaaaaaa aagcaatcgt 2160
ggatctgctg ttcaaaacca accgtaaagt aaccgtgaaa cagctgaagg aagactattt 2220
caagaaaatc gaatgttttg attctgttga aatctccggc gtggaagatc gcttcaatgc 2280
gtccctgggt acgtatcacg acctgctgaa aattatcaaa gacaaagatt ttctggacaa 2340
cgaggaaaac gaagacatcc tggaggatat tgtactgacc ctgaccctgt tcgaagaccg 2400
tgagatgatc gaagaacgcc tgaaaaccta cgcccacctg ttcgatgaca aggtaatgaa 2460
gcagctgaaa cgtcgtcgtt ataccggctg gggtcgtctg tcccgtaaac tgatcaatgg 2520
catccgtgat aaacagtctg gcaaaaccat cctggacttc ctgaaatccg acggtttcgc 2580
gaatcgtaac ttcatgcaac tgattcatga cgattctctg actttcaaag aagacatcca 2640
gaaagcacag gtttccggcc agggtgactc tctgcacgag cacattgcca atctggctgg 2700
ttctccggct attaaaaagg gtattctgca gactgtgaaa gtagttgatg agctggtcaa 2760
agtaatgggc cgtcacaagc cggaaaacat tgtgatcgaa atggcacgtg aaaaccagac 2820
gacccagaaa ggtcagaaaa actctcgtga acgcatgaaa cgtatcgaag aaggcatcaa 2880
agaactgggc tctcagatcc tgaaggaaca ccctgtagaa aatacccagc tgcagaacga 2940
aaagctgtat ctgtattacc tgcagaacgg ccgcgatatg tatgtggacc aggaactgga 3000
tatcaaccgc ctgtccgatt acgatgtaga tcacatcgtg ccgcaaagct tcctgaaaga 3060
cgacagcatt gacaacaaag tactgacccg ttctgataag aaccgtggca aatccgataa 3120
cgtcccgtct gaagaagttg ttaaaaaaat gaaaaactat tggcgtcagc tgctgaacgc 3180
gaaactgatc acccagcgta agttcgacaa tctgactaaa gctgagcgcg gtggtctgtc 3240
cgaactggat aaagcgggtt ttatcaaacg ccagctggtt gaaacccgtc agatcacgaa 3300
gcacgttgcg cagattctgg actctcgtat gaacaccaaa tacgacgaaa acgacaaact 3360
gatccgcgag gttaaggtta tcaccctgaa aagcaaactg gtatccgatt ttcgtaaaga 3420
ctttcagttc tacaaagtgc gcgaaattaa caactatcac cacgctcacg atgcatatct 3480
gaatgcagtt gttggcacgg cgctgatcaa aaagtatccg aaactggaat ctgaattcgt 3540
atacggcgat tacaaagtgt atgacgttcg taagatgatc gcaaaatccg agcaggaaat 3600
tggtaaggcg acggcgaaat acttctttta ttccaatatt atgaactttt tcaaaaccga 3660
aatcaccctg gcgaatggtg aaattcgtaa acgcccgctg atcgaaacca acggtgaaac 3720
tggtgaaatc gtttgggaca aaggccgcga cttcgcgacc gtgcgtaaag ttctgtctat 3780
gccgcaagtg aacatcgtca agaagaccga agtacaaacc ggcggtttta gcaaagagag 3840
cattctgcca aaacgtaact ccgacaaact gatcgcgcgc aagaaagact gggatccgaa 3900
aaaatacggt ggtttcgatt ctccaaccgt tgcttattcc gttctggtgg tagccaaagt 3960
tgagaaaggt aaaagcaaaa aactgaaatc cgtaaaggaa ctgctgggta ttactatcat 4020
ggagcgtagc tccttcgaaa aaaacccgat cgattttctg gaagcgaaag gctataaaga 4080
agtcaaaaag gacctgatca tcaaactgcc aaaatacagc ctgttcgagc tggaaaacgg 4140
ccgtaaacgt atgctggcat ctgcgggcga actgcagaaa ggcaacgagc tggctctgcc 4200
gtccaaatac gtgaactttc tgtacctggc ctctcactac gaaaaactga aaggttcccc 4260
ggaagacaac gaacagaaac agctgttcgt agagcagcac aaacactacc tggacgagat 4320
catcgaacag atttctgaat tttctaaacg tgtgattctg gctgatgcga atctggataa 4380
agttctgtct gcctataaca agcatcgtga caaaccgatc cgcgaacagg ctgagaacat 4440
catccacctg ttcactctga ctaacctggg cgcgccagcg gctttcaagt actttgatac 4500
caccattgac cgcaagcgtt acacctccac taaagaagtg ctggacgcga ctctgatcca 4560
ccagtccatc accggtctgt acgagacccg tatcgatctg agccagctgg gcggtgacaa 4620
aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtgac aaagcccgaa 4680
aggaagctga gttg 4694
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agagcacagt cagcctggcg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cttccagaat tggatctccg 20
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tacggaattg gggagccagc gga 23
<210> 6
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caaagttaac tctctgtgtc t 21
<210> 7
<211> 225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggagagc acagtcagcc tggcggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 8
<211> 225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggcttcc agaattggat ctccggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 9
<211> 937
<212> PRT
<213> 猪(Sus scrofa)
<400> 9
Met Leu Thr Leu Gln Thr Trp Val Val Gln Ala Leu Phe Ile Phe Leu
1 5 10 15
Thr Thr Lys Cys Lys Gly Glu Leu Leu Asp Pro Cys Gly His Ile Ser
20 25 30
Pro Glu Ser Pro Val Ile Gln Leu Gly Ser Asn Phe Thr Ala Val Cys
35 40 45
Val Leu Lys Glu Lys Cys Met Asp His Tyr His Val Asn Ala Ser Tyr
50 55 60
Ile Phe Trp Lys Thr Asn His Val Thr Ile Pro Tyr Glu Gln Tyr Asn
65 70 75 80
Val Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Arg Asp Ile Ser Leu
85 90 95
Leu Asn Ile Gln Leu Thr Cys Asn Ile Arg Thr Phe Gly Gln Ile Asp
100 105 110
Gln Asn Val Tyr Gly Ile Arg Ile Ile Ser Gly Leu Pro Pro Glu Lys
115 120 125
Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Met Cys
130 135 140
Gln Trp Asp Pro Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu
145 150 155 160
Lys Ser Glu Trp Ala Thr Glu Lys Phe Asp Asp Cys Lys Ala Lys Arg
165 170 175
Asp Ile Pro Thr Ser Cys Thr Val Asp Tyr Ser Pro Val Tyr Phe Val
180 185 190
Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr
195 200 205
Ser Asp His Ile Asn Phe Asp Pro Val Asp Lys Val Lys Pro Asn Pro
210 215 220
Pro His Asn Leu Ser Val Ser Asn Ser Glu Glu Leu Ser Ser Ile Leu
225 230 235 240
Lys Leu Thr Trp Ile Asn Ser Ser Ile Arg Asn Phe Ile Arg Leu Lys
245 250 255
Tyr Asn Ile Gln Tyr Arg Thr Lys Ala Ala Ser Thr Trp Asn Gln Ile
260 265 270
Cys Ile Ser Ser Lys Asp Gln Gln Glu Asp Ile Gln Ile Glu Asn Thr
275 280 285
Ala Glu Ile Glu Ile Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser
290 295 300
Phe Thr Val Gln Asp Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile
305 310 315 320
Arg Cys Met Lys Glu Asp Gly Lys Gly Phe Trp Ser Asp Trp Ser Glu
325 330 335
Glu Ala Ser Gly Val Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser
340 345 350
Phe Trp Tyr Lys Ile Glu Pro Ser His Thr His Gly Tyr Arg Ser Val
355 360 365
Gln Leu Met Trp Lys Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile
370 375 380
Leu Asp Tyr Glu Val Thr Leu Thr Arg Trp Lys Ser Arg Leu Gln Asn
385 390 395 400
Tyr Thr Val Asn Asp Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg
405 410 415
Tyr Ile Ala Thr Leu Thr Ala Arg Asn Met Val Gly Lys Ser Asp Ala
420 425 430
Ser Val Leu Thr Ile Pro Ala Cys Asp Phe Gln Ala Thr His Pro Ile
435 440 445
Lys Asp Leu Lys Ala Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp
450 455 460
Thr Ala Pro Asn Glu Ser Val Asn Arg Tyr Val Leu Glu Trp Cys Val
465 470 475 480
Leu Ser Asp Lys Ser Pro Cys Ile Pro Asp Trp Gln Gln Glu Asp Gly
485 490 495
Thr Val His Arg Thr Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys
500 505 510
Tyr Leu Ile Thr Val Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro
515 520 525
Glu Ser Ile Lys Ala Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro
530 535 540
Thr Val Arg Thr Lys Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp
545 550 555 560
Asp Gln Leu Pro Val Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr
565 570 575
Ile Phe Tyr Arg Thr Val Ile Gly Asn Glu Thr Ala Val Asn Val Asp
580 585 590
Ser Ser His Thr Glu Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu
595 600 605
Tyr Met Val Arg Met Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly
610 615 620
Pro Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu
625 630 635 640
Ala Ile Val Val Pro Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu
645 650 655
Gly Val Leu Phe Cys Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile
660 665 670
Trp Pro Asn Val Pro Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser
675 680 685
Pro His Thr Pro Pro Arg His Phe Asn Ser Lys Asp Gln Met Tyr Pro
690 695 700
Asp Gly Asn Phe Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp
705 710 715 720
Lys Lys Pro Phe Pro Glu Asp Leu Lys Ser Leu Asp Ile Phe Lys Lys
725 730 735
Glu Lys Ile Asn Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser
740 745 750
Cys Met Ser Ser Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu
755 760 765
Ser Ala Gln Asn Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His
770 775 780
Ser Gly Tyr Arg His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser
785 790 795 800
Glu Ser Thr Gln Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Glu Leu
805 810 815
Gln Leu Val Asp Asn Val Asp Gly Ser Asp Gly Ile Leu Pro Arg Gln
820 825 830
Gln Tyr Phe Lys Gln Asn Cys Gln His Glu Thr Ser Pro Asp Ile Ser
835 840 845
His Phe Glu Arg Ser Lys Gln Val Ser Ser Val Asn Glu Asp Phe Val
850 855 860
Arg Leu Lys Gln Gln Gln Ile Ser Asp Cys Ile Ser Gln Pro Tyr Gly
865 870 875 880
Ser Gly Gln Met Lys Met Phe Gln Glu Val Ser Ala Thr Asp Ala Phe
885 890 895
Gly Pro Gly Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met
900 905 910
Glu Ala Ala Ile Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr
915 920 925
Val Arg Arg Gly Gly Tyr Met Pro Gln
930 935
<210> 10
<211> 825
<212> DNA
<213> 猪(Sus scrofa)
<400> 10
tatatatatt ataattaata tttataatta ttctagcatt tgtaaatgaa catgcactgt 60
aggtaaataa tctgttttct ctcttttaag cattttaatt caaaagatca aatgtatcca 120
gatggaaatt tcactgatgt aagtgttgtg gaaatagaag caaatgacaa aaaacctttt 180
ccagaagatc tgaaatcatt ggacatattc aagaaggaaa aaattaatac tgaaggacac 240
agtagtggta ttggagggtc ttcgtgcatg tcatcttcta ggccaagcat ttctagcagt 300
gatgaaaatg aatctgcaca gaacacttca agcactgtcc agtattccac tgtggtacac 360
agtggctaca gacaccaggt accatcggtc caagtcttct cacggtccga gtccacccag 420
cccttgttag attctgaaga gcggccagaa gagctacagc tagtagataa tgtagatgga 480
agtgatggca ttttacccag acaacagtat ttcaaacaaa actgtagtca acacgaaacc 540
agtccagata tttcacattt tgaaaggtca aagcaagttt catcagtcaa tgaagatttt 600
gttagactta aacagcagca gatttcagat tgtatttcac agccctatgg atctgggcaa 660
atgaaaatgt ttcaggaagt ttctgcaaca gatgcttttg gtccaggcac tgagggacaa 720
gtagagagat ttgaaacagt tgggatggag gctgcaattg atgaaggaat gcccaaaagt 780
tacttaccac agactgtaag acgaggtggc tacatgcctc agtga 825
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tttcactgat gtaagtgttg tgg 23
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tggactggtt tcgtgttgac t 21
<210> 13
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cactgatgta agtgttgtgg aaa 23
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aatctaacaa gggctgggtg g 21
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tattccactg tggtacacag 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tcatcactgc tagaaatgct 20
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caccgtattc cactgtggta cacag 25
<210> 18
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaacctgtgt accacagtgg aatac 25
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
caccgtcatc actgctagaa atgct 25
<210> 20
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaacagcatt tctagcagtg atgac 25
<210> 21
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ttctagctct aaaacctgtg taccacagtg gaatacctat agtgagtcgt attaatttc 59
<210> 22
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tacacaggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaactt 59
<210> 23
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ttctagctct aaaacagcat ttctagcagt gatgacctat agtgagtcgt attaatttc 59
<210> 24
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aaatgctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaactt 59
<210> 25
<211> 225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggtattc cactgtggta cacaggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 26
<211> 225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggtcatc actgctagaa atgctgtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 27
<211> 183
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aaggacacag tagtggtatt ggagggtctt cgtgcatgtc atcttctagg ccatcgattt 60
ctagcagtga tgaaaatgaa tctgcacaga acacttcaag cactgtccag ttttccactg 120
tagtacacag tggctacaga caccaggtac catcggtcca agtcttctca cggtccgagt 180
cca 183
Claims (16)
1.一种含Cas蛋白的特异融合蛋白,其特征在于所述的特异融合蛋白自N端至C端依次包括如下元件:用于目的蛋白分泌表达的信号肽,增加目的蛋白可溶性的分子伴侣融合蛋白,用于蛋白纯化的标签蛋白,用于去除融合标签,从融合蛋白中获取天然形式Cas蛋白的内切蛋白酶识别位点,引导Cas蛋白进入细胞核的核定位信号,Cas蛋白,引导Cas蛋白进入细胞核的核定位信号。
2.根据权利要求1所述的特异融合蛋白,其特征在于所述的用于目的蛋白分泌表达的信号肽选自大肠杆菌碱性磷酸酶信号肽、金黄色葡萄球菌蛋白A信号肽、大肠杆菌外膜蛋白信号肽或任何其他原核基因的信号肽,优选碱性磷酸酶信号肽;所述的增加目的蛋白可溶性表达的分子伴侣融合蛋白为任何帮助形成二硫键的蛋白,优选为硫氧还原蛋白Trx,进一步优选为TrxA;所述用于蛋白纯化的标签蛋白选自His标签、GST标签、Flag标签、HA标签、c-Myc标签或其他任何蛋白标签,优选为His蛋白标签;所述用于去除融合标签,从融合蛋白中获取天然形式Cas蛋白的内切蛋白酶识别位点选自肠激酶、因子Xa,凝血酶、TEV蛋白酶、HRV3C蛋白酶、WELQut蛋白酶或任何其他内切蛋白酶的识别位点,优选为肠激酶识别位点;所述引导Cas蛋白进入细胞核的核定位信号为任何真核细胞核定位信号,优选为SV40核定位信号和/或nucleoplasmin核定位信号;所述Cas蛋白选自Casl-lO、Cpfl或其他类型Cas蛋白,优选为Cas9,进一步优选为spCas9;所述特异融合蛋白更进一步优选从N端至C端依次包括如下元件:碱性磷酸酶信号肽,硫氧还原蛋白,His标签蛋白,肠激酶酶切位点,核定位信号,Cas9蛋白,核定位信号。
3.编码权利要求1或2所述的特异融合蛋白的特异融合基因;所述的特异融合基因序列优选如SEQ ID NO.1中第5209-9849位核苷酸所示,或者如SEQ ID NO.2所示。
4.一种原核Cas9高效表达载体pKG-GE4,从上游至下游依次包括如下元件:启动子、操纵子、核糖体结合位点、权利要求3所述的特异融合基因、终止子;优选全序列如SEQ IDNO.1所示。
5.权利要求3所述的特异融合基因、权利要求4所述的原核Cas9高效表达载体pKG-GE4在制备Cas9蛋白中的应用。
6.一种制备Cas9蛋白的方法,其特征在于包含以下步骤:
(1)将鉴定正确的pKG-GE4质粒转化入大肠杆菌表达菌株BL21(DE3),培养菌体,加入IPTG,在25℃的温度下诱导所述的基因工程菌表达可溶性目的蛋白,并收集菌体沉淀;
(2)粗提融合蛋白,然后采用Ni-NTA琼脂糖柱进行融合蛋白的纯化;
(3)用带his标签的重组牛肠激酶酶切融合蛋白,并用Ni-NTA树脂纯化得到酶切后去除重组牛肠激酶和TrxA-His的NLS-spCas9-NLS目的蛋白。
7.一种用于构建GP130基因突变的胃癌模型猪核移植供体细胞的基因编辑系统,其特征在于包含按照权利要求6所述方法制备的Cas9蛋白,针对GP130基因的gRNA以及含有GP130突变位点的单链Donor DNA。
8.根据权利要求7所述的基因编辑系统,其特征在于所述的针对GP130基因的gRNA的靶点选自SEQ ID NO.15所示的GP130-E16-gRNA4和SEQ ID NO.16所示的GP130-E16-gRNA7。
9.根据权利要求8所述的基因编辑系统,其特征在于所述的针对GP130基因的gRNA分别由如SEQ ID NO.25所示的GP130-T7-gRNA4转录模板和如SEQ ID NO.26所示的GP130-T7-gRNA7转录模板经体外gRNA转录得到。
10.根据权利要求7所述的基因编辑系统,其特征在于所述的含有GP130突变位点的单链Donor DNA序列如SEQ ID NO.27所示。
11.根据权利要求7-10中任意一项所述的基因编辑系统,其特征在于GP130-E16-gRNA4:GP130-E16-gRNA7:Cas9蛋白:单链Donor DNA的质量比为1:1:4:2。
12.权利要求7-10中任一项所述的基因编辑系统在构建GP130基因突变的猪重组细胞中的应用。
13.一种重组细胞,其特征在于由权利要求7-10中任一项所述的基因编辑系统共转染猪原代成纤维细胞经验证后所得。
14.权利要求7-10中任一项所述的基因编辑系统、权利要求13所述的重组细胞在构建GP130基因突变的胃癌模型猪中的应用。
15.用权利要求13所述重组细胞所制备的胃癌模型猪的猪组织、猪器官和/或猪细胞。
16.权利要求13所述重组细胞、权利要求15所述的猪组织、猪器官、和/或猪细胞,或者用权利要求13所述重组细胞制备的胃癌模型猪在筛选胃癌治疗药物、胃癌治疗药物的药效评价、胃癌基因治疗和/或细胞治疗的疗效评价或胃癌的发病机制研究中的应用。
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