CN116622777A - 基因编辑构建体及其应用 - Google Patents
基因编辑构建体及其应用 Download PDFInfo
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- CN116622777A CN116622777A CN202310645338.1A CN202310645338A CN116622777A CN 116622777 A CN116622777 A CN 116622777A CN 202310645338 A CN202310645338 A CN 202310645338A CN 116622777 A CN116622777 A CN 116622777A
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Abstract
本公开提供一种基因编辑构建体及其应用,所述基因编辑构建体用于将外源基因定点整合入基因组的核糖体DNA(rDNA)区,并能高效地表达其携带的外源基因。
Description
援引加入
本说明书中引用的全部公开出版物、专利和专利申请均通过引用并入本文,就像每个公开出版物、专利和专利申请以其全部内容已经具体记载并分别援引加入本文一样。
技术领域
本发明涉及基因编辑领域,尤其涉及一种基因编辑构建体及其应用。
背景技术
目前,随着人类社会的不断进步,影响疾病的因素也在逐渐的发展变化之中,其中肿瘤成为备受关注的疾病之一,严重影响了人们的身体和心理健康。近年来,基因疗法成为肿瘤治疗过程中的一种常用治疗方法;一般情况下,基因治疗方法是通过一定的供体载体或方式将外源基因导入目标细胞中,使外源基因能够安全有效地表达,从而实现***疾病的目的。
诱导性多能干细胞(iPSCs)是通过实验室技术,将某些特定的遗传基因导入体细胞中,从而使已经终末分化的体细胞重新还原为具有同胚胎干细胞几乎一样功能的多能干细胞。诱导性多能干细胞同样具有自我更新和多向分化潜能,理论上,其可以分化成为外胚层、中胚层以及内胚层等三种胚层的成员,之后再分化成为人体的多种细胞类型。目前诱导性多能干细胞已被应用在神经***疾病、心血管疾病等领域,而在近期的研究中诱导性多能干细胞也越来越多地被应用在肿瘤治疗过程中。
随着研究人员对诱导性多能干细胞技术的探索,研究人员发现将外源基因导入诱导性多能干细胞中进行定点整合时,往往存在外源基因表达不足或不表达的情况,外源基因在诱导性多能干细胞中定点整合时的表达量难以满足肿瘤治疗过程中的需要;而非整合类型的工程化诱导性多能干细胞又面临无法长时间持续表达外源基因的问题,其应用范围有限。
因此如何进一步提高定点整合时外源基因的表达量成为目前诱导性多能干细胞技术中亟待解决的问题之一。
发明内容
本申请发明人在前期研究中构建了一种基因编辑构建体(本文中也称载体)minipHrneo,能够将外源基因表达框通过基因编辑的方式定点整合至细胞rDNA区18S区的5468位点(Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal StemCells for Targeted Cancer Therapy,Zujia Wang,et al,Stem Cells TranslMed.2022Mar 31;11(3):297-309.doi:10.1093/stcltm/szab031.)。针对rDNA区这一多拷贝位点进行基因定点整合时,本发明人自主设计构建了minipHrneo载体和改造的人工核酸酶TALENickases,该酶其中一个Fok1切割域被失活,丧失切割活性,因此不会产生DNA双链断裂,只会产生单链断裂,在降低细胞毒性的同时提高了基因定点整合效率。然而,在细胞rDNA区18S区的5468位点定点整合外源基因后,发现存在外源基因表达不足的问题。
发明人在进一步研究中,意外发现,当将minipHrneo载体中的下游同源臂进行截短,仅保留下游同源臂的266bp时,能显著增强外源基因的表达。基于此,完成本发明。
本发明针对传统供体模板minipHrneo的同源臂做了进一步的优化,在上游同源臂不变的同时缩短下游同源臂,提高了外源基因的表达能力,同时还保持了高水准的定点整合效率。另外,本发明还提供了采用优化后的供体模板制备而成的细胞,所述细胞具有提高的外源基因表达能力。现有技术已经验证了细胞的rDNA区属于安全的基因打靶区域,本发明的实施方式中优选地在rDNA区进行基因打靶,更优选地,在rDNA区的18S区进行基因打靶,最优选地,在rDNA区18S区的5468位点进行基因打靶。
在第一个方面,本发明提供一种基因编辑构建体,所述构建体包括包含上游同源臂、下游同源臂以及上游同源臂和下游同源臂之间多克隆位点的构建体骨架;所述上游同源臂的核苷酸序列如SEQ ID NO:2所示或与SEQ ID NO:2具有至少70%、至少80%、至少90%、至少95%或至少98%的序列同一性,所述下游同源臂的核苷酸序列如SEQ ID NO:4所示或与SEQ ID NO:4具有至少70%、至少80%、至少90%、至少95%或至少98%的序列同一性;所述构建体骨架为非病毒骨架。在本发明的一些实施方案中,所述构建体的出发骨架为minipHrneo,即,基于minipHrneo质粒进行修饰和改造。
在本发明的一些实施方案中,所述上游同源臂的核苷酸序列如SEQ ID NO:2所示和所述下游同源臂的核苷酸序列如SEQ ID NO:4所示。
在本发明的一些实施方案中,所述构建体的核苷酸序列如SEQ ID NO:27所示。
在本发明的另一些实施方案中,所述构建体进一步包含外源基因,所述外源基因***在所述多克隆位点。在一些实施方案中,外源基因编码治疗性肽、DNA结合蛋白、RNA结合蛋白、荧光蛋白或酶。在又一些实施方案中,所述治疗性肽选自人白细胞介素家族成员(例如,IL-2、IL-7、IL-10、IL-11、IL-12、IL-15、IL-23和IL-24)、肿瘤坏死因子家族成员(例如,TNF、LTA、LTB、FASLG、TNFSF8、TNFSF9、TNFSF10、TNFSF11、TNFSF12、TNFSF13、TNFSF14、TNFSF15、TNFSF18和EDA)、干扰素(INF-α、INF-β和INF-γ)、CAR、F8、F9、TNFR和TRAIL。
在本发明的一些实施方案中,所述构建体进一步包含启动子,所述启动子位于所述多克隆位点,优选地,所述启动子为CMV启动子或EF1α启动子。现有技术已知的其它启动子也可以用于本发明。
本发明还提供了包含所述构建体的试剂盒及使用说明。
在第二个方面,本发明提供一种基因编辑方法,包括将第一方面所述的构建体导入细胞,通过基因编辑***将外源基因定点整合入所述细胞的基因组中。
在一些实施方案中,所述基因编辑***选自Cre-lox***、Zinc FingerNucleases(ZFNs)、CRISPR-Cas9或Transcription Activator-Like Effector Nucleases(TALENs),优选为TALENs,更优选为采用人工核酸酶TALENickases进行基因编辑。
在一些实施方案中,所述定点整合位点位于基因组的核糖体RNA转录区(rDNA区)18S rRNA转录区的5468位点。
在一些实施方案中,所述细胞为间充质干细胞、T细胞、B细胞、NK细胞、巨噬细胞或诱导性多能干细胞及其衍生细胞。所述诱导性多能干细胞的衍生细胞为由所述诱导性多能干细胞分化而来的间充质干细胞、T细胞、B细胞、NK细胞、巨噬细胞、造血细胞、内皮细胞、肝细胞、心肌细胞、神经元细胞或胰岛细胞。
在第三个方面,本发明提供一种细胞,其由第二方面所述的方法编辑后获得。
在第四个方面,本发明提供一种药物组合物,其包含第一方面所述的构建体或第三方面所述的细胞和药学上可接受的辅料。
在第五个方面,本发明提供第一方面所述的构建体或第三方面所述的细胞在制备***的药物中的用途。
本发明所提供的经优化后的供体模板、及其制备得到的细胞以及试剂盒,具备提高的外源基因表达能力。本发明所公开的技术方案改善了定点整合后的单克隆细胞外源基因表达不足的缺陷,有利于推动基因治疗技术在肿瘤疾病治疗过程中的应用。
附图说明
图1示本发明基因编辑构建体,即新型定点整合载体骨架266-minipHrneo的构建;其中A:BamHI和NdeI双酶切minipHrneo质粒;B:SHA266 PCR扩增片段。
图2示266-minipHrneo质粒测序结果;其中A:SHA266上游与质粒接头处测序结果;B:SHA266下游与质粒接头处测序结果。
图3示携带外源基因IL15的新型定点整合供体模板质粒266IL15的构建;其中A:BamHI酶切minipHrneo质粒;B:BamHI酶切266-minipHrneo质粒。
图4示实施例中miniIL15、266IL15质粒测序结果;其中A:miniIL15包含外源基因处测序结果;B:266IL15包含外源基因处测序结果。
图5示实施例中miniIL15、266IL15定点整合单克隆鉴定结果;其中A:miniIL15、266IL15定点整合单克隆PCR鉴定电泳结果;B:一个定点整合miniIL15的阳性单克隆上游同源臂接头处测序结果;C:一个定点整合266IL15的阳性单克隆上游同源臂接头处测序结果。
图6示Western Blot检测miniIL15、266IL15定点整合阳性单克隆中外源基因蛋白IL15的表达;图6A:Western Blot结果图;图6B:对Western Blot条带进行的半定量分析结果。*,p<0.05。
具体实施方式
除非另有定义,本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
尽管本发明的广义范围所示的数字范围和参数近似值,但是具体实施例中所示的数值尽可能准确的进行记载。然而,任何数值本来就必然含有一定的误差,其是由它们各自的测量中存在的标准偏差所致。另外,本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围;也就是说,所有以最小值1或更大起始的子范围,例如1至6.1,以及以最大值10或更小终止的子范围,例如5.5至10。另外,任何称为“并入本文”的参考文献应理解为以其整体并入。
另外应注意,如本说明书中所使用的,单数形式包括其所指对象的复数形式,除非清楚且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用,除非上下文另有清楚指明。
本文术语“构建体”(construct)是指人工构建的一系列DNA分子,如扩增得到的基因片段、基因编辑时用于目的基因递送的载体、TALENickase模板等。本文术语“载体”(vector)指运载DNA片段的结构,即将外源基因片段递送到受体细胞中进行表达的工具,常用的载体例如质粒、病毒等。术语“质粒”是指细菌、酵母中能自主复制的环状DNA分子,经人工修饰和构建后,是常用的外源基因运载体,携带遗传信息或目的DNA片段,可被递送到宿主细胞中被表达。本文中,构建体、载体和质粒有时可互换使用,此外,术语“构建体骨架”与“构建体”有时可互换使用。
术语“外源基因”,也称“目的基因”,指经转基因步骤导入受体细胞的基因。根据不同病种或不同目的可以选择不同的外源基因。如针对血友病A基因治疗中的F8基因;血友病B基因治疗中的F9基因;以及肿瘤中相关的抑制基因IL-24、TRAIL等;免疫调节相关基因PD1/PDL1等,优选地,外源基因是能够直接表达出蛋白的基因,例如IL15基因等。
术语“启动子”是指一段能使基因进行转录的DNA序列,可以被RNA聚合酶辨认并开始转录合成RNA。真核生物中使用广泛的启动子有CMV、EF1α、CAG、PGK1、SV40等。现有技术已知的启动子均可用于本发明。
B6-hiPSCs是由一男性供者的尿液细胞诱导而来的诱导性多能干细胞,命名为“B6-hiPSCs”,与人体内天然多能干细胞具有相似性,表达Sox2、Nanog、Oct4等干细胞基因,能在免疫缺陷小鼠体内形成畸胎瘤,具有自我更新和多潜能分化的特征。
本发明的组合物也可包含辅料,例如防腐剂、湿润剂、乳化剂和分散剂。可通过上文中的灭菌程序和通过包含多种抗菌和抗真菌剂,例如对羟基苯甲酸酯、氯代丁醇、苯酚、山梨酸等等双重保证避免微生物的存在。也可能希望在组合物中包含等渗剂,例如糖、氯化钠等等。另外,可通过包含延迟吸收的试剂,例如单硬脂酸铝和明胶实现可注射的药物形式的延长吸收。
实施例
下面结合具体实施例,进一步阐述本发明的一些优选的实施方式和方面,不应被解释为限制其范围。
实施例1定点整合供体模板同源臂设计
(1)利用Primer Premier软件设计引物,对基因定点靶入位点——rDNA区5468位点上下游各2000bp范围内的基因分段进行PCR扩增。(分别使用引物18S-1-F/R、18S-2-F/R、ITS1-1-F/R、ITS1-2-F/R、ITS1-5-F/R进行PCR扩增)。所用到的引物如表1所示。
表1
PCR体系如下:
按以下程序进行PCR反应:
(2)PCR产物送生物公司(北京擎科生物科技有限公司)测序。根据测序结果设计传统定点整合供体模板,具体地,其同源臂为:上游同源臂选择rDNA区5468位点上游933bp的序列(SEQ ID NO:2),下游同源臂选择rDNA区5468位点下游576bp的序列(SEQ ID NO:3)。
(3)在本实施例中对传统定点整合供体模板做了进一步优化,即下游同源臂选取1-266bp的部分,即为新型定点整合载体。
实施例2新型定点整合载体骨架266-minipHrneo构建
(1)酶切现有技术已公开的定点整合供体模板骨架质粒minipHrneo(“Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal Stem Cells forTargeted Cancer Therapy”,Zujia Wang,et al,Stem Cells Transl Med,11(3):297-309,2022.3.10),minipHrneo的序列如SEQ ID NO:1所示(已公开的学位论文(刘博.人iPSCs核糖体基因区IL24基因打靶及其分化MSCs的抗肿瘤研究[D].长沙:中南大学,2017:19-25)中公开了非病毒的人核糖体DNA(hrDNA)靶向载体mini-pHrneo的具体结构)。用BamHI(NEB,货号R0136V)和NdeI(NEB,货号R0111V)双酶切minipHrneo质粒,酶切体系如下:
37℃放置6h。
(2)以minipHrneo为模板,其上游同源臂序列如SEQ ID NO:2所示,下游同源臂序列如SEQ ID NO:3所示。PCR扩增下游同源臂的1-266bp(SEQ ID NO:4)片段(SHA266)。所用到的引物及序列如表2所示。
表2
(3)琼脂糖凝胶电泳
(i)量取0.6g琼脂糖(Biowest,货号111860),倒入锥形瓶,加入60mL 0.5×TBE,煮沸3min。室温冷却10-20min,倒入制胶板,***梳齿,静置待其凝固。
(ii)取下梳齿,将胶放入电泳槽中。向电泳槽内倒入0.5×TBE至完全没过胶块。将(1)中酶切产物和(2)中PCR扩增片段加入点样孔。
(iii)开始电泳(180V,60min)。
(4)DNA回收
(i)将胶块在暗室中浸泡于EB染液中15min。
(ii)取出胶块,流水清洗后在紫外灯下切出目的片段(如图1所示),装入EP管内,做好标记。
(iii)使用FastPure Gel DNA Extraction Mini Kit(Vazyme,货号DC301-01),严格按照说明书回收目的条带DNA。
(5)下游同源臂连入载体
使用II One Step Cloning Kit(Vazyme,货号C112-01)将右同源臂连入带有左同源臂的质粒。
冰上配制以下体系:
在干式恒温仪中37℃反应30min后,立即置于冰上。
(6)转化
取DH5α感受态,置于冰上解冻5min;
将10μL连接产物与50μL DH5α感受态混合,低温静置30min;
打开水浴箱,待温度升高至42℃;
将混合物在水浴箱中加热45s,再次低温静置2min;
在超净台内加入100μL不含抗生素的LB溶液,置于37℃摇床上,190rpm轻摇50min;
于超净台内把液体全部涂在含氨苄的固体LB培养皿上,37℃恒温静置12-16h;
第二天挑取五个白色单个菌落于含氨苄的液体LB中,做好标记,置于37℃摇床上,220rpm轻摇7h;
菌液测序。
(7)266-minipHrneo质粒小量抽提
查看测序序列,选择测序正确菌液(结果如图2所示);完整266-minipHrneo质粒序列如SEQ ID NO:27所示。
取15mL离心管,在超净台中将菌液吸至离心管中,加入8mL含氨苄的液体LB,固定至37℃摇床,220rpm轻摇12h;
使用Endo-free Plasmid Mini Kit II(OMEGA,货号D6950-02),严格按照说明步骤小量抽提质粒,做好标记;
用Nanodrop 1000测定质粒浓度,标记于管壁,-20℃保存用于后续实验。
实施例3包含外源基因IL15的供体模板质粒266IL15的构建
(1)生物公司(上海生工生物工程有限公司)合成含EF1α启动子的人IL15 CDS序列。
(2)按前述步骤分别使用BamHI酶切minipHrneo、266-minipHrneo质粒。
(3)PCR扩增含EF1α启动子的人IL15 CDS序列(SEQ ID NO:16)。所用到的引物及序列如表3所示:
表3
(4)按前述步骤琼脂糖凝胶电泳、DNA回收酶切产物(如图3所示)及PCR产物。
(5)按前述步骤使用II One Step Cloning Kit分别将含EF1α启动子的人IL15 CDS序列连入minipHrneo、266-minipHrneo,分别命名为miniIL15(SEQ ID NO:19)、266IL15(SEQ ID NO:20),并进行转化、测序,选取测序正确的菌液质粒小量抽提(如图4所示),获得miniIL15、266IL15定点整合供体模板质粒。
实施例4将266IL15靶入细胞及外源基因表达鉴定
1.新型定点整合供体模板质粒266IL15靶入B6-hiPSCs
(1)取12孔板,每孔加500μL稀释后的Matrigel(Corning,货号354277),室温静置12h以上。
(2)待B6-hiPSCs细胞生长至80%汇合度,弃上清,加入1mL mTeSR1 Plus(StemCell,货号100-0276),且加入终浓度为10μM的Y27632(Stem Cell,货号72302),将细胞放入培养箱。
(3)2h后取出细胞,吹打细胞后弃上清液。用1×DPBS(Hyclone,货号SH30028.02)润洗细胞一次,加入TrypLE Select(Gibco,货号12563-011)至没过孔底,室温放置3min。同时取出核转试剂盒Amaxa Human Stem Cell Nucleofector Starter Kit(Lonza,货号VPH-5022,内含Supplement 1试剂和Solution 2试剂)
(4)吸弃TrypLE Select,用1mL mTeSR1 Plus培养基轻轻吹打细胞2-3次,使细胞完全脱落。
(5)用改良牛鲍氏计数板计数。
(6)取两个15mL离心管,将细胞悬液从孔板中等分移到离心管内,170g离心5min。
(7)取两个灭菌EP管,各加入18μL Supplement 1和82μL Solution 2,轻轻混匀,静置20min。
(8)向EP管内分别加入5μg miniIL15、266IL15质粒,再各加入5ug本发明人团队构建的人工核酸酶TALENickases-L和TALENickases-R质粒(TALE nickase mediates highefficient targeted transgene integration at the human multi-copy ribosomalDNA locus,Yong Wu,et al,Biochem Biophys Res Commun.2014Mar 28;446(1):261-6.doi:10.1016/j.bbrc.2014.02.099.见“2.2.Plasmids”段落。原文已列于附件。),轻轻混匀,静置10min。
(9)吸弃15mL离心管中的上清液体,用EP管中的混合溶液重悬离心管内的细胞沉淀,之后将细胞悬液小心转移至核转杯中。
(10)打开核转仪,将核转杯放至核转仪中,开始核转。
(11)核转完成后立即加入500μL mTeSR1 Plus,静置5min。
(12)吸弃包被孔板用的Matrigel。
(13)接种细胞悬液,加入10μM的Y27632。
(14)摇匀,放入细胞培养箱内静置,核转12h后换液(使用含10μM Y27632的mTeSR1Plus培养基)。
(15)核转24h后使用mTeSR1 Plus培养基换液。待细胞汇合度至80%时,加入含G418(Sigma,货号A1720)的培养基筛选培养。
2.获得266IL15定点整合的阳性单克隆
(1)取6cm细胞培养皿,加入稀释后的Matrigel至没过底部,室温静置12h以上。
(2)分别取出核转miniIL15、266IL15后的细胞,吹打后弃上清液。用1×DPBS润洗一次,加入TrypLE Select至没过底部,室温放置3min。
(3)吸弃TrypLE Select,用1mL mTeSR1 Plus培养基轻轻吹打细胞2-3次,使细胞完全脱落。
(4)用计数板计数。
(5)取一支15mL离心管,加入700μL cloneR补充剂(Stem Cell,货号05888)和7mLmTeSR1 Plus培养基,颠倒混匀。
(6)弃6cm细胞培养皿中的Matrigel,取3mL含cloneR补充剂的mTeSR1 Plus,向其中加入500个细胞。摇匀,放入细胞培养箱中培养。
(7)6cm培养皿中的细胞长至半个显微镜视野时,取48孔细胞培养板,加入稀释后的Matrigel至没过板底,室温包被过夜。
(8)弃孔板中的Matrigel,加入适量mTeSR1 Plus至没过底部。
(9)镜下选择6cm细胞培养皿中生长状态较好的单个细胞克隆,用小Tip将其挑至48孔细胞培养板内,做好标记,每孔接种一个克隆。放入细胞培养箱中培养。
(10)待单克隆细胞数量增殖较多时传代。使用FastPure Cell/Tissue DNAIsolation Mini Kit(Vazyme,货号DC102-01)提取单克隆细胞gDNA。
(11)按上述方法对跨上游同源臂区域序列进行PCR扩增、琼脂糖凝胶电泳、PCR产物测序。
所用到的引物及序列如下:
18S-2-F:GGCCCGAAGCGTTTACTTTGAA(SEQ ID NO:21)
screenL-R4:CTGCGTGCAATCCATCTTGTTC(SEQ ID NO:22)
根据电泳结果和测序结果(如图5所示),获得在rDNA区5468位点定点靶入含EF1α启动子的人IL15 CDS序列的阳性单克隆。使用G418筛选25天后,对核转miniIL15、266IL15的细胞分别挑取31个单克隆,用PCR做定点整合鉴定,并对PCR鉴定为阳性的单克隆进行测序确认。经鉴定,266IL15的定点整合效率为9/31(29.03%),miniIL15的定点整合效率为9/31(29.03%),即所使用的新型定点整合载体虽然为了提高同源性而缩短了下游同源臂,但定点整合效率并未受到影响。
3.qRT-PCR检测阳性单克隆的外源基因mRNA表达情况
(1)取阳性单克隆细胞用于抽提RNA,吸去上清后,1×DPBS润洗两次,每孔加1mL4℃预冷的Trizol(Invitrogen,货号15596026),等待2min后将细胞轻轻吹打下来,细胞悬液转移到无菌无酶的EP管内。
(2)2min后,向EP管内移入250μL氯仿,混匀后再放置5min。放入离心机内13000g离心10min。
(3)将上层液体移至新的无菌无酶的EP管内,做好标记。加入约0.5倍体积的异丙醇,混匀后再静置10min。放入离心机内13000g离心10min。
(4)吸弃上层液体,向管内加入500μL 75%的乙醇,混匀后静置片刻,放入提前预冷的低温离心机内,13000g离心10min。
(5)吸弃上清,风干沉淀。
(6)加20μL无菌无酶的ddH2O溶解,用Nanodrop 1000测定浓度和OD值,标记于管壁。
(7)将RNA浓度稀释到30ng/μL,使用HiScript II Q RT SuperMix for qPCR(+gDNA wiper)(Vazyme,货号R223-01)按操作说明书进行逆转录反应。
(8)向逆转录产物中每管加入80μL RNase-free ddH2O稀释cDNA,使用ChamQ SYBRqPCR Master Mix(Vazyme,货号Q711-02)进行后续qRT-PCR实验。
(9)qRT-PCR体系如下:
所用到的引物及序列如表4所示:
表4
按以下程序进行qRT-PCR反应:
(10)用Bio-Rad CFX Manager软件分析IL15基因的转录表达情况。
qRT-PCR检测结果显示,使用新型定点整合载体而获取的阳性单克隆外源基因mRNA含量较高,新型定点整合载体提高了外源基因的表达能力。
4.Western Blot检测阳性单克隆的外源基因表达情况
(1)待12孔板中定点整合miniIL15、266IL15的阳性单克隆细胞汇合度达90%,吸弃培养基,1×DPBS洗涤细胞两次;
(2)每孔加100μL细胞裂解液和1μL PMSF,用Tip轻轻刮下细胞,于冰上裂解30min;
(3)将细胞裂解液转移至EP管中。超声裂解,3-4s/次,每次间隔5s,共7-8次;
(4)于4℃下12000g离心10min;
(5)将离心后的上清转移到新的EP管中,用PierceTMBCA Protein Assay Kit(Thermo Scientific,货号23225)试剂盒按说明书进行BCA蛋白定量;
(6)制备SDS-PAGE胶:清洗玻璃板,用洗洁精清洗制胶玻璃板,清水冲洗,再用ddH2O冲洗,最后用电吹风吹干,利用SDS-PAGE凝胶配制试剂盒(翌圣生物科技(上海)股份有限公司,货号20328ES50)按说明书制备分离胶和浓缩胶;
(7)上样:将制好的SDS-PAGE胶置于电泳槽中,并配制新鲜的1×running buffer加入电泳槽中,将准备好的蛋白样品按照顺序加入到孔中;
(8)恒定电流80V电泳30min,之后120V电泳60min;
(9)转膜:预先配制好1×transfer buffer置于-20℃冰箱预冷,裁剪PVDF膜,并用甲醇浸泡5min,再置于转膜液中平衡20min,进行转膜操作;
(10)恒压252mA电泳90min;
(11)封闭:转膜完成后,将PVDF膜用5%脱脂牛奶浸泡,放在摇床封闭1h;
(12)将一抗用TBST稀释至适当浓度,覆盖PVDF膜,4℃孵育过夜,用TBST在室温下脱色摇床洗3次,每次10min;
(13)吸弃一抗,加入二抗,摇床孵育1h,TBST洗3次,每次10min;
(14)蛋白条带显影。
经Western Blot检测,结果如图6所示,以B6-hiPSCs的IL15蛋白表达量为1,其中定点整合266IL15的阳性单克隆266IL15 Clone 19、266IL15 Clone 22、266IL15 Clone 26的IL15蛋白相对表达量较高,分别为1.79±0.06、1.89±0.32、1.83±0.41,定点整合miniIL15的三个阳性单克隆IL15蛋白相对表达量分别为0.88±0.33、0.62±0.43、0.51±0.55。未定点整合IL15的克隆unintegrated Clone 40为阴性单克隆,IL15蛋白相对表达量0.59±0.24。即使用新型定点整合载体而获取的阳性单克隆外源基因蛋白表达量较高。
由此,本发明实施例中通过使用优化后的定点整合模板载体,改善了外源基因的表达能力。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神,本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明,但是应该理解所要求保护的本发明不应该被不适当地局限于这些具体实施方式。事实上,那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。
参考文献:
1.TALE nickase mediates high efficient targeted transgene integrationat the human multi-copy ribosomal DNA locus,Biochem Biophys ResCommun.2014Mar 28;446(1):261-6.doi:10.1016/j.bbrc.2014.02.099.
2.Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal StemCells for Targeted Cancer Therapy,Stem Cells Transl Med.2022Mar 31;11(3):297-309.doi:10.1093/stcltm/szab031。
Claims (15)
1.一种基因编辑构建体,所述构建体包括包含上游同源臂、下游同源臂以及上游同源臂和下游同源臂之间多克隆位点的构建体骨架;
所述上游同源臂的核苷酸序列如SEQ ID NO:2所示或与SEQ ID NO:2具有至少70%、至少80%、至少90%、至少95%或至少98%的序列同一性,所述下游同源臂的核苷酸序列如SEQ ID NO:4所示或与SEQ ID NO:4具有至少70%、至少80%、至少90%、至少95%或至少98%的序列同一性;和
所述构建体骨架为非病毒骨架。
2.根据权利要求1所述的构建体,其中所述上游同源臂的核苷酸序列如SEQ ID NO:2所示和所述下游同源臂的核苷酸序列如SEQ ID NO:4所示。
3.根据权利要求1或2所述的构建体,其包含如SEQ ID NO:27所示的核苷酸序列。
4.根据权利要求1至3任一所述的构建体,所述构建体进一步包含外源基因,所述外源基因位于所述多克隆位点,所述外源基因编码治疗性肽、DNA结合蛋白、RNA结合蛋白、荧光蛋白或酶。
5.根据权利要求4所述的构建体,其中所述治疗性肽选自人白细胞介素家族成员(例如,IL-2、IL-7、IL-10、IL-11、IL-12、IL-15、IL-23和IL-24)、肿瘤坏死因子家族成员(例如,TNF、LTA、LTB、FASLG、TNFSF8、TNFSF9、TNFSF10、TNFSF11、TNFSF12、TNFSF13、TNFSF14、TNFSF15、TNFSF18和EDA)、干扰素(INF-α、INF-β和INF-γ)、CAR、F8、F9、TNFR和TRAIL。
6.根据权利要求1至5任一所述的构建体,所述构建体进一步包含启动子,所述启动子位于所述多克隆位点,优选地,所述启动子为CMV启动子或EF1α启动子。
7.一种基因编辑方法,包括将权利要求1至6任一所述的构建体导入细胞,通过基因编辑***将外源基因定点整合入所述细胞的基因组中。
8.根据权利要求7所述的方法,其中所述基因编辑***选自Cre-lox***、Zinc FingerNucleases(ZFNs)、CRISPR-Cas9或Transcription Activator-Like Effector Nucleases(TALENs),优选为TALENs,更优选为采用人工核酸酶TALENickases进行基因编辑。
9.根据权利要求7或8所述的方法,其中所述定点整合位点位于基因组的核糖体RNA转录区(rDNA区)18S rRNA转录区的5468位点。
10.根据权利要求7至9任一所述的方法,其中所述细胞选自间充质干细胞、T细胞、B细胞、NK细胞、巨噬细胞或诱导性多能干细胞及其衍生细胞。
11.根据权利要求10所述的方法,其中所述诱导性多能干细胞的衍生细胞为由所述诱导性多能干细胞分化而来的间充质干细胞、T细胞、B细胞、NK细胞、巨噬细胞、造血细胞、内皮细胞、肝细胞、心肌细胞、神经元细胞或胰岛细胞。
12.根据权利要求7至11任一所述的方法,其中所述外源基因选自CAR基因、白细胞介素-15、白细胞介素-24、F8、F9、TNFR和TRAIL。
13.一种细胞,其由权利要求7至12任一所述的方法编辑后获得。
14.一种药物组合物,其包含根据权利要求1至6任一所述的构建体或权利要求13所述的细胞和药学上可接受的辅料。
15.根据权利要求1至6任一所述的构建体或权利要求13所述的细胞在制备***的药物中的用途。
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