CN104560864A - 利用CRISPR-Cas9***构建的敲除IFN-β基因的293T细胞系 - Google Patents
利用CRISPR-Cas9***构建的敲除IFN-β基因的293T细胞系 Download PDFInfo
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Abstract
本发明公开了利用CRISPR-Cas9***构建的敲除IFN-β基因的293T细胞系。本发明所提供的敲除IFN-β基因的293T细胞系,具体为人胚肾细胞293T-KO-IFN-β,它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC?No.10096。实验证明,本发明所得的敲除IFN-β基因的293T细胞系不能正确的表达IFN-β蛋白。由于IFN-β蛋白具有抗病毒作用,故B型流感病毒在野生型的293T细胞中不能大量的增殖。而敲除IFN-β基因的293T细胞系由于不能正确的表达IFN-β蛋白,故可用于B型流感病毒在该细胞系中更好的增殖,获得更大量病毒用于实验研究。
Description
技术领域
本发明属于生物技术领域,涉及一种利用CRISPR-Cas9***构建的敲除IFN-β基因的293T细胞系。
背景技术
293T细胞由293细胞衍生而出,293细胞是转染腺病毒E1A基因的人肾上皮细胞系,同时表达SV40大T抗原,含有SV40复制起始点与启动子区的质粒可以复制。用Ca3(PO4)2转染效率可高达50%。蛋白表达水平高,转染后2-3天用碱性磷酸酶分析可较容易地检测到表达的蛋白。瞬时转染293T细胞是过表达蛋白并获得细胞内及细胞外(分泌的或膜)蛋白的便捷方式。
IFN-β是在细菌、病毒、多聚肌苷酸多聚胞苷酸(Poly IC)、核苷酸等刺激物诱导下,主要由成纤维细胞、白细胞等产生的一种糖蛋白。HuIFN-β的基因均定位于人第9号染色体,HuIFN-β蛋白由166个氨基酸组成,属于糖基化蛋白,分子量约23KD,肽链中含3个Cys,分别在17、31和141位,其中31和141位的半胱氨酸之间形成的二硫键对HuIFN-β的生物学活性非常重要。IFN-β具有多种生物学功能,主要包括抗病毒、抑制某些细胞的生长、免疫调节及抑制和杀伤肿瘤细胞的功能。IFN-β的抗病毒效果针对不同的病毒,甚至是同一病毒的不同血清型而不同。其抗病毒的作用机理主要是通过两个方面实现:1)通过抑制某些病毒的吸附、脱衣壳和转录、病毒蛋白合成以及成熟病毒的释放来实现其抗病毒功能;2)通过增强自然杀伤NK细胞,单核巨噬细胞对病毒的吞噬作用来实现其抗病毒功能。IFN-β还可以抑制某些细胞的生长,如成纤维细胞、上皮细胞、内皮细胞以及造血细胞的增殖,其机理可能是通过使细胞***停留在G0/G1期,降低DNA的合成,下调细胞原癌基因的转录水平,下调某些生长因子受体表达。IFN-β还具有免疫调节作用,包括促进大多数细胞MHC-I类抗原的表达,活化NK细胞和杀伤性T淋巴细胞增强巨噬细胞的功能,调节T、B淋巴细胞的功能。IFN-β可抑制和杀伤肿瘤细胞,主要是通过促进机体的免疫功能,提高巨噬细胞、NK细胞和CT L的杀伤水平。
CRISPR-Cas9基因编辑技术是继ZFN和TALEN技术之后迅速发展起来的第三代基因组编辑技术,该技术来源于细菌和古细菌中存在抵抗噬菌体入侵的CRISPR-Cas获得性免疫***,经人工改造而逐渐发展起来。CRISPR是指规律成簇间隔短回文重复序列,Cas是指CRISPR相关蛋白。CRISPR-Cas基因组编辑技术是通过一段RNA来识别打靶位点,通过Cas核酸内切酶对打靶位点附近的核酸进行切割来实现对基因组的定点编辑,因此该技术也叫做RNA指导的核酸内切酶技术。与ZFN和TALEN技术相比,CRISPR-Cas基因组编辑技术在设计、合成与阳性克隆的筛选上都更为便捷,而且可以实现同时在一个细胞内对多个位点进行编辑,提高基因编辑的效率。
发明内容
本发明的一个目的是提供一种敲除IFN-β基因的293T细胞系。
本发明所提供的敲除IFN-β基因的293T细胞系具体为人胚肾细胞293T-KO-IFN-β,它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.10096。
本发明的第二个目的是提供一种基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的方法。
本发明所提供的基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的方法,是以序列表中序列1所示IFN-β基因序列中符合5’-GG-18N-NGG-3’或5’-GG-20N-NGG-3’或5’-CCN-18N-CC-3’或5’-CCN-20N-CC-3’序列排列规则的序列中的“18N”或“20N”所示序列为靶序列的;N为A或T或C或G。其中,18N为18个脱氧核糖核苷酸,20N为20个脱氧核糖核苷酸.
所述靶序列可为1-2个;当所述靶序列为2个时,2个所述靶序列之间的间隔优选为大于200bp。
在本发明的一个实施例中,所述靶序列为序列表中序列1所示IFN-β基因序列的第216-235位,(记为靶序列1);在本发明的另一个实施例中,所述靶序列为序列表中序列1所示IFN-β基因序列的第545-562位(记为靶序列2)。
更加具体的,所述方法为如下(A)或(B):
(A)包括如下步骤(a1)-(a4):
(a1)合成名称为正向单链DNA1和名称为反向单链DNA1的两个单链DNA;所述正向单链DNA1的序列如序列表中序列2所示,为在所述靶序列1的5’端加上ACCG;所述反向单链DNA1的序列如序列表中序列3所示,为在所述靶序列1的反向互补序列的5’端加上AAAC;
(a2)将所述正向单链DNA1和所述反向单链DNA1进行退火反应,得到双链DNA1(特异于靶序列1),该双链具有粘末端,其粘末端与BsaI酶切后的pGL-U6-gRNA质粒的酶切位点互补,可直接进行连接反应;
(a3)将所述双链DNA1连接到pGL-U6-gRNA质粒的限制性内切酶BsaI的切割位点处,得到的重组质粒记为pGL-U6-gRNA-IFNβ-1;
(a4)将所述pGL-U6-gRNA-IFNβ-1(Puromycin抗性)和Cas9质粒(Blasticidin抗性),共转染293T细胞,用Puromycin(3ug/ml)和Blasticidin(3ug/ml)进行抗性筛选,筛选时间为7天,然后换为正常的DMEM培养基,从转染后的293T细胞中获得IFN-β基因被敲除的293T细胞系;
(B)包括如下步骤(b1)-(b4):
(b1)合成名称为正向单链DNA2和名称为反向单链DNA2的两个单链DNA;所述正向单链DNA2的序列如序列表中序列4所示,为在所述靶序列2的反向互补序列的5’端加上ACCG;所述反向单链DNA2的序列如序列表中序列5所示,为在所述靶序列2的5’端加上AAAC;
(b2)将所述正向单链DNA2和所述反向单链DNA2进行退火反应,得到双链DNA2(特异于靶序列2),该双链具有粘末端,其粘末端与BsaI酶切后的pGL-U6-gRNA质粒的酶切位点互补,可直接进行连接反应;
(b3)将所述双链DNA2连接到pGL-U6-gRNA质粒的限制性内切酶BsaI的切割位点处,得到的重组质粒记为pGL-U6-gRNA-IFNβ-2;
(b4)将所述pGL-U6-gRNA-IFNβ-2(Puromycin抗性)和Cas9质粒(Blasticidin抗性),共转染293T细胞,用Puromycin(3μg/ml)和Blasticidin(3μg/ml)进行抗性筛选,筛选时间为7天,然后换为正常的DMEM培养基,从转染后的293T细胞中获得IFN-β基因被敲除的293T细胞系;
在所述方法的步骤(a2)和(b2)中,所述退火反应的条件均可为:97℃作用7min,然后关机,自然降温1h。
在所述方法的步骤(a4)和(b4)中,将所述pGL-U6-gRNA-IFNβ-1(或所述pGL-U6-gRNA-IFNβ-2)和Cas9质粒共转染293T细胞时,所述pGL-U6-gRNA-IFNβ-1(或所述pGL-U6-gRNA-IFNβ-2)和所述Cas9质粒的质量比可为1:1。
利用所述方法制备得到的IFN-β基因被敲除的293T细胞系也属于本发明的保护范围。
所述IFN-β基因被敲除的293T细胞系具体为所述人胚肾细胞293T-KO-IFN-βCGMCC No.10096。
本发明的第三个目的是提供一种成套质粒。
本发明所提供的成套质粒由所述pGL-U6-gRNA-IFNβ-1(或所述pGL-U6-gRNA-IFNβ-2)和所述Cas9质粒组成。
其中,所述成套质粒中的两种质粒可以分别单独包装,也可以按照质量比为1:1的比例混合包装。
所述成套质粒的用途也属于本发明的保护范围。
所述用途具体为基于CRISPR-Cas9敲除293T细胞中IFN-β基因(序列1)。
本发明的第四个目的是提供一种基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的试剂盒。
本发明所提供的基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的试剂盒,含有所述成套质粒及说明书;所述说明书中记载有如上所述的基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的方法。
所述人胚肾细胞293T-KO-IFN-β,或利用所述方法制备得到的IFN-β基因被敲除的293T细胞系在扩增B型流感病毒中的应用也属于本发明的保护范围。
本发明的优点在于:敲除IFN-β基因的293T细胞系,或缺少IFN-β基因的一部分,或由于敲除或***某些片段而引起移码突变,故不能正确的表达IFN-β蛋白。由于IFN-β蛋白具有抗病毒作用,故B型流感病毒在野生型的293T细胞中不能大量的增殖。而敲除IFN-β基因的293T细胞系由于不能正确的表达IFN-β蛋白,故可用于B型流感病毒在该细胞系中更好的增殖,获得更大量病毒用于实验研究。
保藏说明
参椐的生物材料:293T-KO-IFN-β
科学描述:人胚肾细胞
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2014年12月1日
保藏中心登记入册编号:CGMCC No.10096
附图说明
图1为PCR鉴定结果。
图2为Westren-Blotting鉴定结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
pGL-U6-gRNA质粒:将gRNA人工***该质粒后组成的重组质粒具有导向作用,可以与所要编辑的基因组中相应的位置进行结合,起到Guiding的作用。记载于“MaY,Ma J,Zhang X,Chen W,Yu L,Lu Y,Bai L,Shen B,Huang X,Zhang L.2014.Generation of eGFP and Cre knockin rats by CRISPR/Cas9.FEBS J.Jul 17.doi:10.1111/febs.12935.”一文,公众可从中国科学院微生物研究所获得。
Cas9质粒:具有核酸内切酶的功能,该Cas9质粒可以通过与pGL-U6-gRNA的结合后到达所要编辑的基因组的位置实现其对基因组的切割功能。记载于“Ma Y,Zhang X,Shen B,Lu Y,Chen W,Ma J,Bai L,Huang X,Zhang L.2014.Generating ratswith conditional alleles using CRISPR/Cas9.Cell Res.Jan;24(1):122-5.”一文,公众可从中国科学院微生物研究所获得。
pGL-U6-gRNA质粒和Cas9质粒上都带有药物抗性,通过药物筛选,可获得基因敲除的细胞系。
大肠杆菌TOP10:购自Invitrogen公司和货号C4040-10。
293T细胞:记载于“Sh Cao,X-L Liu,M-R Yu,J Li,X-J Jia,Y-H Bi,L Sun,George F.Gao,W-J Liu*.2012.A Nuclear Export Signal in the Matrix Protein of Influenza A Virus IsRequired for the Efficient Virus Replication.Journal of Virology,86(9):4883-91。”一文,公众可从中国科学院微生物研究所获得。
实施例1、敲除IFN-β基因的293T细胞系的构建
一、待敲除基因靶序列的确定及DNA oligo引物的设计
1、待敲除基因靶序列的确定
以HuIFN-β基因序列(序列1,无内含子)为待敲除基因,利用DNAStar软件中的Editseq软件对HuIFN-β基因序列进行分析,在此基础上人工筛选合适的靶序列。对于HuIFN-β基因全序列本发明的发明人最终设计了两个靶序列,分别为序列表中序列1的第216-235位(GAGGCTTGAATACTGCCTCA,记为靶序列1)和序列1的第545-562位(AGGAGTACAGTCACTGTG,记为靶序列2)。
在进行靶序列确定时,本发明的发明人是按照如下原则进行的:
(1)优先选择HuIFN-β基因全序列(序列1)中符合5’-GG-18N-NGG-3’(N代表A或T或C或G)或5’-GG-20N-NGG-3’序列排列规律的序列中的“18N”或“20N”所示序列作为靶序列。若所要敲除的基因的全序列中没有上述两种序列,也可选择上述两种序列的互补序列5’-CCN-18N-CC-3’或5’-CCN-20N-CC-3’基序,将其方向互补后使用。
(2)一般选择外显子上的符合(1)中要求的序列为靶序列,一般同一个敲除的细胞系同时设立两对符合要求的靶序列。
(3)选择两对符合要求的靶序列时两者最好要间隔一定的距离(>200bp)。
(4)所选择的靶序列最好位于所翻译的蛋白质的较重要的功能结构域,这样能更好的保证敲除效果。
2、DNA oligo引物的设计
根据步骤1确定的靶序列,设计两对DNA oligo引物,具体序列如下:
合成了两对寡核苷酸引物用于制备gRNA,序列如下:
针对靶序列1的DNA oligo引物:
HuIFNβ-gRNA-UP1:5’-ACCGGAGGCTTGAATACTGCCTCA-3’(序列2);
HuIFNβ-gRNA-DOWN1:5’-AAACTGAGGCAGTATTCAAGCCTC-3’(序列3)。
针对靶序列2的DNA oligo引物:
HuIFNβ-gRNA-UP2:5’-ACCGCACAGTGACTGTACTCCT-3’(序列4);
HuIFNβ-gRNA-DOWN2:5’-AAACAGGAGTACAGTCACTGTG-3’(序列5)。
二、表达gRNA的重组质粒的构建
1、寡核苷酸单链退火获得双链寡核苷酸
将合成的针对靶序列1的两条DNA oligo单链(序列2和序列3)退火,得到双链双链寡核苷酸1。
将合成的针对靶序列2的两条DNA oligo单链(序列4和序列5)退火,得到双链双链寡核苷酸2。
退火反应体系及退火反应条件均如下:
退火反应体系:HuIFNβ-gRNA-UP1或HuIFNβ-gRNA-UP2(浓度10mM)20μl;HuIFNβ-gRNA-DOWN1或HuIFNβ-gRNA-DOWN 2(浓度10mM)20μl;NEB buffer210μl。
退火反应条件:将上述体系混合均匀后PCR仪中,97℃作用7min,然后关机,自然降温,1h后取出样品进行2%的琼脂糖凝胶电泳检测并切胶回收退火产物,所用回收试剂盒为寡核苷酸回收试剂盒(天根公司),具体操作步骤严格按其说明书进行。
2、酶切及连接反应
用去内毒素的质粒提取试剂盒提取pGL-U6-gRNA质粒,然后用限制性核酸内切酶BSAI进行酶切,酶切条件为37℃切4h。将酶切后的质粒进行1%的琼脂糖凝胶电泳检测酶切效果并进行胶回收,胶回收所用试剂盒为博迈德公司产品。
因退火产物直接含有与pGL-U6-gRNA载体酶切位点互补的粘末端,故可以直接用于连接实验。
将胶回收后的酶切质粒与胶回收退火产物进行连接反应,具体体系及条件如下:
连接体系:胶回收后的退火产物(双链寡核苷酸1或双链寡核苷酸2)6μl;胶回收后的酶切pGL-U6-gRNA载体2μl;连接酶1μl;10×连接酶Buffer 1μl。
连接条件:PCR仪中16℃连接过夜。
3、转化
于超净工作台中将10μl连接产物加入50μl的大肠杆菌TOP10感受态细胞中,冰浴30min,然后42℃水浴中热激90sec,接着冰浴5min,然后加入500μl无抗LB液体培养基后,37℃,200rpm震荡培养40min后,涂布氨苄抗性的LB固体平板,37℃培养箱中培养过夜。待出现单菌落后,挑取5个大小适中的菌落,提取质粒,交由博迈德公司进行测序。
将经测序表明在pGL-U6-gRNA质粒的酶切位点BSAI处***
“5’-GAGGCTTGAATACTGCCTCA-3’”所示DNA片段的重组质粒命名为
pGL-U6-gRNA-IFNβ1;将经测序表明在pGL-U6-gRNA质粒的酶切位点BSAI处***
“5’-CACAGTGACTGTACTCCT-3’”所示DNA片段的重组质粒命名为
pGL-U6-gRNA-IFNβ2。
4、重组质粒的提取
将测序正确的克隆进行扩大培养,用去内毒素的质粒提取试剂盒(天根公司)提取重组质粒并测定质粒浓度,重组质粒的具体操作步骤严格按照说明书进行。
三、转染及单克隆的筛选
将步骤二构建的重组质粒pGL-U6-gRNA-IFNβ1(或pGL-U6-gRNA-IFNβ2)与Cas9质粒共转293T细胞,具体操作步骤如下:
(1)将293T细胞分至10cm细胞培养皿后培养过夜,所用培养基为含10%(体积分数)FBS的DMEM培养基,待次日早细胞汇合度达到70%左右时进行下步操作。
(2)将细胞汇合度达到70%的细胞换液至opti-MEM中,2h后进行转染。然后将两种质粒及Lip2000分别稀释至250μl的opti-MEM中,然后将稀释后的Lip2000加入稀释后的混合质粒中,轻轻混匀,静置20min后,轻轻滴加提前换至opti-MEM培养基的细胞中,置于37℃细胞培养箱中进行培养,4-6h后将细胞换液至含10%(体积分数)FBS的DMEM培养基中继续培养。所转染的两种质粒浓度为分别12μg/10cm皿,质粒与Lip的比例为1:2.5,即加24μg的混合质粒,则需要使用的Lip2000的量为60μl。
(3)待加完质粒24h后,将细胞换液至含3μg嘌呤霉素和3μg杀稻瘟菌素的10%(体积分数)FBS的DMEM培养基中,培养3-5天,每个24h换一次新鲜的抗性培养基。
(4)待3-5天出现单克隆后,换为抗性减半的培养基维持2-3天,之后换为不加抗性含10%(体积分数)FBS的DMEM的培养基进行培养。
(5)待细胞形成单克隆后,有限稀释法将细胞稀释至96孔板中,培养7天后挑取单克隆孔,传代至24孔板中进行扩大培养。24孔板中培养4-5天后,胰酶消化传代至12孔板中,每个单克隆传2个孔,一个孔用于扩大培养至6孔板以方便后续的PCR检测,一个孔用于留存。
(6)PCR鉴定阳性克隆
将扩大至6孔板的单克隆进行细胞基因组DNA的提取,所用试剂盒为天根公司生产。
用于PCR鉴定共转染了重组质粒pGL-U6-gRNA-IFNβ1与Cas9质粒的293T细胞的引物序列如下:
PCR-KOIFN-β-up1:5’-TCTAACTGCAACCTTTCGAAGCC-3’(序列1的第5-27位);
PCR-KOIFN-β-down1:5’-CCAGGACTGTCTTCAGATGGTTT-3’(序列1的第423-445位的反向互补序列)。
采用PCR-KOIFN-β-up1/PCR-KOIFN-β-down1引物对扩增得不到大小为441bp目的条带(序列1的第5-445位)的克隆为阳性。
用于PCR鉴定共转染了重组质粒pGL-U6-gRNA-IFNβ2与Cas9质粒的293T细胞的引物序列如下:
PCR-KOIFN-β-up2:5’-GCATTGACCATCTATGAGATGCT-3’(序列1的第304-326位);
PCR-KOIFN-β-down2:5’-CTTCTAGTGTCCTTTCATATGCAG-3’(序列1的第731-754位的反向互补序列)。
采用PCR-KOIFN-β-up2/PCR-KOIFN-β-down2引物对扩增得不到大小为451bp目的条带(序列1的第304-754位)的克隆为阳性。
实验同时以野生型的293T细胞系基因组为模版作为对照组。
结果显示,以野生型293T细胞系基因组为模版的对照组,采用PCR-KOIFN-β-up1/PCR-KOIFN-β-down1引物对扩增得得到了大小为441bp目的条带(序列1的第5-445位),采用PCR-KOIFN-β-up2/PCR-KOIFN-β-down2引物对扩增得了大小为451bp目的条带(序列1的第304-754位)。而共转染了重组质粒pGL-U6-gRNA-IFNβ1与Cas9质粒的293T细胞,以及共转染了重组质粒pGL-U6-gRNA-IFNβ2与Cas9质粒的293T细胞均获得了阳性克隆。其中,部分采用PCR-KOIFN-β-up1/PCR-KOIFN-β-down1引物对鉴定共转染了重组质粒pGL-U6-gRNA-IFNβ1与Cas9质粒的293T细胞阳性克隆的结果如图1所示,泳道1为以野生型293T细胞系基因组为模版的对照组;泳道2为敲除了部分HuIFN-β基因后的293T细胞系的阳性克隆(即本发明最终获得的保藏细胞293T-KO-IFN-β)的扩增结果。
(7)Western-blot鉴定IFN-β的表达情况
①将所要鉴定的细胞系和野生型的293T细胞传代至24孔板中,培养过夜,所用培养基为含10%(体积分数)FBS的DMEM培养基,待次日早细胞汇合度达到70%左右时进行下步操作。
②用A型流感病毒的PR8株(A/H1N1/PR8)(记载于“钟菊迎,崔晓兰,时宇静等.金柴抗病毒胶囊防治甲型H1N1流感病毒PR8株感染小鼠肺炎的实验研究.世界中西医结合杂志,2010年第5卷第4期”一文)感染所要检测的细胞系,感染剂量为MOI=0.1,感染前和感染后12h分别取样。将细胞沉淀中加入预冷的Lysis buffer(配方:1%体积分数的Triton X100,150mM的NaCl,20mM的Hepes,10%体积分数的甘油,1mM的EDTA,pH7.4),每孔100μl,用细胞刮刮下细胞后,置入1.5ml的EP管中,离心后弃细胞沉淀,上清备用。
③将准备好的样品进行Western-blot分析。具体操作步骤为:
A.按照《分子克隆》所述的方法进行SDS-PAGE。用预染的蛋白质分子量Marker。电泳完毕后,将凝胶放入电转缓冲液中平衡2min后进行转膜。
B.按照凝胶的大小剪好六张3mm厚滤纸,浸泡于转移缓冲液中。剪一张比滤纸稍大的PVDF膜,在甲醇浸泡5min以上,电转液平衡10min后使用。在半干转膜仪中依次放置3层厚滤纸、转印膜、凝胶、3层滤纸,注意赶走各层间气泡。15V电转20min。
C.切勿使转移的转印膜变干,转膜结束后快速放于适量封闭液中封闭。室温轻摇1h。
D.将转印膜放入杂交袋内,按照0.1ml/cm2用量分别加入用封闭液适当稀释的鼠抗HuIFNβ一抗(Santa Cruz公司产品,目录号:sc-17565,1:1000稀释),除尽气泡,封口。在室温下置于脱色摇床上轻摇孵育2h左右(或于4℃轻摇过夜),使抗体充分结合。将膜去除后放入TBST溶液中,洗膜3次,每次7min。
E.将膜放入杂交袋内,加入用封闭液配制的辣根过氧化物酶(HRP)标记的相应羊抗鼠IgG二抗(Santa Cruz公司产品,目录号:sc-166261,1:5000稀释)。赶除气泡封口,室温振荡2h。
F.取出转印膜,用TBST洗三次,每次10min。
G.取发光液(天根公司产品)A液、B液各0.5ml等体积混合均匀。将转印膜轻轻接触滤纸吸干液体,有蛋白质的一面朝上放在保鲜膜上。将ECL混合液滴加到转印膜上,反应3min。立即用保鲜膜包裹PVDF膜,注意表面要平整,放入CLINX化学凝胶成像仪中成像。
结果如图2所示,泳道1为以敲除部分HuIFN-β基因后的293T细胞系感染前0h的结果(无目的条带),泳道2为以敲除部分HuIFN-β基因后的293T细胞系感染后12h的结果(无目的条带),泳道3为野生型的293T细胞系感染前0h的结果(无目的条带),泳道4为野生型的293T细胞系感染后12h的结果(得到大小约为21KD的目的条带,与预期大小一致)。从结果可以看出,敲除部分HuIFN-β基因后的293T细胞系(即本发明最终获得的保藏细胞293T-KO-IFN-β)在病毒刺激后12h仍然检测不到HuIFN-β蛋白的表达,表明该细胞系敲除成功。
本发明的发明人将其中一个经以上PCR鉴定和Western-blot鉴定均为阳性的克隆(共转染了重组质粒pGL-U6-gRNA-IFNβ1与Cas9质粒的293T细胞)于2014年12月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号),参椐的生物材料:293T-KO-IFN-β,科学描述:人胚肾细胞,保藏中心登记入册编号:CGMCC No.10096。
Claims (10)
1.人胚肾细胞293T-KO-IFN-β,它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.10096。
2.一种基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的方法,其特征在于:所述方法以序列表中序列1所示IFN-β基因序列中符合5’-GG-18N-NGG-3’或5’-GG-20N-NGG-3’或5’-CCN-18N-CC-3’或5’-CCN-20N-CC-3’序列排列规则的序列中的“18N”或“20N”所示序列作为靶序列;N为A或T或C或G。
3.根据权利要求2所述的方法,其特征在于:所述靶序列为序列表中序列1所示IFN-β基因序列的第216-235位或第545-562位。
4.根据权利要求3所述的方法,其特征在于:所述方法为如下(A)或(B):
(A)包括如下步骤(a1)-(a4):
(a1)合成名称为正向单链DNA1和名称为反向单链DNA1的两个单链DNA;所述正向单链DNA1的序列如序列表中序列2所示;所述反向单链DNA1的序列如序列表中序列3所示;
(a2)将所述正向单链DNA1和所述反向单链DNA1进行退火反应,得到双链DNA1;
(a3)将所述双链DNA1连接到pGL-U6-gRNA质粒的限制性内切酶BsaI的切割位点处,得到的重组质粒记为pGL-U6-gRNA-IFNβ-1;
(a4)将所述pGL-U6-gRNA-IFNβ-1和Cas9质粒共转染293T细胞,从转染后的293T细胞中获得IFN-β基因被敲除的293T细胞系;
(B)包括如下步骤(b1)-(b4):
(b1)合成名称为正向单链DNA2和名称为反向单链DNA2的两个单链DNA;所述正向单链DNA2的序列如序列表中序列4所示;所述反向单链DNA2的序列如序列表中序列5所示;
(b2)将所述正向单链DNA2和所述反向单链DNA2进行退火反应,得到双链DNA2;
(b3)将所述双链DNA2连接到pGL-U6-gRNA质粒的限制性内切酶BsaI的切割位点处,得到的重组质粒记为pGL-U6-gRNA-IFNβ-2;
(b4)将所述pGL-U6-gRNA-IFNβ-2和Cas9质粒共转染293T细胞,从转染后的293T细胞中获得IFN-β基因被敲除的293T细胞系。
5.根据权利要求4所述的方法,其特征在于:步骤(a2)和步骤(b2)中,所述退火反应的条件均为:97℃作用7min,自然降温1h;或
步骤(a4)中,将所述pGL-U6-gRNA-IFNβ-1和Cas9质粒共转染293T细胞时,所述pGL-U6-gRNA-IFNβ-1和所述Cas9质粒的质量比为1:1;或
步骤(b4)中,将所述pGL-U6-gRNA-IFNβ-2和Cas9质粒共转染293T细胞时,所述pGL-U6-gRNA-IFNβ-2和所述Cas9质粒的质量比为1:1。
6.利用权利要求2-5中任一所述方法制备得到的IFN-β基因被敲除的293T细胞系;
所述IFN-β基因被敲除的293T细胞系具体为权利要求1所述的人胚肾细胞293T-KO-IFN-β。
7.成套质粒,由权利要求4中所述pGL-U6-gRNA-IFNβ-1和所述Cas9质粒组成,或由权利要求4中所述pGL-U6-gRNA-IFNβ-2和所述Cas9质粒组成。
8.权利要求7所述成套质粒的用途,其特征在于:所述用途为基于CRISPR-Cas9敲除293T细胞中的IFN-β基因。
9.基于CRISPR-Cas9构建敲除IFN-β基因的293T细胞系的试剂盒,含有权利要求7所述的成套质粒及说明书;所述说明书中记载有权利要求2-6中任一所述的方法。
10.权利要求1所述的人胚肾细胞293T-KO-IFN-β,或权利要求6所述的IFN-β基因被敲除的293T细胞系在扩增B型流感病毒中的应用。
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